Protein kinase C inhibits BKCa channel activity in pulmonary arterial smooth muscle

Signaling mechanisms that elevate cyclic AMP (cAMP) activate large-conductance, calcium- and voltage-activated potassium (BKCa) channels in pulmonary vascular smooth muscle and cause pulmonary vasodilatation. BKCa channel modulation is important in the regulation of pulmonary arterial pressure, and inhibition (closing) of the BKCa channel has been implicated in the development of pulmonary vasoconstriction. Protein kinase C (PKC) causes pulmonary vasoconstriction, but little is known about the effect of PKC on BKCa channel activity. Accordingly, studies were done to determine the effect of PKC activation on cAMP-induced BKCa channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMC) of the fawn-hooded rat (FHR), a recognized animal model of pulmonary hypertension. Forskolin (10 microM), a stimulator of adenylate cyclase and an activator of cAMP, opened BKCa channels in single FHR PASMC, which were blocked by the PKC activators phorbol 12-myristate 13-acetate (100 nM) and thymeleatoxin (100 nM). The inhibitory response by thymeleatoxin on forskolin-induced BKCa channel activity was blocked by G?-6983, which selectively blocks the alpha, beta, delta, gamma, and zeta PKC isozymes, and G?-6976, which selectively inhibits PKC-alpha, PKC-beta, and PKC-mu, but not by rottlerin, which selectively inhibits PKC-delta. Collectively, these results indicate that activation of specific PKC isozymes inhibits cAMP-induced activation of the BKCa channel in pulmonary arterial smooth muscle, which suggests a unique signaling pathway to modulate BKCa channels and subsequently cAMP-induced pulmonary vasodilatation.     

cAMP activates BKCa channels in pulmonary arterial smooth muscle via cGMP-dependent protein kinase

The signal transduction mechanisms defining the role of cyclic nucleotides in the regulation of pulmonary vascular tone is currently an area of great interest. Normally, signaling mechanisms that elevate cAMP and guanosine-3',5'-cyclic monophosphate (cGMP) maintain the pulmonary vasculature in a relaxed state. Modulation of the large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel is important in the regulation of pulmonary arterial pressure, and inhibition (closing) of the BK(Ca) channel has been implicated in the development of pulmonary hypertension. Accordingly, studies were done to determine the effect of cAMP-elevating agents on BK(Ca) channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMC) of the fawn-hooded rat (FHR), a recognized animal model of pulmonary hypertension. Forskolin (10 micro M), a stimulator of adenylate cyclase and an activator of cAMP-dependent protein kinase (PKA), and 8-4-chlorophenylthio (CPT)-cAMP (100 micro M), a membrane-permeable derivative of cAMP, opened BK(Ca) channels in single FHR PASMC. Treatment of FHR PASMC with 300 nM KT5823, a selective inhibitor of cGMP-dependent protein kinase (PKG) activity inhibited the effect of both forskolin and CPT-cAMP. In contrast, blocking PKA activation with 300 nM KT5720 had no effect on forskolin or CPT-cAMP-stimulated BK(Ca) channel activity. These results indicate that cAMP-dependent vasodilators activate BK(Ca) channels in PASMC of FHR via PKG-dependent and PKA-independent signaling pathways, which suggests cross-activation between cyclic nucleotide-dependent protein kinases in pulmonary arterial smooth muscle and therefore, a unique signaling pathway for cAMP-induced pulmonary vasodilation.     

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.     

The Xanthine Derivative KMUP-1 Attenuates Serotonin-Induced Vasoconstriction and K+-Channel Inhibitory Activity via the PKC Pathway in Pulmonary Arteries

Serotonin (5-hydroxytryptamine, 5-HT) is a potent pulmonary vasoconstrictor that promotes pulmonary artery smooth muscle cell (PASMC) proliferation. 5-HT-induced K⁺ channel inhibition increases [Ca²⁺]_i in PASMCs, which is a major trigger for pulmonary vasoconstriction and development of pulmonary arterial hypertension (PAH). This study investigated whether KMUP-1 reduces pulmonary vasoconstriction in isolated pulmonary arteries (PAs) and attenuates 5-HT-inhibited K⁺ channel activities in PASMCs. In endothelium-denuded PA rings, KMUP-1 (1 μM) dose-dependently reduced 5-HT (100 μM) mediated contractile responses. Responses to KMUP-1 were reversed by K⁺ channel inhibitors (TEA, 10 mM, 4-aminopyridine, 5 mM, and paxilline, 10 μM). In primary PASMCs, KMUP-1 also dose-dependently restored 5-HT-inhibited voltage-gated K⁺-channel (Kv1.5 and Kv2.1) and large-conductance Ca²⁺-activated K⁺-channel (BK_Ca) proteins, as confirmed by immunofluorescent staining. Furthermore, 5-HT (10 μM)-inhibited Kv1.5 protein was unaffected by the PKA inhibitor KT5720 (1 μM) and the PKC activator PMA (1 μM), but these effects were reversed by KMUP-1 (1 μM), 8-Br-cAMP (100 μM), chelerythrine (1 μM), and KMUP-1 combined with a PKA/PKC activator or inhibitor. Notably, KMUP-1 reversed 5-HT-inhibited Kv1.5 protein and this response was significantly attenuated by co-incubation with the PKC activator PMA, suggesting that 5-HT-mediated PKC signaling can be modulated by KMUP-1. In conclusion, KMUP-1 ameliorates 5-HT-induced vasoconstriction and K⁺-channel inhibition through the PKC pathway, which could be valuable to prevent the development of PAH.     

Ca2+ signaling in hypoxic pulmonary vasoconstriction: effects of myosin light chain and Rho kinase antagonists

Antagonists of myosin light chain (MLC) kinase (MLCK) and Rho kinase (ROK) are thought to inhibit hypoxic pulmonary vasoconstriction (HPV) by decreasing the concentration of phosphorylated MLC at any intracellular Ca(2+) concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMC); however, these antagonists can also decrease [Ca(2+)](i). To determine whether MLCK and ROK antagonists alter Ca(2+) signaling in HPV, we measured the effects of ML-9, ML-7, Y-27632, and HA-1077 on [Ca(2+)](i), Ca(2+) entry, and Ca(2+) release in rat distal PASMC exposed to hypoxia or depolarizing concentrations of KCl. We performed parallel experiments in isolated rat lungs to confirm the inhibitory effects of these agents on pulmonary vasoconstriction. Our results demonstrate that MLCK and ROK antagonists caused concentration-dependent inhibition of hypoxia-induced increases in [Ca(2+)](i) in PASMC and HPV in isolated lungs and suggest that this inhibition was due to blockade of Ca(2+) release from the sarcoplasmic reticulum and Ca(2+) entry through store- and voltage-operated Ca(2+) channels in PASMC. Thus MLCK and ROK antagonists might block HPV by inhibiting Ca(2+) signaling, as well as the actin-myosin interaction, in PASMC. If effects on Ca(2+) signaling were due to decreased phosphorylated myosin light chain concentration, their diversity suggests that MLCK and ROK antagonists may have acted by inhibiting myosin motors and/or altering the cytoskeleton in a manner that prevented achievement of required spatial relationships among the cellular components of the response.     

A molecular switch for specific stimulation of the BKCa channel by cGMP and cAMP kinase

The cGMP and the cAMP pathways control smooth muscle tone by regulation of BK(Ca) (BK) channel activity. BK channels show considerable diversity and plasticity in their regulation by cyclic nucleotide-dependent protein kinases. The underlying molecular mechanisms are unclear but may involve expression of splice variants of the BK channel alpha subunit. Three isoforms, BK(A), BK(B), and BK(C), which were cloned from tracheal smooth muscle, differed only in their C terminus. When expressed in HEK293 cells, cGMP kinase (cGK) but not cAMP kinase (cAK) stimulated the activity of BK(A) and BK(B) by shifting the voltage dependence of the channel to more negative potentials. In contrast, BK(C) was exclusively stimulated by cAK. BK(C) lacks a C-terminal tandem phosphorylation motif for protein kinase C (PKC) with Ser(1151) and Ser(1154). Mutation of this motif in BK(A) switched channel regulation from cGK to cAK. Furthermore, inhibition of PKC in excised patches from cells expressing BK(A) abolished the stimulatory effect of cGK but allowed channel stimulation by cAK. cAK and cGK phosphorylated the channel at different sites. Thus, phosphorylation/dephosphorylation by PKC determines whether the BK channel is stimulated by cGK or cAK. The molecular mechanisms may be relevant for smooth muscle relaxation by cAMP and cGMP.     

Protein kinase C regulates vascular myogenic tone through activation of TRPM4

Myogenic vasoconstriction results from pressure-induced vascular smooth muscle cell depolarization and Ca(2+) influx via voltage-dependent Ca(2+) channels, a process that is significantly attenuated by inhibition of protein kinase C (PKC). It was recently reported that the melastatin transient receptor potential (TRP) channel TRPM4 is a critical mediator of pressure-induced smooth muscle depolarization and constriction in cerebral arteries. Interestingly, PKC activity enhances the activation of cloned TRPM4 channels expressed in cultured cells by increasing sensitivity of the channel to intracellular Ca(2+). Thus we postulated that PKC-dependent activation of TRPM4 might be a critical mediator of vascular myogenic tone. We report here that PKC inhibition attenuated pressure-induced constriction of cerebral vessels and that stimulation of PKC activity with phorbol 12-myristate 13-acetate (PMA) enhanced the development of myogenic tone. In freshly isolated cerebral artery myocytes, we identified a Ca(2+)-dependent, rapidly inactivating, outwardly rectifying, iberiotoxin-insensitive cation current with properties similar to those of expressed TRPM4 channels. Stimulation of PKC activity with PMA increased the intracellular Ca(2+) sensitivity of this current in vascular smooth muscle cells. To validate TRPM4 as a target of PKC regulation, antisense technology was used to suppress TRPM4 expression in isolated cerebral arteries. Under these conditions, the magnitude of TRPM4-like currents was diminished in cells from arteries treated with antisense oligonucleotides compared with controls, identifying TRPM4 as the molecular entity responsible for the PKC-activated current. Furthermore, the extent of PKC-induced smooth muscle cell depolarization and vasoconstriction was significantly decreased in arteries treated with TRPM4 antisense oligonucleotides compared with controls. We conclude that PKC-dependent regulation of TRPM4 activity contributes to the control of cerebral artery myogenic tone.     

Enhanced calcium signaling to bradykinin in airway smooth muscle from hyperresponsive inbred rats

Inbred Fischer 344 rats display airway hyperresponsiveness (AHR) in vivo compared with the normoresponsive Lewis strain. Fischer AHR has been linked with increased airway smooth muscle (ASM) contraction ex vivo and enhanced ASM cell intracellular Ca(2+) mobilization in response to serotonin compared with Lewis. To determine the generality of this association, we tested whether bradykinin (BK) also stimulates greater contraction of Fischer airways and greater Ca(2+) mobilization in Fischer ASM cells. Explants of Fischer intraparenchymal airways constricted faster and to a greater degree in response to BK than Lewis airways. BK also evoked higher Ca(2+) transients in Fischer than in Lewis ASM cells. ASM cell B(2) receptor expression was similar between the two strains. BK activated both phosphatidylinositide-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific PLC to mobilize Ca(2+) in Fischer and Lewis ASM cells. PI-PLC activity, as measured by inositol polyphosphate accumulation, was similar in the two strains. PKC inhibition with GF109203X, Go6973, or Go6983 attenuated BK-mediated Ca(2+) transients in Fischer cells, whereas GF109203X potentiated while Go6976 and Go6983 did not affect Ca(2+) transients in Lewis cells. Enhanced Ca(2+) mobilization in ASM cells can arise from variations in PKC and may be an important component of nonspecific, innate AHR.     

Hypoxia divergently regulates production of reactive oxygen species in human pulmonary and coronary artery smooth muscle cells

Acute hypoxia causes pulmonary vasoconstriction and coronary vasodilation. The divergent effects of hypoxia on pulmonary and coronary vascular smooth muscle cells suggest that the mechanisms involved in oxygen sensing and downstream effectors are different in these two types of cells. Since production of reactive oxygen species (ROS) is regulated by oxygen tension, ROS have been hypothesized to be a signaling mechanism in hypoxia-induced pulmonary vasoconstriction and vascular remodeling. Furthermore, an increased ROS production is also implicated in arteriosclerosis. In this study, we determined and compared the effects of hypoxia on ROS levels in human pulmonary arterial smooth muscle cells (PASMC) and coronary arterial smooth muscle cells (CASMC). Our results indicated that acute exposure to hypoxia (Po(2) = 25-30 mmHg for 5-10 min) significantly and rapidly decreased ROS levels in both PASMC and CASMC. However, chronic exposure to hypoxia (Po(2) = 30 mmHg for 48 h) markedly increased ROS levels in PASMC, but decreased ROS production in CASMC. Furthermore, chronic treatment with endothelin-1, a potent vasoconstrictor and mitogen, caused a significant increase in ROS production in both PASMC and CASMC. The inhibitory effect of acute hypoxia on ROS production in PASMC was also accelerated in cells chronically treated with endothelin-1. While the decreased ROS in PASMC and CASMC after acute exposure to hypoxia may reflect the lower level of oxygen substrate available for ROS production, the increased ROS production in PASMC during chronic hypoxia may reflect a pathophysiological response unique to the pulmonary vasculature that contributes to the development of pulmonary vascular remodeling in patients with hypoxia-associated pulmonary hypertension.     

川芎嗪对猪冠状动脉平滑肌细胞大电导钙激活钾通道的作用

本工作旨在研究川芎嗪对猪冠状动脉平滑肌细胞钾通道的作用,为阐明其扩张冠状动脉血管的机制提供实验依据。采用膜片钳细胞贴附式和内面向外式记录方式观察川芎嗪对猪冠状动脉平滑肌细胞大电导钙激活钾通道(large-conductance Ca2+- activated potassium channels,BKCa channels)的作用,分别用蛋白激酶A(protein kinase A,PKA)抑制剂H-89和蛋白激酶G (protein kinase G,PKG)抑制剂KT-5823处理细胞,再观察川芎嗪对BKCa通道作用的变化。结果表明在研究的0．73-8．07 mmol/L浓度范围,川芎嗪可以剂量依赖性地激活BKCa通道,使通道的开放概率从(0．01±0．003)增加到(0．03±0．01)-(．21± 0．18)(P<0．01,n=10),使通道平均关闭时间从(732．33±90．67)ms降低到(359．67±41．30)-(2．96±0．52)ms(P<0．01, n=10)。川芎嗪的这种激活作用在浴液游离钙离子浓度接近0 mmol/L时也存在。PKA的特异性抑制剂H-89(3 μmol/L)和 PKG的特异性抑制剂KT-5823(1 μmol/L)对川芎嗪激活BKCa通道的作用无影响。以上结果提示:川芎嗪能直接激活冠状动脉平滑肌BKCa通道,这种作用可能是川芎嗪扩张冠状动脉血管的一种重要机制。     

Voltage-gated K(+)-channel activity in ovine pulmonary vasculature is developmentally regulated

To examine mechanisms underlying developmental changes in pulmonary vascular tone, we tested the hypotheses that 1) maturation-related changes in the ability of the pulmonary vasculature to respond to hypoxia are intrinsic to the pulmonary artery (PA) smooth muscle cells (SMCs); 2) voltage-gated K(+) (K(v))-channel activity increases with maturation; and 3) O(2)-sensitive Kv2.1 channel expression and message increase with maturation. To confirm that maturational differences are intrinsic to PASMCs, we used fluorescence microscopy to study the effect of acute hypoxia on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in SMCs isolated from adult and fetal PAs. Although PASMCs from both fetal and adult circulations were able to sense an acute decrease in O(2) tension, acute hypoxia induced a more rapid and greater change in [Ca(2+)](i) in magnitude in PASMCs from adult compared with fetal PAs. To determine developmental changes in K(v)-channel activity, the effects of the K(+)-channel antagonist 4-aminopyridine (4-AP) were studied on fetal and adult PASMC [Ca(2+)](i). 4-AP (1 mM) caused PASMC [Ca(2+)](i) to increase by 94 +/- 22% in the fetus and 303 +/- 46% in the adult. K(v)-channel expression and mRNA levels in distal pulmonary arteries from fetal, neonatal, and adult sheep were determined through the use of immunoblotting and semiquantitative RT-PCR. Both Kv2.1-channel protein and mRNA expression in distal pulmonary vasculature increased with maturation. We conclude that there are maturation-dependent changes in PASMC O(2) sensing that may render the adult PASMCs more responsive to acute hypoxia.     

Cyclic strain stimulates monocyte chemotactic protein-1 mRNA expression in smooth muscle cells

Hemodynamic forces are important determinants for the formation of atherosclerotic plaques. The recruitment of circulating monocytes into the arterial wall is an important step during atherogenesis. Monocyte chemotactic protein-1 (MCP-1) has been shown to be a key factor for monocyte transmigration. This study examined the effects of cyclic strain on MCP-1 mRNA expression levels of cultured rat aortic smooth muscle cells. The MCP-1 mRNA levels of aortic smooth muscle cells first increased as the duration of cyclic strain increased, reaching the maximum at 6-12 h, maintained at high levels throughout the 48-h strain period. To explore signaling pathways mediating cyclic strain-stimulated MCP-1 mRNA expression, we examined the involvement of tyrosine kinase and protein kinase C (PKC). Tyrosine kinase inhibitors, genistein and tyrphostin 51, at 50 microM blocked cyclic strain-stimulated MCP-1 mRNA expression. Preincubation with a PKC activator, phorbol 12-myristate 13-acetate (PMA), 2 microM, for 24 h to downregulate PKC did not decrease cyclic strain-induced MCP-1 mRNA expression. A 6-h incubation with 0. 1 microM PMA to activate PKC, which stimulated MCP-1 expression when applied alone, abolished the stimulatory effects of cyclic strain. A specific PKC inhibitor, calphostin C (0.1 microM), diminished cyclic strain-stimulated MCP-1 mRNA expression. Angiotensin II at 10 or 1,000 nM induced a moderate upregulation of MCP-1 mRNA, and no synergistic effects were observed between angiotensin II and cyclic strain. These results indicate that cyclic strain stimulates MCP-1 mRNA expression in smooth muscle cells through signaling pathway(s) mediated by tyrosine kinase activation.     

Inhibition of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilisation by phorbol ester in rat cultured vascular smooth muscle cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 microM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+]i, were 7.8 +/- 0.3 M and 1 microM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 microM PMA for either 1 or 24 h did not significantly change the K(D) and Bmax of the BK receptor for binding (control: K(D) = 1.7 +/- 0.2 nM; Bmax = 47.3 +/- 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs.     

New mechanisms of pulmonary arterial hypertension: role of Ca²⁺ signaling

Pulmonary arterial hypertension (PAH) is a severe and progressive disease that usually culminates in right heart failure and death if left untreated. Although there have been substantial improvements in our understanding and significant advances in the management of this disease, there is a grim prognosis for patients in the advanced stages of PAH. A major cause of PAH is increased pulmonary vascular resistance, which results from sustained vasoconstriction, excessive pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular stiffness. In addition to other signal transduction pathways, Ca(2+) signaling in pulmonary artery smooth muscle cells (PASMCs) plays a central role in the development and progression of PAH because of its involvement in both vasoconstriction, through its pivotal effect of PASMC contraction, and vascular remodeling, through its stimulatory effect on PASMC proliferation. Altered expression, function, and regulation of ion channels and transporters in PASMCs contribute to an increased cytosolic Ca(2+) concentration and enhanced Ca(2+) signaling in patients with PAH. This review will focus on the potential pathogenic role of Ca(2+) mobilization, regulation, and signaling in the development and progression of PAH.     

Cyclic GMP-dependent and cyclic AMP-dependent protein kinases, protein kinase modulators and phosphodiesterases in arteries and veins of dogs. Distribution and effects of arteriovenous fistula and arterial occlusion.

Possible involvement of cyclic GMP-dependent and cyclic AMP-dependent protein kinases, protein kinase modulators and cyclic nucleotide phosphodiesterases in functions of vascular tissues were investigated in the dog. All of the above activities, localized in the smooth muscle-rich inner layer of the blood vessels, were found to be higher in the arteries than in the veins. The peripheral arteries were disproportionately richer in cyclic GMP-dependent protein kinase (as indicated by high ratios of cyclic GMP-dependent to cyclic AMP-dependent protein kinase) than were the veins, with the exception of the pulmonary artery, an atypical arterial tissue exposed to low blood pressure. Interestingly, the protein kinase ratio for the aorta, an artery with no significant role in blood pressure regulation, was not higher than that for the vena cava. Creation of femoral arteriovenous fistulae in the dogs led to preferential reductions in the cyclic GMP-dependent enzyme activity both in the proximal and distal arteries, whereas it was elevated in the stressed vein distal to the anastomotic site. The cyclic GMP-dependent enzyme was preferentially reduced in the saphenous artery distal to occlusion. Changes in the cyclic GMP-dependent enzyme activity appeared to precede gross atrophy or hypertrophy of the vessels. It is suggested that the vascular cyclic GMP-dependent protein kinase may be closely related to peripheral resistance and its regulation.     

Neuropeptide Y-induced intracellular Ca2+ increases in vascular smooth muscle cells

The effect of neuropeptide Y (NPY) on cytosolic free Ca2+ concentration ([Ca2+]i) was studied in cultured smooth muscle cells from porcine aorta (PASMC) and compared with the effect of bradykinin (BK) and angiotensin II (ATII) on [Ca2+]i. All peptides induced dose-dependent and transient rises in [Ca2+]i which were not blocked by extracellular EGTA, but the NPY response was different from the others' as follows. First, the [Ca2+]i rise induced by NPY was not as rapid as that induced by BK or ATII. Second, pertussis toxin abolished the [Ca2+]i rise induced by NPY, but not by BK or ATII. Third, following initial treatment with BK, PASMC were able to respond to NPY, but not to ATII. Finally, BK and ATII, but not NPY, significantly increased inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) generation. Although NPY attenuated forskolin-induced accumulation of cyclic AMP, forskolin- and 3-isobutyl-1-methyl-xanthine-induced alterations in intracellular cyclic AMP did not affect the NPY-induced [Ca2+]i rise. These results suggest that NPY increases [Ca2+]i by a pertussis toxin-sensitive GTP binding protein-involved mechanism which is not mediated by the intracellular messengers such as Ins(1,4,5)P3 and cyclic AMP.     

Cyclic GMP-dependent and cyclic AMP-dependent protein kinases,protein kinase modulators and phosphodieterases in arteries and veins of dogs Distribution and effects of arteriovenous fistula and arterial occlusion

Possible involvement of cyclic GMP-dependent and cyclic AMP-dependent protein kinases, protein kinase modulators and cyclic nucleotide phosphodiesterases in functions of vascular tissues were investigated in the dog. All of the above activities, localized in the smooth muscle-rich inner layer of the blood vessels, were found to be higher in the arteries than in the veins. The peripheral arteries were disproportionately richer in cyclic GMP-dependent protein kinase (as indicated by high ratios of cyclic GMP-dependent to cyclic AMP-dependent protein kinase) than were the veins, with the exception of the pulmonary artery, an atypical arterial tissue exposed to low blood pressure. Interestingly, the protein kinase ratio for the aorta, an artery with no significant role in blood pressure regulation, was not higher than that for the vena cava. Creation of femoral arteriovenous fistulae in the dogs led to preferential reductions in the cyclic GMP-dependent enzyme activity both in the proximal and distal arteries, whereas it was elevated in the stressed vein distal to the anastomotic site. The cyclic GMP-dependent enzyme was preferentially reduced in the saphenous artery distal to occlusion. Changes in the cyclic GMP-dependent enzyme activity appeared to precede gross atrophy or hypertrophy of the vessels. It is suggested that the vascular cyclic GMP-dependent protein kinase may be closely related to peripheral resistance and its regulation.     

Inhibitory and stimulatory effects of phorbol ester on vasopressin-induced cellular responses in cultured rat aortic smooth muscle cells

In rat aortic smooth muscle cells, vasopressin (AVP) induces prostacyclin (PGI2) production, probably as the consequence of phospholipase C activation. Our study analyzes the effects of phorbol 12-myristate 13-acetate (PMA)-induced protein kinase C (PKC) activation on AVP-induced inositol 1,4,5-trisphosphate formation, cytosolic free Ca2+ concentration [( Ca2+]c), and PGI2 production. PMA rapidly decreased PKC activity in the cytosol of smooth muscle cells, while increasing it transiently in the membranes with a maximum around 20 min. Prior exposure of the cells to PMA resulted in a transient inhibition of both AVP-induced inositol 1,4,5-trisphosphate formation and [Ca2+]c rise. This was inversely correlated with membraneous PKC activity and partially reversed by the PKC inhibitor staurosporine. In contrast, pretreating the cells with PMA markedly potentiated A23187 or AVP-induced PGI2 production. Under those conditions, AVP-induced PGI2 production did not correlate either with PMA-induced membranous PKC activity or with AVP-induced PLC activation. However, this potentiating effect of PMA was reversed by staurosporine and was not mimicked by the 4 alpha-phorbol, an inactive analogue of PMA. Thus, the possibility is raised that, while inhibiting AVP-induced PLC activation, PMA-induced PKC activation increases the Ca2+ sensitivity of the cellular signaling system leading to PGI2 production.