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.     

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.     

Ethanol causes desensitization of receptor-mediated phospholipase C activation in isolated hepatocytes.

The effect of ethanol on receptor-mediated phospholipase C-linked signal transduction processes was investigated in isolated rat hepatocytes. Pretreatment of the cells with ethanol (6-300 mM) markedly inhibited a subsequent stimulation of phospholipase C by vasopressin, angiotensin II, or epidermal growth factor. By contrast, the effects of the alpha 1-adrenergic agonist phenylephrine and of glucagon were not affected by ethanol pretreatment. Ethanol inhibited the agonist-induced decrease in polyphosphoinositides, the formation of inositol phosphates, and the increase in cytosolic free Ca2+ levels, as detected with the intracellular Ca2+ indicator indo-1. The effects of ethanol were concentration dependent and were pronounced at low concentrations of agonists but were not significant at saturating levels. Pretreatment of the cells with the protein kinase C inhibitor H7 partly prevented the inhibition by ethanol of vasopressin-induced phospholipase C activation. By contrast, pretreatment of the cells with (Rp)-adenosine cyclic 3':5'-phosphorothioate [Rp)-cAMP-S), a competitive inhibitor of protein kinase A, potentiated the inhibitory effect of ethanol on the Ca2+ mobilization by vasopressin. (Rp)-cAMP-S similarly potentiated the inhibition of phospholipase C by the protein kinase C-activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The kinase A inhibitor also made the Ca2+ mobilization by phenylephrine sensitive to ethanol, indicating that the formation of cAMP in the cells played a role in suppressing the sensitivity to ethanol. Pretreatment of the cells with ethanol enhanced the inhibitory effects of TPA on the vasopressin-induced phospholipase C activation at all concentrations of the hormone; however, these synergistic effects were prevented when TPA was added prior to ethanol, a condition that prevents the activation of phospholipase C by ethanol. The data indicate that ethanol causes desensitization of the receptor-mediated phospholipase C secondary to the ethanol-induced activation of phospholipase C and activation of protein kinase C. Ethanol treatment also affects the sensitivity of the phospholipase C system to control by protein kinases A and C. The data indicate that ethanol can affect the control of intracellular signal transduction processes in liver cells under physiologically relevant conditions.     

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.     

Effect of phorbol ester on stimulus-secretion coupling mechanisms in gonadotropin releasing hormone-stimulated pituitary gonadotrophs

The feedback regulatory control mechanism exerted by activated Ca2+/phospholipid-dependent protein C kinase upon gonadotropin releasing hormone (GnRH) binding, stimulation of phosphoinositide turnover and gonadotropin secretion was investigated in cultured pituitary cells. Addition of the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), at concentrations which activate pituitary protein C kinase, to cultured pituitary cells resulted in up-regulation of GnRH receptors (155% at 4 h). The stimulatory effect of GnRH on [3H]inositol phosphates (Ins-P) production in myo-[2-3H]inositol prelabeled pituitary cells was not inhibited by prior treatment of the cells with TPA (10(-9)-10(-7) M). Higher concentrations of TPA (10(-6)-10(-5) M) inhibited the effect of GnRH on [3H]Ins-P production. Increasing concentrations of TPA or the permeable analog of diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) stimulated luteinizing hormone (LH) release from cultured pituitary cells with ED50 values of 5 x 10(-9) M and 10 micrograms/ml, respectively. No consistent inhibition or additivity of LH release was observed when increasing doses of TPA or OAG were added with a submaximal dose of GnRH. These results suggest that protein C kinase might mediate the known homologous up-regulation of GnRH receptors during the reproductive cycle. Protein C kinase is positively involved in mediating the process of gonadotropin secretion. Unlike many other systems, activation of protein C kinase in pituitary gonadotrophs is not involved in negative feed-back regulation of stimulus-secretion-coupling mechanisms in GnRH-stimulated gonadotrophs.     

Phorbol ester and 1-oleoyl-2-acetylglycerol inhibit angiotensin activation of phospholipase C in cultured vascular smooth muscle cells

Angiotensin II acts on cultured rat aortic vascular smooth muscle cells (VSMC) to induce the rapid, phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate and mobilization of intracellular Ca2+. sn-1,2-Diacylglycerol, the other major product of inositol phospholipid breakdown, is known to activate protein kinase C, but its role in angiotensin II action on VSMC has not been defined. We report herein that, in cultured VSMC prelabeled with [3H]myoinositol, brief incubations (2-5 min) with 4 beta-phorbol 12-myristate 13-acetate (PMA) (1-100 nM) or 1-oleoyl-2-acetylglycerol (10-100 microM), two potent activators of protein kinase C, inhibit subsequent angiotensin II (100 nM)-induced increases in phosphatidylinositol 4,5-bisphosphate breakdown and inositol trisphosphate formation. In addition, pretreatment of VSMC with either PMA (IC50 approximately 1 nM) or 1-oleoyl-2-acetylglycerol (IC50 approximately 7.5 microM) also markedly inhibits angiotensin II (1 nM)-stimulated increases in cytosolic free Ca2+, as measured with the calcium-sensitive fluorescent indicator quin 2, or 45Ca2+ efflux. Neither PMA nor 1-oleoyl-2-acetylglycerol initiated phosphatidylinositol 4,5-bisphosphate breakdown or Ca2+ flux by itself. PMA treatment (10 nM, 5 min) did not influence the number or affinity of 125I-angiotensin II-binding sites in intact cells. These data suggest that one function of angiotensin II-generated sn-1,2-diacylglycerol in vascular smooth muscle may be to modulate, by protein kinase C-mediated mechanisms, angiotensin II receptor coupling to phospholipase C.     

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.     

Regulation of epidermal growth factor-stimulated formation of inositol phosphates in A-431 cells by calcium and protein kinase C

Epidermal growth factor (EGF) treatment of A-431 cells induces a biphasic increase in the levels of inositol phosphates. The growth factor produces an initial, rapid increase in the level of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) due to hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (Wahl, M., Sweatt, J. D., and Carpenter, G. (1987) Biochem. Biophys. Res. Commun. 142, 688-695). The level of inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) also rises rapidly in response to treatment with EGF. The initial formation (less than 1 min) of Ins-1,4,5-P3 and Ins-1,3,4,5-P4 does not require Ca2+ present in the culture medium. However, the addition of Ca2+ to the medium at levels of 100 microM or greater potentiates the growth factor-stimulated increases in the levels of all inositol phosphates at later times after EGF addition (1-60 min). The data suggest that EGF-receptor complexes initially stimulate the enzyme phospholipase C in a manner that is independent of an influx of extracellular Ca2+. The presence of Ca2+ in the medium allows prolonged growth factor activation of phospholipase C. Treatment of A-431 cells with Ca2+ ionophores (A23187 and ionomycin) did not mimic the activity of EGF in producing a rapid increase in the formation of the Dowex column fraction containing Ins-1,4,5-P3, Ins-1,3,4,5-P4, and inositol 1,3,4-trisphosphate (InsP3). However, the initial EGF-stimulated formation of inositol phosphates was substantially diminished in cells loaded with the Ca2+ chelator Quin 2/AM. EGF receptor occupancy studies indicated that maximal stimulation of InsP3 accumulation by EGF requires nearly full (75%) occupancy of available EGF binding sites, while half-maximal stimulation requires 25% occupancy. 12-O-Tetradecanoylphorbol-13-acetate (TPA), an exogenous activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), causes a dramatic, but transient, inhibition of the EGF-stimulated formation of inositol phosphates. Tamoxifen and sphingosine, reported pharmacologic inhibitors of protein kinase C activity, potentiate the capacity of EGF to induce formation of inositol phosphates. Neither TPA nor tamoxifen significantly affects the 125I-EGF binding capacity of A-431 cells; however, TPA appeared to enhance internalization of the ligand. Ligand occupation of the EGF receptor on the A-431 cell appears to initiate a complex signaling mechanism involving production of intracellular messengers for Ca2+ mobilization and activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Angiotensin II causes phosphatidylinositol turnover and increases 1,2-diacylglycerol mass but is not mitogenic in rat liver T51B cells.

Proliferation in rat liver T51B cells has previously been shown to be initiated by the tyrosine-kinase activator epidermal growth factor. We have found that T51B cells also contain angiotensin II receptors, and, as the transforming mas oncogene has been identified as a functional angiotensin receptor [Jackson, Blair, Marshall, Goedert & Hanley (1988) Nature (London) 335, 437-440], we have investigated the possibility that angiotensin II might also regulate proliferation of T51B cells. Angiotensin II at concentrations up to 10 microM did not promote DNA synthesis, even in the presence of the co-mitogens serum (1%) or 12-O-tetradecanoylphorbol 13-acetate (TPA) (50 ng/ml). The addition of 1 microM angiotensin II to myo-[3H]inositol-radiolabelled T51B cells did however result in a rapid accumulation of multiple inositol phosphates as well as in an increase in intracellular Ca2+, demonstrating the coupling of the angiotensin receptor in these cells to a polyphosphoinositide-hydrolysing phospholipase C. The increases in both inositol phosphates and intracellular Ca2+ were lower in cells pretreated for 10 min with 50 ng of TPA/ml and potentiated by a 24 h pretreatment with TPA. In addition, angiotensin II increased 1,2-diacylglycerol levels. These results demonstrate that, although angiotensin II is capable of increasing phosphoinositide-derived second messengers in T51B cells, these responses are not sufficient to trigger DNA synthesis.     

Inhibition of prostaglandin E2-induced phosphoinositide metabolism by phorbol ester in bovine adrenal chromaffin cells.

In bovine adrenal chromaffin cells, prostaglandin E2 (PGE2) stimulates the formation of inositol phosphates and Ca2+ mobilization through its specific receptor [Yokohama, Tanaka, Ito, Negishi, Hayashi & Hayaishi (1988) J. Biol. Chem. 263, 1119-1122]. Here we show that PGE2-induced phosphoinositide metabolism was blocked by pretreatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Using intact cells, we also examined the inhibitory effect of TPA on the individual steps of the activation process of phosphoinositide metabolism. The inhibition was observed within 1 min and complete by 10 min after addition of 1 microM-TPA, and half-maximal inhibition by TPA occurred at 20 nM. TPA prevented Ca2+ mobilization induced by PGE2, but not by the Ca2+ ionophore ionomycin. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not inhibit the formation of inositol phosphates and Ca2+ mobilization by PGE2. TPA treatment affected neither the high-affinity binding of [3H]PGE2 to intact cells and membrane fractions nor the ability of guanosine 5'-[gamma-thio]triphosphate to decrease the binding in membrane fractions. TPA also abolished phosphoinositide metabolism induced by muscarinic-receptor activation. NaF plus AlCl3 and ionomycin caused the accumulation of inositol phosphates, probably by directly activating a GTP-binding protein(s) and phospholipase C respectively; neither accumulation was inhibited by TPA treatment. These results suggest that protein kinase C serves as a feedback regulator for PGE2-induced phosphoinositide metabolism. The site of action of TPA appears to be distal to the coupling of the receptor to GTP-binding protein, but on a component(s) specific to the agonist-induced phosphoinositide metabolism.     

Calcium mobilization in permeabilized fibroblasts: effects of inositol trisphosphate, orthovanadate, mitogens, phorbol ester, and guanosine triphosphate

Utilizing a digitonin-permeabilized cell system, we have studied the release of calcium from a non-mitochondrial intracellular compartment in cultured human fibroblasts (HSWP cells). Addition of 1 mM MgATP to a monolayer of permeabilized cells in a cytosolic media buffered to 150 nM Ca with EGTA rapidly stimulates 45Ca uptake, and the subsequent addition of the putative intracellular messenger inositol trisphosphate (InsP3) induces rapid release of 85% (+/- 6% n = 6) of the 45Ca taken up in response to ATP. Mitogenic peptides (bradykinin, vasopressin, epidermal growth factor [EGF], and insulin) and orthovanadate, which are effective in mobilizing intracellular Ca in intact cells, have little or no effect when added alone to permeabilized cells. However, in the presence of GTP these agents stimulate accumulation of inositol phosphates and release Ca from the InsP3-sensitive pool. These data suggest that a GTP binding protein is involved in receptor mediated activation of phospholipase C, which leads to release of inositol phosphates. The GTP-dependent release of InsP3 and the mobilization of 45Ca from the intracellular compartment are inhibited by pretreatment of cells, prior to permeabilization, with the protein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). TPA pretreatment does not affect the InsP3 stimulated Ca release. These results suggest that protein kinase C is involved in down-regulation or inhibition of phospholipase C, or the GTP binding protein responsible for relaying the mitogenic signal from the cell surface receptor to the phospholipase C activity.     

Synergistic signals in the mechanism of antigen-induced exocytosis in 2H3 cells: evidence for an unidentified signal required for histamine release

The aim of this study was to determine whether the increase in cytosolic free Ca2+ concentration ([Ca2+]i) in response to antigen (aggregated ovalbumin) on IgE-primed 2H3 cells was sufficient to account for exocytosis. When the [Ca2+]i responses to antigen and the Ca2+ ionophore A23187 were compared, A23187 was much less effective at releasing histamine at equivalent [Ca2+]i increases, and little or no stimulated histamine release occurred with A23187 concentrations that matched the [Ca2+]i response to antigen concentrations that stimulated maximal histamine release. The [Ca2+]i response to antigen is not, therefore, sufficient to account for exocytosis, although extracellular Ca2+ is necessary to initiate both the [Ca2+]i response and histamine release: the antigen must generate an additional, unidentified, signal that is required for exocytosis. To determine whether this signal was the activation of protein kinase C, the effects of the phorbol ester 12-0-tetradecanoyl phorbol 13-acetate (TPA) on the responses to antigen were examined. TPA blocked the antigen-induced [Ca2+]i response and the release of inositol phosphates but had little effect on histamine release and did not stimulate exocytosis by itself. The unidentified signal from the antigen is therefore distinct from the activation of protein kinase C and is generated independently of the [Ca2+]i response or the release of inositol phosphates. Taken together with other data that imply that there is very little activation of protein kinase C by antigen when the rate of histamine release is maximal, it is concluded that the normal exocytotic response to antigen requires the synergistic action of the [Ca2+]i signal together with an unidentified signal that is not mediated by protein kinase C.     

Angiotensin II and bradykinin stimulate phosphoinositide breakdown in intact rat kidney glomeruli but not in proximal tubules: glomerular response modulated by phorbol ester

We have investigated the effect of angiotensin II, bradykinin, insulin and insulin-like growth factor I on phosphoinositide turnover in intact rat glomeruli and tubules. Angiotensin II produced a dose-dependent increase in inositol monophosphate formation with an IC50 of 10(-7)M, when added to isolated rat glomeruli. Angiotensin II-stimulated inositol phosphates formation was inhibited by the angiotensin receptor antagonist [Sar-Leu8]angiotensin II, indicating that the above response was mediated through activation of an angiotensin receptor in intact glomeruli. Besides angiotensin, in intact glomeruli, only bradykinin stimulated a phosphoinositide response, while in intact proximal tubules, none of the agonists tested produced an activation of the inositol phosphate formation. Angiotensin II- and bradykinin-stimulated inositol phosphate accumulation in intact glomeruli was inhibited by phorbol myristate acetate, an activator of protein kinase C.     

Endothelial inositol phosphate generation and prostacyclin production in response to G-protein activation by AlF4-.

In order to elucidate the role of guanine-nucleotide-binding proteins (G-proteins) in endothelial prostacyclin (PGI2) production, human umbilical vein endothelial cells, prelabelled with either [3H]inositol or [3H]arachidonic acid, were stimulated with the non-specific G-protein activator aluminium fluoride (AlF4-). AlF4- caused a dose- and time-dependent generation of inositol phosphates, release of arachidonic acid and production of PGI2. The curves for the three events were similar. When the cells were stimulated in low extracellular calcium (60 nM), they released [3H]arachidonic acid and produced PGI2, but depleting the intracellular Ca2+ stores by pretreatment with the Ca2+ ionophore A23187 totally inhibited both events, although the cells still responded when extracellular Ca2+ was added. The Ca2+ ionophore did not inhibit the generation of inositol phosphates in cells maintained at low extracellular Ca2+. Pertussis toxin pretreatment (14 h) altered neither inositol phosphate nor PGI2 production in response to AlF4-. To investigate the functional role of the diacylglycerol/protein kinase C arm of the phosphoinositide system, the cells were pretreated with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the protein kinase C inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7). TPA inhibited the AlF4(-)-induced inositol phosphate generation but stimulated both the release of arachidonic acid and the production of PGI2. H7 had opposite effects both on inositol phosphate generation and on PGI2 production. These results suggest that AlF4(-)-induced PGI2 production is mediated by a pertussis-toxin-insensitive G-protein which activates the phosphoinositide second messenger system. This production of PGI2 can be modulated by protein kinase C activation, both at the level of inositol phosphate generation and at the level of arachidonic acid release.     

Stimulation of glucose production from glycogen by glucagon, noradrenaline and non-degradable adenosine analogues is counteracted by adenosine and ATP in cultured rat hepatocytes.

We have shown previously that exposure of a non-transformed continuous line of rat liver epithelial (WB) cells to epidermal growth factor (EGF), adrenaline, angiotensin II or [Arg8]vasopressin results in an accumulation of the inositol phosphates InsP1, InsP2 and InsP3 [Hepler, Earp & Harden (1988) J. Biol. Chem. 263, 7610-7619]. Studies were carried out with WB cells to determine whether the EGF receptor and other, non-tyrosine kinase, hormone receptors stimulate phosphoinositide hydrolysis by common, overlapping or separate pathways. The time courses for accumulation of inositol phosphates in response to angiotensin II and EGF were markedly different. Whereas angiotensin II stimulated a very rapid accumulation of inositol phosphates (maximal by 30 s), increases in the levels of inositol phosphates in response to EGF were measurable only following a 30 s lag period; maximal levels were attained by 7-8 min. Chelation of extracellular Ca2+ with EGTA did not modify this relative difference between angiotensin II and EGF in the time required to attain maximal phospholipase C activation. Under experimental conditions in which agonist-induced desensitization no longer occurred in these cells, the inositol phosphate responses to EGF and angiotensin II were additive, whereas those to angiotensin II and [Arg8]vasopressin were not additive. In crude WB lysates, angiotensin II, [Arg8]vasopressin and adrenaline each stimulated inositol phosphate formation in a guanine-nucleotide-dependent manner. In contrast, EGF failed to stimulate inositol phosphate formation in WB lysates in the presence or absence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]), even though EGF retained the capacity to bind to and stimulate tyrosine phosphorylation of its own receptor. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate the inhibitory guanine-nucleotide regulatory protein of adenylate cyclase (Gi), had no effect on the capacity of EGF or hormones to stimulate inositol phosphate accumulation. In intact WB cells, the capacity of EGF, but not angiotensin II, to stimulate inositol phosphate accumulation was correlated with its capacity to stimulate tyrosine phosphorylation of the 148 kDa isoenzyme of phospholipase C. Taken together, these findings suggest that, whereas angiotensin II, [Arg8]vasopressin and alpha 1-adrenergic receptors are linked to activation of one or more phospholipase(s) C by an unidentified G-protein(s), the EGF receptor stimulates phosphoinositide hydrolysis by a different pathway, perhaps as a result of its capacity to stimulate tyrosine phosphorylation of phospholipase C-gamma.     

Effects of phorbol ester on mitogen and orthovanadate stimulated responses of cultured human fibroblasts

Mitogenic stimulation of quiescent human fibroblasts (HSWP) with serum or a mixture of growth factors (consisting of vasopressin, bradykinin, EGF, and insulin) stimulates the release of inositol phosphates, mobilization of intracellular Ca, activation of Na/H exchange and subsequent incorporation of [3H]-thymidine. We have determined previously that pretreatment with the tumor-promoting phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) inhibits mitogen-stimulated Na influx in HSWP cells. We report herein that TPA pretreatment also substantially inhibits the mitogen-stimulated release of inositol phosphates in HSWP cells. Half maximal inhibition of mitogen-stimulated inositol phosphate release occurs at 1-2 nM TPA. Treatment of cells with TPA alone has no effect on inositol phosphate release. The effect of TPA pretreatment on inositol phosphate release induced by individual growth factors has also been determined. Orthovanadate, reported by Cassel et al. (1984) to increase Na/H exchange in A431 cells, has been demonstrated to stimulate both Na influx and inositol phosphate release in HSWP cells. TPA pretreatment also inhibits both orthovanadate-stimulated inositol phosphate release and Na influx. In addition, orthovanadate was determined to increase intracellular Ca activity by mobilizing intracellular calcium stores, as determined with the fluorescent intracellular calcium probe fura-2. TPA pretreatment blocks orthovanadate stimulated mobilization of intracellular Ca stores. It appears clear that in HSWP cells pretreatment of cells with phorbol ester is capable of artificially desensitizing the early cellular responses to mitogenic stimuli (growth factors, orthovanadate) by blocking the signal transduction mechanism involved at a point prior to the release of inositol phosphates. We hypothesize that in HSWP cells the normal desensitization of both inositol phosphate release and Na/H exchange is mediated via activation of protein kinase C subsequent to the stimulus-mediated activation of phospholipase C and release of protein kinase C activator diacylglycerol. However it is interesting to note that TPA-mediated inhibition of these early responses in HSWP cells does not inhibit their ability to be stimulated to incorporate [3H]-thymidine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphatidylinositol Metabolism During In Vitro Hypoxia

The effects of in vitro histotoxic hypoxia (0.5 mM KCN) on potassium-stimulated phosphatidylinositol turnover were determined. In rat cortical slices that were prelabeled with [2-3H]inositol, depolarization with 60 mM KCl increased [2-3H]inositol monophosphate and [2-3H]inositol bisphosphate accumulation in a Ca2+-dependent manner. At early times (10 s and 1 min), histotoxic hypoxia enhanced potassium-stimulated [2-3H]inositol monophosphate and inositol bisphosphate accumulation. Under basal conditions, hypoxia did not alter the accumulation of [2-3H]inositol phosphates. These results are consistent with the following hypothesis. The hypoxic-induced increase in cytosolic free calcium that we reported previously may lead to the early stimulation of inositol phosphates formation during hypoxia through activation of phospholipase C. The impairment of inositol phosphates formation during more prolonged hypoxia may be due to negative feedback regulation of the phosphatidylinositol cascade by protein kinase C or to a reduction in ATP levels.     

Long-term phorbol ester treatment down-regulates protein kinase C and sensitizes the phosphoinositide signaling pathway to hormone and growth factor stimulation. Evidence for a role of protein kinase C in agonist-induced desensitization

Exposure of a nontransformed, continuous line of epithelial cells derived from rat liver (WB cells) to epidermal growth factor, angiotensin II, [Arg8]vasopressin, and epinephrine resulted in rapid accumulation of the inositol phosphates (InsP) InsP1, InsP2, and InsP3. Although short-term (5-60 min) pretreatment of WB cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly attenuated InsP accumulation in response to all agonists, the inhibitory effects on the InsP response were lost after 2 h incubation with PMA; and, with extended (6-24 h) preincubation, a time-dependent potentiation of the InsP response to angiotensin II, epidermal growth factor and [Arg8]vasopressin was observed. The InsP response during a 15-min challenge with angiotensin II in cells pretreated for 18 h with 600 nM and 10 microM PMA was increased by 2-3-fold and 4-6-fold, respectively. Long-term (18 h) treatment with 600 nM and 10 microM PMA caused a similar 90-100% loss of measurable Ca2+/phospholipid-dependent enzyme (protein kinase C) activity in cytosolic and soluble particulate fractions. The effects of long-term PMA pretreatment do not represent a general enhancement of hormone responsiveness since the InsP response to epinephrine was not affected. In control cells, the InsP response to angiotensin II and epinephrine desensitized very rapidly. Long-term pretreatment with PMA greatly reduced the contribution of agonist-induced desensitization to the angiotensin II response; in contrast, the extent of desensitization occurring during incubation of WB cells with epinephrine was unaltered by long-term treatment with PMA suggesting that an additional mechanism may be involved in alpha 1-adrenergic receptor desensitization. No PMA-induced change in resting levels of [3H]phosphoinositides or the metabolism of exogenous [3H]inositol 1,4,5-trisphosphate by WB homogenates occurred. Stimulation of InsP formation in intact cells by NaF and activation of phospholipase C by GTP gamma S in membranes both were unaltered by short-term or long-term PMA pretreatment. These data are consistent with the idea that following long-term treatment of WB cells with PMA, the occurrence of agonist-induced desensitization of receptors linked to the phosphoinositide/Ca2+ signaling system is reduced, apparently at least in part due to the loss of contribution of a negative feedback regulatory role of protein kinase C.     

Tumor promoter 12-O-tetradecanoylphorbol 13-acetate inhibits mas/angiotensin receptor-stimulated inositol phosphate production and intracellular Ca2+ elevation in the 401L-C3 neuronal cell line

Stimulation of mas-oncogene transfected 401L-C3 cells by angiotensins leads to the production of inositol phosphates. This response shows dose dependence, and has an apparent rank order of potency angiotensin III greater than or equal to angiotensin II much greater than angiotensin I. Preincubation with 12-O-tetradecanoylphorbol 13-acetate, for 5 min, significantly diminishes both inositol phosphate and intracellular [Ca2+] responses to angiotensins, without affecting those stimulated by the endogenous bradykinin receptor. Incubation of 401L-C3 cells with either phorbol ester or angiotensins leads to elevation of intracellular pH, implying that mas/angiotensin receptor stimulation itself leads to protein kinase C activation. These results suggest the operation of a negative feedback loop specific for the mas/angiotensin receptor signalling pathway, and which may be essential in defining the final biological output response to this receptor stimulation.     

Angiotensin II but not potassium induces subcellular redistribution of protein kinase C in bovine adrenal glomerulosa cells

The distribution of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) between cytosol and membrane fractions was examined in bovine adrenal glomerulosa cells treated with angiotensin II or potassium. Protein kinase C was isolated from cytosol and from detergent-solubilized particulate fractions by DEAE-cellulose chromatography. A major peak of activity for both the soluble and particulate forms of adrenal glomerulosa protein kinase C was eluted at 0.05-0.09 M NaCl. The soluble and particulate forms were found to constitute about 95 and 5%, respectively, of the total enzyme activity in unstimulated cells. A second peak of kinase activity was eluted with 0.15-0.19 M NaCl, which was not dependent on the presence of phospholipids. Exposure of isolated cells for 20 min to 10(-8) M angiotensin II resulted in a decrease in cytosolic activity to 30-40% of control values, and in a corresponding increase in protein kinase C activity associated with the particulate fraction. This hormone-induced redistribution was found to be dose-dependent with an ED50 of 2 nM for angiotensin II, and it occurred rapidly, reaching a plateau within 5-10 min. It was prevented by the specific antagonist [Sar1,Ala8]angiotensin II. By contrast, stimulation with 12 mM KCl did not change the subcellular distribution of protein kinase C activity. These results suggest that redistribution of protein kinase C represents an early step in the post-receptor activation cascade following angiotensin II, but not potassium stimulation of adrenal glomerulosa cells.