WELS~~~~ THE mode of action of acetyl choline upon the isolated ventricular strip

Transcription

1 THE ANTAGONISM OF ACETYL CHOLINE BY ATROPINE. BY A. J. CLARK. (From the Pharmacological Department, University College, London.) THE mode of action of acetyl choline upon the isolated ventricular strip and the Rectus abdominis of the frog has been discussed in a previous paper(l) and the technique used in the experiments described below is the same as there described. The relation between the action (y) expressed as the percentage of maximal possible action, and the concenx x 1'0 I 0-5~~~~~~ WELS~~~~ x Fig. 1. Action of acetyl choline on the heart in presence of atropine. x =molar conc. acetyl choline; y =percentage reduction in isometric contraction. Ordinate: Log loy"_; abscissa: Log x. Molar concentrations of atropine: I, no atropine; II, 10-8; III, 10-7; IV, 10^; V, 10-5; VI, 10-; VII, 10-'. 362

2 548 A. J. CLARK. tration (x) of acetyl choline has been shown (1) to follow the following formula: Formula I. K. x = 100-y. The curves I in Figs. 1, 4 and 6 show this relation. If the formula is expressed as K.xn=-lo--y then the values for n in these three curves are 1-05, 0*85 and 0-75 respectively, but of these numbers the first is based on many more observations than the other two. As I have been unable to prove that n is for certain either more or less than unity I have assumed that this is its value. Fig. 1 shows the effect on the response of the isolated ventricle to acetyl choline of the presence of increasing concentration of atropine, Fig. 2. Action of acetyl choline on the frog's ventricle in presence of atropine. (Isometrib response; stimuli 30 per minute.) A-G, action of acetyl choline in absence of atropine. Molar concentrations: A, 5 x 10-9; B, 10-8; C, 5 x 1O-; D, 10-7; E, 5 x 1O-7; F, 5 x 10; G, 12 xl-5. H-K, action of acetyl choline in presence of atropine 3 x 10-8 molar. Molar concentrations Ac. Ch.: H, 10-7: I, 2 x 10-7; J, 5 x 10-7; K, 10-A. b-n, atropine 3 x 1O-7 molar. Molar concentrations Ac. Ch.: L, 25 x 10-6; M, 5 x 10-6; N, 2-5 x 10-. O, atropine 10-1 molar. Molar concentrations Ac. Ch. 2-5 x 1O-5.

3 ACETYL CHOLINE AND ATROPINE. 549 in the fluid around the tissue, and Fig. 2 shows samples of the curves from which Fig. 1 was constructed. Fig. 3 shows the molar concentrations of atropine and acetyl choline which when present together produce a 50 p.c. reduction in the response of the heart Fig. 3. Concentrations of acetyl choline and atropine which when present together produce 50 p.c. reduction in the response of the heart. Ordinate: Log. of molar conc. of acetyl choline. Abscissa: Log. of molar conc. of atropine. It will be seen that over the greater portion of the curve the relation between the concentrations is Log (molar conc. Acetyl choline) - Log (molar conc. Atropine) = constant. Therefore, when constant effects are produced the following relation holds: Conc. Ac. Ch. Formula H Conc. Atr. = constant. There is naturally a threshold for the concentration of acetyl choline, namely the concentration needed to produce 50 p.c. reduction in the absence of atropine (10-6 molar). The values obtained suggest that there is also a threshold value for the concentration of atropine between 10-9 and 10- molar, below which it produces no demonstrable effect. The figures for the action of acetyl choline when the concentration of atropine is 10-7 molar or less show a deviation from formula II. This slight deviation was also observed in other experiments, and I believe that it cannot be explained as due to experimental errors. I have been unable to devise any simple formula which will cover this deviation, but if the figures for these lowest active concentrations of atropine be ignored, then formula II holds.

4 550 A. J. CLARK. A combination of formulae I and II gives the following: Formula III. K. Conc. Conc. Ac. Atr. Ch. - lo-y' y This formula is the same as that found by HUfner(2) to express the combination of haemoglobin with oxygen in the presence of carbon monoxide, a conclusion confirmed later by Douglas, Haldane and Haldane(3). Figures calculated by means of formula III agree very well with the results shown in Fig. 1; in this experiment K = Table I TABLE I. Concentration of atropine constant at 10-5 molar. Molar conc. acetyl choline x Percentage of action produced (Calculated by acetyl choline ) Observed (Complete arrest = 100) shows the calculated and observed results when the concentration of atropine is taken as constant (10-5 molar). Table II shows the calculated TABLE II. Concentration of acetyl choline constant at 3 2 x 10-8 molar. Molar conc. atropine x Percentage of action produced (Calculated by acetyl choline i Observed (Complete arrest = 100) and observed results when the concentration of acetyl choline is taken as constant at 3*2 x 10-3 molar. Fig. 3 shows the observed results when the action produced is taken as constant. In these experiments the concentrations of acetyl choline used ranged from 0-1 to 10-s molar and the concentrations of atropine from 10-s to 10-7 molar and the agreement between the observed and calculated figures over this very wide range of concentrations must be considered satisfactory. The values obtained for K in experiments on different hearts varied. The figures obtained in four experiments were as follows: 0 5, 0-2, 0-2, 0*028. This variation is probably connected with the great differences observed in the susceptibilities of different hearts to acetyl choline. The experiments show that in any single heart an equal effect is produced as the resultant of the action of the two drugs as long as Conc. Atr. remains constant, provided that the concentration of atropine is sufficient to produce a well-marked effect. This relation is in accordance with that found by Cushny(4) for the antagonism by atropine of the action of pilocarpine on the salivary gland of the dog. He found that one part of atropine antagonised about eight parts of

5 ACETYL CHOLINE AND ATROPINE. 55I pilocarpine, and that this ratio remained constant even when the doses were varied fifty-fold. Figures given by Broom and Clark(5) show a similar relationship for the antagonism of adrenaline by ergotamine in the isolated rabbit's uterus. These figures show that approximately equal effects are produced with the following concentrations of the two drugs: Mol. conc. ergotamine x Mol. conc. adrenaline x The ratio between the concentrations of ergotamine and adrenaline is about 1 to 25 and remains nearly constant. Gaddum(6) gives results that show that the ratio between the concentrations of adrenaline and ergotamine that produce equal actions on the isolated rabbit's uterus remains approximately constant. The rabbit's isolated uterus is not well adapted to accurate quantitative work, but these results show that the antagonism between adrenaline and ergotamine follows a course very similar to the antagonism between atropine and acetyl choline. The results obtained with the Rectus abdominis muscle are not quite Fig. 4. Action of acetyl choline on the Rectus abdominis, in presence of atropine. x =molar conc. acetyl choline; y=contraction produced expressed as percentage of maximal contraction. Ordinate and abscissa as in Fig. 1. Molar concentrations of atropine: I, no atropine; II, 3-3 x 105; HII, 10; IV, 103.

6 552 A. J. CLARK. so simple. Fig. 4 shows a sample of the measurements obtained and Fig. 5 the concentrations of acetyl choline and atropine which, when acting together, produce a con- - 2 traction amounting to 20 p.c. of the maximal. These results follow the formula Formula IV. -3 K.Conc. Ac. Ch. yj K. (Conc. Atr.)'5 1oo - y This formula gives satisfactory results over the range of concentra- 4 tions at which atropine produces a well-marked effect. The following values were found -5 for K with the Rectus abdominis: 0-32, 0'25, 0*16, 0.1. A relation of a somewhat similar type was found by Le Heux, Storm van Leeu- -6 wen and van den Brocke(7), Fig. 5. Concentrations of acetyl choline and who studied the action of atropine atropine which together produce 20 of maximal p.c. and contraction pilocarpine on in the the isolated gut Rectus abdominis. Ordinate and abscissa as in of the rabbit. They showed that Fig. 3. (The curve is constructed from L idth de Jeude (8) was mistaken four different experiments.) in supposing that the action of atropine was not dependent on the concentration of the drug; and that the relation between atropine and pilocarpine could be expressed by the statement that equal effects were produced when Conc. Atr. = K (Conc. Pilo.)". In one of their experiments n was 1-5. The Quantity of Atropine Uniting with the Heart. I have described in the previous paper(1) a method of estimating the amount of drug actually reacting with tissues by adding to the moist ventricular strip a minute quantity of fluid. This method was used with atropine. The reaction of a heart strip to solutions of acetyl choline was first determined, and then the reaction of the preparation to minute quantities of acetyl choline. After these figures had been determined a small quantity of atropine was added to the moist strip in 1 mg. of fluid. After 10 minutes the reaction of the strip to a minute quantity of acetyl choline was measured. The strip was then thoroughly washed and the process recommenced.

7 ACETYL CHOLINE AND ATROPINE. 553 The results obtained are shown in Fig. 6. The addition of grm. mol. of atropine reduces tenfold the sensitivity of the strip to acetyl choline. Accurate measurements are very difficult to obtain with this -7 J1 T TIT[ I -6 I [ II 11 _ ō1 XLiX 0-5 CsEfXIa Z -0.5 x IV _. Fig. 6. Antagonism of small ab8olute quantities of acetyl choline by small ab8olute quantities of atropine in a ventricular strip (weight 15 mg.). Ordinate: Log Y (y=percentage reduction in response). Abscissa: along top-log of molar conc. acetyl choline (curve I); along bottom-log of gram mols. of acetyl choline added (curves II-V). I. Response of heart immersed in 5 c.c. of solution (no atropine). II-V. Response of moist ventricular strip to small quantities of acetyl choline. Gram mol. of atropine present: II, Nil; III, 10-1O; IV, 10'; V, method, and therefore I cannot say definitely whether, when equal effects are produced, the fraction Grm.Mols.Ae.Ch remains constant or not; Fig. 7 suggests that the ratio probably does remain constant. These experiments are quoted merely to show that the amount of atropine which actually unites with the heart tissues is extremely small, and of the same order as the amount of acetyl choline. The Rate of Action ofatropine. When atropine is introduced to a heart depressed by the presence of acetyl choline, the length of time taken by the atropine to produce half its action in antagonising the acetyl choline varies from 20 to 60 seconds. The rate of action of atropine is less than half the rate of action of acetyl choline. Rates of action as rapid as these cannot, however, be determined with any accuracy because of the error

8 554 A. J. CLARK. due to the delay caused by drug diflusing into the sponge-like tissue of the ventricle. The rate of wash-out - of I atropine is very much slower than its rate of action. The rate of wash-out can be calculated by testing the heart repeatedly with acetyl choline after removal of the atropine solution. In this way the concentration of atropine can be calculated that would be needed to produce the observed interference with the action of acetyl choline. Table III shows figures thus obtained, and indicates that about half the drug is removed in about 10 minutes. Acetyl choline is washed out of the heart in about half a minute and therefore the rate of release of atropine is far slower than that of acetyl choline. / 1D -10 _ -111 J ATR. Antagonism of small absolute quantities of acetyl choline by small absolute quantities of atropine. Ordinate: Log. of gram mols. of acetyl choline needed to produce a 50 p.c. reduction in the response. Abscissa: Log. of gram mols. of atropine present. Fig. 7. TABLE III. Effects produced on heart by acetyl choline 10-a6 molar during the wash-out of 1O-5 molar atropine. The above concentration of acetyl choline produced 99-5 p.c. reduction of response in the heart before the introduction of atropine. Time in minutes since atropine removed Percentage reduction in force of response Molar conc. atropine which would diminish the action of molar Ac. Ch. to this response O ^ The Mode of Antagonism of Atropine and Acetyl Choline. The figures in Table III show at once that the antagonism between these two drugs depends upon the atropine being fixed in some manner by the tissues: for the atropine continues to exert its antagonistic action on acetyl choline for a long time after thorough and repeated washing away of all the drug around the heart cells. This excludes the possibility of the antagonism being due to any reaction between the drugs outside the heart cells Ṫhe two drugs do not appear to react when the atropine is fixed on I

9 ACETYL CHOLINE AND ATROPINE. 555 the heart cells, for the application of concentrated solutions of acetyl choline does not hasten the rate at which the atropine is washed out. Straub(9) suggested that atropine rendered tissues impermeable to drugs like acetyl choline. He concluded that in the heart of Aplysia muscarine acted only when entering or leaving the tissues, but he admitted that this was not the case in the frog's heart. There is, indeed, no evidence that the action of acetyl choline depends upon the drug entering the heart cells. I showed in a previous paper(l) that the drug does enter, but that there was no clear relation between the amount entering the heart cells and the amount of action which it produced. The relation found to hold over a wide range of concentrations for the antagonism between acetyl choline and atropine, namely Cone. Ac. Ch. y K. Conc. Atr. 100-y is the same as that found for the antagonism of oxygen and carbon monoxide when the two substances react with haemoglobin. There is, however, a fundamental difference between these two processes because oxygen and carbon monoxide displace each other from combination with haemoglobin. This does not appear to be the case with acetyl choline and atropine, for when a heart which is recovering from atropine is exposed to a concentration of acetyl choline sufficient to produce arrest, this does not increase the rate of recovery of the heart from the atropine. Acetyl choline in excess, therefore, does not react with the atropine to neutralise it, nor does the former drug displace the latter from the heart cells Ȧtropine and acetyl choline, therefore, appear to be attached to different receptors in the heart cells and their antagonism appears to be an antagonism of effects rather than of combination. The evidence available is insufficient to make the publication of further speculation as to the nature of the antagonism profitable. CONCLUSIONS. (1) The action of acetyl choline and atropine on the heart, when both are present, can be expressed by the formula K Cone. Ac. Ch. y * Conc. Atr y (y = action produced by acetyl choline expressed as percentage of maximal possible action, and K is a constant). This formula only holds when atropine is present in a concentration sufficient to produce a wellmarked action.

10 556 A. J. CLARK. (2) The action of the two drugs on the Rectus abdominis can be expressed by the formula: Cone. Ac. Ch. y K (Cone. Atr.)'l y (3) The amount of atropine that unites with the heart muscle is very small. A quantity of 1-4 x grm. mols. per milligram of heart cells suffices to increase tenfold the quantity of acetyl choline needed to produce a given percentage of reduction in the response of the heart. The expenses of this research were covered by a grant from the Government Grants Committee of the Royal Society REFERENCES. 1. Clark. This Journ. 61. p Hufner. Journ. f. prakt. Chem p Douglas, Haldane and Haldane. This Journ. 44. p Cushny. Journ. Pharm. and Exp. Ther. 6. p Broom and Clark. Ibid. 22. p Gaddum. This Journ. 61. p Le Heux, Storm van Leeuwen and van den Brocke. Pfluiger's Arch p Lidth de Jeude. Ibid p Straub. Ibid p