Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Heart failure wikipedia , lookup
Rheumatic fever wikipedia , lookup
Myocardial infarction wikipedia , lookup
Cardiac surgery wikipedia , lookup
Atrial fibrillation wikipedia , lookup
Electrocardiography wikipedia , lookup
Arrhythmogenic right ventricular dysplasia wikipedia , lookup
Acetylcholine and Electrolyte Metabolism in the Various Chambers of the Frog and Turtle Heart By PAUL MAZEL, M.S., AND AVILLIAM C. HOLLAND, M.D. Evidence has accumulated suggesting- ncetylcholine may be involved in initiation of the heart bent. Acetyleholine and electrolyte metabolism in the various chambers of the heart have been studied. The area of highest intrinsic rhythm (sinus) contained greater amounts of acetylcholine equivalents, true cholinesterase, choline acetylase and sodium; all of these decreasing in amount in areas of lower intrinsic rhythm. It is concluded that intrinsic rhythm may be directly correlated with overall acetylcholine metabolism and sodium content and inversely related to overall energy metabolism and potassium concentration. Downloaded from http://circres.ahajournals.org/ by guest on September 11, 2016 O A^ER the past two decades evidence has accumulated which suggests that acetylcholine plays a role in the initiation of the heart beat. As early as 1937, Sachs 1 found that small doses of acetylcholine exerted a stimulant effect on rabbit atria and AVelsh2 in 1938 reported that small amounts of acetylcholine increased the rate of the crab heart. Later Abdon and Hammarskjold 3 ' 4 reported on an acetylcholine precursor which yielded free acetylcholine upon heating or acidification. The mechanical activity of the heart appeared to vary directly with the amount of precursor present. The findings have been extended and confirmed by Burn, SpadoLini and coworkers.5' ° I t has been suggested by the latter group of investigators that acetylcholine induces the automatic rhythm and regulates the rate and force of beat. Receutly, it has been postulated by Holland 7 that acetylcholiue might act by virtue of its ability to increase membrane permeability to sodium aud potassium ions. Thus the intrinsic metabolism of aeetylcholine iu pacemaker tissue may be of such a nature that periodic chauges in permeability occur which give rise to impulses at regular intervals. If the above hypothesis be true then one would expect that the region of the heart with the higher intrinsic rhythm would exhibit a greater overall metabolism of acetylcholine. There have been scattered observations on acetylcholine metabolism in the various regions of the heart. In general, these studies were incomplete in that most of them have been confined to atria and ventricles. In order to pursue the matter further we have made a study of the acetylcholine and electrolyte metabolism in the several chambers of the frog and turtle heart. METHODS The turtle (Psuudemys scripta) and the bull frog (Rana eastesbiana) were used in all experiments. In these studies pooled sinuses, ntriu and ventricles were used. Therefore, in a number of cases, only mean values are presented and no statistical analysis of the data was undertaken. The aeetylcholine content of the various chambers was determined by quickly excising the tissue, weighing and homogenizing in ice cold 10 per cent trichloroacetic acid (TCA) 3 to 4 ml./Gni. tissue. After centrifugation, TCA was removed by extraction with ether and the solution then freed of ether by aeration. Aliquots of the extract were neutralized with a few drops of saturated sodium bicarbonate solution just prior to assay on the eserinized (1.0 X 10~5) frog rectus abdominis muscle. The acetylcholine equivalent was determined using known amounts of acetylcholine bromide as standard. Precautions of Feldberg and Hebb8 were observed and the extract again tested after alkalinization with 1.0 X XaOH and heating. Choline acetylase activity was determined by a modification of the method described by Hebb and AVaites.8 Acetone dried powders were pre- From the Department of Pharmacology, Vanderbilt University School of Medicine, Xnshville, Tenn. Supported by U. S. Public Health Service grant H-1679(c) and the American Heart Association. Keeeired for publication April 9, 1958. 684 Circulation. Research, Volume VI. November 1BSS ACETYLCHOLIXE AND ELECTROLYTE METABOLISM 685 TABLE 1.—Acetylcholine Metabolism of the Sinus, Atria and Ventricle Average of 5 Observations Turtle tissue ACh equlv. /iK./Gm. (Wet weieht) Sinus venosus 5.0 (3.15 - 8.30) Cholinesterase activity p\. COi/Gro./hr. ACh 154 Ventricle 0.99 (.51 • 2.29) 81 Downloaded from http://circres.ahajournals.org/ by guest on September 11, 2016 # Pabgt Laboratories, Milwaukee, Wisconsin. 280 (263 - 291) 52.S 188 (126 - 316) 36.0 104 (54.0 - 160.0) that of the atria while the latter is, in turn, above that of the ventricle. The quantity of acetylcholiue (ACh), cholinesterase and choline acetylase activities in the various chambers of the frog and turtle heart are summarized in tables 1 and 2. It should be noted that the ACh equivalent in the sinus is more than twice that of the atria and the atria, in turn, greater than the ventricle. The cholinesterase activities show a similar distribution pattern as did choline acetylase activity. The results on the frog shown in table 2 presented a similar pattern. Acetylcholine equivalent in the sinus M'as greater than that of the atrium and the atrium above that of the ventricle. The frog ventricle displayed a proportionately greater ACh equivalent than did the turtle ventricle. Table 3 summarizes the studies on cation distribution in the turtle heart. It should be noted that the sodium content of the sinus is much greater than the atria and the atria, TABLE 2.—Acetylcholine Content and Cholinenteraxe Activity of the Sinus, Atria and Ventricle of the Frog Heart. Average of 5 Observations Froff tissue RESULTS The inherent rhythmicity of various heart areas has been studied for many years. It is now generally accepted that the rate of intrinsic beat of the sinus is greater than MeCh 193 2.0 (1.53 • 3.0) Heart. Choline acetylase H%. ACh equiv. formed/Gin, powder/hr. 480 Atria pared by grinding the tissue in 100 volumes of ice cold acetone (—10 C.) in a Waring Blendor. This solution was then filtered through a Buehner funnel and the powder dried in air several min. and stored in vaeuo until ready for use. The powder was then weighed and an extract prepared by grinding in cysteine-saline (3 ing. Z-cysteine/ ml.). The suspension was centrifuged and the supernatant diluted to contain approximately 50 to 250 ing. powder/ml. Coenzyme A was obtained commercially* and prepared so each 0.1 ml. contained 70 units. The incubation mixture was the same as described by Hebb and Waites" except for the frozen liver enzyme. The time of incubation WILS 1 hour at 37 C. The amount of acetylcholine formed was then measured on the frog rectus as previously described. Cholinesterase activity of pooled sinuses, atria and ventricular slices was determined manometrically using Ringer-Bicarbonate buffer at 37 C. in an atmosphere of carbon dioxide and nitrogen. Acetylcholine bromide (ACh) and aeetyl-B-methy] choline (MeCh) were used as substrates in final concentration of 5 X 10~:s M. Sodium and potassium content was determined by analyzing TCA extract of the appropriate tissue by flame speetrophotometry. Magnesium was determined by the Titan Yellow method of Orange and Rhein10 using a Beckman Model B spectrophotometer. Calcium was determined by a colorimeter adaptation of the ammonium-purpurate titration described by Mendelsohn11 using a KlettSummerson colorimeter with blue filter. The indicator was stabilized by dissolving it in a wateralcohol mixture as suggested by Williams.12 of the Turtle ACh equiv. (/ij./Gm,, wet weight) Cholinesterase (/il. COi/Gm./hr.) ACh MeCh Sinus venosus 5.0 (4.78 - 5.34) 794 322.5 Atria 3.1 (2.16-4.01) 280 80.0 Ventricle 2.9 (2.33-3.30) 132 50.0 686 TABLE 3.—Cation Distribution in the Various Chambers of the Turtle Heart, Determinations Made on Trichloroacetic Acid Extract of 6 Pooled Sinuses, Atria and Ventricles Turtle tissue Na+ Sinus venosus 129.5 Atria 64.00 Ventricle 49.8 56.8 57.8 66.0 2.29 1.11 0.75 3.5 3.5 1.4 2.3 3.2 5.0 Downloaded from http://circres.ahajournals.org/ by guest on September 11, 2016 TABLE 4.—Cation Distribution in the Various Chambers of the Frog Heart, Determinations Made on Trichloroacetic Acid Extract of 6 Pooled Sinmes, Atria and Ventricles Sinus venosus Atria Ventricle Na+ 3 24 87 49.7 "I Cations, (mM/Kj., tissue) K+ Na+/K+ Ca++ Mg+ in turn, greater than the ventricles. The reverse is true with potassium; potassium being greatest in the ventricle but approximately the same in the atria and sinus. The sodium-potassium ratio shows a progressive decline from sinus to atria to ventricle. Similar sodium aud potassium distributions were observed in the frog heart (table 4). The high concentration of Na in the sinus could be the result of a large extracellular space in this tissue. This seems unlikely in view of the data presented in table 5. In these experiments, sinuses and atria were incubated for one hour in an Na deficient medium (28 mMolar). Isotonicity was maintained by the addition of sucrose. Even though incubating the tissues in a low Na medium reduces the Na content in both chambers, the ratio of Na concentration in the sinus to that in the atria remains essentially the same. (Compare data, in table 5 with those in table 3.) Calcium was found to be identical in amount in the sinus and atria with far less in the ventricle. With respect to magnesium, the content was similar in the sinus and atria but greater in the ventricle. Figure 1 is a summary of some of the data. Here acetylcholine equivalents, eholinesterase Frog tissue HOLLAND MAZFJJ, Cations K+ 44.5 42.2 68.2 (mM/Kg.) Na+/K* 2.79 1.97 0.73 O IC 20 1.8 1.8 2.0 RHYT™ d'O 50 SO BEATS/VIUMJTE FIG. 1. Summary of data. Intrinsic rhythm of turtle sinus = 46; atria = 29; ventricle = 13. These are mean values obtained from observations on 3 different preparations. and choline acetylase activities and Na/K ratios are plotted against intrinsic rhythm of the various chambers. It should be noted that the tissue with a higher inherent rhythm (sinus) contains a greater amount of ACh equivalents, eholinesterase and choline acetylase activity and higher sodium-potassium ratio; all of these factors decreasing in amount in those areas of lowered inherent rhythm (atria and ventricle). DISCUSSION On more than one occasion, attempts have been made to correlate intrinsic rhythm of the various chambers of the heart with metabolic patterns in these separate regions. For example, glycogen is said to be highest in pacemaker tissue.18 On the other hand, an inverse correlation has been found for succinic dehydrogenase,14 oxygen consumption,15 and content of adenosine and its derivatives.18 TABLE 5.—The Effect of Na Deficient Medium on the Na and K Content of Sinuses and Atria of the Turtle Heart. Temperature 27 C. Na Concentration in Medium: 28m Molar. Duration of Incubation 1 Hour. Medium Changed Every 10 Minutes Cations Ca« 30 INTTHN9C Tissue experiments Sin us Atria 8 8 (Mm/Kg. , Na 68.6 ± 12 36.4 ± 8 wet tissue) Ni K 56 63.6 •+2 7 9 1..22 0..57 AC'ETYU'HOLIXE AXD ELECTROLYTE METABOLISM Downloaded from http://circres.ahajournals.org/ by guest on September 11, 2016 In fact, the work load of the several chambers bears a direct correlation with overall energy metabolism. In the present study evidence is presented which shows a direct relation between the rate of intrinsic rhythm and aeetyleholine metabolism. On the other hand, these investigations may well represent a comparison of the distribution of nervous elements in these regions of the heart. This is reasonable in light of the known role of acetyleholine in nervous activity. However, it has been shown histochemically and enzymatically18 that pacemaker and Purkinje tissue contain large amounts of acetylcholinesterase. In addition, acetyleholine is not always associated with nervous tissue as evident by the large amounts found in the placenta17 and spleen. Burn" has recently reported the presence of the acetylcholinecholinesterase system in the gill plates of the mussel Mytilus edulis, a nerveless structure. Girvin and Stevenson18 have described a choline acetylating system in Lactobacillus plant arum. The presence of large amounts of sodium in pacemaker tissue is very interesting. Similar findings have been described by Davies19 et al. for the ox heart. This could be the result of the high intrinsic metabolism of acetyleholine in the sinus venosus as it is known that acetylcholiue will release potassium from and cause an uptake of sodium by myoeardial tissue.7 However, the full significance of these findings requires further study. Although the evidence at present is curcumstantial in nature, it is quite possible that synthesis and destruction of acetyleholine with its known effects on cell permeability may play a fundamental role in the genesis of the heart beat. SUMMARY Acetyleholine metabolism, including cholinesterase and choline acetylase, in the various chambers of the frog and turtle heart have been studied. Cation distribution (Xa+, K+, Ca<+, Mg'*) have been included. In both species the ACh equivalents (Gm/wet wt.) 6S7 were as follows: turtle, sinus 5.0, atria 2.0, ventricle 0.99; frog, 5.0, 3.1, 2.9. True cholinesterase values (/il./CO2/Gm./hr.) were: turtle, 480, 154, 81; frog, 794, 280, 132, respectively. Choline acetylase activities (^.g./ ACh/formed/Gm. powder/hr.) in the turtle were: 280, 188, and 31.8. Sodium content for both species was highest in the sinus, less in the atria and least in the ventricle. The reverse was true for potassium. Calcium and magnesium contents of both sinus and atria were approximately the same. It is concluded that intrinsic rhythmicity of the various chambers may be directly correlated with over-all ACh metabolism and sodium content, and inversely related to over-all energy metabolism and potassium concentration. SUMMARIO IN INTERLINGUA Esseva studiate le metabolismo de acetylcholina—incluse cholinesterase e cholinacetylase—in le varie cameras del corde de ranas e tortucas. Le distributiones cationic (Xa+, K*, CaT', Mg*+) esseva includite in le studio. Le valores de acetylcholina in sinus, atrios, e ventriculo—exprimite in /,g per g de peso humide—esseva 5,0, 2,0, e 0,99, respectivemente. in le caso del tortuca e 5,0, 3,1, e 2,9 in le caso del rana. Le valores correspondente pro ver cholinesterase—exprimite in fA de CO2 per g per hora—esseva 480, 154, e 81 in le caso del tortuca e 794, 280, e 132 in le caso del rana. Le valores correspondente del activitate de cholinacetylase—exprimite in /xg de acetylcholina formate per g de pulvere per hora—esseva 280, 188, e 31,8 in le caso del tortuca. In ambe species—tortuco e rana —le contento de natrium esseva le plus alte in le sinus e le plus basse in le ventriculo. Le contrario valeva in le caso de kalium. Le contento de calcium e de magnesium in sinus e in le atrios esseva approximativemente le mesme. Es concludite que le rhythmicitate intrinsec del varie cameras es forsan directemente correlationate con le metabolismo general de acetylcholina e le contento de natrium e inversemente con le metabolismo general de energia e le concentration de kalium. MAZEL, HOLLAND 688 REFERENCES 1. SACHS, A.: Effects of choline compounds on auricle. Cardiologia 1: 74, 1937. 2. WELSH, J. H.: Occurrence of acetyleholine in nervous tissue of crustaceans. Nature 142: 151, 193S. 3. ABDON, X. 0 . : On the metabolism of acetyleholine precursor in isolated hearts. Acta pharmacol. et toxicol. 1: 169, 1945. 4. ABDON, X. O., AND HAMMARSKJOLD, S. 5. Downloaded from http://circres.ahajournals.org/ by guest on September 11, 2016 6. 7. S. 11. MENDELSOHN, D.: A micromethod for the photometric determination of calcium in serum with ammonium purpurate. South African J. M. Sc. 21: 57, 1956. 12. WILLIAJIS, M. B., AND MOSER, J. H.: Colori- metric determination of calcium with ammonium purpurate. Anal. Chem. 26: 1414, 1953. 13. DAVIES, F., FRANCIS, E. T. B., AND STONES, A. B.: The distribution of nucleotide, phosphocreatine and glycogen in the heart. J. Physiol. 106: 154, 1947. 0.: Is any free acetyleholine preformed in resting muscles or in the heartT Acta physiol. scandiiiav. 8: 75, 1944. BURN, J. H.: Functions of autonomic transmitters. Baltimore, Williams & Wilkins Company, 1956, pp. 18-61. SPADOLINI, I.: Risposte graduate e risposte massimali cardiache in rapporto al metabolismo awiticolinico della fibra miocardica l'inbizione vagale. Arch, fisiol. 52: 443, 1953. HOLLAND, W. C.: Studies on permeability. VII. Role of acetyleholine metabolism in the genesis of the electrocardiogram. Am. J. Physiol. 170: 339, 1952. FELDBERG, W., AND HEBH, C.: The effects of magnesium ions and of creatine phosphate on the synthesis of aeetylcholine. J. Physiol. 106: S, 1947. 9. HEBB, C. 0., AND WAITES, G. M. H.: Choline acetyla.se in antero and retrograde degeneration of a cholinergic nerve. J. Physiol. 132: 667, 1956. 10. ORANGE, M., AND RHEIN, H. G.: Microestimation of magnesium in body fluids. J. Biol. Chem. 189: 379, 195]. 14. COOPER, W. G., AND COPENHAVER, W. M.: The distribution of snecinic dehydrogena.se within the heart of urodUe amphibian. Anat. Rec. 126: 503, 1956. 15. CLABK, A. J., AND WHITE, A. C.: The oxygen consumption of the auricles of the frog and of the tortoise. J. Physiol. 68: 406, 1930. 16'. MOMMAERTS, W. F. H. M., KHAIRALLAH, P. A., AND DICKENS, M. F . : Acetylcholines- terase in the conductive tissue of the heart. Circulation Research 1: 460, 1953. 17. COMLINE, R. S.: Synthesis of acetyleholine by non-nervous tissue. J. Physiol. 105: 6 P, 1946. IS. GIRVIN, G. T., AND STEVENSON, J. W.: Cell free "choline acetylase" from lactobacillus plantarum. Canad. J. Biochem. Physiol. 32: 131, 3954. 19. DAVIES, F., DAVIES, R. E., FRANCIS, E. T. B., AND WHITTAM, R.: The sodium and potas- sium content of cardiac and other tissues of the ox. J. Physiol. 118: 276, 1952. Acetyicholine and Electrolyte Metabolism in the Various Chambers of the Frog and Turtle Heart PAUL MAZEL and WILLIAM C. HOLLAND Downloaded from http://circres.ahajournals.org/ by guest on September 11, 2016 Circ Res. 1958;6:684-688 doi: 10.1161/01.RES.6.6.684 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1958 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/6/6/684 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/