Desensitization Of Protein Kinase C Research Articles

Mechanism Involved in the Spasmolytic Effect of a Mixture of Two Triterpenes, Cycloartenol and Cycloeucalenol, Isolated fromHerissanthia tiubaein the Guinea-Pig Ileum

The smooth muscle relaxant properties of a mixture of the two triterpenoids cycloeuclalenol and cycloartenol (CC) isolated from Herissanthia tiubae (Malvaceae) were studied in several smooth muscle preparations. CC inhibited contractions induced by carbachol, histamine and KCl in the guinea-pig ileum, but no spasmolytic activity was found in guinea-pig trachea or rat aorta. In guinea-pig ileum, concentration-response curves to carbachol and CaCl (2) in high K(+) were shifted to the right by CC in a concentration-dependent manner with slopes of the Schild plot differing from the unity. IC(50) values were 3.4 +/- 0.8 x 10 (-5) M and 8.44 +/- 1.87 x 10 (-5) M for carbachol and CaCl(2), respectively. The phorbol ester TPA, which activates protein kinase C (PKC), potentiated contractions induced by submaximal concentrations of carbachol. This potentiation was inhibited by CC. Desensitization of PKC by TPA completely abolished the inhibition produced by CC on carbachol-induced contractions. Together, our results indicate that inhibition of PKC is involved in the spasmolytic effect of CC in the guinea-pig ileum.

Transforming Growth Factor-β1 Regulates Kir2.3 Inward Rectifier K+ Channels via Phospholipase C and Protein Kinase C-δ in Reactive Astrocytes from Adult Rat Brain

The multifunctional cytokine, transforming growth factor beta(1) (TGF-beta(1)), exerts complex effects on astrocytes with early signaling events being less well characterized than transcriptional mechanisms. We examined the effect of TGF-beta(1) on the 14-pS Kir2.3 inward rectifier K(+) channel in rat primary cultured reactive astrocytes. Immunofluorescence study showed that cells co-expressed TGF-beta(1) receptors 1 and 2, Kir2.3, and glial fibrillary acidic protein (GFAP). Patch clamp study showed that TGF-beta(1) (0.1-100 ng/ml) caused a rapid (<5 min) depolarization because of dose-dependent down-regulation of Kir2.3 channels, which was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (10-500 nm) and which was inhibited by the PKC inhibitor calphostin C (100 nm), by PKC desensitization produced by 3 h of exposure to phorbol 12-myristate 13-acetate (100 nm), and by the PKC-delta isoform-specific inhibitor rottlerin (50 microm). Immunoblot analysis and confocal imaging showed that TGF-beta(1) caused PKC-delta translocation to membrane, and co-immunoprecipitation experiments showed that TGF-beta(1) enhanced association between Kir2.3 and PKC-delta. Additional electrophysiological experiments showed that Kir2.3 channel down-regulation was blocked by the phospholipase C inhibitors, neomycin (100 microm) and D609 (200 microm). Given the commonality of signaling involving PLC-PKC-delta, we speculate that TGF-beta(1)-evoked depolarization may be an early signaling event related to gene transcription in astrocytes.

Neural grafting reverses prenatal drug-induced alterations in hippocampal PKC and related behavioral deficits

Administration of heroin or phenobarbital to pregnant mice evokes neurochemical and behavioral deficits consequent to disruption of septohippocampal cholinergic innervation. The present study evaluates the relationship between the drug-induced biochemical changes and the behavioral deficits, applying two different approaches: neural grafting and within-individual correlations of biochemistry and behavior. Mice were exposed transplacentally to phenobarbital or heroin on gestational days 9–18 and tested in adulthood. Drug-exposed mice displayed impaired radial arm maze performance, increases in presynaptic choline transporter sites (monitored with [ 3H]hemicholinium-3 binding), upregulation of membrane-associated protein kinase C (PKC) activity, and desensitization of the PKC response to a cholinergic agonist, carbachol. Grafting of cholinergic cells to the impaired hippocampus reversed the behavioral deficits nearly completely and restored basal PKC activity and the PKC response to carbachol to normal; the drug effects on hemicholinium-3 binding were also slightly obtunded by neural grafting, but nevertheless remained significantly elevated. There were significant correlations between the performance in the eight-arm maze and both basal PKC activity and PKC desensitization, and to a lesser extent, between behavioral performance and hemicholinium-3 binding. Taken together, these findings indicate an inextricable link between the biochemical effects of prenatal drug exposure on the PKC signaling cascade and adverse behavioral outcomes. The ability of neural grafting to reverse both the drug-induced changes in PKC and behaviors linked to septohippocampal cholinergic function suggest a mechanistic link between this signaling pathway and neurobehavioral teratology caused by heroin or phenobarbital.

Role of protein kinase C in mediating alpha-1-adrenoceptor-induced negative inotropic response in rat ventricles.

The aim of this study was to determine the effect of protein kinase C (PKC) activation on intracellular Ca(2+) transient and its relation to alpha(1)-adrenoceptor (alpha(1)-AR)-stimulated negative inotropic response in rat ventricles. The electromechanical responses to phenylephrine (PE) in rat ventricular muscles were concomitantly examined using the conventional microelectrode method. The responses of intracellular Ca(2+) transient and cell contractions to PE in the absence of certain pharmacological interventions were ascertained in fura-2-loaded myocytes. The influence of PE on L-type Ca(2+) current (I(Ca,L)) was also examined using a voltage clamp in a whole-cell configuration. PE did not alter the action potential parameters during the negative inotropic phase. The negative inotropic effect (NIE) was inhibited by prazosin, chloroethylclonidine (CEC) and staurosporine, but was insensitive to pertussis toxin. Desensitization of PKC after prolonged pretreatment of rat ventricles with PDBu also abolished the NIE of PE. Caffeine modulated the NIE, but thapsigargin did not. The evoked intracellular Ca(2+) transient and cell contraction were initially decreased by PE, while I(Ca,L) was not altered. Prazosin and staurosporine significantly inhibited the responses. Our data indicated that alpha(1)AR-mediated NIE in rat ventricular muscles was due to the decrease of intracellular Ca(2+) transients by the modulation of PKC on Ca(2+)-releasing channels signaling through a CEC-sensitive alpha(1)AR subtype.

Hydrogen peroxide inhibits the immune response to lipopolysaccharide by attenuating signaling through c-Jun N-terminal kinase and p38 associated with protein kinase C

This study examined the immunomodulatory effects of hydrogen peroxide (H2O2) in B6C3F1 mouse splenic lymphocytes. H2O2 produced a marked and dose-related inhibition of both lipopolysaccharide (LPS)-induced B-cell proliferation and concanavalin A (Con A)-induced T-cell proliferation. Unexpectedly, little effect was observed with H2O2 on the antibody-forming cell (AFC) response to the polyclonal B-cell activator, LPS. It was also observed that H2O2 did not have any detectable effect on forskolin-stimulated adenylate cyclase, indicating that cyclic AMP (cAMP) is not a mediator of H2O2-induced suppression of the immune response. Rather, LPS-induced activation of protein kinase C (PKC) was completely inhibited when cells were pretreated with H2O2 for 18 h, although PKC activity was increased approximately twofold following treatment with H2O2 for 10 min. In addition, H2O2 pretreatment blocked the phosphorylation of two stress-activated mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase (JNK) and p38 by LPS in a concentration-dependent fashion. Therefore, these data suggest that H2O2 suppresses immune response through the desensitization of PKC, which subsequently results in inhibition of JNK and p38.

Involvement of protein kinase C and protein tyrosine kinase in thyrotropin-releasing hormone-induced stimulation of alpha-melanocyte-stimulating hormone secretion in frog melanotrope cells.

We have previously shown that the stimulatory effect of TRH on alpha-MSH secretion from the frog pars intermedia is associated with Ca2+ influx through voltage-dependent Ca2+ channels, activation of a phospholipase C and mobilization of intracellular Ca2+ stores. The aim of the present study was to investigate the contribution of protein kinase C (PKC), adenylyl cyclase (AC), Ca2+/calmodulin-dependent protein kinase II (CAM KII), phospholipase A2, and protein tyrosine kinase (PTK) in TRH-induced alpha-MSH release. Incubation of frog neurointermediate lobes (NILs) with phorbol 12-myristate-13-acetate (24 h), which causes desensitization of PKC, or with the PKC inhibitor NPC-15437, reduced by approximately 50% of the effect of TRH on alpha-MSH release. In most melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca2+ concentration ([Ca2+]i). Preincubation with phorbol 12-myristate-13-acetate or NPC-15437 suppressed the plateau phase of the Ca2+ response. Incubation of NILs with TRH (10(-6) M; 20 min) had no effect on cAMP production. In addition, the AC inhibitor SQ 22,536 did not affect the secretory response of NILs to TRH. These data indicate that the phospholipase C/PKC pathway, but not the AC/protein kinase A pathway, is involved in TRH-induced alpha-MSH release. The calmodulin inhibitor W-7 and the CAM KII inhibitor KN-93 did not significantly reduce the response to TRH. Similarly, the phospholipase A2 inhibitors quinacrine and 7-7'-DEA did not impair the effect of TRH on alpha-MSH secretion. The PTK inhibitors ST638 and Tyr-A23 had no effect on TRH-induced [Ca2+]i increase but inhibited in a dose-dependent manner TRH-evoked alpha-MSH release (ED50 = 1.22x10(-5) M and ED50 = 1.47x10(-5) M, respectively). Taken together, these data indicate that, in frog melanotrope cells, PKC and PTK are involved in TRH-induced alpha-MSH secretion. Activation of PKC is responsible for the sustained phase of the increase in [Ca2+]i, whereas activation of PTK does not affect Ca2+ mobilization.

Inhibition of protein tyrosine kinases or protein kinase C prevents nonspecific killer T lymphocyte-mediated tumoricidal activity

The signal transduction events which govern major histocompatibility complex-unrestricted tumour cell destruction by nonspecific killer T lymphocytes induced with anti-CD3 antibody have not yet been determined. In this study we used pharmacologic inhibitors to investigate the role of protein tyrosine kinases (PTK) and protein kinase C (PKC) in this process. The PTK-inhibitors herbimycin A, genistein, and methyl 2,5-dihydroxycinnamate blocked anti-CD3-activated killer T (AK-T) lymphocyte-mediated killing of tumour target cells. The PKC-inhibitors staurosporine, calphostin C, and myristoylated PKC pseudosubstrate peptide, as well as PKC desensitization by phorbol 12-myristate 13-acetate pretreatment, also suppressed the cytolytic effector function of AK-T lymphocytes. Lack of tumoricidal activity was not due to reduced AK-T lymphocyte binding to tumour target cells but was associated with the abrogation of granule exocytosis, indicating that PTK and PKC are involved in the postbinding process which results in delivery of the `lethal hit' by AK-T lymphocytes. © 1997 Elsevier Science B.V. All rights reserved.

Expression of the nucleoside triphosphate pyrophosphohydrolase PC-1 is induced by basic fibroblast growth factor (bFGF) and modulated by activation of the protein kinase A and C pathways in osteoblast-like osteosarcoma cells.

The closely related cytokines bFGF and aFGF regulate the function of bone cells and mineralization. Osteoblasts express PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH)/nucleotide phosphodiesterase I activity. bFGF and aFGF (10 ng/ml) up-regulated NTPPPH in human SaOS-2 and U2OS osteosarcoma cells, which express osteoblast-like features in culture. The induction was selective as alkaline phosphatase activity was down-regulated and specific as insulin-like growth factor-1 (IGF-1) and interleukin-1 beta (IL-1 beta) were not active. Furthermore, IL-1 beta but not IGF-1 inhibited bFGF-induced up-regulation of NTPPPH. The induced NTPPPH remained predominantly associated with cells. bFGF can induce signaling through pathways including protein kinase A (PKA) and protein kinase C (PKC)-mediated transduction. An activator of the PKA pathway (8-bromo cyclic adenosine monophosphate [cAMP]) induced NTPPPH. Furthermore, pretreatment with the PKC activator phorbol myristate acetate (PMA) (80 nM) markedly increased subsequent NTPPPH induction by both bFGF and cAMP. The PMA effect was associated with morphologic changes characterized by long, thin intercellular extensions. PKC desensitization also potentially contributed to this effect because the PKC inhibitors staurosporine and H-7 enhanced bFGF-induced and cAMP-induced NTPPPH expression in the absence of morphologic changes. We observed that bFGF induced expression of PC-1, a member of the NTPPPH gene family. The majority of NTPPPH activity was depleted by immunoadsorption using a monoclonal antibody to native human PC-1. bFGF- and aFGF-induced production of PC-1/NTPPPH in osteoblastoid cells may contribute to the effects of FGFs on bone metabolism.

Protein Kinase-C Mediates Chicken Vasoactive Intestinal Peptide Stimulated Prolactin Secretion and Gene Expression in Turkey Primary Pituitary Cells

This set of experiments investigated the role of protein kinase-C (PKC) as a second messenger in vasoactive intestinal peptide (VIP)-stimulated prolactin (PRL) secretion and PRL mRNA abundance. Dispersed anterior pituitary cells (5 × 105 or 106 cells/tube) were isolated from laying turkeys and incubated in 1.0 ml of M-199. In Experiment 1, 10-7M VIP increased PRL secretion three- to fivefold. Prolactin mRNA abundance was higher in VIP-treated cells (11.45 ± 2.11 arbitrary optical unit; AOU) than control cells (4.59 ± 1.2 AOU). In Experiment 2, the addition of 10-12, 10-10, 10-8, and 10-6M phorbol 12-myristate 13-acetate (PMA;PKC agonist) increased PRL release from 8.5 ± 0.7 to 14.9 ± 1.1, 17.2 ± 1.3, 18.1 ± 2.2, and 18.7 ± 2.8 μg/106 cells, respectively. PRL mRNA abundance was significantly (P < 0.01) increased in only 10-6M PMA treatmemt. In Experiment 3, PKC desensitization decreased VIP-stimulated PRL release from 10.0 ± 2.3 to 4.2 ± 0.6 μg/5 × 105 cells and PMA-induced release from 7.1 ± 1.3 to 2.7 ± 0.3 μg/5 × 105 cells. VIP and PMA up-regulated PRL mRNA abundance was decreased two- to fourfold by PKC desensitization. In Experiment 4, 10-6M staurosporine (ST; PKC antagonist) decreased both 10-7M VIP-stimulated PRL secretion from 7.86 ± 2.9 to 2.43 ± 0.5 μg/5 × 105 cells and 10-8M PMA-stimulated PRL secretion from 4.26 ± 0.2 to 2.23 ± 0.3 μg/5 × 105 cells (P < 0.01). In addition, ST decreased VIP-stimulated PRL mRNA abundance from 15.95 ± 2.18 to 11.35 ± 2.27 AOU in VIP- and from 6.36 ± 1.21 to 4.57 ± 0.63 AOU in PMA-treated cells. Intracellular pituitary PRL content did not differ in any treatment (P > 0.05). In conclusion, these results suggest that the PKC second messenger system is involved in VIP-stimulated PRL release and gene expression in primary turkey pituitary cell culture.

Protein kinase C in ATP regulation of lung surfactant secretion in type II cells

Previous studies provided indirect evidence for a role for protein kinase C (PKC) in ATP stimulation of surfactant secretion. The present study demonstrates that ATP increases PKC activity in the membrane fraction and decreases PKC activity in the cytosol fraction of alveolar type II cells, indicating translocation of PKC to the membranes. The kinetics of ATP concentration dependence of increases in phosphatidylcholine secretion and diacylglycerol content were similar, suggesting direct correlation between these two parameters. ATP also increased membrane PKC activity in a concentration-dependent manner. Almost one-half of the PKC activity in the cytosol and membrane fractions was Ca2+ independent. The ATP-induced increase was greater in membrane-associated Ca(2+)-dependent enzyme (233%) than in Ca(2+)-independent enzyme (121%). Desensitization of PKC by exposure of cells to phorbol esters decreased PKC activity in the membrane and cytosol fractions. In cells pretreated for 3 h with phorbol esters, PKC activity was near minimum, and ATP-stimulated secretion was lowest (< 40% of that observed in untreated cells). These results indicate that a major part of ATP-stimulated surfactant secretion in type II cells is mediated via activation of PKC.

Thyrotropin-induced hydrogen peroxide production in FRTL-5 thyroid cells is mediated not by adenosine 3',5'-monophosphate, but by Ca2+ signaling followed by phospholipase-A2 activation and potentiated by an adenosine derivative.

The production of hydrogen peroxide (H2O2) as an essential process for iodide organification is a key reaction in TSH-induced thyroid hormone synthesis. Here we characterize the signal transduction pathway involved in TSH-induced H2O2 production in FRTL-5 thyroid cells. At higher than 1 nM TSH, N6-(L-2-phenylisopropyl)adenosine (PIA), an adenosine receptor agonist having, by itself, no influence on H2O2 generation, potentiated this TSH action, whereas the TSH increase and PIA addition reduced cAMP accumulation. RO 20-1724, a phosphodiesterase inhibitor, amplified the TSH-induced cAMP accumulation, but did not change H2O2 generation in the whole range of TSH used. Ca(2+)-mobilizing agonists, GTP and ATP, also induced H2O2 production without stimulating cAMP accumulation. Chelation of intracellular Ca2+ markedly inhibited the TSH action, but intracellular Ca2+ increases by either thapsigargin or ionomycin mimicking it. All of the findings show the participation of Ca2+, but not cAMP, in the action of TSH. Desensitization of protein kinase-C (PKC) did not influence the receptor-mediated H2O2 production, suggesting the reduced importance of PKC activation compared to Ca2+ signaling to the reaction. A rise in intracellular Ca2+ independent of receptor activation also induced H2O2 production as well as arachidonate release, and both were potentiated by PIA. In addition, inhibitors of phospholipase-A2 and the arachidonate metabolic pathway depressed H2O2 generation, suggesting the participation of an arachidonate cascade in the Ca(2+)-dependent H2O2 production. Lipoxygenase inhibitors depressed the Ca2+ action without influencing arachidonate release, suggesting the involvement of a lipoxygenase product(s) of arachidonate in the Ca(2+)-signaling mechanism. In conclusion, in FRTL-5 cells, TSH-induced H2O2 production is mediated not by cAMP, but by the phospholipase-C/Ca2+ cascade, possibly followed by the Ca(2+)-dependent phospholipase-A2/arachidonate cascade. PIA amplifies TSH-induced H2O2 production at the steps of phospholipase-C and phospholipase-A2 activation in a pertussis toxin-sensitive manner.

Role of Ca2+ and protein kinase C in shear stress-induced actin depolymerization and endothelin 1 gene expression.

Vascular endothelial cells adapt to changes in blood flow by altering the cell architecture and by producing various substances. We have previously reported that low shear stress induces endothelin 1 (ET-1) expression in endothelial cells and that this induction is mediated by depolymerization of actin fiber. In the present study, we examined the role of Ca2+ and protein kinase C (PKC) in shear stress-induced actin depolymerization and subsequent ET-1 gene expression. Exposure of cultured porcine aortic endothelial cells to low shear stress (5 dyne/cm2) for 3 hours increased the ratio of G-actin to total actin from 54 +/- 0.8% to 80 +/- 1.0%. This shear stress-induced actin depolymerization was completely blocked by chelation of extracellular Ca2+ with EGTA and partially inhibited by intracellular Ca2+ chelation with the tetraacetoxymethyl ester of BAPTA (BAPTA/AM). Pretreatment with staurosporine, a PKC inhibitor, or desensitization of PKC by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) for 24 hours also resulted in partial inhibition of shear stress-induced actin depolymerization. Although PKC activation by TPA mildly increased G-actin content, the effect of TPA and shear stress on actin depolymerization was not additive. Moreover, shear stress-induced ET-1 gene expression was inhibited by EGTA, BAPTA/AM, and staurosporine to a degree similar to the inhibition of actin depolymerization. In contrast, ET-1 gene expression induced by cytochalasin B, an actin-disrupting agent, was not affected by staurosporine.(ABSTRACT TRUNCATED AT 250 WORDS)

Basic fibroblast growth factor-induced increase in zif/268 and c-fos mRNA levels is Ca2+ dependent in primary cultures of hippocampal neurons.

Basic fibroblast growth factor (bFGF) is present in the developing rat brain and has been shown to provide critical trophic support for hippocampal neurons in culture. The influence of bFGF on the expression of mRNAs encoding the transcription factors zif/268 and c-fos was studied in primary cultures of hippocampal neurons (derived from rat embryos) using reverse transcription-coupled PCR. In these cultures grown for 3 days in the absence of serum, bFGF causes a dramatic and transient increase in the levels of zif/268 and c-fos, within 15 and 30 min, respectively. A similar induction of these two early genes occurs following activation of protein kinase C (PKC). The bFGF-induced activation persists after PKC desensitization but is inhibited by chelation of intracellular Ca2+. These results suggest that in primary cultures of hippocampal neurons, bFGF induces the expression of immediate early genes through a pathway that requires Ca2+ mobilization.

Protein kinase C regulation of prolactin gene expression in lactotroph cells: involvement in dopamine inhibition.

The role of protein kinase C (PKC) on dopamine inhibition of PRL messenger RNA (mRNA) levels was studied in anterior pituitary cells kept in primary culture. PKC was desensitized by long-term exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA). Effectiveness of PKC desensitization was confirmed by the fact that after TPA pretreatment, short-term (1-h) exposure to TPA was no longer able to trigger PRL release. In contrast, the capacity of nonreceptor-mediated secretagogues as depolarization with 48 mM K+ to release the hormone was preserved. Pretreatment with TPA did not affect basal PRL mRNA levels. In contrast, it significantly reduced the dose-dependent inhibition of PRL mRNA induced by 1 nM bromocriptine after a 4-day incubation period. Since dopamine inhibition of PRL release is mediated by several second messager pathways, including cAMP, inositol phosphates, and Ca2+, we investigated whether PKC depletion was able to interact with direct stimulation of these pathways. Pretreatment with PKC suppressed stimulation of PRL mRNA levels induced by either Forskolin (FK) or 8Br-cAMP. In parallel, it reduced basal as well as FK stimulated intracellular cAMP levels. In addition, chronic exposure to TPA completely suppressed PRL mRNA inhibition induced by nifedipine, a dihydropyridine antagonist which blocks voltage-dependent Ca2+ channels. TPA desensitization also affected the action of bromocriptine, FK or nifedipine on PRL release measured under the same conditions. The data indicate that endogenous PKC can interfere with the regulation of PRL gene expression induced by both cAMP and Ca2+ pathways, two second messengers associated with the action of dopamine in lactotroph cells.

PKC modulation of inhibitory coupling of angiotensin II receptors with adenylate cyclase in lactotroph cells

Angiotensin II (AII) and thyreoliberin (TRH) have recently been shown to stimulate intracellular cAMP formation in rat lactotroph cells, in addition to their already documented coupling to phospholipase C. The effect on intracellular cAMP is unaffected by pertussis toxin (PTX) and is not due to a direct coupling to adenylate cyclase (AC); it results instead from a protein kinase C (PKC)-dependent process. In contrast, when tested in membrane preparations, AII, but not TRH, induces a PTX-sensitive inhibition of AC. The present work indicates that AII, but not TRH, is also able to inhibit intracellular cAMP formation in mixed as well as in lactotroph-enriched cells. Two conditions are required to reveal this effect: desensitization of PKC by prior exposure to TPA and concomitant stimulation of CAMP level. This effect is observed only in the presence of vasoactive intestinal peptide, whose receptor is directly coupled to AC, but not in the presence of other AC-stimulating agents such as cholera toxin and forskolin. This AII inhibitory effect is dose dependent and sensitive to PTX as is AII membrane inhibition of AC activity. PTX also reverses DA inhibition of AC, on both membrane preparations and intact cells. However different G proteins seem to be involved in the negative coupling of AII and DA receptors, since both effects do not exhibit the same PKC sensitivity in entire cells and GTP dependency in membrane preparations. An inhibitory coupling of the AII receptor with AC thus exists in intact cells but is masked by PKC interactions. Under specific conditions, this AII inhibition of intracellular cAMP formation might be implicated in the regulation of PRL secretion.

Regulation of proenkephalin expression in cultured skin mesenchymal cells.

Proenkephalin, a classically defined opioid encoding gene, is transiently expressed in nondifferentiated mesodermal cells during organogenesis. We examined the hypothesis that this expression is associated with mesenchymal cell proliferation. For this purpose, we established a cell culture derived from fetal skin mesenchyme that specifically expresses proenkephalin mRNA in correlation with hypodermis development. These mesenchymal cells also produce and secrete significant amounts of proenkephalin-derived peptides. Using this model system, we observed a marked increase in proenkephalin mRNA expression in response to serum. This effect is time dependent and reaches peak levels during the G1/S transition. Similarly, 12-O-tetradecanoyl-phorbol-13-ester, whose biological actions have been shown to be mediated by the activity of protein kinase C (PKC), up-regulates proenkephalin expression. Desensitization of PKC by prolonged exposure of cells to 12-O-tetradecanoyl-phorbol-13-ester attenuates the serum induction of proenkephalin. The results presented in this report demonstrate that proenkephalin expression in mesenchymal cells is regulated by serum factors via mechanisms that involve PKC activity. A possible association between proenkephalin expression and cell proliferation is suggested.

A Ca 2+ calmodulin dependent kinase rather than protein kinase C is involved in up-regulation of the LHRH receptor

Stimulation of protein kinase C (PKC) by phorbol ester (PMA) was reported previously to increase total binding of the peptide in whole rat pituitary cells. The effect could be obtained in cells from intact, not from spayed animals, suggesting a different level of spontaneous phosphorylation in both conditions. In the present work, endogenous PKC was desensitized in pituitary cells sampled from intact or 3 weeks castrated male rats and maintained in primary culture. Desensitization was induced by overnight incubation with 1μM PMA. The maximum number of plasma membrane LHRH receptors (Bmax) present on cells from in intact animals was higher (+98±9%) when binding was performed at 0.5°C instead of 21°C as already observed in non PKC-desensitized cells. PMA (100nM) was ineffective to increase Bmax, suggesting effectiveness of enzyme desensitization. In contrast, ionomycin 1μM increased Bmax (53±10%). This increment was inhibited by W7, a calmodulin inhibitor, with an IC 50=1±0.35 10 −6M. No temperature dependency of the Bmax was observed in cells from castrated rats as already shown in the absence of PKC desensitization. Under these conditions, a Bmax decrease of 34±6% and 36.5±7.5% respectively was observed in the presence of H7, a PKC inhibitor, or of W7 (IC 50 = 1±0.5 10 −5M and IC 50 = 0.8±0.2 10 −6M). We conclude that a Ca 2+ calmodulin dependent protein kinase rather than PKC itself is responsible for unmasking LHRH receptors.

Protein kinase C and cell proliferation in spontaneously hypertensive rats.

Cultured aortic fibroblasts from spontaneously hypertensive rats (SHR) exhibit increased proliferation rate compared with cells from normotensive Wistar Kyoto (WKY) rats. The present study was designed to investigate whether this growth abnormality could be accounted for by alteration in protein kinase C (PKC). The enzyme activation by 12-O-tetradecanoyl phorbol 13-acetate (TPA) promoted 3H-thymidine incorporation which was higher in SHR-derived fibroblasts compared with WKY-derived cells. Likewise, 3H-phorbol 12,13-dibutyrate (PDBu) binding to intact cells was markedly increased in SHR-derived fibroblasts. These findings suggest a difference in PKC activity between the two cell types. In both cell types, serum-induced 3H-thymidine incorporation was enhanced by PKC down-regulation, which was obtained by prolonged treatment of cells with high dose of TPA, whereas it was inhibited in a dose-dependent manner by activation of the enzyme. The changes in serum-induced 3H-thymidine incorporation elicited either by activation or desensitization of PKC, did not differ between SHR and WKY fibroblasts. Our results indicate therefore i) that in the presence of serum PKC exerts an antiproliferative effect in rat aortic fibroblasts and ii) that the increase in PKC activity and in sensitivity to TPA exhibited by SHR-derived fibroblasts, is not involved in the increased proliferation rate displayed by SHR-derived fibroblasts in serum-containing medium.

Activation of the CD3/T cell receptor (TcR) complex or of protein kinase C potentiate adenylyl cyclase stimulation in a tumoral T cell line: involvement of two distinct intracellular pathways.

A monoclonal antibody (OKT3) directed against the T cell receptor (TcR)/CD3 molecular complex, as well as a protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate, PMA) were added to a culture of tumoral Jurkat T cells, in order to precise the sequence of intracellular signals leading to T cell activation. The experiments were performed in the presence or in absence of various stimulators of adenylate cyclase (AC) such as forskolin (FK), cholera toxin (CT) or prostaglandin E2 (PGE2). OKT3 increased inositol phosphate (IP) production; in parallel, it induced a slight accumulation of cAMP. The effect was markedly potentiated in presence of FK or CT, and to a lesser extent in the presence of PGE2. FK stimulated adenylate cyclase of Jurkat cell membranes, but the effect was not potentiated by OKT3, suggesting that potentiation of cAMP accumulation requires intact cells and is not mediated by direct receptor coupling. On the other hand, elevated cAMP accumulation induced a negative feedback on IP production. The effect of OKT3 on cAMP was mimicked by A23187, a Ca2+ ionophore, and abolished in the absence of extracellular Ca2+. PMA had the same effect as OKT3 on basal or FK- and CT-induced accumulation of cAMP. In contrast, it inhibited the PGE2 effect on the cyclic nucleotide. After desensitization of PKC by pretreatment with a high concentration of PMA, the phorbol ester was no longer effective. Under those conditions, facilitation by OKT3 of FK-induced accumulation of cAMP was preserved, whereas potentiation by the monoclonal antibody of the PGE2 stimulation of AC was even enhanced. The data indicate that cAMP accumulation indirectly elicited by phospholipase C activation is, at least partly, mediated by IP-dependent Ca2+ mobilization, while PKC is preferentially effective as an inhibitor of PGE2 stimulation.

Activation of protein kinase-C inhibits vitamin D receptor gene expression.

The abundance of 1,25-dihydroxyvitamin D3 receptors (VDR) in cultured cells has been shown to vary in direct relation to the rate of cell proliferation. This study examines the question of whether the growth-factor mediated up-regulation of VDR is due to direct modulation of VDR gene expression or is secondary to the stimulation of cell cycle events. Mitogenic agents, such as basic fibroblast growth factor and phorbol esters, were found to cause significant decreases in VDR abundance, while substantially stimulating proliferation of NIH-3T3 cells. Potent phorbol esters, such as phorbol myristate acetate (PMA) and phorbol-12,13-dibutyrate, whose biological actions have been shown to be mediated through the activation of protein kinase-C, down-regulated VDR in a time- and dose-dependent manner. An inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate, which does not activate protein kinase-C, did not alter VDR levels. Desensitization of protein kinase-C by prolonged exposure of cells to phorbol esters eliminated the PMA-mediated down-regulation of VDR. Staurosporine, an inhibitor of protein kinase-C, blocked the actions of PMA. Oleoyl acetyl glycerol, a synthetic diacyl glycerol, and A23187, a calcium ionophore, were both able to suppress VDR abundance alone and were additive in combination. The results suggest that activation of the protein kinase-C pathway and elevation of intracellular Ca2+ lead to significant down-regulation of VDR. The inhibitory effect of PMA appears to be exerted at the level of VDR mRNA expression. Northern blot analysis revealed significant decreases in steady state levels of VDR mRNA species that qualitatively corresponded to the decrease in VDR protein concentration seen on a Western blot.(ABSTRACT TRUNCATED AT 250 WORDS)