THE mechanisms by which acetylcholine

The Action of Acetylcholine and Epinephrine on the Cellular Membrane Potentials and Contractility of Rat Atrium By J. LEYDEN WEBB, PHJD. AND PHILIP B. HOLLANDER Acetylcholine diminished the developed tension of contraction in rat atria; simultaneously there was a slight increase in resting potential, no appreciable change in action potential, a decrease in the rate of conduction, only a minor alteration of the form of contraction, and, most strikingly, a decrease in the duration of the action potential or the area enclosed by it. The rate of repolarization subsequent to the action potential was markedly accelerated. Epinephrine, in general, produced the opposite effects and slowed the repolarization rate as it augmented atrial contraction. Cholinesterase does not seem to play a direct role in cardiac membrane potentials or ion transfer since physostigmine had no effect of its own on the electric manifestations. Certain aspects of the mechanisms involved are discussed. THE mechanisms by which acetylcholine and epinephrine alter cardiac function are perhaps related to changes in ionic fluxes across the cell membranes and the resultant disturbances in electric potential. The slight increase of the resting potential following vagal stimulation 1 and the shortening of the duration of the action potential 2 haa'e been adequately confirmed by numerous workers and recently placed on a more quantitative basis by the use of intracellular electrodes, 3 - A applying choline esters as well as vagal stimulation. Alterations in the action potential have been variable but a decrease has most commonly been reported. The effects of epinephrine on the electric characteristics of cardiac muscle are less well understood and in general have been assumed to be rather slight. 6 - The purpose of the present work is to extend our knowledge in these directions in the attempt to understand better the nature of the coupling between the membrane action potential and the contractile elements of the cardiac cell. METHOD The membrane potentials and mechanical activity of mt atria electrically stimulated at a rate of 200/ From the Department of Pharmacology, School of Medicine, University of Southern California, Los Angelas, Calif. This work was supported by a grunt from the Life Inmirunco Medical llesearch Fund. Received for publication January 16, 105C. minute in Krebs-Ringer-bicarbonate medium at ph 7.4 and 30 C. were determined simultaneously using microelectrodes and a sensitive strain gage as previously described. 7 RESULTS Effects of acelylcholine on tlic normal alrium. Acetylcholine was applied in concentrations between 10~ 7 M and 10~ B M after an adequate series of control readings had been made; a second series of control readings was made after the tissue had become stabilized following removal of the acetylcholine. The control readings given are means of these two since this tends to eliminate any drift in the tissue characteristics during the procedure. The results are given in table 1. The number of penetrations to yield statistically valid results required 10 to 15 minutes following addition of acetylcholine; the maximal action occurred between 1 and V/l minutes and partial recovery ensued. The overall action is given for the total interval and for all the concentrations used. The maximal action from the two or three penetrations made during this period is also shown for all concentrations and for the higher range of concentrations. Although not as statistically valid as the overall results, due to the fewer penetrations possible, the results obtained for the maximal action are very similar except more marked in each category. It is also evident that the higher acetylcholine concentrations produced greater alterations in most cases. 332 Cimilalion Reteanh, Volume IV, Man I9S0

WEBB AND HOLLANDER 333 TABLE 1. Effects of Acetylcholine on Hat Atria. Maximal Action (1 to 1H min.) Overall Action (10 to 15 min.) All Concentrations - 6-10 X 10" i\l % ChanRc Resting potential (mv.) Action potential (niv.) Action potential duration Action potential area (mv. sec.) Developed tension (mg.) Developed tension duration Developed tension rise time Conduction time Conduction rate (em./soc.) Excitation-con tract ion time ' Latent period Number of ruts 60.94 72.92 11.98 46.30 1.33 506.2 107.0 44.94 14.76 73.2 10.35 25.H 429 20 62.52 72.93 10.41 21.14 0.644 366.2 103.6 42.95 14.91 72.4 9.89 24.SO 293 +2.6 0.0-54.4-51.5-27.6-3.2-4.4 + 1.0-1.0-4.4-1.2 59.59 72.16 12.57 48.01 1.32 547.0 114.5 47.63 15.44 69.9 9.90 25.34 229 20 62.38 73.35 10.97 17.05 0.532 325.5 10S.2 43.21 15.78 68.5 9.S3 25.61 43 +4.7 + 1.7-64.5-60.0-40.6-5.5-9.3 +2.2-2.2-0.7 + 1.1 57.91 69.27 11.36 50.36 1.35 566.8 115.0 46.51 14.88 72.6 9.S9 24.77 121 9 62.26 71.79 9.53 11.32 0.365 297.1 109.6 41.87 15.79 68.4 10.17 25.96 2S + 7.5 +3.6-77.5-72.9-47.6-4.7-9.9 +6J -6.1 +2.S +4.S There was a slight, though significant, increase in the resting potential, this occurring in 17 out of the 20 preparations, demonstrating the Caskell effect in rat atrium. 1 There was no certain effect on the action potential unless the slight increases noted during maximal action are significant; at least the absence of a depression seems certain. The absolute level of depolarization reached was thus somewhat less in the presence of acetylcholine, as measured by the decrease in the overshoot, but it is doubtful if this is of any importance in altering the mechanical response. The most striking change observed was the decrease in the duration of the action potential and the area enclosed by it. This was almost entirely due to the more rapid rate of repolarization induced by acetylcholine; the rise time of the action potential was only about ]/100th the total duration so that any shortening of it could not have had appreciable effect. The acceleration of the repolarization rate was approximately twofold when the contractile depression was 25 per cent and fivefold when the contractile depression was 50 per cent; higher concentrations of acetylcholine produced up to tenfold accelerations of repolarization accompanied by marked reduction of contraction. The rise time and duration of the contraction were shortened relatively little, indicating that the form of the total contraction was not altered appreciably by acetylcholine although a slight shift of the peak may have occurred. Some shortening would be expected during the depression of developed tension and it would appear that in the rat atrium there is no specific failure of slowly reacting cells as postulated by Burgen and Terroux 1 in the cat atrium, but only a decreased contraction of each cell. The effect of acetylcholine on conduction rate was very slight but is believed to be significant since it occurred in 15 out of the 20 preparations and was more marked with the higher concentrations of acetylcholine. It is surprising that the marked shortening of the action potential had so little effect on the conduction rate and indicates that the factors involved in conduction probably depend primarily on the magnitude of the depolarization and the responsiveness of the normal membrane to depolarization. The slight changes observed in the latent period are probably not significant since half the preparations showed and increase and half a decrease. The characteristic effects of acetylcholine seem to be independent of the substrate from

334 ACETYLCHOLINE AND EPINEPHRINE ACTIONS ON RAT ATRIUM which the cardiac muscle is deriving its energy. Atria allowed to beat in the absence of substrate show a steady decline in developed tension to low values; if either glucose or pyruvate is added at this time, recovery is observed. In such atria beating in the presence of glucose or pyruvate, and presumably deriving most of their energy from these substrates, the effects of acetylcholine were quite comparable to those observed on fresh atria utilizing endogenous substrate. Effect of physosligmine on the response to acetylcholine. In the presence of 10" 1 M physostigmine, the sensitivity to acetylcholine was increased over 100 fold. The results of a single experiment are shown in table 2 and, although not quantitatively reliable, they demonstrate the same changes noted with acetylcholine alone, except to a greater degree. The depression of the magnitude of the action potential began when the action potential had been shortened markedly, as if the repolarization process were beginning before the membrane had time to depolarize normally, and similar depressions of the action potential were observed with very high concentrations of acetylcholine and carbachol when the repolarization rate had been greatly accelerated. More asymmetry of the contraction may be noted, the TABI,E 2. Effects Resting potential (mv.) Action potential (mv.) Action potential duration Action potential area (mv. sec.) Developed tonsion (mg.) Developed tension duration Developed tension rise time Conduction time Excitation-contraction time Latent period Number of rats peak occurring relatively earlier than in the control, and there was definite, although slight, slowing of the rate of conduction. Effect of physostigmine alone. Physostigmine produced a slow depression of rabbit atria, 4 due presumably to accumulation of acetylcholine produced endogenously, and recently such results have been used as evidence for the role of acetylcholine in cardiac contraction. 9 ' 10 The possible role assumed by some investigators for cholinesterase in ion transport across membranes also prompted us to determine if physostigmine exerted any direct effect on the electric behavior of the rat atrium. No immediate effects were observed from 10~ 6 M to lo" 1 M physostigmine. After several minutes a slow depression of the developed tension was seen, reaching a steady level, and the changes in the atria at this time are given in table 2. The results are compatible with a mild acetylcholine effect. It is further interesting that these effects were produced by 10~ 6 M physostigmine and increasing the concentration to 10~' M resulted in no additional action. Effect of carbachol. Carbachol differs primarily from acetylcholine in being resistant to cholinesterase and hence is somewhat more consistent and stable in its cardiac action. 8 The results from seven experiments are given in of Acetylcholine in the Presence of Physostigmine, of Physostigmine and of Carbachol. (10-«M) after physostigmine (10"< M) 65.55 76.11 10.56 44.55 1.21 3S2.3 117.3 50.11 14.55 7.77 22.32 18 1 65.70 68.50 2.SO 8.25 0.269 190.2 107.5 40.25 15.25 5.75 21.00 S +0.2-10.0-81.4-77.8-47.9-8.4-19.7 +4.8-26.0-5.9 PhysoJtigmine (10"*-l 0"» M) 5S.S6 69.92 11.06 51.26 1.505 504.7 115.5 50.05 14.58 11.26 25.84 23 2 Physo 59.98 71.92 11.94 43.55 1.305 429.1 117.9 48.55 13.95 9.S5 23.SO 17 + 1.9 +2.9-15.0-13.3-15.0 +2.1-3.0-4.3-12.5-7.S Carbuchol (10 '-10' M) 59.73 70.04 10.31 48.90 1.29 472.8 102.4 49.61 12.S7 10.74 23.61 109 7 Cch 60.74 69.54 8.SO 21.27 0.614 303.1 94.63 44.33 12.46 10.42 22. SS SI + 1.7-0.7-56.5-52.5-35.9-7.6-10.6-3.2-3.0-3.1

WEBB AND HOLLANDER 335 TABLE 3. Effects of Epiiiephnne and the ftemoval of Acetylcholiiie. Epinephrine (5 X 10"' M) Acetylcholine removal (10"*-10~«Epi Post-waih % ChanRC* Itcsting potential (rav.) Action potential (mv.) Action potential duration Action potential urea (mv. sec.) Developed tension (mg.) Developed tension duration Developed tension rise time Conduction time Excitation - contraction time Latent period Number of rats 60.37 68.65 8.28 41.22 1.12 445.2 105.8 46.84 13.30 10.42 23.72 76 7 57.66 66.07 8.41 46.57 1.34 576.9 108.9 50.65 13.64 9.53 23.17 50-4.5-3.8 + 13.0 + 19.6 +29.5 +2.9 +8.1 +2.6-8.5-2.4 62.61 76.08 13.47 42.17 1.085 424.2 109.4 46.30 16.08 10.90 26.98 142 8 63.39 75.12 11.73 19.07 0.516 305.2 104.1 43.68 16.72 10.17 26.89 132 62.24 74.86 12.62 49.96 1.27 544.3 113.5 49.96 17.27 8.98 26.25 37-0.6-1.6 +18.5 + 17.0 +28.3 +3.7 +7.9 +7.4-17.6-2.7 * Calculated from the control levels. table 2, from which it may be seen that the changes produced were quite similar to those produced by acetylcholiiie. Response to removal of acetylciwline. When acetylcholine is washed out, following an exposure of 10 to 15 minutes, there ensues a temporary stimulation of atrial contractility above the control levels* and it was of interest to determine the changes in electric behavior during this period. It was difficult to obtain more than four to five penetrations during the two to three minutes of heightened contractility, but the averages from eight preparations showed some definite changes (table 3). The rise in developed tension was accompanied by a prolongation of the action potential, or a slowing in the rate of repolarization, without significant changes in the resting or action potentials. A slowing of the conduction rate occurred in seven out of the eight preparations. This increase in conduction time accounted for the decrease in the excitation-contraction time 7 so that the latent period was essentially unchanged. Effect oj epi?iephrine. The action of epinephrine was in general the opposite to that of acetylcholine on the characteristics measured (table 3). The rise in developed tension was accompanied by a slowing of the repolarization rate and slight depression of both the resting and action potentials. The rate of conduction, however, was slightly slowed, as it was with acetylcholine, substantiating the independence of conduction and the duration of the action potential. The slowing most likely resulted from the lowered membrane potentials. DISCUSSION The shortening of the action potential duration produced by vagal stimulation or choline esters has been reported several times, using both contact electrodes and intracellular electrodes, but in no case has the correlation between the contractile depression and this shortening been expressed quantitatively, and hence it is impossible to compare the present results with those obtained in other species. The only demonstrable marked effect of either acetylcholine or epinephrine on the membrane electric properties of the cardiac cells was upon the rate of repolarization subsequent to the development of the action potential and the obvious correlations tempt one to assume a causal relationship. However, it must be realized that the shortened action potential may not be the cause of the diminished contraction, in the case of acetylcholine, but may be the result of an independent action, or an unimportant coincident result of the same action, or the result of the diminished contraction itself.

336 ACETYLCUOLINE AND EPINEPHRINE ACTIONS ON RAT ATRIUM The data of the present study cannot be used to decide between these but results to be published from modifications of atrial activity by other means strongly argue against the last two possibilities. The alterations in the resting and action membrane potentials by acetylcholine and epinephrine, slight though they are, and probably without significance in the contractile effect, are believed to be of great importance in the eventual explanation of the mechanisms involved. The concept that acetylcholine causes a large nonselective increase in ion permeability, such as has been postulated to explain the endplate depolarization in skeletal muscle, 11 certainly cannot be valid for cardiac muscle. Rather there would appear to be a selective increase in permeability to a particular ion following depolarization of the membrane, whereas the permeability properties of the normal polarized membrane do not seem to be greatly affected. The possibility is suggested that there are fixed charges of various types in the membrane and that some of these at least are intimately dependent on metabolic processes. Furthermore, these groups control in part the diffusion rates of ions through the membrane. Reaction of acetylcholine or epinephrine specifically with certain of these charged groups may modify the permeability in the manner suggested by the present results. Discussion of this hypothesis must await further studies on the modifications of cardiac function by a variety of means. SUMMARY The actions of acetylcholine, carbachol, physostigmine and epinephrine on atrial contraction and membrane potentials were investigated. The choline esters increased the membrane potentials, but only slightly, while markedly shortening the duration of the action potential by an acceleration of the rate of rcpolarization. Epinephrine produced opposite actions. The possibility of correlating these changes with the altered contractility was discussed. No evidence for a direct role of cholinesterase in ion transport across the cardiac membrane was found. SUMMARIO IN 1 INTERLINGUA Le action de acetylcholina, carbachol, physostigmina, e epinephrina super le atrial contraction e potentiates membranal esseva investigate. Le esteres cholinic augmentava le potentiales membranal, sed solmente a leve grados, durante que illos reduceva marcatemente le durantia del potential de action per accelerar le repolarisation. Epinephrina produceva le effecto contrari. Es discutite le possibilitate de correlationar iste alterationes con le alterate contractilitate. Esseva trovate nulle indicio de un rolo directe de cholinesterase in le transporto de iones a transverso le membrana cardiac. REFERENCES I GASKELL, \V. H.: On the action of muscarine upon the heart, and on the electrical changes in the non-beating cardiac muscle brought about by stimulation of the inhibitory and uugmentor nerves..1. Physiol. 8: 404, 1887. 1 SAMOJLOFF, A.: Die Vagus- und Muskarin-wirkung auf die Stromkurve des Frosch-herzens. Arch. f. d. ges. Physiol. 165: 471, 1914. 3 BURDEN, A. S. V., AND TERROUX, K. G.: On the negative inotropic effect in the cat's auricle. J. Physiol. 120:449, 1953. HOFFMAN, B. F., AND SUCKLING, E. E.: Cardiac cellular potentials: effect of vagal stimulation and acetylcholine. Am. J. Physiol. 173: 312, 1953. 6 CHURNEY, L.: Effect of epinephrine on monophasic action potential of auricular muscle. Am. J. Physiol. 171: 516, 1952. ' FlNGL, E., WOODBURY, L. A., AND HECHT, H. H.: Effects of innervation and drugs upon direct membrane potentials of embryonic chick myocardium..j. Pharmacol. & Exper. Therap. 104: 103, 1952. 7 HOLLANDER, P. B., AND WEBB, J. L.: Cellular membrane potentials and contractility of normal rat atrium and the effects of temperature, tension and stimulus frequency. Circulation Research 3: 604, 1955. 8 WEBB, J. L.: The action of acetylcholine on the rabbit auricle. Brit. J. Pharmacol. & Chemotherap. 6: 335, 1950. 'BURN, J. H., AND KOTTEGODA, S. R.: Action of eserine on the auricles of the rabbit heart. J. Physiol. 121: 360, 1953. 10 BRISCOE, S., AND BUBN, J. H.: The formation of an acetylcholine-like substance by the isolated rabbit heart. J. Physiol. 126: 1S1, 1954. II FATT, P., AND KATZ, B.: An analysis of the endplate potential recorded with an intra-cellular electrode..j. Physiol. 116: 320, 1951.