Angiotensin II stimulates vimentin phosphorylation via a Ca2+-dependent, protein kinase C-independent mechanism in cultured vascular smooth muscle cells

Intermediate filaments have been proposed, via phosphorylation by protein kinase C, to be involved in sustained contraction of smooth muscle. We examined the effect of angiotensin II on the phosphorylation of the intermediate filament protein, vimentin, in cultured rat aortic vascular smooth muscle cells. Angiotensin II induced phosphorylation of a Triton X-100- and high salt-insoluble protein with a molecular weight of 58,000. This protein was identified as vimentin based on its specific interaction with anti-vimentin antibody as detected by immunoblot analysis. Angiotensin II-induced phosphorylation of vimentin was time- and dose-dependent. Phosphorylation was detectable at 15 s, peaked at 2 min after angiotensin II stimulation, and gradually declined to a new plateau which was sustained for at least 30 min. The threshold, half-maximal and maximal concentrations of angiotensin II that stimulated vimentin phosphorylation were 0.01, 0.1, and 10 nM, respectively. The Ca2+ ionophore, ionomycin, stimulated vimentin phosphorylation to the same extent as angiotensin II, whereas the protein kinase C-activating phorbol ester, phorbol 12-myristate 13-acetate, had only marginal effects on this reaction. Pretreatment of the cells with [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid attenuated angiotensin II- and ionomycin-induced vimentin phosphorylation to the same extent. Down-regulation of protein kinase C induced by prolonged treatment of the cells with phorbol 12,13-dibutyrate did not inhibit angiotensin II-induced vimentin phosphorylation. These results indicate that angiotensin II stimulates vimentin phosphorylation via a Ca2+-dependent, protein kinase C-independent mechanism in vascular smooth muscle cells and suggest that cytoskeletal proteins are major targets for angiotensin II-induced phosphorylation events.     

Angiotensin II stimulates protein-tyrosine phosphorylation in a calcium-dependent manner.

Cellular responses to epidermal growth factor (EGF) are dependent on the tyrosine-specific protein kinase activity of the cell-surface EGF receptor. Previous studies using WB rat liver epithelial cells have detected at least 10 proteins whose phosphotyrosine (P-Tyr) content is increased by EGF. In this study, we have examined alternate modes of activating tyrosine phosphorylation. Treatment of WB cells with hormones linked to Ca2+ mobilization and protein kinase C (PKC) activation, including angiotensin II, [Arg8]vasopressin, or epinephrine, stimulated rapid (less than or equal to 15-s) and transient increases in the P-Tyr content of several proteins (p120/125, p75/78, and p66). These proteins, detected by anti-P-Tyr immunoblotting, were similar in molecular weight to a subset of EGF-sensitive P-Tyr-containing proteins (P-Tyr-proteins). The increased P-Tyr content was confirmed by [32P]phosphoamino acid analysis of proteins recovered by anti-P-Tyr immunoprecipitation. Elevating intracellular [Ca2+] with the ionophore A23187 or ionomycin or with the tumor promoter thapsigargin mimicked the effects of hormones on tyrosine phosphorylation, whereas treatment with a PKC-activating phorbol ester did not. In addition, responses to angiotensin II were not diminished in PKC-depleted cells. Ca2+ mobilization, measured by fura-2 fluorescence, was coincident with the increase in tyrosine phosphorylation in response to angiotensin II or thapsigargin. Loading cells with the intracellular Ca2+ chelator bis-(o-aminophenoxy)ethane-N ,N ,N' , N'-tetraacetic acid (BAPTA) inhibited the appearance of all P-Tyr-proteins in response to angiotensin II, thapsigargin, or ionophores, as well as two EGF-stimulated P-Tyr-proteins. The majority of EGF-stimulated P-Tyr-proteins were not affected by BAPTA. These studies indicate that angiotensin II can alter protein-tyrosine phosphorylation in a manner that is secondary to, and apparently dependent on, Ca2+ mobilization. Thus, ligands such as EGF and angiotensin II, which act through distinct types of receptors, may activate secondary pathways involving tyrosine phosphorylation. These results also raise the possibility that certain growth-promoting effects of Ca2+ -mobilizing agents such as angiotensin II may be mediated via tyrosine phosphorylation.     

Activation of protein kinase C is not required for exocytosis from bovine adrenal chromaffin cells. The effects of protein kinase C(19-31), Ca/CaM kinase II(291-317), and staurosporine

We examined whether protein kinase C activation plays a modulatory or an obligatory role in exocytosis of catecholamines from chromaffin cells by using PKC(19-31) (a protein kinase C pseudosubstrate inhibitory peptide), Ca/CaM kinase II(291-317) (a calmodulin-binding peptide), and staurosporine. In permeabilized cells, PKC (19-31) inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion as much as 90% but had no effect on Ca2(+)-dependent secretion in the absence of phorbol ester. The inhibition of the phorbol ester-induced enhancement of secretion by PKC (19-31) was correlated closely with the ability of the peptide to inhibit in situ phorbol ester-stimulated protein kinase C activity. PKC(19-31) also blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of numerous endogenous proteins in permeabilized cells but had no effect on Ca2(+)-stimulated phosphorylation of tyrosine hydroxylase. Ca/CaM kinase II(291-317), derived from the calmodulin binding region of Ca/calmodulin kinase II, had no effect on Ca2(+)-dependent secretion in the presence or absence of phorbol ester. The peptide completely blocked the Ca2(+)-dependent increase in tyrosine hydroxylase phosphorylation but had no effect on TPA-induced phosphorylation of endogenous proteins in permeabilized cells. To determine whether a long-lived protein kinase C substrate might be required for secretion, the lipophilic protein kinase inhibitor, staurosporine, was added to intact cells for 30 min before permeabilizing and measuring secretion. Staurosporine strongly inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion. It caused a small inhibition of Ca2(+)-dependent secretion in the absence of phorbol ester which could not be readily attributed to inhibition of protein kinase C. Staurosporine also inhibited the phorbol ester-mediated enhancement of elevated K(+)-induced secretion from intact cells while it enhanced 45Ca2+ uptake. Staurosporine inhibited to a small extent secretion stimulated by elevated K+ in the absence of TPA. The data indicate that activation of protein kinase C is modulatory but not obligatory in the exocytotoxic pathway.     

The role of Ca2+ mobilization and heterotrimeric G protein activation in mediating tyrosine phosphorylation signaling patterns in vascular smooth muscle cells

This work investigated the role of Ca²⁺ mobilization and heterotrimeric G protein activation in mediating angiotensin II-dependent tyrosine phosphorylation signaling patterns. We demonstrate that the predominant, angiotensin II-dependent, tyrosine phosphorylation signaling patterns seen in vascular smooth muscle cells are blocked by the intracellular Ca²⁺ chelator BAPTA-AM, but not by the Ca²⁺ channel blocker verapamil. Activation of heterotrimeric G proteins with NaF resulted in a divergent signaling effect; NaF treatment was sufficient to increase tyrosine phosphorylation levels of some proteins independent of angiotensin II treatment. In the same cells, NaF alone had no effect on other cellular proteins, but greatly potentiated the ability of angiotensin II to increase the tyrosine phosphorylation levels of these proteins. Two proteins identified in these studies were paxillin and Jak2. We found that NaF treatment alone, independent of angiotensin II stimulation, was sufficient to increase the tyrosine phosphorylation levels of paxillin. Furthermore, the ability of either NaF and/or angiotensin II to increase tyrosine phosphorylation levels of paxillin is critically dependent on intracellular Ca²⁺. In contrast, angiotensin II-mediated Jak2 tyrosine phosphorylation was independent of intracellular Ca²⁺ mobilization and extracellular Ca²⁺ entry. Thus, our data suggest that angiotensin II-dependent tyrosine phosphorylation signaling cascades are mediated through a diverse set of signaling pathways that are partially dependent on Ca²⁺ mobilization and heterotrimeric G protein activation.     

Selective down-regulation of angiotensin II receptor type 1A signaling by protein tyrosine phosphatase SHP-2 in vascular smooth muscle cells

The heptahelical AT(1) G-protein-coupled receptor lacks inherent tyrosine kinase activity. Angiotensin II binding to AT(1) nevertheless activates several tyrosine kinases and stimulates both tyrosine phosphorylation and phosphatase activity of the SHP-2 tyrosine phosphatase in vascular smooth muscle cells. Since a balance between tyrosine kinase and tyrosine phosphatase activities is essential in angiotensin II signaling, we investigated the role of SHP-2 in modulating tyrosine kinase signaling pathways by stably transfecting vascular smooth muscle cells with expression vectors encoding wild-type SHP-2 protein or a catalytically inactive SHP-2 mutant. Our data indicate that SHP-2 is an efficient negative regulator of angiotensin II signaling. SHP-2 inhibited c-Src catalytic activity by dephosphorylating a positive regulatory tyrosine 418 within the Src kinase domain. Importantly, SHP-2 expression also abrogated angiotensin II-induced activation of ERK, whereas expression of catalytically inactive SHP-2 caused sustained ERK activation. Thus, SHP-2 likely regulates angiotensin II-induced MAP kinase signaling by inactivating c-Src. These SHP-2 effects were specific for a subset of angiotensin II signaling pathways, since SHP-2 overexpression failed to influence Jak2 tyrosine phosphorylation or Fyn catalytic activity. These data show SHP-2 represents a critical negative regulator of angiotensin II signaling, and further demonstrate a new function for this phosphatase in vascular smooth muscle cells.     

Inhibition of amylase secretion from differentiated AR4-2J pancreatic acinar cells by an actin cytoskeleton controlled protein tyrosine phosphatase activity.

Disruption of the actin cytoskeleton in AR4-2J pancreatic acinar cells led to an increase in cytosolic protein tyrosine phosphatase activity, abolished bombesin-induced tyrosine phosphorylation and reduced bombesin-induced amylase secretion by about 45%. Furthermore, both tyrosine phosphorylation and amylase secretion induced by phorbol ester-induced activation of protein kinase C were abolished. An increase in the cytosolic free Ca2+ concentration by the Ca2+ ionophore A23187 had no effect on tyrosine phosphorylation but induced amylase release. Only when added together with phorbol ester, the same level of amylase secretion as with bombesin was reached. This amylase secretion was inhibited by about 40%, by actin cytoskeleton disruption similar to that induced by bombesin. We conclude that actin cytoskeleton-controlled protein tyrosine phosphatase activity downstream of protein kinase C activity regulates tyrosine phosphorylation which in part is involved in bombesin-stimulated amylase secretion.     

Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells.

Protein tyrosine phosphorylation has not been considered to be important for cellular activation by phospholipase C-linked vasoactive peptides. We found that endothelin, angiotensin II, and vasopressin (AVP), peptides that signal via phospholipase C activation, rapidly enhanced tyrosine phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat renal mesangial cells. The phosphorylated proteins were cytosolic or membrane-associated, and none were integral to the membrane, suggesting that the peptide receptors are not phosphorylated on tyrosine. Epidermal growth factor (EGF), which does not activate phospholipase C in these cells, induced the tyrosine phosphorylation of its own 175-kDa receptor, in addition to five proteins of identical molecular mass to those phosphorylated in response to endothelin, AVP, and angiotensin II. This suggests that in mesangial cells there is a common signaling pathway for phospholipase C-coupled agonists and agonists classically assumed to signal via receptor tyrosine kinase pathways, such as EGF. The phorbol ester, phorbol 12-myristate 13-acetate, and the synthetic diacylglycerol, oleoyl acetylglycerol, stimulated the tyrosine phosphorylation of proteins identical to those phosphorylated by the phospholipase C-linked peptides, suggesting that protein kinase C (PKC) activation is sufficient to active tyrosine phosphorylation. However, the PKC inhibitor, staurosporine, and down-regulation of PKC activity by prolonged exposure to phorbol esters completely inhibited tyrosine phosphorylation in response to PMA but not to endothelin, AVP, or EGF. In conclusion, endothelin, angiotensin II, and AVP enhances protein tyrosine phosphorylation via at least two pathways, PKC-dependent and PKC-independent. Although activation of PKC may be sufficient to enhance protein tyrosine phosphorylation, PKC is not necessary and may not be the primary route by which these agents act. At least one of these pathways is shared with the growth factor EGF, suggesting not only common intermediates in the signaling pathways for growth factors and vasoactive peptides but also perhaps common cellular tyrosine kinases which phosphorylate these intermediates.     

CaM kinase II-dependent activation of tyrosine kinases and ERK1/2 in vascular smooth muscle

In vascular smooth muscle (VSM) and manyother cells, G protein receptor-coupled activation of mitogen-activatedprotein kinases has been linked, in part, to increases in freeintracellular Ca²⁺. Previously, we demonstrated thationomycin-, angiotensin II-, and thrombin-induced activation ofextracellular signal-regulated kinase (ERK)1/2 in VSM cells wasattenuated by pretreatment with KN-93, a selective inhibitor of themultifunctional Ca²⁺/calmodulin-dependent protein kinase(CaM kinase II). In the present study, we show that theCa²⁺-dependent pathway leading to activation of ERK1/2 ispreceded by nonreceptor proline-rich tyrosine kinase (PYK2) activation and epidermal growth factor (EGF) receptor tyrosine phosphorylation andis attenuated by inhibitors of src family kinases or the EGF receptor tyrosine kinase. Furthermore, we demonstrate that pretreatment with KN-93 or a CaM kinase II inhibitor peptide inhibitsCa²⁺-dependent PYK2 activation and EGF receptor tyrosinephosphorylation in response to ionomycin, ATP, and platelet-derivedgrowth factor but has no effect on phorbol 12,13-dibutyrate- orEGF-induced responses. The results implicate CaM kinase II as anintermediate in the Ca²⁺/calmodulin-dependent activation of PYK2.

     

Protein phosphorylation and secretion in digitonin-permeabilized adrenal chromaffin cells. Effects of micromolar Ca2+, phorbol esters, and diacylglycerol

The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin cells were investigated. [gamma-32P]ATP was used as a substrate for phosphorylation in the permeabilized cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) enhanced Ca2+-dependent catecholamine secretion from digitonin-permeabilized cells. The enhancement required MgATP. Only those phorbol esters which activate protein kinase C in vitro enhanced both catecholamine secretion and protein phosphorylation. DiC8, which activates protein kinase C in vitro and mimics phorbol ester effects in situ, also enhanced both catecholamine secretion and protein phosphorylation. Preincubation of intact cells with TPA or DiC8 was necessary for maximal effects on both catecholamine secretion and protein phosphorylation in subsequently digitonin-treated chromaffin cells. The TPA-induced enhancement of protein phosphorylation was almost entirely Ca2+-independent, whereas DiC8-induced enhancement of protein phosphorylation was mainly Ca2+-dependent. Micromolar Ca2+ alone also enhanced the phosphorylation of a large number of proteins. Most of the proteins phosphorylated in response to TPA or potentiated by DiC8 in combination with Ca2+ were also phosphorylated by micromolar Ca2+ in the absence of exogenous protein kinase C activators. In intact cells, 1,1-dimethyl-4-phenylpiperazinium (DMPP) induced Ca2+-dependent phosphorylation of at least 17 proteins which were detected by two-dimensional gel electrophoresis. All of the proteins phosphorylated upon incubation with 1,1-dimethyl-4-phenylpiperazinium were phosphorylated upon incubation with micromolar Ca2+ in digitonin-treated cells. These results demonstrate that TPA- or DiC8-enhanced Ca2+-dependent catecholamine secretion is associated with enhanced protein phosphorylation which is probably mediated by protein kinase C and that activation of protein kinase C modulates catecholamine secretion from digitonin-treated chromaffin cells.     

Angiotensin II and phorbol ester enhance isoproterenol- and vasoactive intestinal peptide (VIP)-induced cyclic AMP accumulation in vascular smooth muscle cells

The importance of Ca2+ and cAMP in the regulation of cellular functions has been well demonstrated. We studied the effect of angiotensin II (AII), a potent Ca2+-mobilizing hormone, on cAMP accumulation induced by isoproterenol (ISO) and vasoactive intestinal peptide (VIP) in cultured vascular smooth muscle cells (VSMC). Although the addition of AII alone caused little increase of cAMP, it enhanced ISO- and VIP-induced cAMP accumulations in a dose-dependent manner. This enhancement was mimicked by tumor-promoting phorbol ester but not by Ca2+ ionophore. This observation suggested that AII enhanced agonist-induced cAMP accumulation through the activation of protein kinase C in VSMC.     

Substrates for protein kinase C in a cell free preparation of rat aorta smooth muscles

Protein phosphorylation has been studied in a cell free system of rat aorta smooth muscles. Addition of Ca2+ caused phosphorylation of several proteins. The addition of phosphatidylserine or calmodulin together with Ca2+ further increased the phosphorylation of proteins with apparent molecular weights of 20 and 92.5 kilodaltons. The activators of protein kinase C, 12-0-tetradecanoylphorbol-13-acetate and 1,2-diolein, increased phosphorylation of the protein bands of similar molecular weight to those increased by phosphatidylserine in the presence of Ca2+, whereas the biologically inactive phorbol ester, 4 alpha-phorbol-12,13 didecanoate (4 alpha PDD) failed to change the pattern of protein phosphorylation. These results show that proteins present in smooth muscle of rat aorta with molecular weights of 20 and 92.5 kilodaltons are substrates for protein kinase C.     

Pyk2- and Src-dependent tyrosine phosphorylation of PDK1 regulates focal adhesions

3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a signal integrator that activates the AGC superfamily of serine/threonine kinases. PDK1 is phosphorylated on tyrosine by oxidants, although its regulation by agonists that stimulate G-protein-coupled receptor signaling pathways and the physiological consequences of tyrosine phosphorylation in this setting have not been fully identified. We found that angiotensin II stimulates the tyrosine phosphorylation of PDK1 in vascular smooth muscle in a calcium- and c-Src-dependent manner. The calcium-activated tyrosine kinase Pyk2 acts as a scaffold for Src-dependent phosphorylation of PDK1 on Tyr9, which permits phosphorylation of Tyr373 and -376 by Src. This critical function of Pyk2 is further supported by the observation that Pyk2 and tyrosine-phosphorylated PDK1 colocalize in focal adhesions after angiotensin II stimulation. Importantly, infection of smooth muscle cells with a Tyr9 mutant of PDK1 inhibits angiotensin II-induced tyrosine phosphorylation of paxillin and focal adhesion formation. These observations identify a novel interaction between PDK1 and Pyk2 that regulates the integrity of focal adhesions, which are major compartments for integrating signals for cell growth, apoptosis, and migration.     

Differences in phorbol-dependent phosphorylation of regulatory proteins and contraction of phasic and tonic smooth muscle

Smooth muscles are divided into slowly contracting tonic and relatively fast phasic muscles. In both cases Ca2+ is a key mediator of the contractile response. However, the appearance of a tonic component during sphincter or arterial muscle contraction and its absence in contracting visceral smooth muscle is characteristic of their difference. We have found that in chicken tissues phorbol 12,13-dibutyrate (PDBu) induces a sustained contraction in carotid arterial muscle, but provokes no contraction in phasic gizzard smooth muscle. Next we were aimed to find differences in PDBu-induced phosphorylation of the key proteins involved in regulation of smooth muscle contraction, i.e. caldesmon, myosin light chain kinase (MLCK), and the myosin light chain kinase-related protein (KRP, also known as telokin). Two correlative differences were observed. 1. PDBu stimulated phosphorylation of MLCK in tonic smooth muscle and had no effect on the level of MLCK phosphorylation in phasic muscle. Phosphopeptide mapping suggests the involvement of mitogen-activated protein (MAP) kinases in phosphorylation of MLCK in situ. 2. PDBu induced phosphorylation of MAP-kinase sites in caldesmon in both types of smooth muscle, but this phosphorylation had no significant effect on caldesmon functional activity in vitro. For the first time we have shown that in gizzard PDBu also stimulates a yet unknown transitory caldesmon-kinase different from protein kinase, C, Ca2+/calmodulin-dependent kinase II and casein kinase CK2. 3. No significant difference was found in the kinetics of PDBu-dependent phosphorylation of KRP in tonic and phasic smooth muscles. KRP was also demonstrated to be a major phosphoprotein in smooth muscle phosphorylated in vivo at several sites located within its N-terminal sequence. Protein kinases able to phosphorylate these sites were identified in vitro. Among them, MAP-kinase was suggested to phosphorylate a serine residue homologous to that phosphorylated in MLCK. 4. p42erk2 and p38 MAP-kinases were found in phasic and tonic smooth muscles. Both were responsive to PDBu in cultured chicken aortic smooth muscle cells, and their role in phosphorylation of MLCK and low molecular weight isoform of caldesmon was evaluated.     

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.     

Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells.

Stimulation of tracheal smooth muscle cells in culture with ionomycin resulted in a rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) and an increase in both myosin light chain kinase and myosin light chain phosphorylation. These responses were markedly inhibited in the absence of extracellular Ca2+. Pretreatment of cells with 1-[N-O-bis(5-isoquinolinesulfonyl)-N- methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a specific inhibitor of the multifunctional calmodulin-dependent protein kinase II (CaM kinase II), did not affect the increase in [Ca2+]i but inhibited ionomycin-induced phosphorylation of myosin light chain kinase at the regulatory site near the calmodulin-binding domain. KN-62 inhibited CaM kinase II activity toward purified myosin light chain kinase. Phosphorylation of myosin light chain kinase decreased its sensitivity to activation by Ca2+ in cell lysates. Pretreatment of cells with KN-62 prevented this desensitization to Ca2+ and potentiated myosin light chain phosphorylation. We propose that the Ca(2+)-dependent phosphorylation of myosin light chain kinase by CaM kinase II decreases the Ca2+ sensitivity of myosin light chain phosphorylation in smooth muscle.     

Agonist stimulation of Na+/K+/Cl- cotransport in rat glomerular mesangial cells. Evidence for protein kinase C-dependent and Ca2+/calmodulin-dependent pathways

Studies were performed to investigate regulatory pathways of loop diuretic-sensitive Na+/K+/Cl- cotransport in cultured rat glomerular mesangial cells. Angiotensin II, alpha-thrombin, and epidermal growth factor (EGF) all stimulated Na+/K+/Cl- cotransport in a concentration-dependent manner. Pertussis toxin pretreatment reduced the effects of angiotensin II and alpha-thrombin but not that of EGF. Addition of the protein kinase C inhibitor staurosporine or down-regulation of protein kinase C by prolonged incubation with phorbol 12-myristate 13-acetate partially reduced the effects of angiotensin II and alpha-thrombin and completely blunted the phorbol 12-myristate 13-acetate-induced stimulation of Na+/K+/Cl- cotransport but did not affect EGF-induced stimulation. Exposure of cells to a calcium ionophore, A23187, resulted in a concentration-dependent stimulation of Na+/K+/Cl- cotransport, which was not significantly inhibited by down-regulation of protein kinase C but was completely inhibited by the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Stimulation of the cotransport by angiotensin II or alpha-thrombin was also partially inhibited by W-7. Inhibitory effects of protein kinase C down-regulation and W-7 were additive and, when combined, produced a complete inhibition of angiotensin II-induced stimulation of Na+/K+/Cl- cotransport. In saponin-permeabilized mesangial cells, phosphorylation of a synthetic decapeptide substrate for Ca2+/calmodulin-dependent kinase II, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-NH3, was demonstrated. Maximal activation of the decapeptide substrate phosphorylation required the presence of Ca2+ and calmodulin and was dependent on Ca2+ concentration. These findings indicate that stimulation of Na+/K+/Cl- cotransport by angiotensin II and alpha-thrombin is mediated by protein kinase C and Ca2+/calmodulin-dependent kinases whereas the action of EGF is mediated by other pathways.     

The role of calcium ion as a mediator of the effects of angiotensin II, catecholamines, and vasopressin on the phosphorylation and activity of enzymes in isolated hepatocytes.

Angiotensin II, catecholamines, and vasopressin are thought to stimulate hepatic glycogenolysis and gluconeogenesis via a cyclic AMP-independent mechanism that requires calcium ion. The present study explores the possibility that angiotensin II and vasopressin control the activity of regulatory enzymes in carbohydrate metabolism through Ca2+-dependent changes in their state of phosphorylation. Intact hepatocytes labeled with [32P]PO43- were stimulated with angiotensin II, glucagon, or vasopressin and 30 to 33 phosphorylated proteins resolved from the cytoplasmic fraction of the cell by electrophoresis in sodium dodecyl sulfate polyacrylamide slab gels. Treatment of the cells with angiotensin II or vasopressin increased the phosphorylation of 10 to 12 of these cytosolic proteins without causing measurable changes in cyclic AMP-dependent protein kinase activity. Glucagon stimulated the phosphorylation of the same set of 11 to 12 proteins through a marked increase in cyclic AMP-dependent protein kinase activity. The molecular weights of three of the protein bands whose phosphorylation was increased by these hormones correspond to the subunit molecular weights of phosphorylase (Mr = 93,000), glycogen synthase (Mr = 85,000), and pyruvate kinase (Mr = 61,000). Two of these phosphoprotein bands were positively identified as phosphorylase and pyruvate kinase by affinity chromatography and immunoprecipitation, respectively. Incubation of hepatocytes in a Ca2+-free medium completely abolished the effects of angiotensin II and vasopressin on protein phosphorylation but did not alter those of glucagon. Treatment of hepatocytes with angiotensin II, glucagon, or vasopressin stimulated phosphorylase activity by 250 to 260%, inhibited glycogen synthase activity by 50%, and inhibited pyruvate kinase activity by 30 to 35% (peptides) to 70% (glucagon). The effects of angiotensin II and vasopressin on the activity of all three enzymes were completely abolished if the cells were incubated in a Ca2+-free medium while those of glucagon were not altered. The results imply that angiotensin II, catecholamines, and vasopressin control hepatic carbohydrate metabolism through a Ca2+-requiring, cyclic AMP-independent pathway that leads to the phosphorylation of important regulatory enzymes.     

Angiotensin II induces expression of the c-fos gene through protein kinase C activation and calcium ion mobilization in cultured vascular smooth muscle cells

Incubation of the serum-deprived cultures of rat vascular smooth muscle cells with angiotensin II, a potent vasoconstrictor, caused a rapid and transient increase in the c-fos mRNA level. The doses of this agonist necessary for the increase in the c-fos mRNA level coincided with those for the phospholipase C-mediated hydrolysis of phosphoinositides. Moreover, protein kinase C-activating 12-O-tetradecanoylphorbol-13-acetate and Ca2+-ionophore A23187 increased the c-fos mRNA level in an additive manner. These results suggest that angiotensin II induces expression of the c-fos gene through the activation of protein kinase C and Ca2+ mobilization in cultured vascular smooth muscle cells.     

Modification of myosin light chain phosphorylation in sustained arterial muscle contraction by phorbol dibutyrate

The decrease in phosphorylation of the 20 kDa myosin light chain during prolonged K(+)-stimulation of arterial smooth muscle was counteracted by treating this muscle with phorbol dibutyrate. Quantitative phosphopeptide analysis revealed that phorbol dibutyrate induced phosphorylation of serine and threonine residues in the light chain by protein kinase C and phosphorylation of a threonine residue by myosin light chain kinase. The same residues of light chain were also phosphorylated when phorbol dibutyrate was added to muscles pretreated either with the Ca2(+)-channel-blocking agents nifedipine and verapamil, or with the Ca2(+)-chelating agent ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The results indicate an interrelationship between protein kinase C and myosin light chain kinase phosphorylated sites of light chain in intact arterial smooth muscle.     

Myosin light chain kinase phosphorylation in tracheal smooth muscle

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+.