Biomedical subjects

V Panagia

Publications and source records attributed to V Panagia.

At least 55 records · Page 3Linked to original sources

PubMed1994

Oxidative stress modifies the activity of cardiac sarcolemmal phospholipase C.

We have examined the direct effects of oxidant metabolites on cardiac sarcolemmal phosphoinositide phospholipase C which transduces signals from various receptors for the modulation of intracellular Ca2+ levels. The enzyme activity in rat cardiac sarcolemmal membranes that had been preincubated (10 min; 37 degrees C) with xanthine-xanthine oxidase, a superoxide anion generating system, was not significantly affected. The addition to this system of superoxide dismutase, which converts superoxide anion to hydrogen peroxide (H2O2), resulted in a significant decrease of the enzyme activity in comparison with control values. Such decrease was fully prevented by catalase. Preincubation of sarcolemma with hypochlorous acid also gave a significant inhibition of phospholipase C, which was counteracted by the synthetic thiol reducer dithiothreitol. H2O2-pretreatment induced a concentration-dependent inhibition of the enzyme which was prevented by catalase but not by the iron chelator deferoxamine. Dithiothreitol was able to protect against, as well as to recover the enzyme activity from the H2O2 effects. These data suggest that superoxide anions and hydroxyl radicals did not interfere with phospholipase C activity, and that the nonradical oxidants, H2O2 and hypochlorous acid, may have acted through oxidation of thiol (SH) groups. The existence of reactive SH groups associated with the enzyme was confirmed by the inhibitory effects of SH modifiers (p-chloromercuriphenylsulfonic acid, 5'5'-dithio-bis(2-nitrobenzoic acid), N-ethylmaleimide and methyl methanethiosulfonate), which were prevented and in some cases also reversed by dithiothreitol. The biological reducer glutathione (GSH) was not able to recover the H2O2-induced inhibition of phospholipase C, whereas its oxidized form (GSSG) decreased the enzyme activity both in control and H2O2-pretreated membranes. The enzyme was active in a wide range of GSH/GSSG redox states, but H2O2 pretreatment narrowed this range. The results showed that oxidative stress changed the redox state of sarcolemmal phospholipase C, and this deactivated the enzyme. The oxidants' concentrations that significantly impaired phospholipase C in this study were compatible with those occurring in vivo during ischemia-reperfusion [Am. J. Med. 91(Suppl. 3C):235, 1991]. This supports the possibility that alteration of the receptor-associated phospholipase C may be a factor in the oxidant-related dysfunction of the ischemic-reperfused heart.

Animals↗

PubMed1994

Alpha 1-adrenergic stimulation of phospholipase C activity in purified cardiac sarcolemmal membranes.

Cardiac sarcolemmal (SL) phospholipase C (PLC) is a key enzyme in the signal transduction of several cardiac receptors. Thus, the earlier described Ca(2+)-stimulated SL PLC activity may represent variously coupled enzymes. The present study was undertaken to delineate the alpha 1-adrenoceptor/G protein-stimulated PLC activity in purified cardiac SL vesicles. Although certain detergents and membrane pore formers enhanced SL PLC activity, measured as formation of 3H-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] from 3H-phosphatidylinositol 4,5-bisphosphate, alpha 1-adrenoceptor stimulated activity was not observed. When SL vesicles were preincubated (0-4 degrees C) with substrate in detergent-free buffer, subsequent incubation (37 degrees C; in mM: 100 NaCl, 2 EGTA, 1.8 CaCl2, 10 LiCl) resulted in a time-dependent production of 3H-Ins(1,4,5)P3, that was increased in the presence of 100 microM GTP gamma S. GTP gamma S stimulation of SL PLC activity required the presence of Mg2+ and Ca2+, but was lost at (sub)millimolar concentrations of these bivalent cations. Mg2+ (0.01-10 mM) promoted a 2,3-diphosphoglycerate-insensitive phosphatase activity. GTP gamma S enhanced the sensitivity of SL PLC to Ca2+, but did not increase the maximum Ca2+ (0.1-1 mM) stimulated SL PLC activity. At 5 microM Ca2+, GTP gamma S induced a concentration-dependent rise in inositol phosphates production, which was further elevated by the alpha 1-agonist, phenylephrine (PhE). The PhE-effect was inhibited by the alpha 1-antagonist prazosin, but not by the beta-antagonist atenolol. These results show that the components necessary for the alpha 1-adrenoceptor transmembrane signal are associated with the SL membrane and can be functionally coupled.

Adrenergic alpha-1 Receptor Agonists↗

PubMed1993

Possible role of phospholipase C in the induction of Ca(2+)-paradox in rat heart.

In order to investigate the involvement of phosphoinositide-specific phospholipase C (PLC), an enzyme associated with phosphoinositide signal transduction pathway, for the occurrence of Ca(2+)-paradox (loss of contractile activity associated with contracture), rat hearts perfused with Ca(2+)-free medium (1 to 5 min) were reperfused (5 to 10 min) with medium containing 1.25 mM Ca2+. Crude membranes isolated from hearts perfused with Ca(2+)-free medium exhibited a significantly increased activity of PLC, whereas normal activity was detected in hearts reperfused with Ca(2+)-containing medium. A significant rise in PLC activity was observed at 1 min of Ca(2+)-free perfusion; maximal increase was seen at 4 min of Ca(2+)-free perfusion. Minimal concentration of Ca2+ in the perfusion medium required for showing an increase in PLC activity was 10 microM, whereas that required for the occurrence of Ca(2+)-paradoxic changes in heart function upon reperfusion was 50 microM. Perfusion of the hearts with Ca(2+)-free medium in the presence of low Na+ or at low temperature, which prevents the occurrence of Ca(2+)-paradox upon reperfusion, did not prevent the increase in PLC activity. An increase during Ca(2+)-free perfusion similar to that seen for PLC was also observed for two other enzymes, namely the phosphatidylinositol (PI) 4-kinase and the PI-4-monophosphate (PIP) 5-kinase, which synthesize the PLC substrate, phosphatidylinositol 4,5-bisphosphate (PIP2). No alteration of the alpha-adrenoreceptors was observed after 5 min of Ca(2+)-free perfusion. On the other hand, the observed changes in PLC activity during Ca(2+)-free perfusion appear to be due to some redistribution of the enzyme in the myocardium. These results suggest a possible role of the phosphoinositide/PLC pathway in the induction of Ca(2+)-paradox via mechanisms which do not appear to be associated with changes in the characteristics of alpha-adrenergic receptors.

Animals↗

PubMed1993

Myocardial phosphoinositides do not share the same fatty acid profile.

It is generally assumed that the fatty acid compositions of the phosphoinositides are identical. To investigate this in myocardium, inositol lipids extracted from rat and pig ventricular homogenates were absorbed to neomycin-coated glass beads, eluted and quantitated by fatty acid analysis after thin-layer chromatography. The percentages of stearic, oleic, linoleic and arachidonic acid (20:4n-6) in the rat were, respectively, 49, 4, 7 and 26 for phosphatidylinositol, 62, 1, 4 and 18 for phosphatidylinositol-4-monophosphate and 63, 2, 4, 18 for phosphatidylinositol-4,5-bisphosphate. Equal distribution patterns of fatty acids were found in homogenate and sarcoplasmic reticulum of pig myocardium. Cultured rat ventricular myocytes were utilized to study the incorporation (25 h) of [14C]20:4n-6 relative to that of myo-[3H]inositol into phosphatidylinositol and phosphatidylinositol-4,5-bisphosphate which were, respectively, 1.61 and 1.22. The data indicate that in myocardium phosphatidylinositol-4,5-bisphosphate represents a relatively modest source of 20:4n-6.

Animals↗

PubMed1993

Receptor-mediated signalling pathways acting through hydrolysis of membrane phospholipids in cardiomyocytes.

The aim is to summarize briefly the evidence for the existence and possible functions of receptor-mediated activity of phospholipases C and D in the myocardium. Muscarinic, alpha 1-adrenergic, angiotensin II, endothelin-1, thrombin, adenine nucleotide and opioid peptide receptors are all linked through GTP-binding proteins to phospholipase C which hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) in the myocardium. Events that are not linked to receptors, such as mechanical loading (stretching) of cardiomyocytes, can also activate phospholipase C. The high capacity for resynthesis of PIP2 maintains the pool of PIP2, even during maximal activation of phospholipase C. Activation of phospholipase C by endothelin-1, alpha 1-adrenoceptor and angiotensin II, is subject to different rates of homologous desensitization. Protein kinase C is probably not involved in the desensitization of the response to endothelin-1. One of the products of the hydrolysis of PIP2, inositol 1,4,5-trisphosphate (IP3), releases Ca2+ from the sarcoplasmic reticulum. This intracellular response seems to be causally related to positive inotropy. The phosphorylated product of IP3, inositol 1,3,4,5-tetrakisphosphate (IP4), is believed to play a role in the handling of intracellular Ca2+, as well as in the inotropic response; however, its formation is controversial. At present the oscillations in the level of intracellular Ca2+ underlying, for example, the positive inotropy induced by alpha 1-adrenoceptors or endothelin are not clearly identified. The other product of phospholipase C, 1,2-diacylglycerol, activates Ca(2+)-dependent protein kinase C and potentially controls a wide array of cellular functions such as ion transport, myofibrillar Ca2+ sensitivity, "cross-talk" between phospholipases C and D, gene expression, protein synthesis and hypertrophic cell growth. Alterations in the fatty acid composition, particularly the polyunsaturated fatty acids, modify the phosphoinositide response induced by hormones. Cultured cardiomyocytes, incubated in sera containing the fatty acids 18:2n-6 or 20:5n-3, but not 18:0 and 18:1n-9, show a decrease in the phospholipase C responses mediated by alpha 1-adrenoceptors. The fatty acid composition of myocardial phosphatidyl inositol 4-monophosphate (PIP) and PIP2 differs from that of phosphatidylinositol, which indicates that phosphatidylinositol kinases have a certain substrate specificity or have access to localized phosphatidylinositol molecules. The estimation of the level of stimulated 1,2-diacylglycerol is complicated by the contribution of the activity of receptor-mediated phospholipase D. The identification of the molecular species of 1,2-diacylglycerol is crucial in establishing the roles and the sources of 1,2-diacylglycerol. The fatty acids covalently bound in the membrane phospholipids may also influence phospholipases C and D.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Phosphoinositide kinases in rat heart sarcolemma: biochemical properties and regulation by calcium.

Phosphatidylinositol (PtdIns) kinase and phosphatidylinositol 4-phosphate (PtdIns4P) kinase have been studied in a purified sarcolemmal fraction isolated from rat heart. Both enzymes were Mg(2+)-dependent and their activities were maximal at 2.5 mM Mg2+ and pH 7.5. Kinetic analysis of endogenous substrate phosphorylation by ATP showed that the apparent Km and Vmax values for PtdIns kinase were 292 +/- 17 microM and 1390 +/- 80 pmol.mg-1.min-1, respectively, while the apparent Km and Vmax values for PtdIns4P kinase were 398 +/- 25 microM and 382 +/- 24 pmol.mg-1.min-1. Under normal conditions, the activity of PtdIns4P kinase was lower than that of PtdIns kinase; however, the former activity increased several fold in the presence of PtdIns4P as an exogenous substrate. The enzymatic synthesis of intramembranal PtdIns4P and phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)P2) was maximally enhanced by 0.1% Triton X-100 and inhibited by micromolar concentrations of Ca2+. Inhibition of PtdIns and PtdIns4P kinase showed IC50 values for Ca2+ of 20 and 6 microM, respectively, and was independent of either Ca(2+)-induced activation of phospholipase C and polyphosphoinositide monophosphoesterases or low ATP concentrations. The results indicate that purified rat heart sarcolemmal membranes contain a very active phosphoinositide phosphorylation system which is regulated by micromolar levels of Ca2+. The Ca2+ effect may contribute to the feedback inhibition of the receptor-activated formation of inositol 1,4,5-trisphosphate.

1-Phosphatidylinositol 4-Kinase↗

PubMed1992

The substrate specificity of phosphoinositide-phospholipase C in rat heart sarcolemma.

In rat cardiac sarcolemmal membranes a phosphoinositide-specific phospholipase C (PLC) was found to be present. The enzyme hydrolysed exogenous [3H-]phosphatidylinositol 4,5-biphosphate ([3H-]PtdIns(4,5)P2) in an optimized assay mixture containing 15 micrograms SL protein, 100 mM NaCl, 1 mM free Ca2+, 14 mM Na-cholate and 20 microM [3H-]PtdIns-(4,5)P2 (400-500 dpm/microliter) in 30 mM HEPES-Tris buffer (pH 7.0). The average specific activity was 9.14 +/- 0.55 nmol.mg-1.2.5 min-1. The addition of Mg2+ to the assay mixture did not change PLC activity but increased the relative amounts of dephosphorylated inositol products. In the absence of Na+ and at a low Ca2+ concentration (0.3 microM), Mg2+ also enhanced the intraSL levels of PtdIns4P and PtdIns, and, moreover, inhibited PLC activity (IC50-0.07 mM). PtdIns4P seemed to be a good substrate for th rat SL PLC (23.07 +/- 1.57 nmol.mg-1.2.5 min-1) whereas PtdIns was hydrolysed at a very low rate (0.36 +/- 0.08 nmol.mg-1.2.5 min-1). Unlike PtdIns(4,5)P2, PLC-dependent PtdIns4P and PtdIns hydrolysis was not inhibited by Ca2+ concentrations over 1 mM. The possibility of distinct isozymes being responsible for the different hydrolytic activities is discussed.

Animals↗

PubMed1992

Depression of cardiac sarcolemmal phospholipase D activity by oxidant-induced thiol modification.

Myocardial phospholipase D (PLD) is primarily localized at the sarcolemmal level and selectively hydrolyzes phosphatidylcholine to form phosphatidic acid as part of the signal transduction mechanisms for regulating Ca2+ movements in the heart. Since the myocardial cell damage induced by oxidative stress is associated with abnormalities in Ca2+ homeostasis and thiol status, we examined the thiol group dependence and the effects of oxidant species on this enzyme. Sarcolemmal membranes isolated from rat heart were exposed to several types of thiol group modifiers. Alkylation with N-ethylmaleimide or methyl methanethiosulfonate, mercaptide formation with p-chloromercuriphenylsulfonic acid, and thiol-disulfide exchange with 5,5'-dithio-bis(2-nitrobenzoate) depressed sarcolemmal PLD activity; in all cases the depression was prevented by dithiothreitol. At different concentrations of N-ethylmaleimide the PLD depression correlated well (r = 0.98) with the decrease in total thiol group content of the membrane. The enzyme activity was not affected by xanthine-xanthine oxidase, a superoxide anion-generating system, but was depressed by hydrogen peroxide (H2O2) in a concentration-dependent manner. This inhibitory effect was prevented by catalase as well as by dithiothreitol, but not by D-mannitol. The effect of a hydroxyl radical-generating system (Fenton reaction) could not be assessed because of an interfering direct inhibition by Fe2+. Dithiothreitol was also able to restore PLD activity in H2O2-pretreated membranes and to prevent a severe deactivation of the enzyme by hypochlorous acid (HOCI). Protection by glutathione and inhibition by its oxidized form were also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

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 5 microM CaCl2, 5 mM cholate and 100 nM [3H-]Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5) P2) resulted in the formation of [3H-]InsP3. GTP-gamma-S (125 microM) 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-]PtdIns(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 microsomal GTP-gamma-S-stimulated PtdIns(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 alpha 1-adrenoceptor mediated PtdIns(4,5)P2 hydrolysis by activation of protein kinase C-induced 15 kD protein phosphorylation.

Animals↗

PubMed1991

Phospholipase D activity in subcellular membranes of rat ventricular myocardium.

The phospholipase D (PL D), which catalyzes the formation of phosphatidic acid (PA), was studied in rat myocardium using 14C-labelled phosphatidylcholine (PC) as an exogenous substrate. Subcellular distribution experiments indicated the presence of PL D in particulate fractions only. Different procedures for the isolation of purified cardiac subcellular organelles showed the presence of PL D in sarcolemma (SL), sarcoplasmic reticulum (SR) and mitochondria with 14-, 11- and 5-fold enrichment when compared to the homogenate value, respectively. The activity of SL PL D was observed over a narrow acid pH range with an optimum at 6.5, and it showed a high specificity for PC while phosphatidylethanolamine and phosphatidylinositol showed a low rate of hydrolysis. Under optimal conditions, PA formation was linear for a 90-min period of incubation and the reaction rate was constant for 10 to 100 micrograms SL protein in the assay medium. The SR PL D displayed properties similar to those seen with the SL PL D. In membrane fractions PL D was also found to catalyze a transphosphatidylation reaction for the synthesis of phosphatidylglycerol. Assessment of the intramembranal levels of radioactive 1,2-diacylglycerol (DAG) in the absence or presence of KF suggested the presence of an active PA phosphohydrolase activity. This study indicates that a PC-specific PL D activity is localized in different membrane systems of the myocardium and may be associated with PA phosphohydrolase to act in a coordinated manner. The functional significance of PL D-dependent formation of PA in cardiac membranes is discussed.

Animals↗

PubMed1991

Role of sulfhydryl groups in phospholipid methylation reactions of cardiac sarcolemma.

The effect of reagents that modify sulfur-containing amino acid residues in the phosphatidylethanolamine N-methyltransferase was studied in the isolated rat cardiac sarcolemma by employing S-adenosyl-L-[methyl-3H]methionine as a methyl donor. Dithiothreitol protected the sulfhydryl groups in the membrane and caused a concentration- and time-dependent increase of phospholipid N-methylation at three different catalytic sites. This stimulation was highest (9-fold) in the presence of 1 mM MgCl2 and 0.1 microM S-adenosyl-L-[methyl-3H]methionine at pH 8.0 (catalytic site I), and was associated with an enhancement of Vmax without changes in Km for the methyl donor. Thiol glutathione was less stimulatory than dithiothreitol; glutathione disulfide inhibited the phosphatidylethanolamine N-methylation by 50%. The alkylating reagents, N-ethylmaleimide and methylmethanethiosulfonate, inhibited the N-methylation with IC50 of 6.9 and 14.1 microM, respectively; this inhibition was prevented by 1 mM dithiothreitol. These results indicate a critical role of sulfhydryl groups for the activity of the cardiac sarcolemmal phosphatidylethanolamine N-methyltransferase and suggest that this enzyme system in cardiac sarcolemma may be controlled by the glutathione/glutathione disulfide redox state in the cell.

Animals↗

PubMed1990

Alterations in cardiac membrane Ca2+ transport during oxidative stress.

Although cardiac dysfunction due to ischemia-reperfusion injury is considered to involve oxygen free radicals, the exact manner by which this oxidative stress affects the myocardium is not clear. As the occurrence of intracellular Ca2+ overload has been shown to play a critical role in the genesis of cellular damage due to ischemia-reperfusion, this study was undertaken to examine whether oxygen free radicals are involved in altering the sarcolemmal Ca2(+)-transport activities due to reperfusion injury. When isolated rat hearts were made globally ischemic for 30 min and then reperfused for 5 min, the Ca2(+)-pump and Na(+)-Ca2+ exchange activities were depressed in the purified sarcolemmal fraction; these alterations were prevented when a free radical scavenger enzymes (superoxide dismutase plus catalase) were added to the reperfusion medium. Both the Ca2(+)-pump and Na(+)-Ca2+ exchange activities in control heart sarcolemmal preparations were depressed by activated oxygen-generating systems containing xanthine plus xanthine oxidase and H2O2; these changes were prevented by the inclusion of superoxide dismutase and catalase in the incubation medium. These results support the view that oxidative stress during ischemia-reperfusion may contribute towards the occurrence of intracellular Ca2+ overload and subsequent cell damage by depressing the sarcolemmal mechanisms governing the efflux of Ca2+ from the cardiac cell.

Animals↗

PubMed1990

Alteration of lipid methylation by oleic acid in rat heart sarcolemma.

Incubation of rat heart sarcolemma with the methyl donor S-adenosyl-L-[methyl-3H] methionine resulted in N-methylation of phosphatidylethanolamine and methylation of a heterogenous fraction of nonpolar lipids in the membrane. Oleic acid reduced the synthesis of N-methylated phospholipids and stimulated the methyl group incorporation into nonpolar lipids in a concentration-dependent manner. Both methylation reactions were not affected when oleic acid was substituted by methyl ester of oleic acid or by the detergents sodium deoxycholate or Triton X-100. This study suggests that the enzymatic biosynthesis of the N-methylated phospholipids may be altered by free fatty acids.

Animals↗

PubMed1990

Inhibition of cardiac phosphatidylethanolamine N-methylation by oxygen free radicals.

This study was undertaken to examine the effects of oxygen free radicals on phosphatidylethanolamine (PE) N-methylation in rat heart sarcolemmal (SL) and sarcoplasmic reticular (SR) membranes. Three catalytic sites involved in the sequential methyl transfer reaction were studied by assaying the incorporation of radiolabeled methyl groups from S-adenosyl-L-methionine (0.055, 10, and 150 microM) into SL or SR PE molecules under optimal conditions. In the presence of xanthine + xanthine oxidase (superoxide anion radicals generating system), PE N-methylation was inhibited at site I and III in the heavy SL fraction isolated by the hypotonic shock-LiBr treatment method. In the light SL fraction isolated by sucrose-density gradient, a significant inhibition of PE N-methylation was seen at all three sites. These inhibitory effects of xanthine + xanthine oxidase on PE N-methylation were prevented by the addition of superoxide dismutase. Hydrogen peroxide showed a significant inhibition of PE N-methylation at site I in the heavy SL fraction, and at site I and II in the light SL fraction. Catalase blocked the inhibitory effects of hydrogen peroxide. The effects of both xanthine + xanthine oxidase and hydrogen peroxide on the SR membranes were similar to those seen for the heavy SL fraction. These results suggest that, in addition to lipid peroxidation, the oxygen free radicals may affect the function of cardiac membranes by decreasing the phospholipid N-methylation activity.

Animals↗

PubMed1990

Stimulation of phospholipid N-methylation by isoproterenol in rat hearts.

Phosphatidylethanolamine (PtdEtn) N-methyltransferase activities were studied in rat heart sarcolemmal and sarcoplasmic reticular fractions after a single intraperitoneal injection of isoproterenol (0.5-5.0 mg/kg). Three active sites (I, II, and III) for PtdEtn N-methylation were assayed by measurement of [3H]methyl group incorporation from 0.055, 10, and 150 microM S-adenosyl-L-[methyl-3H]methionine into membrane PtdEtn molecules. Total methylation activity for catalytic site I of both sarcolemma and sarcoplasmic reticulum was stimulated within 2 minutes by isoproterenol in a dose-dependent manner. Although the increased methyltransferase activity in sarcoplasmic reticulum was normalized at 10 minutes, the enzyme activity in sarcolemma was normalized at 5 minutes but was again increased at 10-30 minutes after isoproterenol injection. No changes in response to isoproterenol were seen for site II and III N-methylation activities in either membrane. Individual N-methylated phospholipids (phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine), which specifically formed at each site, showed similar behavior. Pretreatment of the animals with a beta-blocking drug, atenolol, for 2 days prevented the isoproterenol-induced changes in hemodynamic parameters and sarcolemmal methylation without affecting the enhanced methylation activities in sarcoplasmic reticulum. In vitro addition of cyclic AMP-dependent protein kinase (catalytic subunit) plus Mg-ATP enhanced methyltransferase activities in sarcolemma and sarcoplasmic reticulum from control hearts by 2.7- and 2.3-fold, respectively; however, under the same in vitro conditions, only about 20% activation was seen in both subcellular membranes isolated from the heart of isoproterenol-injected animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1990

Alterations in phospholipid N-methylation of cardiac subcellular membranes due to experimentally induced diabetes in rats.

Phosphatidylethanolamine N-methylation was examined in cardiac subcellular membranes after inducing chronic experimental diabetes in rats (65 mg streptozotocin/kg, i.v.). The incorporation of radiolabeled methyl groups from S-adenosyl-L-methionine in diabetic sarcolemma was significantly depressed at all three catalytic sites (I, II, and III) of the methyltransferase system. An increase in methyl group incorporation was evident at site I without any changes at sites II and III in diabetic sarcoplasmic reticulum and mitochondria. Similar changes were also seen for the individual N-methylated lipids (monomethyl-, dimethylphosphatidylethanolamine, and phosphatidylcholine) specifically formed at each catalytic site in all cardiac membranes from diabetic animals. These alterations in N-methylation were reversible by a 14-d insulin therapy to the diabetic animals. In the presence of 10 microM ATP and 0.1 microM Ca2+, N-methylation was maximally activated at site I in both control and diabetic sarcolemma and sarcoplasmic reticulum, but not in mitochondria. Incubation of cardiac membranes with of S-adenosyl-L-methionine showed that Ca2(+)-stimulated ATPase activities in both sarcolemma and sarcoplasmic reticulum were augmented; however, the activation of diabetic sarcolemma was lesser and that of diabetic sarcoplasmic reticulum was greater in comparison with the control preparations. These results identify alterations in phosphatidylethanolamine N-methylation in subcellular membranes from diabetic heart, and it is suggested that these defects may be crucial in the development of cardiac dysfunction in chronic diabetes.

Animals↗

PubMed1989

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 I, II and II, 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 microM)-dependent manner. Methionine (50 microM) 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 microM phenylephrine did not affect the rate of PE-N-methylation in the presence of exogenous methionine (50 microM). These findings support the existence of interactions, although discrete, between the enzymes involved in the PE-N-methylation and PI turnover.

Adrenergic Agonists↗

PubMed1989

Role of phosphatidylinositol in cardiac sarcolemmal membrane sodium-calcium exchange.

The purpose of this investigation was to study the effects of a distinct type of phospholipase C on sarcolemmal Na+-Ca2+ exchange. With this phospholipase C (Staphylococcus aureus), treatment of cardiac sarcolemmal vesicles resulted in a specific hydrolysis of membrane phosphatidylinositol. This hydrolysis of phosphatidylinositol also released two proteins (110 and 36 kDa) from the sarcolemmal membrane. Phospholipase C pretreatment of the sarcolemma resulted in an unexpected stimulation of Na+-Ca2+ exchange. The Vmax of Na+-Ca2+ exchange was increased but the Km for Ca2+ was not altered. This stimulation was specific to the Na+-Ca2+ exchange pathway. ATP-dependent Ca2+ uptake was depressed after phospholipase C treatment, but passive membrane permeability to Ca2+ was unaffected. Sarcolemmal Na+,K+-ATPase activity was not altered, whereas passive Ca2+ binding was modestly decreased after phospholipase C pretreatment. The stimulation of Na+-Ca2+ exchange after phosphatidylinositol hydrolysis was greater in inside-out vesicles than in a total population of vesicles of mixed orientation. This finding suggests that the cardiac sarcolemmal Na+-Ca2+ exchanger is functionally asymmetrical. The results also suggest that membrane phosphatidylinositol is inhibitory to the Na+-Ca2+ exchanger or, alternatively, this phospholipid may anchor an endogenous inhibitory protein in the sarcolemmal membrane. The observation that a transsarcolemmal Ca2+ flux pathway may be stimulated solely by phosphatidylinositol hydrolysis independently of phosphoinositide metabolic products like inositol triphosphate is novel.

Animals↗