Membrane Protein Kinase C Activity Research Articles

Diets enriched with cereal brans or inulin modulate protein kinase C activity and isozyme expression in rat colonic mucosa

The role of dietary fibres in colon carcinogenesis is controversial. To elucidate the mechanisms by which different dietary fibre sources may affect colonic tumour development, we studied the effects of diets enriched with cereal brans or inulin on protein kinase C (PKC) activity and isozyme expression in rat colon. Male Wistar rats (twelve per group) were fed one of the following AIN-93G-based diets (Reeves et al. 1993) for 4 weeks: a non-fibre high-fat diet or one of the four high-fat diets supplemented with either rye, oat or wheat bran or inulin at 100 g/kg diet. The fat concentration (20 g/100 g) and fatty acid composition of the non-fibre high-fat diet was designed to approximate that in a typical Western-type diet. In the proximal colon, rats fed the inulin diet had a significantly higher membrane PKC activity and a higher membrane PKC delta level than rats fed the non-fibre diet In the distal colon, rats fed the inulin and oat bran diets had a higher total PKC activity and a higher membrane PKC beta 2 level than rats fed the wheat-bran diet. Rats in the non-fibre and wheat-bran groups had the lowest concentrations of luminal diacylglycerol. In conclusion, feeding of wheat bran resulted in low distal PKC activity and expression of PKC beta 2, a PKC isozyme related to colonic cell proliferation and increased susceptibility for colon carcinogenesis, which may explain in part the protective effect of wheat bran against tumour development in a number of experimental colon cancer studies. The increase in PKC activity and PKC beta 2 expression by feeding inulin may be a drawback of inulin as a functional food.

A Novel Neuroprotective Mechanism of Riluzole: Direct Inhibition of Protein Kinase C

In addition to its antiexcitotoxic action, the anti-amyotrophic lateral sclerosis (ALS) neuroprotectant riluzole protects against nonexcitotoxic oxidative neuronal injury. In light of evidence that protein kinase C (PKC) mediates oxidative stress in cortical culture, we examined the possibility that riluzole's antioxidative neuroprotection involves PKC inhibition. Riluzole (30 μM) blocked phorbol 12-myristate 13-acetate (PMA)-induced increases in membrane PKC activity in cultured cortical cells. Suggesting a direct action, riluzole also inhibited the activity of purified PKC. Consistently, both PKC depletion and oxidative neuronal death induced by PMA were markedly attenuated by riluzole. The site of action of riluzole on PKC was not likely the diacylglycerol binding site but the catalytic domain, since riluzole did not alter radiolabeled phorbol-12,13-dibutyrate binding, but inhibited PKM, the catalytic domain of PKC. However, increasing ATP concentrations did not alter the inhibition of PKC by riluzole, making it unlikely that riluzole is a competitive inhibitor of ATP binding at PKM. Present results have demonstrated that riluzole directly inhibits PKC, which action may contribute to its antioxidative neuroprotective effects. In addition, it appears possible that PKC inhibition may be able to explain some of its well-known channel inhibitory and neuroprotective effects. Combined with findings that PKC activity is increased in ALS, the present results suggest that PKC may be a potential therapeutic target in ALS.

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

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

Phospholipid fatty acid composition and protein kinase C activity in the large intestine of rats fed on butter and coconut-oil diets.

Protein kinase C (PKC) has been proposed to play an important role in the aetiology of colon cancer. Therefore, we investigated whether the amount and type of saturated fat could affect colonic PKC activity by modifying either mucosal phospholipid fatty acid composition or faecal diacylglycerol production. Male Wistar rats (n 13 per group) were fed on diets containing butter or coconut oil at energy levels of 10% and 43% for 4 weeks. The control group received a low-fat diet providing 10% of energy from sunflowerseed oil. PKC activity was higher in the distal than the proximal colon but the quantity or type of fat did not alter PKC activity in either region of the colon. Saturated fats caused moderate changes in the fatty acid composition of caecal phospholipids, which were more obvious in the phosphatidylethanolamine than in the phosphatidylcholine fraction. A significant correlation was found between fatty acid composition of phosphatidylcholine and membrane PKC activity. In particular, there was a positive correlation between the proportion of saturated 14:0 and 18:0 and increased PKC activity while unsaturated 18:2n-6, 20:4n-6 and 16:1n-7 were inversely correlated with PKC activity. No relationship was found between phosphatidylethanolamine fatty acids and PKC activity. Concentration of faecal diacylglycerol was not affected by the diet. Overall the data suggest that diets high in saturated fat may not alter colonic PKC activity to a significant extent.

Protein kinase C activity is acutely regulated by plasma glucose concentration in human monocytes in vivo.

Activation of protein kinase C (PKC) by hyperglycemia is implicated in the pathogenesis of long-term diabetic complications. Monocyte activation and transformation into macrophages is a key step in the atherosclerotic process. Therefore, in this study, we sought to determine 1) the effect of hyperglycemia on monocyte PKC activity and on the distribution of Ca2+-dependent and diacylglycerol-sensitive PKC isoforms; and 2) whether the effects on these parameters are determined by hyperglycemia per se, independent of the diabetic state. The studies were performed in 19 type 2 diabetic patients and 14 control subjects. Plasma glucose concentration was higher and insulin sensitivity lower (both P < 0.01) in diabetic patients than in control subjects. Monocytes from diabetic patients showed similar cytosol PKC activity to those from control subjects but higher membrane PKC activity (78+/-6 vs. 50+/-5 pmol x min(-1) x mg(-1) protein; P < 0.01). A direct correlation was observed between fasting plasma glucose and membrane PKC activity (r2 = 0.4008, P = 0.0001). In contrast, a reciprocal correlation was observed between membrane PKC activity and insulin sensitivity index (r2 = 0.28, P < 0.05). Using immunoblotting analysis, we found that membrane beta2, but not alpha, isoform of PKC was more abundant in monocytes from diabetic patients. In diabetic patients, when euglycemia was acutely induced, membrane PKC activity decreased by approximately 42% and beta2 isoform by approximately 15%. In two normal subjects in whom hyperglycemia was induced, membrane PKC increased from 63 and 57 to 92 and 128.6 pmol x min(-1) x mg(-1) protein, respectively. This increase was associated with an increase in the membrane isoform beta2; alpha isoform was unchanged. We conclude that 1) monocytes express the glucose-sensitive beta2 isoform of PKC; 2) the prevailing plasma glucose acutely regulates the activity of the membrane PKC and the content of membrane PKC beta2 isoform; and 3) this effect appears to be a direct effect of glucose per se, since the phenomenon was observed in normal control subjects when hyperglycemia was induced. Monocyte PKC activation may account for the accelerated atherosclerosis of patients with type 2 diabetes.

Effect of protein kinase C and insulin on Na+/H+ exchange in red blood cells of essential hypertensives.

The kinetic properties of sodium-proton exchange are abnormal in human red blood cells of hypertensive patients and it has been demonstrated that the transport protein undergoes post-translational modifications able to affect its kinetic properties. Protein kinase C (PKC) activation decreases the affinity constant for intracellular protons while insulin increases the maximal rate of proton translocation. The present study therefore aimed to examine the relationships among PKC activity, fasting insulin levels and the kinetic behaviour of sodium-proton exchange in red blood cells from 20 normotensives and 36 hypertensives. In comparison with normotensive subjects, hypertensive patients had higher body mass index (26.2 +/- 0.7 vs 23.6 +/- 0.6 kg/m2, P < 0.05), higher fasting insulin levels (93.2 +/- 10.8 vs 38.6 +/- 2.9 pmol/L), increased maximal velocity of proton translocation (37.9 +/- 2.7 vs 27.6 +/- 1.9 mmol/L per cell x h, P < 0.05), and reduced Hill's coefficient (1.6 +/- 0.1 vs 2.0 +/- 0.1, P < 0.01) of sodium-proton exchange. Basal PKC activity of the cytosol and membrane was similar in the study groups. However, after treatment with 1 micromol/L phorbol 12-myristate 13-acetate (PMA) for 10 min, membrane PKC activity was stimulated to a larger extent in hypertensives (to 181 +/- 8 pmol/min/mg protein) than in normotensives (to 136 +/- 6 pmol/min/mg protein, P < 0.01). The PMA stimulated PKC activity was positively correlated to fasting insulin levels (r = 0.59, P < 0.01). Stimulation of membrane PKC by PMA corrected the low Hill's coefficient for H(i)+ activation of sodium-proton exchange in the hypertensives, while the constant for half maximal activation for intracellular protons (ie, the affinity for intracellular protons) decreased to a similar extent in both groups. The maximal transport rate was unaffected by PMA. These results indicate that the abnormal proton activation of red blood cell sodium-proton exchange in hypertensives reflects an abnormal regulation of PKC translocation to the cell membrane, associated to hyperinsulinaemia and probably insulin resistance. Therefore, post-translational modifications of the transport protein(s) account for the altered kinetic behaviour of sodium-proton exchange in hypertensives.

Mediation by membrane protein kinase C of zinc-induced oxidative neuronal injury in mouse cortical cultures.

Transsynaptic movement of endogenous zinc may play a key role in selective neuronal death after brain ischemia and prolonged seizures. As to the mechanism, we have reported recently that zinc-induced neuronal death occurs mainly by oxidative stress in cortical cultures. Here we present evidence supporting the idea that activation of membrane protein kinase C (PKC) in neurons is likely to play a key role in zinc-induced oxidative neuronal injury. Exposure of cortical cultures to 300 microM zinc for 15 min induced increases in the activity, without changing the amount, of membrane PKC to two- to threefold of control values, followed by neuronal death over the next day. Addition of a zinc chelator, Ca-EDTA, or PKC inhibitors with zinc completely abolished the zinc-induced increase in the membrane PKC activity. Indicating the participation of PKC in zinc-induced oxidative stress and neuronal death, the selective PKC inhibitor GF109203X attenuated both. Furthermore, as in zinc-induced neuronal death, activation of PKC with phorbol esters induced free radical generation and neuronal death, which were blocked by GF109203X or an antioxidant, Trolox. The present results support the idea that zinc influx activates PKC in the membrane, which contributes to free radical generation and neuronal death. As an increasing body of evidence suggests that zinc neurotoxicity is an important mechanism of pathological neuronal death, timely prevention of PKC activation after acute brain insult may prove useful in ameliorating this type of neuronal death.

Effect of Ageing on the Expression of Protein Kinase C and Its Activation by 1,25(OH) 2-Vitamin D 3 in Rat Skeletal Muscle

To characterize age-induced effects on muscle protein kinase C (PKC) and its regulation by the steroid hormone 1,25(OH) 2-vitamin D 3 [1,25(OH) 2D 3], changes in PKC activity and the expression and translocation of the specific PKC conventional isoforms α and β, novel isoforms δ, ϵ, and θ and atypical isoform ζ were studied in homogenates and subcellular fractions from skeletal muscle of young (3 months) and aged (24 months) rats treated in vitro with 1,25(OH) 2D 3. The hormone (10 −9 M) increased total and membrane PKC activity, within 1 min, and these effects were completely blunted in muscle from aged rats. The presence of PKC isoenzymes was shown by Western blot analysis with the use of specific antibodies. The expression of PKC α, β and δ was greatly diminished in old rats, whereas age-related changes were less pronounced in the isoforms ϵ, θ and ζ. After a short exposure (1 min) of muscle to 1,25(OH) 2D 3, increased amounts of PKC α and β in muscle membranes and reverse translocation (from membrane to cytosol) of PKC ϵ were observed only in young animals. The data indicate that, in rat muscle, ageing impairs calcium-dependent PKC (α and β) and calcium-independent PKC (δ, ϵ, θ and ζ) signal transduction pathways under selective regulation by 1,25(OH) 2D 3.

Protein Kinase Cδ-dependent Induction of Manganese Superoxide Dismutase Gene Expression by Microtubule-active Anticancer Drugs

Bacterial lipopolysaccharide can induce manganese superoxide dismutase (MnSOD) gene expression in a variety of cells. Paclitaxel (taxol) shares many properties of lipopolysaccharide. Here we report that paclitaxel can induce MnSOD gene expression in human lung adenocarcinoma cell line A549 in a time- and dose-dependent manner. Additional anticancer drugs, vinblastine and vincristine, also induced MnSOD gene expression. We have shown previously (Das, K. C., and White, C. W. (1997) J. Biol. Chem. 272, 14914-14920) that these drugs can activate protein kinase C (PKC). The PKC agonists thymeleatoxin (0.5 microM) and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA; 10 nM) potently induced MnSOD gene expression. Calphostin C and GF109203X, both specific inhibitors of PKC, each inhibited MnSOD gene expression by anticancer agents. Down-regulation of PKC by prolonged treatment with phorbol 12-myristate 13-acetate (PMA) also inhibited induction of MnSOD by anticancer drugs, indicating an important role of PKC in MnSOD signaling by these agents. Of 11 PKC isoenzymes, only PKCdelta translocated to the cell membrane after stimulation with anticancer drugs. By contrast, dPPA, PMA, and thymeleatoxin caused translocation of PKCalpha, betaI, delta, and mu isotypes. Anticancer drug-stimulated cells also had increased total PKC activity in membrane and cytosolic fractions. Thus, paclitaxel, vinblastine, and vincristine each specifically activate PKCdelta, whereas PMA, thymeleatoxin, and dPPA activate multiple isoenzymes. PKCdelta was the only isoform activated by each agent in both groups of compounds effective in MnSOD induction.

Human immunodeficiency virus Tat protein induces interleukin 6 mRNA expression in human brain endothelial cells via protein kinase C- and cAMP-dependent protein kinase pathways.

The intracellular signal transduction pathways utilized by the HIV-1-derived protein, Tat, in the activation of human central nervous system-derived endothelial cells (CNS-ECs) were examined using specific enzymatic assays. Tat induced an increase in interleukin 6 (IL-6) mRNA within 1 hr of treatment. This biological effect of Tat involved activation of both protein kinase C (PK-C) and cAMP-dependent protein kinase (PK-A) in CNS-ECs. Tat at 10 ng/ml induced a sharp, transient increase in membrane PK-C activity within 30 sec of incubation, and reached maximum levels at 2 min, declining to control values within 10 min. Tat also induced a sharp increase in intracellular cAMP levels and PK-A activity in these cells, with the PK-A activity reaching a maximum at 10 min and slowly declining to control values in 4 hr of incubation. Activation of PK-A was dependent on a Tat-induced increase in membrane PK-C activity as demonstrated by calphostin C (a PK-C inhibitor) abolishing this effect. Incubation of cells with the cyclooxygenase inhibitor indomethacin did not affect Tat-induced activation of PK-A, indicating that prostacyclins are not involved in this process. Tat-induced increase in IL-6 mRNA was abolished in the presence on PK-A inhibitor H-89, demonstrating that activation of PK-A is necessary and sufficient for the increase in IL-6 production by these cells. Both the Tat-induced increase in intracellular cAMP and IL-6 mRNA levels in CNS-ECs may play a role in altering the blood-brain barrier and thereby inducing pathology often observed in AIDS dementia.

Pre- and postsynaptic alterations in the septohippocampal cholinergic innervations after prenatal exposure to drugs

The present study was designed to evaluate possible presynaptic and postsynaptic alterations in the hippocampal cholinergic innervations that account for the hippocampus-related behavioral deficits found after prenatal drug exposure. Mice were prenatally exposed to either phenobarbital or heroin. On postnatal day 50, the hippocampi were removed and protein kinase C (PkC) activity, the amounts of Gi, Go, and Gq guanosine 5’-triphosphate binding proteins (G-proteins), and choline transports were determined. Basal PkC activity was higher than control levels in both phenobarbital and heroin treated mice, by 41% and 35%, respectively. The increase of PkC activity in response to carbachol was impaired in both treatment groups: in control mice, membrane PkC activity in hippocampal slices increased by 40%–50%, while no such response, or even slight reduction in PkC activity, was observed in the drug-exposed offspring. A significant increase was found in Gi and Gq G-proteins (18%–21%) in mice exposed to phenobarbital or to heroin compared with control levels. The amount of choline transporters, determined by hemicholinium binding, increased by 70% compared with the control level in mice prenatally exposed to heroin, and increased by 71% in mice prenatally exposed to phenobarbital. The alterations in basal and carbachol-stimulated hippocampal PkC activity after prenatal drug exposure may be related to an impairment in long-term potentiation (LTP); which plays an important role in hippocampal related behavioral abilities, changes in which are caused by prenatal drug exposure.

Comparison of the Changes in Protein Kinase C Induced by Glutamate in Primary Cortical Neurons and by in Vivo Cerebral Ischaemia

Changes in protein kinase C (PKC) were compared in primary cortical neurons exposed to glutamate and in the CA-1 hippocampal region of rats subjected to transient cerebral ischaemia. After a 15-min exposure of cortical neurons to excitotoxic levels of glutamate, a 50–60% loss of membrane PKC activity but only about a 20 % loss in the amount of enzyme was observed, suggesting that in addition to enzyme loss other mechanisms also contributed to the overall loss of membrane PKC activity. Glutamate induced a 25–40% decrease in immunodetectable levels of PKC α, β, γ, and λ but no detectable changes in PCK ϵ and ζ. The loss of PKC activity coincided with a shift in electrophoretic mobility of PKC γ, ϵ, and λ, but not of PKC α, β, or ζ, suggesting post-translational modification of some PKC isoforms. By comparison, in rats subjected to transient (15-min) global ischaemia, a similar 50–60% decrease in membrane PKC activity, a 20–25 % loss in the amount of PKC, and a shift in PKC mobility were observed in CA-1 neurons 6 h post-reperfusion. In both the in vivo and the in vitro “ischaemic” models, administration of the AMPA receptor antagonist NBQX prevented the loss of PKC activity. These results indicate that the loss of PKC observed in in vivo ischaemia is likely to be due to excitotoxic damage and that this event can be closely mirrored in primary neuronal cultures damaged by glutamate.

Calcium and protein kinase C mediate high-glucose-induced inhibition of inducible nitric oxide synthase in vascular smooth muscle cells.

Abnormal vascular smooth muscle (VSMC) proliferation is a key feature in diabetes-associated atherosclerotic disease. Since nitric oxide inhibits VSMC tone, migration, adhesion, and proliferation, we examined the effects of high glucose on IL-1beta-induced NO release from VSMCs in culture. Confluent smooth muscle cells, preincubated with either 5 mmol/L (mM) or 20 mmol/L (mM) glucose for 48 hours, were stimulated with IL-1beta. Nitrite was measured in the culture medium after 24 hours. IL-1beta-induced a 15-fold increase in NO production in normal glucose medium. Glucose (10 to 30 mmol/L (mM)) significantly reduced the response to IL-1beta. High glucose (20 mmol/L (mM)) inhibited IL-1beta-evoked NO production by approximately 50%. IL-1beta-stimulated [3H] citrulline-forming activity of the nitric oxide synthase (NOS) was also significantly lower in high-glucose-exposed cells, and this was reflected in diminished cellular levels of NOS protein. To assess the role of protein kinase C (PKC), membrane PKC activity was measured, and glucose (20 mmol/L (mM)) significantly increased it. Immunoblotting of the membranes revealed a glucose-induced increase in the PKC betaII isoform. 1,2-Dioctanoyl-glycerol, a PKC activator, mimicked the high-glucose effect on IL-1beta-induced NO release, while staurosporine, a PKC inhibitor, reversed it. The role of calcium in the glucose-mediated inhibition of cytokine-induced NO release was determined by treatment with BAPTA, an intracellular chelator of calcium. BAPTA partially reversed the inhibitory effects of glucose. Increasing intracellular calcium by A23187, an ionophore or thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, significantly decreased IL-1beta-induced NO release and NOS expression. These results indicate that glucose-induced inhibition of IL-1beta-stimulated NO release and NOS expression may be mediated by PKC activation and increased intracellular calcium.

Evidence of protein kinase C translocation by ischemic preconditioning in global ischemia model.

We tested recent evidence that ischemic preconditioning (PC) involves in translocation of protein kinase C (PKC) from the cytosol to myocyte membrane. Isolated Langendorff-perfused rabbit hearts (n=96) were subjected to 60 or 45 min of ischemia (I) and 120 min of reperfusion (R) with or without PC (4 cycles of 5 min I and 5 min R; or single dose of 5 min I and 10 min R), respectively. Left ventricular function and infarct size (IS) were measured; myocardial cytosolic and membrane PKC activity were determined by 32P-gamma-ATP incorporation into PKC-specific peptide. PC enhanced improvement of functional recovery and reduced IS (26.9+/-1.4% versus 15.3+/-1.9%, p<0.01, in 60 min of I; 18.3+/-2.6% versus 8.6+/-2.5%, p<0.05, in 45 min of I); cytosolic PKC activity decreased 74% of total activity (p<0.05) both in 60 and 45 min of I; membrane PKC activity increased (1.7-fold of baseline, p<0.01, in 60 min of I; 1.8-fold, p<0.01, in 45 min of I; 1.5-fold, p<0.05, in 60 of min I and 120 min of R). From these results, it is concluded that translocation of PKC from the cytosol to myocyte membranes is an important mechanism responsible for PC effect.

Bradykinin-stimulated cPLA2phosphorylation is protein kinase C dependent in rabbit CCD cells

We have used an established cell line of rabbit cortical collecting duct (RCCD) epithelial cells representing a mixed population of principal and intercalated cell types to determine which phospholipase A2 (PLA2) enzyme therein is responsible for bradykinin (BK)-stimulated arachidonic acid (AA) release and how its activation is regulated. BK-stimulated AA release was reduced 92% by arachidonyl trifluoromethyl ketone, an inhibitor of cytosolic PLA2 (cPLA2). Examination of PLA2 activity in vitro demonstrated that BK stimulation resulted in a greater than twofold increase in PLA2 activity and that this activity was dithiothreitol insensitive and was inhibited by an antibody directed against cPLA2. To determine a possible role for protein kinase C (PKC) in the BK-mediated activation of cPLA2, we used the PKC-specific inhibitor Ro31-8220 and examined its effects on AA release, cPLA2 activity, and phosphorylation. Ro31-8220 reduced BK-stimulated AA release and cPLA2 activity by 51 and 58%, respectively. cPLA2 activity stimulated by phorbol ester [phorbol 12-myristate 13-acetate (PMA)] displayed a similar degree of activation and was associated with an increase in serine phosphorylation identical to that caused by BK. The phosphorylation-induced activation of this enzyme was confirmed by the phosphatase-mediated reversal of both BK- and PMA-stimulated cPLA2 activity. In addition, we have also found that PMA stimulation did not cause a synergistic potentiation of BK-stimulated AA release as did calcium ionophore. This occurred despite membrane PKC activity increasing 93% in response to PMA vs. 42% in response to BK. These data, taken together, indicate that cPLA2 is the enzyme responsible for BK-mediated AA release, and, moreover, they indicate that PKC is involved in the onset responses of cPLA2 to BK.

Studies on the inhibitory effects of quercetin on the growth of HL-60 leukemia cells

Quercetin, a naturally occurring flavonoid, has been shown to exert multiple pharmacological effects and to be an anticancer agent or a supplementary anticancer agent. In this report, the human HL-60 promyelocytic leukemia cell line was used to study the effects of quercetin on the growth, cell cycle, activities of cytosolic and membrane protein kinase C (PKC) and tyrosine protein kinase (TPK), and phosphoinositide production of the tumor cells. The results showed that quercetin inhibited the growth of HL-60 cells in a concentration-dependent manner, with an ic 50 value of about 7.7 μM after 96 hr of treatment; when the concentration of quercetin was 10 μM, the percent inhibition on the growth of HL-60 cells was 17.1, 27.3, 40.1, and 52.7% after 24, 48, 72, and 96 hr of treatment, respectively. Flow cytometric analyses showed that quercetin caused an increase in cells in the G 2 M phase and a decrease in cells in the G 0 G 1 phase of the cell cycle in a concentration-dependent manner; these effects were reversed when quercetin was removed from the culture medium. Quercetin strongly inhibited the activities of cytosolic PKC and membrane TPK from HL-60 cells in vitro, with ic 50 values of about 30.9 and 20.1 μM, respectively, but did not affect membrane PKC or cytosolic TPK activity from HL-60 cells in vitro. Quercetin markedly inhibited in a concentration-dependent manner the production of phosphoinositides in intact HL-60 cells. The results provide evidence that the inhibitory effect of quercetin on the growth of HL-60 cells may be related to its inhibitory effects on PKC and/or TPK in vitro and/or on the production of phosphoinositides.

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.

Down-regulation of protein kinase C by parathyroid hormone and mezerein differentially modulates cAMP production and phosphate transport in opossum kidney cells.

We examined the effects of prolonged exposure to parathyroid hormone (PTH) and the protein kinase C (PKC) activator mezerein (MEZ) on cyclic adenosine monophosphate (cAMP) production, PKC activity, and Na(+)-dependent phosphate (Na/Pi) transport in an opossum kidney cell line (OK/E). A 5 minute exposure to PTH stimulated, while a 6 h incubation reduced, cAMP production, Na/Pi transport was maximally inhibited under desensitizing conditions and was not affected by reintroduction of the hormone. MEZ pretreatment (6 h) enhanced PTH-, cholera toxin (CTX)-, and forskolin (FSK)-stimulated cAMP production, suggesting enhanced Gs alpha coupling and increased adenylyl cyclase activity. However, PKA- and PKC-dependent regulation of Na/Pi were blocked in MEZ-treated cells. The PTH-induced decrease in cAMP production was associated with a reduction in membrane-associated PKC activity while MEZ-induced increases in cAMP production were accompanied by decreases in membrane and cytosolic PKC activity. Enhanced cAMP production was not accompanied by significant changes in PTH/PTH related peptide (PTHrP) receptor affinity or number, nor was the loss of Na/Pi transport regulation associated with changes in PKA activity. The results indicate that down-regulation of PKC by PTH or MEZ differentially modulates cAMP production and regulation of Na/Pi transport. The distinct effects of PTH and MEZ on PKC activity suggest that agonist-specific activation and/or down-regulation of PKC isozyme(s) may be involved in the observed changes in cAMP production and Na/Pi transport.

Activation of protein kinase C by intracellular free calcium in the motoneuron cell line NSC-19

The relationship between intracellular free calcium ([Ca 2+] i) and the activation of protein kinase C (PKC) and Ca 2+/calmodulin-dependent protein kinase II (CaMKII) was investigated in the NSC-19 motoneuron cell line. Increased extracellular calcium ([Ca 2+] 0) up to 10 mM resulted in sustained elevations of [Ca 2+] i. Control cell cultures (1.3 mM [Ca 2+] o, [Ca 2+] i = 83 ± 17 nM) contained Ca 2+- and PS/DO lipid-dependent PKC activity predominantly in the cytosol. However, elevation of [Ca 2+] o up to 5 mM ([Ca 2+] i = 232 ± 24 [ rmnM]) resulted in almost complete loss of cytosolic PKC activity. Cells incubated in 10 mM [Ca 2+] o([Ca 2+] i = 365 ± 13 nM) showed increase levels of both cytosolic and membrane PKC activity compared to control. These alterations in PKC activity appeared to be translocation-independent, since PKC protein levels were unchanged as demonstrated by Western blotting analysis. When cells were exposed to 25 or 50 mM [Ca 2+] o, [Ca 2+] i rose transiently to over 600 and 900 nM, respectively, and then returned to near basal values. Under these conditions, total PKC activity decreased, and increased amounts of the catalytic fragment of PKC, protein kinase M, were generated. Extracts from cells exposed to [Ca 2+] o between 1.3 and 25 mM did not differ significantly in the levels of measurable CaMKII activity 10 min following the change in [Ca 2+] o.

Protein kinase C and insulin regulation of red blood cell Na+/H+ exchange.

Insulin activation of red blood cell (RBC) Na+/H+ (NHE) and Na+/Li+ (NLiE) exchanges is mimicked by okadaic acid, thus suggesting that it may change the state of phosphorylation of serine/threonine NHE residues. To investigate the role of the serine/threonine protein kinase C (PKC) in insulin regulation, we evaluated the effect of phorbol 12-myristate 13-acetate (PMA; 1 microM) and insulin on PKC activity, membrane protein phosphorylation, and the activation kinetics of both exchangers. Our studies revealed that PMA decreased cytosolic PKC activity (4.1 +/- 0.6 to 2.3 +/- 0.5 pmol x mg protein(-1) x min(-1), n = 9, P < 0.001), increased membrane PKC activity (42.3 +/- 5 to 132 +/- 12 pmol x mg protein(-1) x min(-1), n = 11, P < 0.001), and enhanced serine phosphorylation of bands 3, 4.1, and 4.9 membrane proteins. PMA markedly reduced the Michaelis constant (Km) for intracellular H+ (415 +/- 48 to 227 +/- 38 nM, n = 11, P < 0.01) but had no effect on the maximal transport rate (Vmax) of NHE and the Km for Na+ of NLiE. NHE activation and PKC activity were affected differently by insulin (100 microU/ml) and PMA. Insulin increased the Vmax of NHE and the Km for Na+ of NLiE but had no effect on the Km for intracellular H+ and membrane PKC activity. These findings lead us to conclude that in the human RBC, NHE is modulated by PKC and insulin through different biochemical mechanisms.