Increase In PKC Activity Research Articles

Synergistic effects of diacylglycerols and fatty acids on membrane structure and protein kinase C activity.

The synergistic effects of diacylglycerol (DAG) and fatty acid (FA) in activating protein kinase C have been investigated by correlating their individual and combined effects on enzymatic activity and on membrane structure in phosphatidylcholine/phosphatidylserine (4:1) lipid mixtures using a combination of specific enzymatic assays and 31P and 2H NMR. Addition of DAGs and unsaturated FAs to the bilayers synergistically increased the tendency of the lipids to form nonbilayer phases with a concomitant increase in PKC activity until a maximum was achieved. Further increases in the DAG/FA concentration led to the formation of the nonbilayer lipid phases under the conditions of the PKC activity assays and correlated with decreased activity. The nonbilayer lipid phases still supported PKC activity, although with less than 50% efficiency as compared with the bilayer lipids. Long-chain saturated FA increased DAG-induced PKC activity by causing a lateral phase separation of gel (Lbeta) and liquid-crystalline (Lalpha) domains. Due to the preferential partitioning of DAGs into liquid-crystalline domains, the local DAG concentration increased in these domains, leading to an increase in PKC activity. Because a wide range of lipophilic compounds is capable of altering curvature stress, and therefore the tendency for nonbilayer phase formation in cellular membranes, these compounds would be expected to modulate PKC activity and the activities of a number of other membrane-associated enzymes that are sensitive to biophysical properties of lipid membranes.

Effect of Developmental Age and Hyperoxia Exposure on Kinase and Phosphatase Activities in Newborn Rat Lungs

To better understand the biochemical events accompanying lung alveolarization and development, we studied the specific activity of the cAMP-dependent protein kinase (PKA) and the type 2A protein phosphastase (PP2A), and the activity and protein content of the calcium- and lipid-dependent protein kinase (PKC) in cytosolic preparations of lungs. Lungs were obtained from rat pups on day 2 of life and on days 7, 14, and 27 from pups exposed to hyperoxia (>95% 02, days 4–14; 65% 02 days 15–27) or normoxia from day 4 onwards. There were no significant changes in PKA specific activity with developmental age or hyperoxic exposure. PKC specific activity increased significantly (P < .05) in normoxic animals from day 2 {64 + 13.5 pmol phosphate released/min/mg protein) to day 14 (105+ 9). The increase was sustained to day 27. There was no effect on PKC activity due to hyperoxia alone (ANOVA). This increase in PKC activity was accompanied by an increase in the mass of the δ, ε and ς, isoforms of PKC in normoxic pups. The y isoform of PKC was undetectable in all samples whereas the α and β isoforms were detectable but showed no changes with developmental age. PP2A specific activity increased significantly (P < .05) from 13.3 ± 0.5 nmol phosphate released/mini mg protein on day 2 to 17.7 ± 0.9 on day 7 in normoxic pups, then returned to day 2 level at advanced developmental age. Hyperoxia exposure prevented the increase in enzyme activity observed on day 7 in normoxic animals. These data suggest that protein phosphorylation May-June be one mechanism by which alveolarization is regulated in developing lungs.

Increased PKC activity in cultured human keratinocytes and fibroblasts after treatment with 1α,25‐dihydroxyvitamin D3

1 alpha,25-Dihydroxyvitamin D3 (10(-12) M to 10(-8) M) caused a dose dependent increase in PKC activity in the solubilized membrane fractions of cultured human keratinocytes and in the cytosolic fractions of cultured human fibroblasts. Maximum activity was induced by 1 alpha,25-dihydroxyvitamin D3 at 24 h. Sphingosine, which is believed to inhibit PKC mediated biological responses, blunted 1 alpha,25(OH)2D3's inducement of PKC activity in both keratinocytes and fibroblasts. Identical hormone treatment of vitamin D receptor deficient fibroblasts did not increase PKC activity. Treatment of keratinocytes and fibroblasts with 1 beta,25-dihydroxyvitamin D3, which is believed to be ineffective in inducing genomic responses, did not induce PKC activity.

Differential regulation of mRNAs encoding protein kinase C isoenzymes in activated human B cells

Stimulation of human B cells via HLA class II antigens leads to an increase of PKC activity as a consequence of a transcriptional upregulation of the PKC. Extending previous data, other known B-cell activators, which include anti-IgM, SAC, and TSST1, are shown here to increase the cytosolic PKC activity significantly. Human B cells express significant mRNA levels of the PKCα, β, δ, ϵ, and ζ species while the γ species is consistently absent. The levels of PKCα and ϵ mRNA are increased by exposure to a nonmitogenic anti-IgM antibody in a lymphoblastoid B-cell line while PKCβ and δ mRNA are instead downregulated by this agent. An anti-HLA class II antibody (D1.12) induced an increase of PKCα, β, and δ mRNA. A time study of PKC mRNA levels in anti-IgM-treated cells showed that the accumulation of the PKCα mRNA precedes the increase of PKC enzymatic activity. Moreover, PKCβ mRNA decreased following treatment with SAC while, on the contrary, it increased following TPA, anti-HLA class II (1.35) mAb, or mitogenic anti-IgM treatment. Our results underline the complexity of signal transduction via the PKC pathway by revealing that the PKC isoforms are differentially regulated and are in keeping with the idea that they may have distinct physiologic roles in human B cells

PKC Activity in Rat C 6 Glioma Cells: Changes Associated with Cell Cycle and Simvastatin Treatment

The parallel effects of simvastatin on cell cycle and PKC activity in rat C 6 glioma cells were investigated. Simvastatin, 2.5 μM, for 24 h resulted in cell growth arrest in early G 1 phase of the cell cycle and in a significant increase of total PKC activity (283 ± 42 vs 470 ± 61 pmoles/min/mg protein p = 0.002 for control cells and simvastatin-treated cells, respectively). The effect of simvastatin was fully prevented by mevalonate. A time dependent increase of PKC activity was observed in control exponentially free-growing C 6 cells approaching confluency: a highly significant negative correlation (r = −0.91 p<0.0001) between PKC activity and growth rate was calculated. PKC activity was high in cells arrested in G 0 by serum starvation (0.4%). Following addition of complete medium (17.5% serum) the PKC activity progressively decreased and reached a minimum when cells traversed the G 2/M phase, as determined by DNA analysis distribution. PKC activity dropped 30% in simvastatin-arrested early G 1 cells; 44% in hydroxyurea-arrested cells at the G 1/S boundary and 73% in Colcemid mitosis-blocked cells. The results show that C 6 glioma cell PKC activity is maximal in a G 0 quiescent state and varies at different points of the cell cycle.

Involvement of protein kinase C and protein tyrosine kinase pathways in tumor necrosis factor-α-induced clustering of ovarian theca-interstitial cells

Tumor necrosis factor-alpha (TNF) induces clustering of theca-interstitial cells (TIC) isolated from immature, hypophysectomized rats, while inhibiting luteinizing hormone (LH)-stimulated androstenedione in vitro. Stimulators of PKC, l-oleoyl-2-acetyl- sn-glycerol (OAG, 50 and 100 μM) and phorbol-12-myristate-13-acetate (PMA, 50 nM), caused TIC clustering by 6 days in vitro. Clustering induced by these compounds resembled that induced by TNF. The protein kinase inhibitor, staurosporine at 1 and 10 nM, impaired TNF-induced TIC clustering for 6 days, as did the protein kinase inhibitor, l-(5-isoquinolinesulfonyl)-2-methylpiperizine dihydrochloride (H-7); conversely, the protein kinase inhibitor, chelerythrine chloride (0.1, 1.0 or 10 μM), did not attenuate TNF-directed clustering. The protein kinase inhibitors did not reverse the suppression of LH-stimulated androstenedione by TNF. Inhibitors of the EGF receptor PTK, A23 (10, 50, or 100 μM) and A46 (0.1, 1.0, 10, or 50 μM), impaired TNF-induced TIC clustering, while TNF suppression of LH-directed androstenedione was unaffected. EGF-induced TIC clustering was also impaired by A46, while A23 was less effective. Both A23 and A46 blocked EGF attenuation of LH-directed androstenedione after 4 days. When challenged with TNF (1 ng/ml) or PMA (50 nM), PKC activity increased in TIC. A23 (50 μM) and A46 (10 μM) each alone blocked the TNF-associated increase in PKC activity; however, PKC activity attributable to PMA was unaffected by A46. Together, these results suggest that TNF-induced TIC clustering involves activation of PTK which directs subsequent increases in PKC activity; however, mechanisms by which TNF inhibits LH-stimulated steroidogenesis remains elusive.

Differential changes of adrenoceptor- and muscarinic receptor-linked prostacyclin synthesis by the aorta and urinary bladder of the diabetic rat.

1. The effect of experimental diabetes mellitus (DM; hyperglycaemic, non-ketototic; 2 months duration) in the rat on receptor-linked prostacyclin (PGI2) synthesis (measured as 6-oxo-PGF1 alpha by radioimmunoassay) was studied in the aorta and urinary bladder using adrenaline, angiotensin II (AII) and acetylcholine (ACh). Signal transduction systems were studied via stimulation of PGI2 synthesis with phorbol ester dibutyrate (PDBU; a protein kinase C activator [PKC]), Ca2+ ionophore A23187 (A23187) and thapsigargin (both elevate intracellular Ca2+, activating phospholipase A2 [PLA2]) and arachidonate (AA; substrate for PGI2 synthesis). 2. In response to adrenaline, AII and phorbol ester, aortic PGI2 release was markedly reduced (all > 75%) in diabetic rats compared to controls. EC50s of the dose-response curves for adrenaline, AII and PDBU were also markedly increased in aortae from DM rats compared to controls. Although there was decreased output of PGI2 in response to A23187 by aortae from diabetic rats compared to controls, there was no difference in the EC50s (mean +/- s.e. mean: diabetic, 2.7 +/- 0.2 x 10(-6) M; controls 2 +/- 0.18 x 10(-6) M). There were no differences in PGI2 release (or in the EC50s) in response to thapsigargin or AA between aortae from diabetic and control rats. 3. In the urinary bladder, there was a marked increase in PGI2 output in response to ACh and a marked decrease in EC50s for the ACh-PGI2 dose-response curves in diabetic rats (EC50 = 5.8 +/- 0.32 x 10(-7) M) compared to controls (EC50 = 2.2 +/- 0.15 x 10(-6) M). Although there was an increase in PGI2 output in the urinary bladders from diabetic rats in response to A23187, there were no differences in the EC50s (control, 1.8 +/- 0.2 x 10-6 M; diabetic, 1.1 +/- 0.15 X 10-6 M). In the urinary bladders, there were no differences in PGI2 output (or the EC50s) in response to PDBU, thapsigargin or AA between diabetic or control rats.4. These data indicate that: (i) reduced PGI2 synthesis coupled to adrenoceptors and AII receptors in the aortae of diabetic rats may be due to diminished PKC activity and not to changes in receptor density and/or affinity, Ca2+ stores, PLA2, cyclo-oxygenase or PGI2 synthase; (ii) the diametrically opposite effect of DM on ACh-stimulated PGI2 synthesis is not due to an increase in PKC activity, but possibly to an increase in muscarine receptor number and/or affinity; (iii) changes in receptor-linked PGI2 synthesis are not ubiquitous in experimental DM and may be organ-specific.

Immunocytochemical expression and localization of protein kinase C in bovine aortic endothelial cells

Total PKC activity in BAEC incubated for 24 hrs in either 10% serum (FBS) or serum-deprived media (SDM) was similar. However, most of the activity (69%) in the FBS group was detected in the particulate fraction, while it was mainly in the cystosolic fraction (66%) in the SDM group. By confocal microscopy, there was diffuse cytoplasmic localization of the antibodies to the α and β PKC isoforms. γ PKC was not detected. Treatment of FBS or SDM cells with a phorbol ester resulted in an increase in PKC activity with translocation to the particulate fraction. PKC α immunofluorescence redistributed to the perinuclear region whereas PKC β staining remained mostly cytosolic. Calphostin C, a PKC inhibitor, prevented the phorbol ester-induced increase in PKC activity and translocation.

Protein kinase C and cyclic AMP regulate reversible exposure of binding sites for fibrinogen on the glycoprotein IIB-IIIA complex of human platelets

Platelet aggregation is mediated via binding of fibrinogen to sites on the membrane glycoprotein IIB-IIIA complex which become exposed when the cells are stimulated. We report here evidence of a dynamic and reversible exposure of binding sites for fibrinogen. In the absence of fibrinogen, exposed sites (B*) gradually lose their capacity to bind fibrinogen and close (Bo). On stimulation with platelet-activating factor (PAF, 500 nM) at 22 degrees C, closing of B* is enhanced by agents that raise cyclic AMP levels (10 ng of prostaglandin I2/ml; 5 mM-theophylline), inhibit protein kinase C (PKC; 25 microM-sphingosine; 1 microM-staurosporine), or disrupt the energy supply (30 mM-2-deoxy-D-glucose + 1 mM-CN-), or by raising the temperature to 37 degrees C. Conversely, activation of PKC 1 microM-1,2-dioctanoyl-sn-glycerol; 55 nM-phorbol 12-myristate 13-acetate) and an increase in intracellular [Ca2+] (100 nM-ionomycin + extracellular Ca2+) oppose the disappearance of B*. Phosphorylation of the 47 kDa protein illustrates the tight coupling between PKC and B* under all conditions tested, except when the cyclic AMP level is raised, and B* is converted to Bo without affecting PKC activity. Although the increase in PKC activity is much smaller with ADP or even absent upon stimulation with adrenaline, the control of B* is equally sensitive to modulation of cyclic AMP and PKC activity. We conclude that PAF, ADP and adrenaline regulate exposure of fibrinogen binding sites through a common mechanism consisting of two independent pathways, one dominated by PKC and the other by an as yet unidentified cyclic AMP-sensitive step.