Phorbol ester modulates serotonin-stimulated phosphoinositide breakdown in cultured vascular smooth muscle cells

Stimulation of cultured rabbit aortic vascular smooth muscle cells (VSMC) with serotonin (5HT) induced a rapid generation of inositol phosphates from receptor-mediated hydrolysis of inositol phospholipids. Pretreatment of these cells with 500ng/ml of pertussis toxin for 24h prior to addition of 5HT reduced 5HT-induced formation of inositol phosphates. Phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol-12,13-dibutyrate (PDBu), are known to activate protein kinase C (PKC), but their role on cultured VSMC stimulated by 5HT has not been defined. TPA exhibited a rapid inhibition of 5HT-stimulated phosphoinositide breakdown, although 4 alpha-phorbol-12,13-didecanoate (4 alpha PDD), an inactive phorbol ester, did not inhibit it. These data suggest that a guanine nucleotide inhibitory (Gi) protein couples 5HT receptor to phospholipase C and TPA modulates 5HT-stimulated hydrolysis of inositol phospholipids in cultured VSMC through activation of PKC.     

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.     

Phorbol ester effects on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured vascular smooth muscle cells

Tumor promoting phorbol esters stimulate Ca++ phospholipid-dependent protein kinase C. It has been suggested that this enzyme regulates the functional properties of different cell membrane receptors. In this study we investigated the effect of phorbol esters on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured smooth muscle cells derived from rabbit aorta. Treatment of these cells with biologically active phorbol esters for 15 min. to 2 hours caused a marked decrease of norepinephrine stimulation of inositol phospholipid metabolism and a 3 fold decrease in agonist affinity for 125I-HEAT binding to alpha 1-adrenoceptors in the intact smooth muscle cells. The ability of phorbol esters to modulate alpha 1-adrenoceptor responsiveness suggests that activation of protein kinase C may represent an important mechanism regulating alpha 1-adrenergic receptor functional properties.     

Phorbol ester downregulates PDGFbeta receptor via PKCbeta1 in vascular smooth muscle cells

The role of protein kinase C (PKC) and their isoforms in cell growth regulation remains elusive. Here we showed that in cultured human vascular smooth muscle cells (SMC), the PKC stimulator phorbol 12-myristate 13-acetate (PMA) inhibited [(3)H]thymidine incorporation in response to the growth factor PDGF associated with downregulation of PDGFbeta (but not alpha) receptors, which was recovered to normal level after PKC was depleted. The changes in PDGFbeta receptor were inversely correlated with PKCbeta1 protein levels regulated by PMA. The downregulation of PDGFbeta receptor by PMA was fully prevented by the PKCbeta inhibitor LY379196, however, without recovery of [(3)H]thymidine incorporation to PDGF. In contrast, [(3)H]thymidine incorporation was fully recovered after depletion of PKCs. These results indicate that in human SMC PKCbeta1 mediates PDGFbeta receptor downregulation. Other PKC isoforms activated by phorbol ester also contribute to the inhibitory effects on cell growth.     

Characterization of phosphatidylinositol-specific phospholipase C from cultured vascular smooth muscle cells.

Phosphoinositide-specific phospholipase C (PLC) activities have been partially purified from cultured vascular smooth muscle cells and analyzed for substrate specificity, calcium and pH requirements, and molecular weight. The purification procedure involved DEAE-cellulose and heparin-Sepharose chromatographies followed by Mono Q and size exclusion high performance liquid chromatography. This technique resolves multiple peaks of activity using phosphatidylinositol (PI) and PI 4,5-bisphosphate (PIP2) as substrates. The major peak was purified to near homogeneity as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PLC activity in vascular smooth muscle cells can be divided into two types based on their calcium and pH requirements, substrate preferences, and molecular weights. The low molecular weight PLC hydrolyzes both PI and PIP2, has a molecular mass of 58 kDa, requires the most calcium for full activation, and has a PI-pH profile that shifts slightly with calcium concentration. Screening a cDNA library with oligonucleotides directed against several of the known PLCs identified a highly expressed PLC cDNA that is 99% homologous to PLC-alpha, suggesting that this low molecular weight peak in fact corresponds to PLC-alpha. The high molecular mass peak (157 kDa) shows much greater activity against PI than PIP2, is active at lower calcium concentrations, and has a PI-pH optimum of 5.0 regardless of calcium concentration. Each of the PIP2 PLC activities is strongly dependent on the relative levels of calcium and pH in the assay buffer. These observations suggest that vascular smooth muscle contains both a high and low molecular weight PLC whose activities are affected markedly by the changes in calcium and pH accompanying hormonal stimulation of the cell.     

Activation of phospholipase A2 by endothelin in cultured vascular smooth muscle cells

The ability of endothelin to promote phospholipid hydrolysis has been studied in myo-[2-3H]-inositol-, [3H]-arachidonic acid- or methyl-[3H]choline chloride-prelabelled cultured vascular smooth muscle cells (VSMC) from rat and bovine thoracic aortae and human omental vessels. The biochemical responses to endothelin were comparable between the different VSMC isolates. Endothelin promoted the accumulation of glycerolphospho[3H]inositol and concomitant loss of [3H]-inositol label from phosphatidylinositol. Exposure of [3H]choline-labelled VSMC to endothelin resulted in a loss of radioactivity from phosphatidylcholine that was inversely parallelled by an increase in water-soluble [3H]-choline metabolites. In [3H]-arachidonic acid ([3H]-AA)-labelled VSMC, endothelin induced extracellular release of [3H]-AA which derived from both phosphatidylcholine and phosphatidylinositol. Half-maximally effective concentrations of endothelin for all these responses were approximately 2-7 nM and did not vary between VSMC types. Endothelin-induced release of [3H]-AA into VSMC medium-overlay was inhibited by quinacrine and nordihydroguaiaretic acid but not by neomycin or indomethacin. The data herein implicate activation of phospholipase A2 by endothelin with subsequent metabolism of arachidonic acid via the lipoxygenase pathway.     

Phospholipase D in cultured rat vascular smooth muscle cells and its activation by phorbol ester

We determined the phospholipase D (PLD) activity in rat vascular smooth muscle cells by the formation of phosphatidylethanol in cells prelabeled with [3H] myristic acid. The enzyme was markedly activated by a phorbol ester (TPA). Down regulation of protein kinase C (PKC) resulted in almost complete inhibition indicating PKC-dependent mechanism of its activation. Depletion of calcium by EGTA and TMB-8 caused 53% inhibition. Chelator-stable association of PKC to membrane by TPA was observed in the absence of extracellular Ca2+. The mitogenic peptide PDGF also caused a marked stimulation of PLD. These results indicate that PLD in vascular smooth muscle cells is stimulated by TPA through the activation of PKC both by calcium-dependent and independent mechanisms.     

Functional angiotensin II receptors in cultured vascular smooth muscle cells

To study cellular mechanisms influencing vascular reactivity, vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the rat mesenteric artery, a highly reactive, resistance-type blood vessel, and established in primary culture. Cellular binding sites for the vasoconstrictor hormone angiotensin II (AII) were identified and characterized using the radioligand 125I-angiotensin II. Freshly isolated VSMC, and VSMC maintained in primary culture for up to 3 wk, exhibited rapid, saturable, and specific 125I-AII binding similar to that seen with homogenates of the intact rat mesenteric artery. In 7-d primary cultures, Scatchard analysis indicated a single class of high-affinity binding sites with an equilibrium dissociation constant (Kd) of 2.8 +/- 0.2 nM and a total binding capacity of 81.5 +/- 5.0 fmol/mg protein (equivalent to 4.5 x 10(4) sites per cell). Angiotensin analogues and antagonists inhibited 125I-AII binding to cultured VSMC in a potency series similar to that observed for the vascular AII receptor in vivo. Nanomolar concentrations of native AII elicited a rapid, reversible, contractile response, in a variable proportion of cells, that was inhibited by pretreatment with the competitive antagonist Sar1,Ile8-AII. Transmission electron microscopy showed an apparent loss of thick (12-18 nm Diam) myofilaments and increased synthetic activity, but these manifestations of phenotypic modulation were not correlated with loss of 125I-AII binding sites or hormonal responsiveness. Primary cultures of enzymatically dissociated rat mesenteric artery VSMC thus may provide a useful in vitro system to study cellular mechanisms involved in receptor activation-response coupling, receptor regulation, and the maintenance of differentiation in vascular smooth muscle.     

Phorbol ester modulates serotonin receptor-mediated increases in inositol phosphate production and calcium mobilization in cultured rat vascular smooth muscle cells

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on serotonin-induced inositol phosphate (IP) accumulation and intracellular free Ca2+ concentrations [( Ca2+]i) was investigated in cultured rat vascular smooth muscle cells. Pretreatment with TPA had no effect on basal levels of both IP production and [Ca2+]i, whereas it significantly attenuated serotonin-induced increases in both IP production and [Ca2+]i. These data suggest that protein kinase C is involved in the negative feedback control of serotonin-induced rises in both IP production and [Ca2+]i.     

Amiloride sensitive activation of S6 kinase by angiotensin II in cultured vascular smooth muscle cells

Angiotensin II was shown to activate S6-kinase in cultured vascular smooth muscle cells (VSMC) in a dose- (10(-9)-10(-6) M) and time-dependent manner. Pretreatment of quiescent cells with 12-O-Tetradecanoylphorbol-13-acetate had no effect on the activation levels of the kinase at the hormone levels used. However, stimulation of S6-kinase activity by angiotensin II was markedly inhibited by the inclusion of amiloride hydrochloride in serum-free medium during activation procedures. Angiotensin was not mitogenic for VSMC at even the highest doses used (10(-6) M). These findings support the notion that raised intracellular pH results in the activation of protein synthesis in quiescent cells.     

Attenuation of hen granulosa cell steroidogenesis by a phorbol ester and 1-oleoyl-2-acetylglycerol

A series of studies was conducted to evaluate the effects of phorbol esters and a diacylglycerol analog on basal and hormone-stimulated steroidogenesis in granulosa cells from the largest preovulatory follicle of the domestic hen. Agents that previously have been shown to activate protein kinase C, such as the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), and the synthetic diacylglycerol analog, 1-oleoyl-2-acetylglycerol (OAG), suppressed luteinizing hormone (LH)-induced progesterone (PMA at levels of 10 and 100 ng/tube; OAG at levels of 10 and 25 micrograms/tube), and androgen (10 and 100 ng PMA; 25 micrograms OAG) production, but had no effect on basal levels of either steroid. Furthermore, PMA decreased the ability of vasoactive intestinal peptide to induce steroidogenesis, suggesting that protein kinase C activation may generally modulate the activity of hormones that act via the adenylyl cyclase/cyclic 3',5'-adenosine monophosphate (cAMP) second messenger system. In further support of this proposal was the finding that PMA and OAG decreased the production of cAMP in response to LH, and attenuated the steroidogenic response in granulosa cells exposed to 10 mM 8-bromo-cAMP. By contrast, the induction of calcium mobilization using a calcium ionophore (A23187; 0.5-2.0 microM) stimulated progesterone and androgen production without increasing intracellular levels of cAMP, and this stimulatory effect on steroidogenesis was not inhibited by the presence of 100 ng PMA/tube. From these data, we suggest that the activation of protein kinase C in granulosa cells of the hen may provide a physiological mechanism by which receptor-mediated steroidogenesis, involving the adenylyl cyclase second messenger system, is modulated.     

Induction of 5-lipoxygenase activation in polymorphonuclear leukocytes by 1-oleoyl-2-acetylglycerol

1,2-Diacylglycerols (DAGs) can prime polymorphonuclear leukocytes (PMNL) for enhanced release of arachidonic acid (AA) and generation of 5-lipoxygenase (5-LO) products upon subsequent agonist stimulation. Here, we demonstrate that in isolated human PMNL, 1-oleoyl-2-acetylglycerol (OAG) functions as a direct agonist stimulating 5-LO product formation (up to 42-fold). OAG caused no release of endogenous AA, but in the presence of exogenous AA, the magnitude of 5-LO product synthesis induced by OAG was comparable to that obtained with the Ca(2+)-ionophore A23187. Interestingly, OAG-induced 5-LO product synthesis was not connected with increased 5-LO nuclear membrane association. Examination of diverse glycerides revealed that the sn-2-acetyl-group is important, thus, also 1-O-hexadecyl-2-acetylglycerol (EAG) stimulated 5-LO product formation (up to 8-fold).Treatment of PMNL with OAG did not alter the mobilization of Ca(2+) but removal of intracellular Ca(2+) abolished the upregulatory OAG effects. Notably, the PKC activator phorbol-myristate-acetate hardly increased 5-LO product synthesis and PKC inhibitors failed to suppress the effects of OAG. Although OAG rapidly activated p38 MAPK and p42/44(MAPK), which can stimulate 5-LO for product synthesis, specific inhibitors of these kinases could not prevent 5-LO activation by OAG. Together, OAG acts as a direct agonist for 5-LO product synthesis in PMNL stimulating 5-LO by novel undefined mechanisms.     

Roscovitine inhibits ERK1/2 activation induced by angiotensin II in vascular smooth muscle cells

Roscovitine is a potent CDK inhibitor often used as a biological tool in cell-cycle studies, but its working mechanism and real targets in vascular smooth muscle cells (VSMCs) remain unclear. In this study, we observed that ERK1/2 phosphorylation induced by Ang II was abrogated by pretreating VSMCs with roscovitine for 15h. Pretreating VSMCs with roscovitine also inhibited Ang II-induced c-Jun expression and phosphorylation. We further demonstrated that roscovitine could suppress the DNA binding activity of c-Jun and activation of angiotensinogen promoter by Ang II. These results suggest that roscovitine represses Ang II-induced angiotensinogen expression by inhibiting activation of ERK1/2 and c-Jun.     

Phorbol ester dissociates endothelin-stimulated phosphoinositide hydrolysis and arachidonic acid release in vascular smooth muscle cells

Endothelin-stimulated [3H]-inositol phosphate formation and [3H]-arachidonic acid release were measured in cultured vascular smooth muscle cells from rabbit renal artery. Both responses were partially inhibited by pretreatment with pertussis toxin, indicating the involvement of pertussis toxin-sensitive guanine nucleotide binding regulatory proteins in the coupling processes. Pretreatment with the phorbol ester PMA inhibited endothelin-stimulated [3H]-inositol phosphate formation, but potentiated endothelin-stimulated [3H]-arachidonic acid release, suggesting that these two coupling processes occur in a parallel and independent manner in vascular smooth muscle cells.     

Phorbol ester inhibits angiotensin-induced activation of phospholipase C in adrenal glomerulosa cells. Its implication in the sustained action of angiotensin.

The present study was undertaken to determine whether an agonist-induced activation of C-kinase leads to an inhibition of phospholipase C in adrenal glomerulosa cells. When cells are treated with 100 nM-TPA (12-O-tetradecanoylphorbol 13-acetate), subsequent angiotensin ('angiotensin II')-induced aldosterone secretion is greatly inhibited. Treatment with TPA completely inhibits the angiotensin-induced increase in both inositol trisphosphate and the cytosolic Ca2+ concentration. The dose-response curve for TPA-induced inhibition reveals that quite a high concentration of TPA is necessary to block angiotensin action compared with that needed to stimulate aldosterone secretion. 1-Oleoyl-2-acetylglycerol has a weak inhibitory effect, whereas neither 4 alpha-phorbol 12,13-didecanoate or 4 beta-phorbol inhibits angiotensin action. When the time course of changes in inositol trisphosphate and diacylglycerol is measured, angiotensin action is sustained for up to 30 min. In addition, 100 nM-TPA added after 20 min of angiotensin addition attenuates production of both inositol trisphosphate and diacylglycerol. These results suggest that high dose of TPA inhibits angiotensin-induced activation of phospholipase C by acting, at least partly, on C-kinase, but that an inhibitory effect of TPA may be a pharmacological effect with little physiological significance in this system.     

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.     

ERK1/2 activation by angiotensin II inhibits insulin-induced glucose uptake in vascular smooth muscle cells

Clinical evidence suggests a relationship between hypertension and insulin resistance, and cross-talk between angiotensin II (Ang II) and insulin signaling pathways may take place. We now report the effect of Ang II on insulin-induced glucose uptake and its intracellular mechanisms in vascular smooth muscle cells (VSMC). We examined the translocation of glucose transporter-4 (GLUT-4) and glucose uptake in rat aortic smooth muscle cells (RASMC). Mitogen-activated protein (MAP) kinases and Akt activities, and phosphorylation of insulin receptor substrate-1 (IRS-1) at the serine and tyrosine residues were measured by immunoprecipitation and immunoblotting. As a result, Ang II inhibited insulin-induced GLUT-4 translocation from cytoplasm to the plasma membrane in RASMC. Ang II induced extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation and IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹⁶. Ang II-induced Ser³⁰⁷ and Ser⁶¹⁶ phophorylation of IRS-1 was inhibited by a MEK inhibitor, PD98059, and a JNK inhibitor, SP600125. Ang II inhibition of insulin-stimulated IRS-1 tyrosyl phophorylation and Akt activation were reversed by PD98059 but not by SP600125. Ang II inhibited insulin-induced glucose uptake, which was also reversed by PD98059 but not by SP600125. It is shown that Ang II-induced ERK1/2 activation inhibits insulin-dependent glucose uptake through serine phophorylation of IRS-1 in RASMC.     

Dexamethasone decreases phospholipase C beta1 isozyme expression in human vascular smooth muscle cells

The molecular characterization of the human PLC beta1 gene was just reported by Peruzzi et al. [Biochim. Biophys. Acta 1582 (2002) 46]. This prompted us to investigate the effects of dexamethasone on PLC beta1 expression in two types of human vascular smooth muscle cells--coronary artery smooth muscle cells (hCASMC) and aortic smooth muscle cells (hAoSMC), since glucocorticoids are known to affect the signaling pathways of Gprotein coupled receptors. Semi-quantitative RT-PCR was used to analyze mRNA expression and Western-blot for protein expression. Dexamethasone treatment in the two types of cells studied decreased (mRNA and protein) PLC beta1 isozyme expression. A rapid (2 h) fall in mRNA occurred in hCASMC after treatment, and hCASMC were more sensitive to dexamethasone (1 nM versus 100 nM) than hAoSMC. The major reduction (80%) was observed after 48 h of exposure in both VSMC. Treatment with mifeprisone, an antagonist of glucocorticoid receptors, blunted the dexamethasone effect on PLC beta1 mRNA and showed that this effect was mediated by glucocorticoids receptors.     

Phorbol 12-myristate 13-acetate prevents isoproterenol-induced morphological change in cultured vascular smooth muscle cells

The effect of phorbol 12-myristate 13-acetate (PMA) on isoproterenol (ISO)- and dibutyryl cAMP (dBcAMP)-induced morphological change and cytoskeletal reorganization was studied in cultured vascular smooth muscle cells (VSMC) using the fluorescence staining of actin and microtubules. The treatment of VSMC with 1.0 μM of ISO or with 1.0 mM of dBcAMP for 90 min induced the disruption of actin-containing stress fibers followed by cytoplasmic arborization. The addition of 100 or 10 nM of PMA prevented both the destruction of actin fibers and cell arborization induced either by ISO or by dBcAMP. However, PMA rather enhanced cAMP production stimulated by ISO. I-Oleoyl-2-acetylsn-glycerol (100 μg/ml) mimicked this inhibitory effect of PMA whereas 4a-phorbol 12,13-didecanoate (100 nM) failed to block the arborization. These results indicated that the inhibition of arborization by PMA was mediated through the activation of protein kinase C. Colchicine at 5.0 μM also had an inhibitory effect on ISO- and dBcAMP-induced cell arborization. However, immunofluorescence studies revealed that colchicine but not PMA elicited the reorganization of microtubules, suggesting that the effect of PMA was mediated through a mechanism different from that of colchicine. These observations indicated that the morphology of VSMC was regulated through the alteration of cytoskeletal organization induced by cAMP-mediated and by protein kinase C-dependent systems.