Platelet-derived growth factor-BB induces pulmonary venous smooth muscle cells proliferation by upregulating calcium sensing receptor under hypoxic conditions

Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling, which exists in both pulmonary arteries and pulmonary veins. Pulmonary vascular remodeling stems from excessive proliferation of pulmonary vascular myocytes. Platelet-derived growth factor-BB (PDGF-BB) is a vital vascular regulator whose level increases in PH human lungs. Although the mechanisms by which pulmonary arterial smooth muscle cells respond to PDGF-BB have been studied extensively, the effects of PDGF-BB on pulmonary venous smooth muscle cells (PVSMCs) remain unknown. We herein examined the involvement of calcium sensing receptor (CaSR) in PDGF-BB-induced PVSMCs proliferation under hypoxic conditions. In PVSMCs isolated from rat intrapulmonary veins, PDGF-BB increased the cell number and DNA synthesis under normoxic and hypoxic conditions, which was accompanied by upregulated CaSR expression. The influences of PDGF-BB on proliferation and CaSR expression in hypoxic PVSMCs were greater than that in normoxic PVSMCs. In hypoxic PVSMCs superfused with Ca²⁺-free solution, restoration of extracellular Ca²⁺ induced an increase of [Ca²⁺]_i, which was significantly smaller than that in PDGF-BB-treated hypoxic PVSMCs. The positive CaSR modulator spermine enhanced, whereas the negative CaSR modulator NPS2143 attenuated, the extracellular Ca²⁺-induced [Ca²⁺]_i increase in PDGF-BB-treated hypoxic PVSMCs. Furthermore, the spermine enhanced, whereas the NPS2143 inhibited, PDGF-BB-induced proliferation in hypoxic PVSMCs. Silencing CaSR with siRNA attenuated the extracellular Ca²⁺-induced [Ca²⁺]_i increase in PDGF-BB-treated hypoxic PVSMCs and inhibited PDGF-BB-induced proliferation in hypoxic PVSMCs. In conclusion, these results demonstrated that CaSR mediating PDGF-BB-induced excessive PVSMCs proliferation is an important mechanism involved in the initiation and progression of PVSMCs proliferation under hypoxic conditions.     

Chronic Hypoxia Increases TRPC6 Expression and Basal Intracellular Ca2+ Concentration in Rat Distal Pulmonary Venous Smooth Muscle

Background

Hypoxia causes remodeling and contractile responses in both pulmonary artery (PA) and pulmonary vein (PV). Here we explore the effect of hypoxia on PV and pulmonary venous smooth muscle cells (PVSMCs).

Methods

Chronic hypoxic pulmonary hypertension (CHPH) model was established by exposing rats to 10% O₂ for 21 days. Rat distal PVSMCs were isolated and cultured for in vitro experiments. The fura-2 based fluorescence calcium imaging was used to measure the basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) and store-operated Ca²⁺ entry (SOCE). Quantitative RT-PCR and western blotting were performed to measure the expression of mRNA and levels of canonical transient receptor potential (TRPC) protein respectively.

Results

Hypoxia increased the basal [Ca²⁺]_i and SOCE in both freshly dissociated and serum cultured distal PVSMCs. Moreover, hypoxia increased TRPC6 expression at mRNA and protein levels in both cultured PVSMCs exposed to prolonged hypoxia (4% O₂, 60 h) and distal PV isolated from CHPH rats. Hypoxia also enhanced proliferation and migration of rat distal PVSMCs.

Conclusions

Hypoxia induces elevation of SOCE in distal PVSMCs, leading to enhancement of basal [Ca²⁺]_i in PVSMCs. This enhancement is potentially correlated with the increased expression of TRPC6. Hypoxia triggered intracellular calcium contributes to promoted proliferation and migration of PVSMCs.     

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca²⁺]_i) and nitric oxide (NO). In order to understand how [Ca²⁺]_i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.     

Growth of aortic vascular smooth muscle cells in lowered oxygen tension

Summary Primary cultures of vascular smooth muscle cells, isolated from rat aorta, were grown under normoxic (20% O₂) and mildly hypoxic (5 % O₂) conditions. Cells from both conditions were compared for growth characteristics, morphology, protein synthesis, lysosomal enzyme activity, and oxygen consumption. In no case was a consistently significant difference observed. These observations indicate that these cells can adapt or are adapted to mildly hypoxic conditions. Moreover, these results may indicate that the culture of vascular smooth muscle cells in mild hypoxia represents a closer approximation of in vivo growth conditions for these cells.Supported by HL19242     

Osteopontin-Stimulated Vascular Smooth Muscle Cell Migration Is Mediated by β3 Integrin

Osteopontin (OPN), a 41-kDa phosphorylated glycoprotein, has been detected in rat aorta and carotid arteries, and expression of its mRNA in blood vessels is strongly increased in response to vascular injury. To investigate the potential role of OPN in vascular pathophysiology, we studied the effect of rat OPN on aortic smooth muscle cell migration and proliferation in vitro. OPN enhanced the migration of rat smooth muscle cells in a time- and concentration-dependent manner with an EC₅₀ value of 46 ± 11 nmol/liter (n = 5). The maximal increase in cell migration by OPN was 29-fold over basal levels. OPN-induced smooth muscle cell migration was inhibited in a concentration-dependent manner by the monoclonal antibody F11, which recognizes the rat integrin subunit β₃. In contrast, polyclonal antiserum recognizing the rat integrin β₁ subunit did not inhibit smooth muscle cell migration in response to OPN, but did block fibronectin-promoted migration. Moreover, OPN-induced smooth muscle cell migration was dependent on the presence of extracellular divalent cations and was significantly inhibited by anti-OPN antibodies. OPN did not stimulate [³H]thymidine incorporation into cultured smooth muscle cells, indicating that it selectively enhanced migration. In view of the pathological significance of arterial smooth muscle cell migration in the formation of intimal thickening, our results suggest that smooth muscle cell recognition of OPN, probably through the vitronectin receptor, α_vβ₃, could play a role in the cells' response to vascular injury and especially neointima formation.     

Vascular smooth muscle cells synthesize,secrete and express coagulation factor V

Expression of cellular procoagulant activity may be one of the more important responses to vascular injury. Because factor V, a coagulation cofactor in the prothrombinase complex, catalyzes the conversion of prothrombin to thrombin, it may be a key to understanding this response. Therefore, we have investigated the synthesis, secretion and expression of factor V by vascular smooth muscle cells, which proliferate at sites of vascular injury. Cultured aortic vascular smooth muscle cells constitutively secreted Factor V activity, as determined by a functional assay. Labeled factor V was immunoprecipitated from conditioned medium of [³⁵S]methionine-labeled cells, indicating that the secreted factor V was synthesized by vascular smooth muscle cells. Treatment of vascular smooth muscle cells with tunicamycin prevented secretion of factor V, suggesting that its secretion was dependent on the presence of N-linked carbohydrate. Factor V activity was also expressed on the vascular smooth muscle cell surface, as indicated by the ability of cultured cells to promote factor X_a-catalyzed prothrombin activation. These data suggest that the proliferation of smooth muscle cells in response to vascular injury may be one mechanism that links vascular disease with thrombosis.     

Sulfated glycoproteins and extracellular matrix of cultured human pulmonary endothelial cells

Endothelial cells derived from human pulmonary arteries incorporate (³H)-glucosamine and ³⁵SO₄ into glycosaminoglycans and into the carbohydrate side chains of glycoproteins. These ³H/³⁵S-carbohydrate chains were isolated from cells and culture medium after Pronase digestion. The ³H/³⁵S-glycosaminoglycans were separated from the ³H/³⁵S glycopeptides by chromatography on Sephadex G-50. The distribution of cellular glycosaminoglycans and glycopeptides indicated that 30–60% of the cellular ³⁵S-glycopeptides may be associated with the matrix components that are synthesized by the cell and attached to a plastic substratum. Human pulmonary arterial endothelial cells were grown on collagen or on a matrix derived from vascular smooth muscle cells in order to investigate how smooth muscle cell extracellular matrix components may regulate the synthesis of endothelial cell glycoconjugates. Endothelial cells grown on plastic release various proportions of the glycoconjugates they synthesize into the culture medium. However, these same cells, when grown on substratum composed of extracellular matrix materials, synthesized altered proportions of cell-associated glycosaminoglycans and reduced the levels of total glycosaminoglycans they released into the culture medium. Thus the growth of endothelial cells on a matrix of smooth muscle cell components indicates that the glycosaminoglycan materials released into the culture medium by cells grown on a plastic substratum may not be an accurate reflection of the levels or composition of extracellular matrix materials made by endothelial cells in vivo.     

Protease-Activated Receptor 2 Activation Inhibits N-Type Ca2+ Currents in Rat Peripheral Sympathetic Neurons

The protease-activated receptor (PAR)-2 is highly expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although several mechanisms have been suggested to explain PAR-2-induced hypotension, the precise mechanism remains to be elucidated. To investigate this possibility, we investigated the effects of PAR-2 activation on N-type Ca²⁺ currents (I_Ca-N) in isolated neurons of the celiac ganglion (CG), which is involved in the sympathetic regulation of mesenteric artery vascular tone. PAR-2 agonists irreversibly diminished voltage-gated Ca²⁺ currents (I_Ca), measured using the patch-clamp method, in rat CG neurons, whereas thrombin had little effect on I_Ca. This PAR-2-induced inhibition was almost completely prevented by ω-CgTx, a potent N-type Ca²⁺ channel blocker, suggesting the involvement of N-type Ca²⁺ channels in PAR-2-induced inhibition. In addition, PAR-2 agonists inhibited I_Ca–N in a voltage-independent manner in rat CG neurons. Moreover, PAR-2 agonists reduced action potential (AP) firing frequency as measured using the current-clamp method in rat CG neurons. This inhibition of AP firing induced by PAR-2 agonists was almost completely prevented by ω-CgTx, indicating that PAR-2 activation may regulate the membrane excitability of peripheral sympathetic neurons through modulation of N-type Ca²⁺ channels. In conclusion, the present findings demonstrate that the activation of PAR-2 suppresses peripheral sympathetic outflow by modulating N-type Ca²⁺ channel activity, which appears to be involved in PAR-2-induced hypotension, in peripheral sympathetic nerve terminals.     

The synthesis and biological activities of some 12-Aza-prostaglandin analogues

12-Aza-prostaglandin (PG) analogues containing the pyrrolidine-2,4-dione ring system have been synthesized from ,2-disubstituted glycine esters via cyclisation of their -ethoxycarbonylacetyl derivatives. 5-(6-Carboxyhexyl)-1-octylpyrrolidine-2,4-dione (5) had little or no PG-like activity on superfused intestinal or vascular smooth muscle preparations but it selectively antagonised smooth muscle responses to PGE₂, PGE₁, PGF_2α and PGA₂in vitro. At a concentration of 10⁻⁵ g/ml it reduced responses of the rat stomach strip to PGE₂ by over 80% but did not affect responses of this tissue to acetylcholine, 5-hydroxytryptamine (5-HT) or bradykinin. Polyphloretin phosphate (PPP), the known PG antagonist, had a similar effect at the same concentration (10⁻⁵ g/ml).5-(6-Carboxyhexyl)-1-(3-hydroxyoctyl)pyrrolidine-2,4-dione (12) had the same profile of activity on superfused smooth muscle preparations as PGE₂ or PGA₂. On intravenous injection into anaesthetised rats it caused dose-dependent falls in arterial blood pressure with associated tachycardias, which is typical of the response to PGE₂. The smooth muscle activity of (12) was not reduced by passage through isolated perfused guinea-pig lungs nor was its potency as a vasodepressor increased when given intra-arterially to rats. These results suggest that, unlike PGE₂, this analogue is not removed by the pulmonary circulation.     

Protein kinase C does not regulate diacylglycerol metabolism in aortic smooth muscle cells

Summary The effect of a reduction in protein kinase C activity on the metabolism of exogenous [³H]diC8 by freshly isolated smooth muscle cells from rabbit aorta and cultured A10 smooth muscle cells was determined. The metabolism of [³H]diC8 by both smooth muscle cell preparations was predominantly by hydrolysis to yield monoC8 and glycerol (lipase pathway); very little radioactivity was incorporated into phospholipids. Diacylglycerol lipase activity measured in vitro with A10 cell homogenates was much greater than diacylglycerol kinase activity. The addition of the protein kinase C inhibitor H-7 to incubations of isolated aortic smooth muscle cells and cultured A10 cells had no significant effect on the metabolism of [³H]diC8. Protein kinase C activity in cultured A10 cells preincubated for 20 h with a phorbol ester was reduced to 14% of control as a consequence of down-regulation, but diC8 metabolism was not changed. Therefore, protein kinase C does not regulate the metabolism of diacylglycerols in aortic smooth muscle cells.Abbreviations IP₃ inositol 1,4,5-trisphosphate - DG diacylglycerol - MG monoacylglycerol - PL phospholipid(s) - diC8 dioctanoylglycerol - H-7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride - monoC8 monooctanoylglycerol - PS phosphatidylserine - PDBu phorbol 12,13-dibutyrate     

Reduction in Gh protein expression is associated with cytodifferentiation of vascular smooth muscle cells

G_h, a high molecular weight GTP-binding protein that couples ₁-adrenoceptors in heart and liver to phosphatidylinositol (Ptdlns)-specific phospholipase C (PLC), has recently been shown to be a tissue transglutaminase type 11. Transglutaminases have been suggested to play a role in the maintenance of blood vessel structure, and therefore it is possible that changes in their expression may accompany pathological states which involve phenotypic modulation of smooth muscle. Hence, we investigated the expression of G_h during differentiation of rat aortic smooth muscle cells in culture. G_h content was reduced markedly in cultured smooth muscle cells compared to freshly isolated cells as determined by Western blotting using a G_h-specific monoclonal antibody. In contrast, the level of G_q, a heterotrimeric G-protein that couples ₁-adrenoceptors to PLC, was maintained throughout the culture period. These findings indicate that changes in G, expression accompany phenotypic modulation of vascular smooth muscle cells. These changes in G_h protein expression may be important in the altered responsiveness of vessels in pathological disease states.     

Lanthanum suppresses osteoblastic differentiation via pertussis toxin‐sensitive G protein signaling in rat vascular smooth muscle cells

A major cellular event in vascular calcification is the phenotypic transformation of vascular smooth muscle cells (VSMCs) into osteoblast‐like cells. After demonstrating that lanthanum chloride (LaCl₃) suppresses hydrogen peroxide‐enhanced calcification in rat calcifying vascular cells (CVCs), here we report its effect on the osteoblastic differentiation of rat VSMCs, a process leading to the formation of CVCs. Cells were isolated from aortic media of male SD rats, and passages between three and eight were cultured in Dulbeccol's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 10 mM β‐glycerophosphate (β‐GP) in the presence or absence of LaCl₃. Exposure of cells to LaCl₃ suppressed the β‐GP‐induced elevations in calcium deposition, alkaline phosphatase (ALP) activity, and Cbfa1/Runx2 expression, as well as the concomitant loss of SM α‐actin. Furthermore, LaCl₃ activated the phosphorylation of extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK), and the blockage of either pathway with a specific inhibitor abolished the effects of LaCl₃. In addition, pretreatment of the cells with pertussis toxin (PTx), an inhibitor of G protein‐mediated signaling pathway, repealed all the changes induced by LaCl₃. These findings demonstrate that LaCl₃ suppresses the β‐GP‐induced osteoblastic differentiation and calcification in rat VSMCs, and its effect is mediated by the activation of both ERK and JNK MAPK pathways via PTx‐sensitive G proteins. J. Cell. Biochem. 108: 1184–1191, 2009. © 2009 Wiley‐Liss, Inc.     

时钟基因Bmal1促进血管平滑肌细胞增殖

摘要 目的：观察时钟基因Bmal1的过表达对血管平滑肌细胞增殖的影响,进一步探讨生物节律对于血管发育的具体影响。方法：采用包装GV341-Bmal1载体的慢病毒转染的方法构建大鼠胸主动脉平滑肌细胞（A7R5）稳定转染Bmal1的细胞系,实时定量PCR和细胞爬片Bmal1的免疫荧光染色的方法判断所构建细胞系是否稳定过表达Bmal1,细胞爬片Ki67的免疫荧光染色的方法观察时钟基因Bmal1的过表达对血管平滑肌细胞增殖的影响。结果：实时定量PCR结果显示稳定转染Bmal1组细胞Bmal1的表达是对照组的11.2倍（P<0.01）;细胞爬片的免疫荧光染色结果显示稳定转染Bmal1组细胞BMAL1的表达明显升高（P<0.05）,且稳定转染Bmal1组Ki67阳性细胞比例明显升高（P<0.05）。结论：通过慢病毒转染的方法成功构建了血管平滑肌细胞稳定转染Bmal1的细胞系,细胞片Ki67的免疫荧光染色结果显示Bmal1的过表达促进了血管平滑肌细胞的增殖。     

Induction of apoptosis in vascular smooth muscle cells by mechanical stretch

We studied the response of porcine vascular smooth muscle cells (PVSMCs) to cyclic sinusoidal stretch at a frequency of 1 Hz. Cyclic stretch with an area change of 25% caused an increase in PVSMC apoptosis, which was accompanied by sustained activation of c-Jun NH(2)-terminal kinases (JNK) and the mitogen-activated protein kinase p38. Cyclic stretch with an area change of 7% had no such effect. Infection of PVSMCs with recombinant adenoviruses expressing constitutively active forms of upstream molecules that activate JNK and p38 also led to apoptosis. The simultaneous blockade of both JNK and p38 pathways with adenovirus-mediated expression of dominant-negative mutants of c-Jun and p38 caused a significant decrease (to 1/2) of the apoptosis induced by 25% cyclic stretch. The 25% stretch also caused sustained clustering of tumor necrosis factor-alpha (TNF-alpha) receptor-1 and its association with TNF-alpha receptor-associated factor-2 (TRAF-2). Overexpressing the wild-type TRAF-2 in PVSMCs caused an increase in apoptosis. In contrast, the expression of a dominant-negative mutant of TRAF-2 attenuated stretch-induced apoptois. These results support the hypothesis that circumferential overload under hypertensive conditions induces a clustering of death receptors that cause vascular smooth muscle cell apoptosis.     

Synthesis and biological activity of flavanone derivatives

A series of new flavanone derivatives of farrerol was synthesized by a convenient method. The in vitro anti-tumor activity of these compounds was evaluated against human Bel-7402, HL-60, BGC-823 and KB cell lines, the protein tyrosine kinase (PTK) inhibitor activity was also tested. Their cytoprotective activity was tested using hydrogen peroxide (H₂O₂)-induced injury in human umbilical vein endothelial cells. Their in vitro anti-atherosclerosis activity was tested on vascular smooth muscle cells by the MTT method using tetrandrine as a positive contrast drug. The structures of all compounds synthesized were confirmed by ¹H, ¹³C NMR and ESI-MS. Most of the compounds exhibited good pharmacological activity and the preliminary structure–activity relationships were described.     

Isolation and culture of rat superior mesenteric artery smooth muscle cells

Summary The structure and function of vascular smooth muscle cells have been extensively investigated with the aid of in vitro culture techniques. The majority of studies have utilized aortic tissue as the source of cells. We present here a method for isolating and culturing smooth muscle cells of the rat superior mesenteric artery, an elasto-muscular vessel that is structurally and functionally different from the aorta. Cells were isolated from partially digested explants and characterized by immunochemical and biochemical techniques. Unlike cultured fibroblasts, the cultured cells stained positive for smooth muscle specific actin. The cells also produced laminin and type IV collagen in culture. This method provides a means for the isolation of large numbers of viable smooth muscle cells from the superior mesenteric artery which can be propagated in culture for in vitro study.     

Cyclic Nucleotide-Gated Channels Contribute to Thromboxane A2-Induced Contraction of Rat Small Mesenteric Arteries

Background

Thromboxane A₂ (TxA₂)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can be partly attributed to TxA₂-induced Ca²⁺ influx, which resulted in vascular contraction via Ca²⁺-calmodulin-MLCK pathway. This study aims to identify the channels that mediate TxA₂-induced Ca²⁺ influx in vascular smooth muscle cells.

Methodology/Principal Findings

Application of U-46619, a thromboxane A₂ mimic, resulted in a constriction in endothelium-denuded small mesenteric artery segments. The constriction relies on the presence of extracellular Ca²⁺, because removal of extracellular Ca²⁺ abolished the constriction. This constriction was partially inhibited by an L-type Ca²⁺ channel inhibitor nifedipine (0.5–1 µM). The remaining component was inhibited by L-cis-diltiazem, a selective inhibitor for CNG channels, in a dose-dependent manner. Another CNG channel blocker LY83583 [6-(phenylamino)-5,8-quinolinedione] had similar effect. In the primary cultured smooth muscle cells derived from rat aorta, application of U46619 (100 nM) induced a rise in cytosolic Ca²⁺ ([Ca²⁺]_i), which was inhibited by L-cis-diltiazem. Immunoblot experiments confirmed the presence of CNGA2 protein in vascular smooth muscle cells.

Conclusions/Significance

These data suggest a functional role of CNG channels in U-46619-induced Ca²⁺ influx and contraction of smooth muscle cells.     

Endothelin-1 Augments Na+/H+ Exchange Activity in Murine Pulmonary Arterial Smooth Muscle Cells via Rho Kinase

Excessive production of endothelin-1 (ET-1), a potent vasoconstrictor, occurs with several forms of pulmonary hypertension. In addition to modulating vasomotor tone, ET-1 can potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. It is well established that changes in cell proliferation and migration in PASMCs are associated with alkalinization of intracellular pH (pH_i), typically due to activation of Na⁺/H⁺ exchange (NHE). In the systemic vasculature, ET-1 increases pH_i, Na⁺/H⁺ exchange activity and stimulates cell growth via a mechanism dependent on protein kinase C (PKC). These results, coupled with data describing elevated levels of ET-1 in hypertensive animals/humans, suggest that ET-1 may play an important role in modulating pH_i and smooth muscle growth in the lung; however, the effect of ET-1 on basal pH_i and NHE activity has yet to be examined in PASMCs. Thus, we used fluorescent microscopy in transiently (3–5 days) cultured rat PASMCs and the pH-sensitive dye, BCECF-AM, to measure changes in basal pH_i and NHE activity induced by increasing concentrations of ET-1 (10⁻¹⁰ to 10⁻⁸ M). We found that application of exogenous ET-1 increased pH_i and NHE activity in PASMCs and that the ET-1-induced augmentation of NHE was prevented in PASMCs pretreated with an inhibitor of Rho kinase, but not inhibitors of PKC. Moreover, direct activation of PKC had no effect on pH_i or NHE activity in PASMCs. Our results indicate that ET-1 can modulate pH homeostasis in PASMCs via a signaling pathway that includes Rho kinase and that, in contrast to systemic vascular smooth muscle, activation of PKC does not appear to be an important regulator of PASMC pH_i.