Ca2+ signalling by endothelin receptors in rat and human cultured airway smooth muscle cells

1. The aim of the current study was to characterize the ET receptor subtypes in cultured airway smooth muscle cells derived from rat trachea and human bronchus using radioligand binding techniques and to investigate the coupling of ET receptors to intracellular calcium signalling mechanisms using endothelin receptor-selective agonists (sarafotoxin S6c) and antagonists (BQ-123, BQ-788) and digital image fluorescence microscopy.
2. Confluent rat airway smooth muscle cells in culture possessed a mixed ET receptor population (30% ET_A : 70% ET_B), with a density of approximately 3400±280 ET_A and 8000±610 ET_B receptors/cell (n=3 experiments). The density of ET_B, but not ET_A receptors increased substantially in serum-containing medium. However, a 2-day period of serum deprivation, which inhibited cellular growth, substantially reduced ET_B receptor density such that the ET receptor subtype proportions were approximately equal (55% ET_A; 45% ET_B) and similar to those previously observed in intact rat tracheal smooth muscle.
3. Challenge of rat airway smooth muscle cells in culture with endothelin-1 elicited a concentration-dependent biphasic increase in [Ca²⁺]_i (EC₅₀: 16 nM), that comprised an initial transient peak [Ca²⁺]_i increase (typically 350 nM) followed by a modest sustained component. The endothelin-1-induced biphasic [Ca²⁺]_i increase was primarily due to ET_A receptor activation, although a modest and inconsistent ET_B response was observed. The ET_A-mediated [Ca²⁺]_i increase was due primarily to the mobilization of IP₃-sensitive and to a lesser extent ryanodine-sensitive intracellular calcium stores. In contrast, ET_B receptor activation was exclusively coupled to extracellular calcium influx.
4. Somewhat surprisingly, human airway smooth muscle cells in culture contained a homogeneous population of ET_A receptors at a density of 6100±800 receptors cell⁻¹ (n=3 experiments). Serum deprivation was without effect on either ET receptor subtype proportion or ET_A receptor density. Challenge of human airway smooth muscle cells with endothelin-1 provoked a concentration-dependent increase in [Ca²⁺]_i (EC₅₀: 15 nM), with a peak [Ca²⁺]_i increase to greater than 700 nM. Furthermore, the ET_A-mediated calcium response in these human airway smooth muscle cells in culture was entirely dependent upon the mobilization of calcium from intracellular stores.
5. In summary, rat cultured tracheal airway smooth muscle cells contained both ET_A and ET_B receptors. ET_A receptors, the numbers of which remained constant during cell growth, were linked to the release of Ca²⁺ from intracellular stores and a strong rise in [Ca²⁺]_i in the majority of airway smooth muscle cells. In stark contrast, the numbers of ET_B receptors increased significantly during cell growth, an effect that was diminished substantially by incubation in serum-free medium. Moreover, despite the greater number of ET_B receptors, their activation in a small number of airway smooth muscle cells produced only a weak rise in [Ca²⁺]_i, which appeared to be attributable to the influx of extracellular Ca²⁺. In contrast, the populations of ET receptors and their linkage to [Ca²⁺]_i were markedly different in the human cultured airway smooth muscle cells used in the current study compared to that previously observed in intact human isolated bronchial smooth muscle.

     

Ca2+ signalling by P2Y receptors in cultured rat aortic smooth muscle cells

Background and purpose:

P2Y receptors evoke Ca²⁺ signals in vascular smooth muscle cells and regulate contraction and proliferation, but the roles of the different P2Y receptor subtypes are incompletely resolved.

Experimental approach:

Quantitative PCR was used to define expression of mRNA encoding P2Y receptor subtypes in freshly isolated and cultured rat aortic smooth muscle cells (ASMC). Fluorescent indicators in combination with selective ligands were used to measure the changes in cytosolic free [Ca²⁺] in cultured ASMC evoked by each P2Y receptor subtype.

Key results:

The mRNA for all rat P2Y receptor subtypes are expressed at various levels in cultured ASMC. Four P2Y receptor subtypes (P2Y1, P2Y2, P2Y4 and P2Y6) evoke Ca²⁺ signals that require activation of phospholipase C and comprise both release of Ca²⁺ from stores and Ca²⁺ entry across the plasma membrane.

Conclusions and implications:

Combining analysis of P2Y receptor expression with functional analyses using selective agonists and antagonists, we isolated the Ca²⁺ signals evoked in ASMC by activation of P2Y1, P2Y2, P2Y4 and P2Y6 receptors.     

Ca2+ localization and sensitivity in vascular smooth muscle

An increase in cytosolic Ca2+ level ([Ca2+]i) is a prerequisite for smooth muscle contraction. Simultaneous measurements of [Ca2+]i and muscle tension give direct information on the Ca2+ regulation of smooth muscle. The photoprotein aequorin and the fluorescent Ca2+ indicator fura-2 are widely used for this purpose. Although there are some inconsistencies between the results obtained with these two indicators, comparison between [Ca2+]i and muscle tension in vascular smooth muscle indicates that stimulation of alpha-adrenoceptors increases, whereas stimulation of beta-adrenoceptors decreases, both the Ca2+ sensitivity of contractile elements and [Ca2+]i. Thus, as Hideaki Karaki explains, contractility of vascular smooth muscle may be regulated not only by [Ca2+]i but also by the Ca2+ sensitivity of the contractile elements.     

Expression and characterization of the human alpha 2B-adrenoceptor in a vascular smooth muscle cell line

A vascular smooth muscle cell line stably expressing the human alpha 2B-adrenoceptor at a density of 1.5 pmol/mg membrane protein was generated by transfection of rat A7r5 cells. [35S]GTPgammaS binding experiments and [3H]thymidine incorporation experiments indicated that the expressed receptors were functional, had the expected pharmacological characteristics and efficiently stimulated smooth muscle cell proliferation. Confocal fluorescence microscopy was used to visualize alpha2B-adrenoceptors in A7r5-alpha 2B cells and indicated that the receptors were mainly localized in the plasma membrane. The expression of the smooth muscle-specific marker alpha-actin was similar in transfected A7r5-alpha 2B cells and in non-transfected A7r5 wild-type cells. The generated A7r5-alpha 2B cell line will be a useful tool for studying the function and regulation of alpha 2B-adrenoceptors in vascular smooth muscle cells.     

Troglitazone and pioglitazone attenuate agonist-dependent Ca2+ mobilization and cell proliferation in vascular smooth muscle cells.

1. The effects of troglitazone and pioglitazone on agonist-induced Ca2+ mobilization and cell proliferation were studied using fluorescent Ca2+ indicator fura-2 AM and incorporation of [3H]-thymidine in rat aortic smooth muscle cells. The patch clamp techniques were also employed. 2. Vasopressin and platelet-derived growth factor-BB (PDGF) caused a transient elevation in [Ca2+]i by Ca2+ mobilization from intracellular stores, followed by a sustained rise due to Ca2+ entry. Nicardipine partly inhibited the sustained phase, but La3+ completely abolished it. 3. Troglitazone and pioglitazone did not significantly affect the transient rise elicited by these agonists, but preferentially inhibited the sustained phase of [Ca2+]i. 4. Under voltage clamp conditions, troglitazone and pioglitazone inhibited voltage-dependent L-type Ca2+ current (ICa.L). They also inhibited nonselective cation channels (Icat) elicited by vasopressin in a concentration-dependent manner. The half maximal inhibitory concentrations of troglitazone on ICa.L and Icat were 4.6 and 5.7 microM, respectively. On the other hand, nifedipine and nicardipine did not inhibit Icat. 5. Vasopressin and PDGF increased incorporation of [3H]-thymidine, and nifedipine and nicardipine partly suppressed it. However, the inhibitory effects of La3+ and exclusion of extracellular Ca2+ were more potent than the Ca2+ blocking agents. Troglitazone and pioglitazone also inhibited it concentration-dependently. 6. These results suggest that troglitazone and pioglitazone preferentially inhibited agonist (vasopressin and PDGF)-induced Ca2+ entry and proliferation in rat vascular smooth muscle cells, where the inhibitory effects of thiazolidinediones on ICa.L and Icat might be partly involved. Thus, thiazolidinediones may exert hypotensive and antiatherosclerotic effects.     

P2Y receptor-mediated Ca2+ signalling in cultured rat aortic smooth muscle cells.

1. ATP, UTP, ADP and ADP-beta-S elicited Ca2+ -signals in cultured aortic smooth muscle cells although ADP, UDP and ADP-beta-S gave approximately 40% of the maximal response seen with ATP and UTP. Adenosine, AMP or alpha,beta-methylene-ATP had no effect. These responses were attributed to P2Y2/4 and P2Y1 receptors, which we assumed could be selectively activated by UTP and ADP-beta-S respectively. 2. The response to UTP was reduced (approximately 50%) by pertussis toxin, whilst this toxin had no effect upon the response to ADP-beta-S. This suggests P2Y2/4 receptors simultaneously couple to pertussis toxin-sensitive and -resistant G proteins whilst P2Y1 receptors couple to only the toxin-resistant proteins. 3. Repeated stimulation with UTP or ADP-beta-S caused desensitization which was potentiated by 12-O-tetradecanoyl phorbol-13-acetate (TPA) and attenuated by staurosporine. 4. TPA completely abolished sensitivity to ADP-beta-S but the response to UTP had a TPA-resistant component. In pertussis toxin-treated cells, however, TPA could completely abolish sensitivity to UTP and so the TPA-resistant part of this response seems to be mediated by pertussis toxin-sensitive G proteins. 5. Loss of sensitivity to UTP did not occur when pertussis toxin-treated cells were repeatedly stimulated with this nucleotide, suggesting that pertussis toxin-sensitive G proteins mediate this effect. The toxin did not, however affect desensitization to ADP-beta-S.     

Intracellular alkalinization augments capacitative Ca2+ entry in vascular smooth muscle cells

Agonist-induced Ca2+ influx of vascular smooth muscle cells is thought to be triggered by depletion of intracellular Ca2+ stores. This study investigated the effects of intracellular alkalinization on capacitative Ca2+ entry in A7r5 rat aortic smooth muscle cells. Intracellular alkalinization was induced by NH(4)Cl. Transplasmalemmal Ca2+ influx due to Ca2+ store depletion induced by thapsigargin, which was abolished by pretreatment of the cells with SKF-96365 but not affected by that with verapamil, was significantly increased by pretreatment with NH(4)Cl. Neither 5-hydroxytryptamine-induced inositol monophosphate accumulation nor intracellular Ca2+ release from its stores was affected by NH(4)Cl. These results suggest that intracellular alkalinization acts on the process(es) after depletion of Ca2+ stores and facilitates capacitative Ca2+ entry in vascular smooth muscle cells.     

Muscarinic receptors in human airway smooth muscle are coupled to phosphoinositide metabolism

In the present study we investigated whether muscarinic receptors in human airway smooth muscle are coupled to phosphoinositide metabolism as a possible transduction mechanism of contraction. Using isolated bronchial smooth muscle preparations, we found that the muscarinic agonist methacholine caused a time- and concentration-dependent accumulation of inositol phosphates in the presence of lithium, an effect which could be inhibited by atropine. Apart from its physiological significance, this finding may have great relevance for the biochemical investigation of cholinergic hyperresponsiveness in the airways of asthmatic patients.     

The long-term inhibitory effect of a Ca2+ channel blocker, nisoldipine, on cytosolic Ca2+ and contraction in vascular smooth muscle.

The mechanism of the long-term inhibitory effect of a dihydropyridine Ca2+ channel blocker, nisoldipine, on contraction and cytosolic Ca2+ level ([Ca2+]i) was examined in isolated rat aorta. Nisoldipine inhibited the [Ca2+]i and muscle tension induced by high K+. The inhibitory effects were antagonized by a Ca2+ channel activator, 100 nM Bay k8644, and by a high concentration of Ca2+ (6.5 mM). Ultraviolet light, which has been shown to decompose dihydropyridines, attenuated the effects of nisoldipine. After nisoldipine had been removed from muscle bath, the inhibitory effect faded away slowly. The residual inhibitory effects on [Ca2+]i and muscle tension were antagonized by Bay k8644, high Ca2+ and ultraviolet light. These results suggest that the inhibitory effect of nisoldipine is caused by a decrease in [Ca2+]i as a result of inhibition of L-type Ca2+ channels, and that the residual inhibitory effects are caused by the same mechanism as the inhibitory effects of nisoldipine, namely the tight binding of nisoldipine to Ca2+ channels even after washout.     

Direct block of Ca2+ channels by calmidazolium in cultured vascular smooth muscle cells.

We investigated the action of calmidazolium (CMZ), an inhibitor of calmodulin (CaM), on the L-type Ca2+ currents (ICa(L)) of cultured vascular smooth muscle (VSM) cells (A7r5 cell line), by using the whole-cell voltage-clamp method. All experiments were conducted at room temperature (24-25 degrees C). The peak IBa (Ca2+ channel current with 5 mM Ba2+ as charge carrier) was evoked every 15 s by a test potential to +10 mV from a holding potential of -60 mV. To elevate intracellular free Ca2+ concentration ([Ca]i) to pCa 6.5, the pipette solution contained a Ca2+-EGTA buffer (pCa 6.5) to allow equilibration with the cells. Bath application of 1 microM CMZ reduced the peak amplitude of IBa to 36.7+/-4.9% (n = 8); maximal effect occurred within 7-8 min. Peak IBa continued to decrease even after washing out the CMZ. Recovery of IBa was not observed even after 10 min of washout. Even in presence of an peptide inhibitor of CaM-dependent protein kinase-II (5.2 microM) in the pipette solution, CMZ inhibited IBa to 27.8 +/-5.3% (n = 7). To exclude the possibility that other Ca2+/ CaM-dependent kinases and phosphatases may regulate Ca2+ channel activity, we examined the effect of CMZ on IBa when [Ca]i was reduced by use of Ca2+/EGTA-buffered pipette solutions. At pCa approximately equal to 10 (10 mM EGTA and only contaminant Ca2+), CMZ inhibited IBa to 33.4+/-5.9% (n = 14) with a median inhibitory concentration (IC50) value of 0.29 microM. The activation curve (pCa approximately equal to 10) was shifted in the positive direction by 6.3 mV; the inactivation curve was shifted in the negative direction by 5.0 mV. CMZ decreased IBa progressively during repetitive step depolarizations. CMZ did not slow the rate of recovery from inactivation. In conclusion, CMZ inhibits Ca2+ channel current in a use-dependent manner. This inhibition is independent of CaMK-II and other Ca2+/CaM-dependent pathways. Therefore it is likely due to direct blockade of Ca2+ channels by CMZ. CMZ may reduce the outer surface charge and block the open state of the Ca2+ channels.     

吸烟、硝酸甘油对动脉粥样硬化家兔血管平滑肌细胞钙浓度的影响

目的研究吸烟、硝酸甘油对动脉粥样硬化血管平滑肌细胞胞内游离钙浓度([Ca2+]i)的影响,探讨吸烟对动脉粥样硬化形成及硝酸甘油的生物效应的作用。方法复制家兔动脉粥样硬化模型,分离血管平滑肌细胞。Fluo-3/AM负载细胞,应用流式细胞仪检测血管平滑肌细胞[Ca2+]i,激光扫描共聚焦显微系统测定单个血管平滑肌细胞内Ca2+的时空变化。结果各组动物主动脉壁均见到程度不同的动脉粥样硬化斑块形成。动脉粥样硬化家兔血管平滑肌细胞[Ca2+]i明显升高(48.45±5.31,与生理盐水对照组38.09±2.57比较,P<0.01);吸烟能增加动脉粥样硬化家兔血管平滑肌细胞[Ca2+]i(56.48±2.99,与单纯动脉粥样硬化组48.45±5.31比较,P<0.01);硝酸甘油则能明显降低动脉粥样硬化家兔血管平滑肌细胞[Ca2+]i(41.91±3.16,与单纯动脉粥样硬化组48.45±5.31比较,P<0.05);吸烟能明显抑制硝酸甘油降低血管平滑肌细胞[Ca2+]i的作用(47.99±5.10,与硝酸甘油组41.91±3.16比较,P<0.05)。结论吸烟能明显增加动脉粥样硬化血管平滑肌细胞[Ca2+]i,硝酸甘油则能明显降低动脉粥样硬化血管平滑肌细胞[Ca2+]i,吸烟能抑制硝酸甘油的生物学效应。     

Different pathways for Ca2+ influx and intracellular release of Ca2+ mediated by muscarinic receptors in ileal longitudinal smooth muscle.

Muscarinic receptor-mediated elevations in intracellular Ca2+ concentration ([Ca2+]i) in the longitudinal smooth muscle of guinea pig ileum were studied by the use of fura-2 fluorescence. Dose-response analysis indicated a difference in the potencies of carbachol (CCh) to increase [Ca2+]i in the presence and absence of extracellular Ca2+. For the increase in [Ca2+]i due to Ca2+ release from intracellular stores in the absence of extracellular Ca2+, the ED50 value of CCh was 3 x 10(-5) M. On the other hand, in the presence of Ca2+, the ED50 value was 2.5 x 10(-7) M, indicating that a low concentration of CCh (less than 10(-7) M) caused influx of extracellular Ca2+ without Ca2+ release. Oxotremorine and pilocarpine induced Ca2+ influx, but were less potent inducers of Ca2+ release. CCh also stimulated the formation of inositol trisphosphates (IP3) with an ED50 value of (4.5 x 10(-5) M), which was similar to that for Ca2+ release from intracellular stores. Treatment of the smooth muscle with neomycin (1 mM), a phospholipase C inhibitor, abolished both CCh-induced IP3 formation and Ca2+ release from intracellular stores, but did not affect CCh-induced Ca2+ influx. These results suggest that the pathway for muscarinic stimulation of Ca2+ influx through plasma membranes is different from that for Ca2+ release from intracellular stores, which seems to be coupled with IP3 formation.     

Serotonin-2 receptors coupled to phosphoinositide hydrolysis in a clonal cell line

A permanent line of cells has been established from the transplantable rat pituitary tumor 7315a. P11 cells have been cloned repeatedly, and after more than 60 passages their growth and characteristics are stable. Results of radioligand binding studies with 125I-lysergic acid diethylamide (125I-LSD) indicate that P11 cells express serotonin-2 (5-HT2) receptors. Analysis of the binding of 125I-LSD to membranes prepared from P11 cells revealed the presence of a single class of high affinity sites (Kd = 1.6 nM; Bmax = 211 fmol/mg of protein). The pharmacological profile of the inhibition of the binding of 125I-LSD by a panel of drugs was consistent with the expected profile of these drugs at 5-HT2 receptors. The affinity of the site for serotonin was in the low micromolar range and was decreased by GTP. Phosphoinositide hydrolysis in P11 cells, measured in the presence of lithium, was stimulated by serotonin. Increasing concentrations of the 5-HT2-selective antagonist ketanserin blocked phosphoinositide hydrolysis stimulated by serotonin, and Schild analysis was consistent with a simple competitive interaction. The Ki for ketanserin derived from Schild analysis was comparable to the Ki for ketanserin at the binding site for 125I-LSD. These results suggest that stimulation of phosphoinositide hydrolysis in P11 cells by serotonin is mediated by 5-HT2 receptors. Pretreatment of P11 cells with pertussis toxin caused ADP-ribosylation of Gi and Go, but did not affect the ability of serotonin to stimulate phosphoinositide hydrolysis. Therefore, the guaninine nucleotide-binding protein involved in the coupling of 5-HT2 receptors to phospholipase C in P11 cells is unlikely to be either Gi or Go. P11 cells expressing 5-HT2 receptors coupled to phosphoinositide hydrolysis will be a useful model system for future studies of the regulation and function of 5-HT2 receptors on cultured cells.     

Diverse effects of monensin on capacitative Ca2+ entry and release of stored Ca2+ in vascular smooth muscle cells

The effects of monensin, an activator of Na(+)/H(+) exchanger (NHE), on capacitative Ca(2+) entry (CCE) were investigated using A7r5 cells. Capacitative Ca(2+) entry was induced by elevation of extracellular Ca(2+) concentrations of A7r5 cells in which stored Ca(2+) had been depleted by previous administration of thapsigargin. Capacitative Ca(2+) entry was abolished by pretreatment of the cells with SKF-96365 (1-[beta-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride) but was not affected by pretreatment with verapamil. Monensin significantly increased capacitative Ca(2+) entry. On the other hand, 5-hydroxytryptamine-induced inositol monophosphate accumulation and subsequent intracellular Ca(2+) release from its stores were significantly inhibited by monensin, while thapsigargin-induced Ca(2+) release was not affected by monensin. These results suggest that monensin has diverse actions on capacitative Ca(2+) entry and agonist-induced release of stored Ca(2+) in vascular smooth muscle cells.     

Ginsenoside-Rd from panax notoginseng blocks Ca2+ influx through receptor- and store-operated Ca2+ channels in vascular smooth muscle cells

Previously, it was found that total saponins from panax notoginseng inhibited Ca2+ influx coupling to activation of alpha1-adrenoceptor. This study was designed to investigate the effects of ginsenoside-Rd from total saponins of panax notoginseng on receptor-operated (ROCC) and store-operated (SOCC) Ca2+ channels in vascular smooth muscle cells using fura-2 fluorescence, whole cell patch clamp ion channel recording, radio-ligand-receptor binding, 45Ca2+ radio-trace and organ bath techniques. It was found that ginsenoside-Rd reduced phenylephrine-induced contractile responses and Ca2+ influx in normal media without significant effect on these responses in Ca2+ -free media. Ginsenoside-Rd also decreased phenylephrine- and thapsigargin-induced inward Ca2+ currents, and attenuated thapsigargin- and 1-oleoy-2-acetyl-sn-glycerol (OAG)-induced cation entries that are coupled to ROCC and SOCC respectively. Ginsenoside-Rd failed to inhibit KCl-induced contraction of rat aortal rings and Ca2+ influx, and did not alter voltage-dependent inward Ca2+ current (VDCC) which was blocked by nifedipine. Also, ginsenoside-Rd did not change binding site and affinity of [3H]-prazosin for alpha1-adrenoceptor in the vascular plasma membrane. These results suggest that ginsenoside-Rd, as an inhibitor, remarkably inhibits Ca2+ entry through ROCC and SOCC without effects on VDCC and Ca2+ release in vascular smooth muscle cells.     

Role of Ca2+ in vascular smooth muscle contractions induced by Phoneutria nigriventer spider venom.

Phoneutria nigriventer venom (PNV) contracts vascular tissues and increases arterial blood pressure. This study aimed to investigate the mechanisms involved on PNV-induced contractions of rabbit mesenteric and celiac arteries. Strips of mesenteric and celiac arteries were suspended in a cascade system and superfused with warmed and oxygenated Krebs solution. PNV was dialyzed in order to exclude the participation of biogenic amines in the contractions elicited by the venom. Noradrenaline (NA, 30-300 pmol), PNV (1-10 microg), Bay K-8644 (0.3-3 nmol) and KCl (10-100 micromol) dose-dependently contracted the preparations. Ca(2+)-free solution reduced by 38 and 83% the PNV-induced contractions of mesenteric and celiac arteries, respectively. Subsequent infusion of EGTA (0.2 mM) suppressed the residual contractions. Nifedipine (1 microM) and verapamil (10 microM) abolished PNV- and Bay K-8644-evoked contractions, whereas those induced by NA were reduced to a lesser extent. Lanthanum chloride (0.2 mM) inhibited by 75-90% the mesenteric and celiac contractions mediated by PNV. Caffeine (2 mM) fully blocked contractions induced by NA (95% mean inhibition), but only partly reduced those induced by PNV (35% mean inhibition). Ryanodine (10 microM) inhibited by 50% the contractions evoked by NA, but had no effect on the PNV-induced contractions in both tissues. Our findings indicate that PNV contracts vascular smooth muscle mainly due to increased influx of Ca(2+) from extracellular sources.     

Endothelin dissociates muscle tension from cytosolic Ca2+ in vascular smooth muscle of rat carotid artery

Endothelin induced rapid increase followed by a decrease in cytosolic Ca2+ [( Ca2+]i) and a slow increase in muscle tension in the vascular smooth muscle strip of rat carotid artery. Thus, the endothelin-induced contraction was smaller, and it became gradually greater than high K-induced contraction at a given [Ca2+]i. In Ca2+-free solution, endothelin induced a transient increase in [Ca2+]i and a sustained contraction. These results suggest that endothelin-induced contraction is due to the increase in [Ca2+]i, the time-dependent change in Ca2+-sensitivity of contractile elements, and the mechanism which is independent of the increment in [Ca2+]i.     

Sildenafil inhibits human pulmonary artery smooth muscle cell proliferation by decreasing capacitative Ca2+ entry

Ca(2+) is a pivotal signal in human pulmonary artery smooth muscle cells (PASMCs) proliferation. Capacitative Ca(2+) entry (CCE) via the store-operated channel (SOC), which encoded by the transient receptor potential (TRP) gene, is an important mechanism for regulating intracellular Ca(2+) concentration ([Ca(2+)](i)) in PASMCs. Sildenafil, a potent type 5 nucleotide-dependent phosphodiesterase (PDE) inhibitor, has been proposed as a therapeutic tool to treat or prevent pulmonary arterial hypertension (PAH); however, the mechanism of its antiproliferative effect on PASMCs remains unclear. This study was designed to investigate the possible antiproliferative mechanism of sildenafil on human PASMCs, namely, its effect on the Ca(2+)-signal pathway. Cultured normal PASMCs were treated with endothelin-1 (ET-1) or ET-1 plus sildenafil separately. Cell number and viability were determined with a hemocytometer or MTT assay. [Ca(2+)](i) was measured by loading PASMCs with fura 2-AM. Expression of the TRPC1 gene and protein was detected by RT-PCR and Western blot, respectively. The results show that sildenafil dose-dependently inhibited the proliferation of PASMCs, the enhancement of basal [Ca(2+)](i) level, increase of CCE, and upregulation of TRPC expression induced by ET-1. These results suggest that sildenafil potently inhibits ET-1-induced PASMCs proliferation and downregulation of CCE and TRPC expression may be responsible for its antiproliferative effect.