THE CONDUCTIVE system in the

Transcription

1 Acetylcholinesterase in the Conductive Tissue of the Heart By \Y. F. H. M. MOMMAERTS, PH.D., P. A. KHAIRALLAH, M.D., AND MARY FLEMING DICKENS, It is shown that the Purkinje system of bovine hearts contains a specific acetylcholinesterase resembling that of nervous tissue, in an amount comparable -to that of-peripheral nerve. Its possible implication in the conduction of impulses in the heart is discussed. B.A. THE CONDUCTIVE system in the mammalian heart is a specialized part of the myocardium, distinguished by its ability to conduct stimuli at the relatively high rate of 4 meters per second, some 10 times the rate of propagation in the unspecific cardiac musculature. 8 ' ' 8 Since in nervous tissue the conduction of the impulse is correlated with the breakdown and resynthesis of acetylcholine, 21 - " 24 ' 25 we raise the question whether in the differentiation of the Purkinje tissue also the cholinergic system has come into prominence. In this study, we have examined the action of the acetylcholine splitting enzyme, postponing the investigation of the synthesis of acetylcholine, and other aspects of the problem, to future work. Two main types of choline esterases have been distinguished. With regard to its substrate specificity, the acetylcholinesterase of nervous tissue differs markedly from the nonspecific cholinesterase of some other sources. 15 ' 29 Several authors found that the enzyme of the myocardium is predominantly of the unspecific type, different from that of nerve 2 ' 13 ' 26 ' 2 7; it was the aim of the present work to characterize the enzyme from the conductive tissue. From tin; Department of Biochemistry, Duke University School of Medicine, Durham, N. C. This investigation was supported by a Research Giant No from the National Heart Institute of the National Institutes of Health, U. S. P. H. S. This work was done during Dr. Mommaerts' tenure of an Established Investigatorship of the American Heart Association, and during Dr. Khairallah's tenure of a Research Fellowship of the American Heart, Association. Received for publication.june 18, METHODS Preparation of Tissues and Enzyme Solutions.. Most experiments were clone with tissues derived from beef hearts, which were received at the slaughter house* within five minutes of the death of the animal and placed on ice. They were dissected in the cold room, usually within three to four hours. Purkinje tissue obtained from hearts kept in ice is still able to show spontaneous activity after isolation. 12 We usually dissected the entire Purkinje system including the auriculoventricular node, the main bundle and the two bundle branches (yielding Gm. of tissue), several moderator bands from the left and right ventricle, and, occasionally, papillary muscle as a sample of pure myocardial tissue. We also worked with pieces of cat heart ventricles as control material. The tissues, fresh or after storage at 20 C, were ground with sand and extracted with cold water, filtered and centrifuged; the extracts were dialyzed against water for 12 hours and centrifuged if turbid. Extraction was usually conducted so that I Gm. of tissue yielded 10 ml. of extract, final volume. The enzymatic activity of such extracts was stable for a week in the refrigerator. Determination of Enzyme Activity. Cholinesterase was measured electrotitrimetrically at constant ph by means of the glass electrode. 1 - " The relation of the amount of base consumed per mole of substrate, the "equivalence ratio," in general a complicated function of ph and ionic composition, 14 is simple in the present case; since, for all choline derivatives investigated, the liberated acids have pk-values below 5, the equivalence ratio equals unity throughout. The measurements were made at 25 to 26 C., since at physiologic temperature the spontaneous decomposition of the substrate introduces a much larger error. The determinations were made on a small scale in a total fluid volume of 2 or 3 ml. (of which, usu- * We are greatly indebted to the Piedmont, Packing Company at Hillsboro, N. C, for most cooperative assistance. 460 Circulation licscarrh. Volume /, September 1953

2 MOMMAERTS, KHAIRALLAH AND DICKENS 461 ally, 0.5 to 1.0 ml. was the enzyme solution) by means of the apparatus pictured infigure1. The electrodes were connected with a Beckman model G or Cambridge model L ph meter; titration with 0.1 N KOH was done with a microburet permitting the delivery of 0.2 microliters. Unless otherwise specified, the final concentration of each substrate was M and the average ph during the run was 8.0 (fluctuations of 0.2 ph units being due to discontinuous addition of base). Of each substrate, the spontaneous decomposition was measured separately under the same circumstances to arrive at a correction value for each run. Substrates investigated were acetylcholine, acetyl-/3-methyl choline, butyrylcholine and benzoylcholine.* to ph meter of about 0.5 micromoles acetylcholine per minute, at 25 C. This corresponds, approximately, to 10 mg. acetylcholine split per gram tissue per hour at 37 C, the same activity as has been found for the sciatic nerve of the dog. 24 Dependence on ph and Tonic Composition. The determination of the ph-activity curve Q. CO to syringe micro-burette inlet nitrogen gas outlet water bath ACE1fYLCHOL.INE CO UJ Kits. 1. Apparatus for t,ho electrotitrimetric assay of cholinest.orase. RESULTS inlet water both Hydrolysis of Acetylcholine. As an example of the experimental results, figure 2 illustrates the hydrolysis of acetylcholine by extracts of Purkinje tissue and of moderator bands, in comparison with the rate of spontaneous decomposition. It is seen that the activity of the conductive tissue is pronounced, while the activity of the moderator bands was usually about half as great. It was found that, per gram tissue, the bundle of His causes the hydrolysis * Butyiyl and benzoyluholiiie were obtained through the generosity of Hoffmann-LaRoche, Inc., Nutley, N..).; the other substrates were obtained commercially. Some purchased samples wore completely unsuitable. CROMOi TIME IN MINUTES FIG. 2. Hydrolysis of acetylcholine as a function of time. Individual curves refer to hydrolysis by extracts from Purkinje tissue (O) and of moderator bands (A), and by spontaneous decomposition (O). Acotylcholine concentration 0.02 M, ph 7.6. (fig. 3) meets with the difficulty that, as the velocity of enzymatic hydrolysis decreases in the range of higher alkalinity, the spontaneous decomposition rises to such an extent that the measurement of the additional enzymatic reaction becomes inaccurate. The optimum is situated between ph 8 and 8.4, under the circumstances at which it was measured (see the legend of fig. 3). These determinations were done for extracts of the bundle of His, not for the enzyme from other parts of the heart.

3 462 ACETYLCHOLINESTERASE IN CONDUCTIVE TISSUE OF HEART The effects of several cations (as chlorides) were studied in view of the alleged difference in this respect between specific and unspecific cholinesterases. 17 ' 20 ' M - 2S We found that in the present case marked activation effects occur. With KC1, both at ph 7.6 and at ph 8, about 50 per cent activation over the activity in water was reached at 0.05 to 0.1 M above which at most a small depression occurred. With MgCl 2, similar activation was obtained in M solution, above which the "activa- Substrate Specificity. The enzyme extracted from the bundle of His always had the highest activity with acetylcholine, and hydrolyzed acetyl-/3-methyl choline at about one-third to one-half the rate displayed toward the firstmentioned substrate. It had practically no effect upon benzoylcholine (fig. 4) and butyrylcholine. The enzyme from the whole cat heart ventricle did not hydrolyze acetyl-/3-methyl choline, but hydrolyzed benzoylcholine (fig. 4) and butyrylcholine rapidly. Extracts from the moderator bands were more variable in their 0.3- FIG. 3. Dependence of the enzymatic hydrolysis of acetylcholineby Purkinje tissue upon ph. Acetylcholine concentration M, medium 0.05 M KC1. tion did not increase greatly. With CaCb the activation reached up to M was about the same as with MgCU, but the rate decreased rather than increased at higher concentration. Salt effects* were studied at one substrate concentration only, 0.01 M. While all ions tried caused moderate degrees of activation of the enzyme from conductive tissue, the activity of extracts from whole cat heart ventricles were inhibited up to 30 per cent by 0.05 M KC1 and by M CaCl 2. * Since the solutions were not dialyzed exhaustively, these activations by salt are somewhat smaller than the maximal effects reported in other cases O TIME IN MINUTES FIG. 4. Hydrolysis of benzoylcholine by extracts of cat heart ventricle (O), moderator bands (A) and Purkinje tissue (D), and by spontaneous decomposition. activity; sometimes they would attack acetyl- /3-methyl but not benzoyl- and butyrylcholine, while in other samples the latter two substrates would be hydrolyzed. We shall propose an explanation for this variability in the discussion. Effect of Substrate Concentration. We have studied this parameter for the hydrolysis of acetylcholine by the enzyme from the bundle of His (fig. 5) and find that the enzyme acts optimally for a substrate concentration slightly above molar. Other cases were investigated less extensively, but it was found that, while inhibition by excess substrate was characteristic for the enzyme of Purkinje tissue, this effect was not observed for the enzyme

4 MOMMAERTS, KHAIRALLAH AND DICKENS o o <r o I LOG. OF SUBSTRATE CONCENTRATION FIG. 5. Dependence of the enzymatic hydrolysis of acetylcholine by Purkinje tissue upon the substrate concentration; ph 8.0, 0.05 M KCI. from whole ventricles, which showed an increased rate at higher substrate concentration. Effects of Some Pharmacologic Agents. Only observations of an exploratory nature were made, but some of these are of sufficient in a. V) </> UJ _( o o 5 O.I TIME IN MINUTES FIG. 6. Effect of some drugs upon the hydrolysis of acetyleholine by extract from Purkinje fibers; pll 8.0, 0.05 M KCI. Individual curves refer to experiments with enzyme extract only (O); enzyme extract with ouabain, 1:400,000 ( ); enzyme extract with 10~ 6 M eserinc (A); enzyme extract with eserine and ouabain (V); enzyme extract with 3 X 10~ 6 M eserine (+); enzyme extract with eserine and ouabain (X); spontaneous decomposition (D). terest to warrant description. All agents were incubated with the enzyme for one-half hour prior to the addition of substrate. Caffeine (10~ 3 M) caused a 40 per cent inhibition of the enzyme from the bundle of His. It also inhibited the enzyme of moderator bands which was active toward acetyl-js-methyl choline and inactive toward benzoyl- and butyrylcholine, but had only a slight effect upon the extract from moderator bands which hydrolyzed the latter two substrates and in which, possibly, muscle tissue predominated. Eserine at a concentration of 10~ 6 M, caused a 40 per cent inhibition, and at 3 X 10~ 8 M, a 90 per cent inhibition of the enzyme from Purkinje tissue (fig. 6). Ouabain (1:400,000) had a slight stimulative effect by itself but potentiated the inhibitory action of eserine (fig. 6). Ouabain likewise increased the inhibition by caffeine. In these respects the enzyme from the conductive tissue behaved differently from the unspecific cholin esterase from the whole heart. 13 DISCUSSION We wish to discuss the present results in the light of the classification of the choline esterases as specific acelylcholinesterasc and unspecific cholinesterase. The former has its highest activity toward acetylcholine, hydrolyzes acetyl-j8-methyl choline at a lower rate 1 and does not split butyryl- 25 and benzoylcholine 18 ' 19 ' 28 ; it is activated by ions 17 ' 20 ' 23 ; it has an optimum near pll 8.2; and it is inhibited by excess substrate, so that the optimal substrate concentration is about 2 X 10~ 8 M 8-4 ' 6 ; it is inhibited by caffeine. The unspecific cholinesterase does not attack acetyl- 3-methyl choline 1 but does split benzoylcholine, 18 ' l9 and it hydrolyzes butyrylcholine more rapidly than acetylcholine 5 ' - 6 ; it is not inhibited but activated by high concentrations of substrate, within the limits of concentrations investigated 5 ; it is inhibited rather than activated by KCI and CaCl >."' 20 The specific acetylcholinestera.se is a characteristic constituent of nervous tissue and of synapses and motor-end plates, where it is believed to play an essential role in the mechanism of transmission. 21 ' - 5

5 464 ACETYLCHOLINESTERASE IN CONDUCTIVE TISSUE OF HEART It has hitherto been held that the heart contains mainly the unspecific cholinesterase, 2 * ' 27 and we have confirmed this for the unspecific myocardial tissue, and for some samples of the moderator bands. We have demonstrated, however, that the Purkinje tissue contains larger amounts of an acetylcholinesterase, which is of the specific type as judged by its substrate requirements, its dependence upon substrate concentration, its dependence on ions, and its inhibition by caffeine. Actually, the effects of substrate concentration and of ions are mutually dependent 17 ' 20 so that our observations do not cover the problem entirely; they seem to suffice, however, for the identification of the enzyme. In the moderator bands, we sometimes found both the specific and the unspecific acetylcholine-splitting enzyme. In the recent experiments, in which pure white bands were selected which were carefully separated from muscular tissue, the unspecific enzyme was greatly reduced. In such preparations, we were able to detect only Purkinje tissue and connective tissue in histologic preparations stained with Masson's stain.* In other cases, we found significant amounts of muscle as well, and, while our observations are not extensive enough to draw this conclusion with certainty, it would seem that the occurrence of the unspecific enzyme might be ascribed to the presence of such muscular tissue. That all types of moderator strands showed less activity than the bundle of His may be ascribed to the larger amounts of connective tissue in the former. The accumulation of the specific acetylcholinesterase in the Purkinje fibers requires comment. Instrumental in the conduction of impulses, this enzyme seems to become concentrated in those parts of the heart where the conductive function becomes pronounced, although embryologically this tissue is of mesodermal, not of ectodermal origin. Thus, the attainment of a specific biochemical architecture is the result not of morphologic homology * We are indebted to Dr. Duncan Hethcringlon, Department, of Anatomy, Duke University School of Medicine, for his advice and assistance in these observations. or embryologic source, but of functional requirements. With respect to the amount of enzyme present, the Purkinje tissue, as pointed out, is quite comparable to peripheral nerve, to which it also shows resemblance with respect to its physiologic characteristics. 7 ' 8 We must point, however, to one possible uncertainty in the preceding interpretations. Presence of nervous tissue in the bundle of His is not uncommon and could be confirmed by us in histologic preparations. We do not believe that this factor will markedly affect our conclusions since in moderator bands, in which no trace of nervous tissue was detectable, the amount of acetyleholinesterase was nevertheless significant. It may be believed (compare Nachmansohn'- 4 ) that the enzyme is present in sufficient excess in the Purkinje tissue to provide a wide margin of safety for all physiologic requirements in normal cases. However, if we are correct in assuming that the activity of acetyleholinesterase may determine the duration of the refractory period, we consider it possible that the phenomenon of physiologic heart block upon increase of heart rate, or, for example, in uremia, might be due to a decreased amount or activity of the enzyme in the conductive tissue. Our pharmacologic observations require further elaboration before inviting comment. Govier and colleagues 13 have claimed, perhaps not convincingly, that ouabain partially counteracts the inhibition of the unspecific cholinesterase by eserine and other drugs and, consequently, relieves the myocardial failure caused by these poisons. We found, on the contrary, that the inhibition of the specific acetyleholinesterase by eserine or caffeine is not undone but strengthened by ouabain. It is suggestive that ouabain enhances the slowing of perfused fetal hearts caused by acetycholine. 6 Further work is necessary to decide whether the biochemical similarity between Purkinje fibers and nerve tissue is restricted to this one enzyme or extends to other factors as well. We obtained, in preliminary analyses, lower amounts of nucleotide and of creatine than in muscular tissue, and refer to the interesting results of Davies and co-workers, 9 showing a

6 MOMMAERTS, KHAIRALLAH AND DICKENS 465 K:Na ratio in the bundle tissue resembling that in nerve. SUMMARY A method is described for the electrotitrimetric determination of cholinesterases in small samples, and its application to different tissues of the heart. It is shown that the conductive system of the heart contains a cholinesterase which is similar to the specific acetylcholinesterase of nervous tissues in all respects: substrate specificity, dependence on substrate concentration, ph optimum, activation by salts, inhibition by caffeine. Effects of some pharmacologic agents are described. The enzyme occurs in the Purkinje tissue in about the same amount as in typical peripheral nerves. REFERENCES 1 ALLES, G. A., AND HAWKS, R. C: Cholinesterases in the blood of man. J. Biol. Chem. 133: 375, ANTOPOL, W., GI.AUBACH, S., AND GLICK, D.: Cholinesterase activity in various portions of the rabbit heart. Proc. Soc. Exper. Biol. & Med. 42: 280, AUGUSTINSSON, K. B.: Cholinesterases, a study in comparative enzymology. Acta physiol. scandinav. 16: suppl. 52, * -: Acetylcholine esterase and choline esterase. In Sumner and Myrback, eds.: The Enzymes. New York, Academic Press, Vol. 1, p : Substrate concentration and specificity of choline ester splitting enzymes. Arch. Biochem. 23: 111, BAKER, Y. B. E.: Some observations upon isolated perfused human foetal hearts. J. Physiol. 120: 122, CORABOEUF, E., AND WEIDMAN, S.: Potentiels de repos et potentiels d'action du muscle cardiaque, mesures a l'aidc d'ele'ctrodes internes. Compt. rend. Soc. biol. 143: 1329, 1360, CURTIS, H. Y., AND TRAVIS, D. M.: Conduction in Purkinje tissue of the ox heart. Am. J. Physiol. 165: 173, DAVIES, F., DAVIES, 11. E., AND FRANCIS, E. T. B.: The sodium and potassium content of cardiac and other tissues of the ox. J. Physiol. 118: 270, FROMMEL, E., HEHSCHBERG, A. D., AND PIGUET, J.: Effets des ions inorganiques sur I'activit6 de la cholinest6rase seyique. Helvet. physiol. et pharmacol. acta 2: 169, GLICK, D.: Properties of choline esterase in human serum. Biochem. J. 31: 521, , GpLDENBERG, M., AND ROTHBERGEH, C. Y.: Ueber das Elektrogramm der Spezifischen Herzmuskulatur. Pfliiger's Arch. ges. Physiol. 237: 295, GOVIER, WM. M., FHEYBUUGEU, W. A., GIBBONS, A. J., HOWES. B. G., AND SMITH, E.: The relation of the choline cycle to cardiac decomposition; acetylcholine metabolism in the dog heart-lung preparation. Am. Heart J. 45: 122, GREEN, I., AND MOMMAERTS, W. F. H. M.: Adenosine triphosphate systems of muscle. I. An electrotitrimetric method of determination. J. Biol. Chem. 202: 541, KOELLE, G. B.: The histoehemical differentiation of types of cholinesterase. 3. Specific tests for true cholinesterase and pseudo-cholinesterase. J. Pharmacol. & Exp. Therap. 100: 158, LEWIS, T.: The mechanism and graphic registration of the heart beat, cd. 3. London, Shaw, VAN DER MEER, C.: Effect of calcium chloride on choline esterase. Nature 171: 7S, MENDEL, B., MUNDBLL, D. B., AND RUDNEY, H.: Studies on cholinesterase. 3. Specific tests for true cholinesterase and pseudo-cholinestcrase. Biochem. J. 37: 473, , AND RUDNEY, H.: Studies on cholinesterase. 1. Cholinesterase and pseudocholinesterase. Biochem. J. 37: 53, , AND : Some effects of salts on true cholinesterase. Science 102: 616, MINZ, B.: La transmission chimique de l'influx nerveux. Paris, Ed. Med. Flammarion, MURALT, A. VON: Observations on chemical wave transmission in excited nerves. Proc. Roy. Soc, London, s. b. 123: 399, NACHMANSOHN, D.: Action of ions on choline esterase. Nature 145: 513, : Chemical control of nervous activity. A. Acetylcholine. In Pincus, G. and Thimann, K. V.: The Hormones. New York, Academic Press, : Chemical mechanisms of nerve activity. In Ban-on, E. S. G.: Modern Trends in Physiology AND Biochemistry. New York, Academic. Press, (1, AND RoTHENBEHG, M. A.: Studies OH cllolinesterase. I. On the specificity of the enzyme in nerve tissue. J. Biol. Chem. 168: 653, ORD, M. G., AND THOMPSON, R. H. S.: The distribution of cholinesterase types in mammalian tissues.jbiochem. J. 26: 346, WHITTAKER, V. P.: Specificity, mode of action and distribution of cholinesterases. Physiol. Rev. 31:312, 1951.