Frequency modulation of Ca2+ sparks is involved in regulation of arterial diameter by cyclic nucleotides

Valerie A. Porter1, Adrian D. Bonev1, Harm J. Knot1, Thomas J. Heppner1, Andra S. Stevenson1, Thomas Kleppisch1, W. J. Lederer2, and Mark T. Nelson1

1 Department of Pharmacology, University of Vermont, Colchester, Vermont 05446; and 2 Department of Physiology and The Medical Biotechnology Center, University of Maryland School of Medicine, Baltimore, Maryland 21201

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Forskolin, which elevates cAMP levels, and sodium nitroprusside (SNP) and nicorandil, which elevate cGMP levels, increased, by two- to threefold, the frequency of subcellular Ca2+ release ("Ca2+ sparks") through ryanodine-sensitive Ca2+ release (RyR) channels in the sarcoplasmic reticulum (SR) of myocytes isolated from cerebral and coronary arteries of rats. Forskolin, SNP, nicorandil, dibutyryl-cAMP, and adenosine increased the frequency of Ca2+-sensitive K+ (KCa) currents ["spontaneous transient outward currents" (STOCs)] by two- to threefold, consistent with Ca2+ sparks activating STOCs. These agents also increased the mean amplitude of STOCs by 1.3-fold, an effect that could be explained by activation of KCa channels, independent of effects on Ca2+ sparks. To test the hypothesis that cAMP could act to dilate arteries through activation of the Ca2+ sparkright-arrowKCa channel pathway, the effects of blockers of KCa channels (iberiotoxin) and of Ca2+ sparks (ryanodine) on forskolin-induced dilations of pressurized cerebral arteries were examined. Forskolin-induced dilations were partially inhibited by iberiotoxin and ryanodine (with no additive effects) and were entirely prevented by elevating external K+. Forskolin lowered average Ca2+ in pressurized arteries while increasing ryanodine-sensitive, caffeine-induced Ca2+ transients. These experiments suggest a new mechanism for cyclic nucleotide-mediated dilations through an increase in Ca2+ spark frequency, caused by effects on SR Ca2+ load and possibly on the RyR channel, which leads to increased STOC frequency, membrane potential hyperpolarization, closure of voltage-dependent Ca2+ channels, decrease in arterial wall Ca2+, and, ultimately, vasodilation.

cAMP; cGMP; vasodilation; sarcoplasmic reticulum; KCa channels

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

LOCAL CALCIUM TRANSIENTS or "Ca2+ sparks" have recently been measured in smooth muscle cells from cerebral arteries (28). Ca2+ sparks in arterial myocytes as well as in cardiac myocytes (6) appear to arise from the opening of one or the coordinated opening of several tightly clustered ryanodine-sensitive Ca2+ release (RyR) channels in the sarcoplasmic reticulum (SR). In arterial myocytes, Ca2+ sparks activate 10-100 nearby sarcolemmal Ca2+-sensitive K+ (KCa) channels to cause an outward K+ current (17, 28), previously referred to as a "spontaneous transient outward current" (STOC) (4). Ca2+ sparks, themselves, contribute little to overall cellular Ca2+ in pressurized cerebral arteries, since their activity is asynchronous (~1/s in a cerebral artery myocyte) (28). Ca2+ spark-activated KCa channels can continuously regulate, as a negative feedback element, the membrane potential of smooth muscle cells in intact pressurized cerebral arteries (7). Thus either blocking Ca2+ sparks or decreasing their frequency of occurrence (5) would lead to membrane potential depolarization and vasoconstriction through a decrease in KCa channel activity (Ref. 28; see also Refs. 7 and 31). Therefore, these results predict that increasing Ca2+ spark frequency could lead to vasodilation through activation of KCa channels.

The cyclic nucleotides, cAMP and cGMP, mediate vasodilation of important endogenous and therapeutic agents (e.g., beta -adrenergic agents, adenosine, calcitonin gene-related peptide, nitric oxide, atrial natriuretic factor, synthetic nitrovasodilators). Diverse mechanisms have been proposed to explain the vasodilator effect of cyclic nucleotides, including the stimulation of Ca2+ uptake by the SR (24), the direct activation of KCa channels (39), activation of ATP-sensitive K+ channels (18, 19, 36, 48), activation of voltage-dependent K+ channels (1), and changes in the Ca2+ sensitivity of the contractile process (11, 15, 41). Relaxation of cerebral and coronary arteries to agents that elevate cAMP or cGMP are inhibited by blockers of KCa channels (29, 32, 33, 35, 42, 47). In fact, in a number of other types of smooth muscle (airway, mesenteric, uterine, pulmonary), blockers of KCa channels (iberiotoxin or charybdotoxin) reduce significantly the relaxing effects of agents that elevate cAMP and cGMP (2, 3, 8, 16, 23). KCa channels can be activated by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) (39, 44). Recent evidence suggests that KCa channels in pressurized arteries with tone are primarily activated by local Ca2+ transients, i.e., Ca2+ sparks, and not directly by average intracellular Ca2+ (28). We therefore explored the possibility that cAMP and cGMP can increase KCa channel activity by affecting Ca2+ sparks. Specifically, our results provide direct evidence that cAMP and cGMP increase the frequency of local Ca2+ release events (Ca2+ sparks) from the SR, which activate KCa channels. Furthermore, we provide evidence that cAMP can act, in part, to dilate pressurized arteries through the Ca2+ spark right-arrow KCa channel pathway.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell isolation. Single smooth muscle cells were enzymatically isolated from cerebral (basilar) and coronary (septal) arteries of rats. The cell isolation procedure was slightly modified from that previously described for cerebral (37) and coronary (38) arteries. Briefly, an artery was dissected out and placed in ice-cold Ca2+-free isolation solution of the following composition (in mM): 60 NaCl, 85 sodium glutamate, 5.6 KCl, 2 MgCl2, 10 glucose, and 10 HEPES (pH 7.4). After 10 min, the artery was transferred to the first of two enzyme solutions, with the same ionic composition as the Ca2+-free isolation solution described above, containing 1 mg/ml albumin, 0.5 mg/ml papain (Worthington, Freehold, NJ), and 1 mg/ml dithioerythritol, and was digested for 25 min at 37°C. The tissue was then transferred into isolation solution containing 0.1 mM CaCl2 and 1 mg/ml of a collagenase mixture of 30% type H + 70% type F (Sigma) and was digested for another 10 min at 37°C. The tissue was then washed twice in Ca2+-free fresh isolation solution without enzymes for 10 min. Single smooth muscle cells were obtained from the digested artery by gentle trituration with a polished wide-bore pipette. After trituration, cells were stored in the same solution at 4°C, to be used the same day. Cells were left to stick to the coverslip in the experimental chamber for 15-20 min.

Ca2+ spark measurements. The procedure for the measurement of sparks is described in Ref. 28. Briefly, the cells were loaded with the Ca2+-sensitive indicator fluo 3 by a 20-min incubation in 5 µM fluo 3-AM and 2.5 µg/ml pluronic acid (Molecular Probes, Eugene, OR) followed by a 20-min wash. All measurements were made within 30 min of the end of the wash. The cells were scanned with a Bio-Rad 1000 laser scanning confocal microscope, which was housed in the University of Vermont Cell Imaging Facility. The resolution was 0.4 µm × 0.4 µm (x and y) × 0.8 µm (z) (28). 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. The average length of the cell through which the scan line passed was 36 µm. 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 passes in the horizontal direction running from left to right, and position along the scan line is given by the vertical displacement. To minimize the possibility of laser damage affecting the Ca2+ handling of the cells, control and test measurements were made from different cells, with each cell being scanned for the same duration, 18 s total (6 consecutive line scan images of 512 lines, at 6 ms/line, were recorded from each cell along a single line). Sparks were analyzed using custom-written (IDL, Research Systems, Boulder, CO) analysis programs. To test the significance in the changes of Ca2+ spark frequency, a one-way ANOVA on ranks (Student-Newman-Keuls) method was used.

Electrophysiological recordings. K+ currents were measured in the whole cell, perforated-patch configuration (14) of the patch-clamp technique (13) with the use of 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 HEPES (pH 7.4). The pipette solution contained (in mM) 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.05 EGTA. All experiments were conducted at room temperature (23°C). Membrane currents were recorded, while holding the cells at membrane potentials of -40 mV, or as indicated. The STOCs were analyzed off-line, with the use of custom analysis programs. Briefly, STOCs were identified by setting a current threshold at three times the mean KCa single-channel amplitude at -40 mV (~5 pA), and all events that were greater than this threshold were included. The mean amplitude and frequency of the STOCs were determined. The large amplitude and low open probability (Po) of the KCa channel permitted the measurement of single KCa channel currents with the use of the perforated-patch configuration of the whole cell voltage clamp. To observe single KCa channel currents, Ca2+ sparks and hence STOCs were prevented by thapsigargin (28), which inhibits the SR Ca2+-ATPase, and the cells were clamped at 0 mV. NPo was calculated over 5-min intervals as Sigma Nj=1 tj · j/T, where tj is the time spent with j = 1, 2, . . . N channels open, N is the maximum number of channels observed, and T is the duration of the recording (5 min).

Intact pressurized artery. Resistance-sized posterior-cerebral arteries from rats were isolated, cannulated, and pressurized to 60 mmHg as described in Refs. 7, 31, and 20. The maximal diameter of the pressurized arteries was estimated from the arterial diameter in 30 µM diltiazem. Arterial diameter was measured with a video dimension analyzer (Living Systems Instrumentations, Burlington, VT). Results are expressed as means (in µm) ± SE for n vessels in diameter experiments. Student's t-test was used to compare values. A value of P < 0.05 was considered significant.

Conventional Ca2+ imaging. Isolated smooth muscle cells and intact arteries 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 for 20 min before measurements were made. Arteries were loaded by incubation with 2 µM fura 2-AM for 45 min. Arteries were then cannulated and continuously superfused with physiological salt solution at 37°C. Ca2+ was measured ratiometrically (340:380 nm) using the IMAGE-1/FL quantitative fluorescence measurement program (Universal Imaging, West Chester, PA).

Chemicals. Unless otherwise stated, all chemicals used in this study were obtained from Sigma Chemical (St. Louis, MO), including the following drugs: forskolin, adenosine, dibutyryl-cAMP (DBcAMP), sodium nitroprusside (SNP), thapsigargin, and diltiazem. H-89 and ryanodine were obtained from Calbiochem (La Jolla, CA), and iberiotoxin was from Peptides International (Louisville, KY). Nicorandil was a gift from Chugai Pharmaceuticals (Tokyo, Japan).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Forskolin, SNP, and nicorandil increase Ca2+ spark frequency. To test the hypothesis that increases in cAMP and cGMP can increase the frequency of Ca2+ sparks, the effects of forskolin (an activator of adenylyl cyclase that elevates cAMP), SNP, and nicorandil (nitric oxide donors, which elevate cGMP) on Ca2+ sparks were examined in single smooth muscle cells isolated from rat cerebral and coronary arteries. Forskolin significantly increased (P < 0.05) the Ca2+ spark frequency 2.3-fold (Fig. 1, A and B), as measured with a laser scanning confocal microscope and the Ca2+ fluorescence indicator fluo 3. This increase was prevented by an inhibitor of PKA, H-89 (1 µM), which binds to the active center of the catalytic subunit and suppresses the kinase activity (Fig. 1, A and B). The nitrovasodilators, SNP and nicorandil, also increased Ca2+ spark frequency 2.2- and 2.6-fold, respectively (Fig. 1C; P < 0.05). Forskolin, H-89, or SNP did not cause statistically significant changes in Ca2+ spark amplitude or rate of decay. Nicorandil did, however, cause a small but significant increase in Ca2+ spark amplitude (8% increase). If, indeed, Ca2+ sparks can activate sarcolemmal KCa channels, then forskolin, SNP, and nicorandil should increase the frequency of STOCs in these preparations, as had been suggested to occur in other preparations (9, 22).


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Fig. 1.   Forskolin, sodium nitroprusside, and nicorandil increase Ca2+ spark frequency in smooth muscle cells from cerebral and coronary arteries. A: forskolin (10 µM) increases Ca2+ spark frequency, and an inhibitor of protein kinase A (PKA), H-89 (1 µM), prevents the effects of forskolin on single smooth muscle cells from rat cerebral arteries. First column shows a series of 6 consecutive line scan images each of 3 s duration from a control cerebral artery myocyte in which sparks were observed in scans 1 and 2. Second column shows 6 line scan images from a different cerebral artery myocyte bathed in forskolin for 20 min, in which sparks were observed in all scans. Third column shows 6 line scans from a 3rd cerebral artery myocyte bathed for 20 min in 10 µM forskolin and the PKA inhibitor H-89, in which sparks were observed in scans 1, 2, 3, and 5. In each of the line scan images, position across scan line is given by vertical displacement, and time progresses from left to right. Inset (left of the line scan images): orientation of scan line through a smooth muscle cell. B: bar graph summarizing number of sparks per cerebral artery myocyte during 18 s of scanning in control (n = 40 cells), forskolin (n = 40 cells), and forskolin + H-89 cells (n = 40 cells). Spark frequency was significantly (* P < 0.05) increased by forskolin. This effect was blocked by presence of H-89. C: bar graph of effect of nitrovasodilators sodium nitroprusside (SNP; 10 µM) and nicorandil (100 µM) on Ca2+ spark frequency in coronary artery myocytes. Ca2+ spark frequency (n = 164 cells) was significantly increased (* P < 0.05) with both SNP (n = 132 cells) and nicorandil (n = 95 cells). Error bars are SE.

Forskolin, SNP, and nicorandil increase STOC frequency and amplitude. KCa channel currents were measured in arterial myocytes clamped at -40 mV, a potential similar to that of intact pressurized cerebral arteries (7, 20, 31). Whole cell currents were measured using the perforated-patch mode of the whole cell configuration, so as not to disturb the intracellular contents of the cells. Forskolin increased STOC frequency by 2.8 ± 0.5-fold (from 0.9 ± 0.2 Hz; n = 9; Figs. 2 and 3A). The PKA inhibitor H-89 (1 µM) completely reversed the effect of forskolin (Figs. 2 and 3A). Forskolin caused a small but significant (P < 0.05) increase in the average STOC amplitude by 1.3 ± 0.1-fold, which was also reversed by H-89 (Figs. 2 and 3B). Neither forskolin nor H-89 affected the decay of the STOCs. Forskolin had no effect on STOC frequency or amplitude in the continued presence of H-89. The nitrovasodilators, SNP (10 µM) and nicorandil (100 µM), also increased STOC frequencies (SNP, 4.0 ± 1.5-fold, n = 4; nicorandil, 3.3 ± 0.8-fold, n = 8) and amplitudes (SNP, 1.2 ± 0.1-fold, n = 4; nicorandil, 1.3 ± 0.1-fold, n = 8). These results are consistent with the idea that forskolin, through cAMP, and SNP and nicorandil, through cGMP, can increase Ca2+ spark and STOC frequency by 200-300% and STOC amplitude by ~30%.


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Fig. 2.   Forskolin increases Ca2+-sensitive K+ (KCa) channel currents in single smooth muscle cells from cerebral arteries. A: forskolin (10 µM) increases spontaneous transient outward current (STOC) frequency in a single smooth muscle cell isolated from a cerebral artery. PKA inhibitor H-89 (1 µM) reversed effects of forskolin. Whole cell currents were recorded with perforated-patch technique at holding potential (Vh) of -40 mV. Also shown on expanded time scale is average current signal of 20 STOCs averaged under control, forskolin-treated, and forskolin + H-89 conditions. B: expanded portion of experiment in A before and after forskolin. C: time course of STOC frequency from experiment in A, plotted in 20-s bins. Mean STOC amplitude and frequencies in this experiment were 25.4 pA and 2.08 Hz (control), 36.5 pA and 4.8 Hz (forskolin), and 28.8 pA and 1.97 Hz (H-89), respectively.


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Fig. 3.   Adenosine, forskolin, and dibutyryl-cAMP (db-cAMP) increase STOC frequency (A) and amplitude (B), and H-89 reversed the effects of forskolin. Changes in STOC frequency and amplitude relative to pretreatment control levels with application of adenosine (10 µM, n = 7), forskolin (10 µM, n = 9), dibutyryl-cAMP (500 µM, n = 5), and forskolin (10 µM) in the presence of H-89 (1 µM, n = 9) are illustrated. Error bars are means ± SE. Statistical significance was tested with a paired Student's t-test (* P < 0.05, ** P < 0.01).

To provide additional evidence that cAMP can increase STOC frequency, the effects of the membrane-permeable cAMP analog DBcAMP (500 µM) on STOC frequency and amplitude were examined. Externally applied DBcAMP increased STOC frequency 2.0 ± 0.2-fold (n = 5; Fig. 3A). In further support of cAMP/PKA increasing STOC frequency, the vasodilator adenosine (10 µM), which activates adenylyl cyclase, significantly (P < 0.05) increased STOC frequency 2.6 ± 0.7-fold (n = 11) and amplitude 1.3 ± 0.1-fold (Fig. 3, A and B).

Forskolin increases the NPo of KCa channels in the absence of Ca2+ sparks. The forskolin-induced increase in Ca2+ spark frequency would explain the observed elevation of STOC frequency. However, the effect of forskolin on STOC amplitude cannot be explained by its action on Ca2+ sparks alone, because the amplitude of the spark was unaffected. A possible explanation for the 1.3-fold increase in STOC amplitude to forskolin is through an elevation of open state probability (Po) of KCa channels caused by PKA phosphorylation of the channel, as has been demonstrated in excised patches by others (23). An elevated Po of KCa channels would lead to higher Po of the KCa channels during a spark and hence increase the STOC amplitude, if it were not already maximal at that voltage. To test the effects of forskolin on KCa channels, single-channel currents through KCa channels were recorded from isolated arterial myocytes in the whole cell mode, using the perforated-patch technique. Single KCa channels were identified by their characteristic large single-channel conductance, voltage dependence, and sensitivity to block by tetraethylamonium and iberiotoxin (7, 23, 28, 31).

Forskolin increased single KCa channel activity (measured as NPo) from 0.041 ± 0.015 to 0.055 ± 0.02 (n = 7) measured over 5 min at 0 mV (Fig. 4). The single-channel current amplitude was not affected by forskolin (at 0 mV: control, 5.0 ± 0.2 pA; forskolin, 5.0 ± 0.2 pA, n = 7). The forskolin-induced increase in NPo did not appear to be the result of an elevation in global Ca2+ in these voltage-clamped cells, since Ca2+ measured with fura 2 did not increase (106.0 ± 5.4% of the control, n = 4). Forskolin would not be expected to decrease average Ca2+ under these conditions, since the voltage of the cells was controlled. These results are consistent with the idea that forskolin increased NPo by 1.3-fold through an effect of cAMP/PKA directly on the KCa channel and thus provide an explanation for the increase in STOC amplitude to forskolin.


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Fig. 4.   Forskolin increases open probability (NPo) of single KCa channels, in absence of Ca2+ sparks, in a single smooth muscle cell isolated from a cerebral artery. Single-channel currents were recorded with the whole cell, perforated-patch technique from a single isolated smooth muscle cell at 0 mV. STOCs were indirectly inhibited by blocking sarcoplasmic reticulum (SR) Ca2+ uptake with thapsigargin (100 nM). Shown is a series of channel openings from a single cell recorded before (NPo of this series = 0.043) and during (NPo of this series = 0.054) application of 10 µM forskolin at 0 mV.

Role of Ca2+ sparks and KCa channels in forskolin-induced dilations. We tested the hypothesis that part of the forskolin dilation of pressurized cerebral arteries is through increasing Ca2+ spark frequency, activating KCa channels. Consistent with this hypothesis, an inhibitor of Ca2+ sparks, ryanodine, inhibited 79.8 ± 8.1% of the forskolin-induced dilation of pressurized cerebral arteries with tone (Fig. 5D). Furthermore, ryanodine constricted pressurized cerebral arteries to a similar diameter in the absence (to 81 ± 7 µm) (28) or presence of forskolin (100 nM; to 85 ± 20 µm, n = 5), consistent with forskolin acting on a ryanodine-sensitive process. The blocker of KCa channels, iberiotoxin, also inhibited 82.3 ± 7.0% of the forskolin-induced dilation (Fig. 5D) (as shown previously, see Ref. 42). The effects of ryanodine and iberiotoxin were not additive (Fig. 5, B and D; see also Ref. 28), consistent with the idea that Ca2+ sparks act to stimulate KCa channels (28). Part of the dilation to forskolin was insensitive to iberiotoxin or ryanodine, particularly at higher forskolin concentrations, suggesting that forskolin also acts through other mechanisms (1, 11, 15, 18, 19, 36, 41, 48). In this preparation, however, forskolin (10 µM or less) was ineffective on pressurized arteries that were constricted with high (60 mM) K+ (Fig. 5, A and D), consistent with forskolin acting in large part through activation of K+ channels (1, 31). Ryanodine and iberiotoxin were ineffective on pressurized arteries previously dilated with the Ca2+ channel blocker diltiazem (1 µM; Fig. 5C), a condition that would lower Ca2+ spark frequency and hence KCa channel activity (7, 20, 28, 31) through reducing average intracellular Ca2+ and SR Ca2+ content. These results suggest that forskolin dilates pressurized cerebral arteries through activation of K+ channels, with part of the dilation due to activation of the Ca2+ sparkright-arrowKCa channel pathway.


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Fig. 5.   Forskolin dilations of pressurized (60 mmHg) cerebral arteries were reversed by ryanodine, but diltiazem dilations were not. Arteries were partially constricted (~35%) by pressure (60 mmHg), as indicated by horizontal dotted line. A: forskolin did not dilate pressurized cerebral arteries constricted by 60 mM K+. Transient dilation to K+ was due to activation of inward rectifier K+ channel present in these arteries (31). B: ryanodine (10 µM) constricted pressurized arteries dilated by forskolin. KCa channel blocker iberiotoxin (IbTx; 100 nM) was without effect in presence of ryanodine. C: ryanodine and iberiotoxin were without effect on pressurized arteries dilated by Ca2+ channel blocker, diltiazem (1 µM). Ryanodine constricted arteries when diltiazem was removed. D: dilations to 30 nM forskolin were inhibited by presence of either ryanodine (10 µM, n = 5) or iberiotoxin (100 nM, n = 4). Ryanodine and iberiotoxin together (n = 5) were no more effective than either agent alone. Forskolin is ineffective in the presence of 60 mM external K+ (n = 4).

Activation of KCa channels leads to membrane potential hyperpolarization of the arterial myocytes, which closes voltage-dependent Ca2+ channels, decreasing Ca2+ entry, which should cause vasodilation through a reduction of arterial wall Ca2+. As predicted, forskolin caused average Ca2+ in the wall of a pressurized artery (60 mmHg) to fall from 184 ± 5 nM (n = 20) to 123 ± 22 nM (n = 7) (Fig. 6, A and B). As expected from the previous diameter measurements (Fig. 5) (28), ryanodine increased arterial wall Ca2+ (to 202 ± 12 nM) through membrane potential depolarization. Caffeine, which opens RyR channels (34), caused a larger release of SR Ca2+ in the presence of forskolin than in its absence (P < 0.05; Fig. 6). In contrast, caffeine released less Ca2+ from arteries dilated by diltiazem (Fig. 6), presumably because SR Ca2+ content had been reduced. Caffeine was relatively ineffective in the presence of ryanodine (Fig. 6). These results taken together support the idea that forskolin (cAMP) can lower arterial wall Ca2+ by increasing Ca2+ spark frequency.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The present study proposes a new mechanism of action for vasodilators that work through cAMP and cGMP (Fig. 7). Our data suggest that cAMP and cGMP can increase KCa channel activity in two ways: 1) by increasing Ca2+ spark frequency and 2) by directly increasing the open probability of the KCa channel. Together these actions lead to increased frequency and amplitude of STOCs, which, when summed across the vessel wall, should cause membrane potential hyperpolarization and, ultimately, relaxation of the artery.


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Fig. 6.   Forskolin decreases and ryanodine increases arterial wall Ca2+ in an intact pressurized (60 mmHg) cerebral artery (A). Ca2+ signal is from an intact artery loaded with fura 2. Elevating pressure from 10 to 60 mmHg increased Ca2+ from ~100 to 200 nM and constricted the artery by ~35%. Rapid application of caffeine (10 mM) to the bath produced a transient increase in cytosolic Ca2+ (arrows). B: ratio images of cerebral artery loaded with fura 2 (same artery as in A) pressurized to 10 mmHg (No tone; a), to 60 mmHg physiological salt solution (PSS tone; b), at 60 mmHg during a caffeine pulse (PSS/Caf pulse; c), at 60 mmHg dilated with forskolin (FSK tone; d), at 60 mmHg with forskolin present during a caffeine pulse (FSK/Caf pulse; e), and at 60 mmHg in presence of forskolin after ryanodine-induced constriction (FSK + RYA tone; f). Diameters of the artery are indicated below images. C: forskolin increases and diltiazem and ryanodine decrease caffeine-induced Ca2+ transients. Peak change in Ca2+ to caffeine is shown for control (PSS) and in presence of 100 nM forskolin, 3 µM diltiazem, and 10 µM ryanodine. * Caffeine transient was significantly increased (P < 0.05) in presence of forskolin and decreased in presence of diltiazem (which reduced arterial wall Ca2+ to 126 ± 4 nM, n = 8) or ryanodine (P > 0.05).


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Fig. 7.   Proposed mechanisms of action of forskolin and nitrovasodilators on Ca2+ sparks and KCa channels in arterial smooth muscle cells. Adenylyl cyclase (AC) produces cAMP when activated by externally applied forskolin or adenosine, which in turn stimulates PKA. Activation of PKA has a direct effect on the KCa channel to increase its open probability, as has been previously demonstrated in inside-out excised patches. However, the predominant effect of PKA is on the SR to increase Ca2+ spark frequency. Possible targets of PKA and PKG are the KCa channel, RyR receptors, and Ca2+-ATPase (phospholamban). Combined effects of increased spark activity and direct effects on the KCa channel lead to significantly increased activity of the KCa channel. Resulting hyperpolarization closes voltage-dependent Ca2+ channels, leading to reduced global intracellular Ca2+ and vasodilation. GC, guanylate cyclase; G, Gs protein.

Comparison of Ca2+ spark and STOC frequency. The mean Ca2+ spark frequency (0.14 Hz) was ~15% of the mean STOC frequency (0.96 Hz). We estimate that we could detect Ca2+ sparks in no more than 23% of cell volume, based on the average spread of a Ca2+ spark (4.8 µm) and maximum width of a cell (10 µm) (28) (assuming a circular cross section of a cell). This detection volume would be less when a scan line is close to the side of the cell and when the length of the scan line is less than the length of the cell, which is the case when the cells are not straight. These results are consistent with the idea that most Ca2+ sparks cause STOCs (17).

Effects on Ca2+ sparks. Forskolin, SNP, and nicorandil increased Ca2+ spark frequency. Forskolin, through cAMP/PKA, could act directly on the RyR channel (43, 46) and/or indirectly through an elevation of SR Ca2+ load, which has been shown to increase the open state probability of RyR channels in skeletal and cardiac muscle (12, 26, 45, 46). cAMP/PKA and cGMP/PKG could increase SR Ca2+ load by enhancing SR Ca2+-ATPase activity by phosphorylating phospholamban (25). The observed increase in caffeine-induced Ca2+ transients in the presence of forskolin (Fig. 6) is consistent with cAMP/PKA increasing SR Ca2+ load. However, forskolin did not cause a detectable increase in Ca2+ spark amplitude, which could be indicative of SR Ca2+ load. Nicorandil did increase Ca2+ spark amplitude by ~10%. Our data do not distinguish between a direct effect on ryanodine receptors and an indirect effect of SR Ca2+ load on Ca2+ spark frequency. cAMP (PKA) and cGMP (PKG) could conceivably act on both the Ca2+-ATPase and the RyR channel to affect SR function and STOC frequency (see Fig. 7) (49).

Contribution of STOCs to overall KCa channel activity. KCa channels do not appear to contribute significantly to regulation of smooth muscle membrane potential in intact pressurized cerebral arteries when Ca2+ sparks are blocked (28). This suggestion is based on the observation that blockers of KCa channels (iberiotoxin) were without effect on pressurized cerebral arteries when inhibitors of Ca2+ sparks (ryanodine, thapsigargin, or cyclopiazonic acid) were present. Furthermore, iberiotoxin was without effect in the presence of ryanodine and forskolin (Fig. 5B), suggesting that, even in the presence of forskolin, KCa channels do not contribute significantly to the regulation of arterial diameter without Ca2+ sparks.

The relative activity (NPo) of KCa channels with and without Ca2+ sparks can be estimated from the data presented here and from Ref. 28. At -40 to -30 mV, NPo contributed by Ca2+ sparks is ~0.4 (assuming peak NPo = 13 lasting 30 ms, frequency 1/s). Baseline NPo is estimated to be in the range of 0.01 at physiological membrane potentials and Ca2+ in pressurized arteries (-40 mV, 200 nM Ca2+), based on extrapolation from direct measurements (Fig. 4). Assuming a slope conductance of KCa channels of 79 pS (estimate at -30 to -40 mV from current-voltage relationship), this would correspond to an average KCa channel conductance of ~32 pS with sparks, consistent with a significant contribution to membrane conductance of smooth muscle cells with a 10 GOmega input resistance (see Ref. 31). However, <5% of the KCa channel conductance would be contributed by channels not activated by Ca2+ sparks. Forskolin would increase NPo caused by sparks two- to threefold or from 0.4 to 0.8-1.2. However, forskolin would directly increase NPo in the absence of sparks by 1.3 or to ~0.013-0.026. Regardless of the uncertainties, these estimates support the idea that Ca2+ sparks should have a profound impact on the overall activity of KCa channels and that forskolin acts largely (99%) on KCa channels through increasing Ca2+ spark frequency.

Proposed mechanism for regulation of arterial diameter by cAMP/PKA or by cGMP/PKG via Ca2+ sparks and KCa channels. Elevation of intravascular pressure causes a graded membrane potential depolarization and vasoconstriction of small myogenic arteries (Fig. 7) (7, 20, 27, 31). The membrane potential depolarization increases the steady-state open probability of voltage-dependent Ca2+ channels (30, 40), which elevates Ca2+ entry and global intracellular Ca2+ (21, 27). Ca2+ channel inhibitors (e.g., nimodipine, diltiazem) prevent pressure-induced constrictions of myogenic cerebral arteries (7, 20, 30). Intracellular Ca2+ could increase Ca2+ spark frequency through cytoplasmic and SR luminal activation of RyR channels. Ca2+ sparks, by themselves, would have little direct effect on intracellular Ca2+, even at the highest observed rate (4/s) (28). Ca2+ sparks would, however, have a profound effect on the activity of KCa channels, which would oppose the pressure-induced membrane potential depolarization. In support of this proposed mechanism, blockers of KCa channels (e.g., iberiotoxin) and Ca2+ sparks (ryanodine, cyclopiazonic acid, thapsigargin) depolarize and constrict pressurized (60 mmHg) myogenic cerebral arteries by ~8 mV and 30% (7, 20, 28). Lowering intravascular pressure (7) or blocking Ca2+ channels (7, 20, 28), which would decrease intracellular Ca2+ and thus KCa channel activity, abolished the effects of iberiotoxin and ryanodine on membrane potential and arterial diameter (also see Fig. 6B with diltiazem). Furthermore, iberiotoxin was without effect on membrane potential and diameter in intact pressurized arteries, when Ca2+ sparks were blocked by ryanodine or thapsigargin (28) (see also Fig. 6A), suggesting that global intracellular Ca2+ does not cause sufficient activation of KCa channels to modulate membrane potential (see above).

Iberiotoxin blocks a significant fraction of the dilation of rat cerebral arteries to forskolin (see Fig. 5 and Ref. 42). However, high extracellular K+ blocked the entire dilation to forskolin. cAMP/PKA has also been shown to activate ATP-sensitive K+ (KATP) channels (18, 19, 36, 48) and voltage-dependent K+ channels (1) in smooth muscle. Forskolin dilations of cerebral arteries were not affected by glibenclamide, an inhibitor of KATP channels (data not shown; also observed in Ref. 42), suggesting that KATP channels do not contribute to the observed dilations. Voltage-dependent K+ channels contribute significantly to the regulation of the membrane potential and diameter of pressurized cerebral arteries (20), suggesting the possibility that forskolin acts in part on this channel (cf. Ref. 1). These results indicate that the forskolin response in our preparation is entirely mediated by effects on the K+ conductance of the cell and that part of this change in K+ conductance is due to effects on the KCa channel.

Here we demonstrate that agents that elevate cGMP (SNP and nicorandil) increase Ca2+ spark and STOC frequency, as well as increase STOC amplitude in coronary arteries from rat. In the same intact coronary artery preparation, SNP (10 µM) has been shown to cause a 13-mV hyperpolarization and almost maximal dilation, which was substantially blocked by iberiotoxin (47). PKG has also been shown to activate KCa channels in excised patches from the same coronary artery myocytes (Ref. 47; see also Refs. 39 and 44). cGMP/PKG can increase SR Ca2+ uptake, presumably through an action on phospholamban (10). Furthermore, smooth muscle relaxations to nitrovasodilators in mesenteric (8, 16), pulmonary (3), and cerebral arteries (33) can be partially inhibited by iberiotoxin, suggesting a role for KCa channels in relaxations of a number of types of vascular smooth muscle to cGMP. These results support the general mechanism presented in Fig. 7 (see also Fig. 4 in Ref. 28).

In conclusion, the present study proposes a new mechanism of action for vasodilators that work through cAMP and cGMP in cerebral and coronary arteries (Fig. 7). Our data indicate that cAMP and cGMP can increase Ca2+ spark frequency two- to threefold as well as have a small, direct effect on KCa channel open probability (23, 39, 44). Together these actions lead to increased frequency and amplitude of STOCs, which, when summed across the vessel wall, should cause membrane potential hyperpolarization and, ultimately, relaxation of the artery through decreasing Ca2+ entry through voltage-dependent Ca2+ channels (7, 28, 31), thereby lowering arterial wall Ca2+ (Fig. 6). The frequency of Ca2+ sparks, even with elevated cAMP or cGMP (2-3 sparks · s-1 · cell-1), would have little direct effect on average cytoplasmic Ca2+. Our results would then explain with one integrated mechanism (Fig. 7) the apparently disparate observations that cAMP and cGMP can increase SR Ca2+ uptake and lower arterial Ca2+, and, yet, relaxations to these cyclic nucleotides are often inhibited by blockers of KCa channels (16, 33, 35, 42). This mechanism (frequency modulation of Ca2+ sparks), along with activation of KATP channels and voltage-dependent K+ channels, inhibition of Ca2+ channels, and changes in Ca2+ sensitivity, would contribute to the actions of cAMP and cGMP. We suggest that frequency modulation of Ca2+ sparks by cAMP/cGMP may be a general mechanism for the SR to regulate plasmalemmal Ca2+ entry through alterations in a cell's membrane potential.

    ACKNOWLEDGEMENTS

We thank the late Dr. Fred Fay and Dr. John Walsh for insights and comments on this study.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-44455, HL-51728, and HL-58231, National Science Foundation Grant IBN-9631416, a grant from the Alexander Von Humboldt Foundation (to T. Kleppisch), and a fellowship (to V. A. Porter) from the American Heart Association, New Hampshire and Vermont Affiliates.

Address for reprint requests: M. T. Nelson, Dept. of Pharmacology, 55A South Park Dr., Univ. of Vermont, Colchester, VT 05446.

Received 9 September 1997; accepted in final form 13 January 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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