Angiotensin II-stimulated Na+/H+ exchange in cultured vascular smooth muscle cells. Evidence for protein kinase C-dependent and -independent pathways

Angiotensin II, a potent vasoconstrictor, is known to stimulate Ca2+ mobilization and Na+ influx in vascular smooth muscle cells (VSMC). The fact that the Na+/H+ exchange inhibitor, amiloride, blocks angiotensin II-stimulated Na+ influx and is itself a vasodilator suggests that Na+/H+ exchange may play a role in the angiotensin II-mediated effects on VSMC. We have used a pH-sensitive fluorescent dye to study Na+/H+ exchange in cultured rat aortic VSMC. Basal intracellular pH was 7.08 in physiological saline buffer. Angiotensin II stimulation caused an initial transient acidification, followed by a Na+-dependent alkalinization. Angiotensin II increased the rate of alkalinization with apparent threshold, half-maximal, and maximal effect of 0.01, 3, and 100 nM, respectively. Angiotensin II stimulation appeared to be mediated by a shift in the Km of the Na+/H+ exchanger for extracellular Na+. Since angiotensin II activates phospholipase C in VSMC, we tested the possibility that angiotensin II increased Na+/H+ exchange by activation of protein kinase C via stimulation of diacylglycerol formation. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated Na+/H+ exchange in VSMC cultured for 24 h in serum-free medium, and the subsequent angiotensin II response was inhibited. However, VSMC grown in serum and treated for 24 h with TPA to decrease protein kinase C activity showed no inhibition of angiotensin II-stimulated Na+/H+ exchange. TPA caused no intracellular alkalinization of VSMC grown in serum, while the angiotensin II response was actually enhanced compared to VSMC deprived of serum for 24 h. We conclude that angiotensin II stimulates an amiloride-sensitive Na+/H+ exchange system in cultured VSMC which is mediated by protein kinase C-dependent and -independent mechanisms. Angiotensin II-mediated Na+ influx and intracellular alkalinization may play a role in excitation-response coupling in vascular smooth muscle.     

Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells: I. Involvement of protein kinase C-dependent and -independent pathways

We recently reported that extracellular ATP was mitogenic for Swiss 3T3, 3T6, and A431 cells (Huang et al.: Proc. Natl. Acad. Sci. USA, 86:7904-7908, 1989). Here we examined the possible involvement of activation of the protein kinase C (PKC) signal transduction pathway in the mechanism of action of extracellular ATP. A potent synergistic stimulation of DNA synthesis in quiescent cultures of 3T3 and 3T6 cells was observed when ATP was presented in combination with growth factors that activate PKC, such as bombesin, vasopressin, or tumor-promoting phorbol esters. This finding suggests that ATP and these mitogens do not act through a common mechanism. In contrast, ATP was unable to show synergism with phorbol esters in A431 cells. We discovered striking differences when we examined the kinetics of formation of diacylglycerol (DAG) stimulated by ATP among these cell lines. Thus, ATP stimulated a sustained biphasic increase of DAG in A431 cells, but only a rapid transient increase of DAG formation was observed in 3T3 and 3T6 cells. The breakdown of phosphatidylcholine was stimulated by ATP in A431 cells; however, a significantly reduced effect was displayed in 3T6 cells. Furthermore, we found that the diacylglycerol-kinase inhibitor, 1-monooleoylglycerol, greatly potentiated ATP-stimulated DNA synthesis in A431 cells. Finally, down-regulation of PKC by long-term exposure to phorbol dibutyrate (PDBu) prevented stimulation of DNA synthesis induced by bombesin, vasopressin, or phorbol esters in 3T3 or 3T6 cells, while it had no such effect on ATP-stimulated mitogenesis in the presence of insulin or epidermal growth factor. On the other hand, PDBu-mediated down-regulation of PKC partially inhibited [3H [thymidine incorporation stimulated by ATP in A431 cells. Taken together, we conclude that a protein kinase C-dependent pathway is partially involved in ATP-stimulated DNA synthesis in A431 cells, but a protein kinase C-independent pathway exists in 3T3 and 3T6 cells. Pertussis toxin (PTX) inhibited the sustained phase of DAG formation and the breakdown of phosphatidylcholine stimulated by ATP in A431 cells. This suggests involvement of a PTX-sensitive G protein.     

The signal transduction pathways of heat shock protein 27 phosphorylation in vascular smooth muscle cells

The objective of this study is to investigate the signal transduction pathways that regulate heat shock protein 27 (HSP27) phosphorylation and migration of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) induced by angiotensin II (AngII) and platelet derived growth factor-BB (PDGF-BB). The activity of HSP27 was evaluated by Western blot with specific phospho-HSP27 antibody. F-actin polymerization was detected by FITC-Phalloidine staining using confocal microscopy. Modified Boyden chamber technique was employed for VSMCs migration assessment. Within a given concentration, the phosphorylation of HSP27 induced by AngII and PDGF-BB was blocked by the specific P38MAPK inhibitor SB202190, the specific PI3K inhibitor LY294002 and the specific ERK1/2 inhibitor U0126 in a concentration-dependent manner, with a peak inhibition rate at 87.2%, 78.4% and 37.3%, respectively, induced by AngII (P < 0.01), with a peak inhibition rate at 85.0%, 55.3% and 41.0%, respectively, induced by PDGF-BB (P < 0.01).The migration of VSMCs induced by AngII and PDGF-BB was inhibited by 100 μmol/l SB202190, 30 μmol/l LY294002, and 30 μmol/l U0126, with a inhibition rate at 60.1%, 71.7% and 47.3%, respectively, provoked by AngII (P < 0.01), with a inhibition rate at 55.3%, 55.6% and 38.1%, respectively, provoked by PDGF-BB (P < 0.01). P38MAPK and PI3 K/Akt are important pathways that contribute to the phosphorylation of HSP27 and migration of VSMCs in response to AngII and PDGF-BB. ERK1/2 might be involved in HSP27 phosphorylation and migration of VSMCs provoked by AngII and PDGF-BB.     

Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.     

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

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

Activation of multiple signal transduction pathways by endothelin in cultured human vascular smooth muscle cells

Cellular responses to the vasoconstrictor peptide, endothelin, have been investigated in quiescent cultured human vascular smooth muscle cells (hVSMC). Endothelin caused intracellular alkalinization and activation of the protein synthetic enzyme S6-kinase, but such responses were not associated with any mitogenic effects of endothelin on hVSMC. In myo-[3H]inositol-prelabelled hVSMC endothelin elicited a rapid increase in inositol bis- and tris-phosphates and concomitant hydrolysis of polyphosphoinositol lipids. In [3H]arachidonate-prelabelled hVSMC endothelin promoted production of diacylglycerol, the early kinetics of which parallelled polyphosphoinositol lipid hydrolysis. Such phospholipase C activation by endothelin was sustained in hVSMC with accumulation of inositol polyphosphates being markedly protracted and the decay of diacylglycerol slow. Endothelin promoted extracellular release of [3H]arachidonate-labelled material from hVSMC which derived via deacylation of both phosphatidylinositol and phosphatidylcholine. This process was inhibited by phospholipase A2 and lipoxygenase inhibitors, but insensitive to phospholipase C and cyclooxygenase inhibitors. Endothelin-induced activation of phospholipase C and phospholipase A2 signal transduction pathways (EC50 approximately 5-8 nM for both) in hVSMC apparently proceed in an independent parallel manner rather than a sequential one.     

Thrombin signal transduction mechanisms in human glomerular epithelial cells.

We have previously shown that alpha-thrombin exerted a mitogenic effect on human glomerular epithelial cells and stimulated the synthesis of urokinase-type (u-PA) and tissue-type plasminogen activator (t-PA) and of their inhibitor, plasminogen activator inhibitor 1 (PAI-1). In the present study, we investigate the signal transduction mechanisms of thrombin in these cultured cells. Thrombin induced an increase in intracellular free calcium concentrations ([Ca2+]i) in a dose-dependent manner, a plateau being reached at 1 U/ml thrombin. A 60% inhibition of this effect was produced by 300 nM nicardipine, a dihydroperidine agent, or by 4 mM EGTA, indicating that increase in [Ca2+]i was due in part to extracellular Ca2+ entry through L-type voltage-sensitive calcium channels. Thrombin also induced an increase in inositol trisphosphate (IP3), suggesting that phospholipase C activation and phosphatidylinositides breakdown were stimulated. Interestingly thrombin-stimulated cell proliferation measured by 3H thymidine incorporation was inhibited by 300 nM nicardipine, and restored by addition of 10(-8) M ionomycin, indicating that calcium entry was critical for the mitogenic signal of thrombin. Conversely, nicardipine did not modify thrombin-stimulated synthesis of u-PA, t-PA, and PAI-1. Both thrombin-stimulated cell proliferation and protein synthesis required protein kinase C activation since these effects were blocked by 10 microM H7, an inhibitor of protein kinases, and by desensitization of protein kinase C by phorbol ester pretreatment of the cells. Interestingly, DFP-inactivated thrombin which binds the thrombin receptor and gamma-thrombin, which has some enzymatic activity but does not bind to thrombin receptor, had no effect when used alone. Simultaneous addition of these two thrombin derivatives had no effect on [Ca2+]i, and 3H thymidine incorporation but stimulated u-PA, t-PA, and PAI-1 synthesis although to a lesser extent than alpha-thrombin. This effect also required protein kinase C activation to occur, presumably by a pathway distinct from phosphoinositoside turnover since it was not associated with IP3 generation. In conclusion, multiple signalling pathways can be activated by alpha-thrombin in glomerular epithelial cells: 1) Ca2+ influx through a dihydroperidine-sensitive calcium channel, which seems critical for mitogenesis; 2) protein kinase C activation by phosphoinositide breakdown, which stimulates both mitogenesis and synthesis of u-PA, t-PA, and PAI-1; 3) protein kinase C activation by other phospholipid breakdown can stimulate u-PA, t-PA, and PAI-1 synthesis but not mitogenesis.     

Platelet-activating factor-stimulated protein tyrosine phosphorylation and eicosanoid synthesis in rat Kupffer cells. Evidence for calcium-dependent and protein kinase C-dependent and -independent pathways.

The lipid mediator platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC) has been shown to elicit several important biochemical signaling responses in mammalian cells, including polyphosphoinositide hydrolysis, arachidonic acid release/eicosanoid production, and protein tyrosine phosphorylation. In the present study, the roles of Ca2+ and protein kinase C (PKC), two signaling components of the phospholipase C pathway, in AGEPC-stimulated eicosanoid production and protein tyrosine phosphorylation, were investigated in cultured rat Kupffer cells. AGEPC at nanomolar concentrations induced an increase in intracellular calcium concentration ([Ca2+]i), stimulated membrane PKC activity, and resulted in protein tyrosine phosphorylation. The maximal increase in [Ca2+]i and membrane PKC activity in response to AGEPC were observed within 30-50 s, whereas the AGEPC-induced protein tyrosine phosphorylation reached maximal levels within 2-5 min. [Ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) but not 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of calcium release from intracellular compartments, nearly abolished the AGEPC-induced increase in [Ca2+]i suggesting involvement of extracellular calcium influx in this event. Both EGTA and TMB-8 abolished or inhibited AGEPC-stimulated protein tyrosine phosphorylation and eicosanoid formation, respectively. The calcium ionophore A23187 alone stimulated eicosanoid production and protein tyrosine phosphorylation with an identical pattern to that of AGEPC. Phorbol myristate acetate (PMA), an activator of PKC, which did not affect [Ca2+]i, mimicked the actions of AGEPC, stimulating eicosanoid production and promoting tyrosine phosphorylation of a set of proteins similar to those phosphorylated following AGEPC stimulation. AGEPC-enhanced tyrosine phosphorylation of some of the protein substrates and eicosanoid production were inhibited in cells "down-regulated" for PKC. Furthermore, both PMA- and AGEPC-stimulated eicosanoid production and protein tyrosine phosphorylation were attenuated or abolished by at least one of the PKC inhibitors, staurosporine, and calphostin C. Taken together, these results are consistent with the conclusions that: (a) AGEPC stimulates the phospholipase-mediated arachidonic acid release/eicosanoid synthesis cascade and protein tyrosine phosphorylation through extracellular Ca(2+)-dependent and PKC-dependent and -independent mechanism(s) and (b) the Ca(2+)-PKC interaction determines the efficacy of the AGEPC-stimulated cellular events.     

Dual mechanisms of platelet hormone receptor desensitization. Differential importance between agonists of protein kinase C-dependent and -independent pathways

Several different agonists, among them alpha-thrombin, platelet-activating factor, vasopressin, thromboxane A2, and endoperoxides, activate platelets to aggregate and secrete granular contents. Each of these agents is thought to act by inducing the turnover of inositol phospholipids and generating the second messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. However, within minutes, the action of these agonists desensitizes. We have studied the characteristics of this desensitization process for the agonists mentioned above in an attempt to clarify the mechanisms involved. Our results show that two different pathways of desensitization exist, one that is mediated by protein kinase C and another that is independent of this enzyme. In addition, the contribution of these pathways to desensitization differs for the agonists studied. Our data suggest that partial agonists and strong agonists differ in the rate at which the primary response is desensitized rather than in their ability to couple to phospholipase C.     

Protein kinase C-dependent and -independent pathways of proto-oncogene induction in human astrocytoma cells

We compared the abilities of the muscarinic agonist carbachol, epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) to induce proto-oncogene mRNA accumulation and other cellular responses in normal and protein kinase C-deficient 1321-N1 human astrocytoma cells. PMA, carbachol, and EGF all stimulated rapid accumulation of mRNA for the proto-oncogenes c-fos and c-myc in the normal cells; in the protein kinase C-deficient cells, carbachol and EGF, but not PMA, retained this effect, which was not mimicked by the calcium ionophore A23187. Both carbachol and PMA activated protein kinase C in these cells, as evidenced by the stimulated phosphorylation of an acidic Mr 80,000 protein kinase C substrate protein with phosphoamino acid and peptide map identity. This response was mimicked by several other neurotransmitters in these cells, including epinephrine, histamine, oxotremorine, and serotonin, and was abolished in cells made protein kinase C-deficient by preincubation with high concentrations of PMA. Both PMA and carbachol promoted the phosphorylation of the ribosomal protein S6 and activated an S6 protein kinase in the normal but not in the protein kinase C-deficient cells. EGF, in contrast, did not appear to activate protein kinase C, but promoted the phosphorylation of S6 and activation of the S6 kinase in both normal and protein kinase C-deficient cells. We conclude that, in 1321-N1 cells, induction of c-fos and c-myc mRNA can occur through a protein kinase C-dependent pathway and one or more independent pathways, exemplified by the responses to carbachol and EGF in the protein kinase C-deficient cells.     

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

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

Growth factor-stimulated protein phosphorylation in 3T3-L1 cells. Evidence for protein kinase C-dependent and -independent pathways

Exposure of serum-deprived 3T3-L1 fibroblasts to phorbol 12-myristate 13-acetate (PMA), synthetic diacylglycerols, platelet-derived growth factor (PDGF), or pituitary fibroblast growth factor (FGF) resulted in stimulated phosphorylation of an acidic, multicomponent, soluble protein of Mr 80,000. Phosphorylation of this protein was promoted to a lesser extent by epidermal growth factor; however, neither insulin nor dibutyryl cAMP was effective. Phosphoamino acid analysis and peptide mapping of the Mr 80,000 32P-protein after exposure of fibroblasts to PDGF revealed identical patterns to those obtained with PMA or diacylglycerols. In contrast to the Mr 80,000 protein, proteins of Mr 22,000 (and pI 4.4) and Mr 31,000 were also phosphorylated in response to insulin as well as to PMA, diacylglycerols, epidermal growth factor, PDGF, and FGF in these cells. Similar findings were noted in fully differentiated 3T3-L1 adipocytes. Preincubation of the cells with high concentrations of active phorbol esters abolished specific [3H]phorbol 12,13-dibutyrate binding, protein kinase C activity, and immunoreactivity and also prevented stimulated phosphorylation of the Mr 80,000 protein by PMA, diacylglycerols, PDGF, or FGF, supporting the contention that this effect was mediated through protein kinase C. The stimulated phosphorylation of the Mr 22,000 and 31,000 proteins in response to PMA was also abolished by such pretreatment. In contrast, the ability of insulin, PDGF, and FGF to promote phosphorylation of the Mr 22,000 and 31,000 proteins was unaffected in the protein kinase C-deficient cells. We conclude that PDGF and FGF may exert some of their effects on these cells through at least two distinct pathways of protein phosphorylation, phorbol ester-like (P) activation of protein kinase C, and an insulin-like (I) pathway exemplified by phosphorylation of the Mr 22,000 and 31,000 proteins.     

The role of protein kinase C in T lymphocyte proliferation. Existence of protein kinase C-dependent and -independent pathways

The mouse cytotoxic T cell clone (CTLL-2) was able to grow in the presence of culture medium supplemented only with transferrin, 2-mercaptoethanol, and recombinant interleukin 2 (IL-2). This lymphokine stimulated the synthesis of DNA in these cells. Similarly, phorbol esters, which activate protein kinase C, induced DNA synthesis in this clone. Furthermore, this later proliferation was not blocked by anti-IL-2 receptor antibodies, which inhibited IL-2-induced proliferation, suggesting that it was not indirectly due to the secretion of IL-2 by the cells. CTLL-2 cells pretreated with high doses of phorbol esters for 48 h down regulated protein kinase C and were depleted of this enzyme. This was shown by: 1) purification and in vitro assay of protein kinase C; 2) the lack of effect of phorbol esters in the stimulation of the Na+/H+ anti-porter which has been directly linked to the activation of protein kinase C. As expected, those protein kinase C-depleted cells no longer synthesized DNA and proliferated in response to phorbol esters. However, they proliferated identically to control cells in response to IL-2. Therefore, our results suggest two different pathways for T cell proliferation, one which involves protein kinase C and the other which does not.     

Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway.

Protein kinase D (PKD) is a serine/threonine protein kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids. Here, we examine the regulation of PKD in living cells. Our results demonstrate that tumour-promoting phorbol esters, membrane-permeant diacylglycerol and serum growth factors rapidly induced PKD activation in immortalized cell lines (e.g. Swiss 3T3 and Rat-1 cells), in secondary cultures of mouse embryo fibroblasts and in COS-7 cells transiently transfected with a PKD expression construct. PKD activation was maintained during cell disruption and immunopurification and was associated with an electrophoretic mobility shift and enhanced 32P incorporation into the enzyme, but was reversed by treatment with alkaline phosphatase. PKD was activated, deactivated and reactivated in response to consecutive cycles of addition and removal of PDB. PKD activation was completely abrogated by exposure of the cells to the protein kinase C inhibitors GF I and Ro 31-8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Co-transfection of PKD with constitutively activated mutants of PKCs showed that PKCepsilon and eta but not PKCzeta strongly induced PKD activation in COS-7 cells. Thus, our results indicate that PKD is activated in living cells through a PKC-dependent signal transduction pathway.     

G-protein-dependent and -independent pathways in denatonium signal transduction

To clarify the involvement of G protein in denatonium signal transduction, we carried out a whole-cell patch-clamp analysis with isolated taste cells in mice. Two different responses were observed by applying GDP-beta-S, a G-protein inhibitor. One response to denatonium was reduced by GDP-beta-S (G-protein-dependent), whereas the other was not affected (G-protein-independent). These different patterns were also observed by concurrently inhibiting the phospholipase C beta2 and phosphodiesterase pathways via G protein. These data suggest dual, G-protein-dependent and -independent mechanisms for denatonium. Moreover, the denatonium responses were not attenuated by singly inhibiting the phospholipase C beta2 or phosphodiesterase pathway, implying that both pathways were involved in G-protein-dependent transduction. In the G-protein-independent cells, the response was abolished by the depletion of calcium ions within the intracellular store. These results suggest that Ca2+ release from the intracellular store is an important factor. Our data demonstrate multiple transduction pathways for denatonium in mammalian taste cells.     

Delineating signal transduction pathways in smooth muscle through focused proteomics

This review will outline examples of the authors' focused proteomics approaches to studying signal transduction pathways in smooth muscle. By focusing the use of traditional proteomics techniques with hypothesis-driven selection methods, this approach efficiently addresses the identification of novel elements in a signal transduction pathway of interest. However, focused proteomics serves only as a starting point in the investigation of novel signaling proteins. While focused proteomics studies can suggest the involvement and general biochemical function of a protein in a signaling pathway, these findings must be further investigated and validated. Through the integrated use of focused proteomics with complementary approaches such as genetics, biochemistry and cell physiology, a complete and detailed mechanism of signal transduction can be determined.     

Activation of mitogen-activated protein kinase pathways by cyclic GMP and cyclic GMP-dependent protein kinase in contractile vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro and in vivo. The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal- regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC.     

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

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