Epoxyeicosatrienoic acids activate Na+/H+ exchange and are mitogenic in cultured rat glomerular mesangial cells

The present study examined responses of cultured rat glomerular mesangial cells to exogenous exposure of epoxyeicosatrienoic acids (EET's), products of cytochrome P450 epoxygenase. One day after administration of 8,9- or 14,15-EET, cultured rat mesangial cells demonstrated significant increases in [3H]thymidine incorporation (10(-7) M 14,15-EET: 120 +/- 7% of control; n = 6; P less than 0.025; 10(-6) M 14,15-EET: 145 +/- 10%; n = 20; P less than 0.0005; 10(-6) M 8,9-EET: 167 +/- 31%; n = 9; P less than 0.05), which was not affected by addition of the cyclooxygenase inhibitor indomethacin. In addition to stimulation of [3H]thymidine incorporation, the epoxides stimulated mesangial cell proliferation. 14,15-EET administration induced intracellular alkalinization of 0.2-0.3 pH units, which was prevented by extracellular Na+ removal and blunted by amiloride (0.5 mM). Following intracellular acidification with NH4Cl addition and removal, greater than 85% of 3 mM 22Na uptake into mesangial cells was inhibited by 1 mM amiloride, indicating Na+/H+ exchange. Under these conditions, 14,15-EET stimulated Na+/H+ exchange by 42% and 8,9-EET stimulated Na+/H+ exchange by 59%. Neither protein kinase C depletion nor addition of the protein kinase C inhibitor, staurosporine, affected this stimulation. In [3H]myo-inositol loaded mesangial cells, no significant stimulation of phosphoinositide hydrolysis was detected in response to administration of 14,15-EET. Twenty-four hours after addition of [14C]14,15-EET, greater than 90% was preferentially esterified to cellular lipids, with predominant incorporation into phosphatidylinositol, phosphatidylethanolamine, and diacylglycerol. Thus, these results demonstrate epoxyeicosatrienoic acids stimulate Na+/H+ exchange and mitogenesis in mesangial cells. These effects do not appear to be mediated via phospholipase C activation. In addition, 14,15-EET was selectively incorporated into cellular lipids known to mediate signal transduction. These observations extend the potential biologic roles of c-P450 arachidonate metabolites to include stimulation of cell proliferation and suggest a role for these compounds in vascular and renal injury.     

Differential recovery of prostacyclin synthesis in cultured vascular endothelial vs. smooth muscle cells after inactivation of cyclooxygenase with aspirin

Conflicting findings from clinical trials on the use of aspirin in preventing myocardial infarction emphasize the importance of understanding the effects of aspirin on vascular cells. Cultured vascular endothelial cells and smooth muscle cells of human, rat and bovine origin synthesized prostacyclin, a key component in vascular homeostasis, when superfused with 14C arachidonic acid. Prostacyclin synthesis was inactivated following brief treatment with aspirin, which irreversibly acetylates cyclooxygenase. Marked differences were observed between endothelial and smooth muscle cells in the recovery of cyclooxygenase after aspirin treatment. Smooth muscle cells recovered within 3 hours by a process that required serum factors replaceable by epidermal growth factor (EGF) and TGF-beta. Recovery in both smooth muscle and endothelial cells was blocked by cycloheximide but not by actinomycin-D. Endothelial cell recovery occurred much more slowly, requiring up to 24 hours and was not dependent on serum factors or EGF. Furthermore, it was suppressed by growth inducing agents such as endothelial cell growth factor (ECGF) and was enhanced by conditions favoring growth arrest and cellular differentiation. Regulation of expression and recovery of cyclooxygenase following inactivation by aspirin thus differs considerably in the endothelial and smooth muscle compartments of the vasculature.     

Inhibition by corticosteroids of epidermal growth factor-induced recovery of cyclooxygenase after aspirin inactivation

Cultures of vascular smooth muscle cells superfused with [14C]arachidonic acid synthesized the antiplatelet substance prostacyclin as the major cyclooxygenase product. Prostacyclin synthesis was inactivated by aspirin, which irreversibly acetylates cyclooxygenase. Aspirin-treated cells recovered within 2 h by a process that was blocked by cycloheximide but not by actinomycin D, and that required a serum component identified as epidermal growth factor (EGF). EGF-induced recovery of cyclooxygenase was greatly potentiated by type beta transforming growth factor (TGF-beta). Incubation with EGF and TGF-beta in the 0.1-1.0 nanomolar range stimulated cyclooxygenase recovery up to 20-fold without increasing [35S]methionine incorporation into other cell proteins. Induction of cyclooxygenase by EGF and TGF-beta also was prevented by cycloheximide but not by actinomycin D. EGF-dependent recovery was blocked by preincubation with dexamethasone (2 microM), an effect that was duplicated by pure lipocortin (2-4 micrograms/ml). Incubation of membrane preparations from these cells with EGF selectively activated phosphorylation of a 35-kDa cellular protein that comigrated with lipocortin. The results suggest that cyclooxygenase recovery in aspirin-inactivated vascular smooth muscle cells is mediated by an EGF-dependent translational control that is inhibited by corticosteroids. The findings also provide a new mechanism whereby corticosteroids suppress inflammatory prostaglandins.     

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.     

Cytosolic and Nuclear Mitogen-Activated Protein Kinases Are Regulated by Distinct Mechanisms

We have investigated the regulation and localization of mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK) in both cytosolic and nuclear fractions of glomerular mesangial cells. p42 MAPK was localized by both immunoblot and kinase activity in both cytosol and nucleus and was rapidly activated, in both fractions, by fetal bovine serum and TPA. Downregulation of protein kinase C (PKC) by TPA inhibited stimulation of cytosolic p42 MAPK, but unexpectedly had no effect on stimulated p42 MAPK in the nucleus. Next we studied the upstream kinase p45 MAPKK by indirect immunofluorescence microscopy, Western blot analysis, and kinase specific activity. Unlike MAPK, p45 MAPKK is almost exclusively cytosolic in resting cells and kinase activity stimulated by TPA is restricted to the cytosol. Interestingly, PKC downregulation for 24 h with TPA dramatically enhanced nuclear MAPKK as assessed by all three techniques. Cytosolic stimulated MAPKK was attenuated in PKC downregulation. Collectively these results show that in mesangial cells: (i) p42 MAPK and p45 MAPKK localize in both the cytosol and the nucleus, and (ii) PKC exerts a negative effect on nuclear MAPKK activity as documented by PKC downregulation, which augments p45 MAPKK nuclear mass and activity. These results indicate that the dual regulation of these two kinases is under differential control in the cytosol and the nucleus.     

Cyclosporin A inhibits protein kinase C activity: a contributing mechanism in the development of nephrotoxicity?

Cyclosporin A modifies many intracellular functions in a variety of different cells. This study investigated the potential interaction between cyclosporin A and protein kinase C, as a possible mechanism for the development of nephrotoxicity. The activity of protein kinase C, in the cytosol of renal epithelial cells, was shown to be significantly inhibited in a dose-dependent manner by CSA. Activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (phorbol ester) in rat mesangial cells in culture leads to an increase in PGE2 release. Phorbol ester stimulated PGE2 release was significantly inhibited by cyclosporin A. These results would suggest that intracellular site of action of cyclosporin A, in producing alterations in intracellular function and toxicity, may be at the level of protein kinase C.     

Time course inhibition of gastric and platelet COX activity by acetylsalicylic acid in humans

Aspirin causes peptic ulcers predominately by reducing gastric mucosal cyclooxygenase (COX) activity and prostaglandin synthesis. Because aspirin circulates for only a few hours, we hypothesized that aspirin's inhibitory effect on gastric COX activity must be prolonged. We performed a placebo-controlled experiment in healthy humans to determine the duration of inhibition of aspirin on gastric mucosal COX activity (PGE(2) and PGF(2alpha) synthesis rates). Recovery of gastric COX activity after stopping aspirin was slow and linear. Seventy-two hours after 325-mg aspirin, gastric COX activity was still reduced by 57% (P < 0.001). Duration of inhibition of gastric COX activity was estimated to be 7-8 days after 325-mg aspirin and 5 days after 81-mg aspirin. Recovery of gastric prostaglandin synthesis after 325-mg but not after 81-mg aspirin occurred at slower rates in subjects with Helicobacter pylori-associated gastritis than in those with normal histology. In conclusion, aspirin inhibits gastric COX activity for much longer than predicted from its pharmacokinetic profile, explaining why aspirin at widely spaced intervals is ulcerogenic.     

Activation of S6 kinase by repeated cycles of stretching and relaxation in rat glomerular mesangial cells. Evidence for involvement of protein kinase C.

Quiescent rat glomerular mesangial cells were exposed to repeated cycles of stretching and relaxation, and the effects on the rate of collagen production, proliferation, and S6 kinase activity were investigated. Stretch/relaxation induced increases in production of both collagen and non-collagenous proteins. Proliferation of mesangial cells was stimulated by stretch/relaxation and epidermal growth factor, but not by angiotensin II; however, administration of angiotensin II augmented stretch/relaxation-induced cell proliferation. Cytosolic S6 kinase activity was stimulated by stretch/relaxation, angiotensin II, epidermal growth factor, or phorbol 12-myristate 13-acetate. The increased S6 kinase activity was detectable within 30 min after initiation of stretch/relaxation and was blocked by either inhibitors of protein kinase C or prior down-regulation of protein kinase C following prolonged incubation with phorbol 12-myristate 13-acetate. Both translocation of protein kinase C from the cytosolic to the membrane fraction and phosphorylation of an endogenous 80-kDa protein were observed within 5 min of initiation of stretch/relaxation. These results demonstrate that in mesangial cells, mechanical factors alone can induce increases in production of collagen and non-collagenous proteins and in cell proliferation. The observation that stretch/relaxation induced stimulation of S6 kinase activity through protein kinase C-dependent mechanisms suggests that activation of protein kinase C may be a key event in initiating adaptive responses of mesangial cells to increased workload.     

Thrombin activation of the Na+/H+ exchanger in vascular smooth muscle cells. Evidence for a kinase C-independent pathway which is Ca2+-dependent and pertussis toxin-sensitive

The mechanism by which human alpha-thrombin activates the Na+/H+ exchanger was studied in cultured neonatal rat aortic smooth muscle cells. Thrombin (0.4 unit/ml) caused a rapid cell acidification followed by a slow, amiloride-inhibitable alkalinization (0.10-0.14 delta pHi above base line). In protein kinase C down-regulated cells (exposed to phorbol 12-myristate 13-acetate for 24 or 72 h), the delta pHi induced by thrombin was only partially attenuated. This protein kinase C-independent activation of the Na+/H+ exchanger was blocked by pertussis toxin (islet activating protein (IAP)), reducing delta pHi by 50%. IAP did not directly inhibit Na+/H+ exchange activity as assessed by the response to intracellular acid loading. Thrombin also stimulated arachidonic acid release by 2.5 fold and inositol trisphosphate release by 6.2 fold. IAP inhibited both of these activities by 50-60%. Intracellular Ca2+ chelation with 120 microM quin2 prevented the thrombin-induced Ca2+ spike, inhibited thrombin-induced arachidonic acid release by 75%, and inhibited thrombin-induced activation of the Na+/H+ exchanger in protein kinase C-deficient cells by 65%. Increased intracellular [Ca2+] alone was not sufficient to activate the Na+/H+ exchanger, since ionomycin (0.3-1.5 microM) failed to elevate cell pH significantly. 10 microM indomethacin inhibited thrombin-induced delta pHi in both control and protein kinase C down-regulated cells by 30-50%. Thus, thrombin can activate the Na+/H+ exchanger in vascular smooth muscle cells by a Ca2+-dependent, pertussis toxin-sensitive pathway which does not involve protein kinase C.     

The effect of vitreous humour on prostaglandin production by cultured rabbit chorioretinal fibroblasts

Factors in vitreous humour which regulate prostaglandin production were investigated using cultured rabbit chorioretinal fibroblasts. These cells produced predominantly prostaglandin E2, 6-ketoprostaglandin F1 alpha, a compound likely to be a metabolite of prostaglandin E2 and 5-hydroxyeicosatetraenoic acid. The synthesis of 6-ketoprostaglandin F1 alpha was nearly completely inhibited by the cyclooxygenase inhibitor aspirin and partially inhibited by 10(-6) M dexamethasone (49%) and 10(-5) M forskolin (68%). Addition of 10% rabbit vitreous humour to subconfluent cells maintained in Dulbecco's modified Eagle's medium plus 1% fetal bovine serum resulted in stimulation of 6-ketoprostaglandin F1 alpha production by as much as 246% as measured by radioimmunoassay. Chorioretinal fibroblasts labelled by [3H]arachidonic acid incorporation into cellular phospholipids synthesised greater amounts of all labelled arachidonic acid metabolites in response to vitreous humour. It was concluded, therefore, that there are factors present in vitreous humour of molecular weight above 10 kDa which are capable of stimulating cellular cyclooxygenase activity. Confluent cells also responded to a factor(s) present in vitreous humour. The fraction of less than 10 kDa inhibited 6-ketoprostaglandin F1 alpha production by 50% when used at a concentration of 10%. Furthermore, 6-ketoprostaglandin F1 alpha production in confluent cells (but not subconfluent cells) was inhibited to 40% of control levels by vitamin C at a concentration of 1 mg/100 ml. The latter result points to an inhibitory role for vitamin C in vitreous humour. We conclude, therefore, that vitreous humour contains factors important for the regulation of prostaglandin metabolism in the eye.     

Extracellular ATP stimulates polyphosphoinositide hydrolysis and prostaglandin synthesis in rat renal mesangial cells. Involvement of a pertussis toxin-sensitive guanine nucleotide binding protein and feedback inhibition by protein kinase C

ATP stimulated a rapid and dose-dependent formation of inositol polyphosphates in rat glomerular mesangial cells. In parallel there was a 80% increase in 1, 2-diacylglycerol (DAG) after 15 s upon stimulation with ATP. The rank order of potency of a series of ATP and ADP analogues for stimulation of inositol trisphosphate (InsP3) formation was ATP greater than ATP gamma S greater than beta gamma-methylene-ATP greater than beta gamma-imido-ATP greater than ADP, while ADP beta S, AMP, adenosine and GTP were inactive, indicating the presence of P2y-purinergic receptors. ATP also stimulated a marked synthesis of prostaglandin E2 (PGE2). The rank order of potency of different ATP and ADP analogues was identical to that of InsP3 generation. Pre-treatment of the cells with pertussis toxin strongly attenuated ATP-induced formation of InsP3 and DAG. Short-term (10 min) pre-treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of protein kinase C, produced a dose-dependent inhibition of the ATP-stimulated InsP3 generation. Furthermore, inhibition of protein kinase C by the potent inhibitor staurosporin, or downregulation of protein kinase C by longterm (24 h) incubation of the cells with TPA, resulted in an enhanced formation of InsP3 towards a stimulation with ATP.     

Effects of TPA, A23187, and prostaglandin F2 alpha on progesterone synthesis by dispersed ovine luteal cells

The possible roles of protein kinase C, intracellular calcium, and oxygen environment in luteal progesterone (P4) production and their interaction with prostaglandin (PGF2 alpha) were investigated in dispersed ovine luteal cells. The following experiments were performed: 1) dose response to TPA and A23187, 2) interactions between the phorbol ester TPA and PGF2 alpha at 5% or 18% O2, 3) effect of TPA and PGF2 alpha on basal and luteinizing hormone (LH)-stimulated P4 secretion, 4) interaction of submaximal inhibitory concentrations of TPA with PGF2 alpha and the effect of indomethacin (IN) on the TPA response. Day 9 (Day 0 = first day of estrus) corpora lutea (CL) from ewes exhibiting estrous cycles of normal duration (15-17 days) were dispersed and 50,000-150,000 cells were cultured for 4 h in Dulbecco's Modified Eagle Medium. The proportion of luteal cells greater than 22 microns in diameter in these preparations averaged 17.8 +/- 2.1%. P4 in medium and cells was measured by radioimmunoassay. Both TPA and A23187 inhibited basal P4 accumulation in a dose-dependent manner. Maximum inhibition (500 nM) by TPA was greater than by A23187 at the same concentration (66.4 +/- 3.4 and 83.2 +/- 7.2% of controls, respectively; p less than 0.05), and the two were not additive in their effects. Reducing O2 did not affect P4 accumulation with or without TPA, PGF2 alpha, or both. Basal P4 accumulation was inhibited 30% by TPA and 10% by PGF2 alpha, but no additivity was seen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol ester TPA inhibits the stimulation of bumetanide-sensitive Na+/K+/Cl- transporter by different mitogens in quiescent BALB/c 3T3 mouse fibroblasts

In this study we examined the effect of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the bumetanide-sensitive Na+/K+/Cl- transporter in quiescent BALB/c 3T3 cells. We have shown that exposure of quiescent BALB/c 3T3 cultures to phorbol ester did not inhibit the basal bumetanide-sensitive Rb+ influx or efflux. In fact, at high concentration (100 ng/ml), TPA slightly stimulated the bumetanide-sensitive Rb+ influx and efflux. However, when the quiescent cultures were stimulated by serum or by defined growth factors, the stimulated fraction of the bumetanide-sensitive Rb+ influx was drastically inhibited by exposure of the cells to the phorbol ester TPA. Based on the above findings, we propose that activation of protein kinase C by the phorbol ester TPA does not inhibit the Na+/K+/Cl- cotransport activity; however it does suppress only the growth-factors-stimulated fraction of the cotransport in quiescent BALB/c 3T3 cells. These data propose that activation of kinase C has a regulatory feedback effect on the stimulation of the Na+/K+/Cl- cotransport activity by growth factors.     

Natriuretic peptide receptor-A negatively regulates mitogen-activated protein kinase and proliferation of mesangial cells: role of cGMP-dependent protein kinase

We have examined the effect of atrial natriuretic peptide (ANP) and its guanylyl cyclase/natriuretic peptide receptor-A (NPRA) on mitogen-activated protein kinase/extracellular signal-regulated kinase 2 (MAPK/ERK2) activity in rat mesangial cells overexpressing NPRA. Agonist hormones such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), angiotensin II (ANG II), and endothelin-1 (ET-1) stimulated 2.5- to 3.5-fold immunoreactive MAPK/ERK2 activity in these cells. ANP inhibited agonist-stimulated activity of MAPK/ERK2 by 65-75% in cells overexpressing NPRA, whereas in vector-transfected cells, its inhibitory effect was only 18-20%. NPRA antagonist A71915 and KT5823, a specific inhibitor of cGMP-dependent protein kinase (PKG) completely reversed the inhibitory effect of ANP on MAPK/ERK2 activity. ANP also inhibited the PDGF-stimulated [(3)H]thymidine uptake by almost 70% in cells overexpressing NPRA, as compared with only 20-25% inhibition in vector-transfected cells. These results demonstrate that ANP/NPRA system negatively regulates MAPK/ERK2 activity and proliferation of mesangial cells in a PKG-dependent manner.     

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.     

Involvement of protein kinase C in pulmonary surfactant secretion from alveolar type II cells

The purpose of this study is to clarify the involvement of protein kinase C in pulmonary surfactant secretion from adult rat alveolar type II cells in primary culture. Surfactant secretion in vitro is stimulated by at least two classes of compounds. One class, (e.g. terbutaline) increases intracellular cyclic AMP, whereas the other class (e.g. 12-O-tetradecanoylphorbol 13-acetate (TPA] does not. TPA has been shown to activate protein kinase C in other cell systems. In our studies, 1-oleoyl-2-acetyl-sn-glycerol (OAG), which is a direct activator of protein kinase C, stimulated [3H] phosphatidylcholine secretion by alveolar type II cells in a dose- and time-dependent manner. Tetracaine, which is an inhibitor of protein kinase C, inhibited the TPA-induced secretion of [3H]phosphatidylcholine from alveolar type II cells in a dose-dependent manner. However, tetracaine had no effect on terbutaline-induced secretion. The effects of terbutaline and OAG upon surfactant secretion were significantly more than additive, but those of TPA and OAG were less than additive. The specific activity of protein kinase C was 6-fold higher than cyclic AMP-dependent protein kinase found in type II cells when both kinases were assayed using lysine-rich histone as a common phosphate acceptor. Ninety-four per cent of protein kinase C activity was recovered in the cytosolic fraction of unstimulated type II cells, and 40% of activity in cytosolic fraction was translocated to particulate fraction upon treatment with TPA. As observed in other tissues, protein kinase C of alveolar type II cells was highly activated by 1,2-dioleoyl-sn-glycerol or TPA in the presence of Ca2+ and phosphatidylserine. These results suggest that pulmonary surfactant secretion in vitro is stimulated by both protein kinase C and cyclic AMP-dependent protein kinase.     

Regulation of the corpus luteum by protein kinase C. I. Phosphorylation activity and steroidogenic action in large and small ovine luteal cells

The activity and steroidogenic action of protein kinase C were evaluated in small and large steroidogenic ovine luteal cells. Protein kinase C activity (per mg protein) was threefold greater in large than in small luteal cells, whereas protein kinase A activity was similar in the two cell types. Phorbol 12-myristate 13-acetate (PMA) activated protein kinase C in luteal cells as demonstrated by membrane association of 91% of available protein kinase C within 15 min of PMA treatment. Longer treatments with PMA produced cells with low protein kinase C activity (protein kinase C-deficient cells) but did not affect cellular viability or protein kinase A activity. Activation of protein kinase C caused an acute, dose-dependent inhibition of progesterone production in unstimulated large and luteinizing hormone (LH)-stimulated small luteal cells. This inhibition by PMA appeared to be specific for protein kinase C since it was greatly attenuated in protein kinase C-deficient cells and since an inactive phorbol ester, 4 alpha-phorbol, had no effect on luteal progesterone production. The inhibitory locus of protein kinase C action in small luteal cells appeared to be distal to the adenylate cyclase enzyme because progesterone production was inhibited similarly in cells stimulated with LH, forskolin, or dibutyryl cyclic adenosine 3',5'-monophosphate. Cholesterol side-chain cleavage activity, as measured by metabolism of 25-hydroxycholesterol, was inhibited by PMA in large, but not in small, luteal cells. These data indicate that activation of protein kinase C specifically inhibits progesterone production in both large and small ovine luteal cells, although the intracellular mechanisms invoked appear to differ in the two cell types.     

p42/p44 mitogen-activated protein kinase activation is involved in prostaglandin F2alpha-induced interleukin-6 synthesis in osteoblasts.

Prostaglandin F2alpha (PGF2alpha) significantly induced p42/p44 mitogen-activated protein (MAP) kinase activity in osteoblast-like MC3T3-E1 cells. PD98059, a selective inhibitor of MAP kinase kinase, inhibited PGF2alpha-induced interleukin-6 (IL-6) synthesis as well as PGF2alpha-induced p42/p44 MAP kinase activation. PD98059 suppressed the IL-6 synthesis induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, or NaF, an activator of heterotrimeric GTP-binding protein, as well as the p42/p44 MAP kinase activation by TPA or NaF. Calphostin C, a highly potent and specific inhibitor of PKC, inhibited the PGF2alpha-induced p42/p44 MAP kinase activity. These results strongly suggest that PKC-dependent p42/p44 MAP kinase activatioin is involved in PGF2alpha-induced IL-6 synthesis in osteoblasts.     

Characterization of protein kinase C activity in interferon gamma treated murine peritoneal macrophages

Macrophage activation for tumoricidal and microbicidal functions can be achieved in part by treatment with recombinant interferon gamma (IFN gamma) in vitro. We have previously demonstrated that IFN gamma treatment of murine peritoneal macrophages results in a two- to five-fold increase in the activity of Ca++, phospholipid dependent protein kinase C (Hamilton et al., J. Biol. Chem., 260:1378, 1985). We now report that this effect was not dependent upon continuing protein synthesis since treatment with cycloheximide under conditions where normal protein synthesis was inhibited by greater than 95% had no effect upon the development of increased enzyme activity. Examination of Ca++ and phospholipid requirements revealed no differences between enzyme isolated from control or IFN gamma treated cells. Similarly, protein kinase C from control and IFN gamma-treated cells could not be distinguished in terms of the diacylglycerol (DG) or phorbol diester (PMA) concentration required for stimulation of activity. Kinetic analysis of the ATP (as substrate) concentration dependence revealed that both control and treated enzyme preparations (either basal or stimulated) had comparable Km values. Maximum velocity (Vmax) was increased both by IFN gamma treatment and also by stimulation with DG or PMA. The major difference which could be discerned between protein kinase C derived from control versus IFN gamma-treated macrophages was the magnitude of the response to DG or PMA; IFN gamma treatment increased the stimulation index (i.e., ratio of basal to stimulated activity) by a factor of two to four fold. These results suggest that IFN gamma treatment leads to reversible modulation of existing protein kinase C resulting in increased catalytic efficiency when exposed to an appropriate stimulant.     

Identification of a ribosomal protein S6 kinase regulated by transformation and growth-promoting stimuli

A serine protein kinase specific for ribosomal protein S6 in 40 S subunits has been identified and purified greater than 15,000-fold (with 18% recovery) from developing chicken embryos. An analogous enzyme has also been detected in serum-stimulated chicken embryo fibroblasts. The S6 kinase was identified as a phosphoprotein of Mr approximately 65,000 based on (i) gel filtration, (ii) apparent autophosphorylation of a 65-kDa protein when several enzyme preparations were incubated with [gamma-32P]ATP in the absence of added substrate, (iii) comigration of S6 kinase activity with the autophosphorylating activity over a variety of chromatographic resins, and (iv) elution and renaturation of S6 kinase activity from the 65-kDa region of a sodium dodecyl sulfate-polyacrylamide gel. The purified protein kinase is highly specific for S6 in 40 S subunits and does not appreciably phosphorylate casein, histone H1, mixed histones, protamine, polyoma virus capsid protein, or phosphorylase a/b. These characteristics suggest that this enzyme is unrelated to other protein kinases believed to be activated in stimulated cells, including cAMP-dependent protein kinase, protein kinase C (Ca2+/phospholipid-dependent enzyme), or Ca2+/calmodulin-dependent protein kinases. In fibroblasts, S6 kinase is activated by a variety of mitogenic agents including the tyrosine-specific protein kinase of Rous sarcoma virus, pp60v-src, phorbol esters, and growth factors. The present identification and purification of the S6 kinase should facilitate future studies aimed at elucidating the molecular mechanisms by which signals from these diverse stimuli rapidly converge upon and activate this enzyme.