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Am J Physiol Cell Physiol 273: C2090-C2095, 1997;
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Vol. 273, Issue 6, C2090-C2095, December 1997

RAPID COMMUNICATION
Activators of protein kinase C decrease Ca2+ spark frequency in smooth muscle cells from cerebral arteries

Adrian D. Bonev1, Jonathan H. Jaggar1, Michael Rubart2, and Mark T. Nelson1

1 Department of Pharmacology, College of Medicine, The University of Vermont, Colchester, Vermont 05446; and 2 Krannert Institute of Cardiology, Indiana University Medical School, Indianapolis, Indiana 46202

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Local Ca2+ transients ("Ca2+ sparks") caused by the opening of one or the coordinated opening of a number of tightly clustered ryanodine-sensitive Ca2+-release (RyR) channels in the sarcoplasmic reticulum (SR) activate nearby Ca2+-dependent K+ (KCa) channels to cause an outward current [referred to as a "spontaneous transient outward current" (STOC)]. These KCa currents cause membrane potential hyperpolarization of arterial myocytes, which would lead to vasodilation through decreasing Ca2+ entry through voltage-dependent Ca2+ channels. Therefore, modulation of Ca2+ spark frequency should be a means to regulation of KCa channel currents and hence membrane potential. We examined the frequency modulation of Ca2+ sparks and STOCs by activation of protein kinase C (PKC). The PKC activators, phorbol 12-myristate 13-acetate (PMA; 10 nM) and 1,2-dioctanoyl-sn-glycerol (1 µM), decreased Ca2+ spark frequency by 72% and 60%, respectively, and PMA reduced STOC frequency by 83%. PMA also decreased STOC amplitude by 22%, which could be explained by an observed reduction (29%) in KCa channel open probability in the absence of Ca2+ sparks. The reduction in STOC frequency occurred in the presence of an inorganic blocker (Cd2+) of voltage-dependent Ca2+ channels. The reduction in Ca2+ spark frequency did not result from SR Ca2+ depletion, since caffeine-induced Ca2+ transients did not decrease in the presence of PMA. These results suggest that activators of PKC can modulate the frequency of Ca2+ sparks, through an effect on the RyR channel, which would decrease STOC frequency (i.e., KCa channel activity).

calcium-dependent potassium channels; caffeine; ryanodine; thapsigargin

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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+ (KCa) channels in smooth muscle (13, 21), which causes outward currents (previously referred to as "spontaneous transient outward currents" or STOCs) (2). An increase in KCa 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 right-arrow KCa 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 KCa 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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Abstract
Introduction
Methods
Results
Discussion
References

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.

Electrophysiological recordings. K+ currents were measured in the whole cell, perforated-patch configuration (11) of the patch-clamp technique (10), using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). The bathing solution (also used for spark measurements) contained (in mM) 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.4). The pipette solution contained (in mM) 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.05 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (pH 7.2). Membrane currents were recorded while holding the cells at a membrane potential of -40 mV. To determine the mean amplitude and frequency of the STOCs, analysis was performed off-line, using a custom analysis program. The threshold of STOCs was set at three times the single channel amplitude at -40 mV. In the presence of ryanodine or thapsigargin, the simultaneous opening of three single KCa channels was not observed at -40 mV. The large amplitude and low open probability of the KCa channel permitted the measurement of single KCa channel currents using the perforated-patch configuration of the whole cell voltage clamp. To observe single KCa channel currents, Ca2+ sparks and STOCs were prevented by thapsigargin (31), which inhibits the SR Ca2+-ATPase, and the cells were clamped at 0 mV. Ca2+ sparks and STOCs were measured in different cells.

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).

Chemicals. Unless otherwise stated all chemicals used in this study were obtained from Sigma Chemical (St. Louis, MO) and Calbiochem-Novabiochem International (La Jolla, CA). All experiments were conducted at room temperature (20-22°C).

Statistical analysis. Results are expressed as means ± SE. Statistical significance was tested at the 95-99% confidence level using a paired or unpaired Student's t-test, where applicable.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 4alpha -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).


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  		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 (4alpha -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 4alpha -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 4alpha -PMA did not. Measurements were made after 15 min of incubation with PMA, 4alpha -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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  		Table 1.   Summarized data of amplitude, spatial distribution, and t1/2 of Ca2+ sparks

PMA decreases STOC frequency and amplitude. Ca2+ sparks activate nearby KCa channels to cause outward currents (STOCs). Therefore, if activators of PKC decrease Ca2+ spark frequency, then these activators should decrease STOC frequency. PMA (10 nM) decreased STOC frequency from 1.23 ± 0.22 (n = 10) to 0.18 ± 0.03 Hz, or by 82.9 ± 3.6%, and decreased STOC amplitude by 22.0 ± 9.7% at -40 mV (Fig. 2, A and B). The nonactive analog 4alpha -PMA (10 nM) had no effect on STOC frequency and amplitude (Fig. 2B). A decrease in STOC amplitude could occur through direct inhibition of KCa channels. To test this possibility, currents through single KCa channels were measured in the whole cell (perforated patch) configuration. Ca2+ sparks, and hence STOCs, were prevented by thapsigargin (100 nM), which depletes SR Ca2+ by inhibiting the SR Ca2+-ATPase. PMA significantly decreased the activity [measured as the product of the number of channels and open probability (NPo)] of KCa channels from 4.71 ± 0.97 × 10-3 to 3.43 ± 0.84 × 10-3 (n = 4 cells; P < 0.01) or by 28.9 ± 4.1% (at 0 mV; Fig. 2C). This effect of PMA on KCa channel NPo should contribute to the decrease in STOC amplitude.


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  		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 4alpha -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 4alpha -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 4alpha -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 × 10-3 to 3.43 ± 0.84 × 10-3 (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 could conceivably decrease Ca2+ spark and STOC frequency indirectly by reducing Ca2+ entry through voltage-dependent Ca2+ channels (17, 28). To exclude this possibility, we examined the effects of PMA on STOC frequency and amplitude in the presence of the inorganic Ca2+ channel blocker, Cd2+ (250 µM) (12). Cd2+ reduced STOC frequency by 43.5 ± 10.8% (n = 4 cells), but it did not affect STOC amplitude (Fig. 2D). STOC frequency, in the presence of Cd2+, remained constant for a relatively long period of time (40-50 min). Nevertheless, PMA, in the presence of Cd2+, reduced STOC frequency by 81.1 ± 5.8% and amplitude by 36.4 ± 1.5% (Fig. 2D), suggesting that the effects of PMA are independent of changes in Ca2+ entry through voltage-dependent Ca2+ channels.

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.


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  		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+ (Delta [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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Abstract
Introduction
Methods
Results
Discussion
References

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 KCa 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 KCa 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 KCa channels is unclear. Activators of PKC have been shown to inhibit KCa 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 KCa 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.

    ACKNOWLEDGEMENTS

We thank Drs. Gary Mawe, Joseph E. Brayden, Valerie A. Porter, and Karen M. Lounsbury for discussion and comments on the manuscript.

    FOOTNOTES

This study was supported by National Institutes of Health Grants HL-44455, HL-51728, and NS-26995, National Science Foundation Grant IBN-9631416, and American Heart Association, Indiana Affiliate, Grant INN-97-700-GIAR.

Address for reprint requests: M. T. Nelson, Department of Pharmacology, College of Medicine, The University of Vermont, 55A South Park Dr., Colchester, VT 05446.

Received 24 June 1997; accepted in final form 12 September 1997.

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Abstract
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Methods
Results
Discussion
References

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AJP Cell Physiol 273(6):C2090-C2095
0363-6143/97 $5.00 Copyright © 1997 the American Physiological Society


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