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The role of sodium in activation of uterine smooth muscle Singh, Harcharan 1958

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THE ROLE OF SODIUM IN ACTIVATION OP UTERINE SMOOTH MUSCLE  A Thesis Submitted i n P a r t i a l Fulfilment of the Requirement f o r the Degree of Master of Arts  in  the Department of Pharmacology We accept t h i s thesis as conforming to "the required standard  fey HARCHARAN SINGH, B. Pharm., Panjab University, India, 1956,  THE UNIVERSITY OF BRITISH COLUMBIA October 1958  ABSTRACT E x t r a c e l l u l a r action potentials and isometric contractile tension have been recorded simultaneously i n v i t r o from uterine longitudinal smooth muscle of the pregnant cat, pregnant rabbit, estrogen-treated rabbit, and estrogentreated r a t . Action potentials were recorded from the surface of the muscle s t r i p s with glass electrodes having a large t i p diameter.  Tension was  recorded with an RCA transducer. —  Spontaneous contractions are associated with a series of action p o t e n t i a l s .  During relaxation no e l e c t r i c a l a c t i v i t y i s observed. E l e c t r i c a l and mechanical a c t i v i t i e s were f i r s t recorded i n Ereb's Ringer medium and then i n sodium-poor media (replacement of sodium chloride with choline chloride or sucrose).  S u f f i c i e n t reduction i n the external sodium concentration  resulted i n increased amplitude (peak to p«iak) of the biphasic action potential spikes.  The duration of the peak to peak d e f l e c t i o n and the maximum rate of  potential, change remained unchanged. However, decrease i n the external sodium concentration reduced the frequency of the action potentials, considerably i n the cat, and l e s s so i n the rabbit and r a t . The external sodium concentration was reduced i n stepwise ; fashion to' \  9  4 and 5 i t s i n i t i a l value.  Each successive decrease i n the external sodium/  concentration was accompanied by a prompt i n i t i a l contraction, followed by very slow relaxation and subsequent resumption of spontaneous contractions accompanied by action potentials.  With cat u t e r i reduction of the sodium  concentration of the medium to a l e v e l of 15-20 mEq/l resulted i n a greatly prolonged contraction with eventual relaxation when tissues f a i l e d to contract.  This paralysis was associated with cessation of action p o t e n t i a l s .  The e l e c t r i c a l responses of u t e r i of the other two species (rabbit and rat)  during exposure t o sodium-poor media were similar to those observed  (ii) wiih the cat uterus.  However, the mechanical a c t i v i t y of r a t and rabbit  u t e r i i n sodium poor media was d i f f e r e n t from that of the cat uterus* Decrease i n the external sodium concentration below 25-30 mEq/l usually resulted i n prolonged contractions, and f i n a l l y to complete f a i l u r e of the tissue to relax (even after 2-2^ hours)©  Outbursts of action potentials  at i r r e g u l a r i n t e r v a l s were seen i n the i n i t i a l stages of t h i s persistent contraction but eventually action potentials also disappeared. I t was d i f f i c u l t to reconcile these facts with the "Sodium Hypothesis". A selective inward flow of sodium ions probably cannot account f o r the i n i t i a t i o n of action potentials i n uterine smooth muscle since reduction of the external sodium concentration  considerable  (down to 15-20 mEq/l i n cat  and 25-30 mEq/l i n the other two species) did not effect the c h a r a c t e r i s t i c s of the action potentials i n the expected manner. However, further reduction i n sodium did r e s u l t i n e l e c t r i c a l and mechanical i n a c t i v i t y .  The view that  an outward flow of i n t r a c e l l u l a r anions might be responsible f o r depolarization (14) receives further support from the present studies. I n addition to many differences from other types of excitable tissue (nerve, cardiac and skeletal muscle) uterine smooth muscle also shows considerable i n t r a - and inter-species v a r i a t i o n .  In presenting the  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  r e q u i r e m e n t s f o r an advanced degree at the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and agree t h a t p e r m i s s i o n f o r e x t e n s i v e f o r s c h o l a r l y purposes may  study.  I  copying of t h i s  be g r a n t e d by the Head o f  Department o r by h i s r e p r e s e n t a t i v e .  be a l l o w e d w i t h o u t my w r i t t e n  Department The U n i v e r s i t y o f B r i t i s h Columbia, Vancouver 8, Canada. Date  £ejj  a L,,.  /^fT  thesis my  I t i s understood  that copying or p u b l i c a t i o n of t h i s t h e s i s f o r g a i n s h a l l not  further  financial  permission.  TABLE OF CONTENTS  ABSTRACT LIST OF TABLES  i i i i  LIST OF FIGURES.  iv  ACKNOWLEDGEMENTS  vi  I  INTRODUCTION  1  II  METHODS  3  III  RESULTS  .'10  IV  DISCUSSION  kl  V  SUMMARY  54  VI  BIBLIOGRAPHY  56  VII  BIOGRAPHICAL INFORMATION  5?  iii LIST OF TABLES  TABLE I - Electrolyte Composition of Various Bathing Media,,  '. u. ...-«..,.-,.-., 5  Characteristics of Action Potentials and Isometric Contractile Tension After Exposure to Bathing Media Having Different Sodium Concentrations i n : II III IV V  Pregnant Cat Uterus  .  11  Pregnant Rabbit Uterus  ,,  21  Estrogen Treated Rat Uterus Estrogen Treated Rabbit Uterus  ........ 27 ...  33  Electrolyte Composition of Bathing Media, Tissues and Their Functional State VI VII VIII IX  Pregnant Cat Uterus  , ..,  36  Pregnant Rabbit Uterus  37  Estrogen Treated Rat Uterus  38  Estrogen Treated Rabbit Uterus  . . . . . . . . . . . 39  iv TABLE OF FIGURES FIGURE I  C i r c u i t diagram of A-C coupled Preamplifier  .... 6  PREGNANT CAT UTERUS; - Specimen record of i n v i t r o tracings of ACTION POTENTIALS and ISOMETRIC CONTRACTILE TENSION obtained i n : I I (a)  Krebs Ringer Medium  .  15  111(b)  Choline substituted sodium-poor medium (sodium concentration 60 mEq/l)  IV (c)  .16  Choline substituted sodium-poor medium (sodium concentration 18 mEq/l)  V  17  Summarized figure of records shown i n Figures I I , I I I , IV and also the tracings i n almost completely sodium free medium  VI (a)  •  18  Summarized record from another experiment i n Krebs Ringer (as a control) and i n choline substituted sodium-poor media with d i f f e r e n t sodium concentrations.  VI  19  Specimen records from another experiment i n sucrose substituted sodium-poor media with different sodium concentrations..........*...«.«......«.«.......  20  PREGNANT RABBIT UTERUS; - Specimen record of i n v i t r o tracings of ACTION POTENTIALS and ISOMETRIC CONTRACTILE TENSION obtained i n : VII (a) Krebs Ringer Medium.......«  ,  22  VTH(b) Choline substituted sodium-poor medium (sodium concentration 60 mEq/l).....  23  V  Table of Figures (cont'd) IX (c)  Choline substituted sodium-poor medium (sodium concentration 29 mEq/l)  X  24  Summarized records represented i n Figures V I I , VIII and LX.. 25 ESTROGEN TREATED RAT UTERUS; - Specimen record of i n v i t r o tracings of ACTION POTENTIALS and ISOMETRIC CONTRACTILE TENSION obtained i n : -  XI (a)  Krebs Ringer Medium  .«.  28  XII(b)  Choline substituted sodium-poor medium (sodium concentration 43 mEq/l)*....«. o.».»......  XIII(c)  «• 29  Choline substituted sodium-poor medium (sodium concentration 27 mEq/l).....•.»...•  ......<,...»......... 30 31  XIV  Summarized records represented i n Figures XI, X I I and XIII* *  XV  ESTROGEN TREATED RABBIT UTERUS; - Specimen records of i n v i t r o tracings of ACTION POTENTIALS AND ISOMETRIC CONTRACTILE TENSION obtained i n Krebs Ringer and choline substituted sodium-poor medium (sodium concentration 29 mEq/l) . s  35"  VI  ACKNOWLEDGMENTS The author wishes to express h i s profound appreciation to Dr. E.E, Daniel and Dr. J.G, Foulks f o r t h e i r keen i n t e r e s t , encouragement and helpful c r i t i c i s m during the course of investigations and the preparation of t h i s report. Thanks are also due to Mr, P„P«, Singh of the Department of Physics U,B«C, Vancouver and Dr. G.E, Dower f o r the helpful discussions. Financial assistance provided by the Banting Research Foundation i also g r a t e f u l l y  acknowledged.  INTRODUCTION  The action potentials of nerve and skeletal muscle are currently considered to be i n i t i a t e d by a selective increase i n the permeability of the c e l l membrane to sodium ions ( l ) . This i s believed to be followed, after a lag period, by a s i m i l a r increase i n the permeability of the membrane t o potassium ions (2). Other sequential events i n the l a t e r phases have been described which permit the maintenance of e l e c t r i c a l n e u t r a l i t y , and bring about recovery of the resting membrane p o t e n t i a l . (1,2,3,4). In many t i s s u e s , a change i n the concentration of sodium i n the external solution e l i c i t s marked changes i n the trans-membrane potential recorded with i n t r a - c e l l u l a r micro-electrode  (the amplitude of the action  p o t e n t i a l , the rate of r i s e of the upstroke of the action p o t e n t i a l , and the rate of conduction of an impulse).  For example, i n the case of (a)  some types of cardiac muscle (5,9); (b) Frog's sartorious muscle, (6); (c) myelinated nerve (7); and (d) giant axon of squid (8), a decrease i n the external sodium concentration r e s u l t s i n marked decreases i n the height of action potentials. I n cardiac muscle decrease i n the external sodium concentration by 2/3 of the i n i t i a l value greatly decreased the p o s i t i v e phase of the i n j u r y potential (9).  I n some cases (5,6) the rate of r i s e  of the upstroke of the action potential was reported to have decreased with decrease i n the external sodium concentration.  Similar decreases i n external  sodium concentration also were shown to cause disappearance of spontaneous a c t i v i t y of Purkinje Tissue (5). Some types of cardiac muscle show l i t t l e a l t e r a t i o n i n the depolarization phase of action potential i n t h i s type of experiment, (9a).  The rate of conduction i n nerve tissue decreases as the s a l i n i t y of the external medium i s reduced. Eventual blockade of nerve conduction occurred on further lowering of the external concentration (6,10). the response of some excitable tissues i s not i n accord with the that sodium ions carry the depolarization membrane currents.  However, hypothesis  Fatt and  Katz (11) have reported an increase i n the amplitude of action potentials i n isolated crustacean muscle f i b r e when sodium chloride i n the perfusion medium was replaced bycholine chloride or c e r t a i n quarternary ammonium ions.  Wood (12) i n a study of neuromuscular transmission i n herbivorous  insects, reported non-specificity of cations i n the external solution as c a r r i e r s of the action currents.  However, r e l a t i v e l y few such studies have  been made on smooth muscle. Holman (13) has shown that action potentials i n guinea pig taenia c o l i are to a great extent independent (down to 17 mEq/l) of the external sodium concentration. In the present i n v i t r o investigation the  effects of a low external  sodium concentration on the e l e c t r i c a l and mechanical a c t i v i t y of uterine smooth muscle have been studied.  The tissues used include uterine myometrium  of pregnant cat, pregnant rabbit, estrogen-treaed rat.  rabbit and  estrogen-treated  The uterine smooth muscle i n these species maintained i t s e l e c t r i c a l  as well as i t s mechanical a c t i v i t i e s i n rather low concentrations of external sodium (15-25 mEq/l). However, when sodium was reduced to extremely low concentsations  (less than 15-20 mEq/l), the action potentials disappeared  and mechanical "paralysis" ensued.  METHODS (a) Recording Systems Isometric contractile tension and action potentials were recorded simultaneously during spontaneous contractions of isolated s t r i p s of uterine longitudinal smooth muscle. Tension was recorded with an RCA transducer (#5734). For recording the action potentials, signals were picked up from the surface of the myometrium. This was accomplished by using a platinum wire lead inserted into a c a p i l l a r y glass electrode with a t i p diameter of about 300 ^u (i.d.) and f i l l e d with the same solution as the external medium. The exploring electrode was arranged to record with respect t o a grounded platinum electrode which also was inserted into the bathing medium. The signals thus picked up, were fed into the input of a pre-amplifier. The pre-amplifier i n turn was connected to a conventional DC-coupled push-pull a m p l i f i e r t o drive the pen motor of a Sanborn-Twin-Viso Recorder, Amplifier tubes were heated by an e l e c t r o n i c a l l y regulated d i r e c t current. (b) Solutions The various perfusion solutions employed consisted of: (1) Krebs Ringer - prepared by d i l u t i n g Deca Krebs 1:10 with double d i s t i l l e d water and adding 160 ml of 2.6% NaHCO^ solution and 100 ml of 20$ f r e s h l y prepared glucose solution per l i t e r of diluted solution: Deca Krebs consisted of: 50 parts 9% NaCl, 4 parts 5.75% KC1, 3 parts 8.06% CaCl„ 1 part 19.1% MgS0 , 1 part 10.55% K P 0 4  2  4  (2) Sodium-free solutions - Due to lack of any suitable substitute f o r NaHCO^, t h i s s a l t was omitted i n the sodium free solution.^ Hence the solution was s l i g h t l y hypotonic as compared with Krebs Ringer.  The chief sodium free solutions employed were ( l ) choline Ringer and (2) sucrose Ringer.  These solutions were identical with Krebs  Singer except that NaCl was replaced by isomotic equivalents of choline chloride o r sucrose respectively.  These solutions are  referred to i n the text as Na-poor media. The composition of the three p r i n c i p l e solutions used i s given i n Table I. A l l the perfusion media were constantly aerated with carbogen (95$ 0^ and 5% CCv,) and were maintained at 34—35° i n a thermostatically controlled water bath. (c) Hormonal Treatment of the Animals: Young rabbits and r a t s were given 100 meg of estrogen per day for six days before the u t e r i were removed. (d) Analytical Methods: (1) For Perfusion Solutions:  Na and K were determined by a method  previously described f o r plasma using a Jahnke flame photometer and employing lithium n i t r a t e as an internal standard (15,16).  Chloride (Cl) was determined  by d i r e c t t i t r a t i o n with Hg (NO^)^ using diphenylcarbazone solution as an indicator (17). (2) Tissues:  The a n a l y t i c a l procedures employed for tissues have been  described i n d e t a i l elsewhere (15). chosen for e l e c t r o l y t e analysis.  Endometrium was scraped from the tissues  The tissue was c a r e f u l l y blotted dry of  external solution, weighed immediately i n a previously weighed beaker, and c a r e f u l l y dried at 95-105° for f i v e days. After re-weighing, the tissues were powdered and f i n a l l y digested with HNO^ (15). potentiometrie  Chloride was determined by  t i t r a t i o n with 0.01N AgNO^ after preliminary cold extraction  of chloride with 0.1N HN0 as outlined by ¥hittam (18). 3  *(Footnote from page 3)« Recently a suitable method of sodium bicarbonate substitution has been reported (28).  TABLE I  Na or Choline or Sucrose  Krebs Ringer mM/L  Choline Ringer mM/L  Sucrose Ringi mM/L  137.4  125.9  248.5  K  5.79  6.23  6.23  Ca  2.47  2.65  2.65  Mg  1.16  lo25  1.25  CI HC0  3  so  4 Glucose  125.1  137.4  11.5  21.9  0.0  0.0  1.16  1.25  1.25  1.16  1.25  1.25  49.2  52.9  52.9  -  6  -  l-ffv II A X - 7  1500 S R . ,  _  Figure I , This figure shows the salient features of the preamplifier assembly. I t consists of ( l ) ACcoupled push-pull amplifying c i r c u i t s and (2) C and coupling condensers which i n conjunction with resistances R ^ and R ^ givenan input time constant of 2.5 seconds. The maximum s e n s i t i v i t y of t h i s system i s 1 mv = 4 cm. SW I i s the c a l i b r a t i o n switch, which gives 1 mv c a l i b r a t i o n signals when the c i r c u i t through the 1.5 V 12Ax-7 battery (Bl) and resistances R and R^ i s closed. A i s the knob f o r f i n e screw adjustments to balance the gain. B i s the knob for fine screw adjustments of the base l i n e . C i s the gain control knob. SW 2 i s the double-pole single-throw (instomatic) switch. When the c i r c u i t through i t i s closed the current can leak r a p i d l y through the path of least resistance.  (e) Calculations Electrolyte concentrations  i n solutions were expressed as mEq/kg fresh  weight of the t i s s u e . Na and CI spaces were calculated on the basis of formulae described by Manery (19). Results treated s t a t i s t i c a l l y were expressed i n terras of + the standard error of the mean. I f a sample varied by more than 2 x the standard deviation i t was rejected and the r e s u l t s re-calculated omitting that p a r t i c u l a r sample. (t) C r i t e r i a of Selection of Action Potentials for S t a t i s t i c a l Treatment Each spontaneous contraction was accompanied by a series of biphasic action potentials.  With such a series, v a r i a t i o n i n amplitude''" i n Krebs  Ringer was very large i n rat and rabbit u t e r i and less so i n the pregnant cat uterus.  I n rabbit and rat u t e r i the amplitude of a considerable  proportion  of action potentials i n a series was too small to be estimated accurately. Consequently i n each series potential with maximum amplitude, and those appearing close to t h i s value, were taken f o r s t a t i s t i c a l evaluation.  The  values obtained under one condition (Krebs Ringer) were compared with the results s i m i l a r l y obtained under other conditions (low external sodium). This provided a uniform though a r b i t r a r y basis for a s t a t i s t i c a l  evaluation  of the c h a r a c t e r i s t i c s of action potentials i n various external sodium concentrations. In sodium-poor media the amplitude of the action potentials was increased considerably.  I n pregnant cat u t e r i the v a r i a t i o n i n amplitude  during a series was much reduced whereas with u t e r i from the other two species variations i n amplitude, though reduced s t i l l persisted.  I f the  *Tn the text amplitude refers to the peak to peak amplitude of biphasic action potentials and the duration and rate of change of potential referes to the duration and the rate of change of the peak to peak d e f l e c t i o n respectively, unless mentioned otherwise.  average amplitude i s calculated f o r a l l of the action potentials i n a series the increase i n the mean amplitude of action potentials i n sodium-poor medium i s f a r greater than that calculated from selected action potentials of near maximum amplitudes.  This i s due to the f a c t that only a small  proportion  of action potentials were near maximum amplitude i n Kreb's solution, but a large proportion of action potentials were near maximum value a f t e r reduction of the external sodium concentration.  Variation i n the duration of action  potentials was not s i g n i f i c a n t and the o r i g i n a l configuration of the action potentials observed i n Kreb's Ringer was maintained i n sodium-poor media. Thus the amplitude was the only major v a r i a b l e , (g) Experimental Procedure Uteri were removed from peegnant cats and rabbits under barbiturate anesthesia (35-40 mg/kg), Estrogen treated rabbits and r a t s were k i l l e d by a blow on the head and the u t e r i rapidly removed. For experimental purposes, 2 or 3 longitudinal uterine s t r i p s , each about 3 cm. i n length, were excised and one of them was mounted i n the medium bath so as to allow the tension to be recorded i s o m e t r i c a l l y with the RCA transducer.  I n i t i a l l y , the medium bath contained Krebs Ringer maintained at  34°- 35° , and aerated with a 95% 0 + 5% C0 mixture. 2  2  Temperature control of the medium was achieved by pumping prewarmed water through the double walled medium bath.  The temperature of the c i r c u l a t i n g  water was controlled thermostatically.  The tissue was mounted and placed  under a basal tension of 4-5 gm. Under these conditions the tissue generally resumed spontaneous contractions after about 30 minutes. At t h i s stage both action potentials and contractile tensions were recorded. At the end of a satisfactory control record, a sample of the bathing mediim was taken for electrolyte analyses.  The bath was then drained and sodium-poor solution  (previously warmed to 3 5 ) was poured into the bath after a preliminary rinse with this solution. After allowing an equilibration period of about 2 0 minutes the activity was recorded as usual.  The total period for which the tissue  was l e f t i n a particular sodium-poor solution varied but i n most cases the minimum duration of exposure was one hour. After satisfactory recording, samples of the medium was taken for electrolyte analyses and the bathing solution was replaced with a medium i n which the sodium concentration was further reduced. In some instances, the tissue was exposed to a medium completely free of sodium i n the f i n a l step.  After the f i n a l step, the tissue  mounted f o r recording and the solution bathing i t were preserved for subsequent electrolyte analyses.  Preliminary observations indicated that mounting per se  did not contribute to alteration i n tissue electrolyte composition i n v i t r o . Unmounted uterine strips were kept i n various media which were warmed and aerated i n the same fashion as the experimental tissues. These unmounted segments kept i n Krebs Ringer served as controls for the estimation of tissue electrolyte changes of the pieces which were subjected to sodium-poor media, control and experimental tissues being removed  from this respective media f o r analysis  simultaneously. For the estimation of the tissue electrolyte changes i n uterine segments which had been exposed to sodium-poor solutions, but were to be subjected to further experiments i n other media, unmounted segments which had been immersed i n similar solutions for the same period of time were employed.  The small  amounts of tissue available i n estrogen-treated rabbits and rats often precluded the p o s s i b i l i t y of analysing unmounted tissue samples for each step i n the reduction of the external sodium concentration.  - 10  -  RESULTS (a) Pregnant Cat Uterus The r e s u l t s obtained from t y p i c a l experiments are summarized i n Table I I . Specimen records of action potentials and contractile tension i n media with various external sodium concentrations  are presented i n Figures  2^-6.  In any p a r t i c u l a r series of r e p e t i t i v e action potentials some degree of v a r i a t i o n usually was observed i n the amplitude of action potentials (Figures 2 and 3).  These variations i n amplitude were somewhat greater i n  Krebs Ringer than i n sodium-poor media. Because of the large v a r i a t i o n i n the amplitude of the action potentials from one experiment to another i n Krebs Ringer, each piece was employed as i t s own control for comparison with the results obtained i n sodium-poor media. The results from four t y p i c a l experiments are arranged i n Table I I . The following findings were consistently tby)observed i n satisfactory experiments. (i)  In a given series of r e p e t i t i v e action potentials, an i n i t i a l action potential prededed contraction and the rest of them occurred during  contractions.  ( i i ) The amplitude of the action potentials was always increased i n sodium-poor media (Figures 4,5 and Table  11)1  * In one of the experiments only s l i g h t e l e c t r i c a l and mechanical a c t i v i t y were observed i n Krebs Ringer medium. Keeping i n view the f a c t that a c t i v i t y generally improved i n sodium-poor solutions, Krebs Ringer was replaced i n part by sucrose Ringer. On reducing the external sodium concentration the muscle contracted and then relaxed gradually. Rhythmic contractions associated with t y p i c a l biphasic action potentials then appeared. In f a c t the peak to peak amplitude of the action potentials i n t h i s series was the highest that we have recorded (up to 5 mv. Table I I series 4 & F i g . 6).  TABLE I I PREGNANT CAT UTERUS No,  l.A  Bath, Medium  Basal p* Maximum Minimum Tension Tension Time-IGm Gm Rise Tension Gm Rise H msec.  Krebs Ringer  Duration of Maximum Ampli- Maximum Rate of Duration of Minimum Contraction tude of Action Change of Action Action poten- Interval sees. Potential Potential tial Between two xx mv/' : - .~mv/100 msecs. msecs. successive Action Potent, msecsp x  4  11.5  60-65  35-40  0.8+ 0.01 (9)  3V4  28-30  300-340  .B-Na-poor medium Na = 60 mEq/l 4  11.5  55-60  75-80  1.2+ 0.92 (9)  5.0  22-25  2400-2500  4  11.5  50-55  90-95  1.0+ 0.02 (10)  5.0  20-24  2400-2500  Krebs Ringer  4  11.6  110-115  57-60  5.3  26-30  1225-1255  B-Na-poor medium Na = 55 mEq/l  2.6 ± 0.03 (9)  4  11.6  80-85  65-70  2.9 + 0.08~(9)  5.8  30-34  3500-4000  C-Na-poor medium Na = 19 mEq/l  4(+l)ll.6  2.9 + 0.1 (9)  6.5  30-35  3700-4060  y  C-Na-poor medium Na = 18 mEq/l 2.A  very prolonged  12.5  375  60-70  1.7 ± 0.6 (9)  9.6  22.24  500-550  B-Na-poor medium/-^ Na = 25 mEq/l 5(+3)l2.5  250  30-35  2.3 + 0.14 (9)  8.3  24-28  800-900  3.A  4.A  Krebs Ringer  Krebs Ringer  5  6  Occasional action potentials of less than 0.2 mv amplitude  Poor Contractions (less than 1 Gm)  B-Na-poor medium/ Na = 23 mEq/l 6(+2)13.5 C-Na-poor medium/ Na = 18 mEq/l 6(+4)13.5  250  15-20  3.1 + 0.18 (12)  11V5  30-32  10,000-12,000  14-16  5.0 + 0.08 (9)  16.5  32-36  10,000-12,000  -  12  TABLE I I (Cont'd) x  The action potentials had biphasic configuration.  X  Substitution with choline ringer medium.  /  Substitution with sucrose ringer medium.  xx  Tissue showed contraction with each reduction i n external sodium, concentration, gradual relaxation and eventual resumption of spontaneous contractions. The duration of contractions i n sodium-poor solution denoted i n the table represents the duration after resumption of spontaneous a c t i v i t y . Unless otherwise mentioned these conditions hold true for subsequent tables.  fi  Figures i n parenthesis indicate the change i n basal tension during the course of an experiment.  H  The time ( i n msecs) during which tension r i s e was fastest was taken. This duration was divided by the number of Gms by which the tension rose to get the value for the Gm maximum tension r i s e as represented i n the t a b l e s e  ( i i i ) The duratiom of peak-to-peak deflections (20-35 msec.) remained s t a t i s t i c a l l y unchanged i n sodium-poor solutions. (iv)  The maximum rate of change of peak-to-peak deflections either remained unchanged or increased (Table I I ) .  (v)  The minimum i n t e r v a l between two successive action potentials (frequency) was increased considerably i n sodium-poor media, sometimes as great as 8 times that observed i n Krebs Ringer.  (vi)  The duration of spontaneous contractions i n sodium-poor madia was more prolonged than i n Krebs Ringer although exceptions sometimes were observed.  ( v i i ) The same r e s u l t s were obtained irrespective of whether choline chloride or sucrose was substituted f o r Na. (Figures 5 and 6, and series 4 i n Table I I ) . ( v i i i ) l n very low concentrations of external sodium (less than 10-15 mEq/L) both e l e c t r i c a l and mechanical a c t i v i t i e s disappear (Fourth tracing from the l e f t i n Figure 5). When the external medium i s changed to a sodium-poor solution, a sudden contraction vtook; place.  The muscle gradually relaxed after a r e l a t i v e l y  long period of time, (about 15-20 mins.) and spontaneous a c t i v i t y eventually was resumed.  However, when the external sodium concentration was lowered to  less than 15-20 mEq/l, the muscle showed an even more prolonged i n i t i a l contraction with intermittent outbursts of action potentials. relaxed gradually but f a i l e d to contract again.  The tissue then  At t h i s stage the complete  cessation of action potentials also occurred. A tendency f o r basal tension to he increased commonly was observed on exposure to moderately sodium-poor solutions, and t h i s affect often became  -  14  -  more marked i n media with lower concentrations of sodium. The e f f e c t of  ,  reduction i n the external sodium concentration on the rate of r i s e of contractile tension was rather variable, although the maximum rate remained f a i r l y constant, (b) Pregnant Rabbit Uterus The r e s u l t s obtained from a single pregnant rabbit uterus are summarized i n Table I I I . Specimen records are presented i n Figures (<7 to ID).  The r e s u l t s  obtained were q u a l i t a t i v e l y the same as i n pregnant cat uterus.  Action  potentials were complex rather than biphasic and tended to be more variable i n shape and magnitude than i n the pregnant cat uterus. Action potentials were associated with contraction but they d i d not always precede the contraction (unlike pregnant cat uterus) The amplitude of action potentials was increased i n sodium-poor media (Table IV and F i g . 10) (from 0.74 + 0.03 i n Krebs Ringer to 1.03 +0.07 i n Na-poor media). While the rate o f change of potential was increased, i t s duration remained p r a c t i c a l l y unaltered (17-20 msec.). The basal contractile tension was increased as the sodium concentration of the external solution was lowered.  When the external  sodium concentration was reduced from 138.5 mEq/l to 29 mEq/l (Table V) the basal tension rose from 5 Gm. to 11 Gm.  The concentration of sodium  waw not decreased any further i n t h i s experiment to avoid the sustained contraction which develops i n very low external sodium concentrations (below 20-25 mEq/l). The maximum rate of tension r i s e remained unaltered but the duration of contraction increased to about 8 to 10 times that observed i n Krebs Ringer medium. The frequency of action potentials was s l i g h t l y decreased.  -  15  PREG-  -  CAT  UTERUS  I  r  IN KREBS RINGER MED-  45-1  Figure 2. This figure shows spontaneous action potentials and isometric Contractile tension rrom the pregnant cat uterus i n Krebs Ringer. Note that the i n i t i a l action potential preceeds the contraction. The amplitude of the action potentials i n t h i s series varied from 0.2 to 0.7 mv. Action potentials ceased to occur before the tension began to f a l l . During the i n t e r v a l of maximum tension, the action potentials occurred at a frequency of about 2/sec. * I n the figures to follow isometric contractile tdnsion i s referred to simply as contraction.  Figure 3. This record i l l u s t r a t e s a spontaneous contraction and accompanying action potential i n a medium i n which sodium was replaced by choline. Sodium concentration i n the medium as subsequently determined was 60 mEq/l. This record should be compared with the control record presented i n Figure 2 which was obtained from the same preparation. Note the increased amplitude of action potentials (0.9 to 1.1 mv) their almost unaltered peak to peak duration, their decreased frequency ( l every 2.5-4.5 seconds) and the very much prolonged duration of contractions This contraction continued over an interval of 70 seconds. The general pattern of action potentials and contraction during t h i s 70 second i n t e r v a l was the same as that i n the portion shown i n t h i s figures (To follow page 16)  -  PREG-  16 ~  CAT  UTERUS  -  PRE6-  17 -  CAT  UTERUS  Figure 4. This record also, i s from the same experiment as the one represented i n Figures 2 and 3. The sodium concentration i n the medium was lowered by further replacement with choline. F i n a l sodium concentration i n the medium was 18 mEq/l, The amplitude of action potentials i s s t i l l increased as compared with the control record (0.6 to 0.8). The unaltered duration and pattern and decreased frequency of action potentials also i s evident (compare to the control records i n Figure 2)„  18  PREG-  CAT  -  UTERUS  i Figure 5. This figure summarizes the effects of d i f f e r e n t external sodium concentrations on the amplitude of action potentials i n the pregnant cat uterus. For effects of d i f f e r e n t external sodium concentrations on other characteristics of action potentials see Figs. 2, 3 and 4 ) . The fourth segment from the l e f t shows the disappearance of action potentials and contractile tension ( which f e l l to the control l e v e l under which the tissue was o r i g i n a l l y placed) when external sodium concentration was approximately 1 mEq/l. The f i f t h portion of the record indicates the reappearance of spontaneous contractions and action potentials five minutes after the sodium concentration was raised from 1 to 14 mEq/l.  -  Figure 5a  19  -  This record summarizes the effect of d i f f e r e n t external sodium concentrations on action potentials and contractions i n prggnant cat uterus from another experiment. Note the s i m i l a r i t y to the records presented i n Figs. 2, 3, 4 and 5,  ~  PREG-  IMV  20  CAT  UTERUS  3IMV  Figure 6 - This figure shows r e s u l t s from another experiment with pregnant cat uterus i n which the effects of lowering external sodium were analyzed. This record lacks the control portion because spontaneous action potentials during a contraction f a i l e d to appear i n t h i s preparation when Krebs Ringer was used as the medium. Replacement of sodium by sucrose i n i t i a t e d spontaneous action potentials and contractions. Action potentials of high amplitude developed when the sodium concentration was lowered to 35 mEq/l ( i n the f i r s t portion on the l e f t ) and to 16 mEq/l ( i n the second portion of the f i g u r e ) . This record however, lacks the t y p i c a l representation of effects of sodium depletion i n the external medium on the frequency of action potentials and the contractile responses of the t i s s u e .  TABLE I I I PREGNANT RABBIT UTERUS No. Bath. Medium  Basal Maximum Minimum Tension Tension Time-IGm Gm. Rise Tension 7 RiseM msec.  Krebs Ringer  5  12.5  B-Na-poor medium Na = 60 mEq/l  5(+2)12.5  C-Na-poor medium Na = 29 mEq/l*  5(+6)12.5  X  x, xx, X, fi, fifi  =  125  250  See Table I I  Duration of Maximum Ampli- Maximum Rate of Duration of Contration tude of Action Change of Action Action potensees. Potential Potential tial xx mv x mv/lOO msecs. msecs.  Minimum Interval Between two successive Action Potent. msecs.  14-16  0.7 + 0.03 (9)  6.0  13.-16  75-100  35-40  0.8 + 0.01 (7)  6.0  15-17  250-300  90-120  1.0 + 0.07 (9)  8.0  17-21  300-350  -  PREGNANT  T  -  _ < ! — -  :;ti-  ---  \  - - r  1 -  — — r  I  M  I i  1 l  1  I l  ----|  i I  ;| ••  z  1  I  I | L1  i1 i  T-T::i:;i:i;i  :]\^xxxxxx\:xxm  7jl;.:;-. !-;-'j;:;:L:;j::: ' L I : 1 i "1 ••.!:;.-ill;-ili.i'.i-j ~:-.'-::lii•!'•' ==.-! _= \\-Xr-X HI-I^T; . l - ' l I'J L -Iv11 I :  :  -jf : :. :  !• • i .-.i  1: 1 i  ;i:;i:  i  iliiH!  KREB'S  :i..i_:;i;;j;i;=ii;; ! l ; l l ! l i r i  ! 1  : :  : : H:  ;  i ] i •I  f  —1-  ! 1 ; ii i ;i .. i : " T T r r r I I .........i • " i T T T " r ' T T T T T T T i...... ' j . ~i i T i n " i " , r am 1 11 I I I !• ! "1 l . l i! ! I 1 i I I l l l l. . . L i M i l .L..I.JIL T:: I ' J....UT j j i iji|!jI i — i 1.....! ! 1 IT ! ! 1 ! ! i 1 1 I i i: i i: !i i: i p - j — j " " | — j - - | ~ r — | — j — j — i 1 ! J 1 !_ii i i .,i ]--!' !-!. . i " T T T T T i ; ; ] ; j j i j j""'; i i ; ; • ! ! 1 : M i l l ! !....!. i l l ! ! ! "i i ! ; ; j P"'p .1 L 1 I I ! i i ; i i i i i ! ! 1 i t ! 1 ! ! i ii .i i ;!i 1 i! I-l I:-..!- ii 1 ••! :•]-.- i -• 1 :• i::„i~ >• :i i i ..1. I - i .. i1 •. L.::: !:! i I 11 1 j 1 1 1 1 ' 1 1 1 ' - i - i I ! I ii i i 1 i i i j. i-i i 1 1 1 1 J \,\ T L.l .! V . xm\,x i . _ . ..... . . i  T  UTERUS  M  s v — ->if !:!!;:; I •  Till  RABBIT  T 1 " ... . 1....! 1 1' ! '!TJIj_.L_.L_T _ 1:11 i -.J....LLLU •i i I i 1 1 1 ! 1 1 1i I I I ! { 1 i i i i ] i II II 1 1 L-.I-: i i i T .... T. -!-]-!• „!.„.!„„! . 1 j . j j1 M i l i . JTLLuJLIT T T T i1 1 t Iil ! i i i i i i I i I I i I I I _ „ . !1 „ 1 11 *£_\ i : i i i t !• i i 1 1 1 1i 1 i I LI LT i i ii A > i__V|j^i m i 1 i l_Jn f ! 1 i Lj^ ^. j" — 1. . L . l ." " T T i »i i i iii r !• • i i"T i- i i _L|_! M l ! ! _]_1 .L.I. ! 1 1 i • I i I I I I i i i i i I T i t j jI •. .! •  iiiiiji  ¥  22 -  RINGER  MEDIUM  60-1  Figure 7 . This figure i l l u s t r a t e s spontaneous action potentials during the contraction 6f pregnant rabbit uterus i n Krebs Ringer medium. Note the v a r i a b i l i t y i n the amplitude of action potentials and the presence of a number of small wavy e l e c t r i c a l fluctuations (compare with figure 2). The f i r s t action potential coincided with the i n i t i a l r i s e of tension (note the s i m i l a r i t y to cat u t e r i i n figure 2 ) .  -  P R E G N A N T  Na  POOR.  23 -  R A B  C H O L I N E  B I T  R I N 6 E R  U T E R U S  MED.  60-2  Figure 8. This record i l l u s t r a t e s action potentials during uterine contraction i n a sodium-poor medium ( i n the same preparation as that i l l u s t r a t e d i n Figure 7 ) . Choline p a r t i & a l l y replaced sodium, the concentration of sodium i n the medium as determined subsequently being 60 mEq/l. Note the rise i n basal tension on exposure to the sodium-poor medium (compare with Figure 3 ) ,  -  PREGNANT  Na.  POOR;  24 -  RABBIT  CHOLINE  RINGER  MED- 60-3  Figure, -9» This record i l l u s t r a t e s spontaneous action potentials during contraction ( i n the same preparation furnishing the records shown i n figures 7 and 8) after further reduction i n external sodium concentration (sodium concentration as determined subsequently was 29 mEq/l). Note the marked increase i n the amplitude of action potentials, t h e i r unaltered frequency and the persistance of small wavy e l e c t r i c a l fluctuations (also 8). Though not very c l e a r l y seen i n the figure, basal contractile tension was increased from 5 to 11 gm during exposure to t h i s medium. The maximum tension r i s e was 12 - 12.5 gm. External sodium concentration was not lowered any further to avoid the persistant contracture which was observed i n some of the similar experiments done previously i n the non-pregnant rabbit uteruag  -  PREGNANT  25 -  RABBIT  UTERUS  Figure 10, This figure summarizes some of the t y p i c a l features of the records presented i n figures 7, 8 and 9, Note the increased amplitude of action potentials and t h e i r unaltered frequency i n sodium-poor media.  »  26  -  (c) Estrogen-Treated Rat Uterus The r e s u l t s obtained from estrogen-treated rat u t e r i are shown i n Table IV. Various specimen-tracings of action potentials and contractile tension are shown i n Figures 11 to IS.  As i n pregnant cat and rabbit u t e r i , the  reduction i n the sodium concentration of the external medium resulted i n an increased amplitude of action potentials. remained almost unaffected.  The duration of action potentials  The maximum rate of change of potentials  was  variable i n d i f f e r e n t experiments, being increased i n some cases (series 1 and 2 i n Table V I ) , but decreased s l i g h t l y i n others (series 4 i n Table V I ) . The frequency of action potentials was not changed s i g n i f i c a n t l y i n sodiumpoor solutions.  In a given chain of r e p e t i t i v e action potentials variations  were considerable and the frequency of spikes also was i r r e g u l a r (Figures 12-14)o  Thus the properties of rat u t e r i resembled  those of the pregnant  rabbit uterus rather closely. Spike discharges (action potentials) always were related to mechanical a c t i v i t y i n some manner. They usually occurred during the r i s i n g phase, on the plateau of contraction, However, i n the r a t uterus, groups of action potentials sometimes occurred when the muscle had j u s t started to relax. The exact significance of the relationship between the occurrence of action potentials and the phase of contraction i s not c l e a r  0  Reduction i n  the external sodium concentration produced very marked effects on the contractile function of t h i s tissue.  With each step i n lowering the  external  sodium concentration there was aI.tendency toward more and more prolonged contractions ( l a s t i n g several minutes as compared with the usual 15-20 i n Krebs Ringer Medium) and incomplete relaxation was common.  sees,  Spastic  contraction, or even complete i n a b i l i t y to relax, developed when the external  - 2.1  TABLE IV ESTROGEN-TREATED RAT UTERUS No,  Bath Medium  Basal Maximum Minimum Tension Tension Time-IGm Gm Rise Rise msec« Gm 1  1. A Krebs Ringer  4  9.5  B-Na-poor medium 4 11,0 (+3) X  90-100 500  V  C-Na-poor medium 4 11,0 (+5.5) 2. A Krebs Ringer 4 11,0 B-Na-poor medium 4 12.0 25 mEq/L (+3)  Duration of Maximum Ampli— Maximum Rate of Duration of Minimum contraction tude of Action Change of Action Action poten-Interval sees. Potential Potential tial Between two mv/lOO msecs. xx mv msecs* successive x Action Potent, Msecs, 10-12 2,0+ 9.7 15-17 350-375 .08 (9) 35-45 1.9+ 9.8 18-20 275 0.04 (9) 120  300  15  400  50-52  2,2+ 0,07 (9) 1.6+ .06 (9) 2.1+ .13~(9)  11.1  20-22  280-295  12.5  20-24  385-295  25-30  380  16.0  In media of lower external sodium concentration, the muscle went into sustained concentration and the action potentials disappeared, 3.A  Krebs Ringer 2.5  B-Na-poor medi C 4.A  2.5  9.5  15  0,7+ 0.01 (9)  16-20  9.5  300-1800  0.8+ 0.23 (9)  18-22  Further muscles passes into sustained contraction with disappearance  X/,X of AP's. Low Na Concentration.  Krebs Ringer ^ 4 7  8  B-Na-poor medium ^ 28 mEq/l 4 9.5 (44.5)  650  20  1,2  6.0  22-24  300-1800  1.0  5.0  20-22  7  # Further decrease i n Na Concentration of the external medium caused sustained spastic contraction i n the muscle with disappearance of action potentials, X/ Action potential had nomophasic (-A-) configuration. /,X,4,^,x,xx = See Table I I .  Figure 11:This record i l l u s t r a t e s spontaneous action potentials during a contraction from an estrogen treated rat uterus i n Krebs Ringer medium. Note the gradual onset of action potentials and t h e i r r e l a t i o n to contraction. Also note the v a r i a b i l i t y i n the amplitude and configuration of the action p o t e n t i a l s 0  (To follow page  28)  -  R A T  •1 i  ii  M  1', r :•!+!: ri r - r -  -  n  •yr  i i|i: if; iiii iii ill!  ::j:.n::  - •- i  _  -  " i  :  j  .  | ^  -!  j |  - ::-  |.  !••!•••!•  1  ...  T 1 Ti "l i i T N T 1 I i"l i T !'[" T ' \ !"""!""' 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L- 1 I I II 1 n ?II1 Ii I!i i.i II iI rM i l l 1  t i  1 I  i  V  1 1  :•_! nr  F  -  I  1  i i iJ i i 1 1 iM ii il il i i 1i i 1 i  i  1  1  ii  -•  1 1  1 1  1  1  1  j  i  i I  1  1  \  —  I l_ ... _ "i _i_ ~ E II I .MM Ml -\rn~r  :  ~  !  1 l  i  _..li i!  i i' "i MM i M i l l . ,,1-1:::  11 i 1 1 l i  1 1  HI  :IH  a  1 1 I- 1 1 1  1 1  l  *  :~.  -Iii 411 411  iH  '/  / r J  f f iiii  m  101  1  iii  iii;  iii  I  IH  —  i i" i r i i r r j' - [ ~i. ! ._!_. I Mil' i[ 1 A l ! I I "T I ! i 11l 1IIlI i i "i1 i 1 1 i I 1 .1. l 1 r n i i i r i i r i" i.  M i l l  iiH  1  ii |i  .-.  L i!if  |||f l 1 1 IIII ;:[; :  r~;  liii  iir i =  =?  1  -M  :S  iiii ~\-  1  i  1 !  :iii M  ,i..|,.  iiii  i  iTi i  i i  M  l M  l  l i  i  •I 1  1  i  i-1 _ _ 1 I i  II ! 1 1 1 1 1 M M . 1 1 M I I 1 1 1 1 1  i  I  t  i 1 1  1  _  1  1  1  1  i i  1  1  i"  I{-'-  i  1 1  i  1 l  l  1  i  i  l  i  i i  l i  I  II  —  II  1 1  4  i  l l  l  I 1 1 1 1  1  1  1 •  M  ::-|:-ii|i-::  :  pH"' i i i  •  1  .if-  ;  7~ Irj L~  l  1  in  4 UU  ii  i|i|i|:i iiiiiiii .il.ii  iiiiiiii  €1  :  i '  1 -  i  = ~i  1 HI!  ; Ei l ~:  mi  f  ll-  iiii  Ij!  !!!II.1 111  ¥ ,;•  ![.  lii-  iir  =H i s  iir  Hi ifr ==  i l HI" HI  i-L ••I'-  V|ii:  HI? •:|  | .  .1...  I  1 I  1  1  • 1 1  i  1  i  1  1  1  1 1 1 1 1 1  ! 1 :•  |.,:|L  i,  "  1  ii-  ::,  • :|'  ;  i:-p: .•liii  iiiiiiii  a?  iiiiP  ~ IH H= HL  ii~  iiii 1  I  1 .1  Kr!  I-.  -1 1  :Mii;;M :: IT •1:,  1 1 1  1  i 1•! w  i' i  i 1  I I I I I 11 11 1  1 1 1  iii  '•i  1 1  1 1  i  I  1 1 1 -  I 1  1  1 i ; 1 . 1 1 1 1  1 1 1 1 1  --  >*  /  • 111  liii ==  ii HI  H nr  li-i  ~  29  ~  Figure 1 2 . This record i l l u s t r a t e s spontaneous action potentials during" a contraction i n a sodium-poblFWdxW"" i n the same preparation from which the record shown i n figure 11 was obtained. Choline was substituted for sodium. The f i n a l concentration of sodium i n t h i s experiment was 43 mEq/l. Note the gradual onset of the action potentials and of contraction (compare with figure 3 and 4). The v a r i a b i l i t y i n the configuration of the action potentials s t i l l persisted (compare with figure l l ) . Note that the action potentials tended to disappear when the contraction reached i t s peak. The frequency of action potentials was not affected s i g n i f i c a n t l y i n t h i s medium as compared to the control i n Krebs Ringer (see figure 11, and compare with figure 3 and 4).  -  R A T  U  30 -  T  E  R  U  S  ffigur.e,.13,, • _ This record i l l u s t r a t e s the changes i n action potentials and contractions i n the same experiment as t h a i represented i n figures 12 and 13 during further lowering of the external concentration of sodium. The sodium concentration of t h i s medium was 27 mEq/l. Note the increase i n basal tension after exposure to the sodium-poor medium (compare with the control i n figure 11). The variations i n the configuration of the action potentials persisted even under these conditions. Note the contrast to the cat u t e r i under similar external sodium concentrations (figures 3 and 4 ) .  -  R A T ilii  hi:  llli  iliiiiliiii il! ii: Iiii Hi: iii: iii; ill: Iiii iiii i llli 'Pi :::: i i i ! ! ! ! ! ! ill iii ii!! iii! ::•: till lijthiiiMl: iiil i:!i ilhhill hu Ii!  it!: ill ii IIII ii; II  iii! iiil iii! i i! Iiii !>'' ilii IMV imi flii iiii III! IIII  ;  iii It!  l/i  1  iiilhiiilii ;  i: i  in:  :  I  iii iii! iiii iii i i i i i i i i i i i 1 I •ii iiti iiii ii! i i i i i i i i i i 1 i i I il iiii iiii iii i i i i i i i i i i ! Ii! H m i Hi) in iiiiliiiilii i 11 i i iiii ii!! iii! iiii i i i l ! ij iii 1 ii ill! iii! Iiii llli i i i i ! ill I ii iiii iiii iiii iii! i i i ! ! ! iii Ii! iii Hi! iii! iii! IiiiI i i i i ! III! ii! iii !!ll iiii iiii iii Ilii ill! iii! 1!! m ill! im in: Iiiiiiiiiimi i l l III!  If!  IIII  :: v.  iiiiiif II  tttf  ii'  iili'i!  iijii  IIi 1  i i! III! ii!! ii'i I II iii II III iiii :i|i t; i ;:t: Ii il i i i i ill! !!!! Ilii ilii il ! ill! 1 :::: :: : :::: Im Hi II i ii nit iiil Iii! ii III .,., .. in: i i ! iii! iiil III! mi iiii i! i Iiii ill! ill! i i i iii! Iiii Iii! iljj i i i Ilii iiii 1!!! 1 ! i i III! III! iiti !!!! iiii II iiiii ' » Hi! iiii iir, iiiii Iiii  iii!  -  iii! Aii (!ii  1 ffi  i  !ii,ii • • |;  i  i* ii;; 3111  1 ilii  iii!  ....  :  iii.  :: r"  ' 11  :::¥  •s  r III  . i::  ''v  iiii  iii^  Iiiii  ,::::  '.llli:  i.... i'.1  •i  <:  II!  i  'iiil .' l !  I'  ii"  .1  iv, i  ,i:  j  V  Iiiii  IS!!  irir  I'M  III!  II  I'M! I  KREB'S .Figure 14:  iiii iiii!  ::::  III! III!  ~\  IMS  T E RU  \  iiii  III! III!  12  U  tl  iiiijiijii I!! f\Iiii TTTT iii! iii! ii'.i iii iiii Iiii i!i iii iiii ;:; i iiii iii iii! iiiiliiii ji ii iii iii! iiii iii ii Iii!iii i i i i i i i i i i i III il! ill! III! Hi! im i! i i i i i i i i i i i Iii .! ii !!!! 1llli  _ ill: IIII mi  31 -  :t::  iiil ill!  Iiil  iiiii Iiiii  ill: Ilii  Na = 2 7 * * ^ L  This figure summarizes the records presented i n figures 11, 12 and 13. Portion 1 on the l e f t shows action potential at the peak of contraction while i n the remaining two portions of the record ( i n sodium-poor medium) the increase i n basal tension should be noted. The amplitude of action potentials i s not so large as that observed i n other experiments with u t e r i of the same species. The frequency of action potentials was almost unaltered.  ill!  sodium concentration was s t i l l i n the v i c i n i t y of 25 mEq/l.  This  behaviour precluded the p o s s i b i l i t y of any further reduction i n the external sodium concentration i f sustained contracture, and subsequent mechanical and e l e c t r i c a l i n a c t i v i t y was to be avoided.  The contraction  which developed i n sodium-poor solutions was found to p e r s i s t f o r as long as 2-2^ hours.  During the f i r s t stage of such contracture  intermittent outbursts of action potentials were recorded, but as the contracture became more prolonged, a l l e l e c t r i c a l a c t i v i t y disappeared. Possibly t h i s behaviour represents another facet of the same paralytic phenomenon which i s seen i n cat u t e r i i n very low concentrations of external sodium. However, cat u t e r i ultimately relaxed after the i n i t i a l prolonged contracture i n sodium-poor solutions, and f a i l e d to contract again, while r a t u t e r i became incapable of relaxing after persistent contraction developed i n sodium-poor media. This type of contraction could not be antagonized by the conventional pharmacological smooth muscle relaxants.  (Sodium-nitrite f a i l e d to i n i t i a t e r e l a x a t i o n  of t h i s type of contracture.) . Some of the i n h i b i t o r y sympathomimetic 1  amines also f a i l e d to bring about relaxation, (d) Estrogen-Treated  Rabbit  Results obtained with estrogen-treated rabbit u t e r i are summarized i n Table V.  Specimen recordings are presented i n Figure IS"* Only two  experiments were carried out. 1  The r e s u l t s obtained were q u a l i t a t i v e l y  This persistent increase i n tension probably should be c l a s s i f i e d as contracture since eventually i t was not accompanied by e l e c t r i c a l activity.  TABLE V ESTROGEN TREATED RABBIT UTERUS No, Bath Medium  A  Basal Maximum Minimum Duration of Tension Tension Jjme-lGm Contraction Gm Rise Tension sees. 7" Ji5} Riseffy^ xx msec.  Krebs Ringer  6  7  1000  Maximum Ampli- Maximum Rate of Duration of tude of Action Change of Action Action potenPotential Potential tial rav > mv/lOO""msecs. msecs,  Minimum Interval Between two successive Action Potent, msecs.  7-8  0,3 + 0.02 (9)  2.0  16-18  450-500  B  Na-poor medium 6 (2.5)  9  _  6-7  0.3 + .01 (10)  1.7  18-20  600-650  C  Na-poor medium 6 (5.5)  12  —  6-7  0.4 + 0.03 (9)  2.4  18-22  650-700  20-25  0.3 + 0.01 (10)  2.2  18-20  Irregular  0.4 + 0.03'(9)  2.7  18^22  600-650  A  Krebs Ringer 5 (+2)  11.5  700  B  Na-poor medium 6 (+4)  11.0  1000  H  %  x, xx, X, / =  See Table I I .  the same as i n other species.  The amplitude of action potentials was  very small (0.1-0.3) i n Krebs Ringer medium, .  In f a c t , e a r l i e r attempts  to record action potentials from t h i s tissue with electrodes having a smaller t i p diameter had always ended i n f a i l u r e .  The increase i n  maximum amplitude of action potentials i n sodium-poor media was 2 slight , but the duration and rate of change of the peak to peak deflection remained unchanged. This tissue showed a tendency toward incomplete r e l a x a t i o n with each decrease i n the external sodium concentration, resembling r a t u t e r i i n t h i s respect.  At a sodium concentration of about 25-30 mEq/l,  the basal tension had increased to approximately the value observed with maximal contractions i n Krebs Ringer, (e) Tissue E l e c t r o l y t e s Tables VI, V I I , VIII and IX summarize the electrolyte analyses of the t i s s u e s , the corresponding perfusion f l u i d s and the functional states of the tissues i n these media. Tissue sodium tended to be l o s t i n sodium-poor media. The greater the reduction i n the concentration of external sodium, the greater was the tissue sodium loss u n t i l a point was reached when further reduction i n the external sodium concentration no longer affected the concentration of tissue sodium to an appreciable extent.  Even when the sodium concentration i n the external medium was  almost n i l (0.2-1'»DmEq/l), and the tissues were i n a c t i v e , electrolyte analysis revealed a tissue sodium concentration which was not s i g n i f i c a n t l y 2  Action potentials within the maximum range of amplitude were considered for s t a t i s t i c a l representation i n the t a b l e . However, the average of a l l action potentials was markedly greater i n amplitude i n sodium-poor media because the proportion of action potentials with maximum amplitude was markedly greater i n sodium-poor media.  -  3T  -  Figure 15. This record i l l u s t r a t e s spontaneous action potentials during a contraction obtained from an estrogen-treated rabbit uterus, f i r s t i n Krebs Ringer and then i n a sodium-poor medium (The sodium concentration of the sodium-poor medium 29 mEq/l). Note the increased amplitude of action potentials, t h e i r unaltered frequency, and the persistance of small wavy e l e c t r i c a l fluctuations even during exposure to the sodium-poor medium. The increase i n basal tension on exposure to a sodium-poor medium also should be noted.  TABLE VI PREGNANT CAT UTERUS Electrolyte Composition of Media, Tissues and Their Functional State Treatment  Functional state  ——  —  Direct Control  —  Krebs Ringer Active n  •H  it  P a r t i a l Chol i n e Ringer Active  — —  —  II  Na  Electrolyte Composition of Tissues mEq/kg G/Kg fresh weight fresh weight K CI H-O  86.7  51.2  82.0  806  85.8  68.8  82.8  820  151.2  5.0  122.5  127.8  51.3  95.2  818  138.5  4.8  128.0  92.2  56.7  101.5  814  60.0  5.5  137.5  79.1  51.2  78.8  834  it  it  n  54.4  5.4  129.4  32.9  14.8  104.4  853  II  ti  n  25.0  5.2  126.3  28.7  15.3  107.5  762  II  n  II  19.0  5.5  133.8  16.7  9.5  112.2  834  6.0  5.0  123.0  26.2  51.2  95.7  3.0  5.5  130.6  19.8  19.0  90.0  70.0  5.1  88.4  44.2  33.3  44.9  100# Choline Ringer ti  II  Inactive II  P a r t i a l Sucrose Ringer Active  837  II  ii  II  23.2  5.8  35.9  15.5  4.8  57.9  801  n  u  II  18.5  5.3  24.5  12.3  3.0  45.5  779  0.2  5.7  8.2  17.5  25.5  21.8  818  1.0  5.8  10.9  18.9  43.5  25.0  760  0  12.5  174.5  10.2  845  Sucrose Ringer II  II  Left i n Isotonic  K S0 2  Electrolyte Composition of media mEq/l Na ^ CI  4  Inactive II  0  167.0  Action potentials and contraction always occurred together although the sequence of occurrence of one with respect to another was variable sometimes (details i n the t e x t ) . However i n the absence of contraction action potentials were not recorded.  TABLE VII Pregnant Rabbit Uterus Electrolyte analysis of tissues and perfusion f l u i d s at d i f f e r e n t functional states of the t i s s u e . -  Treatment  Functional* E l e c t r o l y t e Composition of state media mEq/l  Direct Control II  —  II  Na  K  CI  Electrolyte Composition of Tissues m E q / k g G / K g fresh weight fresh weight Na K CI H-O^  (148)  (4.5)  (110)  71.27 66.35  53.73  817  81.04 29.89  78.94  837  117.80  827  —  —  —  A Krebs Ringer  Active  138.5  4.8  128.07 90.22 31.86  B Choline Ringer  Active  60.0  4.8  131.57  —  C Choline Ringer  Active  29  5.8  127.63 31.74 30.30  94.50  * See Table VI  -  798  38  -  TABLE VIII Estrogen Treated Rat Uterus Electrolyte analysis of tissues and corresponding perfusion solutions at different functional states of the tissues  *  Treatment  Functional state  Electrolyte Composition of media MEq/l Na  CI  2  —  Control  Krebs Ringer Active Part Choline Ringer  Active  11  it  it  n  II  +  10($ Choline Ringer / n  K  II  Inactive n  136.5  5.0  Electrolyte Composition of Tissues mEq/kg G/Kg fresh weight fresh weight Na K CI H 0  130.1  74.5  43.0  105.1  40.5  68.3  798  86.6  804  1 43.0  5.4  126.3  40.6  21.0  87.7  818  30.2  5.7  125.6  43.1  54.4  78.9  818  5.2  126.3  19.5  35.1  103.3  783  1.2  5.8,  123.0  19.0  43.2  74.7  809  4.0  7.0  122.0  22.8  61.2  99.5  818  25.0  Sucro se Ringer**  Active  28.0  6.5  32.5  Sucrose Ringer /  Inactive  16.0  6.0  19.5  14.9  18.0  19.1  802  it  it  2.0  6.5  9.7  15.7  20.9  16.5  806  II  ti  0.5  5.7  7.8  27.5  57.5  14.0  819  * See Table VI ** Tissues showing incomplete relaxation i n spite of increased amplitude of action potential after changing to sodium-poor medium. / Tissue showing i n d e f i n i t e l y prolonged contraction and eventual arrest of action potentials.  4  TABLE IX Estrogen Treated Rabbit Uterus Electrolyte Analysis of Tissues and Perfusion Fluids at Different Functional States of the Tissues  *  Treatment  Functional Electrolyte Composition of State media mEq/l  Krebs Ringer w  Active  II  II  Choline Ringer it  II  Choline Ringer Left i n Choline Chloride * See Table VI  Active II  Inactive  Na  K  CI  Electrolyte Composition of Tissues mBq/kg G/Kg fresh weight Na K CI -2^4  138.5  4.8  128.0  75.8  62.3  72.1  864  140.5  5.0  130.2  81.3  66.3  76.9  866  31.5  5.4  122.8  28.2  56.5  75.0  871  29.0  5.4  125.4  24.5  60.3  77.4  860  1.0  5.3  112.0  29.9  61.1  52.8  850  0  0  167.0  22.7  44.4  101.1  860  -  40  d i f f e r e n t from that which was found i n f u n c t i o n a l l y active tissues i n somewhat higher sodium concentrations (20-25 mEq/l).  Even when tissues were l e f t i n  isotonic solutions of K^SO^, choline chloride or sucrose, over a prolonged period (3-4 hours and sometimes even over night) considerable quantities of sodium  (about 15-20 mEq/kg) were s t i l l found i n the tissues.  Such residual  tissue sodium has been reported previously from t h i s laboratory (15). After exposure to sodium-poor media, tissue potassium concentrations also were lower than the control values. The low concentration of tissue potassium i n the pregnant cat u t e r i was very s t r i k i n g .  Values as low as 4—5 mEq/kg were  obtained while the tissue s t i l l exhibited f u l l mechanical and e l e c t r i c a l a c t i v i t y . In ::>ther species, the l e v e l s of tissue potassium i n sodium-poor media were never as low as i n the cat although a decrease sometimes was noticed. The a l t e r a t i o n i n tissue chloride i n sodium-poor media was not s i g n i f i c a n t when choline chloride was used to replace sodium chloride.  However, tissue  chloride was decreased i n sodium-poor media employing sucrose as a substitute f o r sodium chloride.  The decrease i n tissue chloride i n the l a t t e r case probably was  due primarily to the decrease i n chloride concentration i n the e x t r a c e l l u l a r space.  - 41 DISCUSSION I*  Recording Techniques The microelectrode technique has been employed extensively to record  transmembrane action potentials from many excitable tissues (1-4).  In this  laboratory, attempts to apply t h i s technique to one -of the tissues (uterus of rabbit) employed i n the present investigation have met with  considerable  d i f f i c u l t y , presumably due to small c e l l size and f a i l u r e of microelectrodes to penetrate the c e l l s without appreciable damage. However, action potentials dould be recorded with ease from such tissues using e x t r a c e l l u l a r surface electrodes.  The l i m i t a t i o n s of such recordings i n f a i l i n g to give exact  information regarding transmembrane potentials and rates of depolarization and repolarization were recognized. Concomitant recordings with microelectrodes and e x t r a c e l l u l a r electrodes have been made by other workers (20) from i s o l a t e d single f i b r e s of s k e l e t a l muscle. A comparison of the simultaneous biphasic e x t r a c e l l u l a r action potentials and monophasic i n t r a c e l l u l a r action potentials showed that the positive peak of the biphasic action potentials occurred at the same time as the onset of the r i s i n g phase of the monophasic action p o t e n t i a l , and the negative peak of the biphasic action potential coincided with the peak depolarization of monophasic action potentials.  The peak to peak deflection of biphasic e x t r a c e l l u l a r action  potentials thus corresponds i n time with the upstroke of monophasic action potentials.  However, the precise relationship between transmembrane potentials  and e x t r a c e l l u l a r recordings made from m u l t i c e l l u l a r units (such as the tissues we have studied) has not been c l a r i f i e d .  Various factors can influence the  extracellularly-recorded action potential from m u l t i c e l l u l a r units which have l i t t l e effect on i n t r a c e l l u l a r l y recorded action potentials.  These factors  may be described as follows: An extracellular electrode near an e l e c t r i c a l l y active region i n the tissue records a voltage which i s d i f f e r e n t from that of a distant  electrode  because the two electrodes are at different isopotential l i n e s emanating from the e l e c t r i c a l source and sink*  In practice the distant electrode  usually be regarded as at zero potentials  can  In these studies alterations i n  the position of the distant electrode did not noticeably a l t e r the records obtained, so that i t could be e f f e c t i v e l y regarded as i n d i f f e r e n t . A recordable change e x i s t s somewhere i n the medium throughout the entire period that an impulse i s present i n any part of the tissue (21),  Further  studies (23) have shown that i n uterine muscle using the present recording technique, a c t i v i t y at one electrode had no recordable effect on the second electrode which was 1-2 mm away from i t . Therefore the d i f f e r e n t electrode could be regarded as recording only from the c e l l s which were very close to i t s tip.  According to Lofente De No' • (23) the tracings obtained from a single  c e l l with an external electrode are related to the transmembrane potential curve as i t s second derivative, i . e . amplitude of e x t r a c e l l u l a r l y recorded action potentials related to the rate of change of the slope of the transmembrane potential curve. In m u l t i c e l l u l a r u n i t s , e x t r a c e l l u l a r l y recorded potentials are related to, the sum of the potentials generated by a l l of the c e l l s i n the immediate v i c i n i t y of the recording electrode t i p . The net potential difference recorded might be influenced by the r a p i d i t y with which these c e l l s are activated and  by  sequential pattern of a c t i v a t i o n as well as by the magnitude of the potentials across the individual c e l l s .  'If the! c e l l s  , are activated s u f f i c i e n t l y close  together (synchronously) and i n proper sequence, the sum of t h e i r potentials w i l l allow the recording of larger e x t r a c e l l u l a r action p o t e n t i a l .  With  -  43  -  r e l a t i v e l y asynchronous or nonsequential a c t i v a t i o n , potential produced i n different c e l l s may p a r t l y or completely n u l l i f y one another.  I t appears  obvious from the considerations of the size of the electrode t i p used and of the uterine muscle c e l l s that some summation of potentials must occur to permit the recordings made e x t r a c e l l u l a r l y . Many factors can increase the r e l a t i v e synchrony of the activation of the c e l l s and i n turn might contribute towards an increased amplitude of recorded potentials. 1,  The rate of conduction of action potentials and the pattern of a c t i v i t y  over the entire m u l t i c e l l u l a r area from which the electrode records 8 A f a s t rate of spread of activation w i l l increase the degree of synchrony of activation of f i b r e s , provided that the pathway of spread of a c t i v i t y i s regularly and l i n e r l y maintained throughout the series of action potentials. Moreover, the r e g u l a r i t y and symmetry of the pattern of spread of an impulse into the area which contributes the major potentials may determine the extent to which the potentials generated by asynchronously activated c e l l s w i l l tend to cancel one another.  I f the pattern of spread of a c t i v i t y varies during a  series of impulses, considerable v a r i a b i l i t y i n amplitude of the e x t r a c e l l u l a r potentials may r e s u l t . 2.  The number of c e l l s responding i n the area from which the electrode records; But of those c e l l s which are close enough to the electrode (and can influence  the recordings) the proportion of c e l l s which are actually activated might determine the magnitude of e x t r a c e l l u l a r l y recorded action potentials.  The factors which  might determine the proportion of the responding c e l l s i n the close v i c i n i t y of the electrode are d i f f i c u l t to define i n the absence of conclusive evidence as to whether or not a particular smooth muscle i s e l e c t r i c a l l y excitable(24).  I f there i s an  -  44  -  absolute and r e l a t i v e refractory period as would be expected i n an e l e c t r i c a l l y excitable tissue or i f e x c i t a b i l i t y i s d i r e c t l y related to the recovery of the transmembrane potential following an action p o t e n t i a l , then the rate of repolarization and the frequency of action potentials might influence the magnitude of action potentials recorded by the technique used i n t h i s study* Thus an increase i n the rate of r e p o l a r i z a t i o n might increase the r e l a t i v e number of c e l l s capable of responding during each of a series of action potentials provided that the time i n t e r v a l between each action potential remains constant*  S i m i l a r l y an increase i n the time i n t e r v a l between two  successive action potentials (decreased frequency) might increase the proportion of c e l l s capable of responding by allowing enough time for even those c e l l s which normally recovered slowly to regain t h e i r e x c i t a b i l i t y more completely i n the longer period available* In addition to these factors which i n d i r e c t l y influence the recorded amplitude of e x t r a c e l l u l a r action potentials (by effecting the synchrony of activation of the c e l l s ) the change i n the resistance between the electrode and the medium might also influence the amplitude of e x t r a c e l l u l a r l y recorded action potentials*  Changes i n the composition of the medium may produce changes i n  the resistance between the electrode and the medium which would a f f e c t the current flow through the medium and the potential occurring at the near electrode and would change the recorded amplitude of action potentials* The resistance between the electrode and medium is not expected to change s i g n i f i c a n t l y when sodium chloride i n the medium i s replaced by another e l e c t r o l y t e , e.g. choline chloride*  However, the resistance might increase when  a sucrose containing medium i s used* The implications of these p r i n c i p l e s are applicable t o the observed changes and are discussed i n further d e t a i l i n the following sections*  ~ lis  45 -  Results ( i ) Changes i n Action Potentials i n Reduced External Sodium Concentration; The amplitude of action potentials was increased i n sodium-poor media  (Tables II-V and Figures 2-15), The aslope of the major deflection was s l i g h t l y increased or appeared unchanged and the o r i g i n a l biphasic configuration of action potentials was maintained at a l l stages i n sodium-poor media. However, cessation of action potentials occurred (along with mechanical i n a c t i v i t y ) when the sodium concentration was reduced to the v i c i n i t y of 15-20 mEq/l,  Independent studies on the conduction of action  potentials revealed no decrease i n the conduction rate i n sodium-poor media u n t i l just before mechanical and e l e c t r i c a l i n a c t i v i t y developed (22), The frequency of action potentials i n the cat uterus was decreased considerably i n sodium-poor media (to sbout 3-5 times less frequent than i n Krebs Ringer),  In  the u t e r i of other species the decrease i n frequency i n sodium-poor media was not marked.  The v a r i a t i o n i n amplitude between individual members of a series  of action potentials was quite large i n Krebs Ringer,  I n cat u t e r i ,  such  variations were considerably decreased i n sodium-poor media but were l i t t l e affected i n the u t e r i of other species, (2) Explanation of Observed Changes i n Action Potentials; Microelectrode  studies (14) of uterine smooth muscle i n sodium-poor media have  shown no change i n the rate of depolarization, while the rate of repolarization was actually increased.  The amplitude of the transmembrane action potential  was not appreciably altered i n sodium-poor media and even overshoot sometimes was observed.  This indicates that i n sodium-poor media there was no decrease  i n the transmembrane potential of individual c e l l s .  I t also suggests that the  increased amplitude of e x t r a c e l l u l a r l y recorded action potentials i n sodium-poor  media must be due to increased synchrony of response of m u l t i c e l l u l a r areas and/ or altered pattern of spread of activation. I n a given series of r e p e t i t i v e action potentials uniformly increased synchrony during each of the member impulses might have been brought about by the faster rate of repolarization (14) which presumably decreased the period of refractoriness of the c e l l s .  In  cat u t e r i the decreased frequency (3-5 times less than that i n Krebs Ringer medium) of action potentials might have further contributed to an increased i n the degree of synchronous a c t i v a t i o n of c e l l s i n such a way as to produce action potentials which were of r e l a t i v e l y uniform high amplitude (as discussed i n f i r s t section).  In the other two species almost unaltered high frequency  (150-300 msecs. between two successive impulses) of action potentials i n sodiumpoor media might have accounted for the persistence of aany action potentials of low and variable amplitude (as every new impulse i n a series might f i n d a variable number of c e l l s which were completely excitable). In choline chloride sodium-poor media the s l i g h t change i n conductivity between medium and electrode presumably did not play a s i g n i f i c a n t role i n modifying the amplitude of action potentials. However, i n sucrose containing media the increased resistance could have been s u f f i c i e n t to decrease the amplitude of the action potentials.  This tendency would be opposed by the  reduced electrolyte concentration of the medium which would retard short c i r c u i t i n g through the medium and thus enhance the recorded amplitude of the action potential. Actually, no decrease i n the amplitude of action potentials ever was recorded i n sucrose-containing  sodium-poor media. The s i m i l a r i t y  between the changes observed when sucrose or choline chloride was used to replaced sodium chloride suggest that altered conductivity of the external medium played l i t t l e role i n producing the observed changes.  -  —  III,  The implications of current observations f o r the "Sodium Hypothesis" i n Smooth Muscle. According to the sodium hypothesis ( l ) a selective increase i n the  permeability of the c e l l membrane to sodium ions i s responsible for depolarization and the i n i t i a t i o n of action potentials i n many tissues (1-10), I f t h i s theory were applicable to uterine smooth muscle, the reduction i n the external sodium concentration  should have produced a decrease i n the amplitude  of transmembrane action potentials ( l ) and the rate of depolarization should have increased ( l ) . However, no such changes were observed (14).  This  indicates that the sodium hypothesis cannot be applied to uterine smooth muscle without serious modifications.  Our own observations have shown that  action potentials recorded e x t r a c e l l u l a r l y p e r s i s t at remarkably low external sodium concentrations.  The possible r e c o n c i l i a t i o n of these findings with  the "Sodium Hypothesis" and an explanation of the r e l a t i v e independence of the presence of action potentials and the external sodium concentration could be based on three main arguments. ( l ) Non-equilibration of external sodium i n the medium with e x t r a c e l l u l a r sodium i n the tissue The available evidence i s not i n harmony with t h i s explanation for the r e l a t i v e independence of t h i s presence and amplitude of action potentials and the external sodium concentration.  I f e q u i l i b r a t i o n between the medium and  the e x t r a c e l l u l a r space of the tissue were incomplete, i t would be d i f f i c u l t to account for other change i n function which are observed i n sodium-poor media after a few minutes (5-10 min.).  The disappearance of action potentials i n  tissues whose sodium concentrations were very similar to those of other tissues which under the same circumstances remained active also indicates that the persistence of action potentials i n sodium-poor media i s not due to incomplete e q u i l i b r a t i o n (retention of e x t r a c e l l u l a r sodium).  The tissues immersed  i n solutions with negligible external sodium for prolonged periods of time retained considerable amounts of residual sodium (Tables VI-IX).  The sodium  space under these conditions was enlarged s i g n i f i c a n t l y as compared with the control.  This suggests the p o s s i b i l i t y that a part of the residual sodium  was bound i n a non-diffusable form. The degree of such binding, however important , cannot be determined s a t i s f a c t o r i l y u n t i l suitable d i r e c t methods are used, ( 2 ) Maintenance of Constant Ratio Between the c e l l u l a r and e x t r a c e l l u l a r concentrations of sodium (Na^/Na-) i n various concentrations of external sodium. This explanation of the r e l a t i v e independence of the magnitude of action potentials and the external sodium concentration also appears implausible. Maintenance of a fixed r a t i o Nat /Na.. at various external sodium concentrations o x  would only be possible i f tissue sodium losses from outside and inside of the c e l l s occurred at a constantly proportionate r a t e . The available evidence indicates (15) that extrusion of c e l l u l a r sodium i n sodium-free media i s met with c e r t a i n b a r r i e r s tending to hinder the c e l l u l a r sodium loss.  Sodium loss  i s rapid from the extracellular space. Thus, the r a t i o Nat/Na. must be o 1 decreased at lowered external sodium concentration, at least i n the i n i t i a l stages.  Hence, the amplitude of i n t r a c e l l u l a r l y recorded action potentials  should have decreased and some reduction i n magnitude of e x t r a c e l l u l a r action potentials might have been expected.  However, no decreases i n transmembrane  action potentials were observed i n low external sodium solutions (14.) and e x t r a c e l l u l a r l y recorded potentials actually manifested an increase i n t h e i r amplitude very soon after exposure to such media. (3) Saturation of sodium "carrier-system". I t has been suggested that the mechanism whereby excitation i s accomplished by membrane depolarization may involve a sodium " c a r r i e r system" which i s made  -  49 -  available by excitatory depolarizing processes, permitting an inward current to be carried by sodium ions moving along t h e i r concentration gradient. I t might be possible that such a sodium "carrier-system" i s super-saturated  with  sodium i n smooth muscle so that reduction of external sodium concentration has l i t t l e effect on the quantity of c a r r i e r combined with sodium u n t i l very low levels of external sodium are reached. Evidence has been presented previously (14) which almost excludes t h i s hypothesis as an explanation of the r e l a t i v e independence of the presence, amplitude and pattern of action potentials and the external sodium concentration. Selective sodium currents do not appear adequate to account f o r the depolarization of uterine smooth muscle.  The p o s s i b i l i t y that other ions may  carry the action potential currents i n these tissues should be considered. However, potassium and chloride ionic currents flowing as a r e s u l t of a generalized increased i n membrane permeability would cause hyperpolarization (14),  An increased permeability to a l l ions as a cause of depolarization does  not adequately account for the experimental r e s u l t s (14),  Under these  conditions, an hypothesis based on an outward flow of i n t r a c e l l u l a r anions other than chloride as a possible cause of depolarization and action currents must be considered.  This hypothesis could explain the various findings without  leading to any glaring discrepancies, IV,  Eventual Mechanical and E l e c t r i c a l I n a c t i v i t y i n Extremely Low Sodium Concentration, I t i s d i f f i c u l t to advance an explanation of eventual mechanical and  e l e c t r i c a l i n a c t i v i t y i n extremely low concentrations of external sodium i f the sodium hypothesis i s discarded.  However, the fact remains that mechanical  a c t i v i t y was affected i n some important way i n s u f f i c i e n t l y sodium-poor media.  50  Sudden Contraction, gradual relaxation and resumption of spontaneous activityoccurred when the external sodium concentration was only moderately lowered. When the external sodium concentration was lowered beneath 15-20 mEq/l, the pregnant cat uterus usually showed sudden contraction, gradual relaxation, and f a i l u r e to resume spontaneous a c t i v i t y . at t h i s stage.  The action potentials disappeared  In the other two species, the i n i t a l sudden uterine contraction  on transfer to sodium-poor media was usually followed by incomplete relaxation.  In sodium concentrations as low as 20-25 mEq/l persistent  contraction occurred and the tissue f a i l e d to relax even over a long period (over 3 hours).  Action potentials were recorded i n the i n i t i a l stages but  they disappeared i n the later  stages.  I t i s possible that certain metabolic pathways associated with e l e c t r i c a l and mechanical a c t i v i t y were i n h i b i t e d at extremely low l e v e l s of tissue sodium. I t has been suggested that contraction i n smooth muscle i s associated with break-down of high-energy phosphates (25) as i n other types of tissue (26,27,28), Whether or not sodium i n optimum concentrations plays a role i n the breakdown of high-energy phosphate fractions or t h e i r re-synthesis i s a matter of speculation.  I f the break-down of certain high-energy phosphate bonds were  i n h i b i t e d , or i f t h e i r synthesis were retarded, the energy required to produce mechanical activation might not be available and consequently i n a c t i v i t y would ensue. Differences of i n h i b i t i o n i n the site of these energy sources (during the c y c l i c phases of contraction and relaxation) might possibly account for the two types of eventual mechanical i n a c t i v i t y observed i n d i f f e r e n t species i n extremely low external sodium concentrations:  ( i n a c t i v i t y without exertion  of tension i n the cat uterus; and i n a c t i v i t y with exertion of tension i n rabbit and rat u t e r i ) .  -  51 -  Such an i n h i b i t i o n of metabolism might hinder the repolarization of the c e l l s after excitation and thus could account for secondary e l e c t r i c a l i n a c t i v i t y . I f e l e c t r i c a l activation i s necessary to e l i c i t contraction, then mechanical i n a c t i v i t y might follow secondarily.  Thus i t might be that i n h i b i t i o n of  metabolism d i r e c t l y affects the e l e c t r i c a l a c t i v i t y and only  secondarily  the mechanical a c t i v i t y . V. Tissue Potassium and Chloride D i s t r i b u t i o n After Exposure to Low Sodium Solutions: Quantitatively, the d i s t r i b u t i o n of uterine tissue potassium following immersion i n sodium-poor media was inconsistent, as was noted e a r l i e r for brain s l i c e s (29).  Tissue potassium tended to be l o s t more rapidly i n sodium-  poor media, although the extent of the loss was variable.  Greater quantities  of potassium were l o s t i n solutions poorer i n sodium content, and losses appeared to be greater when choline chloride rather than sucrose was used to replace sodium chloride (see 15).  I n pregnant cat u t e r i tissue potassium  concentrations as low as 4-5 mEq/kg fresh weight were obtained i n some cases (Table V I ) .  Evidently potassium gradients across the C e l l u l a r membranes were  greatly altered from the normal value i n these cases. The altered potassium gradient i n these tissues raised important questions regarding the mechanism of potassium d i s t r i b u t i o n i n smooth muscle and i t s relationship to resting and action potentials.  I t was of i h t e r e s t to determine  i f loss of e l e c t r i c a l a c t i v i t y had any relationship to potassium loss i n Na-poor media.  I f potassium were f r e e l y diffusable across the c e l l membrane and at  equilibrium, i t s d i s t r i b u t i o n would be expected to be related approximately to the resting potentials by the relationship E^p = RT/F log Kc/Ke. This would be approximately true irrespective of whether or not equilibrium was obtained i f i t s permeability were much greater than that of other ion species.  In  calculated Kc, an estimated value for the e x t r a c e l l u l a r space (ECS) must be  -  52  used. The chloride space was often larger than the sodium space and even larger than the ECS conceivably could be.  Therefore, an ECS value was  calculated assuming Kc = 150 mEq/liter for the controls and t h i s value f o r the ECS was applied to the experimental pieces to calculate t h e i r Kc. i n turn was used to calculate  This  the potassium equilibrium potentials, which  are close to the resting transmembrane potential i n these cellw.  These  values indicated that neither the l e v e l of tissue potassium nor the magnitude of the resting membrane potentials calculated from potassium d i s t r i b u t i o n have any d i r e c t relationship to the presence or absence of e l e c t r i c a l a c t i v i t y . Actually there was no correlation between the extent of tissue potassium loss and the incidence of mechanical, and e l e c t r i c a l i n a c t i v i t y i n the various species studied.  Some u t e r i with tissue potassium levels close to t h e i r  controls became inactive i n low external sodium concentrations while other u t e r i with a much lower tissue potassium concentration under similar conditions remained active (Table VI-IX).  Moreover, no relationship seemed  to e x i s t between the degree of c e l l u l a r potassium depletion i n the u t e r i of various species studied and the two different types of mechanical i n a c t i v i t y , which. were observed at extremely low l e v e l s of external sodium. In the potassium depleted c e l l s , e l e c t r i c a l n e u t r a l i t y must be maintained i n some fashion.  This could be accomplished by gain of cations, e.g., sodium,  hydrogen, calcium, and/or magnesium. Alternately loss of i n t r a c e l l u l a r anions could maintain the i o n i c balance.  However, the available data (30)  do not support the view that sodium could substitute for the l o s t c e l l u l a r potassium, A compensating gain of hydrogen would change the c e l l pH to high&y acidic l e v e l s . While the p o s s i b i l i t y of calcium and/or magnesium uptake to offset c e l l u l a r potassium loss cannot be ruled out, the small concentrations i n which these ions exist i n tissueis suggest that thes'e 1  :  -  53  -  ions probably are not involved i n an exchange f o r l o s t c e l l u l a r potassium. The loss of i n t r a c e l l u l a r anions accompanying potassium loss would maintain e l e c t r i c a l balance but would considerably decrease the i n t r a c e l l u l a r osmolarity. cell.  Under these conditions water must be l o s t from within the  I f so, the r a t i o E.C.S./l.C.S. might change under d i f f e r e n t conditions.  However, the answer to these problems must be awaited t i l l further evidence bearing upon these questions i s available. Values f o r tissue chloride and chloride spaces i n sodium-poor media containing choline chloride were comparable to control samples l e f t i n Kreb's Ringer solution. However, when sodium-poor media containing sucrose were used, the tissue chloride l e v e l s decreased (Table VI-IX).  Such  losses probably could be accounted f o r by decreased chloride i n the extracellular space. However, the estimated chloride space i n sodium-poor media containing sucrose were enlarged, i n d i c a t i n g that some of the chloride probably was bound within the tissue or equilibrated very slowly with the external medium.  -  54  -  SUMMARY 1.  E x t r a c e l l u l a r action potentials and isometric contractile tension have been simultaneously recorded i n v i t r o from uterine longitudinal smooth muscle of the pregnant cat, pregnant rabbit, estrogen treated rabbit and estrogen treated r a t . Recordings were at f i r s t made i n Krebs Ringer and then i n sodium-poor media (sodium chloride replaced by choline chloride or sucrose),  2.  Decrease i n the external sodium concentration resulted i n increased peak to peak amplitude of action potentials. The rate of change and the duration of peak to peak deflection of action potentials remained almost unchanged. The frequency of action potential was reduced i n pregnant cat u t e r i but remained almost unchanged i n the u t e r i of other species.  3. In. s u f f i c i e n t l y reduced external sodium concentrations, the muscle went into sudden i n i t i a l contraction, gradual relaxation and resumption of spontaneous contraction and action potentials. However, i n rabbit and rat u t e r i , the relaxation after an i n i t i a l contraction was usually incomplete and even a persistant contracture developed at extremely low external sodium concentrations 4.  (15-20 mEq/l).  In extremely low sodium concentrations  (below 15-20 mEq/l) action  potentials eventually disappeared along with mechanical i n a c t i v i t y . In cat u t e r i mechanical i n a c t i v i t y occurred when the u t e r i were i n the relaxed phase after contraction while i n other species the tissue had f a i l e d to relax after i n i t i a l contraction at such low external sodium concentrations. 5.  Evidence i s presented to indicate that selective inward flow of sodium ions probably cannot account for. i n i t i a t i o n of action potentials i n  (Summary  5»  55  (c ont * d*)  uterine smooth muscle since  considerable reduction of the  external sodium concentration (down to 15-20 mEq/l i n cats and 25-30 mEq/l i n other species) did not affect the c h a r a c t e r i s t i c s of action potentials i n the expected manner. 6.  The view that an outward flow of i n t r a c e l l u l a r anions might be responsible f o r depolarization i n these tissues receives further support from the present studies,  7.  The observed changes i n action potentials i n sodium-poor media and also i n t r a and i n t e r species variations are  8.  discussed.  I t i s suggested that the eventual i n a c t i v i t y ( e l e c t r i c a l as well as two d i f f e r e n t types of mechanical i n a c t i v i t y , i . e . without exertion of tension as i n cats and with exertion of tension as i n rabbits and rats) might be due to altered biochemical processes related to the e l e c t r i c a l and/or mechanical phenomenon i n these tissues.  -  56 -  BIBLIOGRAPHY  1, Hodgkin, A.L.  The Ionic Bases of E l e c t r i c a l A c t i v i t y i n Nerve and Muscle, B i o l . Rev. 26: 339-409, 1951.  2.  Keynes, R.D.  The Ionic Movements During Nervous A c t i v i t y . J . Physiol. 114: 119-150, 1951.  3  Hodgkin, A.L. and Huxley, A.F.  Currents Carried by Sodium and Potassium Ions Through the Membrane of Giant Axon of Loligo J . Physiol. 116: 449-472, 1952.  0  4. Desmedt, J.E.  E l e c t r i c a l A c t i v i t y and I n t r a c e l l u l a r Sodium Concentrations i n Frog Muscle. J . Physiol. 121: 191, 1953.  5. Draper, M.H. and Weidman, S.  Cardiac Resting and Action P o t e n t i a l , Recorded with an I n t r a c e l l u l a r Electrode. J . Physiol. 115: 74-93, 1951.  6. Nastuk, W.L. and Hodgkin, A.L,  The E l e c t r i c a l A c t i v i t y of Single Muscle Fibre. J . C e l l . Comp. Physiol. 35: 39-74, 1950.  7,  Effect of Potassium and Sodium on Resting and Action Potential of. Single Myelinated Nerve Fibre. J . Physiol. 112: 496-508, 1951.  Huxley, A.F, and Stampfli, R«  8. Hodgkin, A.L, and Katz, B.  The E f f e c t of Sodium on E l e c t r i c a l A c t i v i t y of Giant Axion of Squid. J . Physiol. 108: 37-77, 1949.  9. Cranefield, P,F., Eyster, J.A.E., and Gilson, E.  Effect of Reduction of External NaCl on Injury Potential of Cardiac Muscle. Am. J . Physiol. 166: 269-272, 1951.  9a. Cranefield, P.S., and Hofman, B.F.  Electrophysiology of Single Cardiac C e l l s . Physiol. Rev. 38: 41-77, 1958.  10? Katz, B.  The Effect of Electrolyte Deficiencies on the Rate of Conduction i n Single Nerve Fibre. J . Physiol. 106: 411 et seq, 1947.  11.  Fatt, P., and Katz, B,  The E l e c t r i c a l Properties of Crustacean Muscle Fibre. J . Physiol. 120: 171-204, 1953.  12.  Wood, D.W.,  The Effect of Ions upon Neuromuscular Transmission i n a Herbivorous Insect. J . Physiol. 138: 119-139, 1957.  -  Bibliography  57  -  (Cont'd)  13* Holman, M.E,  The Effect of Changes i n Sodium Chloride Concentration on Smooth Muscle of Guinea Pig Taenia C o l i . J . Physiol. 136: 569-584, 1957.  14  The E l e c t r i c a l Properties of Smooth Muscle C e l l Membrane Can. J . Biochem. & Physiol. 36: 959-975, 1958.  6  Daniel, E. and Singh, EV  15. Daniel, E., and Daniel, B.  Effects of Ovarian Hormones on the Content and D i s t r i b u t i o n of Intact and Extracted Rabbit and Cat U t e r i . Can. J . Biochem. & Physiol. 35: 1205-1223, 1957.  16. Spencer, A.G.  Flame Photometry The Lancet, November, 623, 1950.  17. Asper, S.P., and Schales, S.C.  A Simple and Accurate Method f o r Determination of Chloride i n B i o l o g i c a l F l u i d s , J . B i o l . Chem. 140: 879, 1941,  18, Whittam, R.  A Convenient Micro-method f o r Estimation of Tissue Chloride J.., Physiol. 128: 65 P, 1955.  19. Manery, J.F.  Water and Electrolyte Metabolism. Physiol. Rev. 34: 335-417, 1954.  20. Hakansson, C.H.  Action Potential Recorded I n t r a c e l l u l a r l y and E x t r a c e l l u l a r l y from isolated frog muscle f i b r e i n Ringer Solution and A i r . Acta. Physiol. Scand. 39: 291-312, 1957.  21.  Text Book of Physiol. 17th E d i t i o n , 519 et seq, 1957.  Fulton, J.F.  22. Daniel, E.E.  Unpublished results Department of Pharmacology, U.B.C. Vancouver  23. Lorente De No  A Study of Nerve Physiology, Stud. Rockefeller Institue of Medical Research 132: 394-477, 1947o  24. Grundfest, H.  E l e c t r i c a l I n e x c i t a b i l i t y of Synapses and Some Consequences i n Central Nervous System. Physiol. Rev. 37: 337, 1957.  25. Born, G.V.R.  The Relation Between the Tension and the High Energy Phosphate Content of Smooth Muscle. J . Physiol. 131: 704-711, 1956.  S.V.,  58  -  Relation Between Chemical and Contractile Function and Structure of Skeletal Muscle Cell Physiol. Rev. 36: 1, 1956.  26.  Perry,  27.  F r i t z , B., Svensmark, A, and Rosenfalck, P.  Mechanical and Chemical Events i n Muscle Contraction. Physiol. Rev. 36: (4) 503, 1956.  28.  SzentrGyorgyi, H.  Chemical Physiology of Contraction i n Body and Heart Muscle. 2nd E d i t i o n , Academic Press, N.Y., 1951.  29.  Pappius, H.M,, Rosenfeld M., Johnson, D.M., and E l l i o t , K.A.C.  Effects of Sodium-free Media Upon the Metabolism and the Potassium and Water Content of Brain S l i c e s . Can. J . Biochem. & P h y s i o l . 36: 217-226, 1958.  30.  Daniel, E.E.  Unpublished Results. Department of Pharmacology, U.B.C.  

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