pacemaker cells. However, after Ica has been enhanced by the addition of Charlottesville, VA 22908, U.S.A. (Received 4 September 1987)

Transcription

1 Journal of Physiology (1988), 405, pp With 8 text-figures Printed in Great Britain IONIC MECHANISMS OF ADENOSINE ACTIONS IN PACEMAKER CELLS FROM RABBIT HEART BY L. BELARDINELLI*, W. R. GILESt AND A. WEST* From the Departments of Medical Physiology and Medicine, University of Calgary, Calgary, Alberta T2N 1N4, Canada and * Departments of Medicine and Physiology, Division of Cardiology, University of Virginia Medical Centre, Box 456, Charlottesville, VA 22908, U.S.A. (Received 4 September 1987) SUMMARY 1. Whole-cell and patch clamp techniques have been applied to cells isolated from the rabbit sino-atrial (S-A) node to study the ionic mechanism(s) of adenosineinduced slowing of cardiac pacemaker activity. 2. Viable spontaneously active cells were isolated from the central region of the S-A node of the rabbit heart by an enzymatic dispersion procedure similar to that reported by Giles & van Ginneken (1985) and van Ginneken & Giles (1988). In these spontaneously beating cells application of adenosine caused a dose-dependent slowing accompanied by a small hyperpolarization of the maximum diastolic potential. Relatively high doses of adenosine (> 20,M) caused complete arrest, associated with a hyperpolarization of mv. 3. In corresponding whole-cell voltage clamp experiments adenosine activated a time-independent potassium current, IK(ADO)' which at -50 mv is approximately 30 pa in normal Tyrode solution and 50 pa in high [K+]o (20 mm) Tyrode solution. This current is similar to the one identified previously in guinea-pig atrium (Belardinelli & Isenberg, 1983a; Kurachi, Nakajima & Sugimoto, 1986). 4. Patch clamp recordings of the single-channel events underlying IK(ADO) showed that they have a conductance of approximately ps. The whole-cell or macroscopic current, IK(ADO), and the adenosine-induced single-channel events exhibit strong inward-going rectification. 5. Adenosine in doses (10,1M) which significantly activate IK(ADO) failed to produce any measurable effect on the calcium current, Ica in these isolated cardiac pacemaker cells. However, after Ica has been enhanced by the addition of isoprenaline, adenosine (1-10 /Lm) caused a significant inhibition: it reduced Ica back to approximately the control levels. 6. A similar 'indirect' effect of adenosine was observed on If, the slow time- and voltage-dependent inward current which is activated by hyperpolarizing these S-A node cells. Adenosine (10- M) failed to influence the control or basal If; however, after If was enhanced by isoprenaline, adenosine markedly inhibited it. 7. These results provide explanations for both the direct and the indirect effects t To whom all correspondence and reprint requests should be sent.

2 616 L. BELARDINELLI, W. R. GILES AND A. WEST of adenosine in mammalian cardiac pacemaker tissue: activation of IK(ADO), and of a time-independent background potassium current and inhibition of ICa and If, respectively. Since it is known that there is significant adrenergic tone in the mammalian S-A node both the indirect and the direct effects of adenosine may be of physiological importance. INTRODUCTION In the mammalian heart normal primary pacemaker activity arises from the discharge of cells in the sino-atrial (S-A) node. This myogenic spontaneous discharge of the S-A node is modulated by both sympathetic and parasympathetic nerves, as well as metabolic factors. The nucleoside adenosine, which is released by cardiac cells, may be important in the regulation of cardiac pacemaker activity and of conduction through the atrio-ventricular (A-V) node. Adenosine has been shown to slow the heart rate in a variety of species, including dog, cat, rabbit, guinea-pig and human (Drury & Szent-Gyorgi, ; James, 1965; DiMarco, Sellers, Berne, West & Belardinelli, 1983; West & Belardinelli, 1985 a, b). Recent data indicate that adenosine may be the causal factor in slowing A-V node conduction during hypoxia and ischaemia (Clemo & Belardinelli, 1986). The electrophysiological effects of adenosine in the whole heart and in isolated S-A node tissue have been described in detail (West & Belardinelli, 1985 a, b). In isolated S-A node preparations adenosine produces a dose-dependent negative chronotropic effect and it may also cause a reversible shift in leading pacemaker site. In small strands of S-A node tissue adenosine prolongs the spontaneous cycle length, and causes a hyperpolarization of maximum diastolic potential, as well as an increase in rate of rise of the initial depolarization of the action potential. When a S-A strand is arrested with D600 or NiCl2, adenosine produces a dose-dependent hyperpolarization; and when relatively low doses of adenosine and acetylcholine are combined, the hyperpolarizations induced by each agent are additive. However, adenosine has no effect on the maximum hyperpolarization caused by very high doses of acetylcholine. These results suggested that in the S-A node adenosine, like ACh, may cause an increase in a time-independent potassium current. Subsequently, Belardinelli & Isenberg (1983a) showed that adenosine in fact did induce a time-independent (or background) inwardly rectifying outward K+ current in guinea-pig atrial myocytes. Recently, Kurachi et al. (1986) have applied the patch clamp technique to guinea-pig atrial cells and demonstrated that both adenosine and acetylcholine activate K+-selective channels which have very similar single-channel conductances (- 40 ps) and open times (- 1P0 ms). Adenosine -also increases an outward K+ current in neurones (Akasu, Shinnick-Gallagher & Gallagher, 1984; Trussel & Jackson, 1985) and in Xenopus oocytes (Lotan, Dascal, Oron, Cohen & Lass, 1985). In cardiac tissue the activation of a K+ current is thought to underlie the so-called 'direct', or cyclic AMP-independent, effects of adenosine, which are observed in the S-A node, atrium and A-V node. 'Indirect' or cyclic AMP-dependent effects of adenosine have also been described in the heart. For example, adenosine has been shown to inhibit noradrenaline release (Hedqvist & Fredh0lm, 1979; Wakade & Wakade, 1982; Dolphin, Fonda & Scott, 1986), a presynaptic effect which may be

3 ADENOSINE ACTIONS ON RABBIT S-A NODE CELLS due to inhibition of the Ca2+ influx into the nerve varicosities. In addition adenosine attenuates the positive inotropic effect of catecholamines at postsynaptic sites (Schrader, Baumann & Gerlach, 1977; Dobson, 1978). In isolated ventricular myocytes adenosine inhibits the isoprenaline-induced increase in calcium current, ICa (Belardinelli & Isenberg, 1983b; Isenberg & Belardinelli, 1984). At present, the available data indicate that the indirect effect is the only response to adenosine in the mammalian ventricle. In contrast, although both indirect and direct effects of adenosine are thought to be present in S-A node, atrium and A-V node. However, no detailed studies of either the direct and indirect effects of adenosine in mammalian cardiac pacemaker tissue have been published. The main goal of our experiments was to study the ionic mechanism(s) of the negative chronotropic effect of adenosine in the mammalian S-A node. METHODS Cell isolation The procedure for isolation of rabbit S-A node cells has been described in detail by van Ginneken & Giles (1988). In brief, after death by cervical dislocation, hearts were removed from New Zealand White rabbits (1-2 kg), and perfused with normal Ca2+ Tyrode solution for 5-10 min at 37 C using a modified Langendorff apparatus. The heart was then perfused for 10 min with a nominally zero Ca2+ Tyrode solution. This was followed by perfusion for 20 min with 50,uM-Ca2+ Tyrode solution containing collagenase (Sigma, Type I, 0.1 mg/ml). The ventricles and left atrium were removed, leaving the right atrium and surrounding tissue and the right atrium was then opened to expose the crista terminalis, the S-A node region and the interatrial septum. Parallel incisions were made along the crista terminalis and the intercaval region up to the superior vena cava. The resulting small piece of S-A node tissue was trimmed to approximately 3 x 5 mm and was placed in a small vial containing HEPES-buffered low-ca2+ Tyrode solution (100 /M) with 1-33 mg/ml collagenase (Sigma, Type I) and elastase 1 mg/ml (Sigma, Type III). This solution was gently stirred with a 1 x 3 mm Teflon magnet (Microflea) for approximately 20 min at a temperature of 35 'C. Following this the tissue was triturated with a fire-polished pipette (tip diameter approximately 1.5 mm) until the S-A node tissue almost completely dissociated into single cells. The solution containing the dissociated cells was centrifuged at approximately 10 g for 3-4 min. The supernatant was discarded and the cells were re-suspended in 0-5 mm-ca2+ Tyrode solution. This procedure resulted in a consistent yield of spontaneously beating cells of various types, as described previously by van Ginneken & Giles (1988). In contrast to previous descriptions of isolated S-A node cells (Taniguchi, Kokobun, Noma & Irisawa, 1981; Sakmann, Noma & Trautwein, 1983; Nakayama, Kurachi, Noma & Irisawa, 1984) the cells used in this study were spindle-shaped (approximately 7-10,um in width and jum in length) and not rounded-up and spherical. However, the spontaneously beating cells which we recorded from did not have welldefined striations. Aliquots of the cell suspension were transferred to a small volume (approximately 200 dl) recording chamber, which was mounted on the stage of an inverted microscope (Nikon Diaphot). The isolated cells were allowed to settle 5-10 min before superfusion with normal Tyrode solution was started. The temperature of the bath was maintained at 34+1 'C by pre-heating the superfusate and by using a heat-conductive glass plate in contact with the cover-slip (0-1 mm) which formed the bottom of the recording chamber (cf. van Ginneken & Giles, 1988). Solutions were superfused at approximately 1 ml/min, giving an exchange time of approximately 30 s. Solutions were selected using a six-way rotary Teflon valve (Rainin Instruments Woburn, MA, U.S.A.) placed close to the recording chamber. Solutions Normal Tyrode solution for perfusion of whole heart contained (mm): NaCl, 121; KCI, 5; CaCl2, 2-2; MgCl2, 1; sodium acetate, 2-8; NaHCO3, 24; NaH2PO4, 1-0; glucose, 5-5. In the low- Ca2' Tyrode solution, CaCl2 was omitted. These solutions were gassed with 95% % CO2 resulting in a ph of 7*4. During dispersion of isolated S-A node tissue the solution contained (mm): NaCl, 137; KCI, 5-4; CaCI2, 0-1; MgCI2, 06; Na2HP04, 035; KH2PO4, 03; glucose, 5-5; HEPES, 617

4 618 L. BELARDINELLI, W. R. GILES AND A. WEST 20; ph adjusted to 7-4 with NaOH. The HEPES-buffered Tyrode solution for superfusion of cells in recording chamber contained (mm): NaCl, 121; KCI, 5-0; CaCl2, 2-2; MgCl2, 1-0; sodium acetate, 2-8; glucose, 25; HEPES, 10; ph adjusted to 7-4 with NaOH. HEPES-buffered solutions were gassed with 100% 02. Adenosine (Sigma) and isoprenaline bitartrate (Sigma) stock solution were made fresh each day of experiments. N6-R-Phenylisopropyladenosine (L-PIA, Boehringer Mannheim) stock 10 mm was made in 100% dimethyl sulphoxide (DMSO) and diluted to final concentration in Tyrode solution. Unless otherwise noted all bath solutions contained 20 /Mtetrodotoxin (TTX). Electrophysiological techniques The suction microelectrode technique for whole-cell and cell-attached patch recordings was used in these experiments. Microelectrodes were pulled from 1-0 mm square-bore Pyrex glass (Glass Co. of America, Bargaintown, NJ, U.S.A.) and were filled with (mm): potassium aspartate, 130; KCI, 20; Na-ATP, 4; MgCl2, 1-0; HEPES, 10; ph was adjusted to 7-4 with KOH. Only microelectrodes having resistances of 2-5 MQ were used. For single-channel recordings the microelectrodes were filled with 150 mm-kcl, 300,tM-CdCl2, 10 mm-hepes and ph was adjusted to 7-4 with KOH. The voltage clamp electronics used for whole-cell measurements were similar to those described previously (Giles & Shibata, 1985; Clark & Giles, 1987). During the impalement procedure, seal formation was monitored under voltage clamp and only recordings with seal resistances greater than 5 GQ were accepted. After an acceptable seal had been obtained 1-2 min were allowed before applying additional suction to rupture the patch and begin whole-cell recording. Patch clamp recordings were made using an Axopatch Model 1B (Axon Instruments, Burlingame, CA, U.S.A.). Data analysis Recordings of the spontaneous pacemaker activity and action potential waveforms were made directly on a chart recorder (Gould 2400). Current and voltage data were monitored on an oscilloscope (Kikusui, Model 5516T, Tokyo, Japan) and were digitized on-line (using a Data Translation 2801A A/D board) and stored on the hard disc of an IBM PC XT (cf. Robinson & Giles, 1986). Data were also stored on tape using a PCM recorder (Medical Systems Co., Greenville, NY, U.S.A.) and VCR device (Sony SL HF 400, Super Beta). Digitized data were sent from the IBM PC to a minicomputer (VAX 750 Digital Equipment Corp.) for analysis and curve fitting. The single-channel data were analysed on an IBM AT using the IPROC-2 software (Dr C. Lingle, Department of Biology, Tallahasse, FL, U.S.A.). This program provides a means for automated detection of single-channel events. The parameters for the event detection are set by the user, e.g. an opening transition is detected when the current amplitude exceeds 50 % of the estimated singlechannel current selected by the user and a closing transition when the current returns to less than 50% of the selected level. Complete details of this program are given in Sachs, Neil & Barkakati (1982). Single-channel records were displayed on a laser printer (HP 2686D). RESULTS Adenosine-induced changes in pacemaker activity This enzymatic dissociation procedure yielded 5-40% spontaneously beating cells. As shown in Fig. 1 A a variety of cell types were obtained (cf. van Ginneken & Giles, 1988). Only the spontaneously active, spindle-shaped cells which closely resembled descriptions of primary pacemaker cells from the intact S-A node, (Bleeker, MacKay, Masson-Pevet, Bouman & Becker, 1980; Masson-Pevet, Jongsma, Bleeker, Tsjernina, van Ginnenken, Treijtel & Bouman, 1982) were used. An example of this cell type is illustrated in Fig. 1 B. The percentage of cells that beat spontaneously increased when the bath solution was warmed from room temperature, 24 C, to 34 'C. All whole-cell recordings were made from cells that were spontaneously beafing before and after impalement. Stable spontaneous activity could often be recorded for over 45 min. Some of the cells became quiescent after formation of

5 ADENOSINE ACTIONS ON RABBIT S-A NODE CELLS 619 _ '.. i.:.. I ona ( :._ "i. \..': i b.. B :.. 5 gm Fig. 1. A is a phase contrast micrograph of single cells isolated from rabbit S-A node. Only spontaneously beating spindle-shaped cells which did not have regular cross-striations were used in this study. This micrograph was taken at room temperature in order to slow the spontaneous activity. The calibration bar denotes 5 gim. The rounded and 'blebbed' cells are spontaneously active but damaged S-A node cells. The relatively long cylindrical and striated cells are from the crista terminalis. B shows at higher magnification the S-A node cell type used in these electrophysiological experiments. The relatively poor optical resolution is due to imaging through the gold emulsion-covered bottom of the recording chamber (see Methods).

6 620 L. BELARDINELLI, W. R. GILES AND A. WEST the gigaohm seal. This sensitivity to small changes in transmembrane current is expected on the basis of the extremely high input resistance ( P76 GQ, mean +s.e.m., n = 12) of these cells. Integration of the capacitive transients elicited by ± 10 mv clamp pulses yielded an input capacitance of pf (n = 18). A Control B Adenosine (50 ym) C Adenosine (10,M) D Wash 400 ms 30 mv Fig. 2. Dose-dependent slowing and arrest of isolated S-A node pacemaker cells by adenosine. A shows control spontaneous activity having a cycle length of 340 ms. The maximum diastolic potential is -62 mv and the action potential amplitude is 74 mv. B, adenosine (50 #M) caused complete arrest of the cell and a hyperpolarization of -12 mv. C, during subsequent application of 10 SM-adenosine, spontaneous pacing resumed with a cycle length of 960 ms and a maximum diastolic potential of -69 mv. Data in D were recorded after wash-out of adenosine. The cycle length is 290 ms and maximum diastolic potential is -60 mv. Figure 2A shows a representative suction microelectrode recording of the electrophysiological activity in a spontaneously beating S-A node cell. This pattern of pacemaker activity closely resembles conventional microelectrode recordings from cells in the central region of isolated rabbit S-A node preparations (Bleeker et al. 1980; Kodama & Boyett, 1985). Application of 50 /tm-adenosine arrested this spontaneous pacemaker activity and hyperpolarized the cell by approximately 15 mv, as expected from previous work (West & Belardinelli, 1985 a, b). During subsequent application of a lower dose of adenosine (10 /M) pacemaker activity recovered, but its cycle length (960 ms) was significantly longer than control, and the maximum diastolic potential remained negative to the original control recording

7 ADENOSINE ACTIONS ON RABBIT S-A NODE CELLS A Adenosine loomm 50pM 621 J 20 s 10 pa B -100 Voltage (mv) mm-k' o Control * 50 5M-adenosine k C Voltage (mv) , mm-k' o Control * 50 jm-adenosine a +- C -120 I Fig. 3. Adenosine-induced increase in time-independent or background outward K+ current. A, adenosine at 50 and 100 4M caused an increase in the background outward current of 12 and 23 pa respectively. These recordings were filtered at 100 Hz. In B and C, current-voltage relations of the adenosine-induced outward current are shown. Results were obtained in normal (5-0 mm) [K+]0 (B) and at 20 mm [K+]o (C). Note that the adenosine-induced current is larger in 20 mm [K+]0, and that this current, IK(ADO)I exhibits inward rectification. Similar adenosine-induced current changes were observed in five other experiments.

8 622 L. BELARDINELLI, W. R. GILES AND A. WEST A Control B Adenosine (10 MM) 0 mv 0mV -50 mv -50 mv -8OmV -80 mv I V IV 1-1 '%r ~ ~~~~~~~ 'li mm 1 l -100 mv r-t- I- c; L>L 25 ms O AW W f Ir ",,ImI, W 1RP-,-e-II1 q -120 mv Voltage (mv) -50 C c4-4 Fig. 4. Fd-6 For legend see facing page.

9 ADENOSINE ACTIONS ON RABBIT S-A NODE CELLS (Fig. 2C). After adenosine was washed out completely spontaneous pacemaker activity resumed from a maximum diastolic potential that was slightly depolarized to the control recording. This type of experiment involving consecutive application of drug doses is relatively difficult in these isolated cells since any small changes in seal resistance can significantly alter the rate of pacing and the shape of the pacemaker waveform. Adenosine-induced slowing was seen in all cells studied (n = 20) although some differences in sensitivity (dose range) was observed. In most cells 50 jtm-adenosine caused a significant hyperpolarization (11+ 3 mv, mean + S.E.M., n = 10) and complete arrest. However, in three cells even 100 /M-adenosine did not cause complete cessation of spontaneous beating. Adenosine-induced changes in background current Previous work (Belardinelli & Isenberg, 1983 a; Kurachi et al. 1986) has shown that application of adenosine to mammalian atrial tissue activates a time-independent, or background K+ current (IK(ADO)). The data in A and B of Fig. 3 show that a similar K+ current is induced following adenosine application to rabbit S-A node cells. In this experiment cumulative additions of 50 and 100/lM-adenosine activated an outward current of approximately 10 and 23 pa, respectively. After wash-out, the holding current returned to very near the control level. This increase in outward current did not exhibit desensitization in any of these experiments. B and C of Fig. 3 show current-voltage relationships for the adenosine-induced outward current obtained in two different Tyrode solutions, one containing normal (5 mm) [K+]. (Fig. 3B) and the second containing 20 mm [K+]. (Fig. 3C). These findings indicate that the adenosine-induced current is carried largely by K+ and also that its ion transfer mechanism exhibits significant inward-going rectification. However, once again, the extremely high input resistance of these cells made measurements of the reversal potential of the adenosine-induced current somewhat uncertain in normal Tyrode solution. In additional experiments patch clamp recordings were used to identify the singlechannel events underlying this macroscopic adenosine-induced K+ current (IK(ADO)). The data in Fig. 4 were recorded under control (A) conditions and from a different cell after adenosine (10 #m) had been added to the recording pipette (B). In this and all other experiments very few single-channel events were observed under control conditions. This finding is consistent with previous work which showed that the rabbit S-A node exhibits only a very small background K+ conductance (Irisawa, 1984; van Ginneken & Giles, 1988). In contrast, when an effective dose (5-10 /LM) of Fig. 4. Adenosine-induced single-channel events recorded from isolated S-A node pacemaker cells using a cell-attached patch clamp technique. A shows a control experiment i.e. made without adenosine in the patch pipette. Very few single-channel events were-recorded. B, single-channel activity with 10#,M-adenosine in the pipette. Each trace corresponds to 1-5 s of continuous recording. The numbers beside each trace denote the voltages applied to the patch pipette. C is a current-voltage relation: the mean conductance of the adenosine-induced channels (-) is approximately 24 ps (mean+ S.E.M., n = 3 cells) and the apparent reversal potential is approximately +45 mv. The control data (O, n = 2 cells) indicate that the background K+ channels have a similar conductance to those which underlie IK(ADO)' 623

10 624 L. BELARDINELLI, W. R. GILES AND A. WEST A Control B Adenosine (1 0 #M) 0 mv 0 mv_a -50 mv -50 mv rht~f-r~ rer.nrf.2v'f- 1-0.,- -80 mv U' -80 mv 7F--7rT T T---~",7-100 mv -100 mv T T n -1 " I T O'I,-oA -,Ag -120 mv I Voltage (mv) C CL 03 C.). Fig For legend see facing page.

11 ADENOSINE ACTIONS ON RABBIT S-A NODE CELLS adenosine was added to a patch pipette, enhanced single-channel activity, often showing 'bursting' phenomena, was observed consistently (Fig. 4B). The voltage dependence of the adenosine-induced single-channel current is illustrated as a current-voltage plot in C of Fig. 4 (control, 13; adenosine, *). The unitary events underlying the adenosine-induced current have a single-channel conductance of approximately 25 ps at 24 'C. There appears to be no significant difference between the conductance of the single-channel events which underlie the control background current and those which are responsible for IK(ADO) However, because there were so few events the control single-channel activity could not be analysed in detail in this series of experiments. Adenosine-induced K+ current in crista terminalis cells Recent voltage clamp investigations have demonstrated marked differences in the ionic currents which are present in the ventricle, atrium, and S-A node of rabbit and guinea-pig hearts (cf. Irisawa, 1984; Hume & Uehara, 1985; Giles & Imaizumi, 1988). We therefore compared the IK(ADO) currents in single cells from the S-A node and the crista terminalis of rabbit hearts. Figure 5A and B shows patch clamp recordings of single-channel events underlying the control background K+ current and adenosineinduced current in a cell from the crista terminalis. The current-voltage relationship in C indicates that both the control and adenosine-induced single-channel events have a significantly larger conductance in cells from the crista terminalis (- 40 ps) than those from S-A node pacemaker tissue (= 25 ps, Fig. 4). The reason(s) for this difference have not yet been studied in detail. However, these results suggest that (i) the single-channel events underlying the adenosine-induced current differs in the central region of the S-A node compared with the crista terminalis and (ii) the adenosine-induced single-channel events in crista terminalis are very similar to those previously identified in guinea-pig atrium (Kurachi et al. 1986). 625 Adenosine-induced changes in calcium current As described in the Introduction, adenosine exerts both direct and indirect actions in mammalian cardiac muscle. In mammalian ventricle the indirect actions of adenosine are due to an inhibition of the calcium current (ICa) under conditions where it has previously been increased by 8-agonists e.g. isoprenaline (Isenberg & Belardinelli, 1984). Therefore experiments were performed to determine (i) whether adenosine had any direct effects on ICa recorded under control conditions and (ii) whether adenosine could indirectly inhibit the isoprenaline-induced increase in ICa in S-A node cells. Figure 6 illustrates the results of whole-cell voltage clamp measurements in single myocytes from the central region of the S-A node. As shown in A, ICa in these cells is very similar to the ICa which has been described in rabbit Fig. 5. Effect of adenosine on unitary K+ currents in single cells from the crista terminalis of the rabbit heart. A shows control data. B consists of data obtained with adenosine (10,UM) in the recording pipette. The current-voltage relation in C illustrates both control and adenosine data. Note that the single-channel conductance in the crista terminalis cells (approximately 45 ps) is larger than that in the S-A node cells (approximately 25 ps) shown in Fig. 4.

12 626 L. BELARDINELLI, W. R. GILES AND A. WEST A 500 pa B 100 ms 400 pa 20 ms C o Control 400 * 100 pm-adenosine = Fig. 6. For legend see facing page.

13 ADENOSINE ACTIONS ON RABBIT S-A NODE CELLS 627 A 2 < 400 pa 20 ms B Voltage (mv) o 1 ALM-isoprenaline * 1 AM-isoprenaline + 10 ;M-adenosine Fig. 7. Enhancement of ICa by isoprenaline and inhibition of this effect by adenosine. A consists of three superimposed current traces elicited by 150 ms depolarizations to 0 mv from a holding potential of -40 mv. These records illustrate a control (trace 1), an isoprenaline-induced increase in ICa (trace 2) and subsequent inhibition by adenosine (trace 3) in which adenosine (10 #M) decreased ICa from 890 to 602 pa. Control ICa was 525 pa. The horizontal line indicates the zero current level. B shows current-voltage curves of the effects of adenosine on isoprenaline-induced Ca. TTX (10-5 M) was present in the Tyrode solution. Fig. 6. Whole-cell time- and voltage-dependent ionic currents in isolated S-A node cells. A shows control ICa records. 10 mv steps were made between -30 and +50 from a holding potential of -40 mv. Peak calcium current was 450 pa at + 10 mv. B, adenosine (20 and 100 lm) had no significant effect on peak ICa elicited by depolarization to 0 mv from holding potential of -40 mv. TTX (10-5 M) was present in the Tyrode solution. C, current-voltage relation showing the lack of effect of adenosine (100 /LM) on control ICa'

14 628 L. BELARDINELLI, W. R. GILES AND A. WEST atrium (Giles & Imaizumi, 1988) or in rabbit crista terminalis (Giles & van Ginneken, 1985). The threshold for its activation is near -25 mv and largest net inward current was obtained at approximately + 10 mv as shown in the current-voltage curve in Fig. 6C. The two superimposed current traces in B of Fig. 6 illustrate that doses of adenosine (20 and 100 /M) which produce significant changes in the background K+ current (Fig. 3) have no significant effect on the normal (control) ICa in this S-A node single-cell. In contrast, after ICa was increased by bath application of isoprenaline (10-6 M) adenosine produced very marked inhibition of the augmented 'Ca In the experiment shown in A of Fig. 7, 10-6 M-isoprenaline increased the peak 'Ca from approximately 500 to 890 pa (trace 2). This increase in ICa was almost completely inhibited by 10-5 M-adenosine (trace 3). At higher doses, i.e. 5 x 10-5 M, adenosine completely abolished the isoprenaline-induced increase in ICa (not shown). Figure 7B shows a summary of the adenosine effects on isoprenaline-enhanced ICa' and demonstrates that the type of indirect effect of adenosine on ICa which has been described previously in guinea-pig ventricular preparations can also be demonstrated in mammalian cardiac pacemaker tissue. Adenosine-induced changes in the inward current, If DiFrancesco (1985), Nathan (1986) and van Ginneken & Giles (1988) have shown that in single-cell preparations from rabbit S-A node a very slow but relatively large inward current can be activated by hyperpolarizations. An example of this hyperpolarization-activated inward current, If, is shown in Fig. 8A. If was first identified in multicellular 'strip' preparations by Yanagihara & Irisawa (1980) and Brown & DiFrancesco (1980). The selectivity and kinetics of If have been studied in detail (for review see DiFrancesco, 1985; van Ginneken & Giles, 1988). Previous data (DiFrancesco, Ferroni, Mazzanti & Tromba, 1986) have also shown that If is increased in the presence of /?-agonists. We therefore attempted to determine whether adenosine had any significant indirect effects on If. Data from these experiments are shown in Fig. 8B. Isoprenaline (10-6 M) consistently increased the I4, and subsequent application of adenosine (5 0 x 10' M) produced a marked inhibition of this isoprenaline-induced increase. Thus, at least two indirect effects of adenosine can be observed consistently after isoprenaline treatment of cardiac pacemaker cells: (i) an inhibition of ICa and (ii) an inhibition of If. Adenosine had no significant effect on If in the absence of isoprenaline (not shown). DISCUSSION Our results show that adenosine produces both direct and indirect electrophysiological effects in single cells isolated from the central region of the rabbit S-A node. The direct effect is mediated by an adenosine-induced increase in a K+ current which exhibits inward-going rectification. Although this current is small in normal Tyrode solution it is large enough to produce the hyperpolarization which was observed, given the large input resistance (approximately 3 Gn) of these cells. This response to adenosine in the sinus node is very similar to that identified previously in guinea-pig atrial myocytes (Belardinelli & Isenberg, 1983a; Kurachi et al. 1986).

15 ADENOSINE ACTIONS ON RABBIT S-A NODE CELLS The single-channel conductance of the adenosine-induced outward current in atrial myocytes is ps at 37 C (Kurachi et al. 1986). In contrast, the single-channel conductance for the adenosine-induced unitary currents in the S-A node cells is approximately 25 ps at 24 C - significantly smaller than that for the adenosine response in either atrial or crista terminalis cells (Figs 4 and 5). The reasons for this A 629 "M, lw V F -o L4&' -4 P., P. FA 61=4 Nalgowww"" pa 100 ms B 500 pa 100ms Fig. 8. Adenosine effects on If in S-A node cells. In A, the five superimposed current records were elicited by 400 ms hyperpolarizations from a holding potential of -30 mv. The horizontal line denotes the zero current level. Current changes in response to voltage clamp steps to -40, -50, -70, -90 and -10 mv are shown. Note that under control conditions between -70 and -110 mv, If manifests itself as a time- and voltagedependent activation of inward current. In B three If records are superimposed. Each was elicited by a 400 ms hyperpolarization to -90 mv. The control current (trace 1) is enhanced by isoprenaline (10-6 M; trace 2). Subsequent adenosine application (10- M; trace 3) significantly inhibited this isoprenaline-induced increase in If. difference are not known at present but these findings suggest that in the heart the adenosine receptor-activated K+ channels may be heterogeneous. The postsynaptic indirect effects of adenosine in S-A node appear to be mediated by inhibition of ICa and If. However, both of these responses depend upon ICa and If having first been increased by isoprenaline. This implies that the indirect effect of adenosine in physiological situations depends on the existing adrenergic tone.

16 630 L. BELARDINELLI, W. R. GILES AND A. WEST Interestingly, the direct effects of adenosine, i.e. activation Of 'K(ADO)' and its indirect effects, i.e. attenuation of isoprenaline-enhanced ICa and If, exhibit very similar dose dependence. The lack of effect of adenosine on the control, i.e. unstimulated ICa' further suggests that in physiological conditions the indirect effect of adenosine arises from an inhibition of the tonic effects of catecholamines on ICa. The adenosine-induced inhibition of the isoprenaline-stimulated current, If, have not been reported previously, although DiFrancesco & Tromba (1987) has shown a somewhat similar inhibitory effect of acetylcholine on isoprenaline-stimulated If in rabbit S-A node cells. In the intact heart the indirect effect(s) of adenosine and acetylcholine may also involve pre-synaptic inhibition of noradrenaline release (Hedqvist & Fredh0lm, 1979; Wakade & Wakade, 1982). However, our results show that significant indirect effects are also present at postsynaptic sites. S-A node cell preparation As indicated in Results some of our conclusions are dependent on the anatomic source and on the physiological condition of the isolated cells. The cells which we have used are very similar to those described previously by van Ginneken & Giles (1988) and are somewhat similar to those used by DiFrancesco et al. (1986). Cells which were rounded-up or which failed to show spontaneous activity after isolation were not used. Perhaps for that reason the so-called "ICa run-down' phenomena was not a significant problem. The marked heterogeneity, i.e. differences in the sizes and types of ionic currents present in the S-A node versus the crista terminalis or atrium (van Ginneken & Giles, 1988) requires careful attention concerning the source and condition of cells when attempts are made to compare data from one anatomical region of the heart to another. For example, the crista terminalis and atrium exhibit substantial transient outward currents which are not found in the S-A node (cf. Giles, van Ginneken & Shibata, 1985). In addition, it appears that single-channel events underlying the adenosine-induced K+ current in the crista terminalis have a significantly larger conductance than those in S-A node (compare data in Figs 4 and 5). Physiological role of adenosine in rabbit S-A node The ionic mechanism of the pacemaker depolarization in rabbit heart remains incompletely understood (Irisawa, 1984; DiFrancesco, 1985). However, at a minimum it involves interaction of the following currents: (i) activation of ICa' (ii) deactivation of the delayed rectifier potassium current, IK, (iii) activation of If and (iv) the presence of a time-independent inward sodium current. In the present study three prominent effects of adenosine have been identified which, by themselves or in combination, could result in a significant negative chronotropic response. Adenosine, in physiological doses, hyperpolarizes S-A node cells via activation of IK(ADO) and it slows the rate of pacing due to inhibition of Ic. and If. Thus, each of the important components of the negative chronotropic responses to adenosine which has been described in rabbit S-A node by West & Belardinelli (1985 a, b) can be accounted for. It is possible that the known electrophysiological heterogeneity in the anatomical S-A node region is responsible for the frequently observed pacemaker shift after application of adenosine or ACh. The hyperpolarization produced by adenosine and/

17 ADENOSINE ACTIONS ON RABBIT S-A NODE CELLS or ACh may completely inhibit the primary S-A node cells since they do not possess a transient inward sodium current, INa (cf. van Ginneken & Giles, 1988). In contrast, cells from the periphery of the S-A node (Nathan, 1986) or the crista terminalis (Giles & van Ginneken, 1985) become more excitable when hyperpolarized due to removal of INa inactivation. For this reason, the release of ACh or adenosine in the central or primary pacemaker region of the S-A node could cause a shift of the leading pacing site toward the periphery (West & Belardinelli, 1985a; Kodama & Boyett, 1985). This series of experiments is the first to demonstrate unequivocally both direct and indirect effects of adenosine in the same cardiac cell type. Although conclusive evidence is not available from either receptor binding studies or structure-activity relationships, it is reasonable to expect that the adenosine receptors that mediate both the direct and indirect effects are the same, and are of the A1 subtype. However, the final effector pathway for these two types of response is likely to be different. In one case (direct effects) the receptors are linked to K+ channels whereas in the other (indirect effects), the receptors presumably are coupled to adenylate cyclase synthesis of intracellular cyclic AMP. The anti-adrenergic effect of adenosine in the ventricle has been shown to involve modulation of cyclic AMP levels (West, Isenberg & Belardinelli, 1986) which in turn regulate Ca2+ channels. A plausible hypothesis for the indirect effect on If is that it also involves modulation of intracellular cyclic AMP. This mechanism has also been suggested for some ACh effects on If (DiFrancesco et al. 1986). The molecular basis for the difference in the direct and indirect effector mechanisms may reside in the coupling of the receptor to regulatory G-proteins. Recently both direct and indirect effects of adenosine have been shown to involve GTP-binding proteins and both effects have been shown to be inhibited by pertussis toxin (Kurachi et al. 1986; Wilson, West, Hewlett & Belardinelli, 1987). Whether there are different G-proteins coupling the receptor to K+ channels and adenylate cyclase is not yet known. Our results show that an endogenous metabolite, adenosine, can significantly modulate the current, If. This finding in combination with previous reports indicating that (i) adrenaline can augment If, (DiFrancesco et al. 1986) and (ii) that under some circumstances significant activation and deactivation of If can occur within the pacemaker range of potentials (van Ginneken & Giles, 1988) provide a strong motivation for a more quantitative analysis of the ionic mechanism of pacing in mammalian S-A node and its modulation by autonomic transmitters, as well as endogenous metabolites and peptides (cf. Giles et al. 1985). Our work helps to formulate a working hypothesis for the mechanism whereby adenosine may modulate heart rate under physiological and pathophysiological conditions. Since adenosine levels in the heart have been shown to change on a beat-to-beat basis, adenosine levels may be an important factor in determining heart rate. Adenosine could act as a negative feed-back element which slows heart rate and thus reduces oxygen consumption and assists in preserving energy balance. Thus, both the direct (activation of IK(ADO)) and indirect (inhibition ICa and If) mechanisms result in reduced heart rate and contractility. This work was supported by grants from the N.I.H. (DHHS-HL-33662), the Canadian MRC, the Canadian Heart Foundation, and the Alberta Heritage Foundation for Medical Research (AHFMR). Dr Giles is an AHFMR Scholar. We thank Doctors R. B. Clark and Y. Momose for help with the single-channel recordings and analysis, and Ms Lenore Doell for skilled secretarial 631

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