Ethanol increases superoxide anion production stimulated with 4beta-phorbol 12-myristate 13-acetate in human polymorphonuclear leukocytes. Involvement of protein kinase C.

Stimulation of human polymorphonuclear leukocytes (PMNs) with PMA initiates a cascade of events leading to the production and release of superoxide anion (O-2), a major component in anti-bacterial defense. Generation of O-2 by PMA-stimulated PMNs occurs through the translocation and activation of protein kinase C (PKC). In this study, using freshly isolated PMNs, we examined the effect of ethanol on this response to PMA. Our results show that the basal production of O-2 was not affected by ethanol. In contrast, the response induced by PMA was potentiated by ethanol. This potentiation was observed even at high doses of PMA (200 nM) which alone had stimulated the O-2 response maximally. This enhanced response was not due to an increase of PMA uptake by PMNs. The maximal effect was obtained when the cells were preincubated with 80 mM of ethanol before PMA stimulation. Measurement of PKC activity in the cytosolic and membrane fractions showed that pretreatment of PMNs with ethanol increased twofold the PMA-stimulated PKC activity in the membrane fraction. Furthermore, Western blot analysis verified that this increase in PKC activity in the membrane fraction was linked to an increase in the translocation of PKC-alpha and -beta isoforms to the membrane. These results suggest that ethanol potentiates PMA-induced O-2 production through increasing PKC translocation and activity in PMNs.     

Direct activation of a protein kinase activity from rat lacrimal gland by PMA in a phospholipid-free system

The analysis of the cytosolic fraction from rat exorbital lacrimal gland with DEAE-cellulose ion-exchange chromatography showed the presence of a peak of protein kinase activity which was dependent on the presence of phosphatidylserine and diolein as well as calcium. This activity showed the same properties as the previously reported protein kinase C (PKC). Moreover, we have shown for the first time that this kinase or a kinase that coeluted from the column with PKC could be activated by a phorbol ester, PMA, in a phospholipid-free system, i.e. in the absence of any cofactor of PKC. These findings emphasize the need for caution in the interpretation of experimental results obtained when using phorbol esters to probe for a role of PKC in many regulatory processes.     

Protein kinase C (PKC) isoforms translocate to Triton-insoluble fractions in stimulated human neutrophils: correlation of conventional PKC with activation of NADPH oxidase.

The responses of human neutrophils (PMN) involve reorganization and phosphorylation of cytoskeletal components. We investigated the translocation of protein kinase C (PKC) isoforms to PMN cytoskeletal (Triton-insoluble) fractions, in conjunction with activation of the respiratory burst enzyme NADPH oxidase. In resting PMN, PKC-delta (29%) and small amounts of PKC-alpha (0.6%), but not PKC-betaII, were present in cytoskeletal fractions. Upon stimulation with the PKC agonist PMA, the levels of PKC-alpha, PKC-betaII, and PKC-delta increased in the cytoskeletal fraction, concomitant with a decrease in the noncytoskeletal (Triton-soluble) fractions. PKC-delta maximally associated with cytoskeletal fractions at 160 nM PMA and then declined, while PKC-alpha and PKC-betaII plateaued at 300 nM PMA. Translocation of PKC-delta was maximal by 2 min and sustained for at least 10 min. Translocation of PKC-alpha and PKC-betaII was biphasic, plateauing at 2-3 min and then increasing up to 10 min. Under maximal stimulation conditions, PKC isoforms were entirely cytoskeletal associated. Translocation of the NADPH oxidase component p47phox to the cytoskeletal fraction correlated with translocation of PKC-alpha and PKC-betaII, but not with translocation of PKC-delta. Oxidase activity in cytoskeletal fractions paralleled translocation of PKC-alpha, PKC-betaII, and p47phox. Stimulation with 1,2-dioctanoylglycerol resulted in little translocation of PKC isoforms or p47phox, and in minimal oxidase activity. We conclude that conventional PKC isoforms (PKC-alpha and/or PKC-betaII) may regulate PMA-stimulated cytoskeletal association and activation of NADPH oxidase. PKC-delta may modulate other PMN responses that involve cytoskeletal components.     

Translocation of PKC isoforms in bovine aortic smooth muscle cells exposed to strain

Our laboratory has previously reported that the exposure of smooth muscle cells (SMC) to the cyclic strain results in significant stimulation of protein kinase C (PKC) activity by translocating the enzyme from the cytosol to the particulate fraction. We now sought to examine the strain-induced translocation of individual PKC isoforms in SMC. Confluent bovine aortic SMC grown on collagen type I-coated plates were exposed to cyclic strain for up to 100 s at average 10% strain with 60 cycles/min. Immunoblotting analysis demonstrates that SMC express PKC-alpha, -beta and -zeta in both cytosolic and particulate fractions. Especially, PKC-alpha and -zeta were predominantly expressed in the cytosolic fraction. However, cyclic strain significantly (P < 0.05) increased PKC-alpha and -zeta in the particulate fraction and decreased in the cytosolic fraction. Thus, the cyclic strain-mediated stimulation of PKC activity in SMC may be due to the translocation of PKC-alpha and -zeta from the cytosolic to the particulate fraction. These results demonstrate that mechanical deformation causes rapid translocation of PKC isoforms, which may initiate a cascade of proliferation responses of SMC since NF-kappaB, which is involved in the cellular proliferation has been known to be activated by these PKC isoforms.     

Activation of phospholipase D by protein kinase C. Evidence for a phosphorylation-independent mechanism.

The role of protein kinase C (PKC) in the regulation of phosphatidylcholine-hydrolyzing phospholipase D (PLD) was investigated. In membranes from Chinese hamster lung fibroblasts that had been incubated with [14C]choline to label endogenous phosphatidylcholine, phorbol 12-myristate 13-acetate (PMA) failed to stimulate production of [14C]choline. However, stimulation was observed if fibroblast cytosolic fraction or PKC partially purified from this fraction was added. When incubated with membranes in the presence of PMA, pure PKC from rat brain stimulated [14C]choline production in a concentration-dependent manner, with a maximal 2-3-fold effect. PMA similarly stimulated [14C]phosphatidylpropanol formation from propanol using membranes from [14C]myristic acid-prelabeled cells, confirming the activation of PLD. None of the effects described required exogenous ATP. To probe the role of phosphorylation in the PKC effect, we included high concentrations of apyrase in the assay. This ATPase had no effect on the ability of PKC to activate PLD, but under exactly the same conditions, it eliminated autophosphorylation of PKC. The results provide conclusive evidence for the involvement of PKC in the activation of PLD and suggest that ATP-dependent phosphorylation is not required.     

Protein kinase C-stimulated formation of ethanolamine from phosphatidylethanolamine involves a protein phosphorylation mechanism: negative regulation by p21 Ras protein

Mammalian cells express a phospholipase D (PLD)-like enzyme which forms ethanolamine from phosphatidylethanolamine (PtdEtn) by a protein kinase C-alpha (PKC-alpha)-activated, presently unknown, mechanism. Now we report that addition of a PKC-alpha-enriched purified PKC preparation or recombinant PKC-alpha to a plasma membrane-enriched membrane fraction, isolated from leukemic HL60 cells, greatly ( approximately 6.5-fold stimulation) enhanced PtdEtn hydrolysis if the PKC activator phorbol 12-myristate 13-acetate (PMA) and ATP were both present; this was accompanied by PKC-mediated phosphorylation of several membrane proteins. The combined effects of PKC-alpha, ATP, and PMA on [(14)C]PtdEtn hydrolysis were inhibited by GF 109203X (10 microM), an inhibitor of catalytic activity of PKC. In this membrane fraction, PMA alone also had a smaller ( approximately 3.5-fold) stimulatory effect on PtdEtn hydrolysis which was not affected by adding ATP or GF 109203X to the membranes. These results suggest that PMA can stimulate PtdEtn hydrolysis via a PKC-catalyzed phosphorylation mechanism as well as by a phosphorylation-independent process. Transformation of NIH 3T3 fibroblasts by H-ras reduced the effect of PMA on PtdEtn hydrolysis. Furthermore, in NIH 3T3 fibroblasts, scrape-loaded Y13-259 anti Ras antibody enhanced PMA-stimulated hydrolysis of PtdEtn. These results suggest that activation of the PtdEtn-hydrolyzing PLD enzyme by PKC-alpha is inhibited by p21 Ras.     

Protein kinase C-alpha-induced hypertrophy of neonatal rat ventricular myocytes

Protein kinase C (PKC) isoenzymes play a critical role in cardiomyocyte hypertrophy. At least three different phorbol ester-sensitive PKC isoenzymes are expressed in neonatal rat ventricular myocytes (NRVMs): PKC-alpha, -delta, and -epsilon. Using replication-defective adenoviruses (AdVs) that express wild-type (WT) and dominant-negative (DN) PKC-alpha together with phorbol myristate acetate (PMA), which is a hypertrophic agonist and activator of all three PKC isoenzymes, we studied the role of PKC-alpha in signaling-specific aspects of the hypertrophic phenotype. PMA induced nuclear translocation of endogenous and AdV-WT PKC-alpha in NRVMs. WT PKC-alpha overexpression increased protein synthesis and the protein-to-DNA (P/D) ratio but did not affect cell surface area (CSA) or cell shape compared with uninfected or control AdV beta-galactosidase (AdV betagal)-infected cells. PMA-treated uninfected cells displayed increased protein synthesis, P/D ratio, and CSA and elongated morphology. PMA did not further enhance protein synthesis or P/D ratio in AdV-WT PKC-alpha-infected cells. To assess the requirement of PKC-alpha for these PMA-induced changes, AdV-DN PKC-alpha or AdV betagal-infected NRVMs were stimulated with PMA. Without PMA, AdV-DN PKC-alpha had no effects on protein synthesis, P/D ratio, CSA, or shape vs. AdV betagal-infected NRVMs. PMA increased protein synthesis, P/D ratio, and CSA in AdV betagal-infected cells, but these parameters were significantly reduced in PMA-stimulated AdV-DN PKC-alpha-infected NRVMs. Overexpression of DN PKC-alpha enhanced PMA-induced cell elongation. Neither WT PKC-alpha nor DN PKC-alpha affected atrial natriuretic factor gene expression. Insulin-like growth factor-1 also induced nuclear translocation of endogenous PKC-alpha. PMA but not WT PKC-alpha overexpression induced ERK1/2 activation. However, AdV-DN PKC-alpha partially blocked PMA-induced ERK activation. Thus PKC-alpha is necessary for certain aspects of PMA-induced NRVM hypertrophy.     

Translocation and Down-Regulation of Protein Kinase C-α, -β, and -γ Isoforms During Ischemia-Reperfusion in Rat Brain

We investigated the distribution of protein kinase C (PKC) isoforms in the subcellular fractions (P1, 1,000-g pellet; P2, 10,000-g pellet; P3, 100,000-g pellet; S, 100,000-g supernatant) of rat forebrain after ischemia or reperfusion by immunoblotting. PKC-delta and -epsilon isoforms were predominant in the P2 (synaptosome-rich) fraction, whereas PKC-alpha, -beta, -gamma, -epsilon, and -zeta isoforms were rich in the S (cytosolic) fraction. With time of ischemia (5-30 min), PKC-alpha, -beta, and -gamma translocated to the P2 and P3 fractions, whereas reperfusion for 60 min after 30 min of ischemia reduced PKC-beta activity greatly and PKC-alpha and -gamma activities to a lesser extent. There was no redistribution of PKC-delta, -epsilon, and -zeta after ischemia or reperfusion. A calpain inhibitor, acetylleucylleucylnorleucinal, inhibited the down-regulation of PKC-beta, through intravenous injection. The PKC translocation to the P2 fraction was accompanied by their dephosphorylation, transition of PKC-alpha from dimer to trimer, and the decrease in activity. These data show that PKC-alpha, -beta, and -gamma isoforms translocate chiefly to the synaptosome in ischemic brain in association with the dephosphorylation, multimeric change, and inactivation, followed by the proteolysis of PKC-beta by calpain after postischemic reperfusion.     

Cooperation between PKC-alpha and PKC-epsilon in the regulation of JNK activation in human lung cancer cells

Phorbol esters can induce activation of two mitogen-activated protein kinase (MAPK) pathways, the extracellular signal-regulated kinase (ERK) pathway and the c-Jun N-terminal kinase (JNK) pathway. Unlike ERK activation, JNK activation by phorbol esters is somehow cell-specific. However, the mechanism(s) that contribute to the cell-specific JNK activation remain elusive. In this study, we found that phorbol 12-myristate 13-acetate (PMA) induced JNK activation only in non-small cell lung cancer (NSCLC) cells, but not in small cell lung cancer (SCLC) cells, whereas ERK activation was detected in both cell types. In NSCLC cells, PMA induced JNK activation in a time- and dose-dependent manner. JNK activation was attenuated by protein kinase C (PKC) down-regulation through prolonged pre-treatment with PMA and significantly inhibited by PKC inhibitors G?6976 and GF109203X. Subcellular localization studies demonstrated that PMA induced translocation of PKC-alpha, -betaII, and -epsilon isoforms, but not PKC-delta, from the cytosol to the membrane. Analysis of various PKC isoforms revealed that PKC-epsilon was exclusively absent in the SCLC cell lines tested. Ectopic expression of PKC-epsilon in SCLC cells restored PMA activation of JNK signaling only in the presence of PKC-alpha, suggesting that PKC-alpha and PKC-epsilon act cooperatively in regulating JNK activation in response to PMA. Furthermore, using dominant negative mutants and pharmacological inhibitors, we define that a putative Rac1/Cdc42/PKC-alpha pathway is convergent with the PKC-epsilon/MEK1/2 pathway in terms of the activation of JNK by PMA.     

Increased expression of protein kinase C isoforms in heart failure due to myocardial infarction

The activities of cardiac protein kinase C (PKC) were examined in hemodynamically assessed rats subsequent to myocardial infarction (MI). Both Ca(2+)-dependent and Ca(2+)-independent PKC activities increased significantly in left ventricular (LV) and right ventricular (RV) homogenates at 1, 2, 4, and 8 wk after MI was induced. PKC activities were also increased in both LV and RV cytosolic and particulate fractions from 8-wk infarcted rats. The relative protein contents of PKC-alpha, -beta, -epsilon, and -zeta isozymes were significantly increased in LV homogenate, cytosolic (except PKC-alpha), and particulate fractions from the failing rats. On the other hand, the protein contents of PKC-alpha, -beta, and -epsilon isozymes, unlike the PKC-zeta isozyme, were increased in RV homogenate and cytosolic fractions, whereas the RV particulate fraction showed an increase in the PKC-alpha isozyme only. These changes in the LV and RV PKC activities and protein contents in the 8-wk infarcted animals were partially corrected by treatment with the angiotensin-converting enzyme inhibitor imidapril. No changes in protein kinase A activity and its protein content were seen in the 8-wk infarcted hearts. The results suggest that the increased PKC activity in cardiac dysfunction due to MI may be associated with an increase in the expression of PKC-alpha, -beta, and -epsilon isozymes, and the improvement of heart function in the infarcted animals by imidapril may be due to partial prevention of changes in PKC activity and isozyme contents.     

Ahnak protein activates protein kinase C (PKC) through dissociation of the PKC-protein phosphatase 2A complex

We have previously reported that central repeated units (CRUs) of Ahnak act as a scaffolding protein networking phospholipase Cgamma and protein kinase C (PKC). Here, we demonstrate that an Ahnak derivative consisting of four central repeated units binds and activates PKC-alpha in a phosphatidylserine/1,2-dioleoyl-sn-glycerol-independent manner. Moreover, NIH3T3 cells expressing the 4 CRUs of Ahnak showed enhanced c-Raf, MEK, and Erk phosphorylation in response to phorbol 12-myristate 13-acetate (PMA) compared with parental cells. To evaluate the effect of loss-of-function of Ahnak in cell signaling, we investigated PKC activation and Raf phosphorylation in embryonic fibroblast cells (MEFs) of the Ahnak knock-out (Ahnak(-/-)) mouse. Membrane translocation of PKC-alpha and phosphorylation of Raf in response to PMA or platelet-derived growth factor were decreased in Ahnak null MEF cells compared with wild type MEFs. Several lines of evidence suggest that PKC-alpha activity is regulated through association with protein phosphatase 2A (PP2A). A co-immunoprecipitation assay indicated that the association of PKC-alpha with PP2A was disrupted in NIH3T3 cells expressing 4 CRUs of Ahnak in response to PMA. Consistently, Ahnak null MEF cells stimulated by PMA showed enhanced PKC-PP2A complex formation, and add-back expression of Ahnak into Ahnak null MEF cells abolished the PKC-PP2A complex formation in response to PMA. These data indicate that Ahnak potentiates PKC activation through inhibiting the interaction of PKC with PP2A.     

PKC activates BKCa channels in rat pulmonary arterial smooth muscle via cGMP-dependent protein kinase

Normally, signaling mechanisms that activate large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channels in pulmonary vascular smooth muscle cause pulmonary vasodilatation. BK(Ca)-channel modulation is important in the regulation of pulmonary arterial pressure, and inhibition (decrease in the opening probability) of the BK(Ca) channel has been implicated in the development of pulmonary vasoconstriction. Protein kinase C (PKC) causes pulmonary vasoconstriction, but little is known about the effect of PKC on BK(Ca)-channel activity in pulmonary vascular smooth muscle. Accordingly, studies were done to determine the effect of PKC on BK(Ca)-channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMCs) of the Sprague-Dawley rat. The PKC activators phorbol myristate acetate (PMA) and thymeleatoxin opened BK(Ca) channels in single Sprague-Dawley rat PASMC. The activator response to both PMA and thymeleatoxin on BK(Ca)-channel activity was blocked by G?-6983, which selectively blocks PKC-alpha, -delta, -gamma, and -zeta, and by rottlerin, which selectively inhibits PKC-delta. In addition, the specific cyclic GMP-dependent protein kinase antagonist KT-5823 blocked the responses to PMA and thymelatoxin, whereas the specific cyclic AMP-dependent protein kinase blocker KT-5720 had no effect. In isolated pulmonary arterial vessels, both PMA and forskolin caused vasodilatation, which was inhibited by KT-5823, G?-6983, or the BK(Ca)-channel blocker tetraethylammonium. The results of this study indicate that activation of specific PKC isozymes increases BK(Ca)-channel activity in Sprague-Dawley rat PASMC via cyclic GMP-dependent protein kinase, which suggests a unique signaling mechanism for vasodilatation.     

Effects of neurotransmitters and peptides on phospholipid hydrolysis in sympathetic and sensory neurons

The effects of neurotransmitters and peptides on phosphoinositide hydrolysis were studied by measuring [3H]inositol monophosphate ([3H]IP) and protein kinase C (PKC) activity in the sympathetic and sensory neuronal cultures of the chick embryo. [3H]IP was increased in sympathetic neurons by acetylcholine (ACh), muscarine, serotonin (5-HT), and vasoactive intestinal polypeptide. ACh, muscarine, 5-HT, and bradykinin increased [3H]IP in sensory neuronal cultures. Dopamine, norepinephrine, histamine, and nerve growth factor did not stimulate [3H]IP formation in both cultures. ACh and phorbol 12,13-dibutyrate (PDB) increased the PKC activity by two- to sevenfold in the particulate fraction of both cultures. In sympathetic neurons, PKC activity was increased in the particulate fraction; activity in the cytosolic fraction was not affected. There was a 50% decline in the protein kinase C activity of the cytosolic fraction after PDB and ACh treatment of sensory cultures. The decline in PKC activity in the cytosolic fraction was attributed to the presence of nonneuronal cells in sensory cultures. To confirm this, the enzyme activity was determined in tissues that contain a heterogeneous population of cells. PDB activated PKC in the adrenal medulla and the brain of the rat. In both tissues there was a 65% decline in the PKC activity of the cytosolic fraction and about a 75% increase in the particulate fraction. We conclude that the mechanism of activation of protein kinase C in pure cultures of sympathetic neurons is different than in tissues containing a mixed population of neurons and nonneuronal cells.     

Protein kinase A- and C-induced insulin release from Ca2+ -insensitive pools

Insulin secretion is known to depend on an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). However, recent studies have suggested that insulin secretion can also be evoked in a Ca(2+)-independent manner. In the present study we show that treatment of intact mouse islets and RINm5F cells with protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or protein kinase A (PKA) activator forskolin promoted insulin secretion with no changes of [Ca(2+)](i). Moreover, insulin secretion mediated by PMA or forskolin was maintained even when extracellular or cytosolic Ca(2+) was deprived by treatment of cells with ethylene glycol bis(beta-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-amino phenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxy methyl ester) (BAPTA/AM) in RINm5F cells. The secretagogue actions of PMA and forskolin were blocked by GF109203X and H89, selective inhibitors for PKC and PKA, respectively. PMA treatment caused translocation of PKC-alpha and PKC- epsilon from cytosol to membrane, implying that selectively PKC-alpha and PKC- epsilon isoforms might be important for insulin secretion. Co-treatment with high K(+) and PMA showed a comparable level of insulin secretion to that of PMA alone. In addition, PMA and forskolin evoked insulin secretion in cells where Ca(2+)-dependent insulin secretion was completed. Our data suggest that PKC and PKA can elicit insulin secretion not only in a Ca(2+)-sensitive manner but also in a Ca(2+)-independent manner from separate releasable pools.     

Amelioration of denervation-induced atrophy by clenbuterol is associated with increased PKC-alpha activity

Rat soleus muscle was denervated for 3 or 7 days, and total membrane protein kinase C (PKC) activity and translocation and immunocytochemical localization of PKC isoforms were examined. Dietary administration of clenbuterol concomitant with denervation ameliorated the atrophic response and was associated with increased membrane PKC activity at both 3 (140%) and 7 (190%) days. Of the five PKC isoforms (alpha, epsilon, theta, zeta, and mu) detected in soleus muscle by Western immunoblotting, clenbuterol treatment affected only the PKC-alpha and PKC-theta forms. PKC-alpha was translocated to the membrane fraction upon denervation, and the presence of clenbuterol increased membrane-bound PKC-alpha and active PKC-alpha as assayed by Ser(657) phosphorylation. PKC-theta protein was downregulated upon denervation, and treatment with clenbuterol further decreased both cytosolic and membrane levels. Immunolocalization of PKC-theta showed differences for regulatory and catalytic domains, with the latter showing fast-fiber type specificity. The results suggest potential roles of PKC-alpha and PKC-theta in the mechanism of action of clenbuterol in alleviating denervation-induced atrophy.     

A novel succinyl dipeptide stimulates directed cell migration by modulating protein kinase C activity

In determining the mechanism of the chemokinetic action of the thiol protease inhibitor, E-64, in endothelial cell monolayers subjected to wounding, we synthesized succinyl-leucyl-agmatine (SLA), an analogue of E-64 that lacked the epoxy group and protease inhibitory effect. We observed that this analogue retained its chemokinetic effect on wounded endothelial cells. Its stimulatory action on endothelial cell polarization response to wounding was rapid and associated with directed cell migration. Furthermore, its effect on cellular polarization was blocked by protein kinase C (PKC) inhibitors and mimicked by pharmacologic agents that stimulated PKC activity. To determine if SLA's chemokinetic action was mediated by protein kinase C activation, we compared the effects of SLA and the tumor promoter phorbol myristate acetate (PMA) on the translocation of PKC activity in endothelial cells. We observed that both SLA and PMA induced the translocation of PKC activity from the cytosolic to the particulate fraction of the cells. We also observed that both SLA and PMA induced the phosphorylation of two proteins (Mr 23.4 and 36.5 kDa) in intact 32P-labeled cells. Thus, SLA stimulates the endothelial cell locomotor response to wounding by stimulating PKC activity.     

Activity of protein kinase C during the differentiation of chick limb bud mesenchymal cells

Abstract. To investigate the relationship between protein kinase C (PKC) and chondrogenesis, PKC activity was assayed in cultures of stage 23/24 chick limb bud mesenchymal cells under various conditions. PKC activities of cytosolic and particulate fractions were low in 1 day cultured cells. As chondrogenesis proceeds, cytosolic PKC activity increased more than twofold, while that of the particulate fraction increased only slightly. Three days' treatment of cultures with phorbol-12-myristate-13-acetate (PMA, 5 × 10⁻⁸ M ) inhibited chondrogenesis judged by the accumulation of Alcian blue bound to the extracellular matrix and depressed PKC activity in cytosolic fraction. When cells were grown for 3 days in control medium after 3 days' treatment with PMA, chondrogenesis resumed and PKC activity recovered to normal values. PKC activity in cultures plated at low density (2 × 10⁶ cells/ml) where chondrogenesis is reduced was as low as that in 1 day cultured cells plated at high density (2 × 10⁷ cells/ml) or that in PMA treated cells. On the other hand, staurosporine promoted chondrogenesis without affecting PKC activity. Furthermore, reversal of PMA's inhibitory effect on chondrogenesis by staurosporine was not accompanied by recovery of PKC activity. These data indicate that increases in PKC activity is closely related to chondrogenesis and that PMA inhibits chondrogenesis by depressing PKC. However, staurosporine's enhancing effect on chondrogenesis is not related to PKC activity.     

Role of PKC isoforms in glucose transport in 3T3-L1 adipocytes: insignificance of atypical PKC

To elucidate the involvement of protein kinase C (PKC) isoforms in insulin-induced and phorbol ester-induced glucose transport, we expressed several PKC isoforms, conventional PKC-alpha, novel PKC-delta, and atypical PKC isoforms of PKC-lambda and PKC-zeta, and their mutants in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Endogenous expression and the activities of PKC-alpha and PKC-lambda/zeta, but not of PKC-delta, were detected in 3T3-L1 adipocytes. Overexpression of each wild-type PKC isoform induced a large amount of PKC activity in 3T3-L1 adipocytes. Phorbol 12-myristrate 13-acetate (PMA) activated PKC-alpha and exogenous PKC-delta but not atypical PKC-lambda/zeta. Insulin also activated the overexpressed PKC-delta but not PKC-alpha. Expression of the wild-type PKC-alpha or PKC-delta resulted in significant increases in glucose transport activity in the basal and PMA-stimulated states. Dominant-negative PKC-alpha expression, which inhibited the PMA activation of PKC-alpha, decreased in PMA-stimulated glucose transport. Glucose transport activity in the insulin-stimulated state was increased by the expression of PKC-delta but not of PKC-alpha. These findings demonstrate that both conventional and novel PKC isoforms are involved in PMA-stimulated glucose transport and that other novel PKC isoforms could participate in PMA-stimulated and insulin-stimulated glucose transport. Atypical PKC-lambda/zeta was not significantly activated by insulin, and expression of the wild-type, constitutively active, and dominant-negative mutants of atypical PKC did not affect either basal or insulin-stimulated glucose transport. Thus atypical PKC enzymes do not play a major role in insulin-stimulated glucose transport in 3T3-L1 adipocytes.     

Protein kinase C activity is altered in HL60 cells exposed to 60 Hz AC electric fields

We examined the effects of electric fields (EFs) on the activity and subcellular distribution of protein kinase C (PKC) of living HL60 cells. Sixty Hertz AC sinusoidal EFs (1.5–1.000 mV/cm p-p) were applied for 1 h to cells (10⁷/ml) in Teflon chambers at 37 °C in the presence or absence of 2 μM phorbol 12-myristate 13-acetate (PMA). PMA stimulation alone evoked intracellular translocation of PKC from the cytosolic to particulate fractions. In cells that were exposed to EFs (100–1,000 mV/cm) without PMA, a loss of PKC activity from the cytosol, but no concomitant rise in particulate PKC activity, was observed. In the presence of PMA. EFs (33–330 mV/cm) also accentuated the expected loss of PKC activity from the cytosol and augmented the rise in PKC activity in the particulate fraction. These data show that EFs alone or combined with PMA promote down-regulation of cytosolic PKC activity similar to that evoked by mitogens and tumor promoters but that it does not elicit the concomitant rise in particulate activity seen with those agents. © 1996 Wiley-Liss, Inc.