Johanna Ta Meij

Modification of Fatty Acid Composition of the Phospholipids of Cultured Rat Ventricular Myocytes and the Rate of Phosphatidylinositol=4,5=Bisphosphate Hydrolysis

Cultured neonatal cardiac myocytes have been utilized as a model for the study of the role of fatty acids in the alpha 1-adrenoceptor mediated phosphatidylinositol turnover. Experiments were started 24 h after seeding, when there was a confluent monolayer of beating cardiomyocytes. The cells were incubated for 3-4 days in sera containing culture medium with (1) no additives or (2) a mixture of 107 microM 18:0 and 18:1n-9, or (3) only 214 microM 18:2n-6 or (4) 214 microM 20:5n-3. No differences in the cellular content of the various phospholipid classes among the different groups of fatty acid treated cells were found. The predicted elevations of 18:1n-9, 18:2n-6 and 20:5n-3 associated with a partial depletion of 20:4n-6 were confirmed in all phospholipid classes, except for sphingomyelin. The mol% of 18:0, 18:2n-6, 20:4n-6 and 20:5n-3 in the phosphatidylinositol fraction were respectively 39, 4, 30 and 0.6 for the control treated cells, 34, 3, 15 and 0 for 18:0/18:1n-9 treated cells, 40, 17, 24 and 0.2 for the 18:2n-6 treated cells and 41, 3, 13 and 21 for the 20:5n-3 treated cells. Apart from the observed reductions in the basal rates, the phenylephrine (30 microM) stimulated production of inositolphosphates was reduced by 51% and 71%, respectively in the 18:2n-6 and 20:5n-3 treated cardiomyocytes. The basal rate of inositolphosphate formation was 37% increased in the 18:0/18:1n-9 treated cells. The [3H]-inositol incorporation into phosphatidylinositol 4,5-bisphosphate was only slightly reduced by 18:2n-6 and 20:5n-3 treatments (respectively 12 and 28% compared to control treated cells). Prolonged (30 min) alpha 1-adrenergic stimulation did not affect the contents and fatty acid profiles of any class of phospholipid, not even phosphatidylinositol. In conclusion, variations in the polyunsaturated fatty acid composition of membrane phospholipids do affect the basal and the alpha 1-adrenoceptor stimulated rate of phosphatidylinositol-4,5-bisphosphate hydrolysis. The reducing effects of 18:2n-6 and 20:5n-3 treatment on the rate of inositolphosphate production may be partially ascribed to altered levels of phosphatidyl-inositol 4,5-bisphosphate.

Phorbolester inhibits α1-adrenoceptor mediated phosphoinositide breakdown in cardiomyocytes

Abstract The regulation of and the intracellular events following α 1 -adrenergic receptor stimulation of myocardium are not completely understood. The α 1 -adrenergic stimulation of phosphoinositide breakdown was examined in a culture of neonatal rat ventricular myocytes and the influence of a protein kinase C activator, phorbol 12-myristate 13-acetate, on this process was studied. Inositolphosphate accumulation was stimulated by phenylephrine (EC 50 5 μ m ) in the presence of 10 m m LiCl. The increase was antagonized by prazosin (10 −6 m ) but not by propranolol (10 −6 m ). The rate of inositolphosphate accumulation after prolonged α 1 -adrenoceptor stimulation decreased without clear evidence of depletion of the membrane phosphatidylinositolbisphosphate pool. Phorbol ester treatment (IC 50 10 −8 m ) led to a dose-dependent inactivation of α 1 -adrenoceptor stimulated phosphoinositide breakdown. These findings provide evidence that protein kinase C plays a role in the regulation of α 1 -adrenoceptor sensitivity.

Alpha-1-adrenergic stimulation of phosphoinositide breakdown in cultured neonatal rat ventricular myocytes

SummaryThe regulation of and the intracellular events following a,-adrenergic receptor stimulation in the myocardium still remain to be disclosed. The effect of α1- adrenergic stimulation on phosphoinositide breakdown was studied in cultured neonatal rat ventricular myocytes. Phenylephrine (30 μM) stimulated inositolphosphates formation, but only in the presence of 10 mM LiCl this could be measured. The increase was antagonized by prazosin (1 μM) but not by propranolol (1 μM). The variability in proportional distribution of the three inositolphosphates is discussed.

Occurrence and functions of the phosphatidylinositol cycle in the myocardium

In the last decade a great deal of attention was awarded to a signal transduction pathway which is utilized primarily by ‘Ca2+ mobilizing’ signal molecules and which involves the hydrolysis of a quantitatively minor phospholipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by a PtdIns-specific phospholipase C (PLC). The evidence for the existence of receptor-mediated GTP binding protein-coupled PLC in myocardium and its possible functions are briefly summarized. The minireview is concentrated on the following aspects: 1) cellular localization and synthesis of polyphospho-PtdIns from PtdIns, 2) desensitization of the α1-adrenergic agonist and endothelin-1 mediated PtdIns responses, 3) oscillatory Ca2+ transients initiated by Ptdlns(4,5)P2 hydrolysis, 4) polyunsaturated fatty acids as constituents of polyphospho-PtdIns and of the protein kinase C activator 1,2-diacylglycerol (DAG), 5) source other than Ptdlns(4,5)P2 contributing to the stimulated DAG, 6) role of the PtdIns pathway in cardiomyocyte growth and gene expression during the hypertrophic response. (Mol Cell Biochem116: 59–67, 1992)

Phorbol ester and the actions of phosphatidylinositol 4,5-bisphosphate specific phospholipase C and protein kinase C in microsomes prepared from cultured cardiomyocytes

Microsomes were prepared from cultured neonatal rat cardiomyocytes. Incubation of microsomes in buffer containing 5µM CaCl2, 5 mM cholate and 100 nM [3H-]Phosphatidylinosito14,5-bisphosphate (PtdIns(4,5) P2) resulted in the formation of [3H-]InsP3. GTP-gamma-S (125 µM) stimulated the production of [3H-]InsP3. Microsomes prepared from phorbol ester-treated (100 nM phorbol 12-myristate 13-acetate, PMA) cardiomyocytes showed decreased activities of basal as well as GTP-gamma-S-stimulated [3H-]Ptdlns(4,5)P2 hydrolysis. In the microsomes a 15 kD protein was demonstrated to be the major substrate phosphorylated by intrinsic protein kinase C, which was activated by 0.5 mM Ca2+. Addition of phorbol ester (100 nM PMA) enhanced the 32P-incorporation into the 15 kD protein. Protein kinase C, purified from rat brain, in the presence of Ca2+, diglyceride, and phosphatidylserine did not change the phosphorylation pattern any further. In conclusion, it was shown that phorbol ester pretreatment of neonatal rat cardiomyocytes reduces microsomel GTP-gamma-S-stimulated Ptdlns(4,5)P2-specific phospholipase C activity, as estimated with exogenous substrate, and that in cardiomyocyte microsomes phorbol ester activates protein kinase C-induced 15 kD protein phosphorylation. The results indicate that phorbol ester may down-regulate α-adrenoceptor mediated Ptdlns(4,5)P2 hydrolysis by activation of protein kinase C-induced 15 kD protein phosphorylation.

Discrete interactions between phosphatidylethanolamine-N-methylation and phosphatidylinositolbisphosphate hydrolysis in rat myocardium

Both phosphatidylethanolamine(PE)-N-methylation and phosphatidyl-inositol bisphosphate(PI-bisphosphate) breakdown potentially modify the microdomains in the sarcolemmal lipid bilayer. In this study the possibility of a mutual interaction between the enzymes responsible for these phospholipid reactions is examined. In sarcolemma purified from rat heart, prior hydrolysis of PI lipids by exogenous specific phospholipase C inhibited (to 75, 59 and 78% of control for sites 1, 11 and 11, respectively) the PE-N-methyltransferase system. In cultured rat cardiomyocytes the addition of L-methionine, a precursor for the methyl donor S-adenosylmethionine, stimulated PE-N-methylation in a concentration (0.2–300 µM)-dependent manner. Methionine (50 µM) decreased the basal rate of PI-bisphosphate hydrolysis (to 72% of control), but had no effect on the phenylephrine-stimulated PI-bisphosphate hydrolysis. Maximal activation of the PI-bisphosphate breakdown by 30 µM phenylephrine did not affect the rate of PE-N-methylation in the presence of exogenous methionine (50 µM). These findings support the existence of interactions, although discrete, between the enzymes involved in the PE-N-methylation and PI turnover.

Depression of Sarcolemmal Phospholipase C Activity in Congestive Heart Failure

The bioactivc phospholipids of the cardiac cell membrane (sarcolemma) and their signaling pathways are emerging as important mediators of the myocardial response to external stimuli, including catecholamines [1]. It is known that the activity of the sympathetic nervous system is increased in congestive heart failure; this results in elevated levels of plasma catecholamines, which downregulate the β-adrenoceptors in failing hearts, leading to subsensitivity of the β-agonist-mediated biochemical and mechanical responses [2]. The α1-adrenoceptors, which are associated with the positive inotropic effect of catecholamines via activation of the membranal phosphoinositide-phospholipase C (PLC) [1], were found to remain unchanged [3] or to increase [4,5] in heart failure. In view of the downregulation of the β-adrenoceptors, the α1 type reflects a greater proportion of the total adrenoceptor population in the failing ventricle [3–5]. Thus α1-adrenoceptors can be seen to play a dominant role in eliciting the positive inotropic action of catecholamines in failing heart [6].

Alterations in polyunsaturated fatty acid composition of cardiac membrane phospholipids and alpha-1-adrenoceptor mediated phosphatidylinositol turnover

STUDY OBJECTIVE - The aim of the study was to investigate the steps at which polyunsaturated fatty acids are involved in alpha 1 adrenoceptor mediated phosphatidylinositol turnover. DESIGN - Phosphatidylinositol turnover rates were investigated after preincubating neonatal rat ventricular myocytes with culture media enriched with linoleic acid (18:2n-6) or eicosapentaenoic acid (20:5n-3) to change the polyunsaturated fatty acid composition of their membrane phospholipids. EXPERIMENTAL MATERIAL - Cardiomyocytes were isolated from ventricles of 2-4 d old Wistar rats by trypsinization and were then cultured. Experiments were started 48 h after seeding, when there was a confluent monolayer of beating cardiomyocytes. MEASUREMENTS and RESULTS - In 18:2n-6 treated cells the 18:2n-6 content in the total phospholipid fraction rose from 45 to 68 nmol.mg-1 protein; in 20:5n-6 treated cells the 20:5n-3 content rose from 1.5 to 12.5 nmol.mg-1 protein, and the docosapentaenoic acid (22:5n-3) content rose from 5.1 to 14.7 nmol.mg-1 protein. The major n-3 fatty acid, 22:6n-3 (11.4 nmol.mg-1 protein), did not change after 20:5n-3 treatment. Although the phosphatidylinositol fraction showed changes paralleling those in the total phospholipids, none were significant. In this fraction the major n-3 fatty acid appeared to be 22:5n-3 (0.4 nmol.mg-1 protein). The fatty acid treated cells were prelabelled with [3H]-inositol to estimate the rate of phosphatidylinositol-4,5-bisphosphate turnover. There were no differences in the rate of [3H]-inositolphosphate formation between control, 18:2n-6 treated cells, and 20:5n-3 treated cells. Prolonged alpha 1 adrenergic stimulation of control and treated cells did not change the polyunsaturated fatty acid composition of the total phospholipid and phosphatidylinositol fractions. CONCLUSIONS - The alpha 1 adrenoceptor mediated phosphatidylinositol turnover rate is not affected by changes in polyunsaturated fatty acid composition of membrane phospholipids, neither does prolonged alpha 1 adrenergic stimulation lead to significant depletion of any specific or total polyunsaturated fatty acids in the phosphatidylinositol lipids.