Chelerythrine inhibits the secretory response of human blood platelets without specifically inhibiting protein kinase C

Chelerythrine (chloride) has previously been documented to be a potent and selective inhibitor of the serine/threonine-specific protein kinase C (PKC). In this study, it was shown that 10 microM chelerythrine completely inhibited serotonin secretion and partially inhibited phosphatidic acid formation in human blood platelets activated by thrombin (1U/ml). However, there was no effect on PKC activity as assessed by the level of phosphorylation of the 47K protein. Therefore, chelerythrine has been shown not to be a specific inhibitor of PKC. Without specifically affecting PKC activity, it is nevertheless capable of completely inhibiting platelet secretion, indicating that it may affect the signal transduction pathway responsible for platelet secretion at a point downstream or independent of PKC.     

The involvement of protein kinase C in proliferation and differentiation of human keratinocytes--an investigation using inhibitors of protein kinase C

Protein kinase C, the major cellular receptor for tumour-promoting phorbol esters, has been suggested as playing a key role in the regulation of proliferation and differentiation of epidermal cells. In the present study, we investigated the effects of various well-characterized inhibitors of protein kinase C on proliferation and differentiation of SV 40-transformed and normal human keratinocytes. The drugs were found to inhibit cell proliferation in a dose-dependent manner, displaying similar effects in both cell types and reflecting their potencies in inhibiting purified protein kinase C. In contrast, keratinocyte differentiation induced by treatment with a calcium ionophore or spontaneously, i.e. by exposure of cells grown in the presence of low calcium concentration (0.06 mM) to normal calcium concentration (1.6 mM), was not inhibited by the compounds tested. The potent protein kinase C inhibitor, staurosporine, was found even to enhance cell differentiation. Therefore, the present study provides evidence that the classical protein kinase C pathway plays a critical role in the regulation of keratinocyte proliferation rather than in calcium-induced differentiation.     

Activation of protein kinase C delta by the c-Abl tyrosine kinase in response to ionizing radiation

The c-Abl protein tyrosine kinase is activated by ionizing radiation (IR) and certain other DNA-damaging agents. The present studies demonstrate that c-Abl associates constitutively with protein kinase C delta (PKCdelta). The results show that the SH3 domain of c-Abl interacts directly with PKCdelta. c-Abl phosphorylates and activates PKCdelta in vitro. We also show that IR treatment of cells is associated with c-Abl-dependent phosphorylation of PKCdelta and translocation of PKCdelta to the nucleus. These findings support a functional interaction between c-Abl and PKCdelta in the cellular response to genotoxic stress.     

Long-term changes in excitability induced by protein kinase C activation in Aplysia sensory neurons

Protein kinases A (PKA) and C (PKC) play a central role as intracellular transducers during simple forms of learning in Aplysia. These two proteins seem to cooperate in mediating the different forms of plasticity underlying behavioral modifications of defensive reflexes in a state- and time-dependent manner. Although short- and long-term changes in the synaptic efficacy of the connections between mechanosensory neurons and motoneurons of the reflex have been well characterized, there is also a distinct intermediate phase of plasticity that is not as well understood. Biochemical and physiological experiments have suggested a role for PKC in the induction and expression of this form of facilitation. In this report, we demonstrate that PKC activation can induce both intermediate- and long-term changes in the excitability of sensory neurons (SNs). Short application of 4beta-phorbol ester 12,13-dibutyrate (PDBU), a potent activator of PKC, produced a long-lasting increase in the number of spikes fired by SNs in response to depolarizing current pulses. This effect was observed in isolated cell culture and in the intact ganglion; it was blocked by a selective PKC inhibitor (chelerythrine). Interestingly, the increase in excitability measured at an intermediate-term time point (3 h) after treatment was independent of protein synthesis, while it was disrupted at the long-term (24 h) time point by the general protein synthesis inhibitor, anisomycin. In addition to suggesting that PKC as well as PKA are involved in long-lasting excitability changes, these findings support the idea that memory formation involves multiple stages that are mechanistically distinct at the biochemical level.     

Herbimycin A inhibits protein kinase C in vascular smooth muscle cells

A sensitive enantioselective high performance liquid chromatographic assay for determination of the dihydropyridine-type calcium antagonist amlodipine in human plasma samples is described. Chiral chromatography is performed on an alpha 1-acid glycoprotein column (i.e. Chiral-AGP) and the eluted enantiomers are trapped and compressed on two short columns before final achiral chromatography on a narrow bore column (i.e. Zorbax SB-Ph) using electrochemical detection. Both stereoselective quantitative analysis and enantiomeric ratio analysis, for samples with a known total concentration of amlodipine are described. The quantitative assay shows linearity over the range 0.5-10 ng ml-1 for the two enantiomers and the limit of detection is about 0.2 ng ml-1. The method has been applied to a pharmacokinetic study of the enantiomers of amlodipine in human subjects.     

Effect of protein kinase C inhibitors and activators on corneal re-epithelialization in the rat

PURPOSE: To examine the ability of protein kinase C (PKC) inhibitors and activators to influence the rate of corneal re-epithelialization in the rat. METHOD: Rat corneas with 3 mm diameter central epithelial abrasions were organ-cultured in control medium or in medium with inhibitors or activators of PKC. RESULTS: In control corneas, the defect was completely re-epithelialized by 25 hr. In the presence of the PKC inhibitors staurosporine (100 nM), sphinganine (50 mumol/l), or H-7 (100 mumol/l) there were significantly larger epithelial defects than in controls after 5-25 hr of incubation. Re-epithelialization rates were similar to control corneas when the incubation medium contained HA1004 (100 mumol/l), an analogue of H-7 that is a potent inhibitor of cyclic adenosine monophosphate- and cyclic guanosine monophosphate-dependent protein kinases and a weak inhibitor of PKC. Two PKC activators, 1-oleoyl-2-acetyl-sn-glycerol (OAG) and phorbol 12-myristate 13-acetate (PMA), were unable to enhance the rate of epithelial wound healing. CONCLUSIONS: Our results suggest that PKC activity is an important factor in regulating corneal epithelial wound healing, presumably by influencing cell migration. Moreover, the results with OAG and PMA suggest that PKC is maximally activated during re-epithelialization in this organ-culture assay.     

Inhibition of demecolcin-induced DNA synthesis by inhibitors of phospholipase C and protein kinase C

PURPOSE OF STUDY: Interleukin-2 (IL-2) is a potent activator of lymphocytes, but its effectiveness as an anti-cancer agent is compromised by several adverse side effects including pulmonary edema. One explanation for the pulmonary toxicity of IL-2 is that activated lymphocytes directly induce the pulmonary vascular endothelium to become more leaky. METHODS: To test this hypothesis the number of total lymphocytes, gamma delta T cells, and CD2-positive cells (alpha beta T cells and natural killer cells) in peripheral blood and lung lymph of sheep were compared before and after IL-2 infusion. Hemodynamic and lymph dynamic changes were also evaluated. RESULTS: IL-2 decreased mean aortic pressure, increased cardiac output, lowered systemic vascular resistance, and doubled lung lymph flow (P < or = 0.05), but had no effect on plasma or lymph oncotic pressure. The lymph protein concentration and the lymph-to-plasma protein concentration ratio were not different after IL-2 infusion. IL-2 had no effect on the number of total lymphocytes, gamma delta T cells, or CD2-positive cells in the peripheral blood. In contrast, the number of total lymphocytes, gamma delta T cells, and CD2-positive cells in lung lymph decreased significantly (P < or = 0.05). CONCLUSIONS: The lymphocyte populations decreased more than could be explained by the increase in lymph flow, demonstrating that lung lymphocytes were not reduced simply by dilution. These results imply that the pulmonary edema associated with IL-2 is not caused by activated lymphocytes.     

Dephosphorylation of the cadherin-associated p100/p120 proteins in response to activation of protein kinase C in epithelial cells

Protein kinase C signaling pathways have been implicated in the disruption of intercellular junctions, but mechanisms are not clear. p100 and p120 are members of the Armadillo family of proteins and are localized to cellular adherens junctions. In strain I Madin-Darby canine kidney cells, protein kinase C activation leads to disruption of tight junctions and an increase in permeability of cell monolayers. We show that this permeability increase is accompanied by dephosphorylation of p100/p120 on serine and threonine residues. The dephosphorylation of these proteins can also be induced by the kinase inhibitors staurosporine, KT5926, and G? 6976. Treatment of cells with phosphatase inhibitors induced hyperphosphorylation of p100 and p120. Thus, p100 and p120 participate in a regulatable cycle of serine/threonine phosphorylation and dephosphorylation. Protein kinase C must act, directly or indirectly, by perturbing this phosphorylation cycle, by inhibition of a p100/p120 kinase and/or activation of a phosphatase. These data clearly show that p100 and p120 are targets of a novel protein kinase C signaling pathway. Dephosphorylation of these proteins precedes the permeability increase across epithelial cell monolayers seen in response to phorbol esters, raising the possibility that this pathway may play a role in the modulation of intercellular junctions.     

Differential effects of protein kinase C inhibitors on fibronectin-induced interleukin-beta gene transcription, protein synthesis and secretion in human monocytic cells

Tumor vaccination with dendritic cells (DC) presenting tumor antigens to T cells is a promising approach in immunotherapy. The aim of this study was to enhance T cell stimulatory ability of human DC by retroviral expression of the interleukin-7 (IL-7) gene. IL-7 has been shown to provide a potent costimulatory signal for the proliferation of T cells and the generation of cytotoxic T cells (CTL). DC were generated from human peripheral blood mononuclear cells (PBMC). DC were analyzed by light- and electron-microscopy, immunophenotype (CD1a+, CD14-, CD80+, CD86+, HLA-DR+) and functional assays. According to these criteria, 75-85% of the cells were DC. The cells did not produce measurable amounts of IL-7 spontaneously nor did they express the IL-7 receptor. A retroviral IL-7 expression vector was constructed. Retroviral infection was performed with either the LXSN-hIL-7 vector of its variant LXSN. Using the LXSN-hIL-7 vector, IL-7 production of 2296 pg/10(6) cells/24 h could be achieved on average. Transduction of DC was confirmed by RT-PCR in a CD1a-enriched cell fraction. Transduction efficiency by a control virus coding for beta-galactosidase was about 30%. In autologous mixed lymphocyte reaction (MLR), IL-7 transduced DC augmented T cell proliferation by a factor of two compared with unmodified or mock-transfected DC, and in allogeneic MLR there was a 2.7-fold increase in T cell proliferation. The increase in T cell proliferation could be correlated to IL-7 secretion by DC. Dendritic cells that have been simultaneously peptide-loaded and gene-modified to secrete IL-7 are a potential tool to amplify activation of tumor-specific T cells.     

Isoform-specific redistribution of protein kinase C in living cells

As part of a continuing effort to better understand the mechanisms of protein secretion, we compared the mass of pancreatic digestive enzymes, in resting and stimulated states, both in secretion and in the zymogen granule to determine whether their secretion is accompanied by chemical modification. Mass spectra were obtained applying the electrospray method on samples separated by reverse-phase HPLC. We report here our results for alpha-amylase (1,4-alpha-D-glucan glucanohydrolase EC 3.2.1.1). The data illustrate structural differences between states and compartments for this enzyme. Multiple isozymes were identified from the mass spectra, varying roughly from 52 to 60 kDa. On the basis of mass comparisons, not all of the products seen in the zymogen granule were found in secretion, nor were all secreted isoforms in the granule. Stimulation of protein secretion with a cholinergic agonist, led to time-dependent changes in the number and masses of isoforms in secretion, leaving only one of five resolvable forms in the granule. Only one form, 55.5 kDa, was found in all samples, granule and secretion. In addition to these differences, microheterogeneities of 400 Da or less were observed. The data suggest the differential or non-parallel release of different amylase forms and their chemical modification during the secretion process. As such, release appears to involve a third, intermediate compartment, between zymogen granule to ductal space, such as the cytoplasm, in which chemical modification takes place.     

Translocation inhibitors define specificity of protein kinase C isoenzymes in pancreatic beta-cells

The protein kinase C (PKC) family consists of 11 isoenzymes. Following activation, each isoenzyme translocates and binds to a specific receptor for activated C kinase (RACK) (Mochly-Rosen, D. (1995) Science 268, 247-251) that provides an anchoring site in close proximity to the isoenzyme's specific substrate. Pancreatic islet cells contain at least six PKC isoenzymes (Knutson, K. L., and Hoenig, M. (1994) Endocrinology 135, 881-886). Although PKC activation enhances insulin release, the specific function of each isoenzyme is unknown. Here we show that following stimulation with glucose, alphaPKC and epsilonPKC translocate to the cell's periphery, while deltaPKC and zetaPKC translocate to perinuclear sites. betaC2-4, a peptide derived from the RACK1-binding site in the C2 domain of betaPKC, inhibits translocation of alphaPKC and reduces insulin response to glucose. Likewise, epsilonV1-2, an epsilonPKC-derived peptide containing the site for its specific RACK, inhibits translocation of epsilonPKC and reduces insulin response to glucose. Inhibition of islet-glucose metabolism with mannoheptulose blocks translocation of both alphaPKC and epsilonPKC and diminishes insulin response to glucose while calcium-free buffer inhibits translocation of alphaPKC but not epsilonPKC and lowers insulin response by 50%. These findings illustrate the unique ability of specific translocation inhibitors to elucidate the isoenzyme-specific functions of PKC in complex signal transduction pathways.     

Transforming growth factor-beta1 and protein kinase C synergistically activate the c-fos serum response element in myocardial cells

Stimulation of NMDA receptor increases NO-dependent cGMP synthesis. A significantly higher cGMP level was observed in hippocampus (about 8-fold increase) than in cerebral cortex (2.5-fold increase), as compared to basal value. The activity of NO synthase (NOS) and the basal level of cGMP in unstimulated slices were only slightly higher in hippocampus than in the cortex. About 60% of NOS total activity was found in the brain membrane fraction. The enzyme activity was not affected by glucocorticoids, even after 20 days of hydrocortisone treatment in dose of 40 mg/kg b.w. Brain ischemia induced by ligation of the both common carotid arteries in gerbils (Meriones unquiculatus) significantly increased NOS activity as well as cGMP and putrescine concentrations but decreased mono-ADP-ribosolation of proteins. Changes of NOS activity and cGMP concentration evoked by ischemia were decreased by specific inhibitor of the neuronal form of NOS (nNOS), 7-nitrodazole and the inhibitor of guanylate cyclase, LY 83,583 administered respectively in a dose of 25 mg/kg b.w. and 6 mg/kg b.w. 5 min. before ischemia. The inhibitor of nNOS, 7NI, did not change the concentration of putrescine during ischemia and reperfusion. Our results indicated that these inhibitors could protect the brain against excessive production of nitric oxide and biochemical processes dependent on it. In this way they may offer a new strategy in the therapy of brain ischemia.     

Supplementation of MCF-7 cells with essential fatty acids induces the activation of protein kinase C in response to IGF-1

V(D)J recombination consists of a DNA cleavage reaction catalysed by RAG1 and RAG2, followed by an end-joining reaction that utilizes the cell's double-strand break repair machinery. Genes essential for the end-joining reaction include: XRCC4 encoding a protein of unknown enzymatic function; XRCC5 and XRCC6 encoding 86 and 70 kDa subunits of the Ku autoantigen, a DNA end-binding protein that is also the regulatory subunit of DNA-dependent protein kinase (DNA-PK); and XRCC7 encoding the catalytic subunit (DNA-PKcs) of DNA-PK. Recent progress in understanding the cleavage reaction, coupled with what was previously known about Ku, DNA-PK, and double-strand break repair, provide the foundation for a working model of how V(D)J recombination might be catalysed.     

Modulation of regulatory and catalytic subunit levels of cAMP-dependent protein kinase A in anterior pituitary cells in response to direct activation of protein kinases A and C or after GnRH stimulation

We have previously shown that direct activation of protein kinase A (PKA) and protein kinase C (PKC) induced changes in the expression of genes coding for PKA RII beta and C alpha subunit isoforms in cultured anterior pituitary cells, suggesting the possibility of interconnected regulation at this point. To evaluate whether the cell content of PKA protein subunits could be similarly altered, the catalytic (C) and regulatory type I (RI) and type II (RII) subunits were identified by Western blot analysis using specific immunoaffinity-purified antibodies. Activation of PKA by the permeant cyclic adenosine monophosphate (cAMP) analogue 8-Br-cAMP induced a dramatic time- and concentration-dependent decline of C subunit to a residual level that may represent 10-15% of that in untreated cells. The most profound decrease occurred during the first 5 h following treatment with 0.5-2 mM analogue (by 65 +/- 14 and 79 +/- 5%, respectively). Under identical conditions, RII was increased by about 40% at the higher concentrations, while RI increased slightly, but only at low concentrations (below 1 mM 8-Br-cAMP), and then gradually decreased. Exposure of cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) also resulted in decreased levels of the PKA C subunit, however, with a different concentration-dependent profile. In particular, a decline in PKA C was most pronounced (60%) at a low concentration of TPA (10 nM) as compared with the concentrations equal to or above 20 nM (40% decrease). TPA at 10 nM also depressed notably (by 25%) the level of RII subunit, but higher concentrations were essentially ineffective, although a slight and statistically not significant elevation of the cell subunit content was observed as for RI. Simultaneous activation of both PKA and PKC pathways resulted in further depletion of PKA C and an important loss (50%) of RII, a subunit which was enhanced by the activation of either system alone. Finally, gonadotropin-releasing hormone, a neuropeptide that has the potentiality to activate both PKA and PKC signaling in gonadotropes, was able to alter PKA subunit cell content: PKA C was significantly reduced at either a subliminal (0.1 nM) or maximal (10 nM) concentration, whereas RII increased at the low concentration and decreased at the high concentration. In conclusion, these data demonstrate that the pituitary cell contents of RI, RII, and C subunits of PKA are regulated under the activation of PKA itself as well as PKC in a manner that can exhibit further alteration when both systems come simultaneously into play. Changes in the PKA subunit levels in certain cases may correlate with a variation of the mRNAs suggesting multiple control mechanisms, including an alteration of gene expression and changes in subunit degradation, synthesis, and/or turnover. These data, together with those obtained in the presence of gonadotropin-releasing hormone, provide further support for a hormonally induced interplay between PKA and PKC signaling pathways at the crucial level of PKA in the pituitary gland including gonadotropes.     

Signal transduction by protein kinase C in mammalian cells

1. The past two decades have witnessed great advances in our understanding of the role of protein kinase C (PKC) in signal transduction. The Ca(2+)-activated, phospholipid-dependent protein kinase discovered by Nishizuka's group in 1977 is now a family of at least 11 isoforms. Protein kinase C isoforms exist in different proportions in a host of mammalian cells and each isoform has a characteristic subcellular distribution in each cell type. 2. Stimulation of a specific PKC isoform often causes redistribution of the isoform from one subcellular compartment to another compartments where it complexes with and phosphorylates a specific protein substrate. 3. The interaction of a specific PKC isoform with its protein substrate may directly activate a specific function of the cell or may trigger a cascade of protein kinases that ultimately stimulates a specific response in differentiated cells or regulates growth and proliferation in undifferentiated cells.     

The morphology of identified neurons in the abdominal ganglion of Aplysia californica

The morphology of identified neurons and of one multiaction interneuron (L10) of the abdominal ganglion of Aplysia has been studied using cobalt chloride, injected intracellularly. Cells with little synaptic input, R3-R14, had a relatively poorly developed dendritic tree, whereas the dendrite tree of cells L7 and L10, with extensive synaptic input, was highly complex. Cells L1-L6 and the RB cell cluster were found to have intermediate complexity of synaptic inputs and dendritic morphology. Within cell clusters, individual cells were often morphologically distinct. Identified cells have both invariant and variant axonal branches. Variant axons often project down other than their customary nerve trunks or are supernumerary. Three features of neuropil architecture were encountered. (1) When cells from the same cluster send their axons down the same nerve the axons often run in fascicles. (2) Although an identified cell's dendritic geometry varies from preparation to preparation, its dendrites always occupy approximately the same position in the neuropil. (3) The postsynaptic follower cells of L10 send their main axons through the axonal arborization of L10.     

Roles for protein kinase C and mitogen-activated protein kinase in nicotine-induced secretion from bovine adrenal chromaffin cells

Both the Ca2+/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca2+ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca2+ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC50 1-5 microM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC50 approximately 10 microM) than that induced by nicotine (IC50 approximately 30 microM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca2+ ionophore and the participation of both PKC and MEK/MAPK in optimal, secretion induced by nicotine.