Prolactin induces proliferation of vascular smooth muscle cells through a protein kinase C-dependent mechanism

The effects of prolactin (PRL) on A10 (aortic smooth muscle) cell proliferation were examined by measuring both [3H]thymidine incorporation and increases in cell number. PRL induced a significant proliferative response from 10(-11) to 10(-7) M, with optimal activity at 10(-10) M. PRL also enhanced platelet-derived growth factor (PDGF)-induced proliferation. The possibility that PRL induces proliferation through a protein kinase C (PKC)-mediated mechanism was also examined. PRL caused activation of PKC from 10(-12) to 10(-8) M. Antiserum to PRL, a monoclonal antibody directed against the PRL receptor and the immunosuppressive agent cyclosporine A, were able to inhibit PRL-induced proliferation and activation of PKC. The PKC inhibitors, staurosporine, sphingosine, and 1-(-5-iso-quinoline-sulfonyl)-2-methylpiperazine (H-7) also antagonized both proliferation and PKC activation. These data strongly suggest that PRL-induced A10 cell proliferation is mediated through the PKC pathway and that this may play a role in vascular smooth muscle cell hyperplasia, characteristic of the pathogenesis of cardiovascular diseases such as hypertension and atherosclerosis.     

Ca(2+)- and protein kinase C-dependent stimulation of mitogen-activated protein kinase in detergent-skinned vascular smooth muscle

Protein kinase C and mitogen-activated protein (MAP) kinase are expressed in all smooth muscle cells and believed to be important in several physiologically relevant properties of this muscle. Our goal was to determine if protein kinase C and MAP kinase are activated by a simple increase in cellular Ca(2+) and to determine if protein kinase C is an upstream activator of MAP kinase. These studies were performed in the Triton X-100 detergent-skinned preparation of the swine carotid artery, which allows control of the intracellular environment without influence from membrane or receptor-mediated modulation. The p42 and p44 isoforms of MAP kinase were activated in a concentration-dependent fashion by an increase in Ca2+. This was shown by in-the-gel kinase assay and direct measurement of MAP kinase phosphotransferase activity. Protein kinase C was also activated by an increase in Ca2+, as shown by a novel assay that measures total active protein kinase C in the tissue. Inhibition of protein kinase C activity completely abolished MAP kinase activity. Additionally, inhibition of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) also abolished MAP kinase activity. Using intact swine carotid arteries, we showed p42 and p44 MAP kinase to be activated by both histamine and phorbol dibutyrate, but only the p42 isoform was calcium-sensitive. Our results suggest that a Ca(2+)-dependent isoform of protein kinase C and CaM kinase II are upstream activators of MAP kinase in the swine carotid artery.     

Ca2+ antagonist-insensitive coronary smooth muscle contraction involves activation of epsilon-protein kinase C-dependent pathway

Certain angina and coronary artery disease forms do not respond to Ca²⁺ channel blockers, and a role for vasoactive eicosanoids such as PGF₂ in Ca²⁺ antagonist-insensitive coronary vasospasm is suggested; however, the signaling mechanisms are unclear. We investigated whether PGF₂-induced coronary smooth muscle contraction is Ca²⁺ antagonist insensitive and involves activation of a PKC-dependent pathway. We measured contraction in single porcine coronary artery smooth muscle cells and intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in fura 2-loaded cells and examined cytosolic and particulate fractions for PKC activity and reactivity with isoform-specific PKC antibodies. In Hanks' solution (1 mM Ca²⁺), PGF₂ (10^-5 M) caused transient [Ca²⁺]_i increase followed by maintained [Ca²⁺]_i increase and 34% cell contraction. Ca²⁺ channel blockers verapamil and diltiazem (10^-6 M) abolished maintained PGF₂-induced [Ca²⁺]_i increase but only partially inhibited PGF₂-induced cell contraction to 17%. Verapamil-insensitive PGF₂ contraction was inhibited by PKC inhibitors GF-109203X, calphostin C, and -PKC V1-2. PGF₂ caused Ca²⁺-dependent -PKC and Ca²⁺-independent -PKC translocation from cytosolic to particulate fractions that was inhibited by calphostin C. Verapamil abolished PGF₂-induced -but not -PKC translocation. PMA (10^-6 M), a direct activator of PKC, caused 21% contraction with no significant [Ca²⁺]_i increase and -PKC translocation that were inhibited by calphostin C but not verapamil. Membrane depolarization by 51 mM KCl, which stimulates Ca²⁺ influx, caused 36% cell contraction and [Ca²⁺]_i increase that were inhibited by verapamil but not GF-109203X or calphostin C and did not cause - or -PKC translocation. Thus a significant component of PGF₂-induced contraction of coronary smooth muscle is Ca²⁺ antagonist insensitive, involves Ca²⁺-independent -PKC activation and translocation, and may represent a signaling mechanism of Ca²⁺ antagonist-resistant coronary vasospasm. eicosanoids; calcium; vascular smooth muscle     

Angiotensin II stimulates phosphorylation of nuclear lamins via a protein kinase C-dependent mechanism in cultured vascular smooth muscle cells

Angiotensin II (ang II) induces c-fos gene expression in part via a protein kinase C-dependent mechanism in cultured vascular smooth muscle cells (VSMC). However, little is known about the mechanisms by which protein kinase C regulates nuclear functions. We examined the ability of ang II to phosphorylate nuclear lamina proteins in VSMC and the possibility that protein kinase C is involved in these putative phosphorylation events. Ang II stimulated the phosphorylation of Triton X-100- and high salt-insoluble nuclear envelope proteins with molecular weights of 70,000, 67,000, and 60,000. These proteins were identified as lamins A, B, and C, respectively, based on their mobilities on two-dimensional gel electrophoresis and interaction with antibodies to lamins as detected by immunoblot analyses. After a 2-min delay, phosphorylation levels of lamins increased, peaked at 20-30 min, and were sustained for at least 60 min after ang II stimulation. The threshold, half-maximal, and maximal concentrations of ang II which induced phosphorylation of lamins were 0.1, 0.5-1, and 100 nM, respectively. Phorbol 12-myristate 13-acetate also induced these reactions, whereas ionomycin did not. Down-regulation of protein kinase C by prolonged treatment with phorbol 12,13-dibutyrate attenuated ang II-induced phosphorylation of lamins. In vitro phosphorylation of nuclear envelope proteins by protein kinase C revealed that lamins served as substrates for this enzyme. These results indicate that ang II induces phosphorylation of lamins in cultured VSMC and suggest that protein kinase C is either directly or indirectly involved in these reactions. The results raise the possibility that phosphorylation of nuclear proteins is one of the important steps by which the protein kinase C signaling pathway regulates agonist-induced nuclear events.     

Reversible phosphorylation of tonoplast proteins involves tonoplast-bound calcium-calmodulin-dependent protein kinase(s) and protein phosphatase(s)

In highly purified tonoplast fractions from Acer pseudoplatanus cells, the in vitro reversible phosphorylation of proteins affected only a restricted set of polypeptides. The phosphorylation process has been shown to be dramatically stimulated by calcium via the mediation of calmodulin as the transducer. The protein kinase(s) was totally inhibited by micromolar concentrations of a calmodulin antagonist. Tonoplast appears to be potentially a good experimental system for the evaluation of the effects of protein phosphorylation on membrane properties in plants.Abbreviations CaM calmodulin - EGTA ethylene glycol-bis-(-amino ethylether)N,N,NN-tetraacetate - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

蛋白激酶C对大鼠支气管平滑肌KV通道的影响

用全细胞膜片钳、Western印迹法和逆转录—PCR技术,观察蛋白激酶C(protein kinase C,PKC)对大鼠支气管平滑肌细胞(bronchial smooth muscle cells,BSMCs)电压依赖性延迟整流钾通道(Kv)活性及其亚型Kvl．5表达的影响。结果为：(1)PKC激活剂豆蔻酰佛波醇乙酯(phorbol 12-myristate 13-acetate,PMA)显著抑制急性分离大鼠BSMCs的Kv通道电流,该效应被PKC阻断剂Ro31—8220显著抑制;(2)PMA显著抑制体外培养大鼠BSMCs的Kvl．5 mRNA和蛋白质的表达,该效应被Ro31—8220显著抑制。上述观察结果提示,PKC活化可抑制大鼠BSMCs的Kv通道电流活性,下调Kvl．5亚型的表达水平。     

p90(RSK) blocks bad-mediated cell death via a protein kinase C-dependent pathway

Although activation of protein kinase C (PKC) is known to promote cell survival and protect against cell death, the PKC targets and pathways that serve this function have remained elusive. Here we demonstrate that two potent activators of PKC, 12-O-tetradecanoylphorbol-13-acetate and bryostatin, both stimulate phosphorylation of Bad at Ser(112), a site known to regulate apoptotic cell death by interleukin-3. PKC inhibitors but not PI 3-kinase/Akt inhibitors block 12-O-tetradecanoylphorbol-13-acetate-stimulated Bad phosphorylation. PKC isoforms tested in vitro were unable to phosphorylate Bad at Ser(112), suggesting that PKC acts indirectly to activate a downstream Bad kinase. p90(RSK) and family members RSK-2 and RSK-3 are activated by phorbol ester and phosphorylate Bad at Ser(112) both in vitro and in vivo. p90(RSK) stimulates binding of Bad to 14-3-3 and blocks Bad-mediated cell death in a Ser(112)-dependent manner. These findings suggest that p90(RSK) can function in a PKC-dependent pathway to promote cell survival via phosphorylation and inactivation of Bad-mediated cell death.     

Feedback inhibition on cell wall integrity signaling by Zds1 involves Gsk3 phosphorylation of a cAMP-dependent protein kinase regulatory subunit

We report here that budding yeast cAMP-dependent protein kinase (cAPK) is controlled by heat stress. A rise in temperature from 30 to 37 degrees C was found to result in both a higher expression and an increased cytoplasmic localization of its regulatory subunit Bcy1. Both of these effects required phosphorylation of serines located in its localization domain. Surprisingly, classic cAPK-controlled processes were found to be independent of Bcy1 phosphorylation, indicating that these modifications do not affect cAPK activity as such. Alternatively, phosphorylation may recruit cAPK to, and thereby control, a specific subset of (perhaps novel) cAPK targets that are presumably localized extranuclearly. Zds1 and Zds2 may play a role in this process, since these were found required to retain hyperphosphorylated Bcy1 in the cytoplasm at 37 degrees C. Mck1, a homologue of mammalian glycogen synthase kinase 3 and a downstream component of the heat-activated Pkc1-Slt2/Mpk1 cell wall integrity pathway, is partly responsible for hyperphosphorylations of Bcy1. Remarkably, Zds1 appears to act as a negative regulator of cell wall integrity signaling, and this activity is dependent in part on the phosphorylation status of Bcy1. Thus, Mck1 phosphorylation of Bcy1 and Zds1 may constitute an unprecedented negative feedback control on the cell wall integrity-signaling pathway.     

Nitric oxide-induced inhibition of smooth muscle cell proliferation involves S-nitrosation and inactivation of RhoA

Nitric oxide (NO) acts as a vasoregulatory molecule that inhibits vascular smooth muscle cell (SMC) proliferation. Studies have illustrated that NO inhibits SMC proliferation via the extracellular signal-regulated kinase (ERK) pathway, leading to increased protein levels of the cyclin-dependent kinase inhibitor p21^Waf1/Cip1. The ERK pathway can be pro- or antiproliferative, and it has been demonstrated that the activation status of the small GTPase RhoA determines the proliferative fate of ERK signaling, whereby inactivation of RhoA influences ERK signaling to increase p21^Waf1/Cip1 and inhibit proliferation. The purpose of these investigations was to examine the effect of NO on RhoA activation/S-nitrosation and to test the hypothesis that inhibition of SMC proliferation by NO is dependent on inactivation of RhoA. NO decreases activation of RhoA, as demonstrated by RhoA GTP-binding assays, affinity precipitation, and phalloidin staining of the actin cytoskeleton. Additionally, these effects are independent of cGMP. NO decreases SMC proliferation, and gene transfer of constitutively active RhoA (RhoA^63L) diminished the antiproliferative effects of NO, as determined by thymidine incorporation. Western blots of p21^Waf1/Cip1 correlated with changes in proliferation. S-nitrosation of recombinant RhoA protein and immunoprecipitated RhoA was demonstrated by Western blotting for nitrosocysteine and by measurement of NO release. Furthermore, NO decreases GTP loading of recombinant RhoA protein. These findings indicate that inactivation of RhoA plays a role in NO-mediated SMC antiproliferation and that S-nitrosation is associated with decreased GTP binding of RhoA. Nitrosation of RhoA and other proteins likely contributes to cGMP-independent effects of NO. cell signaling; posttranslational modification; vascular disease     

Alpha-tocopherol (vitamin E) regulates vascular smooth muscle cell proliferation and protein kinase C activity

Alpha-Tocopherol (vitamin E) protects against free radical damage, which has been implicated in aging, cancer initiation, and atherosclerosis. We have found that physiological concentrations of alpha-tocopherol specifically inhibited aorta smooth muscle cell (VSMC, line A7r5) proliferation and protein kinase C (PKC) activity. Other water and lipid soluble antioxidants were inactive. alpha-Tocopherol inhibition of PKC and of VSMC proliferation may represent a physiological mechanism, relevant to the onset of diseased states such as atherosclerosis.     

Vasopressin stimulates action potential firing by protein kinase C-dependent inhibition of KCNQ5 in A7r5 rat aortic smooth muscle cells

[Arg(8)]-vasopressin (AVP), at low concentrations (10-500 pM), stimulates oscillations in intracellular Ca(2+) concentration (Ca(2+) spikes) in A7r5 rat aortic smooth muscle cells. Our previous studies provided biochemical evidence that protein kinase C (PKC) activation and phosphorylation of voltage-sensitive K(+) (K(v)) channels are crucial steps in this process. In the present study, K(v) currents (I(Kv)) and membrane potential were measured using patch clamp techniques. Treatment of A7r5 cells with 100 pM AVP resulted in significant inhibition of I(Kv). This effect was associated with gradual membrane depolarization, increased membrane resistance, and action potential (AP) generation in the same cells. The AVP-sensitive I(Kv) was resistant to 4-aminopyridine, iberiotoxin, and glibenclamide but was fully inhibited by the selective KCNQ channel blockers linopirdine (10 microM) and XE-991 (10 microM) and enhanced by the KCNQ channel activator flupirtine (10 microM). BaCl(2) (100 microM) or linopirdine (5 microM) mimicked the effects of AVP on K(+) currents, AP generation, and Ca(2+) spiking. Expression of KCNQ5 was detected by RT-PCR in A7r5 cells and freshly isolated rat aortic smooth muscle. RNA interference directed toward KCNQ5 reduced KCNQ5 protein expression and resulted in a significant decrease in I(Kv) in A7r5 cells. I(Kv) was also inhibited in response to the PKC activator 4beta-phorbol 12-myristate 13-acetate (10 nM), and the inhibition of I(Kv) by AVP was prevented by the PKC inhibitor calphostin C (250 nM). These results suggest that the stimulation of Ca(2+) spiking by physiological concentrations of AVP involves PKC-dependent inhibition of KCNQ5 channels and increased AP firing in A7r5 cells.     

Respiratory epithelium-dependent inhibition of protein kinase C of airway smooth muscle cells

We have examined the effect of phorbol myristate acetate (PMA) on airway smooth muscle (ASM) in the presence and absence of respiratory epithelium (RE) and analyzed the dependence of this response on extracellular sodium, Na+/H+ exchange, calcium, and cyclooxygenase products; we determined both the resting membrane potential and isometric force developed by ASM preparations. Removal of RE had no effect on the values of the resting membrane potential of ASM cells. In the presence of RE in the preparation, both electrical and contractile responses to PMA (10(-5) M) were significantly different compared with the response of ASM to PMA without RE. When the RE was present, stimulation of protein kinase C caused only a biphasic response in both membrane potential and isometric force. In either the presence or absence of RE, amiloride (10(-5) M) and a low-sodium solution inhibited both electrical and contractile changes of ASM cells caused by PMA. In the presence or absence of RE, verapamil (10(-5) M) attenuated (P less than 0.05) both electrical and contractile responses of ASM cells as induced by PMA. Verapamil, however, had no effect on the last phase of PMA-induced response. Pretreatment of preparations with indomethacin (10(-6) M) changed the PMA-induced response of ASM with RE to a response usually observed in ASM without RE. Finally, the incubation of tracheal preparations without RE with prostaglandin E2 (10(-8) M) altered the response of these preparations in such a way that their electrical and contractile response to PMA was essentially identical to the PMA response observed in preparations with an intact RE.(ABSTRACT TRUNCATED AT 250 WORDS)     

EETs relax airway smooth muscle via an EpDHF effect: BK(Ca) channel activation and hyperpolarization

Epoxyeicosatrienoic acids (EETs) are produced from arachidonic acid via the cytochrome P-450 epoxygenase pathway. EETs are able to modulate smooth muscle tone by increasing K(+) conductance, hence generating hyperpolarization of the tissues. However, the molecular mechanisms by which EETs induce smooth muscle relaxation are not fully understood. In the present study, the effects of EETs on airway smooth muscle (ASM) were investigated using three electrophysiological techniques. 8,9-EET and 14,15-EET induced concentration-dependent relaxations of the ASM precontracted with a muscarinc agonist (carbamylcholine chloride), and these relaxations were partly inhibited by 10 nM iberiotoxin (IbTX), a specific large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel blocker. Moreover, 3 microM 8,9- or 14,15-EET induced hyperpolarizations of -12 +/- 3.5 and -16 +/- 3 mV, with EC(50) values of 0.13 and 0.14 microM, respectively, which were either reversed or blocked on addition of 10 nM IbTX. These results indicate that BK(Ca) channels are involved in hyperpolarization and participate in the relaxation of ASM. In addition, complementary experiments demonstrated that 8,9- and 14,15-EET activate reconstituted BK(Ca) channels at low free Ca(2+) concentrations without affecting their unitary conductance. These increases in channel activity were IbTX sensitive and correlated well with the IbTX-sensitive hyperpolarization and relaxation of ASM. Together these results support the view that, in ASM, the EETs act through an epithelium-derived hyperpolarizing factorlike effect.     

Heparin selectively inhibits a protein kinase C-dependent mechanism of cell cycle progression in calf aortic smooth muscle cells [published erratum appears in J Cell Biol 1990 Mar;110(3):863]

The proliferation of arterial smooth muscle cells (SMCs) plays a critical role in the pathogenesis of arteriosclerosis. Previous studies have indicated that the glycosaminoglycan heparin specifically inhibited the growth of vascular SMCs in vivo and in culture, although the precise mechanism(s) of action have not been elucidated. In this study, we have examined the ability of specific mitogens (PDGF, EGF, heparin-binding growth factors, phorbol esters, and insulin) to stimulate SMC proliferation. Our results indicate that SMCs derived from different species and vascular sources respond differently to these growth factors. We next examined the ability of heparin to inhibit the proliferative responses to these mitogens. In calf aortic SMCs, heparin inhibits a protein kinase C-dependent pathway for mitogenesis. Detailed cell cycle analysis revealed several new features of the effects of heparin on SMCs. For example, heparin has two effects on the Go----S transition: it delays entry into S phase and also reduces the number of cells entering the cycle from Go. Using two separate experimental approaches, we found that heparin must be present during the last 4 h before S phase, suggesting a mid-to-late G1 heparin block. In addition, our data indicate that heparin-treated SMCs, while initially blocked in mid-to-late G1, slowly move back into a quiescent growth state in the continued presence of heparin. These results suggest that heparin may have multiple targets for its antiproliferative effect.     

Phosphorylation of smooth muscle caldesmon by mitogen-activated protein (MAP) kinase and expression of MAP kinase in differentiated smooth muscle cells.

Smooth muscle caldesmon was phosphorylated in vitro by sea star p44mpk up to 2.0 mol of phosphate/mol of protein at both Ser and Thr residues. The phosphorylation sites were contained mainly in the COOH-terminal 10-kDa cyanogen bromide fragment which houses the binding sites for calmodulin, tropomyosin, and F-actin. Tryptic peptide maps of 32P-labeled caldesmon by p44mpk and p34cdc2 showed that while both enzymes recognized similar sites of phosphorylation, they have different preferred sites. Phosphorylation of caldesmon attenuated slightly its interaction with actin and had no effect on its binding to calmodulin and tropomyosin. Smooth muscle cell extracts from chicken gizzard and rat aorta contained 42- and 44-kDa proteins, respectively, which were cross-reactive with an antibody to sea star p44mpk. Immunoprecipitates from gizzard and aorta cell extracts, generated with the p44mpk antibody, possessed kinase activities toward myelin basic protein as well as caldesmon. These results suggest that MAP kinase may have functions in the differentiated smooth muscle cells distinct from those involved in the cell cycle.     

Stimulation of arterial smooth muscle L-type calcium channels by hydrogen peroxide requires protein kinase C

Changes in intracellular calcium regulate countless biological processes. In arterial smooth muscle, voltage-dependent L-type calcium channels are major conduits for calcium entry with the primary function being determination of arterial diameter. Similarly, changes in intracellular redox status, either discrete controlled changes or global pathological perturbations, are also critical determinants of cell function. We recently reported that in arterial smooth muscle cells, local generation of hydrogen peroxide leads to colocalized calcium entry through L-type calcium channels. Here we extend our investigation into mechanisms linking hydrogen peroxide to calcium influx through L-type calcium channels by focusing on the role of protein kinase C (PKC). Our data indicate that stimulation of L-type calcium channels by hydrogen peroxide requires oxidant-dependent increases in PKC catalytic activity. This effect is independent of classical cofactor-dependent activation of PKC by diacylglycerol. These data provide additional experimental evidence supporting the concept of oxidative stimulation of L-type calcium channels.     

Staurosporine inhibition of zipper-interacting protein kinase contractile effects in gastrointestinal smooth muscle.

Zipper-interacting protein kinase (ZIPK) is a serine-threonine kinase that has been implicated in Ca2+-independent myosin II phosphorylation and contractile force generation in vascular smooth muscle. However, relatively little is known about the contribution of this kinase to gastrointestinal smooth muscle contraction. The addition of a recombinant version of ZIPK that lacked the leucine zipper domain to permeabilized ileal strips evoked a Ca2+-independent contraction and resulted in myosin regulatory light chain diphosphorylation at Ser19 and Thr18. Neither Ca2+-independent force development nor myosin regulatory light chain phosphorylation was elicited by the addition of kinase-dead ZIPK to the ileal strips. The sensitivity of ZIPK-induced contraction to various kinase inhibitors was similar to the in vitro sensitivity of purified ZIPK to these inhibitors. Staurosporine was the most effective ZIPK inhibitor, with a Ki value calculated to be 2.6 +/- 0.3 micromol/L. Through the use of specific kinase inhibitors, we determined that Rho-associated protein kinase and Ca2+/phospholipid-dependent protein kinase (protein kinase C) do not mitigate ZIPK-induced contraction in ileum. Our findings support a role for ZIPK in Ca2+-independent contractile force generation in gastrointestinal smooth muscle.     

Vasoconstrictor effect of endothelin-1 on hypertensive pulmonary arterial smooth muscle involves Rho-kinase and protein kinase C

Although one of the common characteristics of pulmonary hypertension is abnormal sustained vasoconstriction, the signaling pathways that mediate this heightened pulmonary vascular response are still not well defined. Protein kinase C (PKC) and Rho-kinase are regulators of smooth muscle contraction induced by G protein-coupled receptor agonists including endothelin-1 (ET-1), which has been implicated as a signaling pathway in pulmonary hypertension. Toward this end, it was hypothesized that both Rho-kinase and PKC mediate the pulmonary vascular response to ET-1 in hypertensive pulmonary arterial smooth muscle, and therefore, the purpose of this study was to determine the role of PKC and Rho-kinase signaling in ET-1-induced vasoconstriction in both normotensive (Sprague-Dawley) and hypertensive (Fawn-Hooded) rat pulmonary arterial smooth muscle. Results indicate that ET-1 caused greater vasoconstriction in hypertensive pulmonary arteries compared with the normal vessels, and treatment with the PKC antagonists chelerythrine, rottlerin, and G? 6983 inhibited the vasoconstrictor response to ET-1 in the hypertensive vessels. In addition, the specific Rho-kinase inhibitor Y-27632 significantly attenuated the effect of ET-1 in both normotensive and hypertensive phenotypes, with greater inhibition occurring in the hypertensive arteries. Furthermore, Western blot analysis revealed that ET-1 increased RhoA expression in both normotensive and hypertensive pulmonary arteries, with expression being greater in the hypertensive state. These results suggest that both PKC and Rho/Rho-kinase mediate the heightened pulmonary vascular response to ET-1 in hypertensive pulmonary arterial smooth muscle.