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.     

Different effects of phorbol ester on angiotensin II- and stable GTP analogue-induced activation of polyphosphoinositide phosphodiesterase in membranes isolated from rat renal mesangial cells.

Pretreatment with pertussis toxin inhibits angiotensin II-induced activation of polyphosphoinositide phosphodiesterase in rat renal mesangial cells [Pfeilschifter & Bauer (1986) Biochem. J. 236, 289-294]. Furthermore, activation of protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and by 1-oleoyl-2-acetylglycerol (OAG) abolishes angiotensin II-induced formation of inositol trisphosphate (IP3) in mesangial cells [Pfeilschifter (1986) FEBS Lett. 203, 262-266]. Using membrane preparations of [3H]inositol-labelled mesangial cells we tried to obtain further insight as to the step at which protein kinase C might interfere with the signal transduction mechanism in mesangial cells. Angiotensin II (100 nM) stimulates IP3 formation from membrane preparations of [3H]inositol-labelled mesangial cells with a half-maximal potency of 1.1 nM. The angiotensin II-induced formation of IP3 is enhanced by GTP. This effect of angiotensin II is completely blocked by the competitive antagonist [Sar1,Ala8]angiotensin II. Guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) and guanosine 5'-[beta gamma-imido]triphosphate (Gpp[NH]p), non-hydrolysable analogues of GTP, stimulate IP3 production in the absence of angiotensin II with Kd values of 0.19 microM and 2.4 microM, respectively. Angiotensin II augments the increase in IP3 formation induced by GTP gamma S. However, when mesangial cells were pretreated with TPA there was a dose-dependent inhibition of the synergistic action of angiotensin II on GTP gamma S-induced IP3 production. Comparable results are obtained with OAG, while the non-tumour-promoting phorbol ester 4 alpha-phorbol 12,13-didecanoate is without effect. These results suggest that activation of protein kinase C in mesangial cells does not impair phosphoinositide hydrolysis by stable GTP analogues but somehow seems to interfere with the stimulatory interaction of the occupied angiotensin II receptor with the transducing G-protein.     

Role of protein kinase C in arginine vasopressin-stimulated ERK and p70S6 kinase phosphorylation

We previously showed in rat renal glomerular mesangial cells, that arginine vasopressin (AVP)-stimulated cell proliferation was mediated by epidermal growth factor receptor (EGF-R) transactivation, and activation (phosphorylation) of ERK1/2 and p70S6 kinase (Ghosh et al. [2001]: Am J Physiol Renal Physiol 280:F972-F979]. In this paper, we extend these observations and show that different protein kinase C (PKC) isoforms play different roles in mediating AVP-stimulated ERK1/2 and p70S6 kinase phosphorylation and cell proliferation. AVP treatment for 0-60 min stimulated the serine/threonine phosphorylation of PKC isoforms alpha, delta, epsilon, and zeta. The activation of PKC was dependent on EGF-R and phosphatidylinositol 3-kinase (PI3K) activation. In addition, inhibition of conventional and novel PKC isoforms by chronic (24 h) exposure to phorbol 12-myristate 13-acetate (PMA) inhibited AVP-induced activation of ERK and p70S6 kinase as well as EGF-R phosphorylation. Rottlerin, a specific inhibitor of PKCdelta, inhibited both ERK and p70S6 kinase phosphorylation and cell proliferation. In contrast, a PKCepsilon translocation inhibitor decreased ERK1/2 activation without affecting p70S6 kinase or cell proliferation, while a dominant negative PKCzeta (K281W) cDNA delayed p70S6 kinase activation without affecting ERK1/2. On the other hand, G?6976, an inhibitor of conventional PKC isoforms, did not affect p70S6 kinase, but stimulated ERK1/2 phosphorylation without affecting cell proliferation. Our results indicate that PKCdelta plays an important role in AVP-stimulated ERK and p70S6 kinase activation and cell proliferation.     

Potentiation of angiotensin II-stimulated phosphoinositide hydrolysis, calcium mobilization and contraction of renal mesangial cells upon down-regulation of protein kinase C

Long-term pretreatment of rat mesangial cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) down-regulated protein kinase C activity and potentiated the angiotensin II-induced inositol trisphosphate (InsP3) formation. This increased response to angiotensin II occurred without a significant change in the receptor number or Kd value of angiotensin II binding to the cells. The biologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect on angiotensin II-stimulated InsP3 generation. Long-term pretreatment with TPA also increased the angiotensin II-induced mobilization of Ca2+ and the subsequent contraction of mesangial cells.     

IgA immune complex blunts the contraction of cultured mesangial cells through the inhibition of protein kinase C and intracellular calcium

The effects of IgA immune complex (IgA-IC) on the contractile function of cultured mesangial cells were measured by the changes in planar surface area in response to treatment with agonists. Incubation of mesangial cells with IgA-IC for 24 hours significantly decreased the contractile responses to angiotensin II (10(-6) M) and phorbol 12-myristate 13-acetate (PMA, 10(-6) M). Pretreatment of mesangial cells with the protein kinase C (PKC) inhibitor, chelerythrine (10(-6) M), eliminated the difference in contractile responses to angiotensin II or PMA between the control and IgA-IC groups indicating IgA-IC may inhibit the activity of PKC. The contractile responses to ionomycin were not significantly different between IgA-IC treated and control mesangial cells, suggesting that the contractile machinery is not impaired by IgA-IC. Intracellular calcium, [Ca2+]i measured by changes in fura-2 level in response to ATP or bradykinin, was significantly inhibited in IgA-IC treated mesangial cells, compared to control cells. In contrast, treatment with thapsigargin did not result in significant differences in [Ca2+]i between IgA-IC and control mesangial cells, suggesting that a negligible role of endoplasmic reticulum in the effects of IgA-IC. Using PKC specific antibodies, IgA-IC significantly increased the particulate fraction of PKC-iota of mesangial cells to 141+/-13% of control, without significantly changing the protein content of PKC-alpha, -delta and -lambda in the cytosolic and particulate fractions. In summary, IgA-IC inhibits the contractile responses of cultured mesangial cells to agonists by inhibiting the activation of PKC and [Ca2+]i.     

Phorbol esters and calcium-mobilizing hormones increase membrane-associated protein kinase C activity in rat hepatocytes

Vasopressin, angiotensin II, epinephrine (alpha 1-adrenergic action) and phorbol 12-myristate 13-acetate (PMA) induce increases in membrane-associated protein kinase C activity concomitant with decreases in the cytosolic activity. The data indicate that the calcium-mobilizing hormones and the active phorbol ester induce translocation from the cytosol to the plasma membrane of this protein kinase. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, blocked the translocation to the membrane of this protein kinase induced by PMA and vasopressin.     

Role for protein kinase C in the modulation of glomerular PGE2 production by angiotensin II

Angiotensin II increased PGE2 release from superfused glomeruli, and stimulated labeled inositol phosphate production. 12-O-Tetradecanoyl phorbol -13-acetate (TPA, 10(-7) M), which stimulates protein kinase C activity in soluble fractions of glomerular homogenates, suppressed angiotensin II actions on inositol phosphate production and PGE2. By contrast, 4a phorbol 12,13 di-decanoate and phorbol had no effect on protein kinase C activity or angiotensin II induced increases in inositol phosphate or PGE2. 1-(5-Isoquinolinyl)-2-methylpiperazine (H-7), which inhibits protein kinase C activity in soluble fractions of glomerular homogenates, prevented TPA induced suppression of angiotensin II actions on inositol phosphate production and PGE2. Moreover H-7 prolonged the time course of angiotensin II induced inositol phosphate production and enhanced angiotensin II actions on glomerular PGE2 production. The results support a role for inositol phospholipid hydrolysis through the phospholipase C pathway in the mediation of angiotensin II actions on PGE2 in glomeruli and are consistent with negative modulation of these actions by protein kinase C.     

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.     

Calcium-activated, phospholipid-dependent protein kinase in cultured rat mesangial cells

The analysis of the 100 000 X g supernatant fraction of cultured rat glomerular mesangial cells with DEAE-cellulose ion-exchange chromatography revealed a large peak showing the activity of a protein kinase (protein kinase C) which depended on phospholipid and diolein as well as Ca2+. Furthermore, it was shown that angiotensin II (AII) (10(-6)M) induced rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate, leading to production of diacylglycerol rich in arachidonic acid, in the cultured rat mesangial cells. These results suggest that activation of protein kinase C resulting from enhancement of phosphoinositide metabolism may be important as an intracellular regulatory mechanism of AII upon cultured mesangial cells.     

Regulation of S6 kinase activity in Madin-Darby canine kidney renal epithelial cells

Mitogenic stimulation of mammalian cells results in increased serine phosphorylation of ribosomal protein S6. Phorbol esters, which stimulate protein kinase C activity, can also increase S6 phosphorylation. In order to further investigate the role of protein kinase C in the activation S6 kinase, we studied the stimulation of an S6 kinase activity in response to phorbol ester and epinephrine in a renal epithelial cell line, Madin-Darby canine kidney cells (MDCK). In these cells, S6 phosphorylating activity in cytosolic extracts was increased following the addition of phorbol ester to the intact cells. S6 kinase and protein kinase C activities were measured in separate fractions prepared by DEAE-Sephacel fractionation of cytosolic extracts prepared from the same cells. The time course and dose-response curves for the effects of phorbol 12-myristate 13-acetate (PMA) on S6 kinase activity were similar to those for its effects on protein kinase C binding to the membrane fraction, indicating that S6 kinase activation was correlated with protein kinase C activation. Epinephrine, acting via alpha1-adrenergic receptors, also stimulated S6 kinase activity in MDCK cells; the magnitude of this effect was similar to that of PMA. However, epinephrine causes only a slight and transient association of protein kinase C with the membrane. The effect of epinephrine on S6 kinase activity, unlike that of PMA, was dependent on the presence of extracellular calcium. A23187, a calcium ionophore, could also stimulate S6 kinase activity. These results suggest that S6 kinase can be activated through more than one signaling pathway in MDCK cells. The properties of the PMA-stimulated S6 kinase were further investigated following partial purification of the enzyme. The S6 kinase was distinct from protein kinase C by several criteria. Noteably, the S6 kinase was highly specific for S6 as substrate. These results show that phorbol esters, acting through protein kinase C, stimulate the activity of a unique S6 kinase. This S6 kinase can also be activated through a signaling pathway that appears to be dependent on increased intracellular calcium.     

Protein kinase C from rat renal mesangial cells: its role in homologous desensitization of angiotensin II-induced polyphosphoinositide hydrolysis

Protein kinase C activity towards exogenous histone was found in a cytosolic fraction of rat renal mesangial cells. The analysis of the 100,000 x g supernatant fraction with DEAE-cellulose ion-exchange chromatography gave a protein kinase C preparation that was dependent on Ca2+ and phosphatidylserine for its activity. The addition of diolein decreased the Ca2+ requirement of the enzyme. 1-(5-Isoquinoline-sulfonyl)-2-methylpiperazine (H-7), sphingosine and cytotoxin I potently inhibited the protein kinase C activity prepared from mesangial cells as well as the 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced prostaglandin synthesis in intact mesangial cells. In the second part of the study, the desensitization of angiotensin II-stimulated phospholipase C activity was investigated. Angiotensin II induced a rapid increase in inositol trisphosphate (IP3) formation. Pretreatment of cells with angiotensin II, followed by removal of the hormone, resulted in a decreased response to a second application of angiotensin II. A similar protocol involving pretreatment with angiotensin II had no effect on subsequent responsiveness to [Arg8]vasopressin. The specific antagonist [Sar1, Ala8]angiotensin II did not stimulate IP3 formation neither did it inhibit the response to a subsequent stimulation with angiotensin II. After angiotensin II pretreatment, a prolonged incubation (120 min) restored responsiveness of the cells to angiotensin II. Pretreatment of mesangial cells with H-7, sphingosine or cytotoxin I almost completely diminished the desensitization of angiotensin II-stimulated IP3 generation. These results indicate that, in rat mesangial cells, angiotensin II induces a homologous desensitization of phospholipase C stimulation. It is proposed that protein kinase C activation plays an important role in the molecular mechanism of desensitization of angiotensin II-stimulated polyphosphoinositide metabolism.     

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.     

Down-regulation of protein kinase C potentiates angiotensin II-stimulated polyphosphoinositide hydrolysis in vascular smooth-muscle cells.

In smooth-muscle cells (SMC) isolated from rat aorta, angiotensin II stimulates a phospholipase C with subsequent formation of inositol trisphosphate (InsP3). Short-term (10 min) pretreatment of SMC with 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) decreases the angiotensin II-induced InsP3 formation. However, this inhibition is not observed after incubating the cells for 2 h with TPA. Longer-term pretreatments even lead to an enhanced generation of InsP3. This increased response to angiotensin II occurs without a significant change in the receptor number or Kd value of angiotensin II binding to the cells. The biologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect on angiotensin II-stimulated InsP3 generation, irrespective of the time of preincubation. In parallel with this potentiation of angiotensin II-induced generation of InsP3 by TPA, a down-regulation of protein kinase C activity is observed. A 24 h pretreatment of SMC with TPA decreases protein kinase C activity to less than 10% of that of control cells. Longer-term pretreatment also increases the angiotensin II-induced release of Ca2+ and delays the decay of the transient Ca2+ increase. All these data suggest that protein kinase C exerts a negative feedback control on angiotensin II-stimulated polyphosphoinositide turnover, and that protein kinase C is an important factor in limiting the production of InsP3 in stimulated cells.     

Phorbol esters, but not epidermal growth factor or insulin, rapidly decrease soluble protein kinase C activity in rat hepatocytes

Exposure of freshly isolated rat hepatocytes to tumor-promoting phorbol esters like phorbol 12-myristate 13-acetate resulted in a time- and concentration-dependent translocation of protein kinase C from the soluble to the particulate fraction of the cells. No such disappearance of soluble protein kinase C activity was observed with either epidermal growth factor or insulin, indicating that activation of protein kinase C is not necessarily involved in the short-term metabolic action of physiological growth factors on rat hepatocytes.     

Endothelial cell sodium-potassium-chloride cotransport. Evidence of regulation by Ca2+ and protein kinase C

Previous studies have shown that vascular endothelial cells exhibit a highly active Na-K-Cl cotransport system that is regulated by a variety of vasoactive hormones and neurotransmitters, suggesting that the cotransporter may play an important role in endothelial cell function. In this study, the regulation of endothelial cell Na-K-Cl cotransport was further investigated by probing the stimulus-transfer pathway by which vasoactive agents stimulate the cotransporter. Specifically, three peptides previously shown to stimulate cotransport activity (angiotensin II, vasopressin, and bradykinin) were evaluated. Na-K-Cl cotransport was assessed in cultured bovine aortic endothelial cells as bumetanide-sensitive K+ influx. Stimulation of Na-K-Cl cotransport by angiotensin II, vasopressin, or bradykinin was found to be reduced either by removal of extracellular Ca2+ or by treatment of the cells with 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate or 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In addition, the calmodulin antagonist W-7 was found to prevent stimulation of endothelial cell Na-K-Cl cotransport by the three peptides. These findings suggest that regulation of endothelial cell cotransport by these vasoactive peptides may be both Ca(2+)- and calmodulin-dependent. Angiotensin II, vasopressin, and bradykinin were also found to elevate phosphatidylinositol hydrolysis in the cultured endothelial cells. Thus, the possibility that regulation of endothelial Na-K-Cl cotransport by these vasoactive peptides also involves diacylglycerol activation of protein kinase C was investigated. A 10-min exposure of the endothelial cells to low doses of phorbol 12-myristate 13-acetate was found to reduce Na-K-Cl cotransport whether in the presence or absence of angiotensin II, vasopressin, or bradykinin. However, down-regulation of protein kinase C by a 40-h exposure to higher doses of the phorbol ester was found to elevate Na-K-Cl cotransport activity under both control and agonist-stimulated conditions, indicating that activation of protein kinase C results in inhibition of endothelial cell Na-K-Cl cotransport. Thus, protein kinase C activation may serve as negative feedback in the stimulus-transfer pathway by which these agonists regulate endothelial cell Na-K-Cl cotransport.     

Regulation of protein kinase activities in PC12 pheochromocytoma cells.

Stimulation of serine protein kinase activity (referred to as S6 kinase) occurs within minutes of addition of nerve growth factor (NGF) to PC12 rat pheochromocytoma cells. This enzyme activity is not related to the cAMP-dependent protein kinase (protein kinase A) or the Ca2+- and phospholipid-dependent protein kinase (protein kinase C), two other protein kinases potentially involved in signal transduction. Two peaks of NGF-stimulated S6 phosphotransferase activity are observed upon ion exchange chromatography; one that comigrates with the serine kinase previously described in chicken embryo fibroblasts and another with distinct elution properties. Several other factors are also found to regulate S6 phosphotransferase activity in PC12 cells including epidermal growth factor, insulin, and phorbol myristate acetate. Dibutyryl cAMP stimulates S6 phosphotransferase activity; however, this activity is strongly inhibited by the protein kinase A heat stable inhibitor. At least two mechanisms exist through which the NGF-stimulated S6 kinase activity can be regulated, one that apparently can use protein kinase C whereas the other(s) does not. The potential roles of these protein kinase activities in signal transduction and regulation of cell growth and differentiation is discussed.     

Protein phosphorylation and protein kinase activities in BC3H-1 myocytes. Differences between the effects of insulin and phorbol esters

To determine whether insulin activates protein kinase C in BC3H-1 myocytes, we evaluated changes in protein phosphorylation, protein kinase activities, and the intracellular translocation of protein kinase C activity in response to insulin and phorbol esters. Phorbol 12-myristate 13-acetate (PMA), but not insulin, stimulated the phosphorylation of an acidic Mr 80,000 protein which has been shown to be an apparently specific marker for protein kinase C activation. In addition, PMA, but not insulin, stimulated the rapid association of protein kinase C activity with a cellular particulate fraction. In contrast to these differences, both insulin and PMA stimulated the phosphorylation of ribosomal protein S6 and activated a ribosomal protein S6 kinase in cell-free extracts from cells exposed to these agents. In cells exposed to high concentrations of PMA for 16 h, protein kinase C activity and immunoreactivity were abolished, without changes in cellular morphology. Under these conditions, insulin, but not PMA, stimulated phosphorylation of the ribosomal protein S6 in intact cells and activated the S6 kinase in cell-free extracts derived from insulin-treated intact cells. We conclude that: insulin does not appear to activate protein kinase C in BC3H-1 myocytes, at least as assessed by phosphorylation of the Mr 80,000 protein; both insulin and PMA activate an S6 protein kinase in these cells; and insulin can promote S6 phosphorylation and activate the S6 kinase normally in protein kinase C-deficient cells. Activation of the S6 kinase by insulin and PMA, although apparently proceeding through different mechanisms, may explain some of the similar biological actions of these compounds in BC3H-1 myocytes.     

Activation of protein kinase C by phorbol esters induces DNA synthesis and protein phosphorylations in glomerular mesangial cells

The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is shown to be mitogenic for quiescent glomerular mesangial cells cultured in serum-free conditions. TPA induces DNA synthesis measured by [3H]thymidine incorporation in a dose-dependent manner with an ED50 of 7 ng/ml and an optimal response for 50 ng/ml. The phorbol ester action is potentiated by insulin with an increase of the maximal effect from 232 +/- 15% for TPA alone to 393 +/- 96% for TPA plus insulin. Down-regulation of protein kinase C by prolonged exposure to TPA completely abolishes the mitogenic effect of the phorbol ester. Using a highly resolutive 2D electrophoresis, we have shown that TPA is able to stimulate the phosphorylation of 2 major proteins of Mr 80,000, pl 4.5 (termed 80K) and Mr 28,000, pI 5.7-5.9 (termed 28K). The 80K protein phosphorylation is time- and dose-dependent with an ED50 of 8 ng/ml TPA. Exposure of mesangial cells to heat-shock induces synthesis of a 28K protein among a set of other proteins suggesting that the 28K protein kinase C substrate belongs to the family of low molecular mass stress proteins. Mitogenic concentrations of TPA and phorbol 12,13-dibutyrate inhibit [125 I]epidermal growth factor binding and stimulate the 80K protein phosphorylation with the same order of potency. The inactive tumor-promoter 4 alpha-phorbol was found to be ineffective both on these 2 parameters and on DNA synthesis. These results suggest a positive role for protein kinase C on mesangial cell proliferation and indicate the existence in this cell line of 2 major protein kinase C substrates.     

Calcitonin gene-related peptide stimulates intracellular cAMP via a protein kinase C-controlled mechanism in human ocular ciliary epithelial cells.

Calcitonin gene-related peptides I and II (CGRP I and II) were found to stimulate cAMP levels by approximately 4-6 fold in human nonpigmented ciliary epithelial cells with half-maximal effective concentrations of 20 x 10(-10) and 3 x 10(-10) M, respectively. Prior exposure of cells to 6 x 10(-7) M phorbol 12-myristate, 13-acetate for 15 min resulted in a 40-50% inhibition of CGRP II-dependent cAMP stimulation. Phorbol didecanoate and dioctanoylglycerol also effectively inhibited, whereas 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C, had no effect. Staurosporine, a protein kinase C inhibitor, blocked the inhibition of cAMP formation by phorbol esters. cAMP stimulation by forskolin or cholera toxin was not inhibited by phorbol esters, suggesting that neither a Gs protein nor adenylyl cyclase is the site of inhibition by protein kinase C. These data therefore suggest that CGRP receptors are required for inhibition of adenylate cyclase by protein kinase C.     

Signaling transduction pathway of angiotensin II in human mesangial cells: mediation of focal adhesion and GTPase activating proteins

Human mesangial cells (HMCs) respond to angiotensin II stimulation, which modulates their physiological activities, i.e., contraction and proliferation. It has been revealed that focal adhesion kinase (FAK) and paxillin participate in the angiotensin II-mediated signaling and cytoskeletal rearrangements at focal adhesion. We investigated the influences of cell adhesion upon angiotensin II effects in HMCs. In adherent cells, both FAK and paxillin were tyrosine phosphorylated by angiotensin II, while the cell detachment completely inhibited the tyrosine phosphorylation of paxillin. Activation of p44/42 mitogen-activated protein (MAP) kinase by angiotensin II was accentuated in suspended cells. Moreover, p190, a member of Rho GTPase activating protein (GAP), and RasGAP were coprecipitated with paxillin in adherent cells and angiotensin II stimulation reduced the formation of paxillin-p190 and paxillin-RasGAP complexes. These results suggest that the formation of focal adhesion complexes accelerated by accumulation of mesangial matrices may inhibit the proliferation of HMCs by modulating MAP kinase activity and be related to mesangial cell depletion.