INTRODUCTION

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RYANODINE, A COMPOUND that reduces by twofold the conductance of Ca²⁺-release channels in the sarcoplasmic reticulum (SR) (25), slows the final phase of diastolic depolarization and, therefore, pacemaker activity in a number of cardiac preparations: strips of dilated human right atrium (7), subsidiary pacemaker cells of cat right atrium (26), pacemaker cells of cat (18) and guinea pig (24) sinoatrial (SA) nodes, and pacemaker cells isolated from cat right atrium (32) and rabbit SA node (17). The present study was designed to test the hypothesis that ryanodine's negative chronotropic effect on rabbit SA node cells is due to a reduction of inward currents that are modulated by intracellular Ca²⁺ (Ca²⁺_i). The fluorescent indicator indo 1 was used to monitor Ca²⁺_i, and the perforated-patch configuration of the whole cell patch-clamp technique (15) was employed to record spontaneous electrical activity and ionic currents in single pacemaker cells after 2 or 3 days in culture. Indo 1 allows continuous detection of two emission wavelengths and their ratio, thereby avoiding the switching time required for dual-excitation fluorescent indicators like fura 2. The perforated-patch technique avoids complete dialysis of the cytoplasm by the patch pipette, thereby preventing the loss of compounds that are essential for automaticity (19, 21). 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM), a rapidly acting calcium chelator, was used to test whether reduced intracellular Ca²⁺ concentration ([Ca²⁺]_i) plays a role in the inhibitory effects of ryanodine. Our results confirm that ryanodine inhibits the release of Ca²⁺ from the SR of rabbit SA node pacemaker cells and suggest that reductions of inward Na⁺/Ca²⁺ exchange current (I_Na/Ca) and T-type Ca²⁺ current (I_Ca,T) account for the negative chronotropic effect of ryanodine.

	METHODS

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Cell isolation and culture. Our method for isolating SA node cells from the hearts of male New Zealand White rabbits (1.0-1.5 kg) has been described in detail (19, 21). The cells were plated on glass coverslips (number 0) in 35-mm plastic dishes that contained a culture medium (19, 21) and were stored in an incubator (95% air-5% CO₂) at 37°C.

Measurement of intracellular Ca²⁺. Cultured SA node cells were incubated with 25 µM indo 1-AM for 10-15 min at room temperature and then washed with Tyrode solution for at least 15 min. This concentration and loading period were adopted to optimize the signal-to-noise ratio for indo 1 fluorescence without excessive buffering of Ca²⁺_i. A Photon Technology International (PTI, South Brunswick, NJ) filter-based detection system was used to record indo 1 fluorescence simultaneously at 405 and 485 nm during epifluorescence illumination at 365 nm. Fluorescence was measured as photons per second, and PTI's FELIX software was used to acquire the fluorescence ratio (F₄₀₅/F₄₈₅) and electrophysiological data simultaneously. Fluorescence was measured from a rectangular area roughly the size of the cell under study. The background fluorescence, recorded from a cell-free field of the same size, was subtracted from each of the two signals before the fluorescence ratio was calculated. To minimize photobleaching of indo 1, we exposed the cells to 365-nm light only during the recording period.

Electrophysiological recordings. A model 3900A patch-clamp amplifier (Dagan, Minneapolis, MN) and ruptured-patch or perforated-patch whole cell recordings (19, 21) were used to record ionic currents or spontaneous electrical activity in isolated pacemaker cells after 2 or 3 days in vitro. Cells were superfused with Tyrode solution containing (in mM) 130 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.6 MgCl₂, 0.6 NaH₂PO₄, 18 NaHCO₃, and 5.5 dextrose, pH adjusted to 7.4 with the addition of 95% O₂ or 95% air and 5% CO₂. The pipette solution for perforated-patch recordings with nystatin contained impermeant divalent cations to minimize the Donnan potential [in mM: 75 K₂SO₄, 55 KCl, 7 MgCl₂, 10 dextrose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)] and was titrated to pH 7.2 with KOH. To isolate Ca²⁺ currents, the modified Tyrode solution contained the following blockers (in mM): 0.01-0.03 tetrodotoxin, 2 CsCl, 4 4-aminopyridine, and 2 BaCl₂, and the pipette solution contained Cs⁺ instead of K⁺. Because amphotericin B can achieve a lower access resistance than nystatin, current tail measurements and some Ca²⁺ current measurements employed amphotericin B in a pipette solution that contained (in mM) 5 NaCl, 125 CsCl, 5 MgATP, 10 dextrose, 10 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 10 HEPES and was titrated to pH 7.2 with CsOH. This solution also could be used for ruptured-patch recordings in the same cell. In some experiments, the ruptured-patch technique was used to promote rundown of L-type Ca²⁺ current (I_Ca,L) so as to isolate I_Ca,T. In those measurements, MgATP and HEPES were omitted and the pipette solution contained (in mM) 140 CsCl, 10 NaCl, and 10 dextrose and was titrated to pH 7.2 with CsOH. The calculated corrections for liquid-junction potentials between the modified Tyrode and nystatin or amphotericin B pipette solutions were 2.9 and 5.2 mV, respectively (19, 21). Because these potentials were unchanged by the addition of ryanodine or BAPTA, we did not make such corrections. A stock solution of nystatin [50 mg/ml in dimethyl sulfoxide (DMSO)] or amphotericin B (30 mg/ml in DMSO) was diluted in the appropriate pipette solution and ultrasonicated. The tip of the pipette was filled with antibiotic-free solution and the rest of the pipette with nystatin (300 µg/ml)- or amphotericin B (400 µg/ml)-containing solution. Pipettes were made on a model P80/PC puller (Sutter Instrument, Novato, CA); their resistances ranged from 1 to 5 M. After a pipette-membrane seal had formed (resistance, 10-20 G), we waited 10-15 min for patch perforation before making the electrophysiological measurements. Series resistance compensation was maximized to minimize the time constant for decay of the capacitive transient.

Solutions. The cell isolation solutions and culture medium have been described (19, 21). The indo 1-AM loading solution was made by adding 25 µg indo 1-AM (Texas Fluorescence Laboratory, Austin, TX), 25 µl DMSO, 45 µl fetal calf serum (GIBCO, Grand Island, NY), and 2.2 µl of 25% (wt/wt) Pluronic F-127 (Sigma Chemical, St. Louis, MO) in DMSO to 1 ml of a HEPES-buffered balanced salt solution (19, 21) that contained 1.8 mM CaCl₂. The final concentration of indo 1-AM was 25 µM. Drug-containing solutions were prepared by appropriate dilution of the stock solutions except for caffeine, which was added as a powder directly to the Tyrode solution. BAPTA was loaded into the cells by adding 25 µM BAPTA-AM (Texas Fluorescence Lab) to the Tyrode solution. In each experiment, the isolated pacemaker cells were superfused with Tyrode solution at a rate of 1 ml/min. A control system (model TC-1, Cell Micro Controls, Virginia Beach, VA) was employed to maintain the temperature within ±0.5°C of 34 or 35°C.

Data analysis. Beat rates were calculated from the total period for 10 consecutive action potentials, and pCLAMP 6 software (Axon Instruments, Foster City, CA) was used to measure the characteristics of at least 3 action potentials; then these were averaged. Slopes of the initial and final phases of diastolic depolarization (DD₁ and DD₂, respectively) were obtained from linear fits (Fig. 1C), and the "takeoff" potential (TP) was approximated by the intersection of linear fits of the action potential upstroke and DD₂. Hyperpolarization-activated inward current (I_f) was measured as the difference between the inward current at the end of 300-ms voltage steps, a duration that approximates diastole, and the instantaneous or "background" inward current (I_bg) at the onset of the step. Ca²⁺ currents were measured as the difference between the peak of the transient inward current and the current at the end of 300-ms voltage steps, which was assumed to be leakage current. Currents were normalized by cell input capacitance (C), where C = Q/V, Q is the charge (measured as the area subtended by the capacitive current), and the change in voltage (V) is 10 mV. A Chebyshev-Simplex method (pCLAMP 6) was employed to fit the decay of Ca²⁺_i transients by a single exponential and to fit slow inward current tails by a sum of two exponentials. The tail's peak was determined from the exponential fit. Analog data were digitized at 12-bit resolution by a Labmaster DMA board (Axon Instruments) that was controlled by pCLAMP software and then stored on the hard disk of a computer for later analysis. Data are presented as means ± SE. A paired Student's t-test was used for statistical analyses, and differences between means with P < 0.05 were considered significant.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Simultaneous recordings of electrical activity and action potential-induced intracellular Ca2+ (Ca2+i) transients before and after addition of ryanodine. A: action potentials (Vm, top) and indo 1 fluorescence ratio (F405/F485, bottom) under control conditions. B: action potentials (top) and fluorescence ratio (bottom) 10 min after addition of 10 µM ryanodine (same cell as in A). C: superimposed electrical activity before (solid trace) and after (dashed trace) addition of ryanodine. Note reduced slope of final phase of diastolic depolarization (DD2), but not in initial phase (DD1), and depolarization of "takeoff" potential (arrows) after exposure to ryanodine.

	RESULTS

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Effect of ryanodine on spontaneous electrical activity and Ca²⁺_i transients. During simultaneous recordings of electrical activity and the indo 1 fluorescence ratio, the addition of 10 µM ryanodine slowed the firing rate and reduced the amplitude of action potential-induced Ca²⁺_i transients. For example, in one pacemaker cell, the beat rate (BR) decreased from 86 to 67 beats/min, the peak-to-peak amplitude of F₄₀₅/F₄₈₅ decreased from 0.6 to 0.5, and the time constant for its decay increased from 126 to 176 ms after 10 min (Fig. 1, A and B). The slower firing rate might be explained by the slower DD₂, which decreased from 35.1 to 19.5 mV/s (dashed curve, Fig. 1C), and by depolarization of the TP from 51 to 42 mV (arrows, Fig. 1C). The effects of 10 µM ryanodine on the electrical activity of 15 cultured pacemaker cells are summarized in Table 1. Reductions of BR (30 ± 3%), maximum upstroke velocity (dV/dt_max; 12 ± 3%), DD₂ (37 ± 3%), and TP (11 ± 2%) were all significant (P < 0.01). Results obtained in 10 freshly isolated pacemaker cells (not shown) were not significantly different. The fact that DD₂, but not DD₁, was reduced by ryanodine is consistent with the microelectrode recordings of Rubenstein and Lipsius (18, 26) in cat SA node and subsidiary pacemaker tissue and of Rigg and Terrar (24) in guinea pig SA node tissue. However, in those studies, a significant depolarization of the maximum diastolic potential (MDP) and changes in the action potential overshoot (OS) were also seen. In the present study, ryanodine simultaneously reduced the peak-to-peak amplitude of action potential-induced Ca²⁺_i transients by 19 ± 3% (P < 0.01) and increased their time constant for decay by 51 ± 5% (P < 0.01). Some slowing of BR and reduction of the Ca²⁺_i transient could be seen after just 2 min of exposure to ryanodine, and a maximal effect was reached in <8 min. Thus the results described below were obtained after 8-10 min of exposure.

Blockade of SR Ca²⁺ release by ryanodine. To confirm that the actions of ryanodine were due, in part, to its effects on the SR, we used caffeine to release Ca²⁺ from the SR and then tested whether ryanodine could block this release. Because caffeine enhances I_Ca,L in rabbit SA node tissue (27), it was necessary to block this current before the addition of caffeine to eliminate its contribution to the Ca²⁺_i transient. Exposure of pacemaker cells to Ni²⁺, which inhibits both I_Ca,L and I_Ca,T in this preparation (21), rapidly blocked the action potential-induced Ca²⁺_i transients, and Ca²⁺_i fell below the diastolic level (Fig. 2A). Addition of 10 mM caffeine produced a transient increase in Ca²⁺_i, but a second addition had no effect. This indicates that 10 mM caffeine depleted the Ca²⁺ stores. With Ni²⁺ and caffeine washout, there was complete recovery of the Ca²⁺_i transients. Pretreatment of another pacemaker cell with 10 µM ryanodine prevented the caffeine-induced Ca²⁺_i transient, and pacemaker activity became arrhythmic with Ni²⁺ and caffeine washout (Fig. 2B). Similar results were obtained in another 11 pacemaker cells.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Ryanodine blockade of caffeine-induced release of Ca2+ from sarcoplasmic reticulum (SR). A: blockade of action potential-induced Ca2+i transients by 5 mM Ni2+ followed by caffeine (10 mM)-induced release of SR Ca2+. A second addition of caffeine failed to elicit Ca2+ release. B: pretreatment of another cell with 10 µM ryanodine abolished transient response to caffeine. Note that recordings were interrupted for 1-2 min after addition and washout of Ni2+.

Effect of ryanodine on hyperpolarization-activated and background inward currents. Because the slowing of spontaneous firing induced by ryanodine coincided with a slower DD₂ (Fig. 1C), we tested the hypothesis that exposure to ryanodine leads to a decrement of I_f and I_bg, inward currents that might contribute to this phase of the pacemaker potential. To activate these currents, we applied 300-ms voltage steps from a holding potential of 50 mV to potentials between 50 and 100 mV. The records before and 10 min after exposure to 10 µM ryanodine show that ryanodine did not alter I_f or I_bg (Fig. 3A). In fact, no effect was seen after 2, 5, 10, or 15 min of exposure to ryanodine. In eight pacemaker cells, the current-voltage (I-V) relationships for I_f and I_bg before and 10 min after addition of ryanodine were not significantly different (Fig. 3B).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of ryanodine on hyperpolarization-activated (If) and background (Ibg) inward currents. A: hyperpolarizing voltage steps (300-ms duration) from a holding potential (HP) of 50 mV to potentials between 50 and 100 mV; control (top) and 10 min after addition of 10 µM ryanodine (bottom). B: mean current-voltage (I-V) relationships for If and Ibg (n = 8) before and after addition of 10 µM ryanodine.

Effect of ryanodine on T- and L-type Ca²⁺ currents. Because the slowing of spontaneous firing induced by ryanodine coincided with a slower DD₂ and depolarization of the TP (Fig. 1C), we tested the hypothesis that exposure to ryanodine leads to a decrement of I_Ca,T and I_Ca,L, inward currents likely to contribute to these two phases of the electrical activity (8, 13). As demonstrated previously in SA node pacemaker cells, I_Ca,L and I_Ca,T can be separated by the holding potential (8, 13). Both currents could be elicited from a holding potential of 80 mV, whereas only I_Ca,L could be activated from a holding potential of 40 mV. In the present study, total Ca²⁺ current (I_Ca = I_Ca,L + I_Ca,T) was reduced after just 2 min of exposure to 10 µM ryanodine; however, as recorded in ventricular myocytes (1, 20, 23) and freshly isolated rod-shaped rabbit SA node cells (28), the amplitude of I_Ca,L was not changed significantly. Longer exposures (up to 15 min) also did not alter I_Ca,L significantly, ruling out "rundown" of I_Ca,L as the cause of the decline of I_Ca. Figure 4A illustrates the effects of ryanodine on I_Ca and I_Ca,L (traces labeled R) in a representative pacemaker cell. Figure 4B shows the mean I-V relationships for eight pacemaker cells before and 10 min after addition of 10 µM ryanodine. The perforated-patch technique (with nystatin) was employed to minimize rundown of I_Ca,L. Ryanodine reduced I_Ca significantly at 30, 20, and 10 mV (P < 0.05). In contrast, it had no significant effect on I_Ca,L at any potential. Significant changes in I_Ca were observed at 30, 20, 10, 0, and 10 mV (P < 0.05) in another 10 cells when the patch pipette contained amphotericin B to reduce the series resistance (data not shown). The absence of an effect of ryanodine on the amplitude of I_Ca,L suggests that the attenuation of I_Ca is due to a reduction of I_Ca,T. This could explain the reductions of both DD₂ and TP, because I_Ca,T is activated at those potentials (8, 13).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effect of ryanodine on Ca2+ currents (ICa). A: ICa elicited by depolarizing voltage steps to potentials indicated from HP of 80 mV (left) and 40 mV [L-type Ca2+ current (ICa,L), right], before (C) and 10 min after (R) addition of 10 µM ryanodine. B: mean I-V relationships for 8 cells in absence and presence of 10 µM ryanodine (after 10 min). For ICa (top, HP = 80 mV), mean values at 30, 20, and 10 mV are significantly different. For ICa,L (bottom, HP = 40 mV), no changes are significantly different.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effect of ryanodine on T-type Ca2+ current. A: ICa,L elicited from an HP of 40 mV after rundown. B: total Ca2+ current elicited by depolarizing voltage steps from an HP of 80 mV to potentials indicated before (left) and 10 min after addition of 10 µM ryanodine (right). C: mean I-V relationships for ICa (HP = 80) in absence and presence of 10 µM ryanodine (n = 5). Changes at 30, 20, 10, 0, and 10 mV are all significant (P < 0.05).

Effect of BAPTA-AM on spontaneous electrical activity and Ca²⁺_i transients. To test the hypothesis that a reduction of the amplitude of Ca²⁺_i transients was responsible for ryanodine's slowing of DD₂ and BR and depolarization of the TP, we exposed 14 pacemaker cells to BAPTA-AM, a rapidly acting Ca²⁺ chelator. During simultaneous recordings of electrical activity and the indo 1 fluorescence ratio, action potential-induced Ca²⁺_i transients were eliminated 3-5 min after the addition of 25 µM BAPTA-AM, whereas the spontaneous electrical activity continued (Fig. 6). BAPTA, like ryanodine, had no effect on DD₁, yet it slowed the DD₂ and BR, reduced the dV/dt_max, and depolarized the TP (P < 0.01; Table 2). However, unlike ryanodine, BAPTA prolonged the duration of the action potential measured at 20 mV (Dur; P < 0.05) and depolarized the MDP (P < 0.01). These results suggest that the effects of ryanodine on DD₂, BR, dV/dt_max, and TP might arise, in part, from the reduction of [Ca²⁺]_i that accompanies ryanodine's inhibition of SR Ca²⁺ release. The additional effects of BAPTA on the Dur and MDP might be explained by the likelihood that BAPTA can reduce [Ca²⁺] in the cytoplasm to a greater degree than can ryanodine (see DISCUSSION).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Simultaneous recordings of electrical activity and action potential-induced Ca2+i transients before and after addition of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM). A: Vm (top) and F405/F485 (bottom) under control conditions. B: Vm and F405/F485 10 min after addition of 25 µM BAPTA-AM (same cell as in A). Note that action potential-induced Ca2+i transients were blocked completely.

Effect of ryanodine on Ca²⁺ currents in BAPTA-AM-loaded cells. To determine whether the reduction of I_Ca was due to changes in [Ca²⁺]_i or to ryanodine itself, we first reduced [Ca²⁺]_i with BAPTA and then added ryanodine. After the pacemaker cells had been exposed to 25 µM BAPTA-AM for 10 min, the amplitudes of I_Ca and I_Ca,L were unchanged (Fig. 7). This was confirmed by the mean I-V relationships for five pacemaker cells that were not significantly different at any potential (Fig. 8A). Despite the fact that there was no significant effect on the amplitudes of I_Ca and I_Ca,L, BAPTA did slow inactivation of the two currents (Fig. 7), as would be expected for a reduction of Ca²⁺-induced inactivation (22). With BAPTA-AM still present, 10 µM ryanodine reduced I_Ca after only 2 min, whereas I_Ca,L was not changed, just as we had seen in the absence of BAPTA (Fig. 7). After the pacemaker cells had been exposed to ryanodine for 10 min, the reduction of I_Ca was significant at 30, 20, and 10 mV (P < 0.05); however, there was no significant effect on I_Ca,L (Fig. 8B). These results suggest that ryanodine's reduction of I_Ca might be due to a direct effect of ryanodine on T-type Ca²⁺ channels, independent of changes in [Ca²⁺]_i.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Effect of ryanodine on Ca2+ currents in presence of BAPTA. Ca2+ currents were elicited by depolarizing voltage steps from HP of 80 mV (ICa) and 40 mV (ICa,L) under control conditions (left), 8 min after addition of 25 µM BAPTA-AM (middle), and 10 min after addition of 10 µM ryanodine (right).

View larger version (21K): [in this window] [in a new window]   Fig. 8.   A: mean I-V relationships for ICa (HP = 80 mV) and ICa,L (HP = 40 mV) in absence (Control) or presence of 25 µM BAPTA-AM (n = 5). Mean values for control and BAPTA are not significantly different at any potential. B: mean I-V relationships for ICa and ICa,L in presence of 25 µM BAPTA-AM and in absence (BAPTA) or presence of 10 µM ryanodine (n = 5). Mean values for ICa at 30, 20, and 10 mV are significantly different (P < 0.05). Perforated patches were obtained with amphotericin B.

Effects of BAPTA and ryanodine on slow inward current tails. This set of experiments was designed to test the hypothesis that ryanodine also reduces inward I_Na/Ca in SA node pacemaker cells. By decreasing the amplitude of Ca²⁺_i transients, ryanodine would be expected to diminish the extrusion of Ca²⁺ via the Na⁺/Ca²⁺ exchanger and thereby reduce the net influx of Na⁺, i.e., I_Na/Ca. After repolarization of the membrane potential after a 5-ms test pulse to +20 mV, a slow decay of inward current could be seen (Fig. 9). The amplitude of this slow inward current tail seemed to depend on the amplitude of I_Ca,L elicited during the test pulse, and the tail could be eliminated if I_Ca,L was completely inactivated (at a holding potential of 0 mV). Although this slow decay could be interpreted as an I_Ca,T tail (4), this is highly unlikely, because I_Ca,T would have been completely inactivated at the employed holding potentials of 40, 20, and 0 mV (8, 13). Addition of 25 µM BAPTA-AM blocked the tails completely in seven SA node pacemaker cells; moreover, BAPTA slowed inactivation but did not alter the amplitude of I_Ca,L (Fig. 9A). In nine pacemaker cells, exposure to 10 µM ryanodine also blocked the slow tails and slowed the inactivation of I_Ca,L (Fig. 9B). When the holding potential was 40 mV, ryanodine reduced the peak of the tail from 173 ± 22 to 59 ± 13 pA (P < 0.01) and decreased the time constant of its decay from 13 ± 3 to 7 ± 1 ms (P < 0.05).

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Effect of BAPTA or ryanodine on slow inward current tails. A: current tails elicited by a 5-ms test pulse to +20 mV, followed by repolarization to 80 µV, before (Control) or 10 min after addition of 25 µM BAPTA-AM; prepulses to 40, 20, and 0 mV were 1 s in duration. B: current tails obtained with same protocol as in A before (Control) or 10 min after addition of 10 µM ryanodine.

Effects of lower concentrations of ryanodine. To identify the contribution of each process, a reduction of I_Ca,T and blockade of SR Ca²⁺ release, to the negative chronotropic effect of ryanodine, we used lower concentrations of ryanodine and looked for a dose-dependent difference in its effects. Unfortunately, we could not find such a difference. At concentrations of 5 and 2 µM (6 and 9 cells, respectively), ryanodine continued to slow pacemaker activity, reduce I_Ca,T, partially block inward current tails, and block caffeine-induced Ca²⁺_i transients. At a concentration of 1 µM, ryanodine's effects on I_Ca,T and SR Ca²⁺ release still could not be separated. Ryanodine reduced I_Ca,T in three of four cells, and it blocked caffeine-induced Ca²⁺_i transients in four of four cells. We considered 1 µM ryanodine to be the "threshold" dose, because this concentration slowed pacemaker activity in only two of five cells.

	DISCUSSION

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Previously, we showed that ryanodine reduces the amplitude of action potential-induced Ca²⁺_i transients and slows the firing of cultured SA node pacemaker cells (17). The latter effect could be due to changes in any of the currents that contribute to pacemaker activity. However, the present study was designed to test the hypothesis that ryanodine's negative chronotropic effect is due to a reduction of inward currents that contribute to pacemaker activity and can be modulated by Ca²⁺_i. Such currents include the following: 1) I_f, which may (12) or may not (31) depend on [Ca²⁺]_i; 2) time-independent I_bg or "sustained" inward currents carried by Na⁺ (10, 14); 3) I_Na/Ca, which depends on both the Na⁺ and Ca²⁺ electrochemical gradients (11, 32); and 4) I_Ca,T and I_Ca,L, which also depend on the Ca²⁺ gradient (5, 8, 13).

With the use of 10 µM ryanodine, a concentration sufficient to block SR Ca²⁺ release (Fig. 2), we found no significant effect on either I_f or I_bg (Fig. 3). In contrast, 10 µM ryanodine did reduce I_f significantly in freshly isolated rod-shaped rabbit SA node cells (28). We have no explanation for this discrepancy. In our cultured SA node pacemaker cells, 10 µM ryanodine reduced I_Ca significantly at 30, 20, and 10 mV (Fig. 4B). Even though they were not statistically significant, reductions of I_Ca were also seen at 50 and 40 mV (Fig. 4A), potentials at which I_Ca,T is activated (8, 13) and the takeoff potential is seen (Fig. 1C, Table 1). In rabbit SA node cells, the role of I_Ca,T is to accelerate DD₂ and maintain the action potential threshold at more negative potentials (5, 13). For example, blockade of I_Ca,T by 40 µM Ni²⁺ prolonged DD₂ without changing DD₁ (13), just as we have seen in our own experiments with ryanodine (Fig. 1C). Nevertheless, other mechanisms might also play a role, because ryanodine reduced but did not block I_Ca,T (Fig. 4). With the use of two different recording procedures, the perforated-patch technique (with nystatin in 8 cells and amphotericin B in 10 cells) and the ruptured-patch technique (to promote rundown of I_Ca,L in 5 cells), we observed a consistent decline in the amplitude of I_Ca after addition of ryanodine. This decline cannot be explained by rundown of I_Ca,L, because I_Ca,L was recorded about the same time as I_Ca and was unaffected by ryanodine (Figs. 4, A and B). Given that I_Ca = I_Ca,L + I_Ca,T, these results suggest that ryanodine slows pacemaker activity, in part, by reducing I_Ca,T. The rundown experiment (Fig. 5) provides additional support for this hypothesis, because, in the virtual absence of I_Ca,L, I_Ca was reduced significantly by ryanodine.

Studies of ventricular myocytes (1, 20, 23) and SA node pacemaker cells (28) suggest that ryanodine, at concentrations between 1 and 10 µM, does not act directly on L-type Ca²⁺ channels; instead, its effects are mediated by changes in [Ca²⁺]_i. In fact, privileged cross signaling between ryanodine receptors and L-type Ca²⁺ channels has been suggested to occur in adult rat ventricular myocytes (3, 29). Previously, we showed that I_Ca,L inactivation is dependent on [Ca²⁺]_i in SA node pacemaker cells (22), and those observations are supported by the present results in which BAPTA slowed the inactivation of I_Ca,L (Figs. 7 and 9A). However, little slowing of I_Ca,L inactivation was observed after the addition of ryanodine (Fig. 4A), suggesting that its reduction of [Ca²⁺]_i was small. This was confirmed by the modest decrease (19%) in the amplitude of action potential-induced Ca²⁺_i transients after the addition of ryanodine (Fig. 1B). A similar discrepancy can be seen when one compares the effects of ryanodine and BAPTA on the MDP and Dur of the action potential in Tables 1 and 2. Ryanodine (10 µM) had no significant effect, whereas BAPTA depolarized the MDP and increased the Dur significantly. Interestingly, in freshly isolated rod-shaped rabbit SA node cells, 1 µM ryanodine not only slowed pacemaker activity but also increased the OS and Dur significantly; however, it had no effect on the MDP (28). In contrast, 1 or 2 µM ryanodine depolarized the MDP and changed the OS significantly in cat subsidiary (26) and guinea pig SA node (24) pacemaker cells. Such differences might be explained by the relative changes in [Ca²⁺]_i produced by ryanodine or by the relative abundance of SR in cat, guinea pig, and rabbit pacemaker cells. In the present study, it is possible that ryanodine had a direct effect on I_Ca,T, independent of the changes in [Ca²⁺]_i, considering that ryanodine reduced I_Ca regardless of whether BAPTA was present (Figs. 7 and 8B) or not (Fig. 4B). Although BAPTA blocked the Ca²⁺_i transients (Fig. 6) and slowed the inactivation of I_Ca,L (Figs. 7 and 9), it had no effect on the amplitude of either I_Ca or I_Ca,L (Figs. 7 and 8A). Therefore, it is possible that BAPTA failed to reach the T-type Ca²⁺ channels. Thus we cannot rule out the possibility that ryanodine's reduction of I_Ca,T was due to its attenuation of the Ca²⁺_i transients (Fig. 1B). In fact, a reduction of [Ca²⁺]_i has been shown to decrease I_Ca,T in canine ventricular and Purkinje cells (30).

In our cultured SA node pacemaker cells, we observed a relatively slow inward current tail that consistently followed depolarizing voltage steps that activated I_Ca,L (see Controls, Fig. 9). Similar current tails, recorded in atrial myocytes and pacemaker cells, have been attributed to the Na⁺/Ca²⁺ exchanger (2, 6, 9, 32). BAPTA's blockade of such tails (Fig. 9A) is consistent with previous results, in which cat latent pacemaker cells were dialyzed with EGTA (32), and supports the hypothesis that this current depends on [Ca²⁺]_i. In agreement with some but not all previous studies of rabbit atrial myocytes (6, 9) and cat latent pacemaker cells (32), ryanodine inhibited slow inward current tails in our rabbit SA node pacemaker cells (Fig. 9B). Additional results in cat latent pacemaker cells (32) demonstrated that I_Na/Ca can contribute significantly to the generation of diastolic depolarization, particularly DD₂. Nevertheless, the role of I_Na/Ca in rabbit SA node pacemaker activity remains to be clarified. Although ryanodine (10 µM) had just a small effect on the amplitude of action potential-induced Ca²⁺_i transients that were measured by indo 1 in the cytoplasm (Fig. 1), the slow inward current tails were abolished by the same concentration of ryanodine (Fig. 9B). This suggests that the whole cell spatially averaged [Ca²⁺]_i, measured by indo 1, is an unreliable indicator of the Ca²⁺ that is released from the SR and is most effective in activating Na⁺/Ca²⁺ exchange (16).

In summary, ryanodine's negative chronotropic effect is derived, in part, from a reduction of I_Ca,T. This reduction can be explained by the attenuation of action potential-induced Ca²⁺_i transients or by a direct effect of ryanodine on T-type Ca²⁺ channels. Although it is not known whether I_Na/Ca contributes to the diastolic depolarization of rabbit SA node cells, it is known to contribute to DD₂ in cat latent pacemaker cells (32), and it generates inward current tails (2, 6, 9, 32) much like those we recorded (Fig. 9). Thus, on the basis primarily of theory and results in other preparations, it is possible that, after inhibition of SR Ca²⁺ release by ryanodine, the smaller action potential-induced Ca²⁺_i transients could have reduced I_Na/Ca and, therefore, the beat rate.