INTRODUCTION

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LOCAL RELEASE OF CALCIUM ("Ca²⁺ sparks") through ryanodine-sensitive Ca²⁺ release (RyR) channels in the sarcoplasmic reticulum (SR) have recently been measured in arterial smooth muscle cells, using a laser-scanning confocal microscope and the fluorescent Ca²⁺ indicator, fluo 3 (21). Ca²⁺ sparks arise from the opening of a single or a small number of tightly clustered RyR channels. Ca²⁺ sparks activate nearby Ca²⁺-sensitive K⁺ (K_Ca) channels in smooth muscle (13, 21), which causes outward currents (previously referred to as "spontaneous transient outward currents" or STOCs) (2). An increase in K_Ca channel current causes the membrane potential to hyperpolarize, which closes voltage-dependent Ca²⁺ channels, decreases Ca²⁺ entry, and lowers average global intracellular Ca²⁺, which exerts a vasorelaxing influence (22, 23). Thus activation of the Ca²⁺ spark  K_Ca channel pathway appears to oppose pressure-induced constrictions of myogenic cerebral arteries (5, 21). This work suggests that frequency modulation of Ca²⁺ sparks would alter arterial smooth muscle membrane potential and arterial tone (3, 4, 6, 21, 25).

Ca²⁺ spark frequency (i.e., the open probability of RyR channels) increases with cytoplasmic Ca²⁺ and SR Ca²⁺ load (3, 4). In smooth muscle, STOC frequency, which reflects Ca²⁺ spark frequency, has been shown to increase with membrane depolarization (see, e.g., Refs. 2 and 34) and is associated with elevated cytoplasmic and SR Ca²⁺ (2, 18, 29, 34). The phosphorylation state of the RyR channel and certain drugs (caffeine and ryanodine) may modulate Ca²⁺ spark frequency and thereby its consequences, independent of changes in Ca²⁺. Recent evidence suggests that protein kinase C (PKC) can phosphorylate RyR channels in cardiac muscle (30), although the functional effect of this phosphorylation on RyR channel properties is unknown.

In this study, we explored the possibility that activators of PKC (phorbol ester and a diacylglycerol analog), which are potent vasoconstrictors, could affect Ca²⁺ spark properties. Agents that inhibit Ca²⁺ sparks (ryanodine, thapsigargin, cyclopiazonic acid) have been shown to depolarize and constrict myogenic cerebral arteries (21). We provide the first evidence that activators of PKC can decrease Ca²⁺ spark frequency and, consequently, STOC frequency. Activators of PKC also slightly reduced STOC amplitude, which could be explained by a direct effect on the K_Ca channels. Activators of PKC reduced Ca²⁺ spark frequency, even as they slightly elevated cytoplasmic Ca²⁺ and SR Ca²⁺ load. These results are consistent with the idea that PKC acts directly on the RyR channel to decrease its opening rate (i.e., Ca²⁺ spark frequency) and suggests that frequency modulation of Ca²⁺ sparks may be important in the regulation of cell function.

	METHODS

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Cell isolation. Single smooth muscle cells were enzymatically isolated from rat cerebral (basilar) arteries. The cells were isolated with a papain and collagenase digestion as described in Ref. 26. Only spindle-shaped cells with intact membranes were used for measurements.

Ca²⁺ spark measurements. The procedure for the measurement of sparks is described in Ref. 21. Briefly, the cells were loaded with the Ca²⁺-sensitive indicator fluo 3 with a 20-min incubation in 5 µM of the acetoxymethyl ester (AM) of fluo 3, 2.5 µg/ml Pluronic acid (Molecular Probes, Eugene, OR), followed by a 20-min wash. All measurements were made 15-45 min after the application of compounds. Control and treated cells from the same cell isolation were examined randomly to minimize any bias or time-dependent changes. The cells were scanned with a Bio-Rad MRC 1000 laser-scanning confocal microscope, housed in the University of Vermont Cell Imaging Facility. Images were acquired using the line scan mode of the confocal microscope; this mode repeatedly scans a single line through a cell. A scan duration of 6 ms was used. Cells were positioned so that the line would traverse the long axis of the cell to detect sparks occurring in as much of the cell volume as possible. Scan lines are displayed vertically, and each line is added to the right of the preceding line to form the line scan image. In these images, time is in the horizontal direction running from left to right, and position along the scan line is given by the vertical displacement. Six consecutive 3-s line scan images were recorded from each cell along a single line. Sparks were analyzed using custom-written analysis programs using interactive data language (IDL) software (Research Systems, Boulder, CO). Fractional fluorescence increases >1.3 with spreads (spatial distribution determined as the width of the Gaussian distribution at the half amplitude) of >1.2 µm were analyzed. Such events were not observed in the presence of ryanodine or thapsigargin (21), indicating that these events originated from the SR.

Conventional Ca²⁺ imaging. Isolated smooth muscle cells were loaded with the Ca²⁺ indicator dye fura 2. Cells were incubated with 0.25 µM fura 2-AM for 15 min. Cells were then washed and allowed to sit in the dark for 20 min before measurements were made. Ca²⁺ was measured ratiometrically (340:380 nm) using IMAGE-1/FL quantitative fluorescence measurement software (Universal Imaging, West Chester, PA). Fluorescence ratios were converted to Ca²⁺ concentrations (as described in Ref. 9), using an apparent dissociation constant for fura 2 of 282 nM (15).

	RESULTS

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Activators of PKC decrease Ca²⁺ spark frequency. Activators of PKC, phorbol 12-myristate 13-acetate (PMA; 10 nM) and 1,2-dioctanoyl-sn-glycerol (1 µM), decreased Ca²⁺ spark frequency (determined as sparks per cell) from 2.85 ± 0.40 (n = 86 cells) to 0.80 ± 0.20 (n = 130) and 1.15 ± 0.38 (n = 20) or by 71.9% and 59.7%, respectively (Fig. 1, A and B). The inactive phorbol ester analog 4-PMA (10 nM) had no effect on Ca²⁺ spark frequency (Fig. 1, A and B). PMA caused a small decrease in Ca²⁺ spark amplitude, with no effect on spatial spread or rate of decay (Fig. 1C and Table 1).

View larger version (65K): [in this window] [in a new window]   Fig. 1.   Protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl-sn-glycerol (DOG) decrease frequency of Ca2+ sparks in cerebral artery myocytes. A: line scan images from a control cell (top) and from a cell incubated with PMA (10 nM; middle) or nonactive analog of PMA (4-PMA; 10 nM bottom). Scan lines are displayed vertically in a continuous manner. Inset: orientation of scanning line. B: average data for number of sparks per cell in control (2.85 ± 0.40; n = 86 cells), with PMA (10 nM; 0.80 ± 0.20; n = 130), with 4-PMA (10 nM; 2.76 ± 0.52; n = 50), and with DOG (1 µM; 1.15 ± 0.38; n = 20). PKC activators, PMA and DOG, caused a statistically significant decrease in Ca2+ spark frequency (* P < 0.05; ** P < 0.01), whereas nonactive analog 4-PMA did not. Measurements were made after 15 min of incubation with PMA, 4-PMA, or DOG. C: Ca2+ spark image demonstrating time course of fractional fluorescence (F/Fo; bottom) and spatial distribution of Ca2+ spark (left) fitted with a Gaussian distribution (red line). Gray bar labeled "t" indicates region over which fluorescence time course was averaged.

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View larger version (43K): [in this window] [in a new window]   Fig. 2.   PMA decreases frequency and amplitude of spontaneous transient outward currents (STOCs). A: original records of STOCs recorded from single smooth muscle cells isolated from basilar cerebral artery. Holding potential was 40 mV. PMA (10 nM) decreased frequency and amplitude of STOCs (top trace). Nonactive analog 4-PMA (10 nM) was without effect (bottom trace). B: average changes in STOC frequency and amplitude relative to pretreatment control levels with PMA (10 nM; n = 11) and 4-PMA (10 nM; n = 5). PMA caused a statistically significant decrease in frequency (** P < 0.01), by 82.9 ± 3.6%, and in amplitude of STOCs (* P < 0.05), by 22.0 ± 9.7%. Nonactive analog 4-PMA did not have any significant effects. C: consecutive records of single Ca2+-dependent K+ (KCa) channel openings before (control) and 15 min after application of PMA (10 nM). Thapsigargin (100 nM) was added 10 min before starting the experiment to block Ca2+ sparks and hence STOCs. Currents were recorded with perforated-patch configuration of whole cell voltage-clamp technique. Holding potential was 0 mV. Number of channels times open probability (NPo) of KCa channels was determined by analyzing 5-min sections of data in control and in presence of PMA. PMA caused a small but statistically significant reduction in NPo from 4.71 ± 0.97 × 103 to 3.43 ± 0.84 × 103 (P < 0.01) or by 28.9 ± 4.1% (n = 4). Amplitude of single KCa channel openings was not affected by PMA (control, 4.9 ± 0.4 pA; with PMA, 4.9 ± 0.3 pA). D: average relative changes in STOC frequency and amplitude (n = 4) with CdCl2 (250 mM) and after application of PMA (10 nM) in continued presence of CdCl2. Cd2+ reduced STOC frequency by 43.6 ± 10.9% (* P < 0.05) but did not affect STOC amplitude. PMA reduced STOC frequency by 81.2 ± 5.8% and amplitude by 36.4 ± 1.5% (* P < 0.05), comparable with that observed in absence of Cd2+.

PMA does not decrease caffeine-induced Ca²⁺ transients. PMA could decrease Ca²⁺ spark frequency by decreasing cytoplasmic or SR Ca²⁺, which would decrease the opening rate of RyR channels (8, 19, 33). To test this possibility, global intracellular Ca²⁺ was measured in isolated myocytes, with the use of fura 2. PMA (10 nM) caused a slight elevation of global Ca²⁺ from 105.6 ± 3.8 to 153.1 ± 3.3 nM (n = 7 cells; Fig. 3A). Caffeine (10 mM), which opens RyR channels, caused Ca²⁺ transients of 425.0 ± 56.5 and 419.4 ± 32.4 nM (Fig. 3, A and B). After 30 and 60 min of application of PMA, caffeine-induced Ca²⁺ transients were 507.2 ± 43.2 and 626.3 ± 42.1 nM, respectively. These results argue against changes in cytoplasmic or SR Ca²⁺ load leading to a decrease in Ca²⁺ spark or STOC frequency. The remaining likely possibility is that PKC directly decreases the open rate of RyR channels through channel phosphorylation, a possibility that remains to be explored.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   PMA does not inhibit intracellular Ca2+ transients induced by caffeine (Caff). A: transient increases in cytosolic Ca2+ ([Ca2+]cyt ) in response to application of 10 mM caffeine before and after application of PMA (10 nM). Trace is average of [Ca2+]cyt signals from 7 cells. Cells were loaded with Ca2+ indicator fura 2-acetoxymethyl ester, and Ca2+ was measured ratiometrically (340:380 nm) using IMAGE-1/FL quantitative fluorescence equipment. B: average changes in cytosolic Ca2+ ([Ca2+]cyt) induced by caffeine (10 mM) in control (1 and 2) and 30 min (3) and 60 min (4) after application of PMA (10 nM). C: proposed mechanism for action of PKC on Ca2+ sparks and KCa channels.

	DISCUSSION

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Our results are consistent with activation of PKC decreasing Ca²⁺ spark frequency through a direct action on the RyR receptor channel (Fig. 3C). This would, therefore, be the first functional evidence that PKC can affect RyR channels and is consistent with the observation that PKC can phosphorylate RyR channels (30). It seems very unlikely that PKC activation decreases Ca²⁺ spark frequency through a reduction in cytoplasmic or SR Ca²⁺, since neither cytoplasmic Ca²⁺ nor caffeine-induced Ca²⁺ transients declined over 60 min of exposure to PMA (Fig. 3, A and B). Activators of PKC have been reported to inhibit STOCs in rabbit portal vein (14). This inhibitory effect was ascribed to a depletion of the SR, since caffeine failed to produce an outward current in the presence of PKC activators (14). In our experiments, PMA clearly did not decrease caffeine-induced Ca²⁺ transients. Because PKC activation appears to inhibit K_Ca channels (Fig. 2C), it is conceivable that the inhibitory effects of PKC activators on STOCs and caffeine-induced current transients observed in portal vein (14) were due to inhibition of K_Ca channels and not of Ca²⁺ sparks. Alternatively, PKC activators depleted SR Ca²⁺ in this preparation, which led to a loss of caffeine-induced current transients and STOCs. The mechanism by which PKC activation inhibits K_Ca channels is unclear. Activators of PKC have been shown to inhibit K_Ca channels in cultured and freshly isolated smooth muscle cells (20, 27).

Receptor-mediated vasoconstrictors may have complicated effects on Ca²⁺ sparks. Most receptor-mediated vasoconstrictors can cause membrane depolarization, which increases Ca²⁺ entry through voltage-dependent Ca²⁺ channels. Vasoconstrictors can also directly activate voltage-dependent Ca²⁺ channels (24), which could increase Ca²⁺ spark frequency (1). These effects would increase cytoplasmic Ca²⁺ and SR Ca²⁺ and thus elevate Ca²⁺ spark frequency. Vasoconstrictors also cause a transient increase in inositol trisphosphate (IP₃) production, which would release SR Ca²⁺ through IP₃-sensitive channels. IP₃-induced Ca²⁺ release could increase or decrease Ca²⁺ spark activity (3, 7, 14, 16), depending on the extent of the elevation of cytoplasmic Ca²⁺ near the RyR receptors, which would tend to increase Ca²⁺ spark frequency, and of the depletion of SR Ca²⁺, which should decrease Ca²⁺ spark frequency and amplitude. Vasoconstrictors also activate PKC through diacylglycerol, which, as shown here, could cause a steady-state decrease in Ca²⁺ spark frequency. Furthermore, PKC activation could inhibit IP₃ formation (32). The steady-state effect of vasoconstrictors on Ca²⁺ spark properties would therefore be a function of all these factors.

In conclusion, our results support the concept of frequency modulation (3, 4, 6, 21, 25) of Ca²⁺ sparks regulating K_Ca channels. Vasodilators that elevate adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate (35) have been shown to increase Ca²⁺ spark and STOC frequency (25). In contrast, we demonstrate that activators of PKC decrease Ca²⁺ spark frequency and hence STOC frequency. This effect would tend to depolarize smooth muscle, which would open voltage-dependent Ca²⁺ channels, increase Ca²⁺ entry, and constrict. Our results therefore suggest a new mechanism of control of Ca²⁺ spark frequency, which could contribute to the action of vasoconstrictors.