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     

Protein kinase C inhibits insulin-induced Akt activation in vascular smooth muscle cells.

Protein kinase C (PKC) activation, enhanced by hyperglycemia, is associated with many tissue abnormalities observed in diabetes. Akt is a serine/threonine kinase that mediates various biological responses induced by insulin. We hypothesized that the negative regulation of Akt in the vasculature by PKC could contribute to insulin resistant states and, may therefore play a role in the pathogenesis of cardiovascular disease. In this study, we specifically looked at the ability of PKC to inhibit Akt activation induced by insulin in cultured rat aortic vascular smooth muscle cells (VSMCs). Activation of Akt was determined by immunoblotting with a phospho-Akt antibody that selectively recognizes Ser473 phosphorylated Akt. A PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited insulin-dependent Akt phosphorylation. However, PMA did not inhibit platelet-derived growth factor (PDGF)-induced activation of Akt. We further showed that the PKC inhibitor, G06983, blocked the PMA-induced inhibition of Akt phosphorylation by insulin. In addition, we demonstrated that PMA inhibited the insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). From these data, we conclude that PKC is a potent negative regulator of the insulin signal in the vasculature, which indicate an important role of PKC in the development of insulin resistance in cardiovascular disease.     

Protein kinase C translocation in intact vascular smooth muscle strips.

Using intact muscle strips from the bovine carotid artery, the time course of translocation of protein kinase C (PKC) from the cytosol to the membrane fraction was measured in response to various agonists that induce contractile responses. PKC activity was assessed by Ca2+/phospholipid-dependent phosphorylation of histone. Exposure of the muscle strips to phorbol ester (12-deoxyphorbol 13-isobutyrate) induced a rapid and sustained translocation of PKC from the cytosol to the membrane fraction, and a slowly developing but sustained contractile response. Histamine induced a comparable initial translocation of PKC to the membrane which then decreased somewhat to a stable plateau significantly above basal values. Histamine also led to a rapid and sustained increase in tension. Angiotensin I, which caused a rapid but transient contraction, induced a rapid initial translocation of PKC to the membrane. The membrane-associated PKC then declined to a stable plateau significantly lower than that seen after a histamine-induced response, and only slightly above the basal value. Endothelin, which induced a sustained contraction, caused a sustained translocation of PKC from the cytosol to the membrane. In contrast, although exposure to 35 mM-KCl induced a rapid and sustained contraction, it caused only a transient translocation of PKC; the membrane-associated PKC returned to its basal value within 20 min. These results demonstrate that PKC in intact smooth muscle can be rapidly translocated to the membrane and remains membrane-bound during sustained phorbol ester- or agonist-induced contractions, but that such a sustained translocation of PKC does not occur during prolonged stimulation with KCl.     

Protein kinase C activation stimulates plasma membrane Ca2+ pump in cultured vascular smooth muscle cells

We examined the effect of phorbol myristate acetate (PMA), a potent activator of protein kinase C, on Ca2+ extrusion from cultured vascular smooth muscle cells (VSMCs) incubated in the absence of added extracellular Na+ (Na+o). Previously, strong experimental evidence was presented that the Na+o-independent Ca2+ extrusion from VSMCs is effected by the plasma membrane Ca2+ pump (Furukawa, K.-I., Tawada, Y., and Shigekawa, M. (1988) J. Biol. Chem. 263, 8058-8065). Brief (2 min) pretreatment of VSMCs with 30-300 nM PMA suppressed the intracellular Ca2+ transient induced with 1 microM ionomycin to about 60% of the control, whereas it accelerated the concomitant Na+o-independent 45Ca2+ extrusion by up to 20%. When the Ca2+ transient was induced with 0.1 microM angiotensin II, the PMA pretreatment markedly suppressed it and reduced also the rate of 45Ca2+ efflux from cells slightly. These effects of PMA were mimicked by 1-oleoyl-2-acetylglycerol, another protein kinase C activator, but were abolished by prior treatment of cells with staurosporine, an inhibitor of protein kinase C, or prior long incubation of cells with PMA. Analysis of the effect of PMA on [Ca2+]i dependence of the rate of Na+o-independent 45Ca2+ efflux revealed that PMA increased the maximum Ca2+ efflux rate without a significant change in the affinity for Ca2+. These results strongly suggest that the plasma membrane Ca2+ pump in VSMCs can be stimulated by PMA and that protein kinase C is involved in regulation of [Ca2+]i in intact VSMCs.     

Tenascin C may regulate the recruitment of smooth muscle cells during coronary artery development

Tenascin C (TNC) is an extracellular glycoprotein that is thought to be involved in tissue remodeling during organogenesis and regeneration. Using avian embryonic hearts, we investigated the spatiotemporal expression patterns of TNC during the formation of the proximal coronary artery. Immunohistochemistry showed that TNC was deposited around the developing coronary stem and that TNC colocalized with vascular smooth muscle α-actin. A quail-chick chimera, in which a quail proepicardial organ (PEO) had been transplanted, showed that quail tissue-derived cells contributed to the establishment of the endothelial and mural cells of the proximal coronary artery, and the quail tissue-derived mural cells displayed TNC. Proepicardial cells cultured in TNC showed the myofibroblast/smooth muscle cell phenotype and neutralizing anti-TNC antibody suppressed the expression of smooth muscle markers. These observations suggest that TNC plays a role in the mural smooth muscle development of the nascent proximal coronary artery.     

蛋白激酶C和蛋白酪氨酸激酶介导脂多糖及细胞因子诱导大鼠血管平滑肌细胞一氧化氮的生成

采用Spprague-Dawley大鼠胸主动脉中膜、外膜和培养的血管平滑肌细胞(VSMCs)作材料,鉴定不同类型的血管组织经炎性介质刺激后其一氧化氮(NO)的产生来源,闻明蛋白激酶C(PKC)和蛋白酪氨酸激酶(PTK)介导大鼠VSMCs生成NO的调控机制。大鼠VSMCs经脂多糖(LPG)和细胞因子(TNF-α,IL-1β)处理后,以剂量依赖方式促进NO释放。采用Western Blot证实经刺激的VSMCs伴有iNOS表达上调。进一步实验表明PKC和PTK参与LPS和细胞因子诱导NO生成的胞内信号转导。用PKC抑制剂H7与VSMCs共培育,H7能明显减少LPS、TNF-α和IL-1β诱导细胞NO的形成。白屈菜赤碱亦可抑制NO的生成,但HAl004对VSMCs的NO生成无抑制作用,提示PKC参与NO的生成与调控。PTK抑制剂genistein和tyrphostin AG18均能抑制由LPS、TNF-α和IL-1β引发VSMCs释放NO,同时伴iNOS蛋白表达下调,而PKC抑制剂不能阻断iNOS的表达。上述观察结果提示,PKC介导LPS和细胞因子诱导细胞合成NO可能是通过iNOS翻译后加工;而PTK则以上调iNOS表达而促增NO生成。     

Protein kinase C in the regulation of smooth muscle contraction

The cellular and molecular mechanisms underlying smooth muscle contraction are reviewed in the light of recent studies of smooth muscle ultrastructure and of the role of polyphosphoinositide turnover and protein kinase C function in smooth muscle contraction. A new model of smooth muscle contraction is proposed that differs radically from accepted views, particularly the latch bridge hypothesis, in terms of both Ca2+ messenger function and the molecular events underlying this process. A coordinate fibrillar domain model of contraction is proposed in which the initial and sustained phases of contraction are mediated by different cellular and molecular events. The initial phase of response is mediated by a rise in [Ca2+]c and the resulting calmodulin-dependent activation of both myosin light chain kinase and the dissociation of caldesmon from the actin-caldesmon-tropomyosin-myosin fibrillar domain. These events lead to an interaction between actin and the phosphorylated light chains of myosin just as in previous models. However, this initial phase is followed by a sustained phase in which a rise in [Ca2+]sm stimulates the plasma membrane-associated, Ca2+-sensitive form of protein kinase C that results in the phosphorylation of both structural and regulatory components of the filamin-actin-desmin fibrillar domain. These events underlie the tonic phase of contraction.     

Protein tyrosine kinase activity in cultured vascular smooth muscle cells (VSMC) from rat aorta

• 1.1. Protein tyrosine kinase (PTK) activities were detected in both cytosolic and particulate fractions of cultured vascular smooth muscle cells by using poly (Glu: Tyr; 4:1) as an exogenous substrate.
• 2.2. The percent distribution of the enzyme activity between these two fractions was 70 and 30 respectively.
• 3.3. The particulate and not the cytosolic enzyme activity was stimulated by about 4-fold in the presence of non-ionic detergent, Triton X-100 (0.5% v/v).
• 4.4. The PTK activity in both the fractions was absolutely dependent on the presence of divalent cations such as Mg²⁺ and Mn²⁺ which were equipotent in the activation of the enzyme.These data indicate that PTK activity is expressed in cultured VSMC and provide a basis for further studies to examine a possible role of PTKs in growth and proliferation of VSMC.

     

Endothelin stimulates diacylglycerol accumulation and activates protein kinase C in cultured vascular smooth muscle cells

Endothelin, a novel peptide isolated from the conditioned medium of endothelial cells, causes a slow, sustained contraction of vascular smooth muscle, but its mechanism of action remains unclear. To determine whether the diacylglycerol/protein kinase C signalling pathway is stimulated by endothelin, we exposed cultured rat aortic smooth muscle cells to endothelin and measured diacylglycerol accumulation and protein kinase C-dependent protein phosphorylation. Endothelin stimulated a dose-dependent, biphasic increase in diacylglycerol, which was sustained for at least 20 min. This peptide also induced a prolonged phosphorylation of an acidic protein with a molecular weight of 76,000, which was detectable by 30 s and sustained for at least 20 min. This phosphorylation could be mimicked by phorbol 12-myristate 13-acetate, but not by ionomycin, and was markedly reduced when protein kinase C was down-regulated by a 24-h pretreatment with phorbol 12,13-dibutyrate. These results suggest that endothelin causes a robust stimulation of the diacylglycerol/protein kinase C pathway in cultured vascular smooth muscle cells, and that this mechanism may contribute importantly to the physiologic events stimulated by endothelin in intact blood vessels, including slow, tonic contraction and Ca2+ influx.     

Protein kinase C reduces the KCa current of rat tail artery smooth muscle cells

The hypothesis that protein kinase C (PKC) isable to regulate the whole cell Ca-activated K(K_Ca) current independently of PKC effects on local Ca release events was tested using the patch-clamp technique and freshly isolated rat tail artery smooth muscle cells dialyzed with a strongly buffered low-Ca solution. The active diacylglycerol analog1,2-dioctanoyl-sn-glycerol (DOG) at 10 µM attenuated the current-voltage(I-V)relationship of the K_Ca current significantly and reduced the K_Cacurrent at +70 mV by 70 ± 4% (n = 14). In contrast, 10 µM DOG after pretreatment of the cells with 1 µM calphostin C or 1 µM PKC inhibitor peptide, selective PKCinhibitors, and 10 µM1,3-dioctanoyl-sn-glycerol, aninactive diacylglycerol analog, did not significantly alter theK_Ca current. Furthermore, thecatalytic subunit of PKC (PKC_C)at 0.1 U/ml attenuated theI-Vrelationship of the K_Ca currentsignificantly, reduced the K_Cacurrent at +70 mV by 44 ± 3% (n = 17), and inhibited the activity of singleK_Ca channels at 0 mV by 79 ± 9% (n = 6). In contrast, 0.1 U/mlheat-inactivated PKC_C did notsignificantly alter the K_Cacurrent or the activity of singleK_Ca channels. Thus these resultssuggest that PKC is able to considerably attenuate theK_Ca current of freshly isolatedrat tail artery smooth muscle cells independently of effects of PKC onlocal Ca release events, most likely by a direct effect on theK_Ca channel.     

Gender-related distinctions in protein kinase C activity in rat vascular smooth muscle

Gender differences in vascular reactivity have been suggested; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the gender differences in vascular reactivity reflect gender-related, possibly estrogen-mediated, distinctions in the expression and activity of specific protein kinase C (PKC) isoforms in vascular smooth muscle. Aortic strips were isolated from intact and gonadectomized male and female Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Isometric contraction was measured in endothelium-denuded aortic strips. PKC activity was measured in the cytosolic and particulate fractions, and the amount of PKC was measured using Western blots and isoform-specific anti-PKC antibodies. In intact male WKY rats, phenylephrine (Phe, 10(-5) M) and phorbol 12,13-dibutyrate (PDBu, 10(-6) M) stimulated contraction to 0.37 +/- 0.02 and 0.42 +/- 0.02 g/mg tissue wt, respectively. The basal particulate/cytosolic PKC activity ratio was 0.86 +/- 0.06, and Western blots revealed alpha-, delta-, and zeta-PKC isoforms. Phe and PDBu increased PKC activity and caused significant translocation of alpha- and delta-PKC from the cytosolic to particulate fraction. In intact female WKY rats, basal PKC activity, the amount of alpha-, delta-, and zeta-PKC, the Phe- and PDBu-induced contraction, and PKC activity and translocation of alpha- and delta-PKC were significantly reduced compared with intact male WKY rats. The basal PKC activity, the amount of alpha-, delta-, and zeta-PKC, the Phe and PDBu contraction, and PKC activity and alpha- and delta-PKC translocation were greater in SHR than WKY rats. The reduction in Phe and PDBu contraction and PKC activity in intact females compared with intact males was greater in SHR ( approximately 30%) than WKY rats ( approximately 20%). Phe and PDBu contraction and PKC activity were not significantly different between castrated males and intact males but were greater in ovariectomized (OVX) females than intact females. Treatment of OVX females or castrated males with 17 beta-estradiol, but not 17 alpha-estradiol, subcutaneous implants caused significant reduction in Phe and PDBu contraction and PKC activity that was greater in SHR than WKY rats. Phe and PDBu contraction and PKC activity in OVX females or castrated males treated with 17 beta-estradiol plus the estrogen receptor antagonist ICI-182,780 were not significantly different from untreated OVX females or castrated males. Thus a gender-related reduction in vascular smooth muscle contraction in female WKY rats with intact gonads compared with males is associated with reduction in the expression and activity of vascular alpha-, delta-, and zeta-PKC. The gender differences in vascular smooth muscle contraction and PKC activity are augmented in the SHR and are possibly mediated by estrogen.     

Endothelin stimulates phospholipase C in cultured vascular smooth muscle cells

Cultured vascular smooth muscle cells from bovine and rat thoracic aortae and from human omental vessels have been examined for cellular responses to endothelin. In myo-[3H]-inositol-prelabelled cells endothelin induced a rapid (within 30 sec) and protracted increase of [3H]-inositol content in inositol bis- and tris-phosphates. Concomitantly, significant polyphosphoinositide hydrolysis occurred within 30 sec. Accumulation of [3H]-inositol monophosphate and hydrolysis of phosphatidylinositol were delayed. In cells prelabelled with [3H]-arachidonic acid endothelin promoted rapid production of [3H]-diacylglycerol which decayed slowly toward control values after reaching maximum levels (1-2 min). Half-maximally effective concentrations of endothelin for all these cellular responses were comparable (approximately 3-7 nM) and not significantly different between the vascular cell isolates. The involvement of the phospholipase C-signal transduction pathway in mediating endothelin-induced vasoconstriction is invoked.     

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.     

Age-related decrease of protein kinase G activation in vascular smooth muscle cells

The p21 (cip1/waf1) protein induces cell cycle arrest through inhibition of the activity of cdk (cyclin dependent kinase)/cyclin complexes. Expression of p21 is induced in a p53-dependent manner by DNA damage. p21 can also be induced independently of p53 by phorbol ester or okadaic acid. In this study, we have addressed the role of the PKC (protein kinase C) signaling pathway in the induction of p21 in response to PMA (phorbol myristate acetate) and okadaic acid. Levels of p21 (protein and mRNA) rapidly increased (within approximately 4 h) in U937 cells treated with PMA. The PKC-specific inhibitors RO 31-8220 and GF109203X down-regulated PMA or okadaic acid-induced p21 expression. Following persistent PKC activation, p21 mRNA levels remained elevated, indicating an enhanced stability of the mRNA. Using actinomycin D to measure mRNA stability and p21 promoter luciferase assays to measure activity, we provide evidence to support a role for the PKC signaling pathway in p21 mRNA stability. Thus, PKC regulates the amount of p21 in U937 cells at the level of mRNA accumulation and translation.