Kinetics of activation of a PKC-regulated epithelial calcium channel.

The kinetics of calcium entry through regulated calcium channels in cultured renal proximal tubule cells was studied with Fura-2 fluorescence ratio imaging in single cells. The calcium entry was activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG) and phorbol-12-myristat-13-acetate (PMA), similar to that observed for activation by osmo-mechanical stress. OAG (2.5 microM) or PMA (0.5 microM) activated calcium entry is characterized by a significant latency between agonist application and the response, whereas the effect of osmo-mechanical stress was immediate. This pre-response latency was 260 +/- 70s with OAG stimulation and 79.2 +/- 17.3s with PMA stimulation. Once a cell responds, the intracellular calcium level reaches a peak value within seconds. The cell response to agonist is independent of the response of neighboring cells. The response kinetics resembles those of the calcium sparks in excitable cells, except the response is much slower. In all cases, the response appears to be an all-or-none event, that is characteristics of an elementary binary switch. It is suggested that the binary response and the lack of coordinated response of calcium entry in single cells results from limited availability of the calcium channels and/or PKC that activates the channel. The experimental data could be fit to a single binary response mathematical model assuming each response reflected an elementary event of a single channel opening or a co-ordinated opening of a cluster of several channels.

Protein kinase C-stimulated formation of ethanolamine from phosphatidylethanolamine involves a protein phosphorylation mechanism: negative regulation by p21 Ras protein.

Mammalian cells express a phospholipase D (PLD)-like enzyme which forms ethanolamine from phosphatidylethanolamine (PtdEtn) by a protein kinase C-alpha (PKC-alpha)-activated, presently unknown, mechanism. Now we report that addition of a PKC-alpha-enriched purified PKC preparation or recombinant PKC-alpha to a plasma membrane-enriched membrane fraction, isolated from leukemic HL60 cells, greatly ( approximately 6.5-fold stimulation) enhanced PtdEtn hydrolysis if the PKC activator phorbol 12-myristate 13-acetate (PMA) and ATP were both present; this was accompanied by PKC-mediated phosphorylation of several membrane proteins. The combined effects of PKC-alpha, ATP, and PMA on [(14)C]PtdEtn hydrolysis were inhibited by GF 109203X (10 microM), an inhibitor of catalytic activity of PKC. In this membrane fraction, PMA alone also had a smaller ( approximately 3.5-fold) stimulatory effect on PtdEtn hydrolysis which was not affected by adding ATP or GF 109203X to the membranes. These results suggest that PMA can stimulate PtdEtn hydrolysis via a PKC-catalyzed phosphorylation mechanism as well as by a phosphorylation-independent process. Transformation of NIH 3T3 fibroblasts by H-ras reduced the effect of PMA on PtdEtn hydrolysis. Furthermore, in NIH 3T3 fibroblasts, scrape-loaded Y13-259 anti Ras antibody enhanced PMA-stimulated hydrolysis of PtdEtn. These results suggest that activation of the PtdEtn-hydrolyzing PLD enzyme by PKC-alpha is inhibited by p21 Ras.

Diverse effects of neutrophil integrin occupation on respiratory burst activation.

Integrin occupation can alter the function of neutrophils (PMN), but the mechanism(s) involved is still unclear. This study demonstrated that the occupation of PMN integrins (especially those of the beta(3) subfamily) strongly enhances TNF stimulation of the respiratory burst but down-regulates that induced by PMA, fMLP, Con A, and serum treated zymosan. Treatment of PMN with genistein, staurosporine, and wortmannin, inhibitors of tyrosine kinases, protein kinase C, and phosphotidylinostol 3-kinase (PI 3-kinase) respectively, completely blocked the TNF-stimulated respiratory burst in PMN. Genistein and wortmannin enhanced the PMA-stimulated respiratory burst but only in cells adherent to RGD peptide. These findings suggest that PMN integrins (beta(3) subfamily) can generate signals that regulate the PMN agonist responses, probably through the activities of tyrosine kinases and PI 3-kinase.