UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1958_A8 S48 R6.pdf [ 5.83MB ]
Metadata
JSON: 831-1.0106239.json
JSON-LD: 831-1.0106239-ld.json
RDF/XML (Pretty): 831-1.0106239-rdf.xml
RDF/JSON: 831-1.0106239-rdf.json
Turtle: 831-1.0106239-turtle.txt
N-Triples: 831-1.0106239-rdf-ntriples.txt
Original Record: 831-1.0106239-source.json
Full Text
831-1.0106239-fulltext.txt
Citation
831-1.0106239.ris

Full Text

THE ROLE OF SODIUM IN ACTIVATION OP UTERINE SMOOTH MUSCLE A Thesis Submitted i n Parti a l Fulfilment of the Requirement for the Degree of Master of Arts i n the Department of Pharmacology We accept this thesis as conforming to "the required standard fey HARCHARAN SINGH, B. Pharm., Panjab University, India, 1956, THE UNIVERSITY OF BRITISH COLUMBIA October 1958 ABSTRACT Extracellular 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 estrogen-treated rat. Action potentials were recorded from the surface of the muscle strips 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 potentials. During relaxation no electri 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). Sufficient 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 deflection 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 less so i n the rabbit and rat . 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 level 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 potentials. The e l e c t r i c a l responses of uteri of the other two species (rabbit and rat) during exposure to sodium-poor media were similar to those observed ( i i ) wiih the cat uterus. However, the mechanical a c t i v i t y of rat and rabbit uteri i n sodium poor media was different 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 fail u r e of the tissue to relax (even after 2-2^ hours)© Outbursts of action potentials at irregular intervals were seen i n the i n i t i a l stages of this 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 for the i n i t i a t i o n of action potentials i n uterine smooth muscle since considerable reduction of the external sodium concentration (down to 15-20 mEq/l i n cat and 25-30 mEq/l i n the other two species) did not effect the characteristics of the action potentials i n the expected manner. However, further reduction i n sodium did result 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 intr a c e l l u l a r anions might be responsible for depolarization (14) receives further support from the present studies. In 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 variation. In presenting 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 the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that 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 reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e . I t i s understood tha t 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 f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada. Date £ejj a L,,. /^fT TABLE OF CONTENTS ABSTRACT i LIST OF TABLES i i i LIST OF FIGURES. iv ACKNOWLEDGEMENTS v i I INTRODUCTION 1 II METHODS 3 I I I RESULTS .'10 IV DISCUSSION kl V SUMMARY 54 VI BIBLIOGRAPHY 56 VII BIOGRAPHICAL INFORMATION 5 ? i i i 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 Pregnant Cat Uterus . 11 II I Pregnant Rabbit Uterus ,, 21 IV Estrogen Treated Rat Uterus ........ 27 V Estrogen Treated Rabbit Uterus ... 33 Electrolyte Composition of Bathing Media, Tissues and Their Functional State VI Pregnant Cat Uterus , .., 36 VII Pregnant Rabbit Uterus 37 VIII Estrogen Treated Rat Uterus 38 IX Estrogen Treated Rabbit Uterus . . . . . . . . . . . 39 i v TABLE OF FIGURES FIGURE I Circuit diagram of A-C coupled Preamplifier .... 6 PREGNANT CAT UTERUS; - Specimen record of i n vi 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) .16 IV (c) Choline substituted sodium-poor medium (sodium concentration 18 mEq/l) 17 V 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 • 18 VI (a) Summarized record from another experiment i n Krebs Ringer (as a control) and i n choline substituted sodium-poor media with different sodium concentrations. 19 VI 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) 24 X Summarized records represented i n Figures VII, 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.».»...... «• 29 XIII(c) Choline substituted sodium-poor medium (sodium concentration 27 mEq/l).....•.»...• ......<,...»......... 30 31 XIV Summarized records represented i n Figures XI, XII and XIII* * XV ESTROGEN TREATED RABBIT UTERUS; - Specimen records of i n vi 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" V I ACKNOWLEDGMENTS The author wishes to express his profound appreciation to Dr. E.E, Daniel and Dr. J.G, Foulks for their keen interest, encouragement and helpful criticism during the course of investigations and the preparation of this report. Thanks are also due to Mr, P„P«, Singh of the Department of Physics U,B«C, Vancouver and Dr. G.E, Dower for the helpful discussions. Financial assistance provided by the Banting Research Foundation i also gratefully 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 similar increase i n the permeability of the membrane to potassium ions (2). Other sequential events i n the later phases have been described which permit the maintenance of e l e c t r i c a l neutrality, and bring about recovery of the resting membrane potential. (1,2,3,4). In many tissues, 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 potential, the rate of rise of the upstroke of the action potential, 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 results i n marked decreases i n the height of action potentials. In 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 positive phase of the injury potential (9). In some cases (5,6) the rate of rise 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 alteration i n the depolarization phase of action potential i n this 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). However, the response of some excitable tissues i s not i n accord with the hypothesis that sodium ions carry the depolarization membrane currents. Fatt and Katz (11) have reported an increase i n the amplitude of action potentials i n isolated crustacean muscle fibre when sodium chloride i n the perfusion medium was replaced bycholine chloride or certain 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 carriers of the action currents. However, re 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 rabbit and estrogen-treated rat. The uterine smooth muscle i n these species maintained i t s electr 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 strips 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 capillary 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 to 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 amplifier to drive the pen motor of a Sanborn-Twin-Viso Recorder, Amplifier tubes were heated by an electronically regulated direct current. (b) Solutions The various perfusion solutions employed consisted of: (1) Krebs Ringer - prepared by diluting 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$ freshly 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% MgS04, 1 part 10.55% K 2P0 4 (2) Sodium-free solutions - Due to lack of any suitable substitute for NaHCO^ , this salt was omitted i n the sodium free solution.^ Hence the solution was sl 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 or sucrose respectively. These solutions are referred to i n the text as Na-poor media. The composition of the three principle 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 rats were given 100 meg of estrogen per day for six days before the ut e r i were removed. (d) Analytical Methods: (1) For Perfusion Solutions: Na and K were determined by a method previously described for plasma using a Jahnke flame photometer and employing lithium nitrate as an internal standard (15,16). Chloride (Cl) was determined by direct t i t r a t i o n with Hg (NO^)^ using diphenylcarbazone solution as an indicator (17). (2) Tissues: The analytical procedures employed for tissues have been described i n detail elsewhere (15). Endometrium was scraped from the tissues chosen for electrolyte analysis. The tissue was carefully blotted dry of external solution, weighed immediately i n a previously weighed beaker, and carefully dried at 95-105° for five days. After re-weighing, the tissues were powdered and f i n a l l y digested with HNO^  (15). Chloride was determined by potentiometrie t i t r a t i o n with 0.01N AgNO^ after preliminary cold extraction of chloride with 0.1N HN03 as outlined by ¥hittam (18). *(Footnote from page 3)« Recently a suitable method of sodium bicarbonate substitution has been reported (28). TABLE I Krebs Ringer Choline Ringer Sucrose Ringi mM/L mM/L mM/L Na or Choline or Sucrose 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 125.1 137.4 11.5 HC03 21.9 0.0 0.0 1.16 1.25 1.25 so4 1.16 1.25 1.25 Glucose 49.2 52.9 52.9 - 6 -l-ffv II A X -7 1500 SR., _ Figure I , This figure shows the salient features of the preamplifier assembly. I t consists of (l ) AC-coupled 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 sensitivity of this system i s 1 mv = 4 cm. SW I i s the calibration switch, which gives 1 mv calibration 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 for fine 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 rapidly through the path of least resistance. (e) Calculations Electrolyte concentrations i n solutions were expressed as mEq/kg fresh weight of the tissue. 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 results re-calculated omitting that particular 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, variation i n amplitude''" i n Krebs Ringer was very large i n rat and rabbit uteri and less so i n the pregnant cat uterus. In rabbit and rat ut 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 this value, were taken for 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 similarly obtained under other conditions (low external sodium). This provided a uniform though arbitrary basis for a s t a t i s t i c a l evaluation of the characteristics of action potentials i n various external sodium concentrations. In sodium-poor media the amplitude of the action potentials was increased considerably. In pregnant cat uteri the variation i n amplitude during a series was much reduced whereas with uteri 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 deflection respectively, unless mentioned otherwise. average amplitude i s calculated for 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 far greater than that calculated from selected action potentials of near maximum amplitudes. This i s due to the fact 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 after reduction of the external sodium concentration. Variation i n the duration of action potentials was not significant and the original 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 variable, (g) Experimental Procedure Uteri were removed from peegnant cats and rabbits under barbiturate anesthesia (35-40 mg/kg), Estrogen treated rabbits and rats 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 isometrically 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 2 + 5% C0 2 mixture. Temperature control of the medium was achieved by pumping prewarmed water through the double walled medium bath. The temperature of the circulating 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 this 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 lef t in 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 in which the sodium concentration was further reduced. In some instances, the tissue was exposed to a medium completely free of sodium in the final step. After the final step, the tissue mounted for 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 in tissue electrolyte composition i n vitro. Unmounted uterine strips were kept i n various media which were warmed and aerated in the same fashion as the experimental tissues. These unmounted segments kept in 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 for 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 in 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 possibility of analysing unmounted tissue samples for each step i n the reduction of the external sodium concentration. - 10 -RESULTS (a) Pregnant Cat Uterus The results obtained from typical 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 particular series of repetitive action potentials some degree of variation 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 variation 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 typical 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 repetitive 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 slight 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 fact 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 typical biphasic action potentials then appeared. In fact the peak to peak amplitude of the action potentials i n this series was the highest that we have recorded (up to 5 mv. Table I I series 4 & Fig. 6). TABLE II PREGNANT CAT UTERUS No, Bath, Medium Basal p* Maximum Minimum Duration of Maximum Ampli- Maximum Rate of Duration of Minimum Tension Tension Time-IGm Contraction tude of Action Change of Action Action poten- Interval Gm Rise Tension sees. Potential Potential t i a l Between two Gm Rise H xx mv/' x: - .~mv/100 msecs. msecs. msec. successive  Action Potent,  msecsp l.A Krebs Ringer 4 11.5 60-65 35-40 y .B-Na-poor medium Na = 60 mEq/l 4 11.5 55-60 75-80 C-Na-poor medium Na = 18 mEq/l 4 11.5 50-55 90-95 2.A Krebs Ringer 4 11.6 110-115 57-60 B-Na-poor medium Na = 55 mEq/l 4 11.6 80-85 C-Na-poor medium Na = 19 mEq/l 4(+l)ll.6 65-70 very prolonged 0.8+ 0.01 (9) 1.2+ 0.92 (9) 1.0+ 0.02 (10) 2.6 ± 0.03 (9) 2.9 + 0.08~(9) 2.9 + 0.1 (9) 3V4 5.0 5.0 5.3 5.8 6.5 28-30 22-25 20-24 26-30 30-34 30-35 300-340 2400-2500 2400-2500 1225-1255 3500-4000 3700-4060 3.A Krebs Ringer 5 12.5 375 60-70 B-Na-poor medium/-^ Na = 25 mEq/l 5(+3)l2.5 250 30-35 4.A Krebs Ringer 6 Poor Contractions (less than 1 Gm) B-Na-poor medium/ Na = 23 mEq/l 6(+2)13.5 250 15-20 C-Na-poor medium/ Na = 18 mEq/l 6(+4)13.5 14-16 1.7 ± 0.6 (9) 2.3 + 0.14 (9) 9.6 8.3 Occasional action potentials of less than 0.2 mv amplitude 3.1 + 11V5 0.18 (12) 5.0 + 0.08 (9) 16.5 22.24 24-28 30-32 32-36 500-550 800-900 10,000-12,000 10,000-12,000 - 12 TABLE II (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 (in msecs) during which tension rise 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 rise as represented i n the tables 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 interval 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 results were obtained irrespective of whether choline chloride or sucrose was substituted for 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 re 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. The tissue then relaxed gradually but f a i l e d to contract again. At this stage the complete cessation of action potentials also occurred. A tendency for basal tension to he increased commonly was observed on exposure to moderately sodium-poor solutions, and this affect often became - 14 -more marked i n media with lower concentrations of sodium. The effect of , reduction i n the external sodium concentration on the rate of rise of contractile tension was rather variable, although the maximum rate remained f a i r l y constant, (b) Pregnant Rabbit Uterus The results 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 results obtained were qualitatively 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 did not always precede the contraction (unlike pregnant cat uterus) The amplitude of action potentials was increased i n sodium-poor media (Table IV and Fig. 10) (from 0.74 + 0.03 i n Krebs Ringer to 1.03 +0.07 i n Na-poor media). While the rate of change of potential was increased, i t s duration remained practically 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 this 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 rise 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 sli g h t l y decreased. - 1 5 -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 this 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 interval of maximum tension, the action potentials occurred at a frequency of about 2/sec. * In 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 this 70 second interval was the same as that i n the portion shown i n this figures (To follow page 16) - 16 ~ PREG- CAT UTERUS - 17 -PRE6 - 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. Final 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 different external sodium concentrations on the amplitude of action potentials i n the pregnant cat uterus. For effects of different 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 level under which the tissue was ori 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. - 19 -Figure 5a This record summarizes the effect of different external sodium concentrations on action potentials and contractions i n prggnant cat uterus from another experiment. Note the similarity to the records presented i n Figs. 2, 3, 4 and 5, ~ 2 0 PREG- CAT UTERUS IMV 3IMV Figure 6 - This figure shows results 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 this 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 (in the f i r s t portion on the l e f t ) and to 16 mEq/l (in the second portion of the figure). This record however, lacks the typical representation of effects of sodium depletion i n the external medium on the frequency of action potentials and the contractile responses of the tissue. TABLE I I I PREGNANT RABBIT UTERUS No. Bath. Basal Maximum Minimum Duration of Maximum Ampli- Maximum Rate of Duration of Minimum Medium Tension Tension Time-IGm Contration tude of Action Change of Action Action poten- Interval Gm. Rise Tension sees. Potential Potential t i a l Between two 7 RiseM xx mv x mv/lOO msecs. msecs. successive msec. Action Potent. msecs. Krebs Ringer 5 12.5 125 14-16 0.7 + 0.03 (9) 6.0 13.-16 75-100 B-Na-poor medium Na = 60 mEq/l X 5(+2)12.5 250 35-40 0.8 + 0.01 (7) 6.0 15-17 250-300 C-Na-poor medium Na = 29 mEq/l* 5(+6)12.5 90-120 1.0 + 0.07 (9) 8.0 17-21 300-350 x, xx, X, fi, fifi = See Table II - 22 -P R E G N A N T R A B B I T U T E R U S iiiiiji T 1 " • i i I i ... . .... 1 ! 1 1' ! '! i 1 1 1 ! 1 1 1 TJIj_.L_.L_T -.J....LLLU _ 1 : 1 1 T - T : : i : ; i : i ; i :]\^xxxxxx\:xxm I • . 1 j . L-.I-: 1 1 1 i { 1 T T ...  -!-]-!• i t i „!.„.!„„! j j i : i i i t i i I I I ! 1 M i l i i i i i i i ] i I I J T L L u J L I I I 1 1 T T T T 1 i "1 ••.!:;.-ill ; ili.i'.i-j 7jl:;.:;-. !-;-'j;:;:L:;j::: ' L I : :- ~:-.'-:lii==.-! _= \\-Xr-X HI-I^T; . ¥ s v— -> if _ „ . ! „ 1 *£_\ M 1 1 I l ! ! 1 i Lj^ i i i i i i i i !• i 1 1 1 1 i A > i I i I I i i 1 i I LI i 1 i l_Jn f i _ _ V | j ^ i m III LT l - ' l I' Iv1: J L- 1 •!'•' I T T i l l !:!;:; ^. -!• • — 1. . L . l . " " T T j " i i " T i »i i i i i i r i - i !• • i .-.i : :. : - j f —1- f .L.I. i _ L | _ ! 1 i 1 • I i I I I I i i i i i I T i t j j • ! 1 ; i i i i i ; i . . i : M l ! ! 1 ! 1 _]_ 1: 1 i ; i : ; i : : i . . i _ : ; i ; ; j ; i ; = i i ; ; ! l ; l l ! l i r -. _ . . . . . . . . i z I I | L 1 1 I . .! • ! 1 i i T_ \ ---| - - - - - ! "1 " T T l . l r r r .L..I.JIL I I ..... i ! j j i • " i T T T " r ' T T T T T T T i ' I 1 i I I l l l l i i j i | ! j I i ...... j . . . . L i M i l : i : i : i ~i i T i n " i " , r J....UT 1 1 1 I I I : : !• ! am T : : : I : ' H : < ! 1 I 1 I i M i l l I i ] " T T !-!. i 1 ! ! i 1 . ..... ., ]--!' I T ! ! T T T i 1 ; ! ! i 1 1 I i ; ; ] ; j j i j j""' i i ! i  ; i i ; ; • p - j — j " " | — j - - | ~ r — | — j — j — i 1 ! J 1 !_i i — i i — - - - r - — r — I 1 ;| •  i • I .1 L i i 1 M i l l ! !..!. 1 ! ! I I ! i i ; i i i i i i l l ! ! ! ! ! 1 i t ! "i i ! ; ; j P"'p ! ! 1 : i i i ; ! 1 i : ; t i - iliiH! - i - i I ! I i T L.l .! V i i i 1 i i i j. i-i i 1 1 1 1 J \,\ ...1. I - i . 1 • . i . L ! i . i i i .::::! i I 1 xm\,x i -! I-l I:-..!- i 1 ••! :•]-.- i -• 1 :• i:„i~ >• :i 1 1 j 1 1 1 1 ' 1 1 1 ' . K R E B ' S R I N G E R M E D I U M 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 rise of tension (note the similarity to cat uter i i n figure 2). - 23 -P R E G N A N T R A B B I T U T E R U S N a P O O R . C H O L I N E R I N 6 E R M E D . 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 (in the same preparation as that i l l u s t r a t e d i n Figure 7). Choline parti&ally 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), - 24 -PREGNANT R A B B I T Na. POOR; 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 (in 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, their unaltered frequency and the persistance of small wavy e l e c t r i c a l fluctuations (also 8). Though not very clearly seen i n the figure, basal contractile tension was increased from 5 to 11 gm during exposure to this medium. The maximum tension rise 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 - 25 -PREGNANT RABBIT U T E R U S Figure 10, This figure summarizes some of the typical features of the records presented i n figures 7, 8 and 9, Note the increased amplitude of action potentials and their unaltered frequency i n sodium-poor media. » 26 -(c) Estrogen-Treated Rat Uterus The results obtained from estrogen-treated rat uteri 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. The duration of action potentials remained almost unaffected. The maximum rate of change of potentials was variable i n different experiments, being increased i n some cases (series 1 and 2 i n Table VI), but decreased s l i g h t l y i n others (series 4 i n Table VI). The frequency of action potentials was not changed significantly i n sodium-poor solutions. In a given chain of repetitive action potentials variations were considerable and the frequency of spikes also was irregular (Figures 12-14)o Thus the properties of rat uteri 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 rat uterus, groups of action potentials sometimes occurred when the muscle had just started to relax. The exact significance of the relationship between the occurrence of action potentials and the phase of contraction i s not clear 0 Reduction i n the external sodium concentration produced very marked effects on the contractile function of this tissue. With each step i n lowering the external sodium concentration there was aI.tendency toward more and more prolonged contractions (lasting several minutes as compared with the usual 15-20 sees, i n Krebs Ringer Medium) and incomplete relaxation was common. 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 Basal Maximum Minimum Medium Tension Tension Time-IGm Gm 1 Rise Gm Rise msec« Duration of Maximum Ampli— Maximum Rate of Duration of Minimum  contraction tude of Action Change of Action Action poten-Interval  sees. Potential Potential t i a l Between two mv/lOO msecs. xx mv x msecs* successive  Action Potent, Msecs, 1. A Krebs Ringer 4 9.5 90-100 10-12 B-Na-poor mediumX4 11,0 500 35-45 (+3) V C-Na-poor medium 4 11,0 120 (+5.5) 2. A Krebs Ringer 4 11,0 300 15 B-Na-poor medium 4 12.0 400 50-52 25 mEq/L (+3) 2,0+ .08 (9) 1.9+ 0.04 (9) 2,2+ 0,07 (9) 1.6+ .06 (9) 2.1+ .13~(9) 9.7 9.8 11.1 12.5 16.0 15-17 18-20 20-22 20-24 25-30 350-375 275 280-295 385-295 380 In media of lower external sodium concentration, the muscle went into sustained concentration and the action potentials disappeared, 3.A Krebs Ringer 2.5 9.5 15 0,7+ 0.01 (9) B-Na-poor medi 2.5 9.5 300-1800 0.8+ 0.23 (9) 16-20 18-22 C Further muscles passes into sustained contraction with disappearance of AP's. Low Na Concentration. 4.A Krebs Ringer7^ 4 8 650 20 1,2 6.0 22-24 B-Na-poor medium7^ 28 mEq/l 4 9.5 300-1800 1.0 5.0 20-22 (44.5) # 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 . X/,X 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 their relation 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 potentials 0 (To follow page 28) - 28 -R A T U T E R U S i M ? i-•1 i :•!+!: 1', r - r n "IT i ! i j i : : : " i 1 1" i" : T i ! ! " ! " ! " [ i "i i !'[" ............ j ..p.j. ; ; | ; ; | "j" T ! ; ! j |~f-...... .j. ....j.... . | ^ j | |. , " • • .1 l ! j T T ZiZL 1 ~t_ _j_ r Ti i i n " i "l i i . . . ! i T NT 1 I i"l i T '  :\ !"""!""' "i 1 "j 1 ] i "i '" i M : "•"" - - ri r -\~-TT ::j:.n:: ..Lf.1.!'.'.!. ! ; . .. .1 J_i_j_:_!..!_i_ i M . M • T T mm I'M "I"'!'" - ! !••!•••!• ! - ! - : ! . J | | •! j I- ! •I" _U.... ...j_.j..M....j...j.jj.... -'-H-H-/ '7 .I..J..I.J. ! . l i t . mm i _ •yr -... - -1 ... . . . i rV / T i . . . i> T i ....... y- i - ~/ r r i i i i"/ nr ... ::1 i i|i: -i -:: - . . . . . . i i i i - . . . - i - - i i i i i i II-- i i fl - --i - . T I|/I if; iiii - •- - — iii ill! •|i ill l i l ' 1 -- F iiH HI IH _ _ _ — - -• — _ _ _ -- - •- - -.... - -... -M i l l i -1 i i 1 T I i "IT" I i. . _ ! _ . -1 I ! i i i" i r i i i r r M i l ' j' i "i - [ l ~i. "T I l_ "i _i_ . . . - E : - ~ _ "i ~ _..l ! i i i i' :~. * .... . . . 1 i i l 1 1 i 1 1 l 1 I I l I i i i" [ 1 A ! I I I ! II I . M M -\rn~r M M i :IH ~ l i I.., 1 . I. i 1 i i l 1 r n i i i r i i r 1 i 1 1 i I 1 .1. M l M i l l . ,,1-1::: a 7 ) 1 I : -I-I i U'!"! 1 1 ! L- 1 I j 1 i i i i i J i i 1 i I 1 1 1 1 1 i 1 1 -Iii 411 I v 1 1 ? 1 i ! i. I i r I I II 1 i i i i i i i i 1 1 1 1 1 1 1 I- 1 iH 411 iii- i i i i 1 n II I I i i I I M i l l M i l l 1 1 1 1 j ! l i 1 1 1 i l l: % T i n i/ 1 t i 1 I :•_! nr 1 1 1 1 i 1 l i l 1 m m P w i 1 1 \ — ' / / r J f V r m f iiii iii 1 101 iii iii; ii |i 1 .-. ||f ;:[; : l 1 1 I III I liii r~; iir i = L i!if =? liii 1 -M == :S iiii i 1 ' • i iii Kr! ~\- 1 1 i I 1 1 1 i i i 4 1 I-. 7~ :iii ! i-1 _ i 1 l l l l i ' i 1 M l Irj M i l l •I 1 I _ t _ i 1 l i i l l i i •! w -1 1 1 in L~ iiii ,i..|,. i T i i i 1 I i i 1 i i" 1 i i i I I 1 1 :Mii;;M 1 • ; 4 UU i i i i i ! 1 1 I I 1 1 1 i ; 1 1 1 1 1 1 1 1 • :: :IT ::-|:-ii|i-:: .if- i|i|i|:i iiiiiiii iiiiiiii €1 M l 1 1 1 M M . 1 1 1 1 i 1 1 1 1 • 1 : , .il.ii M M I I 1 1 1 . 1 I I- II I I I I I 1 1 pH"' : 1 1 1 1 1 1 1 {-' I I 1 1 1 1 1 1 1 i i i 1 1 1 — / >* -- 1 i 1 1 1 1 1 i ' i 1 -1 1 1 1 = ~i 1 - HI! mi ; Ei iiii l ~: ll- f ¥ ,;• !!! I I. 11 Ij! ![. 1 lii- i i r = H i s iir Hi ifr == i l HI" HI i-L i i - ~ ••I'- IH H= V|ii: i , iiii 1 HL ii~ HI? | . 1 1 1 1 I ii HI li-i • 111 I I • 1 i 1 1 1 1 " 1 : : , i : -p: a? H nr •:| .1... 1 1 1 1 i 1 1 ! 1 :• |.,:|L .1 1 • :|' ; .•liii iiiiiiii iiiiP ~ 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 this 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 significantly i n this medium as compared to the control i n Krebs Ringer (see figure 11, and compare with figure 3 and 4). - 30 -R A T U 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 thai represented i n figures 12 and 13 during further lowering of the external concentration of sodium. The sodium concentration of this 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 uteri under similar external sodium concentrations (figures 3 and 4). - 31 -R A T U T E R U IMV 12 IMS i l i i hi: it!: IIII ill ii i l i i i i l i i i i il! ii: I i i i Hi: iii: lli III! IIII i i i ! ii; II i l l l i ' P i iii; ill: I i i i iiii iiii i i i l i i i ! i ; i ! iii tl i i i ! ! ! ! ! ! i l l i i i ii!! :::: till i i i ! I i i i !>'' i l i i It! lijthiiiMl: ilhhill hu I i ! ::•: i:!i iiil III! III! imi f l i i i i i i iiilhiiilii ; I!! : f\ I i i i 1 in: TTTT i f _ ill: IIII mi l / i i: i i i i i j i i j i i i i i ! i i i ! ii'.i iii iiii Iiii i! i i i i iiii ;:; i i i i i iiiii II i i i i i i ! iiiiliiii j i i i i i i i i ! iiii III! III! i i l i ' i ! i i i i i Iii! i i i i i i i i i i i i i i i III i l ! i l l ! III! H i ! im iijii i ! ~\ i i i i i i i i i i i I i i I .! ii !!!! 1 l l l i I I i 1 i i i i i i ! i i i i iii i i i i i i i i i i i 1 I i i! III! i i ! ! i i ' i IS!! •ii i i t i i i i i i i ! i i i i i i i i i i 1 i i I I II i i i II I I I i i i i :i|i t; i ;:t: i l i i i i i i i i i i i i i i i i i i i i i ! Ii ! 1 Ii il ii i i i i l l ! !!!! Ilii i l i i i l ! i l l ! H m i Hi) in i i i i l i i i i l i i i 11 i I m Hi II i ii nit I'M :::: :: : :::: ii i i i i i i ! ! i i i ! i i i i i i i l ! i i i i 1 i i i l I i i ! i i I I I III! i i i l l ! i i i ! I i i i l l l i i i i i ! j i l l I III! mi in: i i ! i i i ! iiil I I .,., .. i i i i i i i i i i i i i i i i i ! i i i ! ! ! i i i I i ! i l l ! i l l ! i i i i i i ! Iiii iiii i! i Iiii i i i H i ! i i i ! i i i ! Iii I i i i i ! III! i i ! I i i ! iljj i i i I l i i i i i i i i i ! ! l l i i i i i i i i i i i I l i i i l l ! i i i ! 1!! 1!!! 1 ! i i III! III! iiti !!!! i i i i II i i i i i m i l l ! im in: III! If! Iiiiii IIII mi i l l I'M! Iiii I ' » H i ! iiii iir, :t:: i i i i i KREB'S \ : : : : i i i i i i i i ! :: v. 1 ffi i tttf i i ' !ii,ii • • |; i iii. , : : : : '.ll-li: II! i'. •i1 <: i I' i i " .1 i i i l i i i i i i l l ! I i i l I i i i i iii! iii! Aii - (!ii 1 i * ii;; •s ilii iii! .... 3111 r ' ' :v . i:: ' 11 III :: r" :::¥ iiii iii^ Iiiii i.... 'iiil i v , i V j . ' ! l , i : Iiiii irir ill: Ilii ill! Na = 27**^ L .Figure 14: 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 (in 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 uteri of the same species. The frequency of action potentials was almost unaltered. 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 persist for 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 this behaviour represents another facet of the same paralytic phenomenon which i s seen i n cat uteri i n very low concentrations of external sodium. However, cat ute 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 rat uteri 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 relaxation of this type of contracture.) 1. Some of the inhibitory sympathomimetic amines also f a i l e d to bring about relaxation, (d) Estrogen-Treated Rabbit Results obtained with estrogen-treated rabbit ute r i are summarized i n Table V. Specimen recordings are presented i n Figure IS"* Only two experiments were carried out. The results obtained were qualitatively 1 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 a c t i v i t y . TABLE V ESTROGEN TREATED RABBIT UTERUS No, Bath Basal Maximum Minimum Duration of Maximum Ampli- Maximum Rate of Duration of Minimum Medium Tension Tension Jjme-lGm Contraction tude of Action Change of Action Action poten- Interval Gm Rise Tension sees. Potential Potential t i a l Between two 7" Ji5} Riseffy^ xx rav > mv/lOO""msecs. msecs, successive msec. Action Potent, msecs. A Krebs Ringer 6 7 B Na-poor medium 6 9 (2.5) C Na-poor medium 6 12 (5.5) 1000 7-8 _ 6-7 — 6-7 0,3 + 0.02 (9) 0.3 + .01 (10) 0.4 + 0.03 (9) 2.0 1.7 2.4 16-18 18-20 18-22 450-500 600-650 650-700 A Krebs Ringer 5 11.5 700 20-25 (+2) B Na-poor medium 6 11.0 1000 (+4) 0.3 + 0.01 (10) 0.4 + 0.03'(9) 2.2 2.7 18-20 18^22 Irregular 600-650 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 fact, earlier attempts to record action potentials from this 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 relaxation with each decrease i n the external sodium concentration, resembling rat uteri 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 Electrolytes Tables VI, VII, VIII and IX summarize the electrolyte analyses of the tissues, 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 lost 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 inactive, electrolyte analysis revealed a tissue sodium concentration which was not significantly 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 table. 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, their unaltered frequency, and the persistance of small wavy el 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 Electrolyte Composition of Electrolyte Composition of state media mEq/l Tissues mEq/kg G/Kg Na ^ CI fresh weight fresh weight Na K CI H-O Direct Control — —— — — 86.7 51.2 82.0 806 II — — 85.8 68.8 82.8 820 Krebs Ringer Active 151.2 5.0 122.5 127.8 51.3 95.2 818 •H n i t 138.5 4.8 128.0 92.2 56.7 101.5 814 P a r t i a l Cho-line Ringer Active 60.0 5.5 137.5 79.1 51.2 78.8 834 i t i t 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 9.5 112.2 834 100# Choline Ringer Inactive 6.0 5.0 123.0 26.2 51.2 95.7 II t i II 3.0 5.5 130.6 19.8 19.0 90.0 837 P a r t i a l Sucrose Ringer Active 70.0 5.1 88.4 44.2 33.3 44.9 II i i 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 Sucrose Ringer Inactive 0.2 5.7 8.2 17.5 25.5 21.8 818 II II II 1.0 5.8 10.9 18.9 43.5 25.0 760 Left i n Isotonic K 2 S 0 4 0 167.0 0 12.5 174.5 10.2 845 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 text). 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 different functional states of the tissue. -Treatment Functional* Electrolyte Composition of Electrolyte Composition of state media mEq/l Tissues m E q / k g G / K g fresh weight fresh weight Na K CI Na K CI H-O^  Direct Control — (148) (4.5) (110) 71.27 66.35 53.73 817 II II — — — 81.04 29.89 78.94 837 A Krebs Ringer Active 138.5 4.8 128.07 90.22 31.86 117.80 827 B Choline Ringer Active 60.0 4.8 131.57 — -C Choline Ringer Active 29 5.8 127.63 31.74 30.30 94.50 798 * See Table VI 38 -TABLE VIII  Estrogen Treated Rat Uterus Electrolyte analysis of tissues and corresponding perfusion solutions at different functional states of the tissues Treatment * Functional Electrolyte Composition of Electrolyte Composition of state media Na MEq/l K CI Tissues mEq/kg fresh weight Na K G/Kg fresh weight CI H 2 0 4 Control — 74.5 4 3 . 0 68 .3 798 Krebs Ringer Active 136.5 5 .0 130.1 105.1 40.5 1 86 .6 804 Part Choline Ringer Active 4 3 . 0 5.4 126.3 40 .6 2 1 . 0 87 .7 818 11 i t i t 30.2 5 .7 125.6 43 .1 54.4 7 8 . 9 818 n I I + 25 .0 5.2 126.3 19.5 35 .1 103.3 783 10($ Choline Ringer / Inactive 1.2 5.8, 123.0 19.0 43 .2 7 4 . 7 809 n I I n 4 . 0 7 .0 122.0 22.8 61 .2 99.5 818 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 i t i t 2 . 0 6.5 9 . 7 15 .7 20 .9 16.5 806 I I t i 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 indefinitely prolonged contraction and eventual arrest of action potentials. 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 Electrolyte Composition of State media mEq/l Tissues mBq/kg G/Kg fresh weight Na K CI Na K CI -2^4 Krebs Ringer Active 138.5 4.8 128.0 75.8 62.3 72.1 864 w II II 140.5 5.0 130.2 81.3 66.3 76.9 866 Choline Ringer Active 31.5 5.4 122.8 28.2 56.5 75.0 871 it II II 29.0 5.4 125.4 24.5 60.3 77.4 860 Choline Ringer Inactive 1.0 5.3 112.0 29.9 61.1 52.8 850 Left i n 0 0 167.0 22.7 44.4 101.1 860 Choline Chloride * See Table VI - 40 different from that which was found i n functionally 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 this 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 uteri was very striking. 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 levels of tissue potassium i n sodium-poor media were never as low as i n the cat although a decrease sometimes was noticed. The alteration i n tissue chloride i n sodium-poor media was not significant when choline chloride was used to replace sodium chloride. However, tissue chloride was decreased i n sodium-poor media employing sucrose as a substitute for sodium chloride. The decrease i n tissue chloride i n the lat t e r case probably was due primarily to the decrease i n chloride concentration i n the extracellular 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 th i s laboratory, attempts to apply this 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 failure of microelectrodes to penetrate the cel l s without appreciable damage. However, action potentials dould be recorded with ease from such tissues using extracellular surface electrodes. The limitations 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 extracellular electrodes have been made by other workers (20) from isolated single fibres of skeletal muscle. A comparison of the simultaneous biphasic extracellular action potentials and monophasic intracellular action potentials showed that the positive peak of the biphasic action potentials occurred at the same time as the onset of the ris i n g phase of the monophasic action potential, 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 extracellular action potentials thus corresponds i n time with the upstroke of monophasic action potentials. However, the precise relationship between transmembrane potentials and extracellular recordings made from multicellular 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 multicellular units which have l i t t l e effect on in 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 different from that of a distant electrode because the two electrodes are at different isopotential lines emanating from the e l e c t r i c a l source and sink* In practice the distant electrode can usually be regarded as at zero potentials In these studies alterations i n the position of the distant electrode did not noticeably alter the records obtained, so that i t could be effectively regarded as indifferent. A recordable change exists 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 different electrode could be regarded as recording only from the c e l l s which were very close to i t s t i p . 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 extracellularly recorded action potentials related to the rate of change of the slope of the transmembrane potential curve. In multicellular units, extracellularly 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 rapidity with which these c e l l s are activated and by sequential pattern of activation 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 su f f i c i e n t l y close together (synchronously) and i n proper sequence, the sum of their potentials w i l l allow the recording of larger extracellular action potential. With - 43 -relat i v e l y asynchronous or nonsequential activation, potential produced i n different c e l l s may partly 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 extracellularly. Many factors can increase the relative 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 ac t i v i t y  over the entire multicellular area from which the electrode records 8 A fast rate of spread of activation w i l l increase the degree of synchrony of activation of fibres, 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 regularity 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 extracellular potentials may result. 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 extracellularly 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 relative 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 directly related to the recovery of the transmembrane potential following an action potential, 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 repolarization might increase the relative number of ce l l s capable of responding during each of a series of action potentials provided that the time interval between each action potential remains constant* Similarly an increase i n the time interval 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 their 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 indirectly influence the recorded amplitude of extracellular action potentials (by effecting the synchrony of activation of the cells) the change i n the resistance between the electrode and the medium might also influence the amplitude of extracellularly 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 affect 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 significantly when sodium chloride i n the medium i s replaced by another electrolyte, e.g. choline chloride* However, the resistance might increase when a sucrose containing medium i s used* The implications of these principles are applicable to the observed changes and are discussed i n further detail i n the following sections* ~ 45 -l i s 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 sli g h t l y increased or appeared unchanged and the original 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 inactivity) 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 uteri of other species the decrease i n frequency i n sodium-poor media was not marked. The variation i n amplitude between individual members of a series of action potentials was quite large i n Krebs Ringer, In 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 uteri 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 extracellularly recorded action potentials i n sodium-poor media must be due to increased synchrony of response of multicellular areas and/ or altered pattern of spread of activation. In a given series of repetitive 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 ut 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 activation of c e l l s i n such a way as to produce action potentials which were of rel 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 sodium-poor 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 find a variable number of c e l l s which were completely excitable). In choline chloride sodium-poor media the slight change i n conductivity between medium and electrode presumably did not play a significant role i n modifying the amplitude of action potentials. However, i n sucrose containing media the increased resistance could have been sufficient 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 similarity 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. — -I I I , The implications of current observations for 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), If 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 extracellularly persist at remarkably low external sodium concentrations. The possible reconciliation of these findings with the "Sodium Hypothesis" and an explanation of the relative 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 extracellular  sodium i n the tissue The available evidence i s not i n harmony with this explanation for the relative independence of this presence and amplitude of action potentials and the external sodium concentration. I f equilibration between the medium and the extracellular 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 equilibration (retention of extracellular 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 significantly 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 sa t i s f a c t o r i l y u n t i l suitable direct methods are used, ( 2 ) Maintenance of Constant Ratio Between the cellular and extracellular  concentrations of sodium (Na^/Na-) i n various concentrations of  external sodium. This explanation of the relative independence of the magnitude of action potentials and the external sodium concentration also appears implausible. Maintenance of a fixed ratio Nat /Na.. at various external sodium concentrations o x would only be possible i f tissue sodium losses from outside and inside of the cel l s occurred at a constantly proportionate rate. The available evidence indicates (15) that extrusion of cellular sodium i n sodium-free media i s met with certain barriers tending to hinder the cellular sodium loss. Sodium loss i s rapid from the extracellular space. Thus, the ratio 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 extracellular action potentials might have been expected. However, no decreases i n transmembrane action potentials were observed i n low external sodium solutions (14.) and extracellularly recorded potentials actually manifested an increase i n their 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 "carrier system" which i s made - 49 -available by excitatory depolarizing processes, permitting an inward current to be carried by sodium ions moving along their 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 carrier 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 this hypothesis as an explanation of the relative independence of the presence, amplitude and pattern of action potentials and the external sodium concentration. Selective sodium currents do not appear adequate to account for 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 result 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 results (14), Under these conditions, an hypothesis based on an outward flow of intracellular 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 El e c t r i c a l Inactivity 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 ele 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 ac t i v i t y was affected i n some important way i n suf f i c i e n t l y sodium-poor media. 50 Sudden Contraction, gradual relaxation and resumption of spontaneous activity-occurred 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 failure to resume spontaneous a c t i v i t y . The action potentials disappeared at this stage. 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 inhibited at extremely low levels 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 their re-synthesis i s a matter of speculation. I f the break-down of certain high-energy phosphate bonds were inhibited, or i f their 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 in h i b i t i o n i n the site of these energy sources (during the cyclic 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 different 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 inhibition 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 . If e l e c t r i c a l activation i s necessary to e l i c i t contraction, then mechanical in 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 directly affects the el 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 Distribution After Exposure to Low Sodium  Solutions: Quantitatively, the distribution of uterine tissue potassium following immersion i n sodium-poor media was inconsistent, as was noted earlier for brain slices (29). Tissue potassium tended to be lost more rapidly i n sodium-poor media, although the extent of the loss was variable. Greater quantities of potassium were lost 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). In pregnant cat uteri tissue potassium concentrations as low as 4-5 mEq/kg fresh weight were obtained i n some cases (Table VI). Evidently potassium gradients across the Cellular 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 distribution i n smooth muscle and i t s relationship to resting and action potentials. I t was of ihterest to determine i f loss of el 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. If potassium were freely diffusable across the c e l l membrane and at equilibrium, i t s distribution 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 extracellular 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 this value for the ECS was applied to the experimental pieces to calculate their Kc. This i n turn was used to calculate the potassium equilibrium potentials, which are close to the resting transmembrane potential i n these cellw. These values indicated that neither the level of tissue potassium nor the magnitude of the resting membrane potentials calculated from potassium distribution have any direct relationship to the presence or absence of el 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 uteri with tissue potassium levels close to their controls became inactive i n low external sodium concentrations while other uteri with a much lower tissue potassium concentration under similar conditions remained active (Table VI-IX). Moreover, no relationship seemed to exist between the degree of cellular potassium depletion i n the u t e r i of various species studied and the two different types of mechanical inactivity, which. were observed at extremely low levels of external sodium. In the potassium depleted c e l l s , e l e c t r i c a l neutrality 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 intracellular anions could maintain the ionic 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 levels. While the p o s s i b i l i t y of calcium and/or magnesium uptake to offset cellular potassium loss cannot be ruled out, the small concentrations i n which these ions exist i n tissueis suggest that1 thes'e : - 53 -ions probably are not involved i n an exchange for lost cel l u l a r potassium. The loss of intracellular anions accompanying potassium loss would maintain e l e c t r i c a l balance but would considerably decrease the intr a c e l l u l a r osmolarity. Under these conditions water must be lost from within the c e l l . If so, the ratio E.C.S./l.C.S. might change under different conditions. However, the answer to these problems must be awaited t i l l further evidence bearing upon these questions i s available. Values for 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 levels decreased (Table VI-IX). Such losses probably could be accounted for by decreased chloride i n the extracellular space. However, the estimated chloride space i n sodium-poor media containing sucrose were enlarged, indicating that some of the chloride probably was bound within the tissue or equilibrated very slowly with the external medium. - 54 -SUMMARY 1. Extracellular 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 rat. 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 uteri but remained almost unchanged i n the uteri of other species. 3. In. sufficiently 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 (15-20 mEq/l). 4. 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 uteri mechanical i n a c t i v i t y occurred when the uteri 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 - 55 (Summary (c ont * d*) 5» 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 characteristics of action potentials i n the expected manner. 6. The view that an outward flow of intracellular anions might be responsible for 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 intra and inter species variations are discussed. 8. I t i s suggested that the eventual i n a c t i v i t y (electrical as well as two different 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. 2. Keynes, R.D. 3 0 Hodgkin, A.L. and Huxley, A.F. 4. Desmedt, J.E. 5. Draper, M.H. and Weidman, S. 6. Nastuk, W.L. and Hodgkin, A.L, 7, Huxley, A.F, and Stampfli, R« 8. Hodgkin, A.L, and Katz, B. 9. Cranefield, P,F., Eyster, J.A.E., and Gilson, E. 9a. Cranefield, P.S., and Hofman, B.F. 10? Katz, B. 11. Fatt, P., and Katz, B, 12. Wood, D.W., 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. The Ionic Movements During Nervous A c t i v i t y . J. Physiol. 114: 119-150, 1951. Currents Carried by Sodium and Potassium Ions Through the Membrane of Giant Axon of Loligo J . Physiol. 116: 449-472, 1952. El e c t r i c a l A c t i v i t y and Intracellular Sodium Concentrations i n Frog Muscle. J . Physiol. 121: 191, 1953. Cardiac Resting and Action Potential, Recorded with an Intracellular Electrode. J . Physiol. 115: 74-93, 1951. 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. Effect of Potassium and Sodium on Resting and Action Potential of. Single Myelinated Nerve Fibre. J . Physiol. 112: 496-508, 1951. The Effect of Sodium on El 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. Effect of Reduction of External NaCl on Injury Potential of Cardiac Muscle. Am. J . Physiol. 166: 269-272, 1951. Electrophysiology of Single Cardiac Cells. Physiol. Rev. 38: 41-77, 1958. The Effect of Electrolyte Deficiencies on the Rate of Conduction i n Single Nerve Fibre. J . Physiol. 106: 411 et seq, 1947. The E l e c t r i c a l Properties of Crustacean Muscle Fibre. J. Physiol. 120: 171-204, 1953. The Effect of Ions upon Neuromuscular Transmission i n a Herbivorous Insect. J . Physiol. 138: 119-139, 1957. - 5 7 -Bibliography (Cont'd) 13* Holman, M.E, 146 Daniel, E. and Singh, EV 15. Daniel, E., and Daniel, B. 16. Spencer, A.G. 17. Asper, S.P., and Schales, S.C. 18, Whittam, R. 19. Manery, J.F. 20. Hakansson, C.H. 21. Fulton, J.F. 22. Daniel, E.E. 23. Lorente De No 24. Grundfest, H. 25. Born, G.V.R. 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. The E l e c t r i c a l Properties of Smooth Muscle Cell Membrane Can. J . Biochem. & Physiol. 36: 959-975, 1958. Effects of Ovarian Hormones on the Content and Distribution of Intact and Extracted Rabbit and Cat Uteri. Can. J . Biochem. & Physiol. 35: 1205-1223, 1957. Flame Photometry The Lancet, November, 623, 1950. A Simple and Accurate Method for Determination of Chloride i n Biological Fluids, J. B i o l . Chem. 140: 879, 1941, A Convenient Micro-method for Estimation of Tissue Chloride J.., Physiol. 128: 65 P, 1955. Water and Electrolyte Metabolism. Physiol. Rev. 34: 335-417, 1954. Action Potential Recorded Intracellularly and Extracellularly from isolated frog muscle fibre i n Ringer Solution and A i r . Acta. Physiol. Scand. 39: 291-312, 1957. Text Book of Physiol. 17th Edition, 519 et seq, 1957. Unpublished results Department of Pharmacology, U.B.C. Vancouver A Study of Nerve Physiology, Stud. Rockefeller Institue of Medical Research 132: 394-477, 1947o E l e c t r i c a l Inexcitability of Synapses and Some Consequences i n Central Nervous System. Physiol. Rev. 37: 337, 1957. The Relation Between the Tension and the High Energy Phosphate Content of Smooth Muscle. J . Physiol. 131: 704-711, 1956. - 58 -26. Perry, S.V., Relation Between Chemical and Contractile Function and Structure of Skeletal Muscle Ce l l Physiol. Rev. 36: 1, 1956. 27. F r i t z , B., Svensmark, A, and Rosenfalck, P. 28. SzentrGyorgyi, H. Mechanical and Chemical Events i n Muscle Contraction. Physiol. Rev. 36: (4) 503, 1956. Chemical Physiology of Contraction i n Body and Heart Muscle. 2nd Edition, Academic Press, N.Y., 1951. 29. Pappius, H.M,, Effects of Sodium-free Media Upon the Rosenfeld M., Metabolism and the Potassium and Water Johnson, D.M., and Content of Brain Slices. E l l i o t , K.A.C. Can. J . Biochem. & Physiol. 36: 217-226, 1958. 30. Daniel, E.E. Unpublished Results. Department of Pharmacology, U.B.C. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0106239/manifest

Comment

Related Items