Down-regulation of protein kinase C in rat glioma C6 cells: effects on the beta-adrenergic receptor-coupled adenylate cyclase

Continuous exposure of rat glioma C6 cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a time and dose dependent loss of [3H]phorbol dibutyrate binding sites and protein kinase C activity. Thus, by 24 h, the cells were essentially depleted of protein kinase C activity. In agreement with previous studies, TPA treatment caused a reduction in isoproterenol-stimulated adenylate cyclase activity and a sequestration of beta-adrenergic receptors. Cells were treated with TPA for 24-48 h to completely down-regulate protein kinase C and then exposed to isoproterenol. Agonist-mediated desensitization of adenylate cyclase and sequestration of beta-adrenergic receptors occurred at similar rates in control and TPA-treated cells. In addition, agonist-mediated down-regulation of beta-adrenergic receptors was not impaired by the absence of protein kinase C activity. Although both agonists and phorbol esters cause desensitization of the beta-adrenergic receptor-coupled adenylate cyclase, agonist-mediated events can occur independently of protein kinase C.     

Inhibition of prostaglandin E1-induced elevation of cytoplasmic free calcium ion by protein kinase C-activating phorbol esters and diacylglycerol in Swiss 3T3 fibroblasts

In quiescent cultures of Swiss 3T3 cells, prostaglandin E1 (PGE1) known to elevate cAMP increased rapidly cytoplasmic free Ca2+ concentration ([Ca2+]i) as measured with the fluorescent Ca2+ indicator quin2. The primary source of the PGE1-induced elevation of [Ca2+]i was extracellular. Pretreatment of the cells with various doses of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C-activating phorbol ester, inhibited the PGE1-induced elevation of [Ca2+]i in a dose-dependent manner. Inversely, TPA enhanced slightly the PGE1-induced increase of cAMP. TPA alone did not affect the basal level of [Ca2+]i or cAMP in the absence of PGE1. The inhibitory action of TPA on the PGE1-induced elevation of [Ca2+]i was mimicked by other protein kinase C-activating agents such as phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol. 4 alpha-Phorbol 12,13-didecanoate known to be inactive for protein kinase C was ineffective in this capacity. Prolonged treatment of the cells with phorbol 12,13-dibutyrate resulted in the down-regulation and disappearance of protein kinase C. In these protein kinase C-deficient cells, PGE1 still elevated [Ca2+]i to the same extent as that in the control cells, but TPA did not inhibit the PGE1-induced elevation of [Ca2+]i. These results strongly suggest that protein kinase C serves as an inhibitor for PGE1-induced Ca2+ influx in Swiss 3T3 cells.     

Tumor promoter enhances mitogenesis by PDGF with little effect on PDGF binding

Preincubation of Swiss 3T3 cells with the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) at 37 degrees C is observed to cause only a small (approximately 10%) decrease in maximal binding of 125I-platelet-derived growth factor (125I-PDGF), and does not affect the affinity of 125I-PDGF binding to these cells. Under the same conditions, the affinity of the epidermal growth factor receptor is greatly reduced, possibly resulting from phosphorylation by protein kinase C. TPA is also shown to have no effect on the kinetics of internalization or degradation of bound 125I-PDGF. Although TPA has little or no effect on these properties of the PDGF receptor, it was found to act in a synergistic fashion with low, but not high, concentrations of PDGF to increase DNA synthesis by 3T3 cells. Since TPA has previously been shown to activate protein kinase C, these findings suggest that protein kinase C does not regulate the ligand-binding properties of the PDGF receptor, and that the observed synergism between TPA and PDGF in stimulating mitogenesis reflects effects of TPA on other processes in the mitogenic pathway.     

Abnormal protein kinase C down regulation and reduced substrate levels in non-phorbol ester-responsive 3T3-TNR9 cells.

The cell line TNR9 (E. Butler-Gralla and H. R. Herschman, J. Cell. Physiol. 107:59-67, 1981) in a Swiss 3T3 cell variant that expresses protein kinase C (PKC) but is mitogenically nonresponsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We have found that PKCs purified from variant and parental cells are identical as judged by kinase activity, protease mapping, and column chromatography. We analyzed cellular levels and subcellular location of PKC in TPA-treated 3T3 and TNR9 cells via immunoprecipitation of [35S]methionine-labeled protein and assay of immune-complex PKC kinase activity. TNR9 cells grew to higher densities than parental 3T3 cells. TNR9 cells at maximal density did not down regulate PKC in response to long-term TPA treatment. We compared the 80-kilodalton (kDa) PKC substrate phosphorylation in 3T3 and TNR9 cells by using two-dimensional gels and found that TNR9 cells treated with TPA for 30 min contained only 10 to 15% as much 32Pi associated with the 80-kDa as did parental cells. The TNR9 80-kDa substrate was present at reduced levels compared with the parental-cell 80-kDa substrate as judged by immunoblot and silver staining. Thus, the loss of mitogenic responsiveness to TPA in TNR9 cells is accompanied by resistance to TPA-mediated down regulation of PKC and reduced phosphosubstrate levels.     

Antiproliferative action of protein kinase C in cultured rabbit aortic smooth muscle cells

In rabbit aortic smooth muscle cells (SMC), protein kinase C-activating 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited the whole blood serum (WBS)-induced DNA synthesis. The inhibitory action of TPA was mimicked by another protein kinase C-activating phorbol ester, phorbol-12,13-dibutyrate (PDBu), but not by 4 alpha-phorbol-12,13- didecanoate known to be inactive for this enzyme. Prolonged treatment of the cells with PDBu caused the down-regulation of protein kinase C. In these cells, WBS still induced DNA synthesis but the inhibitory action of TPA was abolished. DNA synthesis started at 18 h and reached a maximal level 24 h after the addition of WBS. TPA inhibited the WBS-induced DNA synthesis even when added 12 h after the addition of WBS. These results suggest that protein kinase C has an antiproliferative action in rabbit aortic SMC and that this action is attributed to the inhibition of the progression from the late G1 into S phase of the cell cycle. TPA also inhibited the phospholipase C-mediated hydrolysis of phosphoinositides which was induced by WBS within several minutes, but the relevance of this effect on the antiproliferative action of TPA is uncertain.     

Inhibition of protein kinase C-dependent cellular proliferation by interaction of endogenous ganglioside GM1 with the B subunit of cholera toxin

In quiescent Swiss 3T3 fibroblasts, the B subunit of cholera toxin, a protein which binds specifically to ganglioside GM1 on the cell surface, stimulates DNA synthesis and potentiates the effects of several other growth factors such as insulin, epidermal growth factor, bombesin, and even unfractionated serum. In contrast to its synergistic effect with other known growth factors, the B subunit markedly inhibited DNA synthesis induced by the phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA). The inhibitory effect of the B subunit was observed even in the presence of insulin, which greatly potentiates the mitogenic response to TPA or the B subunit. In contrast to the effect of the B subunit, calcium ionophores and cholera toxin stimulated DNA synthesis induced by TPA. The antagonism between the B subunit and TPA is not simply due to their abilities to modify their mutual binding sites or known effector systems. TPA did not block the early rise in cytosolic free calcium in response to the B subunit, and conversely, the B subunit did not modify the ability of TPA to activate protein kinase C. However, in protein kinase C-deficient cells, the antagonistic effect between TPA and the B subunit was abolished. In addition, there was no indication for the involvement of a pertussis toxin-sensitive G protein in the antagonism. Maximum inhibition was found when the B subunit was added 2 h after the addition of TPA. Significant inhibition was still evident when the time of addition of the B subunit was delayed until 6 h after the addition of TPA. This suggests that the cross-talk between signal transduction induced through endogenous gangliosides and protein kinase C is a late step in mitogenesis.     

Involvement of protein kinase C in phorbol ester-induced sensitization of HeLa cells to cis-diamminedichloroplatinum(II)

We have investigated the effect of tumor promoting phorbol esters on the antiproliferative actions of several antitumor agents. Pretreatment of HeLa cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu) caused a significant (9-fold) increase in cellular sensitivity to cis-diamminedichloroplatinum(II) (CP). TPA also sensitized HeLa cells to melphalan (2.5-fold) but had no effect on the antiproliferative activity of bleomycin, doxorubicin, vincristine, or mitomycin C. The sensitization of HeLa cells by TPA was concentration-dependent up to 1 nM and paralleled the activation of protein kinase C by TPA measured in vitro. The maximum stimulation of protein kinase C (6-fold) was observed with 10 nM TPA. 4 alpha-Phorbol 12,13-didecanoate neither activated protein kinase C nor sensitized HeLa cells to CP. 4-O-Methyl-TPA, which does not affect cell cycle distribution of HeLa cells, also sensitized these cells to CP by 6-fold and activated protein kinase C by 3-fold. Inhibitors of protein kinase C, such as palmitoylcarnitine and sphingosine, antagonized PDBu-induced sensitization of HeLa cells to CP. The maximum sensitization of HeLa cells to CP required prolonged pretreatment (greater than or equal to 24 h) with phorbol esters but could not be explained by down-regulation of protein kinase C. For example, 4-O-methyl-TPA caused no down-regulation of protein kinase C. Moreover, TPA caused substantial down-regulation of protein kinase C (1% of control) in A-253 cells but failed to sensitize A-253 cells to CP. TPA (100 nM), however, activated protein kinase C in A-253 cells by 5.5-fold. Therefore, activation of protein kinase C by TPA appears to be necessary but not sufficient for cellular sensitization to CP. The sensitization of HeLa cells by TPA was associated with a concentration- and time-dependent increase in cellular platinum content. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) blocked sensitization of HeLa cells to CP as well as the increase in platinum content caused by a 24-h pretreatment with PDBu.     

Phorbol ester-induced down-regulation of the 80-kDa myristoylated alanine-rich C-kinase substrate-related protein in Swiss 3T3 fibroblasts. Inhibition by staurosporine.

A polyclonal antiserum raised against an oligopeptide with an amino acid sequence corresponding to a sequence of the myristoylated alanine-rich C-kinase substrate (MARCKS) from mouse macrophages and rat brain recognizes the 80-kDa C-kinase substrate from Swiss 3T3 fibroblasts. Using this antiserum for quantitative determination of the 80-kDa MARCKS-related protein, we found that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a rapid down-regulation of this protein in the fibroblasts. In accordance with earlier reports, TPA causes phosphorylation of the 80-kDa protein which can be inhibited by staurosporine. Staurosporine also suppresses the TPA-induced down-regulation, possibly indicating that the down-regulation of the MARCKS-related protein is dependent on its phosphorylation by protein kinase C.     

Regulation of VL30 gene expression by activators of protein kinase C

The mouse genome contains a retrovirus-like sequence, designated VL30, which is expressed at high levels in transformed cells and which can be induced by exogenously supplied epidermal growth factor (EGF). Binding of EGF to the EGF receptor produces changes in intracellular calcium levels and phospholipase activity which indirectly lead to activation of protein kinase C. We treated AKR-2B cells, Swiss 3T3 cells, and the 3T3 variants NR6 (EGF receptorless) and TNR9 (phorbol ester nonresponsive) with various phorbol ester tumor promoters and with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol. Tumor-promoting phorbol esters (e.g. 12-O-tetradecanoyl phorbol acetate (TPA] increased the level of VL30 expression. Stimulation with either TPA or EGF produced a similar time course of VL30 expression. TPA induced VL30 expression in the EGF-receptorless NR6 cell line, indicating that neither EGF ligand-receptor binding nor phosphorylation of the EGF receptor was required for induction of VL30 expression. Protein synthesis was not required for the TPA-mediated increase in VL30 expression, as pretreatment with cycloheximide did not block or reduce the TPA effect. VL30 expression was also stimulated by treatment with sn-1,2-dioctanoylglycerol, an analog of a probable endogenous activator of protein kinase C. These results suggest that activation of protein kinase C plays a direct role in regulating VL30 expression.     

Transmodulation of epidermal growth factor binding by platelet-derived growth factor and 12-O-tetradecanoylphorbol-13-acetate is not sodium-dependent in Balb/c/3T3 cells

The addition of platelet-derived growth factor (PDGF) to many types of cells causes a rapid decrease in high affinity binding of 125I-epidermal growth factor (EGF), a process which has been termed transmodulation. Treatment with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) also results in the transmodulation of the EGF receptor in many cell types. PDGF can transmodulate EGF binding through a mechanism that is not dependent on protein kinase C activity. A recent report (Wattenberg, E. V., McNeil, P. L., Fujiki, H., and Rosner, M. R. (1989) J. Biol. Chem. 264, 213-219) described the requirement for a sodium ion influx in the down-modulation of the EGF receptor stimulated by a non-TPA-type tumor promoter, palytoxin, in Swiss 3T3 cells. We tested for a similar sodium requirement in Balb/c/3T3 and Swiss 3T3 cells stimulated by PDGF or TPA in Balb cells treated with TPA for prolonged periods to down-regulate protein kinase C activity. Our results clearly show that the PDGF- and TPA-stimulated transmodulation of the EGF receptor does not require external sodium nor is the process affected by amiloride. In each of these experiments, the loss of 125I-EGF binding occurred to a similar extent and at a similar rate in the presence or absence of sodium. Intracellular pH also did not appear to have a role in the response. The sodium ionophore, monensin, was previously shown to bring about the down-modulation of 125I-EGF binding in Swiss cells. However, our results indicate that monensin-induced transmodulation of the EGF receptor occurs with or without external sodium, suggesting that the loss of binding is not the result of a sodium ion influx. These findings demonstrate that an increase in intracellular sodium does not cause nor is it required for PDGF- or TPA-stimulated EGF receptor transmodulation.     

Tumor promoter-induced disruption of junctional complexes in cultured epithelial cells is followed by the inhibition of cytokeratin and desmoplakin synthesis

The organization and synthesis of proteins involved in the formation and stabilization of desmosome-type junctions was investigated in cultured epithelial cells treated with a tumor promoter (12-O-tetradecanoyl-phorbol-13-acetate (TPA]. In Madin-Darby bovine (MDBK) and canine (MDCK) kidney cell colonies, TPA induced a rapid disruption of desmosomes and marked alterations in cell morphology. Within 4-6 h after TPA treatment, cell shape changed from cuboidal to highly irregular, with some very long extensions that contained cytokeratin fibrils, and many flat lamellar protrusions which were devoid of cytokeratin fibrils. These morphological changes in both MDBK and MDCK cells were followed by a dramatic and coordinated inhibition in the synthesis of all cytokeratins, 14-24 h after the addition of TPA, but without a similar effect on the synthesis of vimentin, which is coexpressed in these cells. In contrast, in dense cultures of MDBK and MDCK cells the synthesis of cytokeratins and the organization of desmosomal contacts were not affected by TPA. In an epithelial cell line derived from the bovine mammary gland (BMGE-H) the synthesis of an acidic cytokeratin of 45 kD, which was previously shown to be synthesized at high levels only in dense cultures, was dramatically inhibited by TPA treatment. Cell-free in vitro translation assays with mRNA from control and TPA-treated cells also demonstrated a decrease in the synthesis of cytokeratins in response to TPA. The inhibition of cytokeratin synthesis after TPA treatment was paralleled by a decrease in the synthesis of a high molecular weight (HMW) desmoplakin protein, which was abundant in dense MDBK and BMGE-H cells. The results with TPA-treated cells are suggestive of a coordinated down-regulation in the synthesis of only those cytokeratins and of a desmoplakin which were shown to be regulated by the extent of cell-cell contact. Cytokeratin phosphorylation in TPA-treated cells was low and reflected the decrease in their total mass, suggesting that it was not altered by TPA treatment. The possible linkage between the regulation of synthesis and organization of proteins involved in desmosome formation is discussed.     

Involvement of protein kinase C in the regulation of ornithine decarboxylase mRNA by phorbol esters in rat hepatoma cells

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates a rapid increase in ornithine decarboxylase (EC 4.1.1.17; ODC) activity in target cells. Here we demonstrate that this process involves a rapid accumulation of ODC mRNA, which is maximal 3 h after treatment (three- to eightfold greater than control cells) and decays to control levels within 18 h. Stimulation of ODC mRNA by TPA is blocked by phorbol dibutyrate down-regulation of protein kinase C (PKC). ODC mRNA was also induced by the PKC activators, phospholipase C and 1-oleoyl-2-acetyl-rac-glycerol, and blocked by kinase inhibitors (trifluoroperazine, H7, and palmitoyl-L-carnitine), consistent with a requirement for PKC activation in the induction mechanism. However, the non-PKC-specific protein kinase inhibitor HA1004 also suppressed expression of ODC mRNA in response to TPA, under conditions where it did not inhibit PKC, suggesting that additional kinases may be involved in the intracellular signalling process. The stability of the ODC mRNA (control value = 6.2 +/- 1.6 h) is not significantly changed by either TPA (5.7 +/- 0.8 h) or by cycloheximide (6.0 h). These results are inconsistent with any contribution from altered mRNA half-life towards the accumulation of ODC mRNA following treatment with phorbol ester tumor promoters.     

Differential Correlation Between Translocation and Down-Regulation of Conventional and New Protein Kinase C Isozymes in C6 Glioma Cells

Abstract: Correlation between translocation and down-regulation of conventional protein kinase Cα (cPKCα) and new PKCδ (nPKCδ) induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) at different time courses (5 min, 30 min, 1 h, 3 h, 6 h, 10 h, 17 h, and 24 h) was studied in C₆ glioma cells. From the dose-dependent translocations of these two isoforms by 10-min treatment with TPA (1, 3, 10, 30, 100, 300, and 1,000 nM), we found that cPKCα was translocated by 3–1,000 nM and nPKCδ was translocated by 10–1,000 nM TPA. Both isoforms were maximally translocated by 100–1,000 nM TPA, whereas 1 nM did not translocate these two isoforms. When the cells were treated with 1,000 nM TPA for 5 min to 17 h, the translocation of these two isoforms occurred rapidly after 5-min treatment and could be sustained for 1 h, whereas down-regulation occurred after 3-h treatment and almost complete down-regulation was observed after 17-h treatment. However, the extent of down-regulation of nPKCδ was greater than that of cPKCα at 3-, 6-, and 10-h treatment. Further studies by using different doses of TPA (100, 10, 3, and 1 nM) and extending the time to 24 h showed that cPKCα was more resistant to down-regulation. This conventional isoform was maintained at a translocation state even after long-term treatment with 3–100 nM TPA, and complete down-regulation was only shown after 1,000 nM treatment. On the other hand, nPKCδ was almost completely down-regulated by long-term treatment with a translocation dose of 10–1,000 nM TPA despite higher membrane content of this new isoform. Therefore, the differential translocation and down-regulation of cPKCα and nPKCδ was demonstrated in C₆ glioma cells and this will be useful for exploring cPKCα- or nPKCδ-specific functional roles in cellular functions and different signal transduction pathways in these cells.     

Identification of multiple PKC isoforms in Swiss 3T3 cells: differential down-regulation by phorbol ester.

The expression of members of the Ca2+ and phospholipid-dependent protein kinase (PKC) family were studied in murine Swiss 3T3 cells. In addition to PKC-alpha, the presence of immunoreactive PKC-delta, -epsilon, and zeta was detected. Treatment with 500 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) led to the down-regulation of alpha, delta, and epsilon isoforms, but not that of zeta. Higher concentrations of TPA similarly had no effect on the level of PKC-zeta. In contrast to PKC-alpha, the membrane localization of PKC-delta, -epsilon, and -zeta was not enhanced by extraction in Ca(2+)-containing buffers, whereas acute TPA treatment increased membrane association of PKC-alpha, -delta, and -epsilon but not that of PKC-zeta.     

Activation of the serum response element and 12-O-tetradecanoylphorbol-13-acetate response element by the activated c-raf-1 protein in a manner independent of protein kinase C

Transfection of the cDNA encoding the activated c-raf-1 protein or addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) or dibutyryl cAMP to NIH/3T3 cells activated the c-fos gene enhancer linked to the chloramphenicol acetyltransferase or luciferase reporter gene. Prolonged treatment of NIH/3T3 cells with phorbol 12,13-dibutyrate caused down-regulation of protein kinase C. In these cells, addition of TPA did not stimulate the c-fos gene enhancer any more, but transfection of the c-raf-1 cDNA or addition of dibutyryl cAMP still stimulated the c-fos gene enhancer to the same extent as those induced in the control cells. Transfection of the c-raf-1 cDNA or addition of TPA to NIH/3T3 cells stimulated the serum response element and TPA response element but not the cAMP response element. In contrast, addition of dibutyryl cAMP to NIH/3T3 cells stimulated the cAMP response element but not the serum response element or TPA response element. These results indicate that the activated c-raf-1 protein stimulates the serum response element and TPA response element in a manner independent of protein kinase C and cAMP-dependent protein kinase. Since the c-fos gene enhancer has been shown to contain the serum response element and cAMP response element, it is most likely that the c-raf-1 protein is involved in the regulation of c-fos gene expression through the serum response element.     

Role of protein kinase C in the translational regulation of lipoprotein lipase in adipocytes

The hypertriglyceridemia of diabetes is accompanied by decreased lipoprotein lipase (LPL) activity in adipocytes. Although the mechanism for decreased LPL is not known, elevated glucose is known to increase diacylglycerol, which activates protein kinase C (PKC). To determine whether PKC is involved in the regulation of LPL, we studied the effect of 12-O-tetradecanoyl phorbol 13-acetate (TPA) on adipocytes. LPL activity was inhibited when TPA was added to cultures of 3T3-F442A and rat primary adipocytes. The inhibitory effect of TPA on LPL activity was observed after 6 h of treatment, and was observed at a concentration of 6 nM. 100 nM TPA yielded maximal (80%) inhibition of LPL. No stimulation of LPL occurred after short term addition of TPA to cultures. To determine whether TPA treatment of adipocytes decreased LPL synthesis, cells were labeled with [35S]methionine and LPL protein was immunoprecipitated. LPL synthetic rate decreased after 6 h of TPA treatment. Western blot analysis of cell lysates indicated a decrease in LPL mass after TPA treatment. Despite this decrease in LPL synthesis, there was no change in LPL mRNA in the TPA-treated cells. Long term treatment of cells with TPA is known to down-regulate PKC. To assess the involvement of the different PKC isoforms, Western blotting was performed. TPA treatment of 3T3-F442A adipocytes decreased PKC alpha, beta, delta, and epsilon isoforms, whereas PKC lambda, theta, zeta, micro, iota, and gamma remained unchanged or decreased minimally. To directly assess the effect of PKC inhibition, PKC inhibitors (calphostin C and staurosporine) were added to cultures. The PKC inhibitors inhibited LPL activity rapidly (within 60 min). Thus, activation of PKC did not increase LPL, but inhibition of PKC resulted in decreased LPL synthesis by inhibition of translation, indicating a constitutive role of PKC in LPL gene expression.     

Down-regulation of protein kinase C in Swiss 3T3 fibroblasts is independent of its phosphorylating activity

The phorbol ester TPA induces down-regulation of protein kinase C (PKC) in Swiss-3T3 fibroblasts, as determined by the use of an alpha, beta, gamma PKC-specific antiserum. PKC is almost completely degraded 10 hours after TPA treatment of the cells and recovers within 72 hours. The staurosporine derivative K252a, known to inhibit PKC activity, causes strong suppression of TPA-induced (PKC-catalyzed) protein phosphorylation in Swiss-3T3 cells. Inhibition of protein phosphorylation by K252a is still effective when the process of down-regulation is completed. However, K252a does not influence TPA-induced down-regulation of PKC at all. Thus, down-regulation of PKC is not dependent on the enzyme's phosphorylating activity and, therefore, most likely not on its autophosphorylation as has been suggested by Ohno et al. [J. Biol. Chem. 265, 6296-6300 (1990)].     

Vasopressin rapidly stimulates protein kinase C in digitonin-permeabilized Swiss 3T3 cells: involvement of a pertussis toxin-insensitive guanine nucleotide binding protein

Guanine nucleotides and pertussis toxin were used to test for the involvement of a guanine nucleotide binding protein in the vasopressin V1 receptor-mediated stimulation of protein kinase C activity in Swiss 3T3 cells. Addition of vasopressin in the presence of [gamma-32P]ATP and digitonin caused a marked and rapid increase (8 +/- 1-fold after 1 min) in the phosphorylation of an Mr = 80,000 cellular protein (80K), a specific marker for protein kinase C activation. This phosphorylation was selectively blocked by the V1 receptor antagonist Pmp1-0-Me-Tyr2 [Arg8] vasopressin, indicating that the effect was mediated through the vasopressin V1 receptor. Down regulation of protein kinase C by prior prolonged pretreatment of intact cells with phorbol 12,13-dibutyrate (PBt2) blocked the ability of vasopressin to stimulate the phosphorylation of 80K in digitonin-permeabilized cells. Addition of a submaximal concentration of vasopressin together with the GTP analogue GTP-gamma-S caused a synergistic stimulation of 80K phosphorylation. The GDP analogue GDP-beta-S caused a 50% inhibition of the phosphorylation of 80K induced by a saturating concentration of vasopressin and shifted the vasopressin dose-response curve to the right. GDP-beta-S had no effect on the dose-response for the stimulation of 80K phosphorylation induced by PBt2. Prior incubation of intact quiescent cultures of Swiss 3T3 cells with pertussis toxin did not impair either vasopressin-induced increase in cytosolic [Ca2+] or activation of protein kinase C. These findings provide functional evidence for the involvement of a pertussis toxin-insesitive G protein in the vasopressin V1 receptor-mediated stimulation of protein kinase C in Swiss 3T3 cells.