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(Circulation Research. 1998;83:1192-1204.)
© 1998 American Heart Association, Inc.

Original Contributions

Bay K 8644 Increases Resting Ca²⁺ Spark Frequency in Ferret Ventricular Myocytes Independent of Ca Influx

Contrast With Caffeine and Ryanodine Effects

Hiroshi Satoh, Hideki Katoh, Patricio Velez, Michael Fill, Donald M. Bers

From the Department of Physiology and Cardiovascular Institute, Loyola University Chicago, Maywood, Ill.

Correspondence to Donald M. Bers, Department of Physiology, Loyola University Medical School, 2160 S First Ave, Maywood, IL 60153. E-mail dbers{at}luc.edu

	Abstract

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Abstract—Bay K 8644, an L-type Ca²⁺ channel agonist, was shown previously to increase resting sarcoplasmic reticulum (SR) Ca²⁺ loss and convert post-rest potentiation to decay in dog and ferret ventricular muscle. Here, the effects of Bay K 8644 on local SR Ca²⁺ release events (Ca²⁺ sparks) were measured in isolated ferret ventricular myocytes, using laser scanning confocal microscopy and the fluorescent Ca²⁺ indicator fluo-3. The spark frequency under control conditions was fairly constant during 20 s of rest after interruption of electrical stimulation. Bay K 8644 (100 nmol/L) increased the spark frequency by 466±90% of control at constant SR Ca²⁺ load but did not change the spatial and temporal characteristics of individual sparks. The increase in spark frequency was maintained throughout the period of rest. The increase in Ca²⁺ spark frequency induced by Bay K 8644 was not affected by superfusion with Ca²⁺-free solution (with 10 mmol/L EGTA) but was suppressed by the addition of 10 µmol/L nifedipine (which by itself did not alter resting Ca²⁺ spark frequency). This suggests that the effect of Bay K 8644 on Ca²⁺ sparks is mediated by the sarcolemmal dihydropyridine receptor but is also independent of Ca²⁺ influx. Low concentrations of caffeine (0.5 mmol/L) increased both the average frequency and duration of sparks. Ryanodine (50 nmol/L) increased the spark frequency and also induced long-lasting Ca²⁺ signals. This may indicate long-lasting openings of SR Ca²⁺ release channels and a lack of local SR Ca²⁺ depletion. In lipid bilayers, Bay K 8644 had no effect on either single-channel current amplitude or open probability of the cardiac ryanodine receptor. It is concluded that Bay K 8644 activates SR Ca²⁺ release at rest, independent of Ca²⁺ influx and perhaps through a functional linkage between the sarcolemmal dihydropyridine receptor and the SR ryanodine receptor. In contrast, caffeine and ryanodine modulate Ca²⁺ sparks by a direct action on the SR Ca²⁺ release channels.

Key Words: dihydropyridine receptor • sarcoplasmic reticulum • confocal microscopy • fluo-3

	Introduction

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Intracellular Ca²⁺ release channels, including both ryanodine and inositol 1,4,5-triphosphate receptors, participate in a variety of signaling pathways including fertilization, neurotransmitter release, hormonal activation, T cell activation, and excitation-contraction (E-C) coupling in muscle. Several modulators, including the activators Ca²⁺, adenine nucleotides, caffeine and ryanodine, and the inhibitors Mg²⁺, ruthenium red, and calmodulin, have been well-characterized in terms of their abilities to directly regulate sarcoplasmic reticulum (SR) Ca²⁺ release channel function.^{1 2 3 4 5}

In cardiac muscle, resuming stimulation after a period of rest causes negative or positive inotropic effects in cardiac muscle, which are referred to as rest decay or rest potentiation.⁶ Furthermore, rest decay is due to gradual SR Ca²⁺ depletion and extrusion from the cell via Na⁺/Ca²⁺ exchange during rest.^{6 7} Caffeine and ryanodine can both abolish rest potentiation and accelerate rest decay. These actions are attributable directly to increased SR Ca²⁺ release channel opening and consequent decline of SR Ca²⁺ content during rest.^{8 9}

The dihydropyridine (DHP) Bay K 8644 (BayK) increases Ca²⁺ influx through sarcolemmal Ca²⁺ channels by increasing the open time of the channel.¹⁰ BayK also has been used widely as a specific agonist of the L-type Ca²⁺ channel. An unexpected action of BayK in canine and ferret ventricular muscle is that BayK accelerates the decline of SR Ca²⁺ content during rest and converts rest potentiation to rest decay.^{11 12 13 14 15} Although this action is reminiscent of the action of ryanodine, which acts directly on the SR Ca release channel, there is no evidence for a direct action of BayK on the SR Ca²⁺ release channels.^{16 17} Furthermore, McCall et al¹⁵ found that BayK increased binding of ryanodine to the SR release channel, but only under conditions in which sarcolemmal-SR junctions may be expected to be intact (ie, not after physical disruption). They proposed that these actions of BayK may be mediated by a functional linkage between DHP receptor and the ryanodine receptor, either directly or via an additional spanning protein (see also Cohen and Lederer¹⁸ ).

Ca²⁺ sparks are viewed currently as the elementary event of SR Ca²⁺ release during E-C coupling in the heart.^{19 20 21 22 23 24} Although the number of individual ryanodine receptors responsible for a characteristic Ca²⁺ spark remains a matter of debate,^{19 25 26} the study of cardiac Ca²⁺ sparks can reveal important fundamental characteristics of the subcellular SR Ca²⁺ release process. Thus, it was considered important to study the effects of BayK on local SR Ca²⁺ release at the subcellular level of Ca²⁺ sparks to obtain a more complete understanding of how BayK may alter the SR Ca²⁺ release process.

In this study, we examined the effect of BayK, caffeine, and ryanodine on the frequency and kinetics of Ca²⁺ sparks during a short period of rest after the interruption of electrical stimulation in ferret ventricular myocytes. Our results indicate that BayK accelerates resting Ca²⁺ loss from the SR by increasing Ca²⁺ spark frequency through a Ca²⁺ influx–independent, functional linkage between the sarcolemmal and SR Ca²⁺ channels. In contrast, caffeine and ryanodine modulate Ca²⁺ sparks in a manner consistent with their direct action on the SR Ca²⁺ release channel.

	Materials and Methods

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Cell Isolation
Ventricular myocytes were isolated from adult male ferrets (1 to 1.3 kg) hearts by an enzymatic Langendorff perfusion procedure, as described previously.²⁷ Animals were anesthetized by intraperitoneal injection of sodium pentobarbital (100 mg/kg). Isolated ventricular myocytes were placed in an experimental superfusion chamber (0.5 mL). Cells were superfused continuously with a standard Tyrode solution at a rate of 2 to 3 mL/min. All experiments were performed at room temperature (23°C).

Drugs and Solutions
The standard Tyrode solution contained (in mmol/L): NaCl 140, KCl 6, MgCl₂ 1, HEPES 5, glucose 10, and CaCl₂ 2. In the Na⁺/Ca²⁺–free solution, LiCl replaced NaCl, CaCl₂ was omitted, and 10 mmol/L EGTA was added. In the Na⁺-free solution, NaCl was replaced by LiCl. The pH of all solutions was adjusted to 7.4 with NaOH or LiOH. (±)BayK (Miles Pharmaceuticals) and nifedipine (Sigma) were dissolved in ethanol (final [ethanol] <0.1%). Ryanodine (Sigma) was dissolved in distilled water and added to the superfusion solution immediately before use. Caffeine (Sigma) was dissolved directly in standard Tyrode solution or Na⁺/Ca²⁺–free solution.

Dye Loading and Fluorescence Imaging With Laser Scanning Confocal Microscopy
Isolated ferret ventricular myocytes were loaded with the fluorescent Ca²⁺ indicator fluo-3/AM (Molecular Probes) for 60 minutes at room temperature. A fluo-3 stock solution prepared from 50 µg fluo-3/AM, 12.5 µg Pluronic F-127, and 44.3 µL DMSO was added to standard Tyrode solution to give a final [fluo-3] of 20 µmol/L. The loading solution contained 1% BSA to enhance fluo-3 loading. Excess extracellular dye was removed by exchanging the bathing medium 3 times, and an additional 30 minutes were allowed for intracellular hydrolysis of fluo-3/AM.

Fluo-3 fluorescence imaging was performed with a laser scanning confocal microscope (LSM 410, Carl Zeiss), coupled to an inverted microscope (Axiovert 100, Carl Zeiss) and equipped with a 40x oil immersion objective (Plan-Neofluar, numerical aperture=1.3; Carl Zeiss). Fluo-3 fluorescence was excited with the 488-nm line of an argon ion laser. Emitted fluorescence was measured at wavelengths >515 nm.

Image acquisition for the quantitative analysis of Ca²⁺ sparks was made in the line scan mode. A single myocyte was scanned repetitively (250 Hz) along a line parallel to the longitudinal cell axis, avoiding nuclei. The line scan image was constructed by stacking 512 lines horizontally with time running from left to right. Magnification was set to give a pixel size of 0.05 µm² (0.23 µmx0.23 µm). Because the z resolution of the confocal microscope was set to 1 µm, the volume of an individually scanned voxel was therefore 0.05 µm³.

[Ca²⁺]_i-images were calculated according to the formula [Ca²⁺]_i=K_dx(F/F₀)/[(K_d/[Ca²⁺]_{i, rest})+1–(F/F₀)], where K_d is the dissociation constant for fluo-3, F is the fluorescence intensity, and F₀ is the intensity at rest, determined as the mean fluorescence intensity of the lowest 50 pixels along the each time line (512 pixels).²¹ The K_d and [Ca²⁺]_{i, rest} were assumed to be 1.1 µmol/L and 150 nmol/L, respectively.^{19 28 29}

Frequency Analysis of Ca²⁺ Sparks and Line [Ca²⁺] Transients
Ca²⁺ sparks were located visually. The fluorescence intensities of 5 adjacent pixels of an individual scan line, centered to the highest pixel value, were averaged and transformed to [Ca²⁺]_i. A local increase in fluo-3 fluorescence was counted as a Ca²⁺ spark when the peak amplitude of the [Ca²⁺]_i transient exceeded 60 nmol/L and the duration of the half amplitude was at least 8 ms. The number of Ca²⁺ sparks counted per line scan image was normalized spatially (per µm³) and temporally (per second) as the spark frequency (pL^-1xs^-1). Thus, 1 spark per line scan image corresponds roughly to 20 sparksxpL^-1xs^-1. The [Ca²⁺]_i transients evoked by electrical stimulation or caffeine application were derived from the changes in averaged fluorescence intensities along the scanned line and converted to [Ca²⁺]_i.

Experimental Protocols
[Ca²⁺]_i transients were elicited by field stimulation with 2-ms voltage pulses of 1.5x threshold amplitude, applied through extracellular platinum electrodes. When [Ca²⁺]_i transients had reached a steady state (1 to 2 minutes), stimulation was stopped, and the spark frequency was analyzed during a 20-s period of rest (10 images). During the rest period, a given cell was superfused with either the same solution or test solution. Rapid solution changes were achieved with a time constant of 300 ms (measured fluorometrically).

The SR Ca²⁺ content was evaluated by rapid application of 10 mmol/L caffeine dissolved in Na⁺/Ca²⁺–free solution with 1 mmol/L EGTA.^{27 30} This solution was introduced into the chamber via a quick-switching device at a flow rate of 5 to 7 mL/min, resulting in a [Ca²⁺]_i transient that reached a peak in <200 ms.

For experiments with BayK, cells were equilibrated for 8 to 10 minutes with 100 nmol/L BayK (sufficient for steady state effects), and paired comparisons were performed using the same cell before and after BayK exposure. Because BayK is notorious for contamination of perfusion lines, a separate tubing line was used for all BayK solutions.

SR Microsome Preparation and Single-Channel Recording
Heavy SR microsomes were prepared from dog cardiac muscle as described previously.³¹ Differential centrifugation of homogenized ventricular tissue was used to isolate SR microsomes. The microsomes were stored at –80°C until used. Planar lipid bilayers were made in a 150-µm–diameter hole in the wall of a Delrin cup. The lipid solution contained a 4:1 (by volume) mixture of phosphatidylethanolamine and phosphatidylcholine (Avanti Polar Lipids) dissolved in decane (50 mg/mL). SR vesicles were added into 1 side of the bilayer (defined as cis). The other side was defined as trans (virtual ground). When SR vesicles were fusing into the bilayer, solutions contained 250 mmol/L CsCH₃SO₃ cis (20 mmol/L trans), 10 mmol/L HEPES (pH 7.4), and 10 µmol/L [Ca]. After single-channel incorporation, the trans CsCH₃SO₃ was adjusted to 250 mmol/L. A custom-made current/voltage conversion amplifier was used to optimize single-channel recording.³² Single-channel data were digitized at 5 to 10 kHz and filtered at 1 kHz. The cytoplasmic side of the channel was determined by ATP sensitivity with the cytoplasmic side of the ryanodine receptor facing the cis compartment.³³

Statistical Analysis
Results were expressed as mean±SEM for the indicated number of myocytes. Statistical significance was determined by Student t test.

	Results

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Effects of BayK on Ca²⁺ Spark Frequency and SR Ca²⁺ Content
We sought to test the hypothesis that the known¹⁵ conversion of post-rest potentiation to post-rest decay by BayK in ferret ventricle is due to enhanced loss of Ca²⁺ from the SR. Thus, we investigated the effect of BayK on the frequency of Ca²⁺ sparks and SR Ca²⁺ content during rest. Under control conditions, the frequency of Ca²⁺ sparks during 20 s of rest remained virtually constant in ferret ventricle (Figures 1A and 2A). In the presence of BayK (100 nmol/L), however, the frequency of Ca²⁺ sparks was significantly larger as shown in the line scan image obtained with confocal microscopy (Figure 1B). This effect was most pronounced immediately after cessation of electrical stimulation but was maintained throughout the 20-s rest period (Figure 2A). The decline in Ca²⁺ spark frequency with BayK during rest may be secondary to the more rapid decline in the SR Ca²⁺ content with BayK (see McCall et al¹⁵ ; see below).

View larger version (89K): [in this window] [in a new window]   Figure 1. BayK increases resting Ca2+ sparks in ferret ventricle. Confocal line scan images obtained after 2 and 20 s of rest under control conditions (A) and in the presence of BayK (B). For each line scan image, a line (512 pixels) was positioned along the longitudinal axis of the cell and scanned repetitively at 250 Hz; thus, each image represents 2 s of continuous data acquisition. Single scan lines were stacked horizontally with time running from left to right. Before the rest period, the cells were stimulated at 1 Hz in control and at 0.5 Hz in BayK (100 nmol/L). Selected line profiles of [Ca2+]i shown underneath the line scan images were recorded from the sites marked as 1, 2, or 3. Dashed lines indicate [Ca2+]i=100 nmol/L. Bar=10 µm.

View larger version (44K): [in this window] [in a new window]   Figure 2. BayK effects on the Ca2+ spark frequency and steady-state SR Ca2+ content. A, Time courses of mean spark frequencies were normalized temporally and spatially (pL-1xs-1) during 20 s of rest in control () and in 100 nmol/L BayK () superfusion. Data represent mean±SEM from 8 paired experiments, and Ca2+ spark frequency is significantly higher in BayK at all time points. Dashed lines indicate simple linear regression. B, Peak [Ca2+]i of caffeine-induced [Ca2+]i transient was used to estimate the steady-state SR Ca2+ content. Caffeine (10 mmol/L) dissolved in a Na+/Ca2+–free, 1 mmol/L EGTA-containing solution was rapidly applied 2 s after the interruption of electrical stimulation. With the stimulation rate protocol (1 Hz in control and 0.5 Hz in BayK), there was no significant difference in the peak [Ca2+]i between control and BayK superfusion (NS; paired t test; n=5) induced by caffeine stimulation. C and D, Histograms of peak amplitude and duration of Ca2+ sparks recorded in the presence and absence of BayK. Curves are simple Gaussian fits to the histogram data that passed normalcy tests. r2 values for the Gaussian fits for amplitude in (C) are 0.86 and 0.67 for BayK and control, respectively, and for duration in (D), the r2 values are 0.93 and 0.91 for BayK and control, respectively.

Because BayK enhances Ca²⁺ influx via L-type Ca²⁺ channels, an increased SR Ca²⁺ load during steady-state stimulation could provide a simple explanation for the observed rise in Ca²⁺ spark frequency, as shown previously.³⁴ Therefore, conditions for experiments like that in Figure 1 were chosen explicitly to ensure that the SR Ca²⁺ load was not increased at the beginning of the rest period in the presence of BayK. Two methods were used to ensure comparable SR Ca²⁺ load in control and the presence of BayK. The SR Ca²⁺ load was adjusted by either decreasing the electrical stimulation frequency in BayK (1 Hz in control versus 0.5 Hz in BayK) or lowering [Ca²⁺]_o in BayK (3 mmol/L in control versus 1 mmol/L in the presence of BayK) at a constant stimulation frequency of 0.5 Hz. The SR Ca²⁺ load was tested by measuring the amplitude of the caffeine-evoked [Ca²⁺]_i transients.

In the first set of experiments, in which the SR Ca²⁺ load was controlled by altering the stimulation frequency, the presence of BayK increased the average Ca²⁺ spark frequency during 20 s of rest by 466±90% of control (P<0.01; paired t test). Figure 2B shows that the SR content, as measured by the amplitude of the caffeine-induced [Ca²⁺]_i transient in the presence of BayK, was not significantly different from control conditions. The peak [Ca²⁺]_i transient evoked by 10 mmol/L caffeine was 900±196 nmol/L in control and 846±151 nmol/L in the presence of BayK (NS; paired t test; n=5).

Similar results were found in the second set of experiments in which the SR Ca²⁺ content was adjusted by changing [Ca²⁺]_o at constant stimulation frequency (data not shown). The peak of [Ca²⁺]_i transients evoked by caffeine in the presence of BayK was 94±15% of that in control (NS; paired t test; n=5). Under these conditions, BayK increased the spark frequency on the average by 429±108% (P<0.01; unpaired t test; n=6). This is similar to when frequency was altered to match SR Ca load above (Figures 1 and 2).

The consequences of the increased spark frequency for the Ca²⁺ content of the SR during rest were tested by applying caffeine after 2 minutes of rest in the presence and absence of BayK. As shown in Figure 3, under both conditions, loss of Ca²⁺ from the SR occurred; however, the decrease in SR Ca²⁺ content was much more pronounced in the presence of BayK (Figure 3B) than in control (Figure 3A). On average, the amplitude of the caffeine-induced [Ca²⁺]_i transient, normalized to the amplitude measured immediately after cessation of stimulation (steady-state caffeine transient), decreased by 36% after 2 minutes of rest under control conditions (n=8) and by 58% in the presence of BayK (n=7). This faster loss of resting SR Ca²⁺ content with BayK seems likely to be due to the higher frequency of Ca²⁺ sparks and also may be the cause of the lower Ca²⁺ spark frequency at the end of the 20-s rest period. Therefore, increased Ca²⁺ spark frequency reduced SR Ca²⁺ load and then that reduced SR Ca²⁺ load decreases Ca²⁺ spark frequency.³⁴

View larger version (19K): [in this window] [in a new window]   Figure 3. BayK accelerates rest-dependent loss of SR Ca2+ content in ferret ventricular myocytes. Caffeine-induced [Ca2+]i transients recorded 2 s after cessation of electrical stimulation (steady-state) and after 120 s of rest (post-rest) in the absence (A) and presence (B) of BayK. Caffeine (10 mmol/L) in Na+/Ca2+–free solution was applied rapidly during the period marked by the solid bar underneath the [Ca2+]i transient. Data in (A) and (B) are from different cells. C, Pooled data of caffeine-induced peak [Ca2+]i transients after 120 s of rest in the presence and absence of BayK. Post-rest values were normalized to the steady-state values. *P<0.01 (paired t test) in relation to steady-state values. BayK was also significantly different from control post-rest (P<0.05; unpaired t test). Data are mean±SEM of 7 to 8 paired experiments.

Because the probability of SR Ca²⁺ release opening depends on local [Ca²⁺]_i, any increase in diastolic [Ca²⁺]_i induced by BayK could contribute to the increase in Ca²⁺ spark frequency. However, diastolic [Ca]_i was not altered significantly by BayK in these experiments (fluorescence intensity, 36.5±5.8 units in control and 37.5±5.2 units in BayK). Moreover, a small increase in diastolic [Ca²⁺]_i could even be expected to occur because of the dramatic increase in Ca²⁺ sparks with BayK (as concluded for rat ventricular myocytes after brief rest intervals).³⁴ Thus, the increase in Ca²⁺ spark frequency with BayK cannot be attributed to either increased SR Ca²⁺ content or increased diastolic [Ca²⁺]_i.

Effects of BayK on the Characteristics of Individual Ca²⁺ Sparks
Although the presence of BayK significantly increased the frequency of Ca²⁺ sparks during rest, it did not affect their spatial and temporal characteristics (Figure 2C and 2D). The average amplitude was 130±9 nmol/L under control conditions (n=39) and 126±7 nmol/L in the presence of BayK (n=54). The average spark duration, measured at half-amplitude, was 29.7±1.6 ms and 30.1±1.2 ms in the absence and presence of BayK, respectively. The differences in amplitude and duration were not statistically significant (unpaired t test). The Gaussian fits shown in Figure 2C and 2D may not be useful mechanistically, especially for the amplitude histogram, in which the frequency for the lower bins is likely to be underestimated because it is close to the detection limit.²⁵

Mechanism of the BayK-Induced Increase in Ca²⁺ Spark Frequency During Rest
Ca²⁺ Influx
BayK could, in principle, enhance the spark frequency by increasing the open probability of sarcolemmal Ca²⁺ channels. Occasional openings of L-type Ca²⁺ channels during rest could be increased by BayK and cause localized release of Ca²⁺ (sparks) via Ca²⁺-induced Ca²⁺ release (CICR). To examine this issue, we removed extracellular Ca²⁺ completely during rest so that no diastolic Ca²⁺ influx could occur. This was achieved by removing extracellular Ca²⁺ immediately (300-ms time constant) after interruption of electrical stimulation. In these experiments, standard Tyrode solution was replaced rapidly with a Na⁺/Ca²⁺–free solution containing 10 mmol/L EGTA. Na⁺ also was removed to inhibit Ca²⁺ extrusion via Na⁺/Ca²⁺ exchange and consequent loss of SR Ca²⁺.⁷ These conditions would lower steady-state free [Ca]_o to 0.1 nmol/L (even with 16 µmol/L contaminant Ca). This would decrease the Ca influx through an open Ca channel by a factor >10⁷, from 10⁶ Ca ions/s to 1 Ca ion every 10 s (based on the Goldman current equation). This also would bring the thermodynamic reversal potential for Ca (E_Ca) to –90 mV, making resting Ca²⁺ influx extremely unlikely.

Figure 4 illustrates the effect of removal of extracellular Ca²⁺ on the spark frequency in the presence of BayK. The line scan images recorded after 2 and 16 s of rest showed similar spark frequencies in the presence (Figure 4A) and absence of extracellular Ca²⁺ (Figure 4B). Figure 5A shows that average spark frequency during 20 s of rest, in the presence of BayK, was 16% higher in Na⁺/Ca²⁺–free solution than in normal Tyrode solution (NS; n=7). This Ca²⁺ spark frequency is 500% of that observed in the absence of BayK.

View larger version (84K): [in this window] [in a new window]   Figure 4. Preventing Ca2+ influx during rest does not alter BayK-induced increase in Ca2+ spark frequency. A, Line scan images of Ca2+ sparks in the presence of BayK recorded at 2 and 16 s of rest, respectively. B, Bath solution was switched rapidly to a Na+-free, Ca2+-free, EGTA-containing solution just after the interruption of electrical stimulation (BayK with Na+/Ca2+–free solution). SR Ca2+ content was not altered on the rapid switch to Na+/Ca2+–free solution. Line recordings of [Ca2+]i shown underneath the line scan images were taken from selected sites marked as 1, 2, or 3 and reveal the typical profile of Ca2+ sparks. Dashed lines indicate [Ca2+]i=100 nmol/L. Bar=10 µm.

View larger version (17K): [in this window] [in a new window]   Figure 5. Effect of BayK on the relative Ca2+ spark frequencies in the presence of nifedipine and in the absence of extracellular Ca2+. A, Normalized spark frequencies in the presence of nifedipine (10 µmol/L; [Na+]o=0; [Ca2+]o=2 mmol/L; n=4 cells) and during Na+/Ca2+–free superfusion (n=7 cells). Data were normalized to the spark frequency measured in the presence of BayK (100 nmol/L) in standard Tyrode solution ([Ca2+]o=2 mmol/L). *P<0.05 paired t test vs exposure to BayK in standard Tyrode solution. B, Ca2+ spark frequency as a function of rest duration in either 100 nmol/L BayK alone or when extracellular Na+ and Ca2+ were removed and 10 mmol/L EGTA was applied immediately after the last stimulated twitch (n=7 cells). Dashed lines indicate simple linear regressions.

Figure 5B shows the time course of Ca²⁺ spark frequency change during rest in the presence or absence of extracellular Ca²⁺. There is a somewhat smaller decline in Ca²⁺ spark frequency during rest with BayK compared with the cells in Figure 2A (which was attributed to gradual SR Ca²⁺ depletion). Switching to Na⁺/Ca²⁺–free solution during the rest did not reduce Ca²⁺ spark frequency at any point, and there was a slight tendency for Ca²⁺ spark frequency to increase during rest. This trend was observed previously for rat myocytes under control conditions and in rabbit myocytes when Ca²⁺ extrusion by Na⁺/Ca²⁺ exchange was prevented so that SR Ca²⁺ load did not decline during rest.³⁴ Limiting SR Ca²⁺ depletion in this manner by Na⁺/Ca²⁺–free solution could easily explain the small overall increase in Ca²⁺ spark frequency with Na⁺/Ca²⁺–free solution in Figure 5A.

T-tubule Ca²⁺ depletion may not be complete for 1 to 2 s after the rapid switch to Ca²⁺-free superfusate (containing 10 mmol/L EGTA).^{35 36 37} Indeed, when 0.5-Hz stimulation was continued on rapid switch to Na⁺-free Ca solution, a very small initial twitch could often be observed within 1 to 2 s of the switch, but there was no detectable twitch or [Ca²⁺]_i transient at the time of the second stimulation (<4 s after the switch; note that action potentials are still activated in this Na⁺-free Ca²⁺ solution with Li⁺ in place of Na⁺.²⁷ Thus for most of the 20-s period in Na⁺/Ca²⁺–free/EGTA solution, [Ca²⁺]_o was low enough to prevent Ca²⁺ influx. Furthermore, if Ca²⁺ influx was involved in the resting spark enhancement with BayK in Na⁺/Ca²⁺–free solution, one would expect a very rapid decline in Ca²⁺ spark frequency during rest ( 200 ms). Such a decline was not observed clearly, and very high Ca²⁺ spark frequencies were observed throughout the 20 s in Na⁺/Ca²⁺–free solution (Figures 4 and 5). On the basis of these experiments, we conclude that BayK did not increase the Ca²⁺ spark frequency through enhanced Ca²⁺ influx, local CICR, and/or increased SR Ca²⁺ content.

DHP Receptor
We examined whether BayK increased the spark frequency through binding to the sarcolemmal DHP receptor. This was tested by comparing the effect of 100 nmol/L BayK in the presence and absence of nifedipine, the competitive blocker of L-type Ca²⁺ channels, at a high concentration (10 µmol/L). The concentration of nifedipine was selected because it is 100 times higher than that used for BayK; this would be required to strongly compete with BayK and largely displace it from the DHP receptor. Furthermore, the block of Ca channels by dihydropyridines is voltage-dependent, and micromolar concentrations are required for block of Ca current even in the absence of channel agonist.

Because nifedipine by itself could affect the Ca²⁺ spark frequency due to a block of Ca²⁺ entry and subsequent loss of Ca²⁺ from the SR, matching the SR Ca²⁺ load under both experimental conditions again was critical. In the presence of nifedipine, the cells were superfused for 20 s with a Na⁺-free solution ([Ca²⁺]_o=2 mmol/L). Under these conditions, SR Ca²⁺ loading occurs by Ca²⁺ influx via Na⁺/Ca²⁺ exchange and any residual sarcolemmal Ca²⁺ currents.³⁸ After 20 s of superfusion with Na⁺-free solution, the SR Ca²⁺ content in the presence of nifedipine was, on the average, 94±7% of that measured in standard Tyrode solution (NS; paired t test; n=4). As before, the SR Ca²⁺ content was estimated by rapid application of caffeine (see Effects of BayK on Ca²⁺ Spark Frequency and SR Ca²⁺ Content). The effect of nifedipine on the BayK-induced increase of spark frequency was measured during a 20-s period of rest. Nifedipine antagonized the BayK effect (Figure 5) and reduced the average spark frequency to 42±13% of that with BayK alone (P<0.05; paired t test; n=4). Figure 6 shows line scan images during rest and 2 and 18 s after the electrical stimulation was stopped. On the basis of these experiments, we concluded that the enhancement of Ca²⁺ sparks by BayK during rest was mediated by the DHP receptor, but not through enhanced Ca²⁺ influx through the L-type Ca²⁺ channel.

View larger version (91K): [in this window] [in a new window]   Figure 6. Ca2+ channel antagonist nifedipine inhibits BayK-induced Ca2+ sparks. A, Line scan [Ca2+]i images of Ca2+ sparks in the presence of BayK at 2 and 18 s of rest. B, Line scan images after the addition of 10 µmol/L nifedipine (BayK with nifedipine). To obtain the same SR Ca2+ loading, the bath solution was switched to Na+-free solution (Li+ substitution) for 20 s before the interruption of electrical stimulation. Superfusate then was switched back to normal Tyrode solution. Line recordings of [Ca2+]i shown underneath the line scan images were taken from the sites indicated as 1, 2, or 3. Dashed lines indicate [Ca2+]i=100 nmol/L. Bar=10 µm.

We also tested the effect of nifedipine alone on the Ca²⁺ spark frequency. Figure 7 shows that when SR Ca load was matched in the presence and absence of 10 µmol/L nifedipine (using the same experimental strategy shown in Figure 6), there was no significant effect on Ca²⁺ spark frequency. Thus, although the Ca²⁺ channel agonist BayK increases Ca²⁺ spark frequency in a nifedipine-sensitive manner, nifedipine by itself does not have a detectable effect.

View larger version (16K): [in this window] [in a new window]   Figure 7. Nifedipine alone does not alter resting Ca2+ spark frequency at constant SR Ca load. Protocols were similar to those described in Figure 6, except that BayK was not used. Ca2+ sparks were recorded after Na+-free 2 Ca2+ Tyrode solution was added for 20 s (before or after equilibration with 10 µmol/L nifedipine). A, SR Ca load assessed by caffeine-induced [Ca2+]i transients were the same before and after nifedipine in paired experiments (control peak [Ca]i=1426±124 nmol/L; n=3). B, Ca2+ spark frequency under these conditions at constant SR Ca load was not altered significantly by 10 µmol/L nifedipine (control spark frequency=36.8±15.4 pL-1xs-1; paired t test; n=4). Data was normalized to the spark frequency measured in the standard Tyrode solution in the same cell. Ctl indicates control.

Agents That Directly Activate Ryanodine Receptor
Caffeine and ryanodine have been shown to alter properties of the SR Ca²⁺ release channel and to modulate its gating. Figure 8 shows the effects of caffeine on the frequency and kinetics of Ca²⁺ sparks in ferret myocytes. Under control conditions, the duration of Ca²⁺ sparks, measured at half-maximal amplitude, was 30 ms. Exposure to a low concentration of caffeine (0.5 mmol/L) caused a characteristic change in the spark pattern. In addition to the type of Ca²⁺ spark typically observed under control conditions (Figure 8A), long-lasting [Ca²⁺]_i signals consistent with a continuous increase of local [Ca²⁺]_i were observed frequently (Figure 8B, left). This effect was most prominent during the initial few seconds of rest. Figure 8B (right) shows the distribution of spark durations (n=68 fluorescence signals). The duration distribution histogram was fit with 2 Gaussian distributions with 2 distinct peaks 30 and 220 ms representing the typical sparks and the long-lasting events induced by caffeine, respectively. In the presence of caffeine at a high concentration (10 mmol/L) that depletes the SR, neither the typical Ca²⁺ sparks nor the long-lasting signals were observed (Figure 8C). Mean Ca²⁺ spark amplitude was not significantly altered by 0.5 mmol/L caffeine in unpaired comparisons (96±4 nmol/L with caffeine, 130±9 nmol/L in control). However, the mean value in caffeine was smaller, perhaps because of lower SR Ca²⁺ content.³⁴

View larger version (70K): [in this window] [in a new window]   Figure 8. Dose-dependent effects of caffeine on Ca2+ sparks. Confocal line scan images of Ca2+ sparks in standard Tyrode solution (A) and in the presence of 0.5 mmol/L (B, left) and 10 mmol/L (C) caffeine. All images represent the first 2 s of rest after interruption of electrical stimulation. Line recordings of [Ca2+]i shown underneath the line scan images were taken from the sites indicated as 1 or 2. Dashed lines indicate [Ca2+]i=100 nmol/L. Bar=10 µm. Data are from different cells. B (right), Distribution of spark durations (measured at half-maximal amplitude) in the presence of 0.5 mmol/L caffeine (n=68 fluorescent signals). Duration distribution histogram was well-matched by 2 Gaussian distributions, representing common Ca2+ sparks and caffeine-induced continuous increases of local [Ca2+]i.

Ryanodine is a more potent and specific modulator of the SR Ca²⁺ release channel than is caffeine, and Figure 9 shows the effects of ryanodine on Ca²⁺ sparks in ferret ventricular myocytes. In the initial several minutes (5 minutes) after the addition of a low concentration of ryanodine (50 nmol/L), the spark frequency dramatically increased, and long-lasting localized increases of [Ca²⁺]_i of variable duration were observed (Figure 9B, left). Figure 9B (right) shows the distribution of spark durations (pooled data from 51 sparks). The duration distribution histogram could be fit by 2 Gaussian distributions. However, the frequency of long-lasting signals was somewhat smaller than that observed with caffeine (0.5 mmol/L). On the basis of these experiments, it appeared that low concentrations of ryanodine primarily increased the number of sparks and to a lesser extent also influenced the kinetics of the individual sparks. These Ca²⁺ sparks in 50 nmol/L ryanodine were highly concentrated during the first 2 seconds after the last stimulation (eg, the 2-s bin had a mean spark frequency of 530 pL^-1xs^-1, compared with 30 to 150 pL^-1xs^-1 in Figure 2A). This is consistent with the effects of ryanodine, because it depletes the SR of Ca²⁺ over a similar short time after a twitch.⁸ Prolonged exposure (>10 minutes) to 50 nmol/L or exposure to 1 µmol/L ryanodine resulted in a decrease of the amplitude of electrically evoked [Ca²⁺]_i transients, an increase in resting [Ca²⁺]_i, and the disappearance of Ca²⁺ sparks presumably because of SR Ca²⁺ depletion. The amplitude of local Ca²⁺ release events early in 50 nmol/L ryanodine was not significantly different from control in unpaired comparisons (169±16 nmol/L in ryanodine; 130±9 nmol/L in control). A slightly higher mean value could be caused by overlapping events (more likely when longer events occur) and little effect of low ryanodine concentration on SR Ca²⁺ load immediately after a twitch,⁸ when most of the events with ryanodine occurred.

View larger version (71K): [in this window] [in a new window]   Figure 9. Dose-dependent effects of ryanodine on Ca2+ sparks. Line scan images of Ca2+ sparks in standard Tyrode solution (A) and in the presence of 50 nmol/L (B, left) and 1 µmol/L (C) ryanodine. All images represent the first 2 s of rest after the interruption of electrical stimulation. Line recordings of [Ca2+]i shown underneath the line scan images were taken from the sites indicated as 1 or 2. Dashed lines indicate [Ca2+]i=100 nmol/L. Bar=10 µm. B (right), Distribution of spark durations (measured at half-maximal amplitude) in the presence of 50 nmol/L ryanodine (n=51 fluorescent signals). Duration distribution histogram was well-matched by 2 Gaussian distributions.

These experiments indicated (albeit indirectly) that pharmacological agents that interact with the SR Ca²⁺ release channel directly change the typical characteristics of individual Ca²⁺ sparks, such as amplitude, duration, and spatial spread. BayK, however, simply increased the frequency of stereotypical Ca²⁺ sparks. The caffeine and ryanodine results do not directly test how BayK works to alter Ca²⁺ sparks. Moreover, a direct effect of BayK on the ryanodine receptor was not ruled out by the foregoing experiments. To evaluate this possibility more directly, the effects of BayK were studied on single ryanodine receptor channels incorporated into lipid bilayers.

Ryanodine Receptor Single-Channel Recordings
Figure 10 shows recordings of single-channel currents through a single SR Ca²⁺ release channel. The channel was partially activated by 10 µmol/L Ca²⁺ on the cytoplasmic side of the bilayer. Figure 10A shows the channel gating in the presence of 1 µmol/L ryanodine and the transition into a stable subconductance state, the hallmark of the effect of ryanodine on the SR Ca²⁺ release channel.^{2 39} Figure 10B compares single-channel currents in the absence and presence of BayK (10 µmol/L). Even in the presence of this very high concentration of BayK, there was no effect of BayK on either the amplitude histograms or the channel open probability. The difference between the means of the 2 peaks of the amplitude histograms represents the mean single-channel current amplitude (I_mean) and the relative areas of the 2 peaks indicate open probability (P_o). The I_mean and P_o were 12.5±0.8 pA and 0.192±0.062, respectively, under control conditions. In the presence of 10 µmol/L BayK, I_mean was 12.3±0.9 pA and P_o was 0.181±0.074 (NS; paired t test; n=4). These experiments provided evidence that BayK did not affect the function of the SR Ca²⁺ release channel directly.

View larger version (41K): [in this window] [in a new window]   Figure 10. Action of BayK on single-channel gating. Single ryanodine receptor channels were reconstituted into planar lipid bilayers. Single-channel openings are shown as upward deflections. c indicates closed; o, open. Charge carrying ion was cesium. Resting free [Ca2+] was 10 µmol/L. A, Action of ryanodine to alter channel gating. B, Representative single-channel records in the absence (control) and presence of BayK (10 µmol/L). B, Double Gaussian fits to all-points amplitude histograms constructed from 240 s of single-channel recording on 4 single channels are shown next to the single-channel recordings. Difference between the means of the 2 peaks represents Imean. Relative areas of the 2 peaks indicates Po. There were no significant differences in Imean and Po between control and BayK (see Ryanodine Receptor Single-Channel Recordings).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ryanodine and Caffeine Directly Modify SR Ca²⁺ Release Channels
The effects of ryanodine and caffeine on Ca²⁺ sparks is entirely consistent with the known actions of these agents on ryanodine receptors in bilayers and SR vesicles^{1 2 3} and also with previous measurements of Ca²⁺ sparks in ventricular myocytes.^{19 40} Low concentrations of ryanodine and caffeine produced long SR Ca²⁺ release events of relatively sustained amplitudes (Figures 8 and 9). This has 2 relevant implications. First, local SR Ca²⁺ must not be depleted greatly during a Ca²⁺ spark (otherwise, the amplitude would decline with time). This must mean that the combination of local Ca²⁺ reuptake and diffusion from other regions of SR must be sufficient to keep pace with the rate of SR Ca²⁺ release. This has implications for the termination of SR Ca²⁺ release during E-C coupling. Because local Ca²⁺ depletion does not occur even with the long release events here, we infer that during normal E-C coupling, SR Ca²⁺ release is unlikely to be terminated by local SR Ca²⁺ depletion.⁴¹ Although this may be true in an absolute sense, this does not preclude a participatory role of SR Ca²⁺ depletion in termination of SR Ca²⁺ release (because Ca²⁺ load is an important factor regulating Ca²⁺ release^{34 42} ; also see Song et al²⁵ ).

Second, the effects of ryanodine may be relevant to the number of ryanodine receptors involved in a Ca²⁺ spark. If a Ca²⁺ spark resulted from a cluster of ryanodine receptors working in concert, one could expect that modification of 1 channel in the cluster by ryanodine may produce a much smaller sustained phase of Ca²⁺ release (which was not observed). However, it is also possible that whenever a single, ryanodine-modified channel opens, it causes activation of the entire cluster as a functional unit. Thus, these results cannot discriminate the number of ryanodine receptors involved in a Ca²⁺ spark.

Effect of BayK on E-C Coupling and Ca²⁺ Flux in Ventricular Myocytes
The ability of the DHP compound BayK to activate sarcolemmal L-type Ca²⁺ channels has been documented extensively (see Bers⁶ ). Recent studies, however have suggested an additional effect of BayK on Ca²⁺ fluxes in ventricular myocytes, which may involve a more direct form of communication between the DHP receptor and the Ca²⁺ release channel (or ryanodine receptor). It has been shown that BayK accelerates rest decay and rest-dependent decline in SR Ca²⁺ content in ferret and canine ventricle and that this action of BayK is independent of the action potential, Ca²⁺ current (I_Ca) or extracellular Ca²⁺.^{11 12 15} Furthermore, a reduction in the efficacy of a given I_Ca to elicit Ca²⁺ release during E-C coupling by BayK has been reported.⁴³ These studies on the whole-cell level led to the working hypothesis that binding of BayK to the DHP receptor may alter the state of the SR Ca release channel, possibly through a functional or physical linkage between DHP and ryanodine receptor.

A physical link between DHP and ryanodine receptor is considered important in E-C coupling in skeletal muscle, where the DHP receptor serves as a voltage sensor for sarcolemmal depolarization.^{44 45} Widely accepted models of E-C coupling in skeletal muscle propose that membrane depolarization through an electromechanical link between DHP receptor and ryanodine receptor triggers Ca²⁺ release from the SR. Experimental evidence for such models stem from the measurement of depolarization-induced membrane charge movements and functional expression of chimeric Ca²⁺ channels.⁴⁶ In heart, this issue is more controversial. The generally accepted model in heart is that charge-coupled SR Ca²⁺ release is not functional and that Ca²⁺ influx is strictly required to trigger SR Ca²⁺ release.^{6 47 48} However, depolarization-dependent charge movement does occur in the heart,^{49 50 51} and a directly voltage-dependent SR Ca²⁺ release mechanism also has been suggested.^{52 53 54} Cohen and Lederer¹⁸ also proposed a model involving a spanning protein between DHP and ryanodine receptor to explain a presumed [Ca²⁺]_i-independent alteration of I_Ca by ryanodine, although these results could also be explained by effects of ryanodine on SR Ca²⁺ release.⁵⁵

The previous studies on a putative direct communication between DHP and ryanodine receptors draw their conclusions from whole-cell parameters such as whole-cell I_Ca and whole-cell [Ca²⁺]_i transients. In the present study, we investigated the effect of BayK on SR Ca²⁺ release on a more "microscopic" level by studying the effect of BayK on the behavior of single ryanodine receptors (planar lipid bilayer studies) and Ca flux through a small group of release channels (Ca²⁺ sparks) in intact myocytes. Using confocal microscopy, we demonstrated that the previously described loss of Ca²⁺ during rest in the presence of BayK^{11 12 15} could be attributed to a significant increase in the frequency of Ca²⁺ sparks.

Mechanism of SR Ca²⁺ Release Modulation by BayK
We tested several hypotheses for the mechanism of the BayK-induced increase in Ca²⁺ spark frequency and accelerated loss of Ca²⁺ from the SR during rest. The possibilities tested were: (1) enhanced Ca²⁺ influx via I_Ca and subsequent SR Ca²⁺ loading, (2) increased resting Ca²⁺ influx triggering Ca²⁺ sparks, (3) direct (intracellular) pharmacological effect of BayK on the SR Ca²⁺ release channel, and (4) mediation of the BayK effect through a functional linkage between DHP and ryanodine receptor.

Ca²⁺ Influx
The possibility that BayK increased Ca²⁺ sparks secondary to resting Ca²⁺ influx via L-type Ca²⁺ channel had to be considered. Talo et al⁵⁶ described a continuous Ca²⁺ window current at membrane potentials more negative than necessary for activation of contraction. Because BayK shifts the current-voltage relationship toward more negative potentials,^{43 57} such a window current could be enhanced by BayK and lead to local Ca²⁺ influx. This Ca²⁺ current might not be readily detectable by conventional electrophysiological techniques but could still be sufficient to induce local SR Ca²⁺ release during rest (through CICR) or to increase Ca²⁺ spark frequency indirectly by increasing SR Ca²⁺ load.

The possibility of increased SR Ca²⁺ content was addressed directly (inherently in the experimental design, in which increased SR Ca²⁺ load with BayK was avoided by altering frequency or [Ca]_o). In addition, the SR Ca²⁺ content was assessed by caffeine-induced Ca²⁺ transients under the conditions used to measure Ca²⁺ sparks. There was no difference in SR Ca²⁺ load at the beginning of the rest interval, ruling out the possibility of increased SR Ca²⁺ load as the cause of the BayK-induced increase in Ca²⁺ sparks. Moreover, the more rapid decline in resting SR Ca²⁺ content with BayK (due to the increased Ca²⁺ spark frequency) may mean that we are underestimating the stimulatory effect of BayK on Ca²⁺ sparks by pooling data during 20 s of rest.

The possibility of Ca²⁺ channel influx causing the increase in Ca²⁺ spark frequency because of increased occasional openings of L-type Ca²⁺ channels with BayK was assessed by complete removal of extracellular Ca²⁺ (with 10 mmol/L EGTA). During the first 1 to 2 s of Ca²⁺-free superfusion, this result might be equivocal because of the time required for depletion of T-tubular Ca²⁺. However, as discussed in Results, the maintained elevation of Ca²⁺ sparks throughout the 20-s rest period in Ca²⁺-free, EGTA buffer assures that this BayK effect occurs in the complete absence of extracellular Ca²⁺. Remarkably, complete removal of extracellular Ca²⁺ did not alter the BayK-induced increase in Ca²⁺ sparks (Figures 4 and 5). These results argue strongly against Ca²⁺ influx playing a role in the increase in Ca²⁺ sparks with BayK. These data are also consistent with the previous observations that BayK accelerated the loss of Ca²⁺ from the SR during rest in the absence of extracellular Ca²⁺.¹⁵

Direct Effect of BayK on the SR Ca²⁺ Release Channel
The possibility that BayK could enter the cytoplasm and act directly on the Ca²⁺ release channel also was considered. However, several lines of evidence argue against this possibility. First, the effect of BayK on the frequency of Ca²⁺ sparks during rest could be antagonized by the DHP receptor blocker nifedipine (Figures 5 and 6). The most straightforward explanation for this observation is a competitive binding of BayK and nifedipine to the sarcolemmal DHP receptor. If BayK acted directly on a novel, nonspecific SR Ca²⁺ release channel site, nifedipine would be unlikely to antagonize the BayK effect. Indeed, ryanodine receptors, as routinely purified, do not copurify with DHP receptors, unless efforts are made to isolate SR-sarcolemmal junctional couplings.⁵⁸ Thus, there is no evidence for high-affinity DHP binding directly to the SR.

Second, caffeine and ryanodine, known to act directly on the ryanodine receptor, had more dramatic effects than BayK on the spatial and temporal pattern of individual Ca²⁺ sparks. That is, caffeine and ryanodine both induced long-lasting elevations of [Ca²⁺]_i, whereas BayK did not alter any characteristics of Ca²⁺ sparks such as amplitude, duration, and spatial spread. By itself, this is a relatively weak argument, but the lack of an effect on the basic characteristics of Ca²⁺ sparks is consistent with a lack of direct effect of BayK on the SR release channel.

Third, a direct effect of BayK on the behavior of the ryanodine receptor was tested directly in bilayer studies. These experiments showed that BayK had no effect on SR Ca²⁺ channel gating, single-channel current amplitude, or open probability even at a concentration of BayK 100 times higher than in the cellular experiments (Figure 10). These findings are also consistent with previous reports on the lack of a direct effect of BayK on SR function. For example, BayK increased ryanodine binding to intact ferret ventricular myocytes but not after SR-sarcolemmal junctions were mechanically disrupted by aggressive homogenization.¹⁵ BayK also had no influence on SR Ca release in skinned guinea pig atrial fibers.¹⁶

Functional Linkage Between DHP and Ryanodine Receptor
A direct signaling pathway between sarcolemmal DHP receptors and the Ca²⁺ release channel/ryanodine receptors through a functional or physical linkage would be consistent with the results obtained in the present study and by McCall et al.¹⁵ In skeletal muscle, a voltage-dependent conformational change of the DHP receptor is postulated to induce the opening of the SR Ca²⁺ release channel.^{44 45} Support for a physical link between DHP and ryanodine receptor comes from ultrastructural studies showing the close arrangements of the 2 receptor types⁵⁹ and also by the apparent functional effects of a central intracellular loop of the skeletal muscle DHP receptor (II-III loop) on the skeletal muscle ryanodine receptor.^{46 60} Conformational models involving close interactions between the inositol 1,4,5-triphosphate receptor and surface membrane ion channels also have been postulated for the mechanism of capacitative Ca²⁺ entry that occurs in many different cell types as a consequence of intracellular store depletion (for review, see Berridge⁶¹ ).

Even if there is some functional linkage between the DHP and ryanodine receptor in ferret ventricular myocytes, there are some intrinsic stoichiometric limitations. That is, because there are 10 times as many ryanodine receptors as DHP receptors in ferret myocytes,⁶² such a linkage, if 1:1, could only be expected to affect 10% of the ryanodine receptors directly.¹⁵ The ratio of ryanodine to DHP receptors varies from 4 to 10 among different mammalian ventricular myocytes.⁶² The number of ryanodine receptors that constitute a functionally cooperative SR Ca²⁺ release unit in heart is not known, but a value in the range of 4 to 10 is plausible (where there is 1 DHP receptor per cluster).

The BayK-induced increase in resting ryanodine receptor gating described here could be via a relatively direct functional linkage. However, this does not imply that membrane depolarization would activate the ryanodine receptor in the manner proposed in skeletal muscle.^{44 45} Indeed, action potential depolarizations in Na⁺/Ca²⁺–free solution in the present study did not induce any [Ca²⁺]_i transient or Ca²⁺ sparks. Thus, the present results are not necessarily related to provocative recent results in cardiac muscle that have raised anew the possibility that membrane depolarization can activate SR Ca²⁺ release without Ca²⁺ entry.^{52 53 54} Indeed, in E-C coupling studies, McCall and Bers⁴³ found that BayK depressed SR Ca²⁺ release in response to a given cellular Ca²⁺ current and SR Ca²⁺ content.

A functional linkage between the DHP and ryanodine receptor that would serve as a 2-way communication pathway between the 2 receptors is an intriguing possibility, although the physical nature of such a linkage remains to be determined. Thus, although there does seem to be some functional, Ca²⁺ influx-independent linkage between the cardiac DHP and ryanodine receptor, additional work will be required to fully elucidate the nature of this interaction and its relevance to cardiac E-C coupling.

	Acknowledgments

 
This work was supported by grants from the United States Public Health Service (AR-41197, HL-57832, and HL-30077) and American Heart Association. Dr Fill is an Established Investigator of the American Heart Association. We wish to thank Dr Lothar A. Blatter for extensive advice, discussions, and thoughtful comments. We also thank Christina Hovance for expert technical assistance and Jaclyn R. Holda for critical reading of an early version of the manuscript.

Received April 21, 1998; accepted September 30, 1998.

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