Decrease In Protein Kinase C Activity Research Articles

Mycobacterium avium inhibits protein kinase C and MARCKS phosphorylation in human cystic fibrosis and non-cystic fibrosis cells.

In cystic fibrosis (CF), there is abnormal translocation and function of the cystic fibrosis transmembrane conductance regulator (CFTR) and an upregulation of the epithelial sodium channel (ENaC). This leads to hyperabsorption of sodium and fluid from the airway, dehydrated mucus, and an increased risk of respiratory infections. In this study, we performed a proteomic assessment of differentially regulated proteins from CF and non-CF small airway epithelial cells (SAEC) that are sensitive to Mycobacterium avium. CF SAEC and normal non-CF SAEC were infected with M. avium before the cells were harvested for protein. Protein kinase C (PKC) activity was greater in the CF cells compared to the non-CF cells, but the activity was significantly attenuated in both cell types after infection with M. avium compared to vehicle. Western blot and densitometric analysis showed a significant increase in cathepsin B protein expression in M. avium infected CF cells. Myristoylated alanine rich C-kinase substrate (MARCKS) protein was one of several differentially expressed proteins between the groups that was identified by mass spectrometry-based proteomics. Total MARCKS protein expression was greater in CF cells compared to non-CF cells. Phosphorylation of MARCKS at serine 163 was also greater in CF cells compared to non-CF cells after treating both groups of cells with M. avium. Taken together, MARCKS protein is upregulated in CF cells and there is decreased phosphorylation of the protein due to a decrease in PKC activity and presumably increased cathepsin B mediated proteolysis of the protein after M. avium infection.

In vivo and in vitro zinc (Zn) deficiency diminished T lymphocyte activity through the modulation of PKC isoenzymes. (87.46)

Abstract Zn is essential to numerous signaling proteins that share Zn-finger structures as a common motif. It is crucial for immunocompetent cells, as its deficiency leads to immune function alterations. The exact mechanisms involved in Zn regulation of lymphocyte activity are still unclear. Here we analyzed both the direct and the in vivo effect of Zn deficiency on normal T cells and the involvement of protein kinase C (PKC) (an enzyme with cysteine-rich domains) and its isoenzyme profile. For this purpose mitogen stimulated normal T cells were cultured in the absence or presence of specific intra- or extracellular Zn chelators. These effects were compared with those obtained in mitogen-stimulated lymphocytes purified from mice fed with Zn deficient diets. Proliferation, PKC activity and the pattern of PKC isoenzymes were evaluated. Zn chelators inhibited normal cell proliferation and their effects were reverted by in vitro Zn addition. Similar effects were observed in lymphocytes from Zn deficient mice. In both experimental conditions, a decrease in PKC activity and in mitogen-induced translocation of the enzyme to cell membranes were observed. These effects were accompanied by a decrease in PKC α and 𝛉 isoforms. These results show that Zn plays an important role in T cell growth and that its deficiency would alter the expression of key PKC isoenzymes that are essential signalling molecules involved in lymphocyte activation.

Development and Validation of a Drug Activity Biomarker that Shows Target Inhibition in Cancer Patients Receiving Enzastaurin, a Novel Protein Kinase C-β Inhibitor

To evaluate the effects of the novel protein kinase C (PKC) inhibitor enzastaurin on intracellular phosphoprotein signaling using flow cytometry and to use this approach to measure enzastaurin effects on surrogate target cells taken from cancer patients that were orally dosed with this agent. The activity of PKC was assayed in intact cells using a modification of published techniques. The U937 cell line and peripheral blood mononuclear cells were stimulated with phorbol ester, fixed, permeabilized, and reacted with an antibody specific for the phosphorylated forms of PKC substrates. The processed samples were quantitatively analyzed using flow cytometry. The assay was validated for selectivity, sensitivity, and reproducibility. Finally, blood was obtained from volunteer cancer patients before and after receiving once daily oral doses of enzastaurin. These samples were stimulated ex vivo with phorbol ester and were assayed for PKC activity using this approach. Assay of U937 cells confirmed the selectivity of the antibody reagent and enzastaurin for PKC. Multiparametric analysis of peripheral blood mononuclear cells showed monocytes to be the preferred surrogate target cell. Day-to-day PKC activity in normal donors was reproducible. Initial results showed that five of six cancer patients had decreased PKC activity following enzastaurin administration. In a following study, a group of nine patients displayed a significant decrease in PKC activity after receiving once daily oral doses of enzastaurin. An inhibition of surrogate target cell PKC activity was observed both in vitro and ex vivo after exposure to the novel kinase inhibitor, enzastaurin.

Protein Kinase C Activity in Developing Rat Brain Cells Exposed to 2.45 GHz Radiation

There is growing concern by the public regarding the potential human health hazard due to exposure to microwave frequencies. 2.45 GHz radiation widespread use in industry, research, and medicine, and leakage into the environment is possible. In order to quantitate this, experiments were performed on developing rat brain. Male Wistar 35-day-old rats (n = 6) were used for this study. Animals were exposed to 2.45 GHz radiation for 2 h/day for a period of 35 days at a power density of 0.344 mW/cm2 (SAR 0.11 W/kg). The control group was sham irradiated. After 35 days these rats were sacrificed and whole brain tissue was isolated for protein kinase C (PKC) assay. For morphological study the forebrain was isolated from the whole brain and PKC activity was measured using P32 labeled ATP. Our study reveals a statistically significant (p < 0.05) decrease in PKC activity in hippocampus as compared to the remaining portion of the whole brain and the control group. A similar experiment conducted on hippocampus and the whole brain gave a similar result. Electron microscopic study shows an increase in the glial cell population in the exposed group as compared to the control group. This present study is indicative of a significant change after exposure to the above-mentioned field intensity. This suggests that chronic exposures may affect brain growth and development.

CHRONIC PROPRANOLOL TREATMENT CAUSES DESENSITIZATION OF THE STEROIDOGENIC RESPONSE IN TESTICULAR INTERSTITIAL CELLS BUT DOES NOT ALTER PROTEIN KINASE C

We investigated effects of chronic propranolol treatment on the secretory response of rat testicular interstitial cells (testosterone secretion) to subsequent in vitro stimulation with activators of protein kinase-C (PK-C) (L-propranolol, phorbol 12, 13-dibutyrate (PDBu), LHRH) or activators of protein kinase A (PK-A), (hCG or dibutyryl cAMP (dbcAMP)). We determined [3H]PDBu binding and PK-C activity in these cells. Treatment of rats with propranolol (Inderal 500 mg/L of water for 5 weeks) reduced by 48%, 50% and 29% the L-propranolol-, LHRH- or PDBu-induced testosterone secretion, respectively, when compared to cells from controls. This desensitization in testosterone secretion in vitro was also present when the testicular interstitial cells were stimulated with hCG or dbcAMP (secretion decreased by 65%/57%, respectively, when compared to cells from control rats). Challenging the cells originated from rats that received propranolol chronically with the addition in vitro of propranolol resulted in an additional reduction of the hCG/dbcAMP-stimulated testosterone secretion. Chronic propranolol-induced desensitization was not associated with a loss in [3H]PDBu binding or a decrease in PK-C activity. Chronic propranolol-induced desensitization can be uncoupled from down-regulation of protein kinase C. The effector responsible for the desensitization could be distal to the protein kinase C and protein kinase A.

Protein Kinase C Activity and Isoform Expression During Early Postnatal Development of Rat Myocardium

Total protein kinase C (PKC) activity, its isoform expression, and concentration and fatty acid (FA) composition of diacylglycerol (DAG) were determined in the left ventricular myocardium of the rat during early postnatal development (d 2, 3, 5, 7, and 10). PKC activity measured by the incorporation of 32P into histone IIIS decreased between d 2 and 10 in the homogenate as well as in cytosolic, membrane (100,000 g), and nuclear-cytoskeletal-myofilament fractions (1000 g). Likewise, the expression of PKC isoforms (alpha, delta, and epsilon) determined by immunoblotting generally declined during the period analyzed, although with a variable pattern. In the membrane and nuclear cytoskeletal myofilament fractions, PKCdelta and PKCepsilon expression decreased markedly by d 3, returning to or close to the d 2 level immediately on d 5. PKCalpha expression in the membrane fraction remained almost unchanged by d 7, declining thereafter. PKCdelta and PKCepsilon were associated predominantly with particulate fractions, whereas PKCalpha was more abundant in the cytosolic fraction. DAG concentration exhibited a significant decline by d 5, consistent with the decrease in maximal PKC activity. The unsaturation index of FA in DAG tended to decrease on d 3 owing to the lowered proportion of all polyunsaturated FA of n-6 and n-3 series. These results demonstrate that the developmental decrease in PKC activity and expression in the rat myocardium is not linear and that subcellular localization of the enzyme exhibits isoform-specific day-by-day changes during the early postnatal period. These changes are compatible with the view that PKC signaling may be involved in the control of a rapid switch of myocardial growth pattern during the first week of life.

Distinct Protein Kinase C Isoforms Mediate Regulation of Vascular Endothelial Growth Factor Expression by A2A Adenosine Receptor Activation and Phorbol Esters in Pheochromocytoma PC12 Cells

Vascular endothelial growth factor (VEGF) stimulates angiogenesis during development and in disease. In pheochromocytoma (PC12) cells, VEGF expression is regulated by A(2A) adenosine receptor (A(2A)AR) activation. The present work examines the underlying signaling pathway. The adenylyl cyclase-protein kinase A cascade has no role in the down-regulation of VEGF mRNA induced by the A(2A)AR agonist, 2-[4-[(2-carboxyethyl)phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680). Conversely, 6-h exposure of cells to either phorbol 12-myristate 13-acetate (PMA) or protein kinase C (PKC) inhibitors mimicked the CGS21680-induced down-regulation. PMA activated PKCalpha, PKCepsilon, and PKCzeta, and CGS21680 activated PKCepsilon and PKCzeta as assessed by cellular translocation. By 6 h, PMA but not CGS21680 decreased PKCalpha and PKCepsilon expression. Neither compound affected PKCzeta levels. Following prolonged PMA treatment to down-regulate susceptible PKC isoforms, CGS21680 but not PMA inhibited the cobalt chloride induction of VEGF mRNA. The proteasome inhibitor, MG-132, abolished PMA- but not CGS21680-induced down-regulation of VEGF mRNA. Phorbol 12,13-diacetate reduced VEGF mRNA levels while down-regulating PKCepsilon but not PKCalpha expression. In cells expressing a dominant negative PKCzeta construct, CGS21680 was unable to reduce VEGF mRNA. Together, the findings suggest that phorbol ester-induced down-regulation of VEGF mRNA occurs as a result of a reduction of PKCepsilon activity, whereas that mediated by the A(2A)AR occurs following deactivation of PKCzeta.

Involvement of nuclear factor-kappaB in lipoteichoic acid-induced cyclooxygenase-2 expression in RAW 264.7 macrophages.

We have investigated the role of protein kinase C (PKC) and nuclear factor-kappaB (NF-kappaB) in cyclooxygenase-2 (COX-2) expression caused by Staphylococcus aureus lipoteichoic acid in RAW 264.7 macrophages. A phosphatidylcholine-phospholipase C (PC-PLC) inhibitor (D-609) and a phosphatidylinositol-phospholipase C (PI-PLC) inhibitor (U-73122) attenuated lipoteichoic acid-induced COX-2 expression, while a phosphatidate phosphohydrolase inhibitor (propranolol) had no effect. Two PKC inhibitors (Go 6976 and Ro 31-8220) and the NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), also attenuated lipoteichoic acid-induced COX-2 expression. Lipoteichoic acid resulted in a decrease in PKC activity in the cytosol and an increase in PKC activity in membranes. The lipoteichoic acid-induced translocation of p65 NF-kappaB from the cytosol to the nucleus was inhibited by D-609, U-73122, Go 6976, Ro 31-8220, and PDTC, but not by propranolol. The results suggested that lipoteichoic acid might have activated PC-PLC and PI-PLC to induce PKC activation, which in turn initiated NF-kappaB activation, and finally induced COX-2 expression in RAW 264.7 macrophages.

Neuropeptide Y Activates Protein Kinase C in Hamster Suprachiasmatic Nuclei Brain Slices

The mammalian circadian clock in the suprachiasmatic nuclei (SCN) can be phase-shifted by neuropeptide Y applied in the subjective day. Previous studies suggested that neuropeptide Y might act through a protein kinase C (PKC)-dependent mechanism. We directly measured PKC activity in suprachiasmatic nuclei brain slices following application of neuropeptide Y. PKC activity increased 5 min after neuropeptide Y application, with a return to baseline levels 15 min after application. An initial small decrease in PKC activity 1 min after neuropeptide Y application was also observed after control applications of artificial cerebrospinal fluid. Our results support the hypothesis that phase shifts induced by neuropeptide Y involve activation of PKC.

Protein Phosphatase-Mediated Regulation of Protein Kinase C during Long-Term Depression in the Adult HippocampusIn Vivo

The neural substrates of learning and memory are thought to involve use-dependent long-term changes in synaptic function, including long-term depression (LTD) of synaptic strength. One biochemical event hypothesized to contribute to the maintenance and expression of LTD is decreased protein phosphorylation, caused by a decrease in protein kinase activity and/or an increase in protein phosphatase activity. We tested whether the activity of protein kinase C (PKC) decreases after the induction of LTD in area CA1 of the adult hippocampus in vivo, and then investigated the mechanism responsible for the LTD-associated alteration in PKC activity. We found that LTD was associated with a significant decrease in both autonomous and cofactor-dependent PKC activity. The decrease in PKC activity was prevented by NMDA receptor blockade and was not accompanied by a decrease in the level of either PKCalpha, beta, gamma, or zeta. Western blot analysis with phosphospecific antibodies revealed that phosphorylation of Ser-657 on the catalytic domain of PKCalpha (Ser-660 on PKCbetaII) was decreased significantly after the induction of LTD, and that this dephosphorylation was prevented by the protein phosphatase inhibitor okadaic acid. The decrease in autonomous and cofactor-dependent PKC activity likewise was prevented by okadaic acid. These findings suggest that LTD in the adult hippocampus in vivo involves a decrease in PKC activity that is mediated, at least in part, by dephosphorylation of the catalytic domain of PKC by protein phosphatases activated after LTD-inducing stimulation. Our findings are consistent with the idea that protein dephosphorylation contributes to the expression of LTD.

Evidence from cultured rat cortical neurons of differences in the mechanism of ischemic preconditioning of brain and heart

Ca 2+ influx and activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) during nonlethal ischemic preconditioning have been implicated in the protection of the heart against subsequent lethal ischemic injury. Thus, we determined if Ca 2+ influx, PKC and MAPK also mediate ischemic preconditioning-induced protection in neurons. Preconditioning by exposure of E18 rat cortical cultures to 90 min of nonlethal oxygen–glucose deprivation (OGD) 24 h prior to 180–240 min of lethal OGD was neuroprotective. Exposure to nominally free Ca 2+, or blockade of the α-amino-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptor with CNQX did not eliminate protection. MAPK activity did not change and PKC activity decreased by 50% relative to normal baseline levels at 0 and 24 h following preconditioning. The sustained decrease in PKC activity was not due to a loss of enzyme as determined from immunoblots using pan and ε-, β- and ζ-specific PKC antibodies. Neuroprotection was maintained with pharmacological inhibition of PKC activity by staurosporine, chelerythrine and calphostin C and MAPK activity by PD 98059 during preconditioning, indicating that activation of these enzymes during preconditioning was not necessary for protection. Therefore, in contrast to cardiac tissue, ischemic preconditioning of neurons does not require activation of PKC and MAP kinase, and protection is maintained with substantial removal of extracellular Ca 2+ or blockade of the AMPA receptor.

α-Tocopherol Inhibits the Respiratory Burst in Human Monocytes: ATTENUATION OF p47phox MEMBRANE TRANSLOCATION AND PHOSPHORYLATION

Vitamin E (alpha-tocopherol), one of the most important natural antioxidants, is assumed to be beneficial in the prevention of cardiovascular diseases. alpha-Tocopherol exhibits acyl-peroxyl-radical scavenger properties and exerts cell-mediated actions in the hemovascular compartment, such as inhibition of superoxide anion (O-2) production by leukocytes. The aim of this study was to examine the mechanism underlying the inhibitory effect of alpha-tocopherol on O-2 production by human monocytes. In activated monocytes O-2 is produced by the NADPH-oxidase enzyme complex. The oxidase activation elicited by phorbol myristate acetate (PMA) requires membrane translocation of several cytosolic factors. We found that in human PMA-stimulated adherent monocytes, alpha-tocopherol (but not beta-tocopherol) inhibited O-2 production in intact cells but had no effect on a membrane preparation containing activated NADPH-oxidase, suggesting that alpha-tocopherol impairs the assembly process of the enzyme complex. We showed that translocation and phosphorylation of the cytosolic factor p47(phox) were reduced in monocytes preincubated with alpha-tocopherol. We verified that the tryptic phosphopeptide map of monocyte p47(phox) was similar to that of neutrophil p47(phox), indicating that several serine residues were phosphorylated. Peptides whose phosphorylation is dependent on protein kinase C (PKC) were phosphorylated to a lesser degree when p47(phox) was immunoprecipitated from alpha-tocopherol-treated monocytes. In vitro, the activity of PKC from monocytes was inhibited by alpha-tocopherol in a specific manner compared with that of beta-tocopherol or Trolox(R). Membrane translocation of PKC was not affected. These results show that alpha-tocopherol inhibits O-2 production by human adherent monocytes by impairing the assembly of the NADPH-oxidase and suggest that the inhibition of phosphorylation and translocation of the cytosolic factor p47(phox) results from a decrease in PKC activity.

Isoform-specific protein kinase C activity at variable Ca2+ entry during coronary artery contraction by vasoactive eicosanoids

Vasoactive eicosanoids have been implicated in the pathogenesis of coronary vasospasms. The signaling mechanisms of eicosanoid-induced coronary vasoconstriction are unclear, and a role for protein kinase C (PKC) has been suggested. Activated PKC undergoes translocation to the surface membrane in the vicinity of Ca2+ channels; however, the effect of Ca2+ entry on the activity of the specific PKC isoforms in coronary smooth muscle is unknown. In the present study, 45Ca2+ influx and isometric contraction were measured in porcine coronary artery strips incubated at increasing extracellular calcium concentrations ([Ca2+]e) and stimulated with prostaglandin F2α (PGF2α) or the stable thromboxane A2 analog U46619, while in parallel, the cytosolic (C) and particulate (P) fractions were examined for PKC activity and reactivity with anti-PKC antibodies using Western blot analysis. At 0-300 µM [Ca2+]e, both PGF2α and U46619 (10-5 M) significantly increased PKC activity and contraction in the absence of a significant increase in 45Ca2+ influx. At 600 µM [Ca2+]e, PGF2α and U46619 increased P/C PKC activity ratio to a peak of 9.52 and 14.58, respectively, with a significant increase in 45Ca2+ influx and contraction. The 45Ca2+ influx - PKC activity - contraction relationship showed a 45Ca2+-influx threshold of ~7 µmol·kg-1·min-1 for maximal PKC activation by PGF2α and U46619. 45Ca2+ influx &gt; 10 µmol·kg-1·min-1 was associated with further increases in contraction despite a significant decrease in PKC activity. Western blotting analysis revealed α-, δ-, ε-, and ζ-PKC in porcine coronary artery. In unstimulated tissues, α- and ε-PKC were mostly distributed in the cytosolic fraction. Significant eicosanoid-induced translocation of ε-PKC from the cytosolic to the particulate fraction was observed at 0 [Ca2+]e, while translocation of α-PKC was observed at 600 µM [Ca2+]e. Thus, a significant component of eicosanoid-induced coronary contraction is associated with significant PKC activity in the absence of significant increase in Ca2+ entry and may involve activation and translocation of the Ca2+-independent ε-PKC. An additional Ca2+-dependent component of eicosanoid-induced coronary contraction is associated with a peak PKC activity at submaximal Ca2+ entry and may involve activation and translocation of the Ca2+-dependent α-PKC. The results also suggest that a smaller PKC activity at supramaximal Ca2+ entry may be sufficient during eicosanoid-induced contraction of coronary smooth muscle.Key words: protein kinase C, isoform, calcium influx, eicosanoids, vascular smooth muscle, contraction.

Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1).

Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop. This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. First, studies in vivo revealed that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKC betaII with a catalytically inactive form of PDK-1 in COS-7 cells resulted in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Second, studies in vitro using purified proteins established that PDK-1 specifically phosphorylates the activation loop of PKC alpha and betaII. The phosphorylation of the mature PKC enzyme did not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggered carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme. We have shown that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation.

Efficient protein transfection of cultured coronary venular endothelial cells.

Although it is well recognized that microvascular endothelial cells play an important role in the local regulation of tissue perfusion and exchange processes, the precise effect of specific endothelial proteins on microvascular function remains to be elucidated. The lack of information is partially due to methodological limitations, because pharmacological approaches that are routinely used in conventional microcirculatory studies produce nonspecific information. The purpose of this study was to develop an efficient method of transfecting endothelial cells with proteins for functional analysis. TransIT, a polyamine reagent, proved very successful for beta-galactosidase (beta-Gal) protein transfection of bovine coronary venular endothelial cells, because time-course and dose-dependent experiments showed that a transfection efficiency of 88 +/- 7% was possible. In control studies, beta-Gal was detected in transfected cells that were trypsinized and washed, indicating that the protein was not merely adhering to the cell surface. Furthermore, transfection of a cell-impermeable peptide inhibitor of protein kinase C (PKC) resulted in a decrease in PKC activity in comparison with control cells. This approach provides a technical basis for further transfection of endothelial cell monolayers with antibodies and constitutively active or dominant-negative proteins to study the molecular control of microvascular function.

Differential effects of phorbol ester on growth and protein kinase C isoenzyme regulation in human hepatoma Hep3B cells.

PMA has both mitogenic and antiproliferative effects on human hepatoma Hep3B cells. In response to low PMA concentration (10 nM), Hep3B cells displayed an increasing proliferation potentiation. At high PMA concentration (1 microM) Hep3B cells exhibited modest cytostatic effects. Determinations of protein kinase C (PKC) activity in PMA-treated cells revealed that alterations in PKC activity are associated with proliferative capacity. The decrease in PKC activity mediated by a high dose of PMA was accompanied by cell growth inhibition. Increases in PKC activity mediated by a low dose of PMA were consistent with proliferation stimulation. Immunoblot analysis showed that there are at least six PKC isoenzymes: alpha, delta, epsilon, mu, zeta and iota/lambda, constitutively expressed in Hep3B cells. Cellular fractionation and immunocytochemical staining results demonstrated that both 10 nM and 1 microM PMA treatments induced a marked translocation of PKC-alpha from cytosol to membrane or nuclear fraction within 5-30 min. At the same time PKC-delta and epsilon were translocated from the membrane to nuclear fraction. In addition, prolonged treatment with 1 microM PMA, but not with 10 nM PMA, selectively mediated the down-regulation of these three PKC isoenzymes. The distinct effects of different concentrations of PMA on cell proliferation and PKC-alpha, delta and epsilon isoenzyme modulation support the involvement of these three PKC isotypes in the mechanism of action of Hep3B cells in cell growth events.

Isoform-specific protein kinase C activity at variable Ca2+ entry during coronary artery contraction by vasoactive eicosanoids.

Vasoactive eicosanoids have been implicated in the pathogenesis of coronary vasospasms. The signaling mechanisms of eicosanoid-induced coronary vasoconstriction are unclear, and a role for protein kinase C (PKC) has been suggested. Activated PKC undergoes translocation to the surface membrane in the vicinity of Ca2+ channels; however, the effect of Ca2+ entry on the activity of the specific PKC isoforms in coronary smooth muscle is unknown. In the present study, 45Ca2+ influx and isometric contraction were measured in porcine coronary artery strips incubated at increasing extracellular calcium concentrations ([Ca2+]e) and stimulated with prostaglandin F2alpha (PGF2alpha) or the stable thromboxane A2 analog U46619, while in parallel, the cytosolic (C) and particulate (P) fractions were examined for PKC activity and reactivity with anti-PKC antibodies using Western blot analysis. At 0-300 microM [Ca2+]e, both PGF2alpha and U46619 (10(-5) M) significantly increased PKC activity and contraction in the absence of a significant increase in 45Ca2+ influx. At 600 microM [Ca2+]e, PGF2alpha and U46619 increased P/C PKC activity ratio to a peak of 9.52 and 14.58, respectively, with a significant increase in 45Ca2+ influx and contraction. The 45Ca2+ influx--PKC activity--contraction relationship showed a 45Ca2+-influx threshold of approximately 7 micromol x kg(-1) x min(-1) for maximal PKC activation by PGF2alpha and U46619. 45Ca2+ influx > 10 micromol x kg(-1) x min(-1) was associated with further increases in contraction despite a significant decrease in PKC activity. Western blotting analysis revealed alpha-, delta-, epsilon-, and zeta-PKC in porcine coronary artery. In unstimulated tissues, alpha- and epsilon-PKC were mostly distributed in the cytosolic fraction. Significant eicosanoid-induced translocation of epsilon-PKC from the cytosolic to the particulate fraction was observed at 0 [Ca2+]e, while translocation of alpha-PKC was observed at 600 microM [Ca2+]e. Thus, a significant component of eicosanoid-induced coronary contraction is associated with significant PKC activity in the absence of significant increase in Ca2+ entry and may involve activation and translocation of the Ca2+-independent epsilon-PKC. An additional Ca2+-dependent component of eicosanoid-induced coronary contraction is associated with a peak PKC activity at submaximal Ca2+ entry and may involve activation and translocation of the Ca2+-dependent alpha-PKC. The results also suggest that a smaller PKC activity at supramaximal Ca2+ entry may be sufficient during eicosanoid-induced contraction of coronary smooth muscle.

Selenocompounds Induce a Redox Modulation of Protein Kinase C in the Cell, Compartmentally Independent from Cytosolic Glutathione: Its Role in Inhibition of Tumor Promotion

Since selenite and other redox-active selenocompounds can modify protein kinase C (PKC) in the test tube, we have determined whether or not this redox regulation occurs inside the cell despite having high concentrations of GSH and the role of this regulation in the inhibition of tumor promotion. By using phorbol ester-promoted JB6 epidermal cell transformation assay, the concentrations of selenite, selenocystine, and selenodiglutathione which are optimal for chemopreventive activity were determined. At such concentrations (0.5 to 2 μm) in the cells treated with these agents, only a slight but transient decrease in PKC activity was observed when measured with a low (5 μm), but not with a high (100 μm) concentration of ATP. However, when the cells were serum starved or pretreated with 2-deoxyglucose, there was a pronounced but transient inactivation of PKC when assayed with both low and high concentrations of ATP. The inactivation was reversed in the cell by an endogenous mechanism or by treatment with thiol agents in the test tube. In spite of a substantial (90%) depletion of GSH in the cells by pretreatment with buthionine sulfoximine, there was no further increase in the redox modification of PKC by selenite as well as no change in the inhibitory effect of selenite on the phorbol ester-stimulated induction of ornithine decarboxylase, which is an intermediate marker related to cell transformation. While GSH is known to influence certain actions of selenium, it may not be required to mediate the effects of selenite tested in this study. The water-soluble cytosolic GSH did not interfere with the redox modification of PKC probably due to the shielding of the cysteine-rich region of the enzyme by a weak hydrophobic association with the membrane. Due to the presence of cofactors in the crude cell extracts, PKC was more sensitive to selenite than in the purified form and was inactivated by low concentrations of selenite (IC50= 0.05 μm). This modification was reversed by thiol agents as well as by NADPH. A protein disulfide reductase, which can regenerate PKC, was present in the homogenate. Conceivably, selenite and other selenocompounds induce a redox modification of cellular PKC, compartmentally independent from the cytosolic GSH, but intimately connected to a NADPH-dependent reductase system, to mediate, at least in part, some of the cancer-preventive actions.

Modulatory effects of PKC activity on increased 92-kDa gelatinase secretion by neonatal alveolar macrophages.

We previously demonstrated that alveolar macrophages (AMs) from neonatal rats can secrete more 92-kDa gelatinase than AMs from adult rats. In this study, we investigated the role of the protein kinase C (PKC) pathway in the transductional regulation of 92-kDa gelatinase secretion by rat AMs, and we also evaluated maturational changes in this role with increasing postnatal age. After AM stimulation by phorbol 12-myristate 13-acetate (PMA), we observed a dose-dependent increase in gelatinase secretion that was significantly more marked in AMs from 6-day-old rats than in AMs from adult rats and that was inhibited by the PKC inhibitor calphostin C. Adenosine 3',5'-cyclic monophosphate mimetics or concanavalin A failed to induce an increase in gelatinase secretion by AMs. Time-dependent variations in PKC activity after PMA stimulation differed significantly between 6-day-old rats and adult rats; PKC activity decreased in adult AMs (50%) but remained stable in 6-day-old AMs. We therefore investigated age-related differences in the intracellular proteolytic degradation of PKC, which is thought to be mediated by calpains. Leupeptin, used as a calpain inhibitor, inhibited the decrease in PKC activity after exposure of adult AMs to PMA and induced a greater than threefold increase in PMA-induced gelatinase secretion. Calpain activity was significantly lower in AM extracts from 6-day-old than from adult rats. The physiological implication of these developmental changes in 92-kDa gelatinase regulation was demonstrated by investigation of AMs from 1-day-old rats that showed a high level of spontaneous PKC-dependent gelatinase secretion coexisting with very low calpain activity. We conclude that sustained PKC activity is a key factor in the increased gelatinase secretion by AMs seen during the postnatal period and is due, at least in part, to reduced PKC degradation.

Hybrid structural analogues of 1,25-(OH)2D3 regulate chondrocyte proliferation and proteoglycan production as well as protein kinase C through a nongenomic pathway

1,25-(OH)2D3 and 24,25-(OH)2D3 mediate their effects on chondrocytes through the classic vitamin D receptor (VDR) as well as through rapid membrane-mediated mechanisms which result in both nongenomic and genomic effects. In intact cells, it is difficult to distinguish between genomic responses via the VDR and genomic and nongenomic responses via membrane-mediated pathways. In this study, we used two hybrid analogues of 1,25-(OH)2D3 which have been modified on the A-ring and C,D-ring side chain (1 alpha-(hydroxymethyl)-3 beta-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D3 (analogue MCW-YA = 3a) and 1 beta-(hydroxymethyl)-3 alpha-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D3 (analogue MCW-YB = 3b) to examine the role of the VDR in response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25-(OH)2D3 and 24,25-(OH)2D3. These hybrid analogues are only 0.1% as effective in binding to the VDR from calf thymus as 1,25-(OH)2D3. Chondrocyte proliferation ([3H]-thymidine incorporation), proteoglycan production ([35S]-sulfate incorporation), and activity of protein kinase C (PKC) were measured after treatment with 1,25-(OH)2D3, 24,25-(OH)2D3, or the analogues. Both analogues inhibited proliferation of both cell types, as did 1,25-(OH)2D3 and 24,25-(OH)2D3. Analogue 3a had no effect on proteoglycan production by GCs but increased that by RCs. Analogue 3b increased proteoglycan production in both GC and RC cultures. Both analogues stimulated PKC in GC cells; however, neither 3a nor 3b had an effect on PKC activity in RC cells. 1,25-(OH)2D3 and 3a decreased PKC in matrix vesicles from GC cultures, whereas plasma membrane PKC activity was increased, with 1,25-(OH)2D3 having a greater effect. 24,25-(OH)2D3 caused a significant decrease in PKC activity in matrix vesicles from RC cultures; 24,25-(OH)2D3, 3a, and 3b increased PKC activity in the plasma membrane fraction, however. Thus, with little or no binding to calf thymus VDR, 3a and 3b can affect cell proliferation, proteoglycan production, and PKC activity. The direct membrane effect is analogue-specific and cell maturation-dependent. By studying analogues with greatly reduced affinity for the VDR, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites.