Stella Clark

The role of protein kinase C in arachidonic acid release and prostaglandin E production from CHO cells transfected with EGF receptors

Arachidonic acid release and prostaglandin production are stimulated by both phorbol esters and growth factors in various cell types. Whereas phorbol esters activate and transmit a signal via protein kinase C, this pathway is not necessarily involved in growth factor signal transduction. We investigated the involvement of protein kinase C in the pathway of arachidonic acid metabolism by CHO cells transfected with full-length EGF receptor (CHOwt). Two isoforms of protein kinase C were identified in CHOwt cells, alpha and zeta. On downregulation, the parallel loss of phorbol ester-stimulated arachidonic acid release and the alpha-isoform suggests a possible involvement of this isoform in phospholipase A2 activation in these cells. In addition, we propose that the zeta-isoform may be separately involved in prostaglandin production as residual phorbol ester-stimulation of PGE production occurs in downregulated cells where PKC zeta is the sole remaining isoform. EGF stimulation of arachidonic acid release, as a measure of phospholipase A2 activation, and subsequent prostaglandin production are unaffected by inhibition of protein kinase C in CHOwt cells. Indeed one such inhibitor, staurosporine, augmented the EGF effect. These results suggest that PKC is not required for EGF activation of phospholipase A2 in these cells.

Glucose-induced phosphorylation and activation of a high molecular weight cytosolic phospholipase A2 in neonatal rat pancreatic islets

Previous studies have shown that stimulus-secretion coupling for the release of insulin from the pancreatic islet is potentiated by phospholipase A2 activity. Several biochemically distinct phospholipase A2 activities have been described in the islet. A recently identified cytosolic high molecular weight phospholipase A2, which requires Ca2+ for association with cellular membranes but not for catalytic activity can be activated in a protein kinase C-dependent manner in other cell-types. We determined its phosphorylation and activation in response to phorbol ester and glucose in cultured islet cells from neonatal rats. Islet cell monolayers were labelled to equilibrium with [32P]orthophosphate. Following stimulation cytosolic phospholipase A2 was immunoprecipitated and, after electrophoretic separation and transfer to nitrocellulose membrane, 32P-labelled protein was detected by autoradiography. Phospholipase A2 activity of islet cell cytosol was determined by hydrolysis of exogenous I-stearyl- 2[14C]arachidonyl phosphatidylcholine substrate. It could be shown that phosphorylation of immunoprecipitated phospholipase A2 was augmented by prolonged glucose exposure (> 1 hr) in a protein kinase C-dependent manner. Phosphorylation occurred concomitant with a glucose-induced increase in total cellular phospholipase A2 activity (177 +/- 3 nmol substrate hydrolysed/mg protein at glucose 5.6 mM vs 267 +/- 32 (SEM, n = 4) at glucose 25 mM, P < 0.05). Both acute protein kinase C (459 +/- 71) and glucose-activated phospholipase A2 activities were reduced in the presence of a specific arachidonic acid analogue inhibitor of cytosolic phospholipase A2 (to 231 +/- 10 and 161 +/- 17, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of insulin receptor level with both insulin action and breakdown of a potential insulin mediator precursor; studies in CHO cell-lines transfected with insulin receptor cDNA

A phosphatidylinositol-glycan (PI-glycan) has been described previously that may serve as the precursor for a mediator of some of insulins actions. The present study further addresses the potential relevance of this compound by correlating its breakdown with other insulin actions in Chinese hamster ovary (CHO) cells which express different levels of insulin receptor. Comparisons were drawn between parent CHO cells expressing 3 x 10(3) receptors/cells and two cell-lines transfected with human insulin receptor cDNA, that expressed 600 (CHO.TH) and 300 (CHO.T) times the parent receptor level. A PI-glycan was isolated from all cells that incorporated [3H]glucosamine, [3H]galactose and [3H]inositol and was rapidly turned over upon insulin stimulation. Maximal turnover by insulin of approx. 20% was achieved in each cell line consistent with the fibroblastic nature of these cells. The effect of increased insulin receptor expression was to increase the sensitivity of the PI-glycan response to insulin. Increasing receptor number from 3 x 10(3) to 0.88 x 10(6) also increased the sensitivity of response of other insulin actions measured in this study, namely activation of pyruvate dehydrogenase (PDH), glucose utilization and transport. Thus turnover of the PI-glycan is linked closely to both metabolic actions of insulin and to cell surface insulin receptor expression further supporting its potential role in insulin action.

Activation of phospholipase D in CHO cells transfected with the human epidermal growth factor (EGF) receptor: differential effects of protein kinase C activation and EGF.

Multiple intracellular signal transduction pathways, including phospholipases A2 and D, can be activated by epidermal growth factor (EGF) in both a protein kinase C (PKC)-dependent and -independent manner. We investigated the activation of phospholipase D (PLD) by a PKC activator, phorbol myristate acetate (PMA) and by EGF in CHO cells transfected with the full-length EGF receptor. In cells labelled with arachidonic acid or linoleic acid, PMA activated a PLD, determined by formation of the transphosphatidylation product phosphatidylethanol in the presence of ethanol. A basal PLD activity was seen in linoleic acid-labelled cells but not in cells labelled with arachidonic acid. This basal activity was augmented by the protein phosphotyrosine phosphatase inhibitor vanadate and reduced by tyrosine kinase inhibition and was contributed to by PKC, as activity could not be elicited following prolonged exposure to phorbol ester, known to down-regulate some PKC isoforms. By contrast, EGF failed to stimulate formation of phosphatidylethanol in cells labelled with either fatty acid species. It is proposed that in the basal condition PKC-dependent PLD activation and protein tyrosine kinase phosphorylation are linked (possibly by a phospholipase C (PLC)-mediated formation of diacylglycerol); EGF which activated a phospholipase A2 (PLA2) but which failed to elicit PLC activation in these cells is without further effect on PLD.

Differential disposition of lysophosphatidylcholine in diabetes compared with raised glucose: implications for prostaglandin production in the diabetic kidney glomerulus in vivo

An early increased formation of renal prostaglandins in diabetes which follows the hydrolysis of cellular phospholipids by cytosolic phospholipase A2 is of considerable importance in determining subsequent cellular function. As the disposition of concomitantly formed lysophosphatidylcholine may also affect cellular function, we investigated the cellular fate of exogenous lysophosphatidylcholine in mesangial cell-enriched glomerular cores and showed that in cells taken from diabetic rats there is an increased net reformation of phosphatidylcholine. Positional distribution of labelled palmitate from sn-1 position palmitate-labelled lysophosphatidylcholine showed distribution to both sn-1 and sn-2 position of the phosphatidylcholine formed with a significantly increased sn-2 position labelling in diabetes. Although both a coenzyme A-dependent acyltransferase activity and a coenzyme A-independent transacylase activity could be shown in these cells, the increased phosphatidylcholine formation in cells taken from diabetic animals was due to an increase in coenzyme A-independent transacylase activity. By contrast, an increase in coenzyme-A independent transacylase activity could not be demonstrated in cultured mesangial cells maintained with prolonged raised glucose concentrations. Cell homogenates possess the ability to transfer fatty acid from lysophosphatidylcholine to lysophosphatidylcholine and lysophosphatidylethanolamine with subsequent formation of phosphatidylcholine and phosphatidylethanolamine, respectively. In preparations from diabetic animals phosphatidylethanolamine formed in this manner was increased in the presence of an inhibitor of cytosolic phospholipase A2, indicating that it may provide a substrate for phospholipase A2 activity; an effect not seen in cultured cells maintained at raised glucose concentrations. It is concluded that one effect of an altered disposition of lysophosphatidylcholine in cells from diabetic animals would be to spare fatty acids released following phospholipase A2 hydrolysis of phospholipid, possibly providing the substrate for prostaglandin production, an effect not seen with raised glucose alone.

Negative cooperativity induced by desoctapeptide insulin unmasked by phospholipase C treatment

Summary Phospholipase C treatment of particulate placental membranes allowed the induction of negatively cooperative interactions by the insulin analogue desoctapeptide insulin, previously thought to be non-cooperative. This induction of negative cooperativity was dose-dependent with 10 μg/ml analogue showing the greatest effect. In untreated placental membranes desoctapeptide insulin induced a postively cooperative effect which was also dose-dependent. 125I-labelled insulin binding to phospholipase C treated membranes was inhibited at lower concentrations of desoctapeptide insulin than was required to achieve the same effect in untreated membranes. These results suggest that the structure of the insulin receptor itself as well as that of the hormone may determine whether negatively cooperative interactions occur under all conditions.