A Study of the Effect of Astringents on the Permeability of Frog Membrane Using Radioactive Tracer Techniques

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PU R D U E UNIVERSITY

T H I S I S T O C E R T IF Y T H A T T H E T H E S I S P R E P A R E D U N D E R M Y S U P E R V I S I O N

_________ R u s s e ll Eugene Lux,

e n tttle d

jJ iv

A STUDY OF THE EFFECT OF ASTRINGENTS ON THE PERME­

ABILITY OF FROG MEMBRANE USING RADIOACTIVE TRACER TECHNIQUES

C O M P L I E S W IT H T H E U N I V E R S I T Y R E G U L A T I O N S O N G R A D U A T I O N T H E S E S

A N D IS A P P R O V E D B Y M E A S F U L F IL L IN G T H IS P A R T O F T H E R E Q U IR E M E N T S

FOR THE DEG REE OF

Doc t o r o f P-h ilo so p h y -

P r o fesso r

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J a n u a ry 14

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T H IS T H E S IS IS N O T TO B E R E G A R D E D A S C O N F ID E N T IA L .

PRO FESSO R

G R A D . SCH O O L, FO R M

O—3 . 4 0 —1 M

OT C H A R G E

A STUDY OF THE EFFECT OF ASTRINGENTS ON THE PERMEABILITY OF FROG MEMBRANE USING RADIOACTIVE TRACER TECHNIQUES

A T h e s is S u b m itted t o t h e F a c u lty of P urdue U n iv e r s ity by R u s s e ll Eugene Lux I n P a r t i a l F u lf illm e n t o f th e R eq u irem ents f o r th e D egree of D o cto r o f P h ilo so p h y

J a n u a ry , 1950

ProQuest Number: 27712246

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted. In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion.

uest ProQuest 27712246 Published by ProQuest LLC (2019). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346

ACKNOWLEDGEMENT The w r i t e r w ish es t o e x p r e s s h i s s in c e r e a p p r e c ia tio n t o P r o f e s s o r Jo h n E . C h r i s t i a n , u n d e r whose s u p e r v is io n t h i s work was c a r r i e d o u t.

H is tim e ly s u g g e s tio n s and co m p lete c o o p e r a tio n were

an in v a lu a b le a id i n c o m p le tin g t h i s w ork. The w r i t e r i s a l s o in d e b te d t o Mr. P a u l S c o t t o f t h e Pharmacy D epartm ent f o r h i s u n s e l f i s h a s s i s t a n c e i n p r e p a rin g th e r a d io ­ a u to g ra p h s , and t o D r. D. J . Tendam o f th e P urdue P h y s ic s D epartm ent f o r h i s p r e p a r a tio n o f N a ^ and r e p a i r o f e l e c t r o n i c eq u ip m en t. The w r i t e r a l s o w ish e s t o e x p r e s s a n o te o f th a n k s t o D r. F . D. M a rtin o f th e C h em istry D ep artm en t, D r. I . W. B u rr o f t h e M ath em atics D ep artm en t, and D r. L . D. Edwards o f th e Pharmacy D ep artm en t. The w r i t e r a l s o w ish es t o e x p r e s s h is g r a t i t u d e t o th e P h a m a c r a f t C o rp o ra tio n whose f i n a n c i a l a s s i s t a n c e made t h i s r e s e a r c h p o s s ib le .

TABLE OF CONTENTS Page 1

INTRODUCTION........................................................................................... LITERATURE.......................................

»............

3

EXPERIMENTAL......................................................................... A.

G e n e ra l and S p e c i f ic C o n s id e ra tio n s i n D evelop­ in g an E x p e rim e n ta l M ethod G en e ra l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S p e c if ic

15 15 17

S ta n d a r d iz a tio n o f t h e I s o to p e and C ounting T echnique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

22

C.

P re v e n tio n

24

D.

C hoice and C are o f E x p e rim e n ta l A nim als . . . . . . . . . . . .

26

E.

D e f i n i ti o n

26

B*

o f C o n tam in atio n . . . . . . . . . . . . . . . . . . . . . . . .

o f Terms

.......

28

THE "NORMAL" PERMEABILITY OF FROG MEMBRANE........................................... THE PERMEABILITY CONSTANT OF FROG MEMBRANE........................................*

36

THE EFFECT OF A SATURATED DEXTROSE SOLUTION ON FROG MEMBRANE PERMEABILITY........................................................... ATTEMPTS TO DEVELOP A TECHNIQUE FOR EVALUATING THE EFFECT OF AN ASTRINGENT ON FROG MEMBRANE PERMEABILITY....................................

45

THE QUALITATIVE EFFECTS OF AN ASTRINGENT ON FROG MEMBRANE PERMEABILITY.............................................................................................

49

A QUANTITATIVE STUDY OF THE EFFECT OF ASTRINGENTS ON FROG MEMBRANE PERMEABILITY ....................................................... THE PERMEABILITY OF FROG MEMBRANE TO Na2^ ................................................

57 79

THE STRUCTURE OF FROG MFMBRANE.....................................................

84

SUMMARY AND CONCLUSIONS........................................................................ BIBLIOGRAPHY..............................................................................................................

92 94

LISTS OF TABLES AND FIGURES L i s t o f T a b le s T able 1

Page The T ra n s p o rt o f I o d id e Io n Through F rog Membrane U sing l l 3 l As T r a c e r . The Normal P e r m e a b ility Of F rog Membrane

29

2

The P e r m e a b ility C o n sta n t o f F rog Membrane

37

3

The E f f e c t o f a S a tu r a te d D ex tro se S o lu tio n on t h e P e r m e a b ility o f F rog Membrane . . . . . . . . . . . . .

....

43

The Q u a l i t a t i v e E f f e c t o f an A s trin g e n t on Membrane P e r m e a b ility i n Osmotic System s . . . . . . . . . . . . . . . . . . . .

43

The E f f e c t o f F iv e P e r c e n t Aluminum C h lo rid e on Membrane P e r m e a b ility

54

6

T a b u la tio n o f E x p e rim e n ta l D ata-2.5% AlClg . . . . . . . . . .

64

7

T a b u la tio n o f S t a t i s t i c a l Q u a n tit ie s Used i n F in d ­ in g S lo p e s o f D e lta P lo ts f o r 2 .5 $ AICI3 . . . . . . . . . . .

69

8

T a b u la tio n o f

E x p e rim e n ta l D a ta -5 ^ AICI3 . . . . . . . . . . . .

70

9

T a b u la tio n o f

E x p e rim e n ta l Data-10% AlCl^ . . . . . . . . . .

71

10

T a b u la tio n o f E x p e rim e n ta l D a ta - 2 . 5% A1 M e th io n a t e ...

71

11

T a b u la tio n o f E x p e rim e n ta l Data-10% A1 M e t h i o n a t e . .. .

73

12

T a b u la tio n o f E x p e rim e n ta l D ata-1.5%

....

73

13

T a b u la tio n o f E x p e rim e n ta l D ata-2.5% AJ^CSO^)3 . . ♦ • • • •

75

14

The T ra n s p o rt o f Sodium Io n Through Frog Membrane U sing N a ^ As T r a c e r . . . . . . . . . . . . . . . . . . . . . . . .

80

4 5

L i s t o f F ig u re s F ig u re 1 2

Page The F am ily o f Curves O b tain ed From M easurem ents o f t h e ”N o m a ln P e r m e a b ility o f 12 F ro g s . . . . . . . . . . . .

31

The L o g a rith m ic P lo t F o r t h e C a lc u la tio n o f t h e P e r m e a b ility C o n stan t o f F rog Membrane . . . . . . . . . . . . . . .

40

L i s t o f F ig u re s (C o n tin u ed ) F ig u re 3

Page A G rap h ic R e p r e s e n ta tio n S u g g e s tin g a M anner By Which an A s tr in g e n t Could I n c r e a s e F rog Membrane P e r m e a b ility .............

55

The S e tu p Used I n S tu d y in g C om parative Perme­ a b ilitie s

58

P lo t o f O r i g in a l D ata O b tain ed i n a T y p ic a l B x p erim en t-2 » 5% AlClg • • • • • • • • • • • • • • • • • • • • • ♦ • • • ♦ • • ♦

60

6

The D e lta P l o t f o r F ig u re 5

62

7

A verage D e lta P lo t f o r T hree P e rc e n ta g e s o f A lC l^.#

63

Ô

The V a r ia tio n o f P e rc e n t A s trin g e n t W ith A verage S lo p e o f t h e D e lta P l o t - A lC lj •♦ ••••••••# # # •

72

The V a r ia tio n o f P e r c e n t A s trin g e n t W ith Average S lo p e o f t h e D e lta P lo t- A l M e th io n a te • « • • • • » • • • • • *

74

The V a r ia tio n o f S m all P e rc e n ta g e s o f A s tr in g e n t W ith Average S lo p e o f th e D e lta P lo t- A l^ ( S O ^ ) ^ .»**

76

11

A H is to l o g i c a l S e c tio n o f Frog Membrane * .* * ..# .» » *

85

12

R a d io a u to g ra p h s Showing L o c a liz a tio n o f R adio­ a c t i v i t y on t h e O u ter Membrane L ay ers • • • • • • • • • • • • •

88

R a d io a u to g ra p h s Showing t h e S i e v e - li k e P e n e tr a tio n o f Frog Membrane; The E f f e c t o f Membrane W rin k lin g .

90

4 5

9 10

13

X INTRODUCTION The p e r m e a b ility o f a membrane i s a phenomena o f p rim ary im p o rta n c e t o a l l b i o l o g i c a l organism s*

The c o n tin u e d e x is te n c e and t h e s u c c e s s ­

f u l f u n c t io n o f a c e l l i s d ep en d en t upon t h e a b i l i t y o f t h e c e l l mem­ b ra n e (p e rh a p s o n e - m illio n th o f a cm* i n t h i c k n e s s ) t o p e rm it t h e p a ssa g e o f some s u b s ta n c e s and p re v e n t t h a t o f o th e rs *

The f u n c tio n in g o f th e

r e d -b lo o d c e l l and t h e g lo m e ru lu s o f th e k id n e y a r e exam ples o f p ro ­ c e s s e s e s s e n t i a l t o l i f e imhich a r e m a n if e s ta tio n s o f th e s e l e c t i v e p e r m e a b ility o f c e r t a i n membranes* By m easu rin g th e p e r m e a b ility o f c e l l s t o v a r io u s s u b s ta n c e s in fo rm a tio n i s o b ta in e d w hich e n a b le s u s t o p r e d ic t th e r a t e s a t which v a r io u s ty p e s o f m o le c u le s p e n e tra te *

T hus, i n c o n s id e rin g a group

o f d ru g s , we a r e g u id ed i n o u r s e l e c t i o n o f th o s e w hich p e n e tr a te r a p i d l y and th o s e n o t a t a l l .

We a r e a b le t o d e f in e t h a t group o f

d ru g s w hich m ust t h e r e f o r e e x e r t t h e i r a c t i o n on a c e l l s u r f a c e and th o s e w hich p ro d u ce t h e i r a c t i o n on t h e i n t e r i o r o f t h e c e l l *

I t is

a p p a re n t t h a t t h e p ro b lem s e n c o u n te re d may b e e x c e e d in g ly com plex and a r e p e c u l i a r l y demanding upon e x p e rim e n ta l m ethods b y v i r t u e o f t h i s c o m p le x ity . There a r e numerous s ta te m e n ts i n t h e l i t e r a t u r e by acknow ledged a u t h o r i t i e s t h a t t h e e a r l i e r m ethods o f s tu d y in g p e r m e a b ility problem s b y c h e m ic a l, e l e c t r i c a l , s p e c tro s c o p ic o r volume change m ethods were o f te n h ig h ly in a c c u r a te and s u b je c t t o p o o r c o n tr o l*

C e r ta in o f th e s e

a u t h o r i t i e s have re c o g n iz e d th e p o t e n t i a l u t i l i t y o f r a d i o a c ti v e t r a c e r s i n s tu d y in g membrane p e r m e a b ility .

The p r e s e n t a v a i l a b i l i t y

o f r a d i o a c t i v e is o to p e s r e p r e s e n ts e s s e n t i a l l y a new i n v e s t i g a t i o n a l

2 t o o l w hich com bines t h e a t t r i b u t e s o f co n v e n ie n c e and h ig h p r e c i s i o n w ith t h e p o s s i b i l i t y o f c a r r y in g o u t e x p e rim e n ts s u b je c t t o a h ig h d e g re e o f c o n t r o l and w ith t h e r a p i d a tta in m e n t o f r e s u l t s . The p r e s e n t s tu d y was u n d e rta k e n t o c o n t r ib u t e t o th e fu n d a m e n ta l knowledge o f membrane p e r m e a b ility , w ith t h e fo llo w in g s p e c i f i c p u r­ p o se s i n minds 1# To e s t a b l i s h t h e q u a l i t a t i v e and q u a n t i t a t i v e e f f e c t s o f s e v e r a l aluminum a s t r i n g e n t s on membrane p e r m e a b ility . 2. To compare t h e r e l a t i v e e f f i c a c i e s o f th e s e a s t r i n g e n t s . 3* To s tu d y t h e p e r m e a b ility b e h a v io r o f a n a t u r a l membrane u s in g r a d i o a c t i v e io d id e and r a d i o a c t i v e sodium . 4* To d e te rm in e t h e p e r m e a b ility c o n s ta n t o f t h e membrane. 5 . To s tu d y th e e f f e c t o f th e tr e a tm e n t o f t h e membrane w ith a s a tu r a t e d d e x tr o s e s o l u t i o n . 6 . To p re p a re r a d io a u to g ra p h s o f t h e membrane i n an e f f o r t t o e l u c i d a t e th e mechanism o f io n p e n e tr a tio n of t h e membrane. 7 . To re v ie w t h e l i t e r a t u r e on p e rm e a b ility *

3 LITERATURE I.

A s tr in g e n ts The a c t i o n o f a s t r i n g e n t s upon t h e human s k in and membranes i n

g e n e r a l i s in c o m p le te ly u n d e r s to o d .

However, i t i s g e n e r a lly a g re e d

t h a t a s t r i n g e n t s have a p r o t e in p r e c i p i t a t i n g a c t i o n .

Goodman and

G ilm an ( l ) s t a t e t h a t t h i s e f f e c t i s a s u r f a c e e f f e c t , and t h a t th e p e r m e a b ility o f t h e c e l l membrane i s g r e a t l y re d u c e d .

SoUmann (2 )

s t a t e s t h a t i f t h e h eav y m e ta l p r o te in a te i s in s o lu b le th e u s u a l a s t r i n g e n t a c t i o n r e s u l t s , b u t i f s o lu b le , a more p e n e tr a tin g i r r i t a n t a c tio n r e s u l t s .

S ollm ann f u r t h e r s t a t e s t h a t th e s tr e n g t h o f a s t r i n ­

g e n t a c t i o n v a r i e s w ith t h e c o n c e n tr a tio n o f a s t r i n g e n t s a l t p r e s e n t w hich may o f te n b e l i m i t e d by i t s s o l u b i l i t y , i . e . th e number o f io n s o f th e m e ta l s a l t i n s o l u t i o n .

The te rm s a s t r i n g e n t and c a u s t i c a r e

r e l a t e d , a s a c a u s t ic may be made an a s t r i n g e n t by d i l u t i o n * McDonough (3 ) f in d s t h a t t h e r e a r e two k in d s o f a s t r i n g e n t s , th o s e w hich have a d e f i n i t e p r e c i p i t a t i n g a c t i o n on p r o te in s and th e p s e u d o a s tr in g e n ts , th e l a t t e r g ro u p n o t h av in g t h e p r o te in p r e c i p i t a ­ tin g a c tio n .

The form er group may b e d iv id e d i n t o c a t i o n i c (e g . Al’1"4"1")

and a n io n ic ( ta n n a t e ) a s t r i n g e n t s . by a d r e n a l i n .

The l a t t e r gro u p i s e x e m p lifie d

I t i s s u g g e s te d t h a t i n t h e c a s e o f t h e aluminum

a s t r i n g e n t s , Al+++ a s a b a se com bines w ith t h e am p h o teric s k in p ro ­ t e i n c o lla g e n , and t h a t o n ly two v a le n c e s o f Al*** e n te r i n t o th e co m b in a tio n .

An a c id i s l i b e r a t e d , w ith t h e fo rm a tio n o f a m e ta l

p ro te in a te . Jannaw ay (4 ) q u o te s D r. Herman Goodman t o t h e e f f e c t t h a t p o re s a r e h id d en o p en in g s t o sw eat d u c t s , re a c h in g t o t h e s u rf a c e betw een th e

4 t r u e (lo w e r) s k in and t h e s u p e r f i c i a l f a l s e s k in .

S o - c a lle d 11e n la rg e d

p o re s " r e f e r t o e n la rg e d o p en in g s o f h a i r f o l l i c l e s w hich do n o t con­ ta in a h a ir.

I t i s s tr o n g ly s u g g e ste d t h a t t h e k e r a tin o u s f o l l i c l e

s u b s ta n c e c a n n o t be sh ru n k by a s t r i n g e n t a c t i o n , b u t t h a t t h e s k in s u r f a c e s u rro u n d in g th e p o re c a n , w ith t h e o v e r a l l r e s u l t t h a t t h e p o re s seem t o be re d u c e d i n s i z e .

The e f f e c t o f pH on th e p r o te i n p r e ­

c i p i t a t i n g a c t i o n o f a s t r i n g e n t s i s e v id e n t from th e f in d in g o f G riev e who r e p o r t s (5 ) t h a t t a n n i n does n o t p r e c i p i t a t e p r o te in s i n a c id s o lu tio n . II,

P re v io u s M ethods o f E s tim a tin g ^ s tr in g e n c y Inasm uch a s a l a r g e p a r t o f t h e r e s e a r c h p r o je c t would be

d ev o ted t o th e m easurem ent o f th e e f f e c t o f d i f f e r e n t p e rc e n ta g e s o f d i f f e r e n t a s t r i n g e n t s on membrane p e r m e a b ility , p re v io u s m ethods o f e s tim a tin g a s tr in g e n c y w ere re v ie w e d .

T h e is (6 ) d ev elo p ed a d i l a t o -

m e tric m ethod o f m easu rin g a s tr in g e n c y q u a n t i t a t i v e l y .

A ccording to

t h i s m ethod t h e e x p a n sio n o f t h e l i q u i d sy stem s c o n ta in e d i n an an im al s k in , a f t e r e x p o su re t o a s t r i n g e n t s , was ta k e n a s a m easure o f a s trin g e n t e f f e c t .

S ch u etz ( ? ) made o b s e r v a tio n s on t h e s u p p re s s io n

o f t h e mucus s e c r e t i o n o f f r o g s a f t e r a p p l i c a t i o n o f t h e a s t r i n g e n t t o t h e b r e a s t o f t h e a n im a l, and c o r r e l a t e d h i s f in d in g s w ith a s trin g e n t a c t i v i t y . M adison (8 ) was a b le t o com pare t h e e f f e c t o f d i f f e r e n t a s t r i n g e n t s b y n o tin g t h e e f f e c t upon a b s o r p tio n o f Na* i n th e g a s tr o ­ i n t e s t i n a l t r a c t o f th e r a t by a t r a c e r te c h n iq u e .

G o v ett and de N av arre (9)

r e p o r t e v a lu a tio n s o f a s t r i n g e n t a c t i v i t y by n o tin g th e s h rin k a g e i n a r e a o f a s e c t io n o f f r o g s k in t r e a t e d w ith a s t r i n g e n t , and a ls o b y

5 n o tin g th e d e c re a s e i n o sm o tic flo w th ro u g h a p o r tio n o f f r o g s s k in a f t e r t r e a t i n g w ith a s t r i n g e n t .

A te c h n iq u e o f lo n g s ta n d in g has been

t o employ t h e q u a n t i t a t i v e p r e c i p i t a t i o n o f p r o te in s a s a m easure o f a s tr in g e n c y , a s n o te d by Sollm ann ( 2 ) # III.

P e r m e a b ility The l i t e r a t u r e on p e r m e a b ility i s co n fu sed an d c o n t r a d i c t o r y .

Much o f t h e c o n fu s io n may be a t t r i b u t e d t o th e u se o f p o o rly c o n t r o lle d e x p e rim e n ta l m ethods and t h e advancem ent o f t h e o r i e s having l i t t l e o r no p h y s ic a l- c h e m ic a l b a s i s . m ere s p e c u la tio n .

Much o f th e e a r ly work b o r d e r s on b e in g

W ith th e r e c e n t ad v an ces i n p h y s ic a l c h e m is try and

modern te c h n iq u e t h e r e h as b een a pronounced r i g o r o f i n t e r p r e t a t i o n o f r e s u l t s w ith t h e r e s u l t a n t abandonment o f u n te n a b le th e o r ie s * Davson an d D a n i e l l i (3 2 ) have g iv e n th e m ost s y s te m a tic , m odem and co m p lete d is c u s s io n o f p e r m e a b ility a v a i l a b l e .

I n t h i s work t h e

te rm p e r m e a b ility i s s t r i c t l y d e f in e d , and e q u a tio n s a r e g iv e n f i t t i n g d iffe re n t c a se s. re c o g n iz e d :

Two ty p e s o f d i f f u s io n o f a m a t e r ia l a r e

( l ) When a s u b s ta n c e d i f f u s e s a c c o rd in g t o th e law s o f

therm odynam ics, from a r e g io n o f h ig h e r t o one o f low er ch e m ic a l p o t e n t i a l , so t h a t c o n c e n tr a tio n s a r e e q u a l on b o th s id e s o f t h e membrane a t e q u ilib r iu m , (2 ) when su ch law s a r e a p p a r e n tly b ro k e n , and m o le c u le s accu m u late on one s id e o f a membrane.

The second c a s e

p ro v e s i n t e r e s t i n g s in c e i t i s commonly fo u n d i n n a t u r e , e g . th e sodium c o n te n t o f c e l l s i s u s u a ll y l e s s t h a n th e sodium c o n te n t o f th e t i s s u e f l u i d i n m ost a n im a ls .

The p e n e t r a t i o n o f a membrane by an

io n ca n be c l a s s i f i e d u n d e r t h r e e g e n e r a l c a s e s :

( l ) th e io n p a s s e s

th ro u g h a homogeneous l i p o i d l a y e r by sim p le d i f f u s i o n , (2 ) th e io n

6 com bines w ith an io n o f th e o p p o s ite s ig n i n th e membrane, and th e r e s u l t i n g d o u b le t d i f f u s e s th ro u g h , (3 ) t h e io n p e n e t r a te s th e p o re s o f t h e membrane.

This g e n e r a l c l a s s i f i c a t i o n i s m ost h e l p f u l i n t h a t

i t f i t s m ost c a s e s commonly e n c o u n te re d .

D ed u ctio n s a s t o t h e perme­

a b i l i t y o f a membrane can b e made by co n d u ctan ce and im pedance m easure­ m e n ts, b u t su ch m easurem ents may b e s u b je c t t o la r g e e r r o r s ( 2 6 ) e

Soak­

in g a f r o g membrane i n R in g e rs was found to d i l a t e th e c a p i l l a r i e s and a l t e r t h e p e r m e a b ility o f t h e membrane.

I t i s n o te d t h a t f r o g membrane

has tw o m ain l a y e r s , t h e c e l l s of w hich a r e bound t o g e t h e r by an i n t e r ­ c e l l u l a r m a t r i x , o r cem en t, w hich i s v e r y s e n s i t i v e t o io n ic e n v iro n ­ m en t.

I t i s n o te d a l s o t h a t i n an abnorm al io n ic environm ent c e l l s

have an abnorm al p e r m e a b ility .

Membranes o f am p h o teric m a t e r i a l s , such

a s g e l a t i n , a r e p re d o m in a n tly perm eable t o c a tio n s on t h e a l k a l i n e s id e o f t h e i s o - e l e c t r i c p o i n t , and t o a n io n s on th e a c id s id e .

At t h e i s o ­

e l e c t r i c p o in t su ch a membrane i s s e l e c t i v e l y perm eable t o n e i t h e r . Loeb (3 3 ) s tu d ie d t h e e f f e c t o f d i f f e r e n t io n ic en v iro n m en ts on t h e p e r m e a b ility o f f r o g membrane.

Johnson and Lee (35) found t h a t

unbroken human s k in i s p e r a e a b le t o NaCl, when th e l a t t e r i s in c o r ­ p o ra te d i n t o v a r io u s o in tm e n t b a s e s . M ic h a e lis an d Weech (36) s t a t e t h a t i t i s f a l l a c i o u s t o speak o f t h e d e f i n i t e p e m e a b i l i t y o f any ty p e o f membrane f o r e l e c t r o ­ l y t e s , and s u g g e st t h a t t h i s f a c t i s n o t s u f f i c i e n t l y re c o g n iz e d by many i n v e s t i g a t o r s . B a r n e tt (3 7 ) s u g g e s ts t h a t th e norm al p e r m e a b ility o f a membrane may be a l t e r e d by r a d i a t i o n , p o in tin g o u t th e e f f e c t o f e l e c t r i c a l l y ch arg ed p a r t i c l e s on p o la r l i p o i d a l s u b sta n c e s i n th e membrane w a lls and a l s o th e phenomenom o f p r o te in d é n a tu r a tio n by b e ta r a y s .

T h is

7 s u g g e s tio n i s so u n d , b u t p ro b a b ly has no s ig n if ic a n c e a t low l e v e l s o f r a d i a t i o n b y weak e m i t t e r s , Lucke and McCutcheon (3 8 ) n o te t h a t c e l l p e r m e a b ility i s in d e ­ p e n d e n t o f th e o sm o tic p r e s s u r e o f t h e s o l u t i o n , a f in d in g w hich m ight b e q u e s tio n e d . a b ility ,

They n o te t h a t c a t io n s te n d to d e c re a s e c e U perme­

I t i s a l s o n o te d t h a t a dead c e U r a p i d l y lo s e s i t s p r o p e r t i e s

o f a n o sm o tic system * S t e e l (3 9 ) n o te s t h a t th e h ig h e r t h e te m p e ra tu re th e more r a p id t h e d i f f u s i o n o f a m a te r ia l*

He a ls o n o te s t h a t th e s e l e c t i v e perme­

a b i l i t y o f a c e l l i s u s u a ll y l o s t when th e c e l l d i e s .

Httber (4 0 )

d is c u s s e s t h e am p h o teric n a tu r e o f f ro g membrane. There a r e num erous t h e o r ie s on t h e p e r m e a b ility o f membranes. Much o f th e work d e a ls w ith th e n a tu r e o f th e c e U membrane, w hich i s t h e i n f i n i t e s i m a l p a r t o f t h e g r o s s s tr u c t u r e s t h a t we s tu d y .

I n th e

fo llo w in g s u rv e y i t i s d i f f i c u l t t o e s t a b l i s h th e p r i o r i t y o f i d e a s , hence some o f th e c i t a t i o n s may be g en ero u s t o c e r t a i n men.

However,

th e v a r io u s id e a s advanced a r e lin k e d w ith th e w orkers i n t h e manner g e n e r a lly p r a c t i c e d i n t h e l i t e r a t u r e *

O verton (3 2 ,4 0 ,4 1 ) n o te d t h a t

s a l t s e n t e r t h e p ro to p la s m ic membrane s lo w ly , b u t f a t s o lv e n ts pene­ t r a t e d ra p id ly .

Hence he b e lie v e d t h a t th e p ro to p la s m ic membrane i s

c h ie fly lip o id .

However, i t i s n o te d t h a t u r e a p e n e tr a te s t h i s mem­

b r a n e , and o th e r an o m alie s a r e found t o o c c u r.

O verton h as a ls o found

t h a t o n ly such f a t s o lv e n ts a s a r e w a te r s o lu b le can p e n e tr a te th e membrane.

T h is th e o r y may be c o n s id e re d a p a r t o f t h e g e n e r a l

“s o l u b i l i t y ” t h e o r y o f p e r m e a b ility , s in c e i t i s a d v o cated i n th e l a t t e r t h a t t h e p e n e t r a t i n g su b sta n c e m ust be s o lu b le i n t h e membrane b e f o re i t c a n p e n e t r a t e i t *

Ô The s o - c a l l e d “s ie v e " th e o r y of p e r m e a b ility h a s b een v a r io u s ly a t t r i b u t e d t o T raube (39*41) and t o R uhland, M ic h a e lis and C o lla n d e r (3 2 ).

A ccording t o t h i s t h e o r y t h e membrane a c t s a s a " s i e v e ," and

d is c r im in a te s a g a i n s t p e n e t r a tin g p a r t i c l e s a c c o rd in g t o t h e i r m olec­ u l a r d im en sio n s ( 4 0 ) .

Davson and D a n ie lli ( 3 2 ) n o te t h a t th e s ie v e

th e o r y m ost a c c e p ta b ly f i t s i n w ith th e co n c e p t t h a t th e membrane has a p r o te in s t r u c t u r e , an d t h e c r o s s - lin k e d p e p tid e bonds form a s ie v e ­ l i k e membrane.

The same a u th o r s (32) a ls o p o in t o u t , i n t h e l i g h t of

r e c e n t e v id e n c e , t h a t c e r t a i n membranes may be inhomogeneous and t h a t p a r t o f t h e e f f e c t may be e x p la in e d by assum ing a s ie v e s t r u c t u r e .

It

i s n o te d t h a t su ch a th e o r y h as d e f i c i e n c i e s i n th e c a se o f th e Begg i a t o a c e l l , f o r exam ple. R ecent t h e o r i e s on membrane s t r u c t u r e and th e n a tu r e o f perme­ a b i l i t y a r e more com plex.

Many i n v e s t i g a t o r s have assumed t h a t c e r t a i n

membranes have a m osaic s t r u c t u r e .

N athanson (32) b e lie v e s t h a t a

m a t e r i a l p e n e tr a tin g a membrane i s d is c r im in a te d a g a in s t on th e b a s i s o f t h e p h y s ic a l and c h e m ic a l p r o p e r t i e s o f th e m a t e r i a l . i n a t o r y u n i t s o f t h e membrane may be v e ry l a r g e . s e n ts an inhom ogeneous p a t t e r n o f c o n s tr u c tio n . b e lie v e f r o g membrane t o b e a m osaic ty p e .

The d is c r im ­

Such a membrane p re ­ Davson and D a n ie lli (3 2 )

C o lla n d e r and B arlu n d (40)

s t a t e t h a t a m osaic membrane has b o th l i p o i d a r e a s and s i e v e - l i k e a re a s. C o lla n d e r (3 2 ) n o te s t h a t c e r t a i n membranes seem t o have t h e s t r u c t u r e o f a p r o t e i n l a y e r ad so rb ed on a l i p o i d s u r f a c e , w hich g iv e s r i s e to c e r ta in s ie v e -lik e p ro p e rtie s .

Davson and D a n i e l l i (3 2 ) have

m o d ifie d t h i s th e o r y t o t h e " p a u c im o le c u la r" th e o r y , w hich f i t s th e e v id e n c e g a th e re d fro m a v a r i e t y o f c a s e s .

A ccording t o th e th e o r y o f

9 t h e l a t t e r , t h e c e l l membrane h a s a s t r u c t u r e c o n s is tin g o f a l i p o i d l a y e r w hich i s a few f a t t y m o le c u le s t h i c k , w ith an ad so rb e d p r o te in l a y e r a t e a c h i n te r f a c e * The f o re g o in g t h e o r i e s c o n t r ib u t e t o th e g e n e r a l knowledge o f membrane s t r u c t u r e and a r e p e rh a p s s tim u la tin g i n u n d e rs ta n d in g th e m echanism o f p e n e t r a t i o n o f a membrane.

However, th e v a r io u s t h e o r i e s

o f p e r m e a b ility a r e o f more i n t e r e s t i n t r e a t i n g th e c a s e o f p e n e tr a ­ t i o n o f a membrane by an e l e c t r o l y t e .

M ic h a e lis ( 4 2 ) , i n e x p e rim e n tin g

w ith a r t i f i c i a l m aribranes, s u g g e s ts t h a t io n s may be ad so rb ed on t h e w a lls o f t h e c a p i l l a r y c a n a ls o f th e membrane, and t h a t su ch a d s o r p tio n may be s e l e c t i v e f o r a c e r t a i n io n .

The n e t e f f e c t i s th u s t o a f f e c t

t h e io n m o b i l i t i e s f o r t h e c a tio n s and a n io n s d i f f e r e n t l y i n th e s e c a n a l s , g iv in g r i s e t o an o v e r a ll " s e l e c ti v e " p e r m e a b ility . re v ie w s t h e g e n e r a l e l e c t r i c a l th e o r y o f p e r m e a b ility .

S e i f r i z (4 1 )

I n g e n e r a l,

t h e i d e a i s advanced t h a t a membrane i s charged and p e rm its o n ly io n s o f t h e o p p o s ite s ig n o f c h arg e t o p a s s ; o r io n s o f o n ly one s ig n o f c h a rg e p a s s , g iv in g r i s e t o a p o t e n t i a l e f f e c t , t h e r e f o r e s im u la tin g an e l e c t r i c a l membrane c h a r g e .

The same a u th o r r e f e r s t o m isc e lla n e o u s

t h e o r i e s o f p e r m e a b i l it y , such a s s u r f a c e t e n s i o n t h e o r i e s , and emul­ s io n h y p o th e s e s , f o r each o f w hich t h e b u lk o f e v id en ce i s in c o n s id e r ­ a b le . Amber so n and K le in have c o n tr ib u te d a m ost i n t e r e s t i n g p ap er on t h e e l e c t r i c a l th e o r y o f th e p e r m e a b ility of f r o g membrane (43)* The s u g g e s tio n o f Q stw ald (4 4) t h a t e le c tr o m o tiv e phenomena i n t i s s u e m ig h t a r i s e s in c e membranes a r e s e l e c t i v e l y perm eable t o io n s i s n o te d . These c o l l a b o r a t o r s found t h a t th e o u te r e p i t h e l i a l l a y e r o f th e f r o g membrane seems t o c o n t r o l e l e c t r i c a l phenomena.

They found t h a t pH

c o n t r o l l e d th e e l e c t r i c a l c h a r a c t e r o f t h e m oribund membrane w ith which th e y ex p erim e n ted i n a m anner w hich s u g g e s ts t h e p re s e n c e o f am pholytes i n t h e membrane#

These w o rk ers o b se rv e d t h a t “t h e r e i s some fu n d am en tal

r e l a t i o n b etw een th e e l e c t r i c a l c h a rg e p r e s e n t upon a s u r f a c e and p o re w a lls o f an o r g a n is e d membrane and t h e a b i l i t y o f io n s t o p e n e tr a te t h e s tr u c t u r e # "

When t h e c h a rg e on t h e membrane i s n e g a tiv e , eg . above th e

i s o e l e c t r i c p o in t o f t h e membrane, c a t io n s p e n e tr a te more e a s ily #

These

w o rk ers d is a g r e e w ith th e s u g g e s tio n o f M ic h a e lis (42)# K a tz in (4 5 ) has s tu d ie d th e p e r m e a b ility o f f ro g membrane u s in g N a ^ and K42.

He fo u n d t h e a b s o lu te r a t e o f p assag e f o r p u re N a ^ C l i s

4»7X10"12 mol# cm.""^ s e c e™^.

H auser ( 4 6 ) h a s n o te d t h a t th e osm otic

e f f e c t o f a g lu c o se s o l u t i o n on a p r o te in membrane i s in c r e a s e d by th e a c t i o n o f ta n n in s *

I t i s su g g e ste d t h a t t h i s e f f e c t i s due t o th e narrow ­

in g o f th e p o re s o f th e membrane# Adolph (2 1 ) h as made a th o ro u g h s tu d y o f th e e f f e c t s o f d i f f e r ­ e n t s a l t s on t h e t r a n s p o r t o f w a te r th ro u g h f ro g membrane#

He f in d s

t h a t th e p e r m e a b ility o f f r o g membrane i s in flu e n c e d by ch e m ic a l e n v ir ­ onm ent, p re v io u s h a n d lin g , o r i e n t a t i o n o f th e membrane, and h y d r o s ta tic p re ssu re .

He s u g g e s ts t h a t th e p e r m e a b ility b e h a v io r o f f r o g membrane

c a n b e e x p la in e d on an e l e c t r i c a l b a s i s .

He f u r t h e r s u g g e s ts t h a t Na*

c o n t r o ls th e e x te n t o f t h e p o t e n t i a l d i f f e r e n c e e x i s t i n g a c r o s s a fro g membrane, and t h i s i n t u r n c o n tr o ls t h e d i r e c t i o n and r a t e o f p a ssa g e o f e i t h e r w a te r o r s a l t s .

The m o rp h o lo g ic a l o u te r s id e o f f r o g membrane

seems t o b e n e g a t i v e , and w a te r n o rm a lly t r a v e l s tow ard th e n e g a tiv e , w h ile s a l t s t r a v e l i n t h e r e v e r s e d i r e c t i o n . t h i s l a t t e r s ta te m e n t needs q u a l i f i c a ti o n #

I t i s to b e n o te d t h a t

11 R u b e n s te in (47) s t a t e s t h a t f ro g membrane i s an alo g o u s t o mucous membrane and s p e c u la te s on t h e e f f e c t o f s a l t s on t h e perme­ a b i l i t y o f t h i s membrane. The f o re g o in g p r e s e n ta tio n o f th e l i t e r a t u r e i s th e m ost com­ p l e t e a v a i l a b l e on t h e s u b je c t o f p e r a e a b i lity * IV*

The I s o to p e Used The en e rg y sp ectru m o f 1^31 was e s ta b li s h e d by Downing,

D eu tsch and R o b e rts ( 1 0 ) .

The b e ta r a y spectrum was i n v e s tig a te d

u s in g a m ag n etic le n s sp e c tro m e te r* V.

The In s tru m e n ts Used There i s much i n t h e r e c e n t l i t e r a t u r e on t h e th e o r y and o p er­

a t i o n o f G e ig e r-M u e lle r (G-M) c o u n te r s . fo llo w *

A few s e le c te d r e f e r e n c e s

Brown (1 1 ) h as an e x c e l le n t d is c u s s io n o f t h e th e o r y o f

c o u n te r s and r e l a t e d e l e c t r o n i c phenomena.

B ousquet (1 2 ) d is c u s s e s th e

p e rc e n ta g e p ro b a b le e r r o r i n c o u n tin g r a t e m easurem ents, and d is c u s s e s v a r io u s s c a lin g c i r c u i t s .

Brown (1 3 ) d is c u s s e s t h e p r in c ip l e s o f

o p e r a tio n and d i f f e r e n c e s i n gamma and b e ta r a y c o u n te r s .

Lapp and

Andrews (14) have w r i t t e n an a d m ira b le i n t r o d u c to r y t e x t on r a d i a t i o n p h y s ic s , w hich in c lu d e s a s e c t io n on c o u n tin g d e v ic e s .

Brown and

G h e la rd i (1 5 ) d is c u s s t h e v a r i a b l e s i n c o u n tin g equipm ent and o p e r a tio n w hich m ust b e c o n t r o ll e d i n t h e a tta in m e n t o f r e p r o d u c ib le r e s u l t s . VI*

The Animal Used The f r o g h as b een u sed as a n e x p e rim e n ta l anim al i n numerous

p h y s io lo g ic a l and p h a rm a c o lo g ic a l s t u d i e s .

However, many o f th e

l i t e r a t u r e r e p o r t s a r e w o r th le s s s in c e th e y la c k a com plete d e s c r ip tio n

12 o f e x p e rim e n ta l m eth o d s> o r s u b je c t t h e a n im al membrane t o u n p h y s io lo g i c a l c o n d itio n s .

An a tte m p t h as b een made t o a v o id r e c o g n itio n o f

q u e s tio n a b le w ork i n t h e fo llo w in g s e c tio n ; how ever, where i t h as been n e c e s s a r y t o c i t e such work th e o b v io u s sh o rtco m in g s w i l l b e p o in te d o u t. E.

A. Adolph has p u b lis h e d e x te n s iv e ly h i s s tu d i e s o f i n t a c t

f r o g s and a ls o s t u d i e s o f i s o l a t e d f r o g membrane.

Adolph (1 6 ,1 ? ) h as

o b se rv e d t h a t th e p i t h i n g o f a f r o g in c r e a s e s tre m e n d o u sly th e r a t e o f w a te r u p ta k e o f f ro g s k i n , c a u s in g th e a n im a l t o s w e ll r a p id ly * A dolph (1 8 ) c o n clu d es t h a t th e n erv o u s system c o n t r o ls t h e r a t e o f e n tr a n c e o f w a te r i n t o t h e i n t a c t f r o g , a s a r e s u l t o f h is p ith in g e x p e rim e n ts , and s u g g e s ts t h a t t h e c i r c u l a t i o n o f th e b lo o d i s n o t n e c e s s a r y t o t h e r e g u l a t i o n o f w a te r in ta k e .

He f u r t h e r s u g g e s ts t h a t

t h e environm ent o f th e f r o g s k in d e te rm in e s i t s p e r m e a b ility t o w a te r. Adolph (1 9 ) s t a t e s t h a t a f r o g d rin k s th ro u g h i t s w hole body s u r f a c e . The l i t e r a t u r e on i s o l a t e d f r o g membranes i s e x te n s iv e .

In

t h e work which was u n d e rta k e n i t was n e c e s s a ry t o e s t a b l i s h t h a t i s o ­ l a t e d f r o g membrane s u rv iv e s s a t i s f a c t o r i l y and hence r e t a i n s i t s perme­ a b ility c h a r a c te ris tic s .

B efo re c i t i n g such r e f e r e n c e s , i t i s w e ll t o

s t a t e K ro g h 's d e f i n i t i o n o f a membrane ( 2 0 ):

A membrane i s a " s t r u c t u r e

w hich w i l l r e s t r i c t t h e f r e e movement o f m o le c u le s o r p a r t i c l e s , b u t w i l l n o t by e x p e n d itu r e o f e n erg y b r in g about any t r a n s p o r t o f sub­ s ta n c e a c r o s s i t s t h i c k n e s s ."

He n o te s t h a t s o - c a l l e d " a c tiv e t r a n s ­

p o r t" i s a c h a r a c t e r i s t i c o f l i v i n g m em branes, and hence i s a c o r­ r e l a t i v e c h a r a c t e r i s t i c o f some membranes (e g . s u rv iv in g f r o g membranes)*

13 Adolph (2 1 ) s t a t e s t h a t i s o l a t e d f r o g membrane from f r o g s p ith e d w ith o u t a n e s t h e s i a w i l l s u rv iv e 16-90 h o u rs a f t e r re m o v a l, a s a s c e r t a i n e d i n O2 consum ption s t u d i e s .

W ertheim er (2 2 ) d is c u s s e s th e

O2 consum ption m ethod o f a s c e r t a i n i n g w h eth er a f ro g membrane i s l i v ­ in g o r m o rib u n d , and s t a t e s t h a t th e r e a r e a p p a r e n tly no b ro a d d i f ­ f e r e n c e s i n t h e p e r m e a b ility o f th e l i v i n g and dead membrane.

However,

i t m ight be n o te d t h a t t h i s s ta te m e n t i s e x c e e d in g ly g e n e r a l, f o r c e r t a i n l y d i f f e r e n t i a l p e r m e a b ilit ie s t o e l e c t r o l y t e s would b e e x p e c te d .

U ssin g (23) s t a t e s t h a t fro g membrane can b e k ep t a l i v e

24 h o u rs w ith o u t s p e c i a l p r e c a u tio n s .

R eid (24) s t a t e s t h a t f r o g

membranes re m a in no m a l 7 0 -8 0 h o u rs when p r e s e r v e d i n norm al s a l i n e , b u t s u g g e s ts 24 h o u rs m ig h t b e ta k e n a s a r e l i a b l e e x p e rim e n ta l l i m i t . Krogh (2 5 ) n o te s t h a t i s o l a t e d f r o g membrane e x h i b i t s "norm al'1 perme­ a b ility . Krogh (2 5 ) f i n d s t h a t much o f t h e l i t e r a t u r e on th e p e r m e a b ility o f f r o g membrane i s c o n fu sed and c o n f l i c t i n g s in c e th e power o f th e membrane t o a d so rb io n s was e i t h e r unknown o r o v e rlo o k e d .

Dean and

G a tty ( 2 6 ) n o te t h a t a p o t e n t i a l o f p lu s 40 mv. e x i s t s a c r o s s a s e c tio n o f i s o l a t e d f r o g membrane when b o th s id e s a r e b a th e d i n R in g e rs .

They

a l s o n o te Motokawa1s f in d in g s t h a t th e r a t e o f d i f f u s i o n o f NaCl o u t­ ward th ro u g h th e s k in i s in c r e a s e d by s t r e t c h i n g t h e membrane. S ollm ann and H e n zlic k (2 7 ) r e p o r t on osm o tic e x p erim en ts u s in g f ro g membrane.

Moore (2 8 ) p r e s e n ts an e x c e l l e n t draw ing o f a s e c tio n o f

f r o g membrane, w hich shows c l e a r l y t h e d i f f e r e n t c e l l u l a r s t r u c t u r e s . Moore a l s o d e t a i l s a s e c t io n o f t i s s u e from t h e r a t , w hich a f f o r d s a co m p ariso n o f t h e two ty p e s o f membranes.

C a lv e ry , D raize and Laug ( 2 9 )

14 d is c u s s th e c o m p o sitio n o f human s k in i n d e t a i l , w hich makes p o s s ib le a com parison w ith th e membranes found i n Moore. S e v e r a l w o rk ers have b een i n t e r e s t e d i n t h e t r a n s p o r t o f io n s a c r o s s f r o g membrane.

Ja c o b s f in d s t h a t t h e r e i s an a c t i v e t r a n s p o r t (30)

o f io n s i n f ro g membrane, i n th e d i r e c t i o n m o rp h o lo g ic a l o u te r t o in n e r .

U ssing (2 3 ,3 1 ) h as been t h e fo re m o st w orker i n r e c e n t i n v e s t i ­

g a tio n s on th e a c t i v e t r a n s p o r t phenomena.

I n a c t i v e t r a n s p o r t o f an

io n a c r o s s a membrane work i s d one, hence en erg y m ust b e s u p p lie d . U ssing v i s u a l i z e s t h e mechanism of such a c t i v e t r a n s p o r t a s b e g in n in g w ith th e f o r a a t i o n o f a complex betw een th e d i f f u s in g io n and th e c e l l m a t e r i a l , fo llo w e d by t r a n s p o r t th ro u g h t h e membrane, and c o n clu d in g i n d i s s o c i a t i o n o f th e complex on t h e o th e r membrane s u r f a c e .

I t is

p o s tu la te d t h a t t h e a fo re m e n tio n ed complex d i s s o c i a t e s by a n o th e r io n r e p la c in g th e fo rm er i n t h e com plex.

U ssing n o te d t h a t t h e pH o f th e

medium c o n ta c tin g t h e m o rp h o lo g ic a l in n e r s id e o f th e f ro g membrane had a la r g e e f f e c t on t h e t r a n s p o r t o f N a ^ io n s , w h ile th e o u te r pH has l i t t l e in f l u e n c e .

U ssing (23) has commented on t h e r e l a t i o n

betw een Na+ t r a n s p o r t , p o t e n t i a l d if f e r e n c e a c r o s s t h e membrane and pH* Meyer and B e rn fe ld (34) s tu d ie d th e s e l e c t i v e p e r m e a b ility o f f r o g membrane*

15 EXPERIMENTAL A*

G e n e ra l and S p e c if ic C o n s id e ra tio n s i n D eveloping An E x p e rim e n ta l Method# G e n e ra l The problem posed f o r th e in te n d e d r e s e a r c h was a s tu d y o f t h e

e f f e c t o f d i f f e r e n t m a t e r ia ls ( p r im a r ily a s t r i n g e n t s ) on th e p e r m e a b ility o f f r o g membrane#

O b v io u sly , such a s tu d y c a n b e c a r r i e d o u t on th e

i n t a c t an im al ( i n v iv o ) o r i n an i n v i t r o ty p e o f s tu d y .

The r e l a t i v e

m e r its o f b o th ty p e s o f sy stem s m ust th e r e f o r e b e examined# Numerous problem s a r i s e i n a tte m p tin g t o s tu d y t h e membrane p e r m e a b ility o f a l i v i n g i n t a c t anim al#

A s a lt in je c te d in to th e

t i s s u e s o f a l i v i n g an im al i s im m ed iately s u b je c te d to a l l th e r e s o u r c e s o f t h e a n im al body p ro c e s s e s i n an e f f o r t t o m a in ta in h o m e o sta sis; t h u s , an abnorm al s i t u a t i o n h as b een c r e a te d .

E f f o r t s t o com pensate f o r th e

d i f f e r e n t p H 's and osm o tic p r e s s u r e s o f t h e aluminum s a l t s b y u s in g b u f f e r s o r ad d in g p resum ably i n a c t i v e s a l t s in tro d u c e new f a c t o r s o f t h e i r own, f u r t h e r c o m p lic a tin g th e s tu d y .

Such r e s u l t s m ust o f n e c e s s ity

b e s u b je c te d t o a r i g i d i n t e r p r e t a t i o n t o be v a l i d .

I t would ap p e a r

t h a t th e l a r g e number o f v a r i a b l e s p re s e n te d by an a c t i v e p h y s io lo g ic a l system w ould make a stu d y u n d er such c o n d itio n s e x c e e d in g ly d i f f i c u l t . The p o s s i b i l i t y o f u s in g an e x c is e d membrane was t h e r e f o r e e x p lo re d .

I n v e s t i g a t i o n s b a se d on a la r g e number o f d i f f e r e n t ty p e s

o f m em branes, b o th n a t u r a l and a r t i f i c i a l , a r e r e p o r te d i n th e l i t e r ­ a tu re .

Among th o s e u sed w ere c e l l u l o s e e s t e r , f e r r i c y a n i d e , p i g 's

b la d d e r , i n t e s t i n a l membranes and fro g membrane♦ work has been done u s in g th e l a t t e r membrane.

The b u lk o f t h i s

C o n s id e ra tio n s in v o lv in g

16 a v a i l a b i l i t y # c o s t , e a s e o f p r e s e r v a tio n and co n v en ien ce i n h a n d lin g d e c id e d i n f a v o r o f f ro g membrane.

I t th e n rem ained t o show t h a t

e x c is e d f r o g membrane e x h i b i t s what m ight b e c a l l e d "norm al" perme­ a b i l i t y c h a r a c t e r i s t i c s , and t h i s p o in t h a s b een amply co n firm ed (s e e L ite r a tu r e s e c tio n ). The p rim a ry v a r i a b l e s a f f e c t i n g th e p e r m e a b ility o f f ro g membrane a r e :

I o n ic en v iro n m e n t, pH, osm otic p r e s s u r e , ti m e , tem per­

a t u r e and membrane a r e a .

By c o n d u c tin g e x p e rim e n ts i n a p a r a l l e l

f a s h i o n , u s in g an a d e q u a te ly s ta n d a rd iz e d te c h n iq u e , th e v a r i a b l e s o f io n ic e n v iro n m e n t, osm otic p r e s s u r e , tim e and te m p e ra tu re can be elim ­ in a te d ,

The v a r i a b l e s o f pH and membrane a r e a can th e n b e m inim ized by

a c o n t r o ll e d e x p e rim e n ta l m ethod t o an e x te n t a t w hich th e y a r e no lo n g e r s i g n i f i c a n t . The e f f e c t o f r a d i a t i o n on membrane p e r m e a b ility m ust a ls o be ta k e n i n t o a c c o u n t.

The e f f e c t o f r a d i a t i o n on a membrane having

p r o t e i n c o n s t i t u e n t s i s n o t c l e a r l y u n d e rs to o d .

I t th e r e f o r e seems

w ise t o conduct e x p e rim e n ts i n such a m anner t h a t th e membrane i s exposed t o o n ly low l e v e l s o f r a d i a t i o n .

Inasmuch a s 1^-31

a weak

e m i t t e r , and th e a b s o rb in g a b i l i t y o f an aqueous system i s h ig h , one would e x p e c t t h a t any r a d i a t i o n e f f e c t on membrane s t r u c t u r e i s l i k e l y t o b e s m a ll. The c h o ic e o f th e p ro p e r is o to p e i s b a s e d on s e v e r a l fu n d a­ m e n ta l c o n s id e r a t i o n s .

The t r a c e r u sed sh o u ld p e n e tr a te th e membrane

e a s i l y and sh o u ld have no d e l e t e r i o u s e f f e c t s on t h e membrane, such a s th e p o iso n o u s e f f e c t s o f ON"", f o r exam ple. were P ^ , N a ^ and I^-31e

The is o to p e s c o n s id e re d

Of t h e t h r e e p32 h as a r a t h e r h ig h en erg y

17 b e t a p a r t i c l e (B“ « 1*69 M*E.Ve ) w hich m ight a d v e r s e ly a f f e c t th e mem­ brane*

The sodium is o to p e h as an u n d e s ir a b ly s h o r t h a l f l i f e (T^ = 14*8

h o u rs)*

I M s c h a r a c t e r i s t i c would make N a ^ u n s u ita b le f o r ex ten d ed

r e p e t i t i v e e x p e r im e n ta tio n , and would make c a l c u l a t i o n s u n n e c e s s a r ily in v o lv ed *

The d e c is io n t o u se l ! 3 l was t h e r e f o r e b ased on i t s co n v en i­

e n t h a l f l i f e and th e weak e n erg y o f i t s r a d i a t i o n (s e e P a r t B t h i s s e c tio n ) * A t e n t a t i v e mode o f a t t a c k was c o n s id e re d t o be alo n g th e fo llo w in g l i n e s :

A f r o g membrane would b e exposed t o t h e a s t r i n g e n t ,

o r any o th e r c h em ic al a g e n t , by some m eans.

The m a t e r ia l a p p lie d would

th e n b e washed f r e e o f t h e membrane, and th e e x c is e d membrane, a tta c h e d t o a g l a s s t u b e , would b e immersed i n a s o lu t i o n of r a d io a c t iv e io d id e* By com paring r a t e s o f p a ssa g e o f t h e t r a c e r io n betw een a c o n tr o l membrane and th e t r e a t e d membrane some d e d u c tio n s as t o t h e e f f e c t o f tre a tm e n t on membrane p e r m e a b ility c o u ld b e made* W ith t h e f o re g o in g g e n e r a l c o n s id e r a tio n s i n m ind, s e v e r a l s p e c i f i c c o n s id e r a tio n s w hich a r o s e i n making w orkable th e method o f i n v e s t i g a t i o n o u tlin e d a re co n sid e red * S p e c if ic A sim p le ty p e o f s e tu p w hich f u l f i l l e d i n p r in c ip l e th e r e q u ir e ­ m ents o f t h e t e n t a t i v e mode o f a t t a c k was t h e f o llo w in g : were a tta c h e d t o r i n g s ta n d s i n an u p r ig h t p o s itio n *

G lass tu b e s

The e x c is e d mem­

b ra n e was th e n a t t a c h e d o v e r t h e ends o f t h e s e g l a s s t u b e s , and th e mounted membrane lo w ered i n t o a r a d io a c tiv e s o lu t i o n .

The r a t e o f

d i f f u s i o n o f io n s th ro u g h th e membrane i n t o a s o lu tio n w hich had b een p la c e d i n t h e tu b e c o u ld th e n b e fo llo w e d u s in g t r a c e r ions*

W hile

18 su ch a s e tu p was s u p e r f i d a l l y s im p le , s e v e r a l problem s im m ed iately a ro se . S in c e th e r a t e o f p e n e t r a tio n o f f r o g membrane by io d id e io n h a s n o t been r e p o r te d t h i s had t o b e i n v e s t i g a t e d e x p e r im e n ta lly .

A

s e tu p was u sed i n w hich two m l. o f t a p w ater was p la c e d i n th e i n n e r com partm ent o f t h e tu b e w ith i t s a tta c h e d membrane and t h i s p r o je c te d i n t o a b e a k e r c o n ta in in g f o u r m l. o f r a d io a c tiv e i o d id e .

V arying

t h e a c t i v i t y o f t h e l a t t e r s o lu t i o n betw een 2 -3 ,0 0 0 and 200,000 c .p .m . / m l. e s ta b li s h e d t h e l a t t e r l e v e l a s optimum.

T h is a c t i v i t y gave an

a s s a y a b le a c t i v i t y of t h e s o l u t i o n i n t h e in n e r com partm ent i n a re a s o n a b le tim e , an d a t t h e same tim e was n o t s u f f i c i e n t l y h ig h to s u b je c t t h e membrane t o e x c e s s iv e r a d i a t i o n . The s m a ll amount of membrane o b ta in a b le fro m t h e back o f th e f r o g n e c e s s i t a t e d t h e u se o f 12 mm. g l a s s tu b e s on w hich t o a tt a c h t h e f ro g m embranes. W hile e a r l y i n v e s t i g a t o r s fo u n d th e o r i e n t a t i o n o f f r o g membrane in f lu e n c e s i t s p e r m e a b ility , r e c e n t i n v e s t i g a t o r s (2 6 ,4 8 ) have found th e s e r e p o r t s u n r e l i a b l e .

The o r i e n t a t i o n o f m o rp h o lo g ic a l in n e r s id e

c o n ta c tin g t h e r a d i o a c t i v e s o lu t i o n i n th e b e a k e r was t h e r e f o r e ch o sen f o r co n v en ien ce i n t h i s w ork. I t would a p p e a r n e c e s s a r y , from th e r e p o r ts o f Adolph ( 2 1 ) , t o p re v e n t a h y d r o s t a t i c head o f p r e s s u r e from b e in g e x e r te d on th e mem­ b ra n e d u rin g e x p e rim e n ts .

U n f o rtu n a te ly , i t i s n e c e s s a ry t o t o t a l l y

immerse th e membrane and b in d in g i n o b v ia tin g t h i s d i f f i c u l t y by e q u a l­ iz i n g l i q u i d l e v e l s ( s e e F ig u re 4 , S e c tio n V I).

T h is p r a c t i c e le d to

a co m p le te l a c k o f r e p r o d u c i b i l i t y i n e x p erim en ts and th e p r a c tic e was

19 t h e r e f o r e abandoned.

The a l t e r n a t i v e p ro c e d u re f i n a l l y ch o sen was to

e x te n d t h e membrane i n t o t h e r a d io a c t iv e s o lu t i o n t o a d e p th a s n e a r t h r e e mm. a s p o s s i b l e , and n o t t o c o v e r t h e membrane b in d in g .

A

co m p ariso n o f t h i s te c h n iq u e w ith t h a t o f e q u a liz in g l i q u i d l e v e l s in d i c a t e d t h a t w h ile th e r a t e o f p a ssa g e o f io d id e io n th ro u g h th e membrane was e s s e n t i a l l y t h e sam e, i n e i t h e r c a se t h e l a t t e r method was much more r e p r o d u c ib le . By s l i g h t l y f l a r i n g th e ends o f t h e g l a s s tu b e s u se d s lip p a g e o f t h e membrane d u rin g th e e x p e rim e n ts was p re v e n te d .

The amount o f

f l a r e was n o t m e a su ra b le w ith o r d in a r y c a l i p e r s , and was n e c e s s a r ily s m a ll t o p re v e n t u n d e s ir a b le s tr e t c h i n g o f th e membrane. The m ethod of a t t a c h i n g th e membrane t o th e tu b e s u s in g ru b b e r b an d s was found t o damage o r s t r e t c h t h e membrane.

V ario u s methods

o f a t t a c h i n g t h e membrane were i n v e s t i g a t e d , in c lu d in g u s in g c a t g u t , ru b b e r t a p e , heavy shoem aker’ s th r e a d and o r d in a r y c o tto n t h r e a d . The e f f i c a c i e s o f th e s e v a r io u s m a t e r ia ls c o u ld be compared by d e v is in g an e x p e rim en t o f t h e p ro p e r k in d , w ith th e r e s u l t t h a t o r d in a ry c o tto n th r e a d was u se d i n a l l l a t e r w ork.

There were f u r t h e r b a s e s i n d isc o n ­

t i n u i n g t h e u s e o f ru b b e r b ands f o r t h i s p u rp o se i n t h e r e p o r ts o f Motokawa (4 9 ) t h a t s t r e t c h i n g o f a membrane in c r e a s e d t h e r a t e o f d i f f u s i o n o f N aC l, and Krogh ( 2 0 ) , who s t a t e s t h a t th e m ech an ical s t r e t c h i n g o f t h e c a p i l l a r y w a ll o f a membrane b r in g s about in c r e a s e d p e r m e a b ility . Though membrane from t h e abdomen o f t h e f r o g i s more unifo rm i n c h a r a c t e r , i t was fo u n d t h a t th e amount was i n s u f f i c i e n t f o r c o m p a ra tiv e s tu d i e s on t h e same a n im a l.

I t was t h e r e f o r e n e c e s s a r y t o

20 u s e membrane ta k e n from th e f r o g back and w hich c o u ld b e d iv id e d i n h a lf * Thus a l l o b s e r v a tio n s o f p e r m e a b ility changes were made u s in g th e p e r m e a b ility o f th e u n tr e a te d membrane p o r tio n from th e same a n im a l a s a c o n tr o l*

Such a m ethod i s in h e r e n tly more v a l i d th a n com paring perme­

a b i l i t y ch an g es betw een d i f f e r e n t a n im a ls, among which th e “norm al" p e r m e a b ility v a r i e s tre m e n d o u sly ( s e e S e c tio n I ) , The above p a r t i c u l a r s have been e la b o r a te d i n o r d e r t o show how th e v a l i d i t y o f th e e n t i r e m ethod was e s t a b li s h e d .

The p r e c is io n o f t h e

co m p lete te c h n iq u e c o u ld b e e v a lu a te d by rem oving th e membrane from a t e s t a n im a l, d iv id in g t h e membrane i n t o s im ila r p o r tio n s and th e n com­ p a r in g t h e p e r m e a b i l i t i e s o f th e tw o p o r tio n s .

S in c e each h a l f was

t r e a t e d th e same th e r a t e o f p e n e t r a t i o n o f I " sh o u ld b e th e same i n b o th c a ses*

The p r e c i s i o n o f p i p e t t i n g and c o u n tin g sam ples had been

shown t o be w ith in 1+10#.

The p r e c is io n o f th e e x p e rim e n ta l method was

found t o be r e p r o d u c ib le w ith in seven p e r c e n t, w hich in c lu d e d e r r o r s i n p i p e t t i n g and c o u n tin g o f sa m p le s, v a r i a t i o n s i n a tta c h in g th e mem­ b r a n e , and s l i g h t d i f f e r e n c e s i n t h e e x te n t th e membranes were p ro ­ j e c t e d i n t o t h e r a d i o a c t i v e s o l u t i o n , a s w e ll a s unknown v a r ia b le s * R in g er* s s o lu t i o n was n o t u sed i n th e f o re g o in g m ethod s in c e c o n t r o l ru n s showed t h a t f o r t h e s h o r t p e r io d o f tim e th e s e ex p erim en ts were co n d u cted no o b s e rv a b le e r r o r would be in tro d u c e d .

Davson and D a n ie l li

(3 2 ) n o te t h a t so ak in g f r o g membrane i n R in g e rs ca u se s d i l a t i o n o f th e c a p i l l a r i e s , t h e p e r m e a b ility o f which i s q u i t e d i f f e r e n t from t h a t o f norm al t i s s u e . lib r iu m i s a l t e r e d .

I n any c a s e , th e norm al Gibbs-Donnan membrane e q u i­ S in c e t h e Gibbs-Donnan membrane e q u ilib riu m i s o f

im p o rtan c e i n t h i s w ork, i t i s w e ll t o g iv e b r i e f c o n s id e r a tio n to th e

21 phenomena*

Due t o t h e p re se n c e o f n o n - d if f u s ib le io n s i n th e membrane,

an e q u ilib r iu m o f t h e fo llo w in g k in d i s e s ta b lis h e d : Na

BT

Na+

Clw

ci

®i

c2

c2

Na+

R“

Cl” x

(a )

Na*

01"

C2-X

Cg—x

(b )

I f a i s t h e i n i t i a l c o n d i t i o n , i n w hich th e sodium c o l l o i d i s s e p a ra te d from a d i l u t e s a l t s o l u t i o n (R *= a n o n - d if f u s i b l e a n io n ) , t h e f i n a l e q u ilib r iu m c o n d itio n may b e r e p r e s e n te d by b .

T hus, b o th Na* and Cl""

have d i f f u s e d i n t o t h e l e f t com partm ent, d e p le tin g th e r i g h t s id e * I f t h e o r i g i n a l c o n c e n tr a tio n s on th e l e f t a r e Na*=R~=C^ and a ls o N a * = C 1 ~ o n th e r i g h t , and x m oles o f Na*=Cl— d i f f u s e th ro u g h t h e membrane, t h e f i n a l c o n d itio n may b e fo rm u la te d m a th e m a tic a lly a s b e in g (C-L + x ) x » (C2 - x ) 2 s in c e a c c o rd in g t o Donnan t h e e q u ilib riu m c o n c e n tr a tio n s o f d i f f u s i b l e io n s m ust be eq u al*

I t fo llo w s t h a t x -

.(C.2)Cl+2C2

-iU x C? c2 C1+2C2 where

i s t h e f r a c t i o n o f t h e NaCl o r i g i n a l l y p r e s e n t t h a t has d i f c2 fu s e d th ro u g h * I t can be shown t h a t th e c o n c e n tr a tio n s o f d i f f u s i b l e io n s a t t h e s te a d y s t a t e c o n d itio n a r e u n e q u a lly d i s t r i b u t e d a c ro s s

t h e membrane*

The c o n c e n tr a tio n o f Na* on t h e l e f t i s a p p r e c ia b ly

d i f f e r e n t from t h a t on th e r i g h t a t e q u ilib riu m *

I f a la r g e ex cess

o f NaR i s p r e s e n t , a s compared t o th e NaCl i n t h e o p p o s ite com partm ent, l i t t l e d i f f u s i o n o f NaCl o c c u rs th ro u g h th e membrane*

Donnan membrane

22 e q u i l i b r i a may b e e lim in a te d b y u s in g h ig h c o n c e n tr a tio n s o f e l e c t r o ­ l y t e s , b u t t h i s e x p e d ie n t c an r a r e l y be a p p lie d i n e x p e rim e n ta l work o f t h i s k in d .

I t i s e v id e n t t h a t such a n io n a d ju stm e n t o c c u rs when­

e v e r a membrane i s removed from i t s n a t u r a l environm ent# A w orkable method o f stu d y in g th e p e r m e a b ility c h a r a c t e r i s t i c s o f e x c is e d f ro g membrane was th u s d ev elo p ed .

The s o - c a l l e d "norm al"

p e r m e a b ility o f f ro g membrane could be d e te rm in e d by m easu rin g th e r a t e o f p e n e tr a tio n o f I ” a c c o rd in g t o th e method e la b o ra te d #

The

e f f e c t t h a t v a r io u s tr e a tm e n ts had on membrane p e r m e a b ility c o u ld b e s tu d ie d by t r e a t i n g o n e - h a lf th e membrane s e c tio n and u s in g th e o th e r h a l f a s t h e u n tr e a te d c o n tr o l#

By th e l a t t e r means a l l m easurem ents

b a se d on th e one t e s t an im al were c a r r i e d o u t i n s t r i c t l y p a r a l l e l f a s h io n i n t h a t each membrane p o r tio n was t r e a t e d e x a c tly t h e same, e x c e p t f o r t h e v a r i a b l e u n d er s tu d y .

Such an arrangem ent p e r m itte d a

h ig h d e g re e o f c o n tro l#

B#

S ta n d a r d iz a tio n o f th e Is o to p e and C ounting Technique The is o to p e s u sed i n th e fo llo w in g d e s c rib e d work were o b ta in e d

and s ta n d a rd iz e d a c c o rd in g t o t h e fo llo w in g p ro c e d u re .

The l a b e l l e d

sodium u sed i n s e v e r a l s h o r t ex p erim en ts was produced by t h e c y c lo ­ t r o n bombardment o f o r d in a r y NaCl a c c o rd in g t o t h e n u c le a r r e a c t io n N a ^ ( d ,p ) M a r­ th e c h a r a c t e r i s t i c s o f N a ^ a r e th e fo llo w in g T^ = 14#8 hours B” = 1 ,3 9 0 M.E.V# V » 1 .3 8 0 , 2*758 M.E.V#

23 L a b e lle d io d id e was o b ta in e d from th e p i l e r e a c t o r o f Oak R idge N a tio n a l L a b o r a to r ie s a s a p ro d u c t o f uranium f i s s i o n , and was r e c e iv e d i n t h i s l a b o r a t o r y a s a b i s u l f i t e s o lu t i o n o f N al.

The c h a r a c t e r i s t i c s

o f 1^31 a r e t h e fo llo w in g Ti = 8 days B” « 0 . 5 9 M .E .V .

V= 0 ,0 8 , 0 ,3 7 M.E.V, P r i o r t o s ta n d a r d i z a t io n o f t h e is o to p e i t was u s u a lly n e c e s s a ry t o d i l u t e th e Oak R idge shipm ent t h r e e o r f o u r f o ld w ith d i s t i l l e d w a te r , w hich t h e n gave a s to c k s o lu t i o n having a d e s ir a b le ra n g e o f a c t i v i t y (50 - 100 x 10^ c ,p ,m ,/m l, ) .

F or s ta n d a r d iz a tio n p u rp o se s a one m l,

sam ple o f t h e s to c k s o lu t i o n was p la c e d i n a 0 ,5 1 , v o lu m e tric f l a s k , d i l u t e d t o m ark , and one m l, o f t h i s s o lu ti o n was p la c e d i n a 100 m l, v o lu m e tric f l a s k and a g a in d i l u t e d to m ark.

T hree 0 ,1 m l, sam ples o f

t h e l a s t d i l u t i o n w ere d e p o s ite d on s t r i p s o f o r d in a r y f i l t e r p a p e r c u t t o a s i z e o f 1 cm. x 5 c m ., which had p r e v io u s ly been im p reg n ated w ith 10% NaOH.

The f i l t e r p a p e r s t r i p s (w hich had been a tta c h e d t o

Dennison* s Gummed L a b e ls #217 f o r e ase i n h a n d lin g ) were th e n d r ie d f o r 30 m in u te s a t 90° un d er an i n f r a - r e d lam p.

The s t r i p s were mounted

w ith a s c o tc h ta p e b ac k in g and were c o v e re d by c e llo p h a n e s t r i p s on t h e exposed s i d e .

The s t r i p was th u s e f f e c t i v e l y s e a le d and co u ld be

h a n d le d w ith im p u n ity .

F o r co n v en ien ce i n c o u n tin g t h e s e a le d s t r i p

was a f f i x e d to a sm a ll r e c t a n g u la r p ap er b a c k in g , upon w hich t h e sam ple number was i n s c r i b e d . The f i l t e r s t r i p s were th e n co u n ted b y p la c in g th e s t r i p i n t h e c l o s e s t p ro x im ity p o s s i b l e , d i r e c t l y u n d er and p a r a l l e l t o an Bek

24 and K rebs tu b e .

I n a l l c o u n tin g work a C y c lo tro n S p e c i a l t i e s S c a le - Q f-

E ig h t c i r c u i t was u s e d .

The c o u n ts o b ta in e d were c o r r e c te d f o r back­

g ro u n d , and t h e a c t i v i t y o f th e s to c k s o lu t i o n was d e te rm in e d by m u lt i­ p ly in g th e c o r r e c te d c o u n t by th e d i l u t i o n f a c t o r (5 x 10^ i n th e p r e ­ c e d in g ex am p le).

The f i l t e r s t r i p s so p re p a re d were th e n r e t a i n e d a s

perm anent s ta n d a r d s f o r t h i s p a r t i c u l a r s to c k s o l u t i o n .

The a c t i v i t y

o f th e s to c k s o lu t i o n was e a s i l y d e te rm in e d a t any tim e by c o u n tin g th e s ta n d a rd f i l t e r p a p e r s t r i p s ,

C.

P re v e n tio n o f C o n tam in atio n W hile th e is o to p e s i n th e amounts u sed i n t h i s work were n o t

e s p e c i a l l y d an g ero u s from a h e a lth h a z a rd s ta n d p o in t, c e r t a i n p re ­ c a u tio n s w ere ta k e n .

Such p r e c a u tio n s w ere j u s t i f i a b l e inasm uch a s

th e e f f e c t s o f lo w - le v e l r a d i a t i o n have n o t b een s u f f i c i e n t l y s tu d ie d as to

be

l a b e l e d u n e q u iv o c a lly a s h a rm le s s . P ro b ab ly o f more im por­

ta n c e

is

t h e f a c t t h a t th e u s e o f m a te r ia ls d e te c ta b le i n m in u te

amounts p o se s th e problem o f c o n ta m in a tio n o f s e l f and th e w orking a r e a , th u s making a c c u r a te m easurem ents d i f f i c u l t . The p r e c a u tio n s ta k e n m ig h t b e c l a s s i f i e d c o n v e n ie n tly i n t o two g ro u p s.

These a r e :

(a ) P erso n al 1 . When h ig h ly a c t i v e m a t e r ia ls w ere h an d led i n d i r e c t m ethods were u s e d .

Incom ing shipm ents o f io d id e were

removed from th e o r i g i n a l c o n ta in e r by a rem ote c o n tro l p ip e t.

2 . S a f e ty b ad g es w ere worn c h e s t h ig h on th e l a b o r a t o r y c o a t th ro u g h o u t t h e w orking day.

These b adges were

m o n ito re d w eekly by th e Argonne N a tio n a l L a b o ra to ry and were f o r th e p urpose o f d e te c tin g e x c e s s iv e r a d i ­ a t i o n d o s a g e s, a c c o rd in g to th e s ta n d a rd s s e t up by th e Atomic Energy Commission, 3 . Rubber g lo v e s were worn a t a l l tim e s when c o n ta m in a tio n o f t h e hands by r a d io a c t iv e m a t e r ia ls was l i k e l y , 4 , Hands w ere washed th o ro u g h ly a f t e r e v e ry o p e r a tio n in v o lv in g th e h a n d lin g o f a r a d io a c tiv e m a t e r i a l . Once c o n ta m in a te d , i t was found u s e le s s t o a tte m p t t h e rem oval o f r a d i o a c t i v i t y from t h e hands by a l t e r n a t e tr e a tm e n ts w ith o x id iz in g and re d u c in g a g e n ts , 5, Lead s h ie ld in g was u sed i n th e c a s e s where i t was n e c e s s a r y t o be i n th e v i c i n i t y o f a h ig h ly a c t iv e s o u rc e .

N o rm ally , h ig h ly a c t i v e so u rc e s were s to r e d

away from th e w orking a r e a , (b ) Environm ent a l 1 , Heavy brown w rapping p ap er was u sed to c o v e r a l l w orking s u r f a c e s .

I n c a s e o f a c c id e n ta l s p i l l a g e

such w rapping p a p e r co u ld be d is c a r d e d . 2 , A ll co n ta m in a te d g la ss w a re was washed c o n tin u o u s ly f o r t h r e e days b e f o r e b e in g r e u s e d .

The w ashing equipm ent

was f r e q u e n t l y in s p e c te d d u rin g t h i s tim e t o in s u r e t h a t i t was w orking e f f i c i e n t l y . 3 , The w orking a r e a was f r e q u e n tly m o n ito re d t o d is c o v e r any a c c i d e n t a l c o n ta m in a tio n , 4 , A ll c o u n tin g equipm ent was lo c a te d away from th e la b o r a to r y w orking a r e a .

26 D.

C hoice and Care Of E x p e rim e n ta l A nim als The f r o g s u sed i n t h i s work were o f t h e s p e c ie s Rana P ip i e n s ,

o r t h e common g r a s s f r o g .

As each shipm ent o f f r o g s was r e c e iv e d , th e

a n im a ls w ith damaged membranes were d is c a rd e d and th e h e a lth y u n in ju re d a n im a ls w ere s to r e d i n g a lv a n iz e d t a n k s ,

A f r e s h w a te r s u p p ly was main­

t a i n e d a t a l l tim e s b y a s u i t a b l e in flo w and o v e rflo w arra n g e m e n t.

The

an im a ls r e q u ir e d no f e e d , inasm uch a s th e y were u sed w ith in two o r t h r e e w eeks.

W ith su ch an arran g em en t th e an im als u sed i n t h e e x p e r i­

m e n ta l work were f r e s h and a t f u l l v i t a l i t y . Anim als i n th e w eig h t ran g e o f 30-40 grams w ere u s e d .

U s u a lly ,

an e f f o r t was made t o s e c u re a com plete s e t o f d a ta from, an im a ls o f th e same sh ip m en t.

I n g e n e r a l , i t was o b se rv e d t h a t w in te r shipm ents o f

f r o g s had low v i t a l i t y , w h ile s p rin g and summer f r o g s had a h ig h v i t a l ­ ity ,

E.

D e f i n i ti o n Of Terms c ,p ,m ,/m l. « c o u n ts p e r m in u te p e r m l, o f r a d io a c tiv e sam ple. Background = c o u n ts o b ta in e d from a l l s o u rc e s o f r a d i a t i o n

o th e r th a n t h e sam ple. C o rre c te d co u n t « th e co u n t o f t h e sample a f t e r h av in g been c o r r e c te d f o r t h e background c o u n t.

When th e h a l f - l i f e o f th e is o to p e

was s h o r t , a s i n t h e c a se o f N a ^ , i t was a ls o n e c e s s a r y t o c o r r e c t th e co u n t o b ta in e d f o r decay o f th e is o to p e .

The d e cay c o r r e c t i o n was made

b a ck t o th e z e ro tim e f o r th e p a r t i c u l a r e x p e rim e n t.

C o rre c tio n f o r

decay o f th e is o to p e was made u t i l i z i n g th e fo rm u la I « I 0e” ^ , where I = t h e co u n t o b ta in e d a t any tim e t , I q = th e o r i g i n a l co u n t o f th e sam p le, e ■ th e n a t u r a l lo g b a s e , k * th e d ecay o f t h a t p a r t i c u l a r

27 i s o t o p e , t * tim e e la p s e d ( i n th e same tim e u n i t s a s k ) betw een I 0 and I . Z ero tim e « t h e tim e a t w hich t h e ex p e rim e n t was s t a r t e d .

In

t h i s work t h e z e ro tim e was c o n v e n ie n tly assumed t o b e t h e tim e a t w hich t h e membrane was lo w ered i n t o th e r a d io a c tiv e s o l u t i o n . S p e c if ic a c t i v i t y * th e r a t i o o f ta g g e d atom s t o u n tag g ed atom s. T reatm en t tim e * t h e le n g th o f tim e th e membrane was exposed t o th e a c tio n o f th e a s tr in g e n t.

28 I.

THE "NORMAL" PERMEABILITY OF EXCISED FROG MEMBRANE B efo re c o n s id e r in g changes i n f r o g membrane p e r m e a b ility cau sed

by d i f f e r e n t a g e n ts i t i s w e ll t o e s t a b l i s h t h e p e r m e a b ility re s p o n s e o f presu m ab ly n o m a l membrane. re s p o n s e b etw een a n im a ls .

I t i s lo g ic a l t o expect v a ria tio n s in

A p e ru s a l o f th e l i t e r a t u r e in d ic a te s th a t

t h e r e a r e no e x te n d e d q u a n t i t a t i v e i n v e s t i g a t i o n s p u b lis h e d w hich would answ er t h i s q u e s tio n .

The fo llo w in g d a ta c l e a r l y show th e rem arkable

v a r i a t i o n i n th e re s p o n s e o f a group o f an im als o f a homogeneous pop­ u l a t i o n (a g e , w e ig h t, p re v io u s t r e a t m e n t) .

'While t h e s e m easurem ents

have b een made on h u ndreds o f a n im a ls , o n ly d a ta f o r th o s e o f a r e p r e ­ s e n t a t i v e group a r e g iv e n . E x p e rim e n ta l M ethod.

The s k in o f th e f r o g Rana P ip ie n s was

removed from f r e s h l y k i l l e d and p ith e d an im als and a p o r tio n ta k e n from t h e f r o g back i s p la c e d i n t a p w a te r p r i o r t o a tta c h in g t o th e g la s s tu b e .

I n a l l c a s e s t h e membranes were a tta c h e d o v e r t h e ends o f

12 mm. g l a s s tu b in g c u t i n le n g th s 15 cm. lo n g , th e ends o f w hich had been v e ry s l i g h t l y f l a r e d t o p re v e n t s lip p a g e o f th e membrane a f t e r a tta c h m e n t• The membranes w ere l o o s e ly a tta c h e d u s in g number e ig h t c o tto n th re a d .

I n a H c a s e s t h e membrane was o r ie n te d i n a manner so t h a t th e

m o rp h o lo g ic a l i n n e r s id e c o n ta c te d th e r a d io a c t iv e s o lu t i o n .

A f te r

trim m in g away s u r p lu s membrane, two m l. o f t a p w a te r (342 p.p*m. o f t o t a l h a rd n e s s , e x p re ss e d a s c a rb o n a te ) was p la c e d i n th e tu b e .

A fte r

b l o t t i n g away any e x c e s s m o is tu re from th e exposed membrane, th e tu b e w ith membrane was lo w ered i n t o f o u r m l. o f r a d io a c ti v e io d id e h av in g an a c t i v i t y o f 200,000 c .p .m ./m l.

The io d id e s o lu tio n s u sed i n t h i s

work had a pH w ith in th e ra n g e o f 7 .4 -8 * 3 , stnd t h e ex p e rim e n ts were

29 c a r r i e d o u t a t room te m p e ra tu re *

The tu b e w ith a tta c h e d f r o g membrane

was lo w ered i n t o t h e r a d i o a c ti v e s o lu t io n to a d e p th a s c lo s e a s p o s s i­ b l e t o t h r e e mm* (s e e F ig u re k . S e c tio n V I ) , when s t a r t i n g th e e x p e ri­ ment ( z e ro tim e )*

U s u a lly 80 m in u te s was allo w ed f o r e q u ilib riu m

c o n d itio n s t o be e s t a b l i s h e d b e f o r e 0*1 ml* sam ples were w ithdraw n from th e in n e r com partm ent f o r a c t i v i t y m easurem ents, and sam ples w ere ta k e n e v e ry 40 m in u te s t h e r e a f t e r * R e s u lts .

I t w i l l b e se e n t h a t th e t r a c e r m ethod o f i n v e s t i g a t i o n

b r in g s o u t i n a r a t h e r s t a r t l i n g m anner t h e v a r i a t i o n s i n p e r m e a b ilitie s betw een an im als*

T h is v a r i a t i o n i n re sp o n s e i n d i c a t e s c l e a r l y th e need

f o r c a r r y in g o u t e x p e rim e n ts on a number o f an im als so t h a t r e s u l t s o b ta in e d may b e s t a t i s t i c a l l y s i g n i f i c a n t , and th e av o id an ce o f b a s in g c o n c lu s io n s upon i s o l a t e d ex perim ents* T able 1 The T ra n s p o rt o f Io d id e Io n Through Frog Membrane Using I ^ " ^ As T r a c e r . The Normal P e r m e a b ility Of Frog Membrane 80 m in u te s

120 m in u te s

160 m in u te s

45 50 65 70 70 95 75 90 95 95 140 130

65 80 100 100 102 H5 137 125 162 164 203 227

95 120 150 150 155 155 215 178 253 258 280 340

200 m in u te s 132 165 212 200 205 200 300 237 354 360 363 462

132

196

266

Average A c tiv ity . .,8 5 _______

T ab le 1* The co m p lete r e s u l t s f o r one an im al a r e p re s e n te d h o r i z o n t a l l y a c r o s s t h e t a b u l a t i o n . Time g iv e n r e f e r s t o tim e e la p s e d b etw een im m ersion o f t h e membrane i n t h e r a d i o a c tiv e s o lu t io n and th e tim e a t w hich sam ple was tak en * Average a c t i v i t i e s r e f e r t o th e av erag e

30 c o r r e c te d co u n t o b ta in e d from 12 an im als a t t h e tim e s i n d i c a t e d . A ll c o u n ts a r e b a sed on th e a c t i v i t y o f 0 .1 m l. a l i q u o t s ta k e n from th e i n n e r com partm ent, e x p re s s e d a s c .p .m . The a c t i v i t y o f sam ples a t a mean tim e o f 80 m in u te s i s se e n t o v a r y from 45 t o 1 4 0 , o r an extrem e v a r i a t i o n o f 65$ from t h e mean v a lu e . S in c e t h e i n d i v i d u a l m easurem ents were made w ith in a p r e c i s io n o f seven p e r c e n t , th e v a r i a t i o n i n v a lu e s can o n ly be a s c r ib e d t o v a r i a t i o n s i n t h e m em branes.

From T able 1 i t i s p o s s ib le to c o n s tr u c t a f a m ily o f

c u rv e s w hich show g r a p h i c a l l y th e v a r i a t i o n i n re sp o n s e o f a group o f an im a ls ( c . f . F i g u r e ; i ) .

I t i s d i f f i c u l t , t h e r e f o r e , t o e n v is io n a

s o - c a l l e d "norm al" p e r m e a b ility o f f r o g membrane t o io d id e io n , and w herever t h i s te rm h as been used i t has b een e n c lo s e d i n q u o ta tio n m a rk s. The e x te n t o f t h e v a r i a t i o n s in d ic a te d by t h e fo re g o in g r e s u l t s have n o t been re c o g n iz e d by many w o rk ers.

The l i t e r a t u r e i s r e p l e t e

w ith c o n c lu s io n s b ased on t h e r e s u l t s o f b u t s e v e r a l a n im a ls , o r com­ p a r is o n s o f t h e a b s o lu te r e s u l t s from s in g l e a n im a ls.

Such a p r a c t i c e

i s e x tre m e ly unsound, s in c e t h e in h e r e n t v a r i a t i o n i s g r e a t enough to e i t h e r mask o r e x a g g e ra te l i m ite d e x p e rim e n ta l f in d in g s .

Adolph (21)

re c o g n iz e d th e v a r i a t i o n s among f ro g s b u t was n o t aware o f t h e g r e a t d i f f e r e n c e s i n b e h a v io r w hich have b een fo u n d .

F u rth e rm o re , A d o lp h 's

m easurem ents w ere made u s in g ru b b e r— bands t o b in d th e membrane i n a s tr e t c h e d c o n d i t i o n , a c o n d itio n which m ight be c r i t i c i z e d on th e grounds p o in te d o u t i n t h e p re c e d in g d is c u s s io n . An a n a l y s is o f th e p ro c e d u re used from th e s ta n d p o in t o f d i f f u s i o n k i n e t i c s w i l l s e rv e t o e x p la in th e slow r a t e o f p e n e tr a t io n

31 F ig u re 1

Counts

per 0*1 ml* aliquot

from inner

s o lu tio n

The F am ily o f C urves O btained From M easurem ents Of th e "Normal" P e r m e a b ility of 12 F rogs

500

400

300

200

100

80

90

100 110

120

130

140 150

160

170

180 190

200

F ig u re 1* The wide v a r i a t i o n i n p e r m e a b il it ie s o f e x c is e d f r o g membrane t o I ” i s r e p re s e n te d by th e above fa m ily o f c u r v e s . The r a t e o f p e n e tr a tio n o f a membrane i s ev idenced by th e r a t e a t w hich th e a c t i v i t y o f th e in n e r s o lu tio n i n ­ c r e a s e s w ith tim e*

32 o f io d id e i o n th ro u g h f ro g membrane.

I t i s a p p a re n t t h a t a s te e p con­

c e n t r a t i o n g r a d ie n t e x i s t s i n i t i a l l y betw een t h e o u te r s o lu t i o n and t h e s o lu t i o n i n t h e in n e r com partm ent.

E x p erim en ts have shown t h a t a p lo t

o f a c t i v i t y i n th e in n e r com partment v s . tim e r e s u l t s i n a smooth cu rv e up u n t i l 14-16 h o u rs o r lo n g e r .

The d i f f u s i o n o f an io n th ro u g h s o lu ­

t i o n i s f a i r l y a c c u r a t e l y d e s c rib e d by P i c k 's law , and one would a n t i c i p a t e on t h e b a s is o f t h i s law t h a t th e above p l o t t e d c u rv e would f l a t t e n o u t a s t h e c o n c e n tr a tio n i n t h e in n e r com partment becomes a p p re ­ c ia b le . I t i s e v i d e n t , i n a c c o rd an ce w ith th e Debye-Htickel p i c t u r e , t h a t each io n moving th ro u g h t h e s o lu tio n h a s a s s o c ia te d w ith i t an 11atm o sp h ere11 o f io n s o f th e o p p o s ite c h a rg e .

( I f a p o t e n t i a l g r a d ie n t

e x i s t e d t h i s atm o sp h ere would assum e an a sy m m etrical c o n f ig u r a tio n ) . F u rth e rm o re , t h e modern i n t e r p r e t a t i o n o f io n s i n s o lu tio n p ro p o ses t h a t io n s a r e h y d r a te d , g iv in g i n e f f e c t a body o f more s iz e a b le p r o p o r tio n s th a n t h e o r i g i n a l i o n .

T hus, t h e d i f f u s io n o f an io n

th ro u g h s o lu t i o n m ust p ro ceed w ith t h e f o r c e s o f v i s c o s i t y o f th e medium a s w e ll a s t h e f a c t o r s e la b o r a te d above o p e r a tin g a g a in s t i t . I n p e n e tr a tin g t h e membrane io n s p a s s from a r e g io n o f h ig h ch em ica l p o t e n t i a l t o one o f low ch em ical p o t e n t i a l , and su ch a t r a n s ­ f e r would b e e x p e c te d t o p ro c e e d ( i f t h e membrane rem ained p a s s iv e ) u n t i l e q u ilib r iu m c o n c e n tr a tio n s w ere re a c h e d . Ih e mechanism o f p e n e tr a tio n o f a membrane by an io n i s n o t c l e a r l y u n d e rs to o d .

In b r o a d e s t o u t l i n e s , i t i s b e lie v e d t h a t th e

membrane i s u n d e r c o n tin u a l bombardment by th e d if f u s in g p a r t i c l e s . An io d id e io n s t r i k i n g a p o re a r e a can p a ss th ro u g h ; how ever, t o m eet

33 t h e demands o f e l e c t r i c a l n e u t r a l i t y r e q u ir e d i n sim p le c a s e s o f d i f ­ f u s io n th e s im u lta n e o u s c o l l i s i o n o f Na* and I™ w ith t h e p o re a r e a m ust o c c u r.

The p r o b a b i l i t y o f such a c o l l i s i o n i s dependent on th e concen­

t r a ti o n s o f th e io n s . (l“ ).

The r a t e o f p assag e i n one d i r e c t i o n i s

(Na*)

However, a s th e io n c o n c e n tr a tio n i n c r e a s e s i n t h e in n e r com part­

m e n t, t h e r a t e becomes a p p r e c ia b le i n th e r e v e r s e d i r e c t i o n , and i s e q u a l t o kg (Na*) ( I - ) .

I t i s e v id e n t t h a t when k^ * kg e q u ilib riu m

has been a t t a i n e d , and t h e r e i s no n e t t r a n s f e r o f io d id e i o n .

In th e s e

ex p erim e n ts t h i s c o n d itio n i s n o t a t t a i n e d , s in c e th e e x p erim en ts a r e o n ly o f s e v e r a l hours* d u ra tio n * The above s i t u a t i o n i s o n ly p a r t i a l l y c o r r e c t i n d e s c rib in g t h e d i f f u s i o n th ro u g h f r o g membrane, a s c a s e s a r e d e s c rib e d i n th e l i t e r a t u r e w here p o t e n t i a l m easurem ents a c r o s s t h e membrane i n d i c a t e t h a t demands o f e l e c t r i c a l n e u t r a l i t y a r e a p p a r e n tly n o t s a t i s f i e d . I n t h e s e c a s e s t h e membrane seems t o be s e l e c t i v e l y perm eable t o e i t h e r th e a n io n o r c a t i o n , w ith t h e r e s u l t t h a t th e io n " l e f t b eh in d " d e te r ­ m ines t h e s ig n o f t h e p o t e n t i a l e x i s t i n g on t h a t s id e o f t h e membrane. However, th e membrane p o t e n t i a l may a ls o b e a f f e c t e d b y io n s i n t e r ­ a c tin g w ith p o l a r g ro u p s and io n s p r e s e n t i n th e membrane ( th e c a s e c o n s id e re d l a t e r ) ; o r t h e io n may d is s o lv e i n t h e membrane s t r u c t u r e and a f f e c t t h e membrane p o t e n t i a l i n d i r e c t l y .

The agn and m agnitude

o f t h e membrane p o t e n t i a l d eterm in e to a s i g n i f i c a n t e x te n t th e perme­ a b i l i t y o f a membrane ( 2 1 ) . Us s in g (3 1 ) ad v an ces th e id e a t h a t an io n m ust combine w ith an io n o f th e o p p o s ite s ig n w hich i s p r e s e n t i n th e membrane, and th e r e s u l t i n g io n i c d o u b le t d i f f u s e s th ro u g h .

T h is b e l i e f i s e s p e c i a l l y

34 accommodated t o c a s e s where t h e membrane i s a c t i v e tow ard t h e p e n e tr a ­ t i n g s u b s ta n c e . The e f f e c t o f pH on membrane p e r m e a b ility i s a ls o o f im p o rta n c e . W hile t h i s e f f e c t i s d is c u s s e d a t g r e a t e r le n g th i n a l a t e r S e c tio n , i t m ight b e n o te d h e re t h a t t h e s t a t e o f d i s s o c i a t i o n o f th e p r o te in c o n s t i t u e n t s o f a membrane have a m odifying e f f e c t on membrane perme­ a b ility . U ssin g (2 3 ) h as p o in te d o u t th e sh o rtco m in g s o f th e t r a c e r m ethod o f i n v e s t i g a t i o n a s a p p lie d t o p e r m e a b ility problem s*

I t is

w e ll t o n o te t h a t even w ere a l l o f h i s o b je c tio n s v a l i d , t h e method would s t i l l be c o n s id e ra b ly s u p e r io r t o o ld e r m ethods.

Any c r i t i c i s m

o f t h e m ethod i s l i k e l y t o stem from th e i n a b i l i t y o f many w o rk ers t o a g re e on w h eth er th e p e r m e a b ility o f a membrane sh o u ld in c lu d e c a s e s where t h e membrane i s a c t i v e tow ard t h e io n b e in g in v e s t i g a t e d .

Thus,

t o d e c id e w hether a membrane i s s t i l l a c t i v e l y m e ta b o liz in g and w hether s e c r e t o r y a c t i v i t y i s s t i l l p r e s e n t i s e x trem ely d i f f i c u l t ; and t o e v a lu a te how th e s e f a c t o r s c o n tr ib u te t o membrane p e r m e a b ility i s a problem t h a t can o n ly be s o lv e d by s p e c i f i c enzyme p o iso n in g c a t a l y s t s and su p p lem e n tary i n v e s t i g a t i o n s .

However, t o t r y t o a r t i f i c i a l l y

s e p a r a te t h e c o m p le x ity o f phenomena w hich c o n tr ib u te t o t h e p e r m e a b ility o f a membrane and e v a lu a te t h e i r im p o rtan ce s in g ly i s a t a s k o f alm o st im p o ss ib le d i f f i c u l t y .

The la c k o f knowledge o f c o n c e n tr a tio n d i f f e r ­

en ces i n m ost p e r m e a b ility d e te rm in a tio n s u s in g t r a c e r s can n o t be term ed a f a u l t o f th e m ethod b u t i s r a t h e r one o f te c h n iq u e .

S im i la r ly ,

a l a c k o f knowledge o f t h e e l e c t r i c a l phenomena im p o rta n t i n membrane p e r m e a b ility ca n n o t b e term e d a c r i t i c i s m o f t h e method b u t more p ro p -

35 e r l y a p p l i e s t o t h e f a i l u r e t o s ta n d a r d iz e m ethods. A more r e a s o n a b le l i m i t a t i o n o f t h e m ethod would seem t o r e s u l t from t h e e f f e c t o f r a d i a t i o n on membrane s t r u c t u r e , a f a c t o r d i f f i c u l t t o e v a lu a te p r o p e r ly . in d ic a te d e a r l i e r .

T h is e f f e c t has b een n o te d by B a r n e tt ( 3 7 ) , a s

I t seems e n t i r e l y p r o b a b le , how ever, t h a t th e r e l ­

a t i v e l y low s p e c i f i c a c t i v i t i e s u s e d , combined w ith t h e low en erg y o f t h e r a d i a t i o n and th e h ig h a b s o rb in g power o f th e s o lu tio n d im in is h t h e s ig n i f i c a n c e o f t h i s d a n g e r.

The f a c t t h a t many o f th e r e s u l t s g iv e n

by t h i s m ethod a r e co n firm ed by o th e r m ethods o f i n v e s t i g a t i o n o f a v a s t l y d i f f e r e n t n a tu r e le n d s f u r t h e r a u t h o r i t y t o t h i s a p p l ic a tio n o f ra d io a c tiv e t r a c e r s .

36 II.

THE PERMEABILITY CONSTANT OF FROG MEMBRANE Inasm uch a s t h e p r e s e n t i n v e s t i g a t i o n was co n cern ed p r im a r ily

w ith membrane p e r m e a b ility i t was b e lie v e d t h a t some fu n d am en tal a s p e c ts o f t h e s u b je c t c o u ld b e i n v e s t i g a t e d by t h e t r a c e r m ethod.

R a d io a c tiv e

t r a c e r s a f f o r d a r a p i d and c o n v e n ie n t means o f d e te rm in in g th e perme­ a b i l i t y c o n s ta n t.

I t i s p o s s ib l e t o c o n t r o l a l l v a r i a b l e s i n such an

ex p erim en t so t h a t th e o n ly f a c t o r w hich g o v e rn s th e r a t e o f p assag e o f a m a t e r i a l i s t h e membrane i t s e l f * S in c e s e v e r a l i n v e s t i g a t o r s have n o te d t h a t th e p e r m e a b ility o f a membrane seems t o v a ry w ith e x p e rim e n ta l c o n d itio n s i t i s obv io u s t h a t th e r e s u l t s o f a p e r m e a b ility c o n s ta n t d e te r m in a tio n a r e v a l i d o n ly i f such c o n d itio n s a r e r i g i d l y d e f in e d . Exne rim en t a l .

The e x p e rim e n ta l m ethod d e s c rib e d u n d er S e c tio n

I was u se d w ith s l i g h t m o d if ic a tio n .

D e ta ils a s t o tu b e s i z e , mode o f

a tta c h in g membrane, pH o f t h e r a d io a c tiv e s o l u t i o n , e t c . w ere th e same. The n e c e s s a ry m o d if ic a tio n s w ere t h e fo llo w in g :

th e volume o f th e

r a d i o a c t i v e io d id e s o lu t i o n i n t h e b e a k e r was made la r g e (200 m l*, a s s a y in g 200,000 c .p * m ./m l,) so t h a t i t s c o n c e n tr a tio n d id n o t change a p p r e c ia b ly d u rin g th e e x p e rim e n t.

The r a d i o a c t iv e s o lu ti o n i n th e

b e a k e r was s t i r r e d c o n s t a n t l y i n t h e a r e a o f th e membrane t o p re v e n t th e e s ta b lis h m e n t o f an io n i c c o n c e n tr a tio n g r a d i e n t .

The membrane was

a tta c h e d a s u s u a l , and e x te n d e d in to th e s t i r r e d r a d i o a c ti v e io d id e s o lu t i o n .

At f r e q u e n t I n t e r v a l s sam ples w ere ta k e n s im u lta n e o u s ly from

th e in n e r com partm ent and t h e b e a k e r.

The sam ples from th e b e a k e r were

p la c e d i n 25 m l. v o lu m e tr ie s , d i l u t e d t o m ark, and a l i q u o t s o f t h i s d i l u t i o n were co u n te d w ith t h e o th e r sam ples*

37 R e s u lts .

U sing d a t a g a th e r e d i n t h e above m anner i t was p o s s ib le

t o c a l c u l a t e t h e p e r m e a b ility c o n s ta n t.

The d a ta o b ta in e d i n t h i s

ex p erim en t i s t a b u l a t e d i n T a b le 2* T ab le 2 The P e r m e a b ility C o n stan t o f Frog Membrane Z ero Time, M in u tes

In n e r S o lu tio n c .D .m ./m l.

75

200

182,800

105

650

182,723

165

1370

182,568

195

1530

230

2540

B eaker S o lu tio n c .o .m ./m l.

182,400

A p l o t o f th e raw d a ta showed t h a t th e p o in ts from m easurem ents

o f a c t i v i t y o f t h e in n e r com partm ent f a l l on a s t r a i g h t l i n e , w h ile th e p l o t o f a c t i v i t y o f th e b e a k e r s o lu tio n v s . tim e was n o t a s good, a s would b e e x p e c te d .

I t was t h e r e f o r e n e c e s s a r y t o t r e a t s t a t i s t i c a l l y

th e f o u r p o in ts o b ta in e d from t h e la b o r a to r y m easurem ents.

U sing th e

m ethod d e s c rib e d i n th e s t a t i s t i c a l example g iv e n i n S e c tio n V I, th e y i n t e r c e p t a t tim e = 75 m in u te s was found t o be 1 8 2 ,8 0 0 .

Thus, by

"w eighing" th e e x p e rim e n ta l v a lu e s by s t a t i s t i c a l m ethods, th e a c t i v i t y o f th e b e a k e r s o lu t i o n a t 75 m in u te s was found t o b e 182,800 c*p*m*/ml* S in c e th e p o in ts w ere assum ed t o f a l l on a s t r a i g h t l i n e ( t h i s assum ption i s v a l i d f o r s h o r t p e rio d s o f tim e ) and th e l i n e has a n e g a tiv e s lo p e , th e e q u a tio n f o r t h i s l i n e m ust conform t o t h e g e n e r a l e q u a tio n y = mx + b

38 The s lo p e m can be found by t h e e q u a tio n f o r c a l c u l a t i n g s lo p e s u s e d i n t h e s t a t i s t i c a l example i n S e c tio n V I, and was com puted t o be -2 * 7 6 . The c o n s ta n t b can now be e v a lu a te d from known v a lu e s o f x and y , nam ely when x « 75 (m in u te s) y * 1 8 2 ,8 0 0 ( c .p * m ./m l.) •

Thus

18 2 ,8 0 0 » - 2 .7 6 (75) * b b = 183,007 I t i s e v id e n t t h a t b i s t h e y - i n t e r c e p t a t z e ro tim e (x = 0 ) . e q u a tio n f o r t h e

The

s t r a i g h t l i n e was t h e r e f o r e y = - 2 .? 6 x + 183,007

U sing t h i s e q u a tio n ,

i t was fo u n d t h a t when x = 230, y ■ 1 8 2 ,4 0 0 .

now p o s s ib le t o p l o t

th e d e r iv e d v a lu e s f o r y a t tim e s when x » 7 5 ,2 3 0 ,

w hich gave th e e x a c t

li n e f o r th e p lo t o f th e a c tiv ity o f th e beaker

v s . tim e .

By i n t e r p o l a t i o n th e v a lu e s

I t was

f o r x = 105,165 were found and a r e

g iv e n i n t h e p re c e d in g t a b u l a t i o n . Davson and D a n i e l l i (3 2 ) have d e r iv e d from P i c k ’ s law t h e perme­ a b i l i t y e q u a tio n w hich was u sed i n making t h e c a l c u l a t i o n o f t h e perme­ a b i l i t y c o n s ta n t.

The f i n a l f o r a o f t h e e q u a tio n u se d was

w hich may b e tra n sfo rm e d i n t o 2 .3 0 3 lo g

uo~Gi

- m . V

where C0 = c o n c e n tr a tio n o f r a d i o a c t i v e io d id e i n t h e b e a k e r i n c .p e iiu /m l., 0±

m c o n c e n tr a tio n o f r a d i o a c t i v e io d id e a t tim e

t in

com partm ent; t

= tim e i n m in u te s; A « membrane a r e a i n cm. and

in n e r V = v o l­

ume o f s o l u t i o n i n t h e in n e r com partm ent i n l i t e r s . I t i s e v id e n t t h a t Q

a p l o t o f 2.303 lo g

U0-C i

v s . t sh o u ld g iv e a s t r a i g h t l i n e o f s lo p e

39 kA. By d e te rm in in g t h e s lo p e o f t h i s p l o t t e d l i n e k can b e e v a lu a te d , V and i s k ■ B3L» A The lo g a r ith m ic p l o t i s g iv e n i n F ig u re 2 , and w ith th e e x c e p tio n o f th e f i r s t p o in t (w hich one w ould e x p e c t t o f a l l o f f t h e s t r a i g h t l i n e s in c e com p lete e q u ilib r iu m had n o t y e t b een a t t a i n e d ) i s se e n t o g iv e a f a i r l y good s t r a i g h t l i n e .

The s lo p e o f t h i s l i n e was fo u n d t o b e

m * • P 1( § ô ïl0 5 ? 6 0 S = 1 ' ^

= I 0"6

The v a lu e s o f A and V i n t h i s work were A = 1*192 cm*2 and V = *002 1* Using th e e q u a tio n k = mV and s u b s t i t u t i n g v a lu e s , k = XtASft,# I P ^

T

1.192

2 ,4 4 x 10“ ^ m ole io d id e /c m * ^ /s e c ,

A d im e n sio n a l a n a l y s is o f th e co m p lete

e q u a tio n u s e d co n firm ed th e u n i t s o f k g iv e n . v a lu e i s t h e f o llo w in g :

The s ig n i f ic a n c e o f t h i s

Under u n i t c o n c e n tr a tio n g r a d i e n t , 2 .4 4 x 10w9

mole io d id e io n p a s s e s th ro u g h a sq u a re c e n tim e te r o f membrane a r e a i n one se c o n d .

T his v a lu e o f k h o ld s f o r a te m p e ra tu re o f 26*5° C. and

i s c e r t a i n w ith in a p r e c is io n o f seven p e r c e n t. The o n ly u n c e r t a i n t y in v o lv e d i n t h i s c a l c u l a t i o n was th e assu m p tio n t h a t th e volume o f s o lu t i o n i n t h e in n e r com partment rem ained c o n s ta n t a t .0 0 2 l i t e r .

T h is assu m p tio n was n o t u n r e a s o n a b le ,

s in c e no volume change was o b s e rv a b le f o r 12 h o u rs , and m easurem ents were ta k e n f o r o n ly h a l f t h i s tim e .

However, assum ing a volume change

o f 0 .2 m l. o c c u r r e d , and V = .0 0 1 8 1 . , k was c a l c u l a te d t o be 2*20 x 10"9 m ole io d id e /c m * ^ /s e c . , o r an e r r o r o f 9.84$*

Thus t h e

e r r o r in v o lv e d i n t h i s h y p o t h e tic a l c a s e u s in g an e x c e s s iv e o r d e r o f volume change was s t i l l l e s s th a n 10% . The o n ly v a lu e s a v a i l a b l e i n th e l i t e r a t u r e f o r com parison (4 5 ) w ere th o s e o f K a tz in , who d e te rm in e d th e r a t e s o f p a ssa g e o f Na+ and

Figure 2

AO

The L o g a rith m ic P lo t F or t h e C a lc u la tio n o f t h e P e r m e a b ility C o n stan t o f F rog Membrane ux » Rl LT\ a Lf\ ir \

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4 #5 >6 ) s u g g e s ts t h a t an a s tr i n g e n t s h o u ld d e c re a s e membrane p e r m e a b ility . The q u a l i t a t i v e e f f e c t s o f a s t r i n g e n t s on membrane p e r m e a b ility were s tu d ie d b y in c o r p o r a tin g t h e a s t r i n g e n t i n a w a te r w ashable cream b a s e (shown t o have no e f f e c t o f i t s e l f on p e r m e a b ility ) and a p p lie d t o o n e - h a lf o f a s p e c i f i e d a r e a o f t h e l i v i n g im m obilized f ro g b y means o f a s m a ll b r u s h .

The a s t r i n g e n t was a llo w e d t o rem ain on th e f r o g 1 h r . ,

a t t h e end o f w hich tim e th e f r o g was k i l l e d and p i th e d , a f t e r w hich t h e a s t r i n g e n t was r in s e d o f f and t h e f ro g membrane rem oved.

The membrane

p o r tio n from th e b ack o f t h e f r o g was d iv id e d i n t o two c i r c u l a r p o r­ t i o n s (one t r e a t e d and one u n tr e a te d ) and th e s e were a tta c h e d o v er th e ends o f t h e g l a s s tu b e s i n t h e manner d e s c rib e d p r e v io u s ly and m easure­ m ents ta k e n . The r e s u l t s o f ex p erim en ts on a l a r g e number o f a n im a ls i n d i ­ c a t e s w ith o u t e x c e p tio n t h a t a s tr i n g e n t s in c r e a s e th e p e r m e a b ility o f e x c is e d f r o g membrane.

T his p e r m e a b ility in c r e a s e was found to o c c u r

i r r e s p e c t i v e o f th e o r i e n t a t i o n o f th e fro g membrane i n t h e e x p e rim e n t, th e a c t i v i t y o f t h e r a d i o a c t i v e s o lu tio n o r t h e p a r t i c u l a r is o to p e u s e d , o r t h e m anner i n w hich th e a s t r i n g e n t was a p p lie d t o t h e membrane. The a s t r i n g e n t s AICI 3 ,

and aluminum m e th io n a te w ere e x te n s iv e ly

7 te s te d . The r e s u l t s o b ta in e d u s in g f i v e p e rc e n t A lCl^ a r e t y p i c a l and a r e g iv e n i n T ab le 1 , P a r t I -C .

A com parison o f A and C o f P a r t I o f

t h i s t a b l e shows t h e s i g n i f i c a n t o r d e r o f in c r e a s e d p e r m e a b ility c a u se d . I n o r d e r t o t e s t w h e th e r th e in c r e a s e d p e r m e a b ility e f f e c t was o n ly c h a r a c t e r i s t i c o f io d id e i o n , a s tu d y was c a r r i e d out u s in g sodium io n .

The sodium io n e x p e rim e n ts were perform ed i n a manner s t r i c t l y

com parable t o t h e io d id e io n ex p erim en ts a lr e a d y d e s c r ib e d . I t i s e v id e n t from T able 1 , P a r t II-B t h a t sodium io n a l s o i n d i c a t e s in c r e a s e d membrane p e r m e a b ility fo llo w in g a s t r i n g e n t tr e a tm e n t. H ence, i t would a p p e a r t h a t t h e in c r e a s e d membrane p e r m e a b ility does n o t depend on t h e io n u s e d .

A com parison betw een av erag e a c t i v i t i e s o f

P a r t s I-A and II-A o f T ab le 1 shows t h a t th e d i f f u s i o n o f io d id e io n i s v e r y n e a r ly f o u r tim e s a s r a p id a s t h a t o f sodium io n . I t was now p o s s ib le t o make a q u a n t i t a t i v e s tu d y o f t h e e f f e c t s o f a s t r i n g e n t s on f r o g membrane p e r m e a b ility .

I t i s common knowledge

t h a t a s t r i n g e n t s f in d im p o rta n t a p p lic a tio n s i n v a r io u s m e d ic in e s , a n t i p e r s p i r a n t p r e p a r a tio n s and t h e l i k e .

In c e r ta in cases i t i s h e lp fu l

t o have a means o f e v a lu a tin g q u a n t i t a t i v e l y th e a s tr in g e n c y o f th e s e compounds.

On th e b a s i s o f th e in c r e a s e d p e r m e a b ility e f f e c t o f

a s t r i n g e n t s on f r o g membrane th e fo llo w in g m ethod was d e v ise d f o r e s tim a tin g a s t r i n g e n t e f f e c t s . The g e n e r a l e x p e rim e n ta l method a s d e s c rib e d was m o d ifie d a s f o llo w s :

t h e l i v e im m o b ilized f ro g was t r e a t e d w ith 250 mg. o f th e Q a s t r i n g e n t p r e p a r a tio n b y b ru sh in g t h i s amount on a s i x cm# a r e a o f t h e fro g back.

A s i x cm#2 a r e a a d ja c e n t t o th e t r e a t e d a r e a was l e f t u n -

8 t r e a t e d and s e rv e d a s t h e c o n tr o l*

At t h e end o f 60 m in u te s th e an im al

was k i l l e d , p i t h e d , and t h e a s tr i n g e n t r in s e d o f f th e membrane p o r tio n t h a t had b e e n t r e a t e d .

The membrane was d iv id e d in to two s im i l a r p o r­

t i o n s , t h e t r e a t e d and t h e u n tr e a te d .

The p e r m e a b ility experim ent was

th e n c a r r i e d o u t on th e two membranes a s u su al*

A com parison o f t h e

r a t e s o f p e n e t r a t i o n o f I " b etw een th e t r e a t e d and th e c o n t r o l membrane p e r m itte d d e d u c tio n s a s to t h e e f f e c t o f d i f f e r e n t c o n c e n tr a tio n s o f a s t r i n g e n t on membrane p e r m e a b ility . T h is m ethod was e x te n s iv e ly u sed i n t e s t i n g AICI3 , AlgCSO^)^ and aluminum m e th io n a te i n v a r io u s p e r c e n ta g e s . I t was a p p a re n t t h a t p e r m e a b ility o f t h e membrane was in c r e a s e d i n a m anner c h a r a c t e r i s t i c o f each p e rc e n ta g e o f t h e a s t r i n g e n t .

T his

g r a d a tio n o f e f f e c t s o f d i f f e r e n t p e rc e n ta g e s i s e x p re ss e d by a s o c a l l e d 11d e l t a " p l o t , w hich i s o b ta in e d by p l o t t i n g th e d if f e r e n c e i n p e r m e a b ility o f t r e a t e d and u n tr e a te d membrane p o r tio n s a t tim e s (min­ u t e s ) o f 8 0 , 12 0 , l6 0 and 200*

By d e te rm in in g t h e s lo p e o f th e s e d e l t a

p l o t l i n e s o b ta in e d from one p e rc e n ta g e o f a s tr i n g e n t f o r each an im al and a v e ra g in g th e v a lu e s from 1 0 -1 2 a n im a ls , a v a lu e c h a r a c t e r i s t i c o f t h a t p e rc e n ta g e o f a s t r i n g e n t i s o b ta in e d and i s r e f e r r e d t o a s "m u n i t s " .

These c h a r a c t e r i s t i c v a lu e s a re t a b u l a t e d i n T able 2 and

a r e a c t u a l l y t h e a v e ra g e s lo p e o f t h e d e l t a p l o t s f o r each p e rc e n ta g e of a s tr in g e n t. The d a ta i n T ab le 2 on th e fo llo w in g page i s p l o t t e d . F ig u re 1 , t o g iv e a w orking c u rv e f o r e a c h a s tr i n g e n t •

These cu rv es a re o f

u t i l i t y i n r a p i d l y and c o n v e n ie n tly d e te rm in in g th e e f f e c t o f a p a r­ t i c u l a r p e rc e n ta g e o f a s t r i n g e n t on membrane p e r m e a b ility .

I n s p e c tio n

o f t h i s f i g u r e shows t h a t t h e d e te c tio n o f p e rc e n ta g e d if f e r e n c e s a s

9 T a b le 2 The V a r ia tio n o f P e rc e n ta g e C o n c e n tra tio n o f A s trin g e n t W ith A verage S lo p e o f t h e D e lta P l o t s

C o n c e n tra tio n o f A s trin g e n t

m -U n its

2.5% AICI 3

2 .6 2 4

5

% AICI3

5.393

10

% AICI3

1 2 .3 9

2.5% A l m e th io n a te 10

% Al m e th io n a te

2 .2 2 2

9 .1 1 8

1.5% A l 2 (S 0 4 ) 3

2 .9 6 1

2.5% A12 (S0 4 ) 3

3 .3 4 2

T able 2 . I n ea c h c a se th e r e s u l t s g iv e n r e p r e s e n t av erag e v a lu e s from 1 0 -1 2 a n im a ls . M -u n its r e f e r s t o t h e a v e ra g e s lo p e o f th e d e l t a p l o t s f o r each p e rc e n ta g e o f t h a t a s t r i n g e n t . s m a ll a s one p e r c e n t i s p o s s ib le i n c e r t a i n c a s e s .

The p r e c is io n o f

t h e m ethod i s e s ta b li s h e d t o b e w ith in 10%* The t r a c e r m ethod a s d eveloped f o r p e r m e a b ility s tu d i e s a f f o r d s a r a p i d and c o n v e n ie n t means o f d e te rm in in g t h e p e r m e a b ility c o n s ta n t o f f r o g membrane.

I n t h i s d e te r m in a tio n th e te c h n iq u e d e s c r ib e d u n d er

"E x p e rim e n ta l" was u s e d , w ith s l i g h t m o d if ic a tio n .

D e t a i l s a s t o tu b e

s i s e , mode o f a t t a c h i n g membrane, pH o f t h e r a d i o a c tiv e s o lu t i o n , e t c . were t h e sam e.

The n e c e s s a r y m o d if ic a tio n s were t h e f o llo w in g :

th e

volume o f t h e r a d i o a c t i v e io d id e s o lu t i o n i n th e b e a k e r was made l a r g e (2 0 0 m l . , a s s a y in g 2 0 0 ,0 0 0 c .p .m ./ m l .) so t h a t i t s c o n c e n tr a tio n d id n o t change a p p r e c ia b ly d u rin g th e e x p e rim e n t.

The o u te r r a d io ­

a c t i v e s o lu t i o n was s t i r r e d c o n s ta n tly i n th e a r e a o f t h e membrane t o

10 p re v e n t th e e s ta b lis h m e n t o f an io n ic c o n c e n tr a tio n g r a d i e n t . By ta k in g c o n c u r re n t sam ples from th e b e a k e r and t h e in n e r com partm ent and r e c o rd in g tim e s a t w hich th e s e sam ples were ta k e n , s u f f i c i e n t d a ta was g a th e re d t o make th e c a l c u l a t i o n a c c o rd in g to th e m ethod o f Davson and D a n i e l l i ( ? ) •

The a b s o lu te r a t e o f p assag e o f

io d id e io n f o r a t y p i c a l a n im al membrane was found t o b e 2 ,4 4 x 10 “ ^ m ole T~/cm*

2

o f membrane a r e a p e r seco n d .

Inasm uch a s some o f t h e f in d in g s p r e v io u s ly c i t e d w ere u n ex p ected and c r e a t e d new p ro b le m s, a fu n d am en tal i n v e s t i g a t i o n o f t h e s t r u c t u r e o f f ro g membrane was c a r r i e d o u t .

I t was found t h a t t h e f r o g membrane

c o n s i s t s o f a l a r g e number o f d i f f e r e n t ty p e s o f c e l l s .

H is to lo g ic a l

s e c tio n s show f o u r la y e r s o f c e l l s ; s t a r t i n g w ith t h e in n erm o st l a y e r s , th e se a re :

1 , m uscle and c o n n e c tiv e t i s s u e , 2 , pigm ented m elan o p h o res,

3* s tr a tu m g e rm in a tiv u m , 4» p a r t i a l l y and f u l l y k e r a t in iz e d c e lls *

The

s t r u c t u r e i s honeycombed w ith g la n d s , a l l o f w hich may open on th e s u r­ f a c e o f th e membrane, A r a d io a u to g ra p h ic stu d y o f f r o g membrane was u n d e rta k e n t o e l u c i d a t e f u r t h e r t h e mechanisms o f io n p e n e tr a ti o n o f a membrane. I n g e n e r a l , t h i s work in d ic a te d t h a t t h e r e was no s e l e c t i v e avenue o f p e n e t r a t i o n o f t h e membrane by io d id e io n .

I n a l l c a s e s t h e membrane

was p e n e t r a t e d a s th o u g h i t were a s ie v e .

Inasmuch a s th e r a d io a u to ­

g ra p h ic te c h n iq u e h a s n o t y e t been made f u l l y q u a n t i t a t i v e , a tte m p ts t o show t h a t a t r e a t e d membrane was more f r e e l y p e n e tr a te d b y I*" th a n u n tr e a te d membrane were u n s u c c e s s f u l.

T h is f a c t , how ever, i s ab u n d a n tly

e v id e n t from t h e m ass o f e x p e rim e n ta l d a t a t h a t h as b e e n g a th e r e d , A l o c a l i z a t i o n o f a c t i v i t y was found i n and around th e k e r a -

11 t i n i z e d c e l l s , i n m ost c a se s*

T his f in d in g was u n e x p e c te d , s in c e th e

o p p o s ite membrane s u r f a c e was i n c o n ta c t w ith t h e h ig h ly r a d io a c tiv e s o l u t i o n s th ro u g h o u t t h e c o u rs e o f t h e ex p erim en ts*

D is c u s s io n o f R e s u lts The t r a c e r m ethod cf i n v e s t i g a t i o n b r in g s o u t i n s t a r t l i n g f a s h io n t h e f a c t t h a t fro g membranes v a ry w id ely i n t h e i r p e r m e a b ility . The s i g n i f i c a n c e o f t h i s v a r i a t i o n h as n o t b een r e a l i z e d by o th e r w o rk ers i n c e r t a i n c ases*

Adolph ( l ) re c o g n iz e d t h e v a r i a t i o n s among

f r o g s b u t was n o t aw are o f th e g r e a t d if f e r e n c e s i n b e h a v io r in d ic a te d by t h e s e r e s u l t s *

Many o f h is m easurem ents w ere made u s in g ru b b e r

b an d s t o b in d t h e membrane i n a s tr e t c h e d c o n d itio n , a method found t o b e u n r e l i a b l e i n t h i s and o th e r (Ô, 9 ) work* The work on t h e r a t e s o f p e n e t r a tio n o f sodium and io d id e io n s le a d s t o some i n t e r e s t i n g s p e c u la tio n r e g a rd in g th e n a tu r e o f t h e e l e c t r o s t a t i c c h arg e on t h e f r o g membranes u s e d .

E le c tr ic a l p o te n tia ls

have b ee n shown t o e x i s t i n c a s e s where t h e io n c o n c e n tr a tio n s a r e u n e q u a lly d i s t r i b u t e d a c r o s s th e membrane, and many i n v e s t i g a t o r s ( l , 7 , 1 0 , 1 1 , 1 2 ) s u g g e st t h a t e l e c t r i c a l phenomena a r e im p o rta n t i n reg u ­

l a t i n g membrane p e r m e a b ility .

W hile p ro o f o f th e o ry on th e l a t t e r

p o in t i s u n d e v e lo p e d , t h e d i f f e r e n t i a l r a t e s o f p e n e tr a t io n o f Na+ and I " fo u n d p e rm it d e d u c tio n s a s to t h e r e l a t i o n betw een io n p e r m e a b ilitie s and membrane charge* Amberson and K le in ( l l ) , i n t h e i r i n v e s t i g a t i o n s o f m oribund f r o g s k in , su g g e st t h a t when t h e membrane c h a rg e i s n e g a tiv e , above th e i s o e l e c t r i c p o in t o f t h e am pholytes p r e s e n t i n t h e membrane, t h e p en e-

12 t r a t i o n o f c a tio n s i s p r e fe r e n tia l* I f t h e f r o g membranes u se d i n t h i s work behaved a s sim p le p ro ­ t e i n membranes t h e v a r i a t i o n o f p e r m e a b ility w ith pH would be e x p e c te d t o f o llo w th e d e d u c tio n s o f Amberson and K lein*

These w orkers found

t h e i s o e l e c t r i c p o in t o f f r o g membrane t o be 5*1*

S in ce t h e pH o f t h e

a l k a l i n e io d id e s o l u t i o n s and t h e n e u t r a l NaCl s o lu t i o n s a r e c l e a r l y on t h e b a s ic s id e o f t h e i s o e l e c t r i c p o i n t, one m ight ex p ect t h a t th e membrane w ould be n e g a tiv e ly c h a rg e d , fo llo w in g t h i s l i n e o f reaso n in g * The f a c t t h a t t h e membrane i s more r a p i d ly p e n e tr a te d by t h e n e g a tiv e ly c h arg e d io d id e i o n d is c o u n ts t h i s p o s s i b i l i t y * S in c e a l a r g e body o f e v id en ce i s accu m u latin g w hich su p p o rts t h e v ie w o f t h e p r o t e i n n a tu r e o f a n a t u r a l membrane, th e m ost p rob­ a b le c o n c lu s io n i s t h a t th e f r o g membrane b e a r s a sim u la te d p o s it i v e charge*

Such b e h a v io r may b e e x p la in e d on th e b a s i s o f t h e o r e t i c a l

c o n s id e r a tio n s advanced i n Davson and D a n ie lli ( ? ) .

One can c o n s id e r

t h a t t h e n e g a tiv e ends o f t h e d ip o le s p r e s e n t i n t h e membrane phase m ust be o r ie n te d tow ard th e w a te r l a y e r .

I t i s e v id e n t t h a t t h e f o r ­

m a tio n o f a H elm holtz e l e c t r i c a l double l a y e r can p ro ceed b y th e g ro u p in g o f sodium io n s (common i n b o th c a s e s ) i n th e neighborhood o f th e s e n e g a tiv e d ip o le s a t t h e membrane s u r f a c e .

Such a membrane

s t r u c t u r e would t h e r e f o r e s im u la te b e a r in g ,a p o s it i v e c h a rg e , and would be more r a p i d l y p e n e tr a te d b y n e g a tiv e io n s . The b e h a v io r o b se rv ed c an n o t b e e x p la in e d b y sim p le c o n s id e r ­ a t i o n s o f io n ic s i z e .

P a u lin g (13) g iv e s th e io n ic r a d iu s o f I™ a s

2*16 8 , and of Na* a s 0*95 8 , hence t h e h y d ra te d I " would be e x p e c te d t o p e n e t r a t e a membrane w ith more d i f f i c u l t y th a n t h e h y d ra te d Ha*, w hich i s n o t th e case*

13 S e v e r a l e x p la n a tio n s can be t e n t a t i v e l y advanced f o r t h e in c r e a s e d membrane p e r m e a b ility fo llo w in g a s t r i n g e n t tr e a tm e n t.

I t i s p o s s ib le

t h a t an a s t r i n g e n t c a u s e s an e f f e c t i v e in c r e a s e i n th e s iz e o f p o re s i n th e membrane.

I t i s a ls o p o s s ib le t h a t th e a s tr i n g e n t i s damaging to

th e g r o s s membrane s t r u c t u r e by w eakening th e i n t r a c e l l u l a r cem ent, r e s u l t i n g i n in c r e a s e d membrane p e r m e a b ility . The m ethod d ev elo p ed f o r th e q u a n t i t a t i v e e v a lu a tio n o f a s t r i n ­ g ency sh o u ld be c a p a b le o f wide a p p l i c a t i o n s .

I t sh o u ld be p o s s ib le to

m easure th e i r r i t a b i l i t y o f s k in , f o r exam ple, a problem o f v i t a l i n t e r ­ e s t t o m a n u fa c tu re rs o f s o a p s, s y n th e tic d e te r g e n ts , l o t i o n s , cream s, e tc . A lthough a com parison o f a s tr i n g e n t e f f i c i e n c i e s i s an u n c e r ta in p r o c e s s , t h e te c h n iq u e d e s c rib e d g iv e s th e fo llo w in g o rd e r o f d e c re a s ­ in g a s tr in g e n c y a t low l e v e l s o f p e rc e n ta g e c o n c e n tr a tio n s î A lCl^ y

A l m e th io n a te .

A ^ C S Û ^ )^

On a b a s is o f low l e v e l m olar c o n c e n tr a tio n s ,

th e o r d e r o f d e c re a s in g a s tr in g e n c y i s :

Al m e th io n a te ^

A l^ (O O ^ )^

AICI 3 . The f a c t t h a t th e fro g membranes were p e n e tr a te d i n a s ie v e ­ l i k e f a s h io n can p ro b a b ly be e x p la in e d by th e r e l a t i v e l y few l a y e r s o f c e l l s o f each ty p e t h a t com prise th e membrane and th e porous n a tu r e o f th e membrane.

The c o n c e n tr a tio n o f r a d i o a c t i v i t y t h a t was found t o

o c c u r a t th e l a y e r o f k e r a t i n iz e d c e l l s i n th e membrane i s l e s s r e a d i l y e x p la in e d .

Two p o s s i b i l i t i e s may be t e n t a t i v e l y advanced:

(a ) th e

k e r a t i n i z e d c e l l s s e l e c t i v e l y adsorb io d id e i o n , eg. a r e more e a s i l y i o d i n a t e d , (b ) th e i n e r t l a y e r o f c e l l s a c t a s a b a r r i e r t o th e pene­ t r a t i o n by I " , c o n s e q u e n tly c a u sin g a " b o ttle n e c k " t o d ev elo p a t t h i s membrane s u rfa c e *

14 I t m ig h t g e n e r a lly b e co n clu d ed t h a t t h i s work on f r o g membrane shows t h a t i t i s u n s a fe t o e x tr a p o la t e th e b e h a v io r o f o th e r membranes i n p r e d i c t i n g what w i l l happen w ith fro g membrane.

The e f f e c t s t h a t

d i f f e r e n t m a t e r i a l s were o b se rv e d t o have on f r o g membrane p e r m e a b ility r e s u l t e d from t h e c h a r a c t e r i s t i c s t r u c t u r e o f t h i s membrane.

Summary and C o n clu sio n s 1*

A te c h n iq u e f o r stu d y in g fu n d am en tal p e r m e a b ility problem s

h as s u c c e s s f u l l y b ee n e s t a b l i s h e d , u s in g an e x c is e d fro g membrane, and f o llo w in g p e r m e a b ility changes by means o f r a d io a c tiv e t r a c e r s . The te c h n iq u e has b een t e s t e d i n some hundred odd o rg a n iz e d e x p e r i­ m e n ts , u t i l i z i n g a p p ro x im a te ly 600 f r o g s . 2.

The "norm al" p e r m e a b ility b e h a v io r o f a homogeneous g roup

o f f r o g s o f s p e c ie s Rana P ip ie n s was shown t o v a r y w id e ly .

V a r ia tio n s

a s ex trem e a s 65% from a mean v a lu e w ere found i n one r e p r e s e n t a t i v e g ro u p o f 12 an im als* 3.

A m ethod was d ev elo p ed f o r th e e s tim a tio n o f t h e e f f e c t s

o f d i f f e r e n t p e rc e n ta g e s o f a s t r i n g e n t s p r e s e n t i n a s tr i n g e n t cream p re p a ra tio n s .

The p r i n c i p l e o f th e m ethod was b ased on t h e f a c t t h a t

t h e m ag n itu d e o f t h e p e r m e a b ility change i n f ro g membrane was p ro p o r­ t i o n a t e l y r e l a t e d t o t h e c o n c e n tr a tio n o f a s tr i n g e n t p r e s e n t.

The

te c h n iq u e p ro m ises t o d i f f e r e n t i a t e betw een t h e e f f e c t s o f u n i t p e r­ c e n ta g e d i f f e r e n c e s i n c o n c e n tr a tio n , w ith a maximum e r r o r o f 10 $ . 4*

T r e a tin g f ro g membrane i n v iv o and i n v i t r o w ith d i f f e r ­

e n t a s t r i n g e n t s was found t o in c r e a s e membrane p e r m e a b ility i n v it r o * T his f in d in g was in d e p e n d e n t o f th e o r i e n t a t i o n o f t h e membrane, th e ty p e o f t r a c e r u s e d , o r t h e means o f a p p ly in g th e a s t r i n g e n t .

15 5*

F rog membrane was found, t o b e p e n e tr a te d f o u r tim e s a s

r a p i d l y b y I " a s Ha*.

I t i s su g g e ste d t h a t th e f ro g membrane b e a rs

a s im u la te d p o s i t i v e c h a r g e , th u s p e r m ittin g t h e e a s i e r p a ssa g e o f th e n e g a tiv e io n . 6,

A r a d io a u to g ra p h ic i n v e s t i g a t i o n o f f r o g membrane, u t i l ­

i z i n g I ^ l j d id n o t d i s c l o s e any s e l e c t i v e avenue o f p e n e tr a ti o n o f t h e membrane by I " . ion*

F rog membrane was p e n e tr a te d a s a s ie v e by t h i s

A l o c a l i z a t i o n o f a c t i v i t y was fo u n d t o o c c u r on t h e membrane

s u r f a c e i n t h e a r e a o f t h e k e r a t i n iz e d c e l l s * 7*

The p e r m e a b ility c o n s ta n t o f a f r o g membrane t o I™ u n d er

th e c o n d itio n s o f t h e s e e x p e rim en ts was found t o be 2*44 x 10 “^ mole io d id e /c n u ^ /s e c .

T his v a lu e h o ld s f o r a te m p e ra tu re o f 26*5° C. and i s

p r e c is e w it h i n seven p e r c e n t. 3.

The p e r m e a b ility o f f r o g membrane t o I " i s in c r e a s e d approx­

im a te ly tw o fo ld by s u b je c tin g th e membrane t o a s a tu r a te d d e x tr o s e s o l u t i o n d u rin g t h e tim e o f th e experim ent*

B ib lio g ra p h y (1 )

A dolph, E. A ., Am. J . P h y s i o l . ,

85 (1925)*

(2 )

Goodman, L. and G ilm an, A ., The P h arm a co lo g ic al B a s is o f Thera­ p e u t i c s , M acm illan C o ., New Y ork, 1941*

(3 ) G o v e tt, T. and de N a v a rre , M« G ., American P erfu m er, 4 2 , 3&5 (1 9 4 7 ). (4 )

H a u se r, W., P ro to p la sm a , 2 3 , 200 (1937)*

(5 )

S c h u e tz , E . , A rch, e x p t l . P a th . Pharm akol*, 2%,202 (1 3 9 0 ).

(6 )

M adison, ¥ . L . , M.S. T h e s is , Purdue U n iv e r s ity S chool o f Pharm acy, 1948.

(7 )

D avson, H. and D a n i e l l i , J . F . , The P e r m e a b ility o f N a tu r a l Mem­ b r a n e s , M acm illan C o ., New Y ork, 1943*

16 (8)

Motokawa, J a p . J . Med. S c i . B io p h y s ic s , 2 , 117, 145

(9 )

K rogh, A ., T ra n s. F arad ay S o c ., 2 1 , 912 (1 9 3 7 ).

(1933)#

(10)

O stw ald , W., Z. P h y sik . Chem., 6 , 71 (1 8 9 0 ).

(1 1 )

Amberson, M. R . and K le in , H ., J . Gen. P h y s i o l ., l ^ , 823 (1 9 2 8 ).

(1 2 )

M ic h a e lis , L . , J . Gen. P h y s i o l . , 8 , 33 (1 9 2 5 ).

(1 3 )

P a u lin g , L . , N atu re o f th e Chem ical Bond, C o rn e ll U n iv e r s ity P r e s s , I t h a c a , 1948.

VITA R u s s e ll Eugene Lux, J r . was b o r n i n H opkinton, Iowa Septem­ b e r 2 7 , 1922.

H is p r e - c o lle g e e d u c a tio n was o b ta in e d i n t h e p u b lic

s c h o o ls o f H opkinton, Iow a.

He o b ta in e d th e B a c h e lo r o f A r ts d eg ree

from C o r n e ll C o lle g e , M t. V ernon, Iowa i n Ju n e o f 1943. S h o r tly t h e r e a f t e r M r. Lux was c a l l e d i n t o t h e armed s e r v i c e , and s e rv e d i n t h e Navy u n t i l J u n e , 1946.

H is w ar s e r v ic e was sp e n t

p r i n c i p a l l y i n t h e m e d ic a l b ra n c h ; and h i s n a v a l c a r e e r was te r m i­ n a te d a s he s e rv e d a s a r e s e a r c h ch em ist a t th e N aval M ed ic al R esearch I n s t i t u t e , N a tio n a l N aval M edical C e n te r, B e th e s d a , M aryland. I n S eptem ber o f 1946 M r. Lux e n te r e d t h e S ch o o l o f C hem istry o f P urdue U n iv e r s ity t o do g ra d u a te w ork. O rganic C hem istry i n F e b ru a ry , 1948.

He o b ta in e d h i s M. S . i n

His d o c to r a te i n P h a rm a c e u tic a l

C h em istry was co m pleted F e b ru a ry , 1950 under a g r a n t g e n e ro u sly s u p p lie d by P h a r a a c r a f t C o rp o ra tio n . Mr. Lux i s a member o f t h e fo llo w in g o r g a n iz a tio n s :

American

P h a rm a c e u tic a l A s s o c ia tio n , Am erican Chem ical S o c ie ty , P h i Lambda U p s ilo n , Sigma X i, and Kappa Mu E p silo m .