INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

MUSCLE CONTRACTION IS initiated by a global increase in cytosolic Ca²⁺ resulting from influx from extracellular compartments and release from intracellular stores. In cardiac and skeletal muscles, it has been demonstrated that the global Ca²⁺ transient activated during muscle contraction is generated by the summation of thousands of discrete local Ca²⁺ release events, or "Ca²⁺ sparks," originating from ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR; see Refs. 9, 12, 36). Similar Ca²⁺ sparks have been identified from various types of smooth muscle cells (2, 6, 16, 21, 23, 28, 35, 39-41, 72). Evidence from systemic vascular smooth muscle cells indicates that Ca²⁺ sparks cause local increases of Ca²⁺ concentration ([Ca²⁺]) in subsarcolemmal spaces, activating nearby Ca²⁺-activated K⁺ (K_Ca) channels, leading to membrane hyperpolarization, reduction of Ca²⁺ influx via L-type Ca²⁺ channels, and vasodilation (27, 29, 39, 40, 43). Therefore, they act as negative feedback modulators of membrane potential, rather than direct activators of myofilaments and the contractile apparatus (29, 40). On the other hand, Ca²⁺ sparks of tracheal smooth muscle cells have been shown to activate both K_Ca and Ca²⁺-activated Cl (Cl_Ca) channels (72), depending on the membrane potential. Activation of Cl_Ca channels can cause membrane depolarization, increased Ca²⁺ influx, and cell contraction. Hence, the net physiological effects of Ca²⁺ sparks in these myocytes depends on the relative activities of these counteracting Ca²⁺-activated channels and on the membrane potential at which the Ca²⁺ sparks are generated.

Despite the studies in systemic vascular smooth muscle cells, there is only minimal information on Ca²⁺ sparks in pulmonary arterial smooth muscle cells (PASMCs). A recent study on the heterogeneity of ryanodine- and inositol trisphosphate (IP₃)-sensitive Ca²⁺ stores showed that Ca²⁺ sparks are present in canine PASMCs (30); another study reported that both Ca²⁺ sparks and spontaneous transient outward currents (STOCs) are present in fetal rabbit PASMCs (45). In the present study, we sought to provide a comprehensive characterization of the biophysical properties and physiological functions of Ca²⁺ sparks in intralobar PASMCs from adult rats. We have 1) quantified the spatiotemporal properties of spontaneous Ca²⁺ sparks in rat PASMCs, according to the criteria established in cardiac myocytes, 2) determined the nature of the intracellular Ca²⁺ store(s) from which they originate, 3) examined their modulation by enhanced Ca²⁺ influx, 4) determined their role in modulating resting membrane potential, and 5) investigated their regulation by norepinephrine (NE) and endothelin-1 (ET-1). Our results indicate that, despite similarities in the basic properties of Ca²⁺ sparks in PASMCs and systemic vascular smooth muscle cells, the physiological functions are remarkably different. Inasmuch as the pulmonary circulation is often regulated differently from the systemic circulation (e.g., responses to hypoxia), the present study provides unique information on pulmonary Ca²⁺ sparks fundamental for future studies of local Ca²⁺ signaling in the pulmonary vasculature.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Isolation and culture of PASMCs. PASMCs were enzymatically isolated and transiently cultured as previously described (58). Briefly, male Wistar rats (150-200 g) were injected with heparin and anesthetized with pentobarbital sodium (130 mg/kg ip). They were exsanguinated, and the lungs were removed and transferred to a petri dish filled with HEPES-buffered salt solution (HBSS) containing (in mM) 130 NaCl, 5 KCl, 1.2 MgCl₂, 1.5 CaCl₂, 10 HEPES, and 10 glucose, pH 7.4 (adjusted with NaOH). Second- and third-generation intrapulmonary arteries (~300-800 µm) were isolated and cleaned free of connective tissue. The endothelium was removed by gently rubbing the luminal surface with a cotton swab. Arteries were then allowed to recover for 30 min in cold (4°C) HBSS, followed by 20 min in reduced-Ca²⁺ (20 µM) HBSS at room temperature. The tissue was digested at 37°C for 20 min in 20 µM Ca²⁺ HBSS containing collagenase (type I, 1,750 U/ml), papain (9.5 U/ml), BSA (2 mg/ml), and dithiothreitol (1 mM), then removed and washed with Ca²⁺-free HBSS to stop digestion. Single smooth muscle cells were gently dispersed by trituration with a small-bore pipette in Ca²⁺-free HBSS at room temperature. The cell suspension was then placed on 25-mm glass coverslips and transiently (16-48 h) cultured in Ham's F-12 medium (with L-glutamine) supplemented with 0.5% FCS, 100 U/ml streptomycin, and 0.1 mg/ml penicillin.

Isolation of ventricular myocytes. Excised hearts were cannulated and perfused retrogradely through the aorta with prewarmed (37°C) Ca²⁺-free Tyrode solution containing (in mM) 137 NaCl, 5.4 KCl, 1 MgCl₂, 10 HEPES, and 10 glucose, pH 7.4 (adjusted with NaOH). After 5 min, the heart was perfused with Ca²⁺-free Tyrode solution containing collagenase (type 1, 560 U/ml) and protease (type XIV, 0.28 mg/ml). After a 12-min enzyme-recirculating period, the heart was washed with 0.2 mM Ca²⁺ Tyrode solution for 5 min to stop the enzymatic digestion. The left ventricle was then cut from the heart and placed in 20 ml of prewarmed 0.2 mM Ca²⁺ Tyrode solution. Myocytes were dispersed by gentle agitation and stored at room temperature (~22°C) before use.

Measurement of Ca²+ sparks. Ca²⁺ sparks were visualized using the membrane-permeable Ca²⁺-sensitive fluorescent dye fluo 3-AM. PASMCs and ventricular myocytes were loaded with 5-10 µM fluo 3-AM [dissolved in dimethyl sulfoxide (DMSO) with 20% pluronic acid] in normal Tyrode solution for 30-45 min and 15 min at room temperature (~22°C), respectively. Cells were then washed thoroughly with 2 mM Ca²⁺ Tyrode solution to remove extracellular fluo 3-AM and rested for 15-30 min in a cell chamber to allow for complete deesterification of cytosolic dye. Confocal images were acquired using a Zeiss LSM-510 inverted confocal microscope (Carl Zeiss) with a Zeiss Plan-Neofluor ×40 oil immersion objective (numeric aperture = 1.3). The confocal pinhole was set to render a spatial resolution of 0.4 µm in the x-y axes, and 1.7 µm in the z-axis. Fluo 3-AM was excited by the 488-nm line of an argon laser, and fluorescence was measured at >505 nm. Images were acquired in the linescan mode (digital zoom rendering a 38-µm scan line), scanning at 0.075 µm/pixel, 512 pixels/line at 2-ms intervals for 512 lines/image from different cells within the same culture dish before and after drug application. Photobleaching and laser damage to the cells were minimized by attenuating the laser to ~1% of its maximum power (25 mW) with an acousto-optical tunable filter. Only 10 images (once every 10 s) were taken for each cell. Cells that did not respond to an external solution containing 10 mM Ca²⁺ and 0.5 mM caffeine applied at the end of experiments were discarded. All experiments were performed at room temperature.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Measurement of Ca2+ sparks. A: spark amplitude is defined as the change in peak fluorescence (F) normalized by the basal fluorescence (F0). Points a  a' and b  b' refer to the temporal and spatial profiles, respectively, for the determination of the full-duration half-maximum (FDHM) and full-width half-maximum (FWHM). B: FDHM, the duration of the spark that exceeds 50% of peak amplitude. C: FWHM, the width of the spark that exceeds 50% of the peak amplitude.

Measurement of membrane potential. Membrane potential of PASMCs was measured using the perforated-patch technique to avoid disturbance of Ca²⁺ dynamics resulting from intracellular dialysis. Gigaohm seals were first established with patch pipettes (~3 M) tip-filled with a solution containing (in mM) 35 KCl, 90 potassium gluconate, 10 NaCl, and 10 HEPES, pH 7.2 (adjusted with KOH) and were backfilled with the same solution containing 300 mg/ml amphotericin B. After an access resistance of 30 M was achieved with the cells voltage-clamped at a holding potential of 50 mV with an Axopatch 200B amplifier (Axon Instruments, Union City, CA), membrane potential was recorded by switching the amplifier to current-clamp mode (I = 0). Data were filtered at 5 kHz, digitized with a Digidata 1200 analog-to-digital converter, and analyzed with pCLAMP software. Complete exchanges of external solution were achieved in <1 s using a rapid concentration-clamp system.

Statistical analysis. Throughout this paper, data are expressed as means ± SE. The number of cells or Ca²⁺ sparks was specified in the text. Statistical significance (P < 0.05) of the changes in spark characteristics was assessed by paired or unpaired Student's t-tests or by one-way ANOVA with Newman-Keuls post hoc analyses, nonparametric Mann-Whitney U-tests, or Kolmogorov-Smirnov tests, wherever applicable.

Chemicals and drugs. Collagenase, protease, papain, serum albumin, dithiothreitol, caffeine, 2-aminoethyl diphenyl borate (2-APB), amphotericin B, and antibiotics were purchased from Sigma Chemical (St. Louis, MO). FCS and Ham's F-12 medium were purchased from Mediatech (Herndon, VA). Fluo 3-AM and pluronic acid were purchased from Molecular Probes (Eugene, OR). BAY K 8644, nifedipine, and ryanodine were purchased from Calbiochem (La Jolla, CA). Stock solutions of BAY K 8644, nifedipine, ryanodine, amphotericin B, and 2-APB were prepared in DMSO and diluted 1:400 or 1:1,000 in 2 mM Ca²⁺-Tyrode solution. High-K⁺ (20 mM) solution was prepared by equimolar replacement of NaCl.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Biophysical characterization of Ca²+ sparks in PASMCs. To characterize the biophysical properties of Ca²⁺ sparks in PASMCs and to allow for comparisons with Ca²⁺ sparks recorded in other tissues, the settings for linescan confocal imaging were first verified using spontaneous Ca²⁺ sparks in rat ventricular myocytes as the standard, since they have been characterized extensively in different laboratories. Random spontaneous Ca²⁺ sparks were observed in ~80% of ventricular myocytes during the 10-s recording period, giving an overall average frequency of 1.31 ± 0.21 s¹ (40 cells, 511 sparks; Fig. 2). The amplitude of sparks (F/F₀) was 0.93 ± 0.03, corresponding to a [Ca²⁺]_i increase of 217.4 ± 25.5 nM. The size (FWHM) and duration (FDHM) of Ca²⁺ sparks were 1.76 ± 0.03 µm and 33.9 ± 0.7 ms, respectively, values highly consistent with those reported previously (9, 12, 20), indicating that both the acquisition parameters and the analysis algorithm used in the present study are comparable with other studies.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Comparison of spontaneous Ca2+ sparks from pulmonary artery smooth muscle cells (PASMCs) and cardiac cells. A: representative line-scan images generated from PASMCs (left) and ventricular cells (right) under control conditions. B: bar graphs summarizing spark characteristics obtained from PASMCs (open bars) and cardiac cells (filled bars). Change () in intracellular Ca2+ concentration ([Ca2+]i) values calculated from the change in the ratio of fluorescence (F) to baseline fluorescence (F0) are given in the text. * Significant difference from PASMC sparks.

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View larger version (31K): [in this window] [in a new window]   Fig. 3.   Frequency distributions of different parameters of Ca2+ sparks of PASMCs and cardiac myocytes. No. of events is reported as a function of amplitude (A and B, bin size: 0.1), FDHM (C and D, bin size: 5 ms), and FWHM (E and F, bin size: 0.2 µm) for populations of PASMCs (A, C, and E) and cardiac cells (B, D, and F). Group means are in Fig. 2.

Spontaneous PASMC Ca²+ sparks and ryanodine-sensitive Ca²⁺ stores. To examine if spontaneous Ca²⁺ sparks in PASMCs were due entirely to the activation of RyRs, Ca²⁺ sparks were recorded in the same population of PASMCs before and after exposure to 10 or 50 µM ryanodine for 15 min to inhibit RyRs. Ryanodine reduced spontaneous spark frequency in a concentration-dependent manner from a control value of 0.39 ± 0.06 s¹ (75 cells, 305 sparks) to 0.20 ± 0.04 s¹ (47 cells, 93 sparks; P < 0.05) and 0.05 ± 0.02 s¹ (27 cells, 14 sparks; P < 0.05) with 10 and 50 µM ryanodine (Fig. 4A). Ryanodine did not evoke any changes in spark amplitude, duration, or size (Table 1). In a separate series of experiments (Fig. 4B, Table 1), 50 µM 2-APB, an inhibitor of IP₃-sensitive Ca²⁺ stores, had no effect on the frequency of spontaneous sparks (control = 0.40 ± 0.05 s¹, 53 cells, 216 sparks; 2-APB = 0.40 ± 0.10 s¹, 37 cells, 154 sparks) nor on their amplitude, size, or duration. In the presence of 50 µM ryanodine, coapplication of 50 µM 2-APB did not cause further reduction of spark frequency (ryanodine: 0.06 ± 0.02 s¹, 24 cells, 15 sparks; 2-APB + ryanodine: 0.02 ± 0.01 s¹, 21 cells, 5 sparks). Moreover, 100 µM xestospongin C, another inhibitor of IP₃ receptors, also failed to inhibit Ca²⁺ sparks (Zhang and Sham, unpublished data).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effect of ryanodine and inositol trisphosphate (IP3)-sensitive Ca2+ store inhibition on spontaneous Ca2+ spark frequency in PASMCs. A: PASMC spark frequency is reported in the absence (open bar) or presence incremental concentrations of ryanodine (Ryan; 10 µM, filled bars; 50 µM, hatched bar). B: cells were exposed to 2 mM Ca2+ (control, open bar), 50 µM 2-aminoethyl diphenyl borate (2-APB; filled bar), 50 µM ryanodine (hatched bar), and 50 µM 2-APB + 50 µM ryanodine (striped bar). * Significant difference from control. Significant difference from 2-APB. Corresponding FDHM, FWHM, and amplitude values are in Table 1.

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View larger version (37K): [in this window] [in a new window]   Fig. 5.   Stimulation of Ca2+ sparks by a subthreshold concentration of caffeine (caff). A: caffeine (0.5 mM) elicited an increase in Ca2+ sparks (left), clustered Ca2+ sparks (middle), or global Ca2+ elevations (G-Ca; right). B: summarized data obtained from control cells (open bars) and cells exposed to caffeine (0.5 mM, filled bars) and ryanodine + caffeine (10 µM and 0.5 mM, respectively, hatched bars). * Significant difference from control. Significant difference from caffeine-treated cells.

Effects of Ca²+ channel activity and SR loading on Ca²⁺ sparks. Under physiological conditions, Ca²⁺ is the major stimulus for RyR activation, and the L-type voltage-gated Ca²⁺ channel is the major pathway of Ca²⁺ influx in PASMCs. To understand the relationship between sarcolemmal Ca²⁺ channel activity and Ca²⁺ sparks, we first examined if spontaneous Ca²⁺ spark activity was related to the activation of RyRs by Ca²⁺ influx via Ca²⁺ channels. Inhibition of Ca²⁺ channels by 10 µM nifedipine (>15 min exposure; Fig. 6A) did not alter the frequency (control = 0.38 ± 0.07 s¹, 38 cells, 142 sparks; nifedipine = 0.24 ± 0.06 s¹, 42 cells, 110 sparks) or duration of spontaneous Ca²⁺ sparks (control = 41.8 ± 2.0 ms; nifedipine = 40.3 ± 2.1 ms) but slightly augmented the amplitude (control = 0.46 ± 0.01; nifedipine = 0.54 ± 0.02; P < 0.05) and size (control = 1.62 ± 0.05 µm; nifedipine = 1.99 ± 0.08 µm; P < 0.05) of Ca²⁺ sparks. The increase in F/F₀ could be because of a decrease in the resting [Ca²⁺] (F₀) in the presence of nifedipine, in which case an equivalent Ca²⁺ release (F) would result in a greater F/F₀. Nonetheless, under resting conditions, Ca²⁺ influx via Ca²⁺ channels did not seem to contribute to the activation of spontaneous Ca²⁺ sparks in PASMCs.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Effects of Ca2+ influx via L-type Ca2+ channels on spontaneous Ca2+ spark frequency. A: effect of 10 µM nifedipine on resting spark frequency. B: enhanced spark frequency by the L-type Ca2+ channel agonist BAY K 8644 (BAYK; 3 µM). C: enhanced spark frequency by elevation of extracellular K+ concentration ([K+]o) from 5.4 to 20 mM. D: enhanced spark frequency by increasing external [Ca2+] from 2 to 10 mM. * Significant difference from control. Significant difference from BAY K 8644-treated group.

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Ca²+ sparks and membrane potential. The modulatory effect of Ca²⁺ sparks on the membrane potential of PASMCs was examined using the perforated-patch technique to avoid disturbing subcellular Ca²⁺ dynamics. Under control conditions, the membrane potential of PASMCs appeared to be less quiescent when measured using the perforated-patch technique compared with those measured under the conventional whole cell configuration in our previous studies (58); small sporadic depolarizations were occasionally observed. Application of a subthreshold concentration of caffeine (0.5 mM) to PASMCs, which had enhanced the occurrence of Ca²⁺ sparks in prior experiments, caused an immediate membrane depolarization in 8 out of 10 cells (Fig. 7), from an average resting membrane potential of 45.2 ± 5.4 mV to a slightly depolarized potential of 37.8 ± 5.9 mV (10 cells, P < 0.003). In contrast, in PASMCs pretreated with 50 µM ryanodine for 15 min to inhibit Ca²⁺ sparks, application of caffeine did not alter membrane potential in all six cells tested (before caffeine: 38.5 ± 5.8 mV; with caffeine: 40.3 ± 6.0 mV). These results suggested that activation of Ca²⁺ sparks in PASMCs could lead to membrane depolarization, as opposed to the hyperpolarization observed previously in systemic arterial smooth muscle cells (23, 27, 29, 39, 40, 43).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Effects of enhancing Ca2+ spark activity by subthreshold concentration (0.5 mM) of caffeine on membrane potential (MP) of PASMCs in the absence (A) and presence (B) of 50 µM ryanodine. Representative traces of membrane potentials measured by the perforated-patch technique are on left, and bar graphs of averaged membrane potential before and during application of caffeine are on right. * Significant difference from control tested by paired t-test.

Vasoconstrictors and Ca²+ sparks. Because PASMCs are under the influences of a wide variety of vasoactive factors, some of these vasoactive factors may exert their effects in part by modulating Ca²⁺ sparks. To test this possibility, the effects of two vasoconstricting agents, NE and ET-1, on Ca²⁺ sparks were examined. Application of 10 nM NE (Fig. 8A) elicited a global increase in [Ca²⁺]_i; however, under steady-state conditions, there was a decrease in the spark frequency from a control of 0.39 ± 0.09 s¹ (40 cells, 151 sparks) to 0.17 ± 0.06 s¹ (30 cells, 53 sparks, P = 0.053). There was also a slight increase in the amplitude (control = 0.45 ± 0.02; NE = 0.55 ± 0.03, P < 0.005) and the size (control = 1.60 ± 0.04 µm; NE = 1.86 ± 0.1 µm, P < 0.005) but no significant change in the duration (control = 31.3 ± 1.5 ms; NE = 36.5 ± 3.5 ms) of Ca²⁺ sparks. In contrast, 3 nM ET-1 (Fig. 8B) caused a dramatic three- to fourfold increase in spark frequency from 0.48 ± 0.07 s¹ (61 cells, 293 sparks) to 1.77 ± 0.16 s¹ (61 cells, 1,080 sparks, P < 0.001), in addition to an increase in global [Ca²⁺]. The Ca²⁺ sparks recorded in the presence of ET-1 had a smaller F/F₀ (control = 0.425 ± 0.01; ET-1 = 0.33 ± 0.004, P < 0.001) and a longer duration (control = 41.5 ± 2.0 ms; ET-1 = 50.9 ± 1.0 ms, P < 0.001) but were of similar size (control = 1.77 ± 0.4 µm; ET-1 = 1.75 ± 0.03 µm). These results suggest that PASMC Ca²⁺ are modulated differentially by various vasoconstrictors independent of the agonist's effects on global [Ca²⁺]. In the presence of NE or ET-1, large global increases in [Ca²⁺] or Ca²⁺ waves occurred occasionally, and those images were excluded from Ca²⁺ sparks analyses.

View larger version (48K): [in this window] [in a new window]   Fig. 8.   Effects of 10 nM norepinephrine (NE; A) and 3 nM endothelin-1 (ET-1; B) on global Ca2+ concentration ([Ca2+]) and spark frequency in PASMCs. Top: representative line-scan images in the absence (left) and presence (right) of the agonist. Bottom: averaged time courses of changes in global [Ca2+] induced by NE (23 cells) and ET-1 (30 cells; left) and bar graphs of averaged spark frequency before and during application of the agonists (right). * Significant difference from control. #P = 0.53.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Pulmonary vs. cardiac sparks. In the present study, we have provided definitive evidence showing that Ca²⁺ sparks are present in rat intralobar PASMCs. The biophysical characteristics of these pulmonary Ca²⁺ sparks are first evaluated with those of cardiac Ca²⁺ sparks as the benchmark for comparison (12). The major differences between spontaneous Ca²⁺ sparks of PASMCs and cardiac myocytes were the significantly lower spark frequency and amplitude in PASMCs. The 70-80% lower spark frequency in PASMCs is likely related to a lower number of individual or clustered RyRs. In cardiac myocytes, diadic junctions are abundant, covering ~50% of the cytoplasmic surface of the transverse tubules (42), and the number of RyRs is estimated to be as high as 10⁶/cell (12). In contrast, RyRs are distributed diffusely in vascular smooth muscle, associating with the sparsely located peripheral junctions and the mesh-like central SR network (33). Radioligand binding studies have shown that the number of RyRs per gram of tissue is several times lower in smooth muscle than in cardiac muscle (38, 66). Electron micrographs have also revealed that the density of "foot processes" (RyRs) in the diadic junctions of arterial smooth muscle is less than in striated muscles (33), suggesting that there are fewer RyRs within a release unit. Moreover, electron-probe X-ray microanalyses have indicated that SR Ca²⁺ content is less in vascular smooth muscle than in cardiac muscle (31, 32). The combination of fewer RyRs and lower SR Ca²⁺ content in PASMCs could restrict the activity of RyRs and the amount of Ca²⁺ released from an SR Ca²⁺ release unit, resulting in lower spark frequency and amplitude (50). Furthermore, differential modulation of basal RyR activity and open time duration by kinase-dependent processes (7, 44) might also contribute to the differences in the frequency and amplitude of PASMC and cardiac sparks, even though the RyR isoforms involved are presumably the same.

PASMCs vs. other smooth muscle cells. Even though Ca²⁺ sparks have not been studied in detail in PASMCs (30), they have been examined in a variety of vascular smooth muscle cells from the cerebral arteriole (28, 35, 40), coronary artery (16), portal vein (2, 39), and mesenteric artery (6), as well as nonvascular smooth muscle cells from the trachea (41, 72), ileum (6, 21), and urinary bladder (23). Similar to our results in rat PASMCs, resting spontaneous spark frequency is generally low in smooth muscle cells. It ranges between 0.1 and 0.4 s¹ [with the exception of Löhn et al. (35), ~0.9 s¹; see Refs. 7, 39, 44] when measured using a scan line of length similar to our study and between 0.1 and 0.7 cells/s when recorded using high-speed frame scanning (19, 23, 28, 30, 41, 43). A somewhat higher spark frequency has been recorded in intact mesenteric arteries (0.025 µm/s; see Ref. 37). In addition, Ca²⁺ spark frequency may be further enhanced by an increase in intraluminal pressure, as reported in cerebral artery preparations (40). The spark frequency (~0.3 s¹ or 0.01 µm/s) recorded in our rat PASMCs is well within the range reported in the above-mentioned studies, suggesting that the number of Ca²⁺ release sites and the activity of RyRs are similar in PASMCs and other systemic smooth muscle cells.

Origin of spontaneous Ca²+ sparks. Consistent with previous studies in other cell types (9, 12, 27, 40), our results indicate that spontaneous Ca²⁺ sparks in rat PASMCs originate exclusively from ryanodine-sensitive SR Ca²⁺ stores. This is based on evidence that 1) ryanodine concentration dependently abolished spontaneous Ca²⁺ sparks, and 2) enhancement of RyR activity using a subthreshold concentration of caffeine increased Ca²⁺ spark frequency, an effect reversed by ryanodine. The IP₃-sensitive Ca²⁺ store is supposed to be the major intracellular Ca²⁺ store in smooth muscle cells (63, 66), and local Ca²⁺ release events (known as "Ca²⁺ puffs") originating from IP₃ receptors have been identified in nonvascular smooth muscle cells (5). The finding that ryanodine completely abolishes spontaneous Ca²⁺ sparks and that 2-APB or xestospongin C does not significantly alter spark frequency suggests that IP₃-sensitive Ca²⁺ stores do not contribute to the spontaneous local Ca²⁺ release in PASMCs under resting conditions. However, enhanced IP₃ production during an agonist stimulation in smooth muscle may elicit additional local Ca²⁺ release events via IP₃ receptors in the form of Ca²⁺ puffs or Ca²⁺ waves (5, 30), depending on the spatial association, activation, and coordination of IP₃ receptor release channels (8). We noted that a higher concentration of ryanodine (50 µM) was required to completely block PASMC sparks. This may be because of the lower activity of RyRs in resting PASMCs than in cardiac myocytes, since ryanodine blocks the receptors only at the open state. A similar high concentration of ryanodine was found necessary to block ryanodine-sensitive stores in other smooth muscle preparations (30).

Sarcolemmal Ca²+ channel activation and Ca²⁺ sparks. In cardiac myocytes, L-type Ca²⁺ channels are closely associated with and are functionally coupled to a cluster of RyRs, forming a Ca²⁺ release unit (52, 53). Upon stimulation, Ca²⁺ influx through these Ca²⁺ channels increases local [Ca²⁺] in the junctional clefts, activating RyRs via the Ca²⁺-induced Ca²⁺ release mechanism and generating Ca²⁺ sparks (59). In quiescent cardiac cells, however, the generation of spontaneous sparks does not appear to require Ca²⁺ influx through L-type Ca²⁺ channels (12). In our experiments, the occurrence of Ca²⁺ spark was not significantly affected by nifedipine, suggesting that spontaneous Ca²⁺ sparks in resting PASMCs are not triggered by an L-type Ca²⁺ current but are initiated by the spontaneous stochastic activity of RyRs instead. This concurs with previous observations in cardiac and other smooth muscle cells (2, 6, 14, 21, 39-41, 60).

Physiological functions of Ca²+ sparks in PASMCs. Despite the similarity in their basic properties, our results suggest that the physiological functions of Ca²⁺ sparks may be quite different in pulmonary and systemic vascular myocytes. In systemic vascular smooth muscle cells, it has been proposed that Ca²⁺ sparks modulate membrane potential by activating K_Ca channels to generate STOCs, leading to membrane hyperpolarization, closure of L-type Ca²⁺ channels, decreased [Ca²⁺]_i, and vasodilation (23, 27, 29, 39, 40, 43). In contrast, we found that activation of Ca²⁺ sparks in PASMCs by a subthreshold concentration of caffeine elicited a small but consistent membrane depolarization, which was completely abolished by ryanodine. This raises the possibility that sparks may contribute to vasoconstriction, rather than vasorelaxation, in PASMCs. The disparity from systemic vessels could be because of a diminished influence of K_Ca channels on membrane potential in adult PASMCs. Previous studies showed that inhibition of K_Ca channels with charybdotoxin or tetraethylammonium had no effect on resting membrane potential in adult rat PASMCs (1, 56, 68), resting tension of isolated vessels (67), or baseline perfusion pressure in isolated perfused lungs (22). Recent developmental studies have shown that Ca²⁺ sparks and STOCs are very active in fetal PASMCs (45, 46). However, the occurrence of STOCs, the responses of [Ca²⁺]_i to iberiotoxin, and the expression of K_Ca channel proteins and mRNA in distal PASMCs diminish with maturation (46, 48). The lower K_Ca channel expression in adult PASMCs may therefore compromise the ability of the Ca²⁺ spark to induce membrane hyperpolarization. However, K_Ca currents have been unequivocally demonstrated in adult PASMCs of different species, including rats (57, 70). It is possible that K_Ca channels, in addition to their lower channel protein expression, are coupled less efficiently to RyRs in adult PASMCs. Moreover, prominent Cl_Ca currents (STICs) have been found in PASMCs (69). Activation of Cl_Ca channels similar to those observed in tracheal smooth muscle cells (72), and, perhaps, modulation of other Ca²⁺-sensitive ion transport processes [such as inhibition of voltage-gated K⁺ (K_V) channels (18) and Ca²⁺ extrusion by Na⁺/Ca²⁺ exchange (4)] by Ca²⁺ sparks might override the compromized hyperpolarization of K_Ca channels leading to membrane depolarization. However, the exact contributions of K_Ca, Cl_Ca, and other channels or transporters to the membrane depolarization induced by Ca²⁺ sparks in PASMCs require future investigation. Additionally, it has been shown that expressions of K_V and Cl_Ca channels are more prominent and K_Ca channel expression is much reduced in PASMCs of the distal resistant artery (1, 61). Because our PASMCs were isolated from relatively proximal arteries, it is possible that Ca²⁺ spark may elicit an even greater depolarization in distal pulmonary arteries.