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A STUDY OP THE EFFECTS OF SEVERAL ENZYME INHIBITOR DRUGS UPON ELECTRICALLY STIMULATED EXCISED PORCINE CAROTID ARTERIAL SMOOTH MUSCLE
A Thesis Presented to the Faculty of the School of Medicine University of Southern California
In Partial Fulfillment of the Requirements for the Degree Master of Science
fey Howard Irving Jacobs August 1950
UMI Number: EP60454
All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion.
UMI Dissertation Publishing
UMI EP60454 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code
ProQuest ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48 10 6 - 1346
This thesisj w ritten by
Ho war d __Irv i
under the guidance of h%.§.
Facu lty Com mittee,
and approved by a ll its members, has been presented to and accepted by the C ouncil on Graduate Study and Research in p a rtia l f u lfill ment of the requirements fo r the degree of
Master of Science________________
To the Library Bindery: The form of this thesis is acceptable to the Department of Pharmacology and Toxicology in lieu of Campbell's Thesis Form*
Clinton H. Thienes, M.D. Head, Department of Pharmacology and Toxicology*
The tissues were obtained from the Cudahy Packing Company through the courtesy of Mr. B. A • Sandberg, Superintendent• We are indebted to Mr. Cecil Saunders and to Mr. Edmund Prescott for valuable preliminary data related to this problem. This investigation was supported by a grant from the Life Insurance Medical Research Fund to the Depart ment of Pharmacology and Toxicology, School of Medicine, University of Southern California.
TABLE OP CONTENTS CHAPTER I.
INTRODUCTION AND STATEMENT OP PROBLEM
. . . .
Classification of smooth muscle into groups
Previous investigation of the metabolism of smooth m u s c l e .......................
History of the pharmacological study of arterial spiral strips ................... Statement of the scope of this investigation II.
A METHOD OP ELECTRICALLY STIMULATING SMOOTH MUSCLE OP EXCISED PORCINE CAROTID ARTERY . .
Materials and procedure
Preparation of arterial spiral strip . . .
Procedure for stimulation
Balanced ion m e d i u m .....................
Square wave electronic stimulator
. . . .
Special silver electrode clips ..........
Results and discussion of m e t h o d ...........
Effects of repetitive stimulation
C o n c l u s i o n ..................................
THE EFFECTS OP AZIDE, CYANIDE, AND MALONATE ON THE RESPONSE OP EXCISED PORCINE CAROTID SMOOTH MUSCLE TO ELECTRICAL STIMULATION
Materials and p r o c e d u r e ............
R e s u l t s ....................................
PAGE A z i d e ....................................
C y a n i d e ..................................
M a l o n a t e ..................................
D i s c u s s i o n ..................................
S U M M A R Y ......................................
B I B L I O G R A P H Y ........................................
LIST OF GRAPHS AND RECORDS GRAPH I.
PAGE Decrement of amplitude with repetitive s t i m u l a t i o n ...........................
Changes in amplitude per cent and relaxation time in per cent with repetitive stimulation
Relationship of amplitude changes to relaxa tion time during repetitive stimulation
Effects of exposure to 10~3 and 10~4 M sodium azide on the amplitude response of elec trically stimulated excised arterial smooth m u s c l e ..................................
Effects of exposure to 10“^ and 10"^ M sodium cyanide on the amplitude response of elec trically stimulated arterial muscle....
Effects of exposure to 10“^ M sodium malonate on the amplitued response of electrically stimulated excised arterial muscle ........
Sodium A z i d e ...........................
10“3 m Sodium A z i d e ...........................
Sodium A z i d e ...........................
10“^ M Sodium C y a n i d e .........................
LIST OP FIGURES AND TABLES FIGURE
1* Tissue B a t h ............................
Circuit diagram of square wave stimulator
Diagrammatic sketch of the construction
special electrode clips and electrode
Photograph of the electrode clip-tissue ment .
Photograph of the arrangement of upper electrode and lead to s t i m u l a t o r ...............
Typical record of one c o n t r a c t i o n .....
Effects of Repetitive Stimulation
CHAPTER I INTRODUCTION AND STATEMENT OF PROBLEM In the past few years the major advances in the knowledge of the cardio-vascular system have been related to its reflex and humoral control and to cardiac function, but the physiology and pharmacology of the vascular muscula ture are as yet largely unexplored* Bozler (2) has classified smooth muscle into two groups which differ from each other in functional organiza tion*
In one group are those smooth muscles which are
organized into many small, discrete motor units, and which have a true motor innervation, much in the same manner of skeletal muscle.
This group, termed the ,fmulti-unit type,11
includes the muscles of the blood vessels, the pilomotor muscles, and the nictitating membrane of the cat *s eye* The evidence for including the smooth muscle of blood vessels in this category comes from the work of Fulton and Lutz (11), who were able to demonstrate functional discontinuity in the vascular bed of the retrolingual membrane of the frog.
observed that when the small nerves lying in the membrane were stimulated, the smooth muscle response was sharply limited to a small area of the vascular bed.
group of smooth muscles, which Bozler has called ’’visceral,” is automatically active as is cardiac muscle*
and uterine muscles are typical examples of this group# The metabolic study of multi-unit smooth muscle has been neglected in the past, and investigations of the elec trical properties of isolated preparations of this type of smooth muscle have been restricted to the nictitating mem brane of the cat (7).
It is apparent then that there is a
need for an investigation of arterial musculature.
edge of the metabolism and pharmacodynamics of the arterial system may provide clues Tflh ich might possibly be applicable in the rational treatment of such diseases as hypertension, arteriosclerosis, and other vascular disturbances. One method of approach to the problem Is to study excised arteries in an isolated organ bath.
developed the original method, and Cow (6) and others (1, 6, 9, 10, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 28, 29, 32, 36) have extended this work to some degree.
vestigators reported the changes in tonus produced in arterial musculature as a response to various mechanical, thermal, and chemical agents.
However, it is a possibility
that these agents which effect the tonus level of resting arterial muscle, may have an entirely different effect upon the contractibility of the tissue when it is stimulated# Intestinal smooth muscle exhibits sufficient spon taneous rhythmical activity as to constitute an actively and rhythmically contracting system.
Rona and Neukirch (27)
and others (12, 35) have made use of this spontaneous activity to investigate the effect of intermediate metabolic compounds, such as pyruvate, succinate, etc., upon substrate depleted isolated smooth muscle of the rabbit intestine* Although many workers have reported spontaneous con tractions of arterial muscle (1, 6, 9, 10, 15, 17, 19, 22, 23, 24, 25, 32), we have found that such contractions are neither frequent nor regular enough to enable one to study arterial muscle metabolism in a manner comparable to that used with isolated rabbit intestine* As a result we have investigated the possibility of producing an actively contracting system of excised arterial smooth muscle by means of electrical stimulation.
part of this study is an investigation of the effects of several enzyme inhibitor drugs upon such a system.
CHAPTER II A METHOD OP ELECTRICALLY STIMULATING SMOOTH MUSCLE OF EXCISED PORCTNF. CAROTID ARTERY I.
MATERIALS AND PROCEDURE
Specimens of hog carotid artery were obtained ap proximately 45 minutes after death of the animal*
specimens chosen were from the portion of the carotid which lies from 6 to 10 cm. cephalad of the aorta.
of artery were partially stripped of excess tissue and im mediately placed in cold, oxygenated Locke!s solution. Atzler and Lehman (1) stated that the tissue can be stored in the refrigerator without deterioration from 4 to 7 days.
This was confirmed in the present experiments but
it was found necessary to add fresh Lockefs solution and to oxygenate the medium slowly for ten minutes each day.
practice followed was to add approximately twenty per cent of the original volume of Lockefs solution daily.
experiments reported in this investigation, no tissue over four days old was used. The arteries were prepared by manually stripping the readily separable portion of the adventitia from the artery.1 ^ The histological appearance of the portion of the adventitia remaining on the Artery is described on page 14.
5 The muscle wall was then cut into a flat spiral from right to left beginning at the most cephalad portion of the artery, in a manner similar to that reported by Lewis and Koessler (19).
The angle of the cut was approximately thirty degrees
to the transverse axis of the vessel.
Viable, spirally cut
strips coiled tightly when returned to the Lockefs solution, whereas flaccid strips showed little irritability.
strip was flattened by running gently between the fingers; it was then mounted between the special electrodes^ and placed in oxygenated Locke !s solution at 37° C. using a modified Dale tissue bath apparatus. is illustrated in Figure 1.
The tissue bath used
The arterial spiral strip was
then allowed to remain undisturbed in the bath for at least one hour before starting an experiment. The procedure for stimulation was as follows:
solution was drained from the bottom of the tissue bath, until both the tissue and the electrodes were exposed to air. The preparation was immediately stimulated for 10 seconds at a frequency of 20 impulses per second.
A stopwatch and
a hand-operated knife switch were used to time the duration of stimulation.
When the amplitude of contraction had
reached a maximum, usually within 30 seconds, the tissue
The special electrodes are described on page 8
Figure 1. Tissue Bath; A = fill arm, B = oxygen inlet, C = drain arm, D = sintered glass gas dispersion disc.
7 bath was allowed to fill gently from the bottom, until the upper electrode was below the level of the solution*
strip was not stimulated again until it had spontaneously relaxed to the starting tonus level* In order to assure that the stimulus was not supra maximal, a preliminary contraction was elicited by a stim ulation voltage of between 100 to 125 volts, and then an appropriate voltage level was chosen so as to cause a con traction 20 per cent less in amplitude than that caused by the preliminary stimulation.
This second stimulus level was
used as the standard stimulus for the given strip. The Locke’s solution used in this investigation was prepared as follows: N a C l ............ * . • •
K C 1 ......................0.42 gms CaClg (CaClg-SHgO Glucose
0.24 gms ...
0.32 gms) 1.00 gms
N a H C O g ................... 0.15 gms
Q.S. to one liter with commercially obtained distilled water. (Fresh Puro brand) Experiments were carried out using Locke’s solution prepared with water which had been redistilled from glass apparatus.
No changes in either the maintenance of
viability, or in the form and characteristics of the con traction response of the tissue were noted. The electronic stimulator-^- used produced a square wave impulse having a pulse width of one millisecond and a variable voltage range from 0 to 125 volts.
of impulse was variable from 1 to 20 per second.
stimulator a relaxation oscillator was used to generate a spike potential in the power amplifier.
Rather than use a
clipper circuit which would produce a square wave and also be able to deliver power, a relay was used.
By this means,
when the spike potential reaches the relay threshold, the relay closes and remains closed until the spike potential drops below the relay threshold, whereupon the relay opens. During the time the relay is closed, an impulse is being delivered.
A circuit diagram of this stimulator is present
ed in Figure 2. Special electrode clips were constructed from wide strip silver,
A diagramatic sketch of their construc
tion is shown in Figure 3.
The tension, necessary to hold
the tissue firmly, was provided by a small piece of rubber
The square wave electronic stimulator was designed and constructed by Mr. Robert Leyden in the Precision Instrument Laboratory under the supervision of Dr. John P. Meehan of the Department of Physiology, School of Medicine, University of Southern California.
Figure 2. Circuit diagram of square wave stimulator.
STIMULATOR WAVE SQUARE
Figure 3* Diagrammatic sketch of the construction of the special electrode d i p s , and electrode support; A = assembled electrode, B= silver jaws and coupl ing of electrode clip, C = rubber tubing, D = elec trode support (1 = glass tube, 2 = copper wire from stimulator lead, 3 = mercury, 4 = platinum wire hook sealed into the glass tube)*
The arterial strip was suspended between the elec
trode clips and the lead from the upper electrode was pro vided for by the use of an almost tensionless coil made by winding a very fine enameled copper wire around a pencil.1 This wire was soldered to the electrode and the joint was insulated with collodion.
The bottom electrode was held by
a platinum wire hook embedded into a mercury filled glass tube.
Leads from the stimulator were attached to the mercury
filled glass tube and to the fine enameled copper wire.
entire electrode-tissue arrangement is illustrated in Figure 5. The contractions were recorded on a long paper kymo graph rotating at the rate of 2.4 cm. per minute by means of a front-writing inkwriter.
A lever magnification of 14 to 1
was used and the lifting load at the point of attachment of the thread to the lever was 6.5 gms. II.
RESULTS AND DISCUSSION
Attempts to elicit electrically stimulated contrac tions of an arterial spiral while it was immersed in Locke's solution have proven fruitless.
This is not entirely un
expected if one considers the histology of the arterial muscle spiral strip.
^ See Figure 4.
Hausler (15) reported that in the
Figure 4* Photograph of the arrangement of upper electrode and lead to stimulator; A= thread to muscle lever, B = coil of thin enameled wire, C = silver elec trode clip, D = lead from stimulator*
Figure 5. Photograph of the electrode clip-tissue arrange ment; A = coil of thin enameled wire, B = electrode clip, G = arterial spiral strip.
14 whole artery the muscle fibers lie in a spiral pattern. However, this has recently been challenged by Paskil (26), who claims that in the intact artery it is the elastic fibers which spiral and that the muscle fibers themselves are ar ranged in short sections between these elastic fibers* In the course of our investigation, histological examinations were made of two arterial spiral strips which had been prepared as previously described.^
showed that the muscle fibers of the flattened spiral strips were arranged obliquely to the longitudinal axis of the preparation.
It was further observed that the portion of
the adventitia, which had remained attached to the stripped artery, appeared to be atypical since it was densely filled with elastic tissue and contained some scattered muscle fibers• Therefore, it can be assumed that the resistance to electrical conduction from one muscle bundle to the other is high, and it is reasonable to postulate that when the pre paration is immersed in a fluid bath which is high in elec trolyte content, the current would shunt from one electrode to the other through the Immersion fluid. This explanation is in line with the findings of
**■ The preparation of the arterial spiral strip is described on page 4 .
15 Bozler (3) who, In experiments with uterine muscle cut in a zig-zag manner, reported that near threshold electrical stimulation does not stimulate those muscle bundles which lie perpendicular to the direction of the current.
those muscle fibers which lay in the direction of the course of the stimulus responded.
However, no attempt has been
made in the course of this present study to establish this hypothesis. Since we were unable to produce electrically stim ulated contractions of arterial smooth muscle while it was immersed in Lockefs solution, a method wherein the fluid was drained from the tissue prior to stimulation was suc cessfully developed.
Kymograph records of contractions of
arterial spiral strips thus obtained usually show a drain and a fill artifact.^ tissue,
When the solution is drained from the
there appears to be a small rise in tonus level;
this rise is believed to be an artifact, at least, as regards the actual tonus level of the muscle strip, and is considered to be due to the loss of buoyancy accompanying the removal of the fluid medium.
Conversely, when the bath is filled,
there is a sudden drop in "tonus11 due to the suddenly in creased buoyancy and decrease in effective weight of the
See Figure 6, page 16, for illustration of these artifacts•
Figure 6 Typical record of one contraction; A == peak of contraction, AB = contraction amplitude, BC = relaxation time, D = drain artifact, F = fill artifact, R = resting tonus level.
17 electrodes. When the maximum rise in Mtonus” due to drainage had been reached, the muscle strip suspended in air showed no change in length during the short period of exposure. The temperature change within the tissue bath, con current with the removal fluid, was less than 2 degrees, and averaged less than one degree during the first minute of ex posure,
Since the exposed tissue was covered with a film of
fluid, and was in a warm, moist oxygen-saturated atmosphere, it is probable that the effect of such small temperature changes was negligible.
At least, with the recording ap
paratus used, no effects were seen. It has been found that the handling incident to the stripping of the adventitia from the viable artery produce a state of high tonus.
When the arterial strip was allowed
to equilibrate for one hour at 37° C, in oxygenated Lockefs solution, this high tonus decreased and the tissue arrived at a steady middle tonus state.
However, it has been noted
that even with an equilibration period of one hour or longer, the first one or two contractions of an occasional strip were erratic.
Thereafter, the unstimulated tissue usually
reached a steady physiological state, lasting from 4 to 24 hours• Spontaneously occurring contractions of arterial
18 muscle have been reported by many workers (1, 6, 9, 10, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 28, 29, 32, 36).
however, have been observed during the course of this study. Perhaps this is due to the lack of protein material in our solution, since Atzler and Lehman (1), Loening (20), Macht (21), Weiss (36), and Cow (6) have all noted that the pres ence of serum or peptone in the medium enhances the occur ence of spontaneous contractions* Investigators who have used isolated arterial spiral strips in the past have experimented with contractions as a response to chemical stimulation.
was not reported by any of these workers.
most commonly used was to place the strip under a 20 gram stretching load until the original tonus level had been reached (19).
In our experience, using the previously
described method of electrical stimulation, spontaneous relaxation always occured when the strip was normally viable.
After cessation of the stimulus, the amplitude of
contraction rapidly reached a peak and then slowly fell off until the equilibrium middle tonus level was reached.
relaxation time varied from 1 to 10 minutes. The voltage necessary to elicit a submaximal contrac tion varied widely from specimen to specimen, the range being approximately from 25 to 125 volts.
variation did not appear to be a function of the length of
19 time the tissue had been stored.
This high stimulus voltage
requirement was probably related to the anatomical character istics of the arterial preparation, since Bozler (3) had shown that muscle bundles which lie perpendicular to the direction of electrical stimulus had a considerable higher threshold than did muscle bundles oriented along the path of stimulus.
In our preparation of arterial muscle, almost
no muscle bundles lie in the direction of stimulus. The choice of 20 impulses per second was made, be cause It was found that more consistent results were obtained if the frequency range used was between 10 and 20 Impulses per second, rather than between 1 and 10 impulses per second, and because of the greater ease In the manipulation of the stimulator at this frequency.
The stimulus duration was
standardized at 10 seconds in order to reduce the error in the duration of stimulation attendant to the use of a stop watch and a hand-operated knife switch as a timing device. Longer periods of stimulation produced greater amplitudes of contraction, but also made the relaxation times inconven iently long.
Very short stimulus durations were not in
vestigated. The nature of the response obtained by electrically stimulating an arterial spiral strip was typical of smooth muscle contractions in general.
The form of the contraction
and the time relations were almost identical with those
20 obtained by electrically stimulating the circular muscle of the turtle intestine (13).
EFFECTS OF REPETITIVE STIMULATION
The effects of repetitive stimulation upon undrugged isolated arterial strips were studied.
were carried out, each using a different arterial strip.
these experiments the tissue bath was filled with fresh L o c k e ’s solution after each stimulation.
Table I shows the
decrement in amplitude^ In both millimeters and in per cent occuring with each of sixteen repetitive stimulations of each of the three arterial strips.
The last column of the
table averages the results for all three arterial strips, and these averages were plotted as shown in Graph I. Since the procedure in which fresh Locke’s solution was used after each stimulation necessitated large volumes of solution, a fourth experiment was performed re-using the same solution throughout the entire course of the stimula tions.
The results of this experiment are also shown in
1 In this report the amplitude of contraction was measured by dropping a perpendicular from the highest point on the record of the contraction to a line parallel to the base line and passing through the tonus level of the drain artifact. This is illustrated in Figure 6, page 16.
21 TABLE I EFFECTS OF REPETITIVE STIMULATION
Experimant I with fresh Lookers solu tion after Stim each ulus stimulation Response mm % 100
Experiment II same condi tions as in Experiment I
Experiment III same condi tions as in Experiment I Average response
0 0 > O O r - M > I O ^ * f O C M
GRAPH I Decrement of amplitude with repetitive stimulation; • ■ average per cent amplitude, experiments 1, 2, and 3, X per cent amplitude from experiment 4.
23 Graph I.
It is evident that such re-use of the Locke fs
solution shows no untoward effects,
since the amplitudes of
the contractions in this experiment decrease at essentially the same rate as in the previous three experiments. In the fourth experiment the relaxation time^ as well as the amplitude of contraction was measured.
It was found
that the relaxation time tended to increase with successive stimulations (see Graph II).
However, when the per cent of
relaxation time was plotted against the per cent of ampli tude of the contractions, no clear relation was evident (see Graph III).
Repetitive stimulation did not alter the form
of the contractions. IV.
In view of these experiments, we conclude that the method for electrical stimulation of isolated arterial smooth muscle spiral strips presented in this report is practical and reliable, and that future experiments using this method are fully warranted.
The term relaxation time as it has been used here may be defined as the time necessary for the lever to fall from the peak of contraction to the baseline. See Figure 6, page 16.
GRAPH II Changes in amplitude per cent and relaxation time in per cent with repetitive stimulation, )( = amplitude per cent, • = relaxation time in per cent.
O H < X < _J UJ
aanxndwv GRAPH III Relationship of amplitude changes to relaxation time during repetitive stimulation.
CHAPTER III THE EFFECTS OF AZIDE, CYANIDE, AND MALONATE ON THE RESPONSE OF EXCISED PORCINE CAROTID ARTERIAL SMOOTH MUSCLE TO ELECTRICAL STIMULATION I.
MATERIALS AND PROCEDURE
In these experiments all of the inhibitor solutions were made up in Locke*s solution*
The inhibiting agents
used were as follows: 1.
Sodium Azide--Paragon #2781
Sodium Cyanide— Baker & Adamson Reagent (granular)
Sodium Malonate— Eastman #934
One-tenth molar stock solutions of these compounds were appropriately diluted as needed*
The highest concentra
tions used in these experiments of the above inhibitors did not change the pH of the oxygenated medium more than 0.1 pH unit • In order to conserve chemicals, the tissue was ex« posed to the same 100 ml. of inhibitor solution throughout the course of an experiment, in a manner similar to that used In experiment number 4, relating to repetitive stimu lation.The fluid from the tissue bath was drained into reservoir
before each stimulation, and the same solution was
used to refill the tissue bath after the contraction had
reached a maximum. The preparation was stimulated with either two or three submaximal control stimuli and then the inhibitor drug was added.
Repetitive stimulations were given over the
course of 45 minutes or longer. II. Sodium Azide.
Sodium azide in 10"^, 10~^, and 10"^ M
concentrations produced depression of the amplitude of con tractions of electrically stimulated isolated arterial smooth muscle• Graph IV shows the changes in percentage amplitude produced by two concentrations of sodium azide, plotted against the length of time the tissues was exposed to the in hibitor.
These changes in amplitude response are qualita
1 0 "^
ana io~3 ^ concentra
tions, 10~4 yi sodium azide reduced the amplitude of contrac tion with apparently no other effect.^
This effect may be
reversed by washing with L o ckefs solution.
exposure to 10"^ M sodium azide caused spontaneous irrevers ible shortening of isolated arterial muscle.2
would be a more apt term, yet, this shortening, although
^ See Record 1, page 30. ^ See Record 2, page 31.
GRAPH 131 140
100, Id O 3
20 ** * *
*m * #• * —
GRAPH IV Effects of exposure to 10“3 and 10"4 M sodium azide on the amplitude response of electrically stimulated excised arterial smooth muscle; 9 and x = 10“4 M azide, * = 10"® M azide• Three experiments are represented in this graph.
10 M SODIUM
Record. 1 10~4 M_sodium azide; C 1, C 2, and C 3 = control stimuli, x — addition of drug. Drum in constant motion throughout record, speed 2,4 cm/min.
10 M. SODIUM AZII
Record 2 10“^ M sodium azide; C 1, and C 2 = control stimuli, x = addition of drug, SC = start of spontaneous shortening. Drum in constant motion throughout record, speed 2,4 cm/min.
32 apparently irreversible, did not visibly alter the contrac tion response.
In spite of the increase in tonus level,
there was no reduction of contraction amplitude for the first 15 minutes after onset of the shortening.
tissue for 15 minutes with Locke*s solution showed no reversal of the shortening. In one experiment with 10“3
sodium azide it was
possible to demonstrate that the reduction of the amplitude of contraction was accompanied by a rise in stimulus threshold.
Whether or not any causal relationship exists
between these two phenomena has not been determined. The nature of the effect on 10"^ M sodium azide on relaxation and on the production of spontaneous shortening was essentially the same as that observed with 10“*^ M sodium azide.
However, the effects of the former concentration
were more rapid.
The tissue failed to relax completely after
the first stimulation--after one minute exposure to azide— and spontaneous shortening started after the lowest point of the relaxation curve was reached--two minutes after the peak of contraction.
A spontaneous further shortening occured
after approximately seven minutes.^ Sodium cyanide.
Graph V represents the effects of
1 See Record 3, page 33.
10 M. SODIUM
A SCA LE
Record 5 10"^ M sodium azide; C 1, and C 2 = control stimuli, x = addition of drug. Drum in constant motion throughout record, speed 2.4 cm/min.
GRAPH 121 140
100 ui o
H _l CL