EFFECTS OF CAFFEINE ON THE MEMBRANE POTENTIALS, MEMBRANE CURRENTS AND CONTRACTILITY OF THE BULLFROG ATRIUM

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1 Jap. J. Physiol., 24, , 1974 EFFECTS OF CAFFEINE ON THE MEMBRANE POTENTIALS, MEMBRANE CURRENTS AND CONTRACTILITY OF THE BULLFROG ATRIUM Yosiko KIMOTO, Masahiko SAITO, Masayosi GOTO Department Physiology, School Medice, Kyushu University, Fukuoka, Japan Summary 1. Effects caffee on action potentials, s contractile responses bullfrog atrial muscle were vestigated by means double glycerol-gap technique. 2. Caffee, 3, 10, 50 mm concentration, caused a potentiation twitch contraction without producg contracture. potentiation larger higher concentration. duration action potential as well as relaxation time contraction prolonged. 3. Under voltage clamped conditions constant tetrodotox (2.5 ~10-7g/ml), 5 mm caffee produced an augmentation a rapid onset active slow ward, as well as contractile, decreased cord resistance. 4. curve voltage- relationship shifted to left upward by 5 mm caffee. Maximum augmentation apped for depolarizg mv. 5. In a preparation pretreated with 4-fold Ca Rger solution, 10 mm caffee duced a contracture which not abolished by 1 mm La In 1 mm La+++, 10 mm caffee produced an augmentation twitch contraction a transient contracture. Caffee also augmented a sodium-free contracture under La se results terpreted as dicatg that: a) Caffee acts on both surface sarcoplasmic reticulum. b) It enhances ward calcium augmentg contraction directly directly through sarcoplasmic reticulum. c) In normal Rger solution, amount accumulated calcium is very small but is creased by pretreatment calcium-rich solution. d) La+++ hibits both ward outward trans calcium movements. In skeletal muscle, caffee, 2-4 mm concentration, causes a prolongation active state (RITCHIE, 1954) an crease rate develop- Received for publication June 22,

2 532 Y. KIMOTO, M. SAITO, M. GOTO ment (SANDOW et al., 1965), but does not significantly alter restg potential shape action potential (SANDOW et al., 1964; SANDOW, 1965). In higher concentrations, above 5 mm, its predomant effect is to cause contracture. Effects caffee on mechanical electrical properties cardiac muscle have also been vestigated by several authors (SUZUKI, 1962; NAYLER, 1963; DE GUBAREFF SLEATOR, 1965; CLARK OLSON, 1968; CARMELIET VEREECKE, 1969), it has been shown that caffee not only potentiates contraction but alters duration action potential, dicatg a defite action on surface. However, little has been studied about relationship between alterations action potential contractility, while site action caffee has not yet been fully vestigated. In our precedg report (KIMOTO, 1972), effects caffee on action potential contractile response were studied on atrial muscle guea pig, prolongation action potential duration decrease slope resistance were observed, which dicate an enhancement ward calcium movement occurs. refore, present experiments were carried out to vestigate effect caffee on s, action potentials contractile responses bullfrog atrial muscle, which s were well analysed by voltage clamp technique (ROUGIER et al., 1969; VASSORT ROUGIER, 1972; LEOTY RAYMOND, 1972). METHODS atrial muscle bullfrog, Rana catesbiana, used. When left atrium opened, auricular trabeculae which run to atrio-ventricular valves were exposed. A th strip those fiber bundles which apped as parallel as possible isolated under a bocular microscope. length strip about 5 mm, diameter mm. preparation mounted a perfusion bath at room temperature (17-25 Ž). action potentials contractile responses muscle were measured usg double glycerol-gap technique. In se cases, width central compartment 2 mm for -obtag larger. Details experimental setup were described our previous paper (GOTO et al., 1972). Occasionally potentials, s, contractile responses muscle were also measured under voltage clamped conditions usg a narrower central compartment mm width. methods for voltage clampg those for measurg voltage,, were same as before (GOTO et al., 1974). composition normal Rger solution, mm/liter: NaCl, ; KCl, 2.56; NaHCO3, 2.38; CaCl2 2H2O, 2.18; KH2PO4, 0.34; Glucose, ph this solution 7.4. Sodium-free solution prepd by

3 CAFFEINE ACTION ON MYOCARDIUM 533 substitutg Tris-chloride for sodium chloride, sodium bicarbonate potassium monophosphate. ph adjusted to 7.4 by addg hydrochloride. RESULTS I. Effects caffee on contractility Figure 1 shows effects caffee on twitch contraction bullfrog atrial muscle. Caffee at any concentrations 3, 10, 50mM caused a potentiation twitch contraction. itiation potentiation rapid, degree potentiation larger, higher concentration caffee. However, potentiation followed by a slightly depressed state which most proment case 50mM caffee. When 50mM caffee concentration hed out by normal Rger solution, a transient crease twitch contraction educed (Fig. lc). Fig. 1. Effects caffee on twitch contraction. (A) 3mM, (B) 10mM (C) 50mM caffee were applied. Arrows denote time application ( «) hout ( ª) caffee. In each record, upper tracg is twitch contraction lower tracg, time mark. Rate stimulation 0.1 shock/sec. time mark is 0.2 sec 5 m for fast slow records, respectively. Vertical bars calibrations, 400mg for all records. Figure 2 shows simultaneous records twitch contraction time derivative before addition 10mM (A) 50mM (B) caffee. As can be seen, caffee produced a proment crease maximum rate rise total duration contraction. maximum rate fall did not crease parallel with maximum rate rise, but rar decreased at depressed state 50mM caffee. Figure 2 (C-F) shows relative changes se parameters which plotted agast time addition caffee. time to peak not changed or rar shortened

4 Y. KIMOTO, 534 somewhat contraction M. 10 creased, SAITO, M. GOTO 50 mm caffee. relaxation Sce time evidently total duration creased by caffee. A B Fig. 2. Changes (A, B) time 500, (B) course (b, c, d, D) 0.5 Those ( œ), %. trace), e). (a) In B, C) contraction. mm caffee. peak All values durg b, 70, c, calibrations time E) circles d, rate expressed 800 rise sec mg. 560 each 10 percentages, (A) records 200, 300, addition Horizontal bar is application, its mm effects caffee. figure) e peak show addition 420, to ( ) trace 210,, Open (lower A, bars Changes contraction Figure those Vertical sec. twitch contraction before Figure respectively. duration as time records sec, caffee. taken F caffee 600 caffee, circles D (upper calibration total E Simultaneous derivative 50 mm C 10 mm control F) closed beg

5 CAFFEINE ACTION ON MYOCARDIUM Effects caffee on gap potentials contractile Figure 3 shows superimposed records action potentials isometric contractile s before (control) durg 3 mm (a), 10 mm (b), 50 mm (c) caffee. As can be seen, most proment effect caffee on action potential a prolongation later phase repolarization. Correspondgly total duration contraction creased. degree prolongation repolarization larger higher concentration caffee. Gap-restg potential not significantly altered under fluence 3 10 mm caffee, but it slightly decreased case 50 mm caffee. This can be seen as broadness basal le superimposed records potential. Overshoot action potential not altered or slightly decreased with restg potential. rate rise depolarization, even when examed faster records, not changed cases 3 10 mm caffee, but it decreased case 50 mm caffee. Fig. 3. Effects caffee on gap potentials twitch contractions. potential contraction were recorded simultaneously. rate stimulation 1/sec. (a) Superimposed records control 4th, 8th, 10th, 12th, 14th responses addition 3 mm caffee. (b) Control 4th, 6th, 10th, 12th responses 10 mm caffee. (c) Control 4th 8th responses 50 mm caffee. Vertical bars calibrations 50 mv 100 mg. Horizontal bar is time calibration 1 sec. 3. Effects Figure contractile constant 50 When enhanced contractile held mv reduction sec duration 5 mm wardly-directed by sec restg an rate 10-7 level (-63 mv) applied. durg In this rate depolarizg caffee case, (Fig. crease rise rectangular troduced 5 mm condition apped, augmented. ward clamped g/ml). were slow addition caffee contractile voltage (2.5 ~ enhancement fall markedly records under tetrodotox at produced simultaneous caffee a concentration slow, before, hout on shows potential caffee 4 pulse, 90 sec depolarizg 4b, c, d), a strongly correspondgly, contractile development, consequent pro-

6 536 Y. KIMOTO, Fig. 4. Simultaneous normal (a records addition clamped under tracgs constant 1 sec given at 20mg. (b, sec is record tervals. Vertical is upper lower depolarizg bars a calibration, before voltage voltages, is -63mV bar a (2.5 ~10-7g/ml). tracgs, Horizontal contractile d). hout. potential 10 c, tetrodotox middle Holdg GOTO voltage caffee record s, 100mV, 5mM s. A, e, M. calibrations 1 sec. contraction duration. latencies both slow ward curcontraction tended to be shortened with time addition caffee. forward peaks both slow ward contraction apped earlier than those control. outward 50mV contractile 5 ~10-6 longation rent caffee, tracgs, e) M. SAITO, at end depolarizg 5mM caffee, suggestg a decrease also shifted pulse creased cord resistance. 4. Effects he caffee relation control (2.5 ~10-7g/ml) held is not much which were duration short 150mV or, responses superimposed. rise for (one examed. first phasic can be upward by potential seen, curve addition near Maximum curve threshold augmentation 60-80mV mV tetrodotox broken curve control. step ward ROUGIER, control depolarizg threshold 180mV) left As caffee. above paired 1.0sec, calcium depolarizg (VASSORT muscle, case, applied. to augmented atrial this potential rectangular mechanical depolarizg frog were constant In figure, 5A. shifted change for development 1 sec under Figure -70mV this rapid for tions excess above level clear relationship caffee relationship maximum 5mM more Sce restg apped shows tion a Although caffee voltage- developed voltage- caffee. case tensities is illustrated at various on between 1972; for 20mM is almost LEOTY 0.17 sec Figure caffee known at become disappears RAYMOND, 75mV 5B by for 1972), depolariza- shows contag produced to Rger short results solu

7 CAFFEINE ACTION ON MYOCARDIUM 537 A Fig. 5. A) Voltage- contag relationships 5 mm caffee (2.5 ~ 10-7 g/ml). Mean ference between open circles as Duration tonic depolarizg (1.0 sec, 180 A, my). records 50 (c), mv respectively. prcipally beg for Horizontal B) Effects is 20 tonic, a a calibration on a long (i), bars voltage 0.4 with scale (-70 short contag Vertical caffee dif- curve voltage level by by caffee dots. mm potential elicited solution tetrodotox plotted. le. caffee. holdg were a broken with bar with experiments from control labeled hout phasic (squs) constant is shown sec. mv) responses mg series solution depolarization sec, latter pulse markedly tion apped pulse (0.17 four curves components. pulse superimposed, 10-5 step two recovery step control under a values (triangles) values dicates expressed weak B strong is mv). phasic weak pulse solutions calibrations (v) sec. augmented caffee dependg on crease rate augmenta rise, while second tonic for longer stronger pulse as well as rate rise tonic not altered. outward at end second depolarization, however, considerably creased by caffee. Similar results were also obtaed case solution contag 5 mm caffee. 5. effect caffee on contractile La+++ Lanthanum ions (La+++) known to block Ca entry various preparations, e.g., barnacle muscle fibers (HAGJWARA TAKAHASHI, 1967) mammalian cardiac tissue (SANBORN LANGER,1970). Figure 6 shows effect La+ + + on action potential twitch contraction bullfrog atrial muscle. As seen, 1 mm La+ + + shortened plateau, decreased overshoot action potential depressed markedly twitch contraction. followg examations were carried out La+ + +, order to vestigate characteristics caffee action elimation Ca entry.

8 538 Y. KIMOTO, M. SAITO, M. GOTO Fig. 6. Effects 1 mm La+ + + on action potential twitch contraction. upper tracg is potential (V), middle, contractile (C) lower, time mark (T). right-h figure shows superimposed tracgs action potentials control 1 mm La Vertical bars calibrations 50 mv 400 mg. time marks dicate 1.0, 0.2 sec 1.0 m for fast, middle, slow records, respectively. Fig. 7. Effects 1 mm La mm caffee on contractile. Arrows denote time application ( «) hout ( ª) drugs. A) effects on normal preparation. B) Those on preparation pretreated with 4 ~ Ca Rger solution. Vertical bars calibrations 400 mg. time marks dicate 0.2 sec for fast 5 m for slow records. Furr explanations given text. Figure 7A shows effect caffee on contractile La An addition 10 mm caffee 1 mm La+ + + produced a transient contracture, which unexpected, sce caffee alone, even 50 mm, never elicited contracture (Fig. 1). A potentiation twitch contraction also apped duration contraction creased markedly. Figure 7B shows effect caffee La+ + + on contractile preparation pretreated with 4-fold CaCl2-contag Rger solution. pretreatment produced a more-than-twold augmentation twitch contraction. In this preparation, 10 mm caffee duced a small but sustaed

9 CAFFEINE ACTION ON MYOCARDIUM 539 contracture which not abolished by an addition 1mM La+++. twitch contraction which no longer augmented by caffee completely elimated by 1mM La Effects caffee on sodium free contracture La+++ Several authors have reported effects caffee sodium-free solutions (SUZUKI, 1962; CHAPMAN MILLER, 1971; KIMOTO, 1972). Furr examation carried out La+++ order to vestigate mode action caffee on sodium-free contracture. Fig. 8. Sodium-free contracture effects La+++ caffee. Arrows denote time troduction ( «) drugs or different solutions hg ( ª) with normal solution. A) Effects 1mM La+++ on contracture. B) Those 10mM caffee. C) Comparison effects La+++ with those La+++ plus caffee. Rate stimulation is 0.1 shock/sec. Vertical bars on right side calibrations 400mg for all records. time mark is 0.2 sec for fast 5 m for slow records. Figure 8A shows effect La+++ on sodium-free contracture. As can be seen, contracture as well as twitch contraction markedly depressed 1mM La+++. Figure 8B dicates effects caffee on sodium-free contracture. In 10mM caffee, contracture markedly augmented. Figure 8C shows effect La+++ on sodiumfree contracture left side, that La+++ with caffee on right side. Aga, La+++ depressed contracture markedly, however, when 10mM caffee added 1mM La+++, sodium-free contracture not depressed but conspicuously augmented. DISCUSSION In skeletal muscle, caffee at a concentration 1-4mM produces a

10 540 Y. KIMOTO, M. SAITO, M. GOTO marked augmentation twitch contraction without affectg surface (SANDOW et al., 1964; SANDOW, 1965), Our results on bullfrog atrium, however, dicate that caffee at comparable concentrations creases duration action potential slow ward decreases cord resistance. creased slow ward found to be closely associated with potentiation twitch contraction. With regard to action potential contractility, se results cocide well with those on guea pig atrium (DE GUBAREFF SLEATOR, 1965 ; KIMOTO, 1972) on kitten papillary muscle (CLARK OLSON, 1968). slow ward, which is maly responsible for plateau phase cardiac action potential hibited by Mn, is carried by Na Ca ions through slow channel (ROUGIER et al., 1969 ; MASCHER PEPER, 1969; REUTER BEELER, 1969). Thus, crement slow ward duced by caffee could be considered to be due to crease sodium /or calcium. Recent voltage clamp experiments on dog ventricular muscle have shown that calcium contributes to activation twitch contraction (REUTER BEELER, 1969; BEELER REUTER, 1970). A more direct couplg between slow ward noticed bullfrog myocardium (EINWACHTER et al., 1972; GOTO et al., 1972, 1974). present data that show that potentiation twitch contraction under fluence caffee strongest for clamp mv depolarization (Fig. 5A), where calcium became maximum (LEOTY RAYMOND, 1972; VASSORT ROUGIER, 1972), clearly dicate that potentiation contraction is maly due to crease calcium ward. BASSINGTHWAIGHTE REUTER (1972) proposed that specialized conductive sites associated with activation contraction present mammalian heart just as y frog skeletal muscle. This is region where sarcoplasmic reticulum apposes sarcolemma, se sites apposition occur heart on sarcolemma both T tubules external surface. In frog ventricle, a small amount sarcoplasmic reticulum is present, flattened reticular tubules (sarcolemmal cisternae) were noticed just beneath surface sarcolemma (WINEGRAD, 1971). Thus, if se facts hold our case, it may be that a part crease ward calcium caused by caffee could flow through this region which sarcoplasmic reticulum apposes surface. In present experiments, however, it found that caffee, even 50 mm concentration, evoked no contracture atrial muscle. In preparation pretreated with a 4-fold calcium-rich solution, on or h, 10 mm caffee duced contracture which not abolished by La+ + +, failed to produce a potentiation twitch contraction (Fig. 8). In such preparations, amount calcium sarcoplasmic reticulum is thought to be creased under calcium-rich condition, sce La+ + + ions hibits calcium entry (SANBORN

11 CAFFEINE ACTION ON MYOCARDIUM 541 LANGER, 1970; GOTO et al., 1974), se results could best be explaed by direct action caffee on sarcoplasmic reticulum. CHAPMAN OCHI (1972) reported that frog auricle sodium-free contracture hibited by Mn. In present experiments on bullfrog atrium sodium-free contracture depressed markedly by 1 mm La results clearly dicate that appearance sodium-free contracture is dependent on calcium entry from outside medium. Addition caffee, however, augmented sodium-free contracture even La+ + + (Fig. 9). It might be thought that a lack sodium hibits calcium extrusion usual way but that caffee antigonizes action La+ + + for recovery calcium entry mechanism. With this assumption, however, explanation appearance a simple contracture due to La+ + + caffee (Fig. 8) becomes hardly possible. Thus, it must be presumed aga that trans calcium movements depressed La+ + +, caffee acts on tracellular store muscle to release calcium, n sodium-deficiency hibits calcium extrusion to produce a large contracture In conclusion, it is proposed that effects caffee on cardiac muscle fundamentally same as those on skeletal muscle. However, strong modification calcium through surface affects contractility directly as well as directly through sarcoplasmic reticulum. REFERENCES BASSINGTHWAIGHTE, J. B. REUTER, H. (1972) Calcium movements excitationcontraction couplg cardiac cells. In Electrical Phenomena Heart, ed. by W. C. DE MELLO. Academic Press, New York London, pp BEELER, G. W., Jr. REUTER, H. (1970) Membrane calcium ventricular myocardial fibres. J. Physiol., 207: CARMELIET, E. VEREECKE, J. (1969) Adrenale plateau phase cardiac action potential. Pflugers Arch., 313: CHAPMAN, R. A. MILLER, D. J. (1971) action caffee on frog myocardial contractility. J. Physiol., 217: 64-66p. CHAPMAN, R. A. OCHI, R. (1972) effects manganese ions on contractile responses isolated frog atrial trabeculae. J. Physiol. Soc. Japan, 34: 612 ( Japanese). CLARK, A. OLSON, C. (1968) Effects caffee on action potentials on mammalian ventricular cells. Fed. Proc., 27: 304. DE GUBAREFF, T. SLEATOR, W. (1965) Effects caffee on mammalian atrial muscle its teraction with adenose calcium. J. Pharmacol. Exp. r., 148: EINWACHTER, H. M., HAAS, H. G., KERN, R. (1972) Membrane contraction frog atrial fibres. J. Physiol., 227: GOTO, M., KIMOTO, Y., SAITO, M., WADA, Y. (1972) Tension fall contraction bullfrog atrial muscle examed with voltage clamp technique. Jap. J. Physiol., 22: GOTO, M., WADA, Y., SAITO, M. (1974) Tension components fall bullfrog atrial muscle durg depolarization. Jap. J. Physiol., 24: HAGIWARA, S. TAKAHASHI, K. (1967) Surface density calcium ions calcium spikes

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