Decrease In Cytosolic Protein Kinase C Activity Research Articles

Amended Final Report on the Safety Assessment of Glyceryl Dilaurate, Glyceryl Diarachidate, Glyceryl Dibehenate, Glyceryl Dierucate, Glyceryl Dihydroxystearate, Glyceryl Diisopalmitate, Glyceryl Diisostearate, Glyceryl Dilinoleate, Glyceryl Dimyristate, Glyceryl Dioleate, Glyceryl Diricinoleate, Glyceryl Dipalmitate, Glyceryl Dipalmitoleate, Glyceryl Distearate, Glyceryl Palmitate Lactate, Glyceryl Stearate Citrate, Glyceryl Stearate Lactate, and Glyceryl Stearate Succinate1

Amended Final Report on the Safety Assessment of Glyceryl Dilaurate, Glyceryl Diarachidate, Glyceryl Dibehenate, Glyceryl Dierucate, Glyceryl Dihydroxystearate, Glyceryl Diisopalmitate, Glyceryl Diisostearate, Glyceryl Dilinoleate, Glyceryl Dimyristate, Glyceryl Dioleate, Glyceryl Diricinoleate, Glyceryl Dipalmitate, Glyceryl Dipalmitoleate, Glyceryl Distearate, Glyceryl Palmitate Lactate, Glyceryl Stearate Citrate, Glyceryl Stearate Lactate, and Glyceryl Stearate Succinate1

Role of protein kinase C in endothelin-1-induced contraction of human myometrium.

The role of protein kinase C (PKC) in the contraction of human myometrium induced by endothelin-1 was investigated. The PKC inhibitor, calphostin C, reduced the sustained phase of endothelin-1-induced contraction. The expression and subcellular distribution of PKC isoforms were determined in unstimulated myometrium by Western blotting using isoform-specific antisera. At least five PKC isoforms (PKCalpha, PKCbeta1, PKCbeta2, PKCzeta, and trace amounts of PKCepsilon) were detected. Quantitative immunoblotting revealed that all these isoforms were diversely distributed between the cytosolic and particulate fractions. After stimulation with phorbol 12,13-dibutyrate (PDB) and endothelin-1, differential redistribution occurred, suggesting a selective role of these isoforms in the physiological function of the myometrium. Biochemical assay confirmed that PDB as well as endothelin-1 evoked a decrease in cytosolic PKC activity. Taken together, these results suggest that PKC may play a role in endothelin-1-induced contraction of human uterine smooth muscle.

Increased Natural Killer Cell Cytotoxicity in Alzheimer’s Disease May Involve Protein Kinase C Dysregulation

Increased cytokine-mediated cytotoxic natural killer (NK) cell activity has recently been demonstrated in patients with senile dementia of the Alzheimer’s type (SDAT). In the present study, we evaluated whether protein-kinase C (PKC), a main regulatory enzyme involved in the mechanism of exocytosis by NK cells, has a role in the cytotoxic response of NK cells (during IL-2 and IFN-β exposure) from SDAT patients. Our data demonstrate the presence of an increased cytotoxic response by NK cells to IL-2 (mean increase +102%) and IFN-β (mean increase +132%) in SDAT patients in comparison with healthy elderly subjects (+75% and +88% for IL-2 and IFN-β, respectively). A smaller suppression of NK cytotoxicity after cortisol was also observed in SDAT (mean decrease −24%) than in the control group (−44%). The NK cell activity of SDAT patients was inversely correlated with the cognitive status as evaluated by the analysis of MMSE (Mini Mental State Examination) score. A comparison of young and elderly healthy subjects revealed no variations in NK cell activity. A physiological decrease in cytosolic PKC activity was demonstrated in healthy old subjects after IL-2 and IFN-β incubation, but not in SDAT patients, while no variations in kinase activity were observed after cortisol incubation. The decreased activity with cytokines was associated with reduced levels of PKC α and βII isoforms. An alteration in cytokine-mediated NK cell activity associated with PKC dysregulation is therefore suggested to occur in patients with SDAT. These changes may indicate the existence of an immunological component to the pathogenesis and progression of the disease.

Protein kinase C activity following exposure to magnetic field and phorbol ester

We examined the separate and combined effects of 60 Hz sinusoidal magnetic fields (MFs) and a phorbol ester on protein kinase C (PKC) activity in HL60 cells. No enhancement in PKC activity was observed when a cell culture was exposed to a 1.1 mT (rms) MF alone or to a combination of MF and 2 microM phorbol 12-myristate 13-acetate (PMA) for 1 h. In a second set of experiments, cells were preexposed to a less than optimal concentration of PMA (50 nM) for 45 min, followed by a 15 min exposure to both PMA and MF. The data showed a greater decrease in cytosolic PKC activity and a larger increase in membrane activity than was induced by either 1 h PMA treatment alone or PMA and sham MF exposure. One logical conclusion from these data is that MFs may be acting in a synergistic manner on a pathway that has already been activated. Therefore, we suggest that MFs, rather than producing biological effects by a new pathway or mechanism of interaction, exert their effect(s) by interacting with already functioning reactions or pathways. If correct, the question of an MF's mechanism of interaction refocuses on how weak fields might enhance or depress a molecular reaction in progress, rather than on finding a new transduction pathway.

Protein kinase C activity following exposure to magnetic field and phorbol ester

We examined the separate and combined effects of 60 Hz sinusoidal magnetic fields (MFs) and a phorbol ester on protein kinase C (PKC) activity in HL60 cells. No enhancement in PKC activity was observed when a cell culture was exposed to a 1.1 mT (rms) MF alone or to a combination of MF and 2 μM phorbol 12-myristate 13-acetate (PMA) for 1 h. In a second set of experiments, cells were preexposed to a less than optimal concentration of PMA (50 nM) for 45 min, followed by a 15 min exposure to both PMA and MF. The data showed a greater decrease in cytosolic PKC activity and a larger increase in membrane activity than was induced by either 1 h PMA treatment alone or PMA and sham MF exposure. One logical conclusion from these data is that MFs may be acting in a synergistic manner on a pathway that has already been activated. Therefore, we suggest that MFs, rather than producing biological effects by a new pathway or mechanism of interaction, exert their effect(s) by interacting with already functioning reactions or pathways. If correct, the question of an MF's mechanism of interaction refocuses on how weak fields might enhance or depress a molecular reaction in progress, rather than on finding a new transduction pathway. Bioelectromagnetics 19:469–476, 1998. © 1998 Wiley-Liss, Inc.

Activation of host cell protein kinase C by enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) consists of a group of diarrhea-producing E. coli strains, common in developing countries, which do not produce classical toxins and are not truly invasive. EPEC strains adhere to mammalian cells in an intimate fashion, trigger a localized increase in intracellular calcium levels, and elevate inositol phosphate production. We hypothesized that these mediators could activate host cell protein kinase C (PKC) and tested this idea in vitro with two cultured human cell lines, HeLa cells and T84 cells. Using a recently described subculturing protocol to "induce" or accelerate EPEC adherence, we infected the cells with EPEC at a multiplicity of infection of approximately 100:1 for 30 to 60 min. Under these conditions, EPEC E2348 increased membrane-bound PKC activity 1.5- to 2.3-fold in HeLa cells and T84 cells, respectively. The increase in membrane-bound PKC activity was accompanied by a decrease in cytosolic PKC activity in EPEC-infected HeLa cells. Nonadherent laboratory E. coli strains such as HB101 and H.S. failed to trigger any consistent change in PKC production, similar to the nonadherent mutant strains derived from E2348, JPN15 (plasmid cured) and CVD206 (eaeA). In addition, immunoblots performed on extracts of T84 cells with a monoclonal antibody against PKC-alpha showed an increased PKC content in membranes of EPEC-infected cells. Finally, EPEC-infected T84 cells showed a 60% increase in responsiveness to the E. coli heat-stable toxin. We conclude that mediators produced in response to EPEC adherence activate PKC in intestinal and nonintestinal cells.

Subcellular Distribution of Protein Kinase Cα and β1 in Bovine Spermatozoa, and their Regulation by Calcium and Phorbol Esters1

Protein kinase C (PKC), the major cell target for tumor-promoting phorbol esters, is central to many signal transduction pathways. Previously we have demonstrated the presence of PKC in ram and bovine spermatozoa. However, the relative distribution of various PKC isozymes in the cytosolic and membrane fractions and their regulation by calcium and phorbol esters have not been elucidated. Immunocytochemical studies and Western blotting with antibodies specific for individual isoforms revealed that at least two PKC isoforms, cPKC alpha and cPKC betaI, are found in bovine sperm cells. We demonstrate, by Western blotting analysis, that both PKC isozymes were predominantly localized in the cytosol when subcellular fractionation was carried out in the presence of EGTA. When cell lysis was carried out in the presence of Ca2+, most PKC alpha and PKC betaI redistributed to the particulate fraction. Treatment of sperm cells with the biologically active phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) resulted in a rapid and extensive translocation of cytosolic PKC alpha and cytosolic PKC betaI to the membrane fraction within 1 min. Furthermore, PKC's total activity was measured as a calcium- and phospholipid-dependent phosphorylation of a synthetic peptide in the cytosolic and membrane fractions derived from control and TPA-treated spermatozoa. TPA evoked a decrease in cytosolic PKC activity, accompanied by an increase in the activity associated with the plasma membrane fraction. This translocation of PKC enzymes may ensure their binding to intracellular receptor proteins ("RACKs") and the phosphorylation of specific substrates, which appears to determine their physiological function. The presence of RACK in the membrane fraction of bovine sperm cells was confirmed with use of an antibody directed against the RACK protein. Previously we demonstrated the involvement of PKC in sperm acrosomal exocytosis, a process induced by signal transduction events. Thus, our results suggest that the rapid association of PKC alpha and PKC betaI with the sperm plasma membrane, as shown in the present work for the first time, may be an early event in sperm cell regulation, leading to acrosomal exocytosis and fertilization.

Correlations between hippocampal protein kinase C activity and learning abilities in a spatial reference memory task

Partial learning of a reference memory task induces a decrease in hippocampal cytosolic protein kinase C (PKC) activity with no concomitant changes in the particulate fraction. The particulate PKC activity is correlated with performance (Noguès, Micheau, & Jaffard, 1994). The relationship between the level of training and PKC activity was investigated in the present study. Hippocampal PKC activity was measured after mice were trained for 2, 5, or 12 sessions in a spatial discrimination task in an 8-arm radial maze. No increase in membrane-bound fraction or decrease in cytosolic PKC activity was observed at any stage of learning, although performance was correlated with PKC activity in each subcellular fraction. We sought correlations between performance and the magnitude of the decrease in cytosolic PKC activity, with reference to previously obtained data. The extent of the fall in cytosolic PKC was found to be related to learning abilities rather than to the maximal performance level. Causal explanatory models are proposed to explain these somewhat contradictory data.

Protein kinase C activity in blood vessels from normotensive and spontaneously hypertensive rats

This study investigates the effects of phorbol dibutyrate (PDB) on protein kinase C (PKC) activation, as assessed by the translocation of PKC activity from the cytosolic to the paniculate fraction, in aortas and mesenteric arteries from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). The basal distribution of PKC activity between the cytosolic and particulate fractions of SHR and WKY aortas, and mesenteric arteries, was not significantly different. PDB induced a concentration-dependent decrease in cytosolic PKC activity in SHR and WKY aortas. PDB (0.01 μ,M) decreased cytosolic PKC activity to a greater magnitude in SHR aorta as compared to WKY aorta, while 1.0 μM PDB decreased cytosolic PKC activities to similar magnitudes in SHR and WKY aortas, and mesenteric arteries. These results suggest that the increased sensitivity of SHR vessels to contraction by phorbol esters may be due, at least in part, to the greater sensitivity of PKC in these vessels to phorbol ester activation.

Translocation of protein kinase C in bovine tracheal smooth muscle strips: the effect of methacholine and isoprenaline

Investigations into the mechanisms involved in the contraction of smooth muscle have suggested that the generation of diacylglycerol and the activation of protein kinase C (PKC) may be important in the generation or maintenance of smooth muscle tone. The present study examined the possible role of PKC in the contraction of bovine tracheal smooth muscle. Methacholine (10 μM) induced a rapid elevation in PKC activity associated with the membrane fraction. PKC levels were significantly elevated in the membrane fraction 30 s after agonists addition, reached a maximum at 1 min and then declined to and remained at a lower level which was still elevated above basal. A concomitant decrease in cytosolic PKC activity of smaller magnitude was observed during this period of stimulation. This methacholine-induced re-distribution of PKC from the cytosol to the membrane was concentration-dependent and was blocked by atropine. Pre-treatment of tissues for 2 min with 100 μM isoprenaline prevented both the re-distribution of PKC and the contraction produced by 1 μM methacholine. Addition of 1 μM isoprenaline to tissues pre-contracted with 1 μM methacholine reversed the re-distribution of PKC produced during contraction and completely relaxed the tissues. Thus, under these conditions, translocation of PKC from the cytosol to the membrane seems to be well correlated with contractions of bovine tracheal smooth muscle. Whether the PKC translocation is responsible for the observed changes in muscle tone or whether the enzyme translocation is a result of drug-induced changes in [Ca 2+] i remains to be determined.

Alpha 1-adrenergic and cholinergic agonists use separate signal transduction pathways in lacrimal gland.

The cellular transduction pathways used by alpha 1-adrenergic and cholinergic agonists were compared in isolated acini from rat exorbital lacrimal glands. Peroxidase secretion was the index of protein secretion. Inositol phosphates were measured by anion exchange chromatography, intracellular free Ca2+ concentration ([Ca2+]i) by fluorescence methods using fura-2, cellular adenosine 3',5'-cyclic monophosphate (cAMP) levels by protein binding radioassay, and protein kinase C (PKC) activity by [32P]ATP incorporation into exogenous substrate. Protein secretion stimulated by simultaneous addition of the alpha 1-adrenergic agonist phenylephrine and the cholinergic agonist carbachol was additive. Carbachol (10(-3) M) significantly increased the ratios of inositol phosphates to inositol during a 1- or 20-min incubation in contrast to phenylephrine (10(-5) to 10(-2) M), which did not. Phenylephrine (10(-3) M) significantly increased the [Ca2+]i by a maximum of 15 +/- 3 nM compared with carbachol (10(-4) M), which increased [Ca2+]i to a maximum of 90 +/- 14 nM. Phenylephrine (10(-4) M) did not increase cAMP levels. Phenylephrine (10(-5) to 10(-3) M) decreased cytosolic PKC activity in a concentration-dependent manner. Carbachol (10(-3) M) transiently caused a slight decrease in cytosolic PKC activity. Our results indicate that alpha 1-adrenergic and cholinergic agonists use separate and different pathways to stimulate the lacrimal gland.

Activation of protein kinase C modulates dihydroxycholecalciferol synthesis in rat renal tubules

1,25(OH) 2D 3, the biologically active metabolite of vitamin D, is produced from 25(OH)D 3, by the renal mitochondrial 25(OH)D 3 1α-hydroxylase. Several studies have implicated reversible phosphorylation and a possible role for protein kinase C (PKC) in acute regulation of 1,25(OH) 2D 3 production. In the experiments described here, we studied 1,25(OH) 2D 3 production in freshly isolated rat renal tubules treated with activators and inhibitors of PKC. In this mammalian system, TPA, but not its inactive analogue 4αPDD, inhibited 1,25(OH) 2D 3 production in a dose-dependent fashion within 20 min. The acute inhibition of 1,25(OH) 2D 3 production by TPA exposure was preceded by an increase in membrane associated PKC activity, which was paralleled by a decrease in cytosolic PKC activity. Pre-incubation of tubules with staurosporine, a PKC inhibitor, abolished the inhibitory effect of TPA on 1,25(OH) 2D 3 production. Chronic (18 h) exposure of tubules to high dose TPA resulted in down regulation of both membrane and cytosolic PKC activity and re-exposure to TPA did not affect PKC translocation or 1,25(OH) 2D 3 production in down regulated tubules. Our data strongly suggest that modulation of renal PKC activity may be an important mechanism for acute regulation of 1,25(OH) 2D 3 production.

Role of protein kinase C in insulin's regulation of c-fos transcription.

One of insulin's actions is the induction of DNA synthesis and cell division, but little is known about the molecular mechanisms involved. Previous studies indicate that insulin stimulates cell division and regulates the expression of several genes in rat H4IIE (H4) hepatoma cells. One of these genes is the proto-oncogene c-fos, a cellular gene whose deregulation has been implicated in the process of cellular differentiation and division. We have shown that insulin induces transcription of the c-fos gene in H4 cells. In the present study, the phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated c-fos transcription in a rapid and dose-dependent manner with an 800% increase in transcription following 15-30 min of addition. This increase in c-fos transcription was transitory, returning towards baseline transcription rates within 120 min. PMA stimulated the translocation of protein kinase C (PKC) from the cytoplasm to the membrane in H4 hepatoma cells, as evidenced by a 77% decrease in cytosolic PKC and a 29% increase in membrane PKC activity following 10 min of treatment. Insulin addition to H4 cells for 10 min also resulted in a 31% decrease in cytosolic PKC activity, suggesting a translocation response. When H4 cells were pretreated with PMA for 24 h, there was a decrease of 20-45% in both cytosolic and membrane PKC activity and a complete loss of PMA's induction of c-fos transcription. Thus, the cells were functionally desensitized to further PMA addition. When cells were pretreated with PMA for 24 h, the insulin-induced increase in transcription of c-fos was reduced by 50%. Western blot analysis indicated that the PKC-beta isozyme followed a translocation pattern almost identical with that of total PKC activity. These results suggest that a PMA-sensitive form of PKC is preferentially lost upon PMA pretreatment and that this PKC subtype may be necessary for insulin to fully induce c-fos gene expression.

Structural requirements of lyngbyatoxin A for activation and downregulation of protein kinase C.

Structure-activity studies of novel synthetic analogues of lyngbyatoxin A reveal that the lactam ring but not the 7-linalyl moiety of lyngbyatoxin A is essential for the in vitro stimulation of protein kinase C (PKC). (-)-Indolactam V (ILV), which contains no hydrophobic substituent at C-7, or analogues containing either a linalyl or n-hexyl group at C-7 were equally efficacious in stimulating HeLa cell PKC in vitro and in competing with phorbol 12,13-dibutyrate for binding to PKC in intact cells. The hydrophobicity of alkyl groups at C-7, however, influenced the potency of these compounds to bind to and activate PKC. In addition, these compounds exhibited differences in their ability to translocate PKC. Lyngbyatoxin A (0.1 microM) like TPA induced a rapid translocation of PKC from the cytosol to the membrane and subsequently led to a sustained decrease in both cytosolic and membrane PKC activity. In contrast, (-)-n-hexylILV (0.1 microM) and (-)-ILV (1 microM) produced a transient and attenuated decrease in cytosolic PKC activity. At concentrations that produced half-maximal PKC stimulation, (-)-ILV did not cause any downregulation of PKC whereas lyngbyatoxin A and (-)-n-hexylILV led to 60% and 40% PKC downregulation, respectively. Western blot analyses with monoclonal antibodies to PKC isoforms indicated that reduction in PKC activity by chronic exposure to TPA or lyngbyatoxin A analogues could be explained by downregulation of PKC alpha. Constitutive expression of PKC beta and PKC gamma isoforms was low in HeLa cells and was not affected significantly by TPA or lyngbyatoxin A analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelin inhibits the atrial natriuretic factor stimulated cGMP production by activating the protein kinase C in rat aortic smooth muscle cells

Preincubation of rat thoracic aortic smooth muscle cells with endothelin inhibits the atrial natriuretic factor (ANF)-induced cGMP accumulation in these cells in a concentration dependent manner. The maximal inhibition of 64% was afforded by 1 x 10(-6) M endothelin and the half maximal inhibition (IC50) was achieved with 1 x 10(-9) M endothelin. Endothelin (1 x 10(-6) M) also increased the plasma membrane bound protein kinase C (PKC) activity by 4 fold. Hormone-dependent increase in PKC activity was limited to plasma membranes only and some decrease in cytosolic PKC activity was observed. However, phorbol 12-myristate 13-acetate (PMA) (1 x 10(-6)M) provoked a total loss of cytosolic PKC activity and a net gain in membranous PKC activity indicative of the translocation of the enzyme. Pretreatment of these cells with H-7, a PKC inhibitor, released the endothelin and PMA-mediated attenuation of ANF-stimulated cGMP formation. These results suggest that PKC is involved in the regulation of ANF-induced cGMP accumulation and that the vasoconstrictor activity of endothelin might involve inhibition of the vasorelaxant activity of ANF through the inhibition of cGMP accumulation in smooth muscle cells (SMCs) of the rat aorta.

1,25(OH) 2D 3 increases membrane associated protein kinase C in MDBK cells

To determine whether 1,25-dihydroxycholecalciferol [1,25(OH) 2D 3] affects protein kinase C (PKC) activity in kidney, as has benn demonstrated in HL-60 cells we measured 1,25(OH) 2D 3 binding, PKC activity and PKC immunoreactivity in Madin Darby bovine kidney (MDBK) cells, a normal renal epithelial cell line derived from kidney. Our data demonstrate that MDBK cells exhibit specific high affinity binding for 1,25(OH) 2D 3, indicating the presence ofthe vitamin D receptor (VDR). Treatment of MDBK cells with 1,25(OH) 2D 3 for 24 h increased membrane PKC activity and immunoreactivity. The effect of 1,25(OH) 2D 3 was dose-dependent, with a peak effect observed at 10 −10 M 1,25(OH) 2D 3. The 1,25(OH) 2D 3 induced increase in membrane PKC was paralleled by a comparable decrease in cytosolic PKC activity and amount. Although time course studies were consistent with a VDR mediated effect of 1,25(OH) 2D 3 on PKC protein synthesis, total PKC activity was not increased by 1,25(OH) 2D 3, suggesting an effect on PKC translocation of localization. These results suggest that 1,25(OH) 2D 3 modulates PKC mediated events in kidney, a classic target for this steroid hormone.

A comparison of the priming effect of phorbol myristate acetate and phorbol dibutyrate on fMet-Leu-Phe-induced oxidative burst in human neutrophils

Phorbol esters such as phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBU) are generally considered to have similar effects through a similar mechanism, i.e. protein kinase C (PKC) activation. We recently suggested that this was not the case in human neutrophils. To identify further differences between the two phorbol esters, we compared their priming effects on fMet-Leu-Phe-induced superoxide anion (O2−) production, cytosolic PKC activity and binding of fMet-Leu-Phe. Priming could be initiated with a low (0.2 nM) concentration of both PDBU and PMA. Their effects on the pattern of fMet-Leu-Phe-induced superoxide production were similar in both Ca2+-containing and Ca2+-free medium. PDBU, like PMA, abolished the Ca2+ dependency of fMet-Leu-Phe-induced O2− production in a dose-dependent manner. In cytochalasin B-treated fMet-Leu-Phe-induced O2− production. In Ca2+-free medium, priming abolished the Ca2+ dependency of fMet-Leu-Phe stimulation in cytochalasin B-treated cells. Cytochalasin B, however, enhanced the effect of PMA but not that of PDBU. Priming with PDBU was not associated under any experimntal conditions with a decrease in cytosolic PKC activity, or an increase in PKM activity before or after fMet-Leu-Phe stimulation. Furthermore, priming effects were abolished by cell washing but not by H-7 or staurosporine, which are potent PKC inhibitors. PDBU, in contrast to PMA, increased fMet-Leu-Phe binding to PMNs through a decrease in the dissociation constant and induced degranulation of specific granules as measured by the release of vitamin B12 binding protein. These findings show that the priming effects of PDBU differ in certain respects from those of PMA, namely with regard to its synergism with cytochalasin B and the expression of fMet-Leu-Phe receptors. In addition, priming concentrations of PDBU, like PMA, did not alter cytosolic PKC activity in fMet-Leu-Phe-stimulated neutrophils.

Dissimilarities in superoxide anion production by human neutrophils stimulated by phorbol myristate acetate or phorbol dibutyrate

Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBU) are known to translocate protein kinase C (PKC) and to induce superoxide anion (O2⨪) production in human neutrophils. They are thus currently used to probe the role of PKC in O2⨪ production. We show here that under certain conditions, O2⨪ production induced by PMA is not associated with a decrease in cytosolic PKC activity, whereas these two events are associated after PDBU stimulation. (1) In the presence of extracellular calcium (1 mM), O2⨪ production was related to the concentration of PMA. PMA induced O2⨪ production at all the concentrations studied, but this was not associated with a decrease in cytosolic PKC levels up to 5 ng/ml PMA (50% maximum O2⨪ production). (2) Under PDBU stimulation, even at very low O2⨪ production levels, cytosolic PKC decreased and the decrease as well as the O2⨪ production were related to the concentration of PDBU. (3) For a given decrease in cytosolic PKC, O2⨪ production induced by PMA was much greater than that induced by PDBU. (4) In calcium-free medium, O2⨪ production induced by low concentrations of PMA (up to 5 ng/ml) was lower than that observed in the presence of 1 mM calcium, whereas modifications of cytosolic PKC activity were similar. (5) Cytochalasin B had no effect of PMA-induced O2⨪ production, regardless of the calcium content of the medium, and had no effect on the decrease in cytosolic PKC. On the contrary, following PDBU stimulation, cytochalasin B increased O2⨪ production, regardless of the medium, but induced a larger decrease in cytosolic PKC when Ca2+ was present. (6) Preincubation of PMN with 100 μM H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) before stimulation with PMA or PDBU led to similar inhibition of O2⨪ production whatever the degree of decrease in cytosolic PKC activity. These findings show that, in contrast to PDBU, O2⨪ production by PMA is not always related to cytosolic PKC activity.

Chemotactic Peptide Enhancement of Phorbol Ester-Induced Protein Kinase C Activity in Human Neutrophils

Chemotactic factors and phorbol esters may act synergistically to evoke neutrophil responses, but the mechanism of such interaction is not entirely clear. We investigated the combined effects of the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA) on protein kinase C (PKC) activity in human neutrophils. FMLP had little effect on the sharp decrease in cytosolic PKC activity induced by PMA. However, cells exposed to FMLP and PMA exhibited a synergistic increase in particulate PKC activity (1 +/- 1 pmol 32P/10(7) PMNs/min in unstimulated cells, 53 +/- 12 pmol 32P with 20 ng/ml PMA, 6 +/- 3 pmol 32P with 10(-7) M FMLP, and 191 +/- 17 pmol 32P with FMLP and PMA). FMLP also markedly increased calcium/phospholipid-independent protein kinase activity in particulate fractions of control and PMA-treated cells. Enhancement of PKC activity required the presence of cytochalasin B during cell stimulation. Cellular calcium was crucial to the FMLP effect since enhancement was decreased in cells incubated with EGTA or Quin2. These results suggest that chemotactic factors and phorbol esters may mediate synergistic effects on neutrophil responses through enhancement of particulate PKC activity. The enhancing effect is probably mediated through chemoattractant-mediated increases in intracellular calcium.

Nicotinic and muscarinic agonists stimulate rapid protein kinase C translocation in PC12 cells

Phosphoinositide hydrolysis, a major mechanism for signal transduction in neural cells, generates diacylglycerol, which can in turn activate protein kinase C (PKC). Although cholinergic agonists elicit phosphoinositide hydrolysis in neural tissues, little is known about activation of PKC by cholinergic agonists. PKC requires phosphatidylserine for activation, and in intact cells this lipid requirement is satisfied by binding of the enzyme to cell membranes. Therefore, in intact cells, activation of PKC is often associated with a decrease in cytosolic PKC activity accompanied by an increase in membrane-associated activity. We studied cholinergic-induced activation of PKC by examining changes in the subcellular distribution of the enzyme in PC12 cells treated with cholinergic drugs. Carbachol (1 mM) induced large and rapid increases in membrane-associated PKC activity; a maximal increase of 460% occurred after 5 sec of incubation. Carbachol-induced PKC translocation was concentration-dependent, with a biphasic dose-response curve yielding approximate EC50 values of 10(-6) M and 10(-4) M for the high- and low-affinity components, respectively. Experiments with selective cholinergic agents demonstrated that both muscarinic and nicotinic receptors are involved in carbachol-induced PKC translocation, but the response is predominantly mediated by nicotinic receptor stimulation. Muscarinic-induced association of PKC with cell membrane fractions was resistant to extraction by chelators, whereas nicotinic-mediated membrane binding was partially reduced by homogenization of cells in the presence of EGTA. Omission of calcium from the incubation medium or chelation of calcium with EGTA completely blocked muscarinic- and nicotinic-induced translocation. In addition, the calcium channel blocker nifedipine reduced the nicotinic response by 60%. (ABSTRACT TRUNCATED AT 250 WORDS)