zation. since it is generally considered that a membrane potential

474

J. Physiol. (I958) I43, 474-485

EFFECTS OF DRUGS ON DEPOLARIZED PLAIN MUSCLE

By D. H. L. EVANS, H. 0. SCHILD AND S. THESLEFFFrom the Departments ofAnatomy, Pharmacology and Biophysics,

University College London

(Received 20 March 1958)

It has been briefly reported (Evans & Schild, 1957) that mammalian plainmuscle retains its ability to contract in response to drugs when suspended inRinger's solution in which the sodium ions have been replaced by potassiumions. This observation suggested that drugs can affect the contractile elementsof plain muscle by a mechanism which is not mediated by membrane depolari-zation. Since it is generally considered that a membrane potential change is anessential step in setting off the contractile process both in striated (Huxley,1957) and smooth (Csapo, 1954) muscle, these findings were considered towarrant further study. In this paper the effects upon their reactions to drugsof immersing smooth muscles in 'potassium-Ringer' are now described morefully, and their responses to electrical and to mechanical stimulation are alsoreported. To test whether smooth muscle cells are in fact completely depolar-ized by the external application of potassium, membrane potentials havebeen recorded with intracellular micro-electrodes.

METHODS

Isolated plain-muscle preparations were used from chick amnion, rat uterus, guinea-pig ileum,longitudinal strip of cat intestine and retractor ofthe byssus of Mytilus. The chick amnion prepara-tions were obtained on the 12th-15th day of incubation. Thin threads were tied through thecranial and caudal ends of the amniotic membrane, which was then cut out and suspended as astrip in a bath. Rat uteri were obtained from animals which had received an injection of 0-1 mg/kg stilboestrol on the previous day. The longitudinal strip of cat intestine was prepared as.described by Evans & Schild (1953).Normal 'NaCl-Ringer' solution of the following percentage composition was used: NaCl 0-9, KCI

0-042, CaCl2 0-012 (MgCl2 0.02), NaHCO3 0-03, glucose 0-1. In the 'potassium-Ringer' solutionsNaHCO3 was replaced by KHCO3 0-36, and NaCl was replaced as follows: In 'KCl-Ringer', byKCI 1.25; in 'K2SO4-Ringer' by K2SO4 2-2; in 'K2SO4-Na2SO4-Ringer' by K2SO4 1-38 +Na2S04(anhyd.) 0-82. The concentration of K2SO4 in 'K2SO4-Ringer' was computed from Landolt-Bornstein tables to be equi-osmotic with 0-9 NaCl. Since this gives an excess of extracellularpotassium ions we used the 'K2S04-Na2SO4' solution in some experiments with unchanged results.MgCl2 was used only in the later experiments.

DRUGS ON DEPOLARIZED PLAIN MUSCLEThe temperature was controlled by means of a jacketed isolated-organ bath. For isotonic

recording light frontal-writing levers were used. For recording tension a spring was used con-sisting of a flat steel strip. The deflexion of the spring was recorded either through a linear dif-ferential transformer and ink recorder (Evershed & Vignoles Ltd.) or simply by attaching a lightlever to the spring and recording on a smoked drum. The shortening due to deflexion of the springwas about 5% of the greatest isotonic shortening. At the beginning of each tension recording themuscle was gently stretched until a definite tension increment was seen to occur; the stretch wasthen slightly diminished to give zero tension.For electrical stimulation a spindle-shaped bath was used having circular platinum-black

electrodes at each end. Trains of impulses at 50 c/s were administered, usually of 0 5 sec durationat 1-15 V/cm.The resting membrane potential of the amnion was recorded by conventional capillary micro-

electrodes filled with 3 m-KCI. A freshly dissected chick amnion preparation of 11-13 days ofage was mounted in a transparent Perspex dish and the preparation was viewed with a binoculardissection microscope. Attempts were made to insert the tip of the micro-electrode in those regionsof the preparation in which, as described by Pierce (1933), smooth muscle fibres could be observedto radiate from discrete foci. The temperature of the bath was kept at 25-30o C and oxygenationwas effected by intermittent bubbling of a 95% O2-5% CO2 gas mixture through the fluid.

RESULTS

Effects on smooth muscle of immersion in 'potassium-Ringer'When a smooth muscle preparation is placed in Ringer's solution in whichthe NaCl has been completely replaced by KCl or K2SO4 it contracts immedi-ately, but the contraction is not maintained. The shortening remains maximalfor a few seconds and is followed by relaxation, although the preparationremains immersed in the potassium solution. This initial contraction and thesubsequent relaxation in potassium-Ringer has been observed in smooth-muscle preparations of chick amnion, rat uterus, separated strips of catileum longitudinal muscle, guinea-pig and rabbit ileum and in the retractorof the byssus of Mytilus.The extent of both the contraction and relaxation, but particularly of the

relaxation, depends on the temperature. As shown in Fig. 1 for the rat uterusthe extent of contraction is decreased, and that of relaxation increased, bylowering the bath temperature. At 350 C the contraction was near maximumand was followed by only a small and transient relaxation. At lower tempera-

. tures the contraction became progressively less and the relaxation morecomplete.Rhythmic activity invariably disappeared when smooth muscle prepara-

tions were immersed in potassium-Ringer. This occurred over the wholerange of temperatures (9-38° C) at which the investigations were carried out.

Resting membrane potential of smooth muscle cells in potassium-RingerThe purpose of these experiments was to investigate the changes in mem-

brane potential of smooth muscle when the preparation was transferred fromsodium- to potassium-Ringer. Of the various muscle preparations used, the

475

476 D. H. L. EVANS, H. 0. SCHILD AND S. THESLEFF

chick amnion proved to be the most suitable for intracellular recording. Theamnion also has the advantage of being a thin sheet of smooth muscle andendothelial cells in which diffusion equilibrium can be expected to be rapidlyreached.

't 0 .t 10min

Fig. 1. Rat uterus. Isotonic recording. At the arrows NaCi-Ringer was replaced by K2SO4-Ringer. Consecutive records at about 30 min intervals with intervening 50 C drops intemperature. Note increase in relaxation phase at lower temperatures.

mv20

- 40- 6080

._l-_.___4

- 20- 40>j60 E_ 80

0

10 5 6NaCI-Ringer sec

b

S20E 20 E

10 5 0K2SO4-Ringer sec

Fig. 2. Intracellular recordings from isolated chick amnion in NaCl-Ringer (a) and K,SO4-Ringer (b); negative deflexion downward; records read from right to left.

In experiments on seven preparations membrane potentials could berecorded from amnions immersed in sodium-Ringer. In about 40% of at-tempts (57 successes out of 138 attempts) membrane potentials of 30-70 mVwere recorded. As a criterion of an intracellular insertion a sudden negativepotential change was taken, which persisted, and reverted to zero upon with-drawal of the micro-electrode (Fig. 2a). In K2SO4-Ringer it was not possibleto record any definite resting potentials although in some instances the prod-

DRUGS ON DEPOLARIZED PLAIN MUSCLEding with the micro-electrode gave rise to positive or negative potentials,presumably due to resistance changes at the tip of the electrode (Fig. 2b).As an illustration of the procedure used, in one of the experiments in

sodium-Ringer, ten resting potentials (negative inside) were recorded out ofnineteen trials with a mean of 51 + 4 (s.E.) mV. After changing to potassium-Ringer definite membrane potentials could not be recorded in sixteen trials.In another experiment no membrane potentials were recorded in the courseof thirteen trials in potassium-Ringer, but after reverting to sodium-Ringerresting potentials were recorded in ten out of thirty-one trials with a mean of39 + 2 (S.E.) mV.

Effects of stimulant drugs on depolarized smooth muscleAll the smooth-muscle preparations tested, with the exception ofthe Mytilus

muscle, contracted in response to acetylcholine after immersion in potassiumRinger. The contractions were sustained as long as the acetylcholine re-mained in the bath and were fully reversible when the drug was removed. Theywere graded according to dose and could be obtained repeatedly over a periodof several hours without deterioration. They were best seen in preparationswhich had relaxed to a considerable extent in potassium-Ringer, either spon-taneously or after the administration of isoprenaline (isopropylnoradrenaline).Examples of graded acetylcholine contractions as recorded with an isotonic

lever in preparations suspended in potassium-Ringer solutions are shown inFigs. 3 and 4. In the experiment of Fig. 3 on a chick amnion preparationgraded responses were recorded in sodium-Ringer and at a later stage verysimilar responses were obtained in potassium-Ringer. The record in Fig. 4shows the contractions of a longitudinal muscle strip of cat intestine. In thiscase graded, acetylcholine-induced, contractions were first recorded in potas-sium-Ringer and afterwards in sodium-Ringer. The main difference betweenthe two records is the raised base line in the potassium solution, but therange of effective concentrations of acetylcholine in the two solutions is thesame.The contractions produced by acetylcholine in potassium-Ringer were

always slow and sustained. Preparations such as the rat uterus and chickamnion, which normally respond to acetylcholine with a series of rapid inter-mittent contractions, gave only a slow type of response, usually of less ampli-tude, in potassium-Ringer. When these preparations were tested at low tem-peratures, below about 280 C, they gave slow responses even in sodium-Ringerand in that case the general character of the response in the two solutions wassimilar.

Fig. 5 shows the tension developed by the rat uterus immersed in K2S04-Ringer after application of graded doses of acetylcholine. It is seen thatgraded responses were obtained both in sodium- and in potassium-Ringer but

477


I - NaCI-Ringer - I - KCi-Ringer- I

Fig. 3. Contractions of chick amnion (12 days old) to acetylcholine;isotonic recording at 320 C.

r- KCI-Rincer - r-NaCI-Ringer-

Fig. 4. Acetylcholine-induced contractions of longitudinal muscle strip of cat intestine;isotonic recording.

A B C D E F G A B C D E F G

02-L

4

LNaCI-Ringer * -K2SO4-RingerFig. 5. Rat uterus at 200 C; tension recording. Concentration of ACh, A =10-8, B=2 x 18,

C=4x10-8, D=110-7, E=2x10-7, F=4x10-7, G=10-6 replaced the bath fluid at (4);this was replaced by fresh bath fluid at (t ). Between the two records the uterus wasimmersed for 1 hr in K2SO4-Ringer.

DRUGS ON DEPOLARIZED PLAIN MUSCLE

that the maximum tension developed in the presence of acetylcholine inpotassium solution was only about half that in sodium. On the other hand, thethreshold for acetylcholine is lowered. Both these effects are illustrated inFig. 6 which represents the average of five experiments. With low concentra-tions of acetylcholine greater tensions are developed in potassium-Ringer,whereas with high concentrations the reverse is true. Both the lowering ofthreshold and reduction in tension are reversible, i.e. when the preparationis transferred back to sodium-Ringer the threshold rises and the maximumtension returns to its normal values.

100 XNaCI-Ringer

80 _ xSt0I- /Xc 60 x0

40~~~~~~40K2SO4-Ringer

20-20

0-, lo0-7 10-6 10-5Acetylcholine

Fig. 6. Rat uterus at 200 C. Tensions produced by acetylcholine in NaCI-Ringer and in K2SO4-Ringer. Each point represents the mean of five experiments. The tensions are expressed aspercentages of the maximum tension produced by acetylcholine in NaCl-Ringer.

It seemed possible that the diminished tension response to acetylcholine inpotassium solution could be due to a reduced initial length of the smooth-muscle preparation. To test this point experiments were made in which theinitial length of the preparation (rat uterus at 200 C) was restored by the useof isoprenaline. Table 1 shows that although the maximal tension in potas-sium-Ringer is reduced when the initial length is shorter, the tension inpotassium solution does not exceed about 50% of that in sodium solutioneven when the preparation is relaxed to its normal length.

Fig. 7 shows that 25 min after immersion in potassium-Ringer the acetyl-choline effect becomes stabilized and does not change further. That noirreversible change has been produced is apparent from the fact that thetension response returns to normal after replacing in sodium-Ringer.Graded and reversible contractions of various smooth-muscle preparations

in potassium-Ringer were also obtained with histamine, 5-hydroxytrypt-amine and oxytocin. The drug responses were generally reduced as comparedwith those in normal Ringer's solution, but were clearly demonstrable. The5-hydroxytryptamine response of the rat uterus was particularly reduced,an indication that this may largely be a conducted response. Fig. 8 shows

479

480 D. H. L. EVANS, H. 0. SCHILD AND S. THESLEFFcontractions of the rat uterus in K2SO4-Ringer in response to posteriorpituitary extract, acetylcholine and 5-hydroxytryptamine, and Fig. 11 corre-sponding histamine effects in the guinea-pig ileum preparation.TABLE 1. Effects of maximum doses of acetylcholine on the length (1) and tension (t) of the rat

uterus immersed in K2SO4-Ringer at 200 C. The results obtained in five experiments beforeand after relaxation of the preparation by isoprenaline are shown. The values are expresssedas percentages of the corresponding measurements obtained in NaCl-Ringer

I t I t 1 t I t I tBefore isoprenaline 66 47 88 40 69 31 97 43 91 30After isoprenaline 96 56 100 42 94 49 100 43 100 43

K2SO4-RingerI

100 000

0_~~~~~~~O~

Co 00 NaCI-Ringer- 50

o o

0 50 100 150Time (min)

Fig. 7. Rat uterus at 200 C, showing the effect of time on tension produced by the samedose of acetylcholine in K2SO4- and in NaCl-Ringer.

Inhibitory drugs and antagonistsIsoprenaline, which is a potent smooth-muscle relaxing drug, retains its

inhibitory effect in depolarized preparations (rat uterus and chick amnion).Fig. 9 shows a complete relaxation to base line by isoprenaline (10-4) of a ratuterus preparation which had been made to contract by immersion in K2SO4-Ringer. After washing with potassium-Ringer the preparation remainedrelaxed, but it contracted again when acetylcholine was added to the bath.It was repeatedly observed that the relaxation produced by isoprenaline inpotassium-Ringer was maintained even after the drug had been washed out.The inhibitory effect of isoprenaline was most marked for 10-20 min afterthe immersion of the smooth muscle in potassium-Ringer and graduallydiminished after longer immersion.

Antagonistic drugs also retain their activity in potassium-Ringer. Prepara-tions which are contracted by acetylcholine are relaxed by atropine, as isshown for the chick amnion in Fig. 10. The specificity of drug antagonists isalso maintained as shown by the observation that antihistamine drugs preventthe stimulant effect of histamine but not that of acetylcholine (see Fig. 11).


Pit.0-05 u. ml.

Fig. 8. Rat uteiacetylcholix

Fig. 9. Rat ute

K2SO4-Rinj

K2So4- Ringer

ACh 5-HT10-7 10-7

Fig. 8

rus in K2S04-Ringer; isotonic recordinjie and 5-hydroxytryptamine.Xrus at 30° C; isotonic recording. Effger. At W the isoprenaline was washec

KCI-Ringer

L NaC1- JW K2SO4-Ringer ..JRinger 0 5 10

minFig. 9

g. Effects of posterior pituitary extract,

ects of isoprenaline and acetylcholineinI out.

Mk ..n IV Atr. -IU

Fig. 10. Chick amnion at 320 C; isotonic recording. Slight stretches were applied intermittentlyto the preparation. Atropine was added to the bath without removing the acetylcholine.

481

Ir-


H H H H ACh

L M J2Fig. 11. Guinea-pig ileum in K2SO4-Ringer at 240 C; isotonic recording. The horizontal line

represents the base line in NaCI-Ringer. Three doses of histamine (5 x 10-7) are followed by adose of mepyramine, M (2 x 10-6), which produces a relaxation. In the presence of mepyr-amine a further dose of histamine is ineffective but a dose of acetylcholine (10-6) is effective.

Mechanical and electrical stimulationEvans & Schild (unpublished observations) have found that the nerve-

free chick amnion preparation when suspended in sodium-Ringer responds toa local pinch by a transient generalized contraction, which presumablyrepresents a conducted response of plain muscle (Prosser & Rafferty, 1956)since the chick amnion contains no nerves or ganglion cells. This contraction inresponse to pinch disappears in potassium-Ringer and reappears when thepotassium is replaced by sodium as shown in Fig. 12.The contraction produced by electrical stimulation of smooth muscle is

greatly reduced or abolished in potassium solution although a large increase inthe strength of the electrical stimulus may still produce a contraction. Asis shown in Fig. 13 the response of a rat uterus to electrical stimulation is re-versibly abolished in potassium-Ringer while the contraction produced byacetylcholine is maintained.

DISCUSSION

It has been shown that smooth-muscle preparations immersed in isotonicpotassium-Ringer solutions can still contract and relax in response to drugs,and can develop considerable tensions though less than in normal Ringer'ssolution. The responses to electrical stimulation are greatly reduced and the


Ringerr-NaCI-11 KCI -7r NaCI KC} rNaCI-1

r A-Cn -Cn r iCn r ACn r A&Cn r A'..n

Fig. 12. Chick amnion (12 days old) at 300 C; isotonic recording. Contractions due to crushingedge of preparation with forceps, P, compared with contractions due to acetylcholine(2 x 10-5). Reversible abolition in KCl-Ringer of the contraction produced by the localcrush injury.

L NaCI IL K ,SO4 IJL N-CI JRinge r

Fig. 13. Rat uterus at 300 C; isotonic recording. Contractions due to electrical stimulation,E (8 V/cm, 0 5 sec) and acetylcholine (10-7).

PHYSIO. CXLIII

483

31

484 D. H. L. EVANS, H. 0. SCHILD AND S. THESLEFFconducted responses to an applied mechanical stimulus are abolished. Ourresults thus confirm the general view that conducted responses do not occurin depolarized structures.The present experiments were carried out in potassium chloride or potas-

sium sulphate solutions. The former probably does not completely abolishthe membrane potential of smooth muscle (Burnstock & Straub, 1958) butthe latter, judging from the experiments with direct intracellular recording,seems to produce a complete depolarization of the smooth muscle cells. Itshould be mentioned, however, that our method does not make it possible toexclude completely the presence of a small residual membrane potential evenin the potassium sulphate solution.

There is strong, though not entirely conclusive, evidence that the activa-tion of the contractile elements in striated muscle is linked to membranedepolarization. This evidence is based on the findings that striated musclecontracts when immersed in potassium solutions, that contractions occur atthe cathode of an electric current and that, if a contraction is produced by thelocal application of a drug (Kuffler, 1946) to the surface of a striated musclefibre, this is accompanied by membrane depolarization. In smooth muscle,tension development is also accompanied by depolarization of the musclemembrane (Biilbring, 1955).The present experiments suggest that a contraction in smooth muscle may

be produced by an alternative mechanism to membrane depolarization.Although our results do not provide any new explanation for the mechanismby which drugs contract plain muscle, they are open to a number of interpreta-tions. The following mechanisms can be envisaged, to explain the continuedeffectiveness of drugs in depolarized plain muscle.

(1) The activation of the contractile elements may be brought about by asubstance or ion which is released by the drug from the cell membrane.

(2) Drugs may produce a permeability change in the depolarized smoothmuscle membrane just as acetylcholine produces an increase in membranepermeability in the depolarized motor end-plate (del Castillo & Katz, 1955).The permeability change may cause diffusion of some substance through themembrane which directly or indirectly activates contraction.

(3) The permeability change may produce a membrane potential change,even though the cell membrane is depolarized.

(4) Drugs may act directly on the contractile elements. It would then haveto be assumed that even large molecules such as oxytocin or quaternarysubstances such as acetylcholine can penetrate through the cell membrane.

These hypotheses are all speculative but some at least could be experi-mentally tested. For example, it might be possible to record changes inmembrane resistance and membrane potential of smooth muscle cells inpotassium solutions.


SUMMARY

1. Smooth muscle preparations immersed in Ringer's solutions in whichsodium is replaced by potassium retain their responsiveness to drugs.

2. When immersed in potassium-Ringer (KCI or K2SO4) the smooth musclepreparations contract and subsequently relax.

3. Membrane potentials of smooth muscle cells (chick amnion) were re-corded with intracellular micro-electrodes in sodium-Ringer, but none couldbe recorded after transferring the preparation to K2SO4-Ringer.

4. When smooth muscle preparations have relaxed in potassium solutionsthey contract in response to acetylcholine and relax again when the drug isremoved. In the rat uterus the threshold concentration of acetylcholine is ifanything lowered, but the maximum tension is only half the normal.

5. Smooth muscle stimulant drugs such as histamine, 5-hydroxytryptamineand oxytocin, inhibitory drugs such as isoprenaline, and drug antagonistssuch as atropine and mepyramine retain their activity in potassium-Ringer.

6. Responses to electrical stimulation are greatly reduced in potassium-Ringer and the conducted response to a mechanical stimulus is abolished.

7. The results suggest that drugs may, without the mediation of membranedepolarization, activate the contractile elements of plain muscle.

REFERENCESBULBRrNG, E. (1955). Correlation between membrane potential, spike discharge and tension in

smooth muscle. J. Physiol. 128, 200-221.BURNSTOCK, G. & STRAUJB, R. W. (1958). A method for studying the effects of ions and drugs on

the resting and action potentials in smooth muscle with external electrodes. J. Physiol. 140,156-167.

CsApo, A. (1954). A link between 'Models' and living muscle. Nature, Lond., 173, 1019-1021.DEL CASTMLLO, J. & KATZ, B. (1955). Local activity at a depolarized nerve-muscle junction.

J. Physiol. 128, 396-411.EVANS, D. H. L. & SCHILD, H. 0. (1953). The reactions ofplexus-free circular muscle ofcat jejunum

to drugs. J. Physiol. 119, 376-399.EVANS, D. H. L. & SCHILMD, H. 0. (1957). Mechanism of contraction of smooth muscle by drugs.

Nature, Lond., 180, 341-342. /HUXLEY, A. F. (1957). Muscle structure and theories of contraction. Recent Advances,in Bio-

physics, 7, 257-318. London: Pergamon Press.KUFFLER, S. W. (1946). The relation of electric potential charge to contracture in skeletal muscle.

J. Neurophy8iol. 9, 367-379.PIERcE, M. E. (1933). The amnion of chick as an independent effector. J. exp. Zool. 65, 443-473.PROSSER, C. L. & RAFFERTY, N. S. (1956). Electrical activity in chick amnion. Amer. J. Physiol.

187, 546-548.

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zation. since it is generally considered that a membrane potential

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