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(Circulation Research. 2000;87:1034.)
© 2000 American Heart Association, Inc.

Cellular Biology

Ignition of Calcium Sparks in Arterial and Cardiac Muscle Through Caveolae

Matthias Löhn, Michael Fürstenau, Victoriya Sagach, Marlies Elger, Wolfgang Schulze, Friedrich C. Luft, Hermann Haller, Maik Gollasch

From the Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Charité University Hospitals, Humboldt University of Berlin, Germany, and Medical School Hannover, Department of Nephrology, Hannover, Germany.

Correspondence to Maik Gollasch, MD, PhD, Franz Volhard Clinic, Wiltbergstrasse 50, 13125 Berlin, Germany. E-mail gollasch{at}fvk-berlin.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Ca²⁺ sparks are localized intracellular Ca²⁺ events released through ryanodine receptors (RyRs) that control excitation-contraction coupling in heart and smooth muscle. Ca²⁺ spark triggering depends on precise delivery of Ca²⁺ ions through dihydropyridine (DHP)-sensitive Ca²⁺ channels to RyRs of the sarcoplasmic reticulum (SR), a process requiring a very precise alignment of surface and SR membranes containing Ca²⁺ influx channels and RyRs. Because caveolae contain DHP-sensitive Ca²⁺ channels and may colocalize with SR, we tested the hypothesis that caveolae are the structural element necessary for the generation of Ca²⁺ sparks. Using methyl-ß-cyclodextrin (dextrin) to deplete caveolae, we found that dextrin dose-dependently decreased the frequency, amplitude, and spatial size of Ca²⁺ sparks in arterial smooth muscle cells and neonatal cardiomyocytes. However, temporal characteristics of Ca²⁺ sparks were not significantly affected. We ruled out the possibility that the decreases in Ca²⁺ spark frequency and size are caused by changes in DHP-sensitive L-type channels, SR Ca²⁺ load, or changes in membrane potential. Our results suggest a novel signaling model that explains the formation of Ca²⁺ sparks in a caveolae microdomain. The transient elevation in [Ca²⁺]_i at the inner mouth of a single caveolemmal Ca²⁺ channel induces simultaneous activation and thus opens several RyRs to generate a local Ca²⁺ release event, a Ca²⁺ spark. Alterations in the molecular assembly and ultrastructure of caveolae may lead to pathophysiological changes in Ca²⁺ signaling. Thus, caveolae may be intimately involved in cardiovascular cell dysfunction and disease.

Key Words: caveolae • arterial tone • excitation-contraction coupling • ryanodine receptor • L-type calcium channel

	Introduction

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Intracellular Ca²⁺ signaling has been viewed in terms of global changes in cytosolic [Ca²⁺]. However, Ca²⁺ ions are local signals by virtue of their participation in a wide variety of cellular processes.¹ Recently, these local [Ca²⁺]_i changes have been visualized with fluorescent Ca²⁺-sensitive indicators and laser-scanning confocal microscopy. The application of this technique to cardiac, skeletal, and arterial smooth muscle cells (SMCs) has resulted in the observation of transient (30 to 100 ms), spatially localized (2 to 4 µm) elevations in [Ca²⁺]_i, termed Ca²⁺ sparks.^{2 3 4} Ca²⁺ sparks arise from the openings of sarcoplasmic reticulum (SR) Ca²⁺ release channels of the ryanodine receptor (RyR) family, which are also present in neurons, endothelial cells, and other cells.⁵ In cardiac muscle, Ca²⁺ sparks are induced by membrane potential (V_M)-dependent entry of Ca²⁺ through sarcolemmal voltage-dependent, dihydropyridine (DHP)-sensitive (L-type) Ca²⁺ channels at transverse tubules.^{6 7 8 9} In skeletal muscle, Ca²⁺ sparks are activated by voltage sensors in the transverse tubular membrane that connect physically with the RyRs and secondarily by Ca²⁺.^{4 10} Ca²⁺ entry through a single voltage-dependent, DHP-sensitive (L-type) Ca²⁺ channel is the activator of a Ca²⁺ spark (elementary Ca²⁺-induced Ca²⁺ release).^{6 7 11 12} Ca²⁺ sparks probably constitute the elementary Ca²⁺ release events underlying cardiac excitation-contraction coupling.^{2 13}

In arterial SMCs, Ca²⁺ sparks indirectly cause vasodilatation through activation of plasmalemmal K_Ca channels but have little direct effect on spatially averaged [Ca²⁺]_i that regulates contraction.^{3 14 15} In cardiac ventricular and skeletal muscle cells, Ca²⁺ sparks originate at transverse tubules.^{8 9} How cells provide efficient Ca²⁺ entry to induce Ca²⁺ sparks in cells lacking the transverse tubular membrane system is unknown. Arterial SMCs, mammalian atrial cells, and neonatal cardiomyocytes lack the transverse tubular membrane system (References 16 and 17^{16 17} and unpublished data, April 2000) but have been shown to generate spatially localized, subsarcolemmal, transient Ca²⁺ release events (Ca²⁺ sparks).^{3 14 17} In these cells, infoldings of the surface membrane (caveolae) are abundant.¹⁸ Caveolae contain DHP-sensitive Ca²⁺ channels¹⁹ and may colocalize with junctional SR.²⁰ Thus, caveolae may play an important role in providing the structural relationship between caveolemmal Ca²⁺ channels and RyR Ca²⁺ release channels located in the adjacent junctional SR. Caveolae would thereby be essential to generate local Ca²⁺ release events (Ca²⁺ sparks).

We monitored elementary ryanodine-sensitive Ca²⁺ release events (Ca²⁺ sparks) by measuring rapid local changes in [Ca²⁺]_i in SMCs isolated from resistance-sized cerebral arteries and cardiomyocytes obtained from neonatal hearts, respectively. We provide evidence that the transient elevation in [Ca²⁺]_i triggering SR Ca²⁺ release is established locally in the caveolae microdomain between the caveolemmal Ca²⁺ channel and multiple RyR Ca²⁺ release channels located in the adjacent junctional SR.

	Materials and Methods

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Single SMCs were isolated enzymatically from myogenic cerebral (100 to 800 µm in diameter posterior and basilar) arteries from adult Sprague-Dawley rats (12 to 14 weeks; 200 to 280 g), as previously described.¹⁴ Single cardiomyocytes were isolated enzymatically from newborn rats.²¹ For Ca²⁺ imaging, the cells were incubated with the Ca²⁺ indicator fluo-3-AM (5 µm) and pluronic acid (0.005% wt/vol) for 30 minutes at room temperature in Ca²⁺-free Hanks solution.^{3 14} SMCs and cardiomyocytes were imaged using a BioRad laser scanning confocal microscope attached to a Nikon Diaphot microscope. Whole-cell membrane currents and potentials in freshly isolated cerebral artery myocytes were measured using the perforated patch configuration of the patch-clamp technique configuration with amphotericin B or nystatin.²² Currents were recorded from holding potentials of -80 mV (-100 mV) during lineage voltage ramps at 0.67 V/s from -100 to +100 mV or 300-ms step pulses to different potentials; pulse frequency 0.2 Hz.^{22 23}

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
We used a laser scanning confocal microscope and the Ca²⁺ indicator fluo-3/AM to measure Ca²⁺ sparks in single cells bathed in physiological salt solution.³ In arterial SMCs and neonatal cardiomyocytes, Ca²⁺ sparks were detected in close proximity to cell-surface membrane.^{3 14 24} Figure 1A shows Ca²⁺ sparks in an arterial SMC. In quiescent SMCs (Figure 2), frequency of Ca²⁺ sparks was 0.025±0.003 events · µm^-1 · s^-1 (n=956 cells); peak [Ca²⁺]_i amplitude of Ca²⁺ sparks (measured as F/F_o) was 2.1±0.2 (n=81 sparks); the width at half-maximal amplitude (FWHM₅₀) was 2.89±0.21 µm (n=44 sparks); and the duration of Ca²⁺ sparks at FWHM₅₀ was 45.2±2.6 ms (Figure 2, n=81 sparks).

View larger version (28K): [in this window] [in a new window]   Figure 1. Figure 1. Effects of dextrin on spatial-temporal characteristics of Ca2+ sparks in freshly isolated rat arterial SMCs. A, Confocal line-scan image of a fluo-3–loaded control SMC and, with the time course of Ca2+ sparks indicated below, determined over the line indicated by the 2 arrows. Line-scan image duration was 1 second, and each line was 2 ms. Spatial-temporal characteristics measured at FWHM50 (B) or at FWHM100 (C) of the middle spark shown in panel A. Spatial distribution of Ca2+ fit by a Gaussian (solid line), relative to the initiation point of the spark. Shown are the spatial distributions 1 ms at the first indication of an increase in [Ca2+]i (left), 15 ms at the peak Ca2+ (middle), and 50 ms later (right). FWHM50 at the 3 points of the spark life cycle were 2.4, 2.7, and 4.2 µm. FWHM100 at the 3 points of the spark life cycle were 5.8, 6.8, and 9.5 µm. D, Confocal line-scan image of fluo-3–loaded arterial SMCs after treatment with dextrin (1 mmol/L, 1 hour of incubation) with the time course of a Ca2+ spark indicated below. E, Spatial-temporal characteristics of the spark shown in panel D. Spatial distribution of Ca2+ fit by a Gaussian (solid line) relative to the initiation point of the spark. Shown are the spatial distributions at 1 ms (left), 15 ms at the peak Ca2+ (middle), and 50 ms later (right). The widths at FWHM50 at the 3 points of the spark life cycle were 2.2, 2.4, and 3.8 µm. FMHM100 were 5.2, 5.9, and 8.6 µm. Both sparks shown in panels A and D had a similar duration at FWHM50 of 45 ms. Ca2+ sparks were inhibited by the RyR inhibitor ryanodine (10 µmol/L, not shown).

View larger version (25K): [in this window] [in a new window]   Figure 2. Figure 2. Effects of cholesterol depletion and destruction of caveolae by dextrin on frequency, amplitude, width, and duration of Ca2+ sparks in rat arterial SMCs (filled bars) and rat neonatal cardiomyocytes (open bars). The cells were incubated with dextrin (1 mmol/L, 37°C) or control bath solution (pH 7.4; 37°C) for 1 hour before being examined for sparks. Each cell was scanned for a total time of 30 seconds. The total number of cells examined for the effects of dextrin on Ca2+ spark frequency (left upper panel) is indicated in the text. Ca2+ spark amplitudes were measured as local fractional fluorescence increases (F/Fo) (right upper panel). Width and duration of Ca2+ sparks were determined at FWHM50 (bottom). The total number of sparks examined for the effects of dextrin on Ca2+ spark amplitude, width, and duration is indicated in the text; only 1 randomly selected spark per Ca2+ spark site was used in this analysis.

To test whether the analysis of Ca²⁺ spark properties on different amplitude levels introduced changes and, therefore, different results (Figures 1B, 1C, 1E, and 1F; red lines within the Figures indicate the level used for the analysis of Ca²⁺ spark amplitude), 2 modes of analysis were compared. Ca²⁺ spark widths were analyzed either at FWHM₅₀ (Figures 1B and 1E) or at the beginning of the Ca²⁺ spark amplitude (FWHM₁₀₀ [Figures 1C and 1F]). The analysis revealed a consistent reduction of Ca²⁺ spark width at FWHM₅₀ and FWHM₁₀₀. To obtain the exact start of the Ca²⁺ spark amplitude is often complicated because of fluorescence noise. Therefore, Ca²⁺ spark width was measured continuously at FWHM₅₀.

In neonatal cardiomyocytes (Figure 2), frequency, peak [Ca²⁺]_i amplitude of Ca²⁺ sparks (measured as F/F_o), width, and duration of Ca²⁺ sparks at FWHM₅₀ were 0.071±0.008 events · µm^-1 · s^-1 (n=60 cells), 1.7±0.06 µm (n=47 sparks), 3.55±0.44 µm (n=19 sparks), and 43±3.8 ms (n=24 sparks), respectively. Ryanodine, which inhibits SR Ca²⁺ release channels at micromolar concentrations, blocked Ca²⁺ sparks in both arterial SMCs and adult cardiac muscle cells.^{3 14 15 25} The Ca²⁺ spark frequency in both arterial SMCs and neonatal cardiomyocytes was increased by the DHP agonist BayK8644 (1 µmol/L, n=14 cardiomyocytes) (see Figure 1 online, available at http://www.circresaha.org) by membrane depolarization (using 60 mmol/L external K⁺) or by the RyR activator caffeine (300 µmol/L) (see also References 3 and 25^{3 25} ). In contrast, the frequency of Ca²⁺ sparks was inhibited by the DHP antagonist nimodipine (100 nmol/L, n=21 neonatal cardiomyocytes) or by ryanodine (10 µmol/L, n=22 neonatal cardiomyocytes, n=22 arterial SMCs) (see Figure 1 online). Thus, there was no difference between elementary Ca²⁺ signaling in both arterial SMCs and neonatal heart muscle. These results support previous findings showing that most Ca²⁺ sparks in both cell types result from the openings of ryanodine-sensitive Ca²⁺ release channels in SR in close proximity to the cell-surface membrane^{3 15 25} and that Ca²⁺ entry through a single DHP-sensitive Ca²⁺ channel is the activator of a Ca²⁺ spark.^{6 7 11}

The proximity of the Ca²⁺ sparks to the cell surface raises the possibility that caveolae serve as microdomains to provide efficient, local, and stable Ca²⁺ signal transmission in releasing SR Ca²⁺ sparks (elementary Ca²⁺ induced Ca²⁺ release). Caveolae are 20- to 50-nm invaginations of the plasma membrane that are abundant in arterial SMCs²⁰ and neonatal cardiomyocytes²⁶ and colocalize with the subsarcolemmal occurrence of Ca²⁺ sparks in those cells (this study). Caveolae are rich in cholesterol and sphingolipids; the cholesterol-to-protein ratio of the caveolae fraction is 4 to 5 times higher than in the surrounding plasma membrane.²⁷ Experimentally lowering the cholesterol level of the caveolar fraction disrupts the molecular assembly and ultrastructure of the caveolae domain.^{27 28} We acutely lowered the cholesterol level of the caveolae fraction by incubating arterial SMCs and neonatal cardiomyocytes in the presence of 1 mmol/L methyl-ß-cyclodextrin (dextrin) for 1 hour at 37°C. After incubation, we studied the ultrastructure and measured Ca²⁺ sparks in single cells in PSS. Figure 3 shows that caveolae are abundant in neonatal cardiomyocytes (upper panel). Incubation of neonatal cardiomyocytes with dextrin resulted in a dose-dependent destruction of caveolae. Similar results were obtained in arterial SMCs, as reported by others^{18 28} (see Figure 2 online, available at http://www.circresaha.org).

View larger version (113K): [in this window] [in a new window]   Figure 3. Figure 3. Ultrastructural changes in neonatal rat cardiomyocytes after dextrin incubation. Shown are electron microscopic photographs of a representative control cell (top) and a representative cell after incubation with dextrin at 10 mmol/L (37°C, bottom). The panels show that caveolae are abundant in freshly isolated neonatal rat cardiomyocytes and were destructed by dextrin. Caveolae are shown by arrowheads. Magnification, x63 000. Ultrastructural changes in arterial SMCs after dextrin incubation are shown in Figure 2 online, available at http://www.circresaha.org.

Dextrin treatment had major effects on Ca²⁺ sparks in arterial myocytes. As shown in Figure 2, the frequency of Ca²⁺ sparks in SMCs was significantly decreased from 0.025±0.003 events · µm^-1 · s^-1 (n=956 cells) to 0.017± 0.004 events · µm^-1 · s^-1 (n=214 cells; P<0.05). The peak [Ca²⁺]_i Ca²⁺ spark amplitudes (measured as F/F_o) decreased from 2.1±0.2 (n=81 sparks) to 1.64±0.06 (n=92 sparks; P<0.05). The Ca²⁺ sparks after incubating cells with dextrin were smaller in spatial size. As shown in Figure 1B and 1E, the full width at FWHM₅₀ of the sparks was reduced at all time points of the spark life cycle (2.89±0.2 µm, n=44 sparks versus 2.29±0.2 µm, n=45 sparks for control versus dextrin-treated cells, respectively; P<0.05) (see Figure 2). Similar results were observed when Ca²⁺ spark width was analyzed at 100% (Figures 1C and 1F) and 25% of peak F/F_o amplitude, respectively (not shown). However, the duration of Ca²⁺ sparks was not different between control cells and dextrin-treated arterial SMCs (45.2±2.6 ms, n=81 sparks versus 44.8±1.9 ms, n=93 sparks for control versus dextrin-treated cells, respectively; P>0.05) (see Figure 2).

Similar results were observed in neonatal cardiomyocytes. Figure 2 shows that dextrin treatment of cardiomyocytes decreased the frequency of Ca²⁺ sparks from 0.071±0.008 events · µm^-1 · s^-1 (n=60 cells) to 0.024±0.003 events · µm^-1 · s^-1 (n=98 cells; P<0.05), the peak [Ca²⁺]_i amplitude of Ca²⁺ sparks (measured as F/F_o) from 1.7±0.06 (n=47 sparks) to 1.4±0.03 (n=52 sparks; P<0.05), and the spatial size (full width at FWHM₅₀, 3.55±0.44 µm, n=19 sparks versus 1.6±0.3 µm, n=20 sparks for control versus dextrin-treated cells, respectively; P<0.05). The duration of Ca²⁺ sparks in control cardiomyocytes (43±3.8 ms, n=24 sparks) was not different from cardiomyocytes treated with dextrin (41±3.9 ms, n=23 sparks; P>0.05). Thus, we conclude that cholesterol depletion by dextrin destroys caveolae and decreases frequency, amplitude, and spatial size of Ca²⁺ sparks in arterial SMCs and cardiomyocytes lacking the transverse tubular membrane system. The temporal characteristics of Ca²⁺ sparks are not significantly affected. That pixel filtering is responsible for the observed changes in Ca²⁺ spark properties by dextrin is unlikely. As shown in Figure 3 online (available at http://www.circresaha.org), Ca²⁺ spark amplitudes processed with 1-, 3-, or 5-pixel filtering were reduced by only 3%, 3.5%, and 3.7%, respectively. Because of these small effects, 1-, 3-, and 5-pixel filtering increased Ca²⁺ spark duration in nontreated and dextrin-treated cells by <2%. The effects of dextrin on Ca²⁺ sparks were dose- and time-dependent. Figure 4 shows that dextrin at 1 µmol/L and 1 mmol/L reduced the Ca²⁺ spark frequency in arterial SMCs (P<0.03).

View larger version (12K): [in this window] [in a new window]   Figure 4. Figure 4. Dose-dependent effects of dextrin on Ca2+ sparks in arterial SMCs. Incubation of cells with dextrin decreased frequency of Ca2+ sparks (left) and percentage of cells with Ca2+ sparks (right). Two concentrations of dextrin (1 µmol/L and 1 mmol/L) were tested. Time of incubation is indicated (30 minutes or 60 minutes). n=35 to 211.

To examine the possible direct effects of dextrin on voltage-dependent, DHP-sensitive (L-type) Ca²⁺ channel currents, we measured whole-cell Ca²⁺ channel currents in fresh isolated single vascular SMCs and neonatal cardiomyocytes.^{22 23} Membrane capacitance of both vascular SMCs (7.8±0.3 pF, n=12 versus 7.5±0.4 pF, n=14 for control versus dextrin-treated cells, respectively; P>0.05) and neonatal cardiomyocytes (3.44±0.31 pF, n=20 versus 2.73±0.37 pF, n=15; P=0.05) were unchanged after treatment with dextrin. Figure 5A shows that the whole-cell Ca²⁺ channel current in SMCs was not affected by dextrin pretreatment. Dextrin-treated cells exhibited Ca²⁺ channel currents that were not different in amplitude, activation, and inactivation kinetics than control cells. Figure 5B shows the current-voltage relation of peak Ca²⁺ channel currents without (•) and after () incubation of cells with dextrin (1 mmol/L, 37°C). Solid lines are drawn according to the following equation, assuming a Boltzmann type of channel activation: I_Ca=g_max(V-V_rev){1+exp[(V_0.5-V)/h]}^-1, where I_Ca (pA) is the peak Ca²⁺ channel current, V_0.5 (mV) is the potential of half-maximal activation, V (mV) is the test potential, g_max (pS) is the maximal conductance, V_rev (mV) is the extrapolated reversal potential, and h is the slope factor of Ca²⁺ channel activation. Statistical analysis of the fits revealed that g_max, V_0.5, h, and V_rev were not significantly altered (g_max, 1.6±0.1 and 1.8±0.2 pS; V_0.5, 0.22±0.9 and 1.6±0.4 mV; h, 5.9±0.4 and 4.3±0.45 mV; V_rev, 49±1.9 and 51±0.7 mV for control [n=11] and dextrin-treated [n=13] cells, respectively; P>0.05). Similar results were obtained for L-type Ca²⁺ channel currents in neonatal cardiomyocytes (P>0.05) (Figure 5C).

View larger version (33K): [in this window] [in a new window]   Figure 5. Figure 5. Effect of dextrin application on voltage-dependent Ca2+ channel (VDCC) currents in arterial SMCs and cardiomyocytes. A, Inward VDCC currents (ICa) in freshly isolated rat arterial SMCs were not affected by cholesterol depletion and destruction of caveolae with dextrin. Shown are superimposed current records at different test potentials of a representative control cell (top) and a representative cell after incubation with dextrin (1 mmol/L, 37°C) (bottom). Ca2+ channel currents were evoked by 300-ms depolarizing voltage step pulses to test potentials of -20, 0, and 10 mV (holding potential, -80 mV; pulse frequency 0.33 Hz). The dotted lines represent the zero current levels. Leakage currents were not subtracted. Ca2+ channel currents were measured using 10.8 mmol/L Ba2+ as a divalent charge carrier using the whole-cell configuration. B, Current-voltage relation of peak Ca2+ channel currents without (•) and after () incubation of arterial SMCs with dextrin (1 mmol/L, 37°C). The inward Ca2+ channel currents were reversibly blocked by 1 µmol/L nimodipine or 100 µmol/L Cd2+ and reversibly increased by 1 µmol/L (±)-Bay K 8644 (not shown). C, Effect of dextrin on L-type VDCC currents (ICa) in neonatal rat cardiomyocytes. Current-voltage relation of peak Ca2+ channel currents without (circles) and after (triangles) incubation of cells with dextrin (1 mmol/L, 37°C). ICa was not affected by cholesterol depletion and destruction of caveolae with dextrin (n=22, control cells; n=14, dextrin-treated cells at +10 mV).

To examine whether dextrin had an effect on V_M and thus might indirectly affect voltage-dependent Ca²⁺ entry through DHP-sensitive Ca²⁺ channels, we next measured V_M by the whole-cell perforated patch-clamp technique in single SMCs.²² Steady-state V_M was not different between control cells (-18±4 mV, n=7) and dextrin-treated arterial SMCs (-18±3 mV, n=7, P>0.05) (Figure 6). These results indicate that the changes of Ca²⁺ spark properties in dextrin-treated cells occurred without alteration of the properties of DHP-sensitive Ca²⁺ channels or of cell V_M, indicating that the trigger for SR Ca²⁺ release is unaltered after caveolae depletion.

View larger version (19K): [in this window] [in a new window]   Figure 6. Figure 6. Effect of dextrin application on VM in arterial SMCs. VM of freshly isolated rat arterial SMCs is not affected by cholesterol depletion and destruction of caveolae with dextrin. Left, VM records of a representative control cell (top) and a representative cell (bottom) after incubation with dextrin (1 mmol/L, 1 hour, 37°C). In both cells, the steady-state VM was -18 mV. However, spontaneous, transient membrane hyperpolarizations occurred, which result from the opening of Ca2+-activated K+ channels by Ca2+ sparks (STOCs)22 23 and that can be blocked by RyR blocker (ryanodine, 10 µmol/L) or KCa channel blockers (tetraethylammonium, 1 mmol/L, or iberiotoxin, 100 µmol/L, not shown). These data are consistent with the idea that Ca2+ sparking RyRs of the junctional SR in caveolae-depleted cells are still apposed to the surface membrane. Statistical analysis revealed that the steady-state VM was not significantly different between cells incubated with dextrin (1 mmol/L, 37°C) or control bath solution (pH 7.4; 37°C) for 1 hour before being examined for VM (right panel). VM was measured using the whole-cell perforated-patch configuration.

To explore possible changes in the function of RyRs or the SR content, the effects of 10 mmol/L caffeine were examined. This caffeine concentration was previously shown¹⁴ to cause a typical whole-cell global Ca²⁺ transient in adult rat cerebral myocytes and neonatal rat cardiomyocytes because of the rapid activation of RyRs. Such caffeine-induced Ca²⁺ transients have been used routinely to measure SR Ca²⁺ content in muscle cells.^{14 15} Rapid application of caffeine (10 mmol/L) to dextrin-treated arterial SMCs caused a global Ca²⁺ transient with an amplitude (F/F_o=5.4±0.5, n=20) similar to nontreated cells (F/F_O=5.3±0.5, n=17) (Figures 7A and 7B). Similar results were obtained in neonatal cardiomyocytes (Figure 7C). These results indicate that RyRs are functional and that differences in SR Ca²⁺ load are not responsible for the observed differences in Ca²⁺ spark frequency and characteristics after treatment of cells with dextrin.

View larger version (14K): [in this window] [in a new window]   Figure 7. Figure 7. Influence of dextrin on SR Ca2+ release in rat arterial SMCs and rat neonatal cardiomyocytes. A, Caffeine induced similar Ca2+ transients in both freshly isolated rat arterial SMCs incubated in control bath solution (pH 7.4, 37°C; 1 hour) with or without dextrin (1 mmol/L, 37°C, 1 hour). Time course of fluorescence changes during a bolus addition of caffeine (10 mmol/L) in a control myocyte and a myocyte treated with dextrin are shown. B and C, Averaged peak fluorescence changes in control and dextrin treated cells from arterial SMCs (B) and neonatal cardiomyocytes (C) were not statistically different.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our data indicate that caveolae may have a key function in controlling the formation of local SR Ca²⁺ release events in smooth and cardiac muscle cells lacking the transverse tubular system. The results support a model for discrete Ca²⁺ release events from the SR that unifies diverse observations about local SR Ca²⁺ signaling and that may be applicable to other excitable cells. In this model (Figure 8), the transient elevation in [Ca²⁺]_i that triggers SR Ca²⁺ release is established locally in a caveolar microdomain between a single caveolemmal Ca²⁺ channel and multiple RyR Ca²⁺ release channels located in the adjacent junctional SR. The transient elevation in [Ca²⁺]_i at the inner mouth of the caveolemmal Ca²⁺ channel induces simultaneous activation, thereby opening of multiple RyRs to generate a local Ca²⁺ release event (Ca²⁺ spark). Once triggered, Ca²⁺ sparks have a brief lifespan (40 ms) that is regulated by the intrinsic properties of activated RyRs. This multi-RyR channel model explains how destruction of caveolae decreases frequency, amplitude, and spatial size of Ca²⁺ sparks in arterial SMCs and cardiomyocytes without significantly changing the temporal characteristics of a Ca²⁺ spark. According to this model, destruction of caveolae enhances the distance (by up to 20 to 50 nm) between the caveolemmal Ca²⁺ channel and multiple RyR Ca²⁺ release channels. Thus, the transient elevation in [Ca²⁺]_i at the inner mouth of the caveolemmal Ca²⁺ channel that triggers SR Ca²⁺ release activates a smaller number of RyRs located in the adjacent junctional SR, producing a smaller Ca²⁺ spark without changing their duration. In sharp contrast, Ca²⁺ sparks based on individual RyRs would have been expected to exhibit spatial-temporal characteristics that are not affected by destruction of caveolae. The multi-RyR hypothesis supports the idea that an individual RyR may release a fundamental Ca²⁺ release event, ie, Ca²⁺ quark.²⁹ Because Ca²⁺ quarks are not detectable by presently available confocal laser scanning microscopy,^{14 29} we conclude that the Ca²⁺ sparks (elementary Ca²⁺ release units) observed in dextrin-treated cells represent Ca²⁺ release events by simultaneous opening of at least 2 RyRs. Our interpretation of caveolar Ca²⁺ signaling is strengthened by the observation that Ca²⁺ sparks occur repeatedly at defined discharge sites with relatively stable spatial-temporal characteristics (usually at 2 to 3 Ca²⁺ spark sites per cell) despite the large number of RyRs along the cell membrane in arterial SMCs.¹⁴ Our interpretation is consistent with recent findings suggesting that the simultaneous opening of multiple RyRs is responsible for the generation of a Ca²⁺ spark event despite large uncertainties in the suggested number of RyRs involved. The number may range from <8 RyRs³⁰ to 8 RyRs³¹ to >8 RyRs.³² Nevertheless, we observed a modal amplitude distribution of Ca²⁺ sparks in both arterial SMCs and neonatal cardiomyocytes (data not shown), clearly indicating that Ca²⁺ sparks represent Ca²⁺ release events by simultaneous opening of multiple RyRs but not of a single RyR.³²

View larger version (31K): [in this window] [in a new window]   Figure 8. Figure 8. Proposed model of the organization of caveolemmal Ca2+ channels and RyRs in small resistance-sized arteries and neonatal cardiomyocytes to cause Ca2+ sparks. This model proposes that RyRs cluster in terminal SR plaques in close proximity to caveolae and that Ca2+ sparks arise from the synchronized opening of more than 2 RyRs acting in concert. Ca2+ influx through an individual caveolemmal Ca2+ channel (possibly the DHP receptor) is the trigger Ca2+ to activate colocalized RyRs (filled in red) acting in concert to release a Ca2+ spark. The proposed model implies that changes in the integrity of caveolae has significant impact on Ca2+ sparks. Destruction of caveolae enlarges the diffusion distance between the caveolemmal Ca2+ channel and RyRs and, thus, is expected to recruit of a lower number of RyRs, which consequently generate Ca2+ sparks with decreased frequency, amplitude, and spatial size. However, because Ca2+ sparks still arise from the synchronized opening of multiple RyRs, enlargement of the diffusion distance between the caveolemmal Ca2+ channel and RyRs is expected to have no significant effect on Ca2+ spark duration, as observed in this study.

In conclusion, our data indicate that in SMCs and cardiomyocytes caveolae appear to be membrane hot spots for local Ca²⁺ signaling. Caveolae have been implicated in signal transduction, vesicular trafficking, lipid metabolism, and cellular growth control in adipocytes, endothelia, neuronal cells, and fibroblasts.²⁷ We propose that caveolae perform localized and very specific Ca²⁺ signaling functions in the absence of global cytosolic Ca²⁺ elevations. Our data suggest that Ca²⁺ sparks are of multi-RyR channel origin and that the spatial characteristics of Ca²⁺ sparks are controlled by caveolar microarchitecture, assembled by a mixture of lipids and proteins (eg, caveolins and cavatellins) unlike those found in the plasma membrane proper.²⁷ Alterations in the caveolar microarchitecture may lead to pathophysiological changes in Ca²⁺ signaling. Thus, caveolae may be intimately involved in cardiovascular cell dysfunction and disease.

	Acknowledgments

 

This work was supported by a Deutsche Forschungsgemeinschaft grant (M.G.) and by Boehringer Ingelheim Fonds (M.F. and M.L.). We are grateful to Dr T. Kurzchalia for advice in using methyl-ß-cyclodextrin. We thank Mrs Kott and Dr H. Haase for preparing neonatal cardiomyocytes.

Received August 4, 2000; revision received September 28, 2000; accepted September 28, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
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