Biomedical subjects

V Panagia

Publications and source records attributed to V Panagia.

At least 73 records · Page 4Linked to original sources

PubMed1989

Alterations of phosphatidylethanolamine N-methylation in rat heart by quinidine.

To elucidate the molecular mechanism underlying the adverse depression of myocardial contractility observed during antiarrhythmic therapy of quinidine, we investigated its action on the phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17) activities of cardiac subcellular membranes. Rat heart sarcolemma, mitochondria, and microsomes (sarcoplasmic reticular fragments) were isolated, and the three catalytic sites for N-methylation activities were examined with 0.055 (site I), 10 (site II), and 150 (site III) microM concentrations of S-adenosyl-L-[methyl-3H]methionine as a methyl donor. Total methyl group incorporation into sarcolemmal phosphatidylethanolamine was depressed by 10(-6)-10(-3) M quinidine at sites II and III. The activity of site I was stimulated at low (10(-9) M) concentrations and inhibited at high concentrations of the drug. A similar behaviour was observed with procainamide, although the inhibitory effect was less pronounced and was not additive with quinidine. Quinidine-induced inhibition was associated with a depression of Vmax, while the apparent affinity for S-adenosyl-L-methionine was unaltered. Analysis of individual methylated phospholipids confirmed inhibition by quinidine at sites II and III in sarcolemma. Microsomal phosphatidylethanolamine N-methylation was affected by 10(-6) M quinidine only at site II, whereas no changes were noted in mitochondria. Quinidine also inhibited both the positive inotropic response and concomitant increase in tissue N-methylated phospholipids observed upon L-methionine perfusion of rat heart. These results suggest that quinidine alters the intramembranal level of N-methylated phospholipids, and this may serve as a biochemical mechanism contributing to its negative inotropic effect.

Animals↗

PubMed1988

Alterations in heart membrane calcium transport during the development of ischemia-reperfusion injury.

Global ischemia in guinea-pig hearts for 60 to 90 min depressed microsomal and mitochondrial Ca2+ uptake activities. Reperfusion of the 60 min ischemic hearts resulted in incomplete recovery of contractile function and calcium uptake activities of both mitochondrial and microsomal fractions. On the other hand, reperfusion of the 90 min ischemic hearts further depressed the microsomal Ca2+ uptake activity. Coronary occlusion for 90 min in dog hearts was found to decrease microsomal Ca2+-pump and sarcolemmal Na+-K+ ATPase activities. Reperfusion of these regional ischemic hearts further depressed the microsomal Ca2+ uptake and Ca2+-stimulated ATPase as well as sarcolemmal Na+-K+ ATPase activities whereas mitochondrial Ca2+ uptake was increased. Perfusion of rat hearts for 60 min with hypoxic medium resulted in depression of the sarcolemmal Na+-dependent Ca2+ uptake and ATP-dependent Ca2+ uptake activities. Reperfusion of these hypoxic hearts failed to recover the sarcolemmal Na+-Ca2+ exchange and Ca2+-pump activities. These results demonstrate that membrane defects with respect to Ca2+ transport processes in ischemic/hypoxic hearts may be associated with irreversible injury.

Animals↗

PubMed1988

Increased SR phospholipid N-methylation in skeletal muscle of diabetic rats.

Phosphatidylethanolamine (PE) N-methylation was studied in skeletal muscle sarcoplasmic reticulum (SR) 6 wk after the induction of experimental diabetes in rats by an injection of streptozocin (65 mg/kg iv). A significant increase in the incorporation of radiolabeled methyl groups from S-adenosyl-L-methionine (AdoMet) into intramembranal PE was observed in diabetic preparations at 0.055 microM AdoMet, whereas the methylation activity was unaltered at 10 and 150 microM AdoMet concentrations. The increase in PE N-methylation activity was not evident until 28 days after streptozocin injection and was normalized by a 2-wk treatment of diabetic animals with insulin. In the presence of 10 microM of ATP and low concentrations of Ca2+ (0.1 microM), PE N-methylation was maximally activated, but the percent increase was similar in control, diabetes, and insulin-treated diabetes; at 100 microM Ca2+, however, N-methylation activity was depressed only in diabetic preparations. Calmodulin inhibitors such as compound 48/80 and calmidazolium (compound R24571) abolished the effect of Ca2+ and ATP on PE N-methylation in all three groups. Sarcolemmal (SL) PE N-methylation in diabetic skeletal muscle was also found to be increased at 0.055 microM AdoMet. The results suggest that intramembranal calmodulin may participate in regulating PE N-methylation in skeletal muscle membranes, but it may not be responsible for the high N-methylation activity in diabetic rats.

Adenosine Triphosphate↗

PubMed1988

Methionine-induced positive inotropic effect in rat heart: possible role of phospholipid N-methylation.

Perfusion of isolated rat heart with L-methionine produced a positive inotropic effect that was temporally preceded, as well as accompanied, by an increase of methyl group incorporation into N-methylated phospholipids of the myocardium. Maximal increase in contractile force development was associated with maximal methyl group incorporation. Both parameters showed a dose-related dependence on methionine and correlated positively (r = 0.965) upon regression analysis of the data. The presence of adenosine, L-homocysteine thiolactone and erythro-9-(2-hydroxy-3-nonyl) adenine in the perfusion medium inhibited the positive inotropic effect as well as the incorporation of methyl groups into phospholipids. Cycloleucine, an inhibitor of S-adenosylmethionine synthetase, also reduced the increase in contractility by methionine. Methionine-induced positive inotropic effect could be modulated by varying Ca2+ concentration in the perfusate and was inhibited by ryanodine, a blocker of sarcoplasmic reticular Ca2+ release. These observations indicate that L-methionine may serve as a powerful positive inotropic agent and suggest that phospholipid N-methylation plays an important role in functional activity of rat heart.

Adenine↗

PubMed1988

Defects in sarcolemmal Ca2+ transport in hearts due to induction of calcium paradox.

Na+-Ca2+ exchange and Ca2+-pump activities were studied in sarcolemmal vesicles isolated from rat hearts subjected to "calcium paradox" on perfusion with Ca2+-free medium followed by reperfusion with medium containing 1.25 mM Ca2+. Perfusion of hearts with Ca2+-free medium for 5 minutes did not affect the Na+-dependent Ca2+ uptake, ATP-dependent Ca2+ uptake, or Ca2+-stimulated ATPase activities in sarcolemma. Reperfusion of the Ca2+-deprived hearts with medium containing Ca2+ for 1-2 minutes increased Na+-dependent Ca2+ uptake, whereas reperfusion for 5-10 minutes decreased Na+-dependent Ca2+ uptake in sarcolemmal vesicles. Both ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase activities in sarcolemma were depressed on reperfusion of Ca2+-deprived hearts for 2-10 minutes. Reperfusion of Ca2+-deprived hearts for 5 minutes, which failed to generate contractile force, resulted in contracture without any recovery of the contractile force development. These changes in sarcolemmal Ca2+ transport and contractile function were prevented when hearts were perfused with Ca2+-free medium either in the presence of low sodium (35 mM) or at a low temperature (21 degrees C) before starting the reperfusion. No alterations in the purity of the preparation or permeability of sarcolemmal vesicles with respect to Na+ or Ca2+ were detected in hearts perfused with Ca2+-free medium or on reperfusion with medium containing calcium. The results indicate abnormalities in sarcolemmal Na+-Ca2+ exchange and Ca2+-pump mechanisms on reperfusion of Ca2+-deprived hearts with medium containing Ca2+, and such changes may partly account for the occurrence of intracellular Ca2+ overload during the development of calcium paradox.

Adenosine Triphosphate↗

PubMed1988

Phospholipid N-methylation-dependent alterations of cardiac contractile function by L-methionine.

Isolated rat, rabbit and guinea pig hearts exhibited an initial negative inotropic (20-30%) effect followed by a positive inotropic response (60-80%) upon perfusion with 300 microM L-methionine. In contrast, frog hearts did not show any delayed positive inotropic effect, whereas initial negative inotropic effect (25%) of L-methionine was seen. In subsequent studies using rat hearts, methionine was found to induce a dose-dependent increase in contractile force which correlated linearly (r = 0.93) with incorporation of methyl groups into tissue N-methylated phospholipids. The presence of adenosine, L-homocysteine thiolactone and erythro-9-(2-hydroxy-3-nonyl) adenine mixture in the perfusion medium inhibited the contractile effects of L-methionine as well as the incorporation of 3H-methyl groups by about 75%. Cycloleucine, an inhibitor of S-adenosylmethionine synthase, and methyl acetimidate, a blocker of the phosphatidylethanolamine polar groups, inhibited phospholipid N-methylation and prevented the contractile changes due to L-methionine. The initial negative inotropic effect of methionine was attenuated by lowering the concentration of Na+, whereas the delayed positive inotropic effect was dependent on the concentration of Ca++ in the perfusion medium. Ryanodine, a blocker of the sarcoplasmic reticular Ca++ release, prevented the positive inotropic effect of methionine whereas verapamil, a well known Ca++ antagonist, blocked the initial depressant effect and reduced the delayed positive inotropic response. Marked alterations in the sarcolemmal and sarcoplasmic reticular calcium transport activities were seen upon perfusing the hearts with methionine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Inhibition of Na+-Ca2+ exchange in heart sarcolemmal vesicles by phosphatidylethanolamine N-methylation.

The effect of phosphatidylethanolamine N-methylation on Na+-Ca2+ exchange was studied in sarcolemmal vesicles isolated from rat heart. Phosphatidylethanolamine N-methylation following incubation of membranes with S-adenosyl-L-methionine, a methyl donor for the enzymatic N-methylation, inhibited Nai+-dependent Ca2+ uptake by about 50%. The N-methylation reaction did not alter the passive permeability of the sarcolemmal vesicles to Na+ and Ca2+ and did not modify the electrogenic characteristics of the exchanger. The depressant effect of phosphatidylethanolamine N-methylation on Nai+-dependent Ca2+ uptake was prevented by S-adenosyl-L-homocysteine, an inhibitor of the N-methylation. Pretreatment of sarcolemma with methyl acetimidate hydrochloride, an amino-group-blocking agent, also prevented methylation-induced inhibition of Ca2+ uptake. In the presence of exogenous phospholipid substrate, the phospholipid N-methylation process in methyl-acetimidate-treated sarcolemmal vesicles was restored and the inhibitory effect on Ca2+ uptake was evident. These results suggest that phosphatidylethanolamine N-methylation influences the heart sarcolemmal Na+-Ca2+ exchange system.

Animals↗

PubMed1987

Sarcolemmal phospholipid N-methylation in genetically determined hamster cardiomyopathy.

The heart sarcolemmal phosphatidylethanolamine N-methylation in UM-X7.1 strain of cardiomyopathic hamsters was examined by using 0.055, 10 and 150 microM S-adenosyl-L-(methyl-3H) methionine as methyl donor for sites I, II and III, respectively. In comparison with control values, methylation activities at site I was increased in 40, 120 and 250 days old cardiomyopathic hamsters. On the other hand, methylation activities at sites II and III in 120 and 250 days old cardiomyopathic animals were depressed without any change in the 40 days old group. The alterations in N-methylation activities were associated with kinetic changes in apparent Vmax values without any changes in the apparent Km. These results indicate a defect in the phospholipid N-methylation process in heart sarcolemma during the development of genetically determined cardiomyopathy.

Aging↗

PubMed1987

Decreased Ca2+-binding and Ca2+-ATPase activities in heart sarcolemma upon phospholipid methylation.

Heart sarcolemma has been shown to possess three catalytic sites (I, II and III) for methyl transferase activity (Panagia V, Ganguly PK and Dhalla NS. Biochim Biophys Acta 792:245-253, 1984). In this study we examined the effect of phosphatidylethanolamine N-methylation on ATP-independent Ca2+ binding and ATPase activities in isolated rat heart sarcolemma. Both low affinity (1.25 mM Ca2+) and high affinity (50 microM Ca2+) Ca2+ binding activities were decreased following incubation of sarcolemmal membranes with AdoMet under optimal conditions for site II and III. Similarly, Ca2+ ATPase activities measured at 1.25 mM and 4 mM Ca2+ were depressed by phospholipid N-methylation. S-adenosyl homocysteine, a specific inhibitor of phospholipid N-methylation, prevented the depression of low affinity Ca2+ binding and Ca2+ ATPase activities, whereas the methylation-induced effect on the high affinity Ca2+ binding was not influenced by this agent. Pretreatment of sarcolemma with methyl acetimidate hydrochloride, an amino group blocking agent, also prevented the methylation-induced inhibition of both Ca2+ binding and Ca2+ ATPase. A further decrease in Ca2+ binding and Ca2+ ATPase activities together with a marked increase in the intramembranal level of PC was seen when membranes were methylated under the site III conditions in the presence of phosphatidyldimethylethanolamine as exogenous substrate. There was no effect of phospholipid methylation on sarcolemmal Na+-K+ ATPase and Mg2+ ATPase activities. These results indicate a role of phospholipid N-methylation in the regulation of sarcolemmal Ca2+ ATPase and low affinity ATP-independent Ca2+ binding.

Animals↗

PubMed1987

Biphasic changes in the sarcolemmal phosphatidylethanolamine N-methylation activity in catecholamine-induced cardiomyopathy.

Phosphatidylethanolamine (PE) N-methylation activity was studied in rat heart sarcolemma at 1, 3, 9 and 24 h after an intraperitoneal injection of isoproterenol (40 mg/kg). Three reaction sites for PE N-methylation were examined by assaying the incorporation of radiolabeled methyl groups from S-adenosyl-L-methionine (AdoMet) into sarcolemmal PE molecules under optimal conditions. Total methylation activity at catalytic site I (studied by employing 0.055 microM AdoMet) was increased at 1 and 3 h after the isoproterenol injection and depressed at 24 h; 9 h samples showed no change. Similar biphasic alterations were seen for phosphatidyl-N-monomethylethanolamine, the major methylated product formed at site I. Alterations in the methylation activity at site I were associated with changes in Vmax values but the apparent affinity for AdoMet remained unaltered. No alterations were found in total methylation activities at sites II and III in isoproterenol treated preparations when studied by employing 10 and 150 microM AdoMet, respectively. An increase and a decrease in the PE N-methylation activity at site I were also observed in the sarcoplasmic reticular (microsomal) fraction from experimental hearts after 1 h and 24 h of the isoproterenol injection respectively, without changes at sites II and III. On the other hand, no changes were seen in the mitochondrial fraction. These results indicate biphasic alterations in the sarcolemmal and microsomal PE N-methylation activities during the development of catecholamine-induced cardiomyopathy.

Animals↗

PubMed1987

Modification of sarcolemmal phosphatidylethanolamine N-methylation during heart hypertrophy.

To evaluate changes in heart sarcolemmal phosphatidylethanolamine (PE) N-methylation, left ventricular hypertrophy was induced in rabbits by banding the abdominal aorta for 4, 8, 14, and 22 wk. The degree of cardiac hypertrophy did not change over the period of time studied. Three catalytic sites involved in the sequential methyl transfer reactions were examined by assaying the incorporation of radiolabeled methyl groups from S-adenosyl-L-methionine (0.055, 10, and 150 microM) into sarcolemmal PE molecules under optimal conditions. Total N-methylation activity at all three sites was significantly increased at 4 wk, unaltered at 8 and 14 wk, and depressed at 22 wk after banding the aorta. Similar biphasic changes were seen for the individual methylated lipid products (monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, and phosphatidylcholine) specifically formed at each catalytic site. At all three sites, alterations in PE N-methylation at 22 wk were associated with changes in Vmax values without any change in the apparent affinity for S-adenosyl-L-methionine. In contrast to sarcolemma, a significant increase of the PE N-methylation activity at sites I and III was observed in the sarcoplasmic reticular (microsomal) fraction from 22-wk hypertrophied hearts; the increase in site II was not significant. On the other hand, no changes in the N-methylation activity of the mitochondrial fraction were seen at 22 wk after banding. These findings indicate the occurrence of biphasic alterations in the sarcolemmal PE N-methylation activity during the presence of a stable degree of hypertrophy.

Animals↗

PubMed1986

Stimulation of Ca2+-pump in rat heart sarcolemma by phosphatidylethanolamine N-methylation.

Incubation of purified cardiac sarcolemmal vesicles (SL) in the presence of S-adenosyl-L-methionine, a methyl donor for the enzymatic N-methylation of phosphatidylethanolamine (PE), increased the Ca2+-stimulated ATPase and ATP-dependent Ca2+ accumulation activities. Quantitative analysis of the methylated phospholipids revealed that maximal increase of Ca2+-pump activities was associated with predominant synthesis and intramembranal accumulation of phosphatidyl-N,N-dimethylethanolamine. The stimulation of SL Ca2+-pump activities was prevented by inhibitors of PE N-methylation such as S-adenosyl-L-homocysteine and methyl acetimidate hydrochloride. The results suggest a possible role of PE N-methylation in the regulation of Ca2+-transport across the heart SL membrane.

Animals↗

PubMed1986

Subcellular distribution of cardiac 5'-nucleotidase: alteration of microsomal pool in hypertrophied pig heart.

The subcellular distribution of the 5'-nucleotidase activity was investigated in normal and hypertrophied pig hearts; normal rat hearts were used for comparison. The left ventricular hypertrophy was induced in pigs by banding the supravalvular aorta for 4, 8 and 12 weeks. By employing different procedures for the isolation of cardiac membranes, a major catalytic site for 5'-nucleotidase was found to be located at sarcolemma in rat heart and microsomes (sarcoplasmic reticulum) in pig heart. A progressive decrease in the homogenate and microsomal 5'-nucleotidase activity occurred upon the development of myocardial hypertrophy in pigs. This reduction in microsomal 5'-nucleotidase activity was characterized by a depression in both apparent Vmax and Km values. These results indicate that a primary 5'-nucleotidase pool is present in the intracellular compartment of the pig heart and is altered during the development of hypertrophy.

5'-Nucleotidase↗

PubMed1986

Reversibility of ultrastructural, contractile function and Ca2+ transport changes in guinea pig hearts after global ischemia.

To understand the subcellular basis of contractile failure due to ischemia-reperfusion injury, effects of 20, 60, and 90 min of global ischemia followed by 30 min of reperfusion were examined in isolated guinea pig hearts. Cardiac ultrastructure and function as well as Ca2+ transport abilities of both mitochondrial and microsomal fractions were determined in control, ischemic, and reperfused hearts. Hearts were unable to generate any contractile force after 20 min of ischemia and showed a 75% recovery upon reperfusion. However, there were no significant changes in the subcellular Ca2+ transport in the 20-min ischemic or reperfused hearts. When hearts were made ischemic for 60 and 90 min, the recovery of contractile force on reperfusion was 50 and 7%, respectively. There was a progressive decrease in mitochondrial and microsomal Ca2+ binding and uptake activities after 60 and 90 min of ischemia; these changes were evident at various times of incubation period and at different concentrations of Ca2+. Mitochondrial Ca2+ transport changes were only partially reversible upon reperfusion after 60 and 90 min of ischemia, whereas the microsomal Ca2+ binding, uptake and Ca2+ ATPase activities deteriorated further upon reperfusion of the 90-min ischemic hearts. Ultrastructural changes increased with the duration of the ischemic insult and reperfusion injury was extensive in the 90-min ischemic hearts. These data show that the lack of recovery of contractile function upon reperfusion after a prolonged ischemic insult was accompanied by defects in sarcoplasmic reticulum Ca2+ transporting properties and structural damage.

Animals↗

PubMed1985

Activation of Ca2+-stimulated ATPase by phospholipid N-methylation in cardiac sarcoplasmic reticulum.

Incubation of cardiac sarcoplasmic reticulum (SR) in the presence of S-adenosyl-L-methionine, a methyl donor for the enzymatic N-methylation of phosphatidylethanolamine, increased Ca2+-stimulated ATPase activity. The increase in Ca2+-ATPase activity was not due to changes in the affinity for Ca2+ and was prevented by methyl acetimidate, an inhibitor of phospholipid N-methylation. The results suggest a possible regulatory role of phospholipid N-methylation in SR Ca2+-pump mechanism.

Animals↗

PubMed1985

Subcellular localization of phosphatidylethanolamine N-methylation activity in rat heart.

Synthesis of phosphatidylcholine (PC) by S-adenosyl-L-methionine (AdoMet)-dependent methylation of phosphatidylethanolamine (PE) has been recently characterized in rat heart sarcolemma obtained by hypotonic shock-LiBr treatment method. The present study, employing different procedures for the isolation of purified cardiac sarcolemmal membranes in rat, confirms the existence of three catalytic sites which are specifically involved in the sequential methyl transfer reactions from PE to PC. Other subcellular organelles such as sarcoplasmic reticulum (microsomes) and mitochondria showed methyltransferase activity which was absent in myofibrils and in cytosolic fraction. Experiments with several concentrations of AdoMet revealed that the kinetic pattern of methyltransferase activity in both microsomes and mitochondria was comparable to that obtained in sarcolemma. In addition, the characteristics of three catalytic sites as identified by the synthesis of phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine and PC in these subcellular organelles were similar to those of sarcolemma. The results are consistent with the view that methyltransferase activity is localized in different membrane systems of the myocardium.

Animals↗

PubMed1985

Adaptive changes in subcellular calcium transport during catecholamine-induced cardiomyopathy.

Rats were injected intraperitoneally with isoproterenol in dosage of 40 mg/kg body weight and heart microsomal and mitochondrial fractions were isolated 3, 9 and 24 h later. The heart/body weight ratio increased at 9 and 24 h after injection without any changes in the yield of subcellular organelles. Microsomal calcium uptake was significantly elevated at 3 h but returned to normal at 9 h and then became depressed 24 h post-injection. Mitochondrial calcium uptake was significantly increased 9 and 24 h after isoproterenol administration. Kinetic parameters of the calcium transport function indicated that the apparent affinity for Ca2+ remained unchanged, whereas Vmax values were altered in the experimental groups. Although there was no significant change in the phospholipid composition, the total phospholipid contents were increased (at 3, 9 and 24 h for microsomes; 3 and 9 h for mitochondria) in both types of organelles. The protein composition, as determined by gel electrophoresis, was altered in microsomes, but not in mitochondria. These results demonstrate rapid structural and functional changes in subcellular organelles. Such alterations may play an adaptive role in maintaining the intracellular calcium homeostasis during the development of catecholamine-induced cardiomyopathy.

Animals↗

PubMed1985

Evidence for three catalytic sites in heart sarcolemmal phospholipid N-methylation.

Conversion of phosphatidylethanolamine to phosphatidylcholine through sequential transmethylation reactions was studied in rat cardiac sarcolemmal membrane prepared by the hypotonic shock-LiBr treatment method. Three catalytic sites were identified on the basis of their requirement for divalent cations and kinetic parameters. Site I (Km = 0.1 +/- 0.01 microM) required Mg2+ as a cofactor and had a very high affinity for [3H]-S-adenosyl-L-methionine (AdoMet). Sites II (Km = 2.9 +/- 0.3 microM) and III (Km = 112 +/- 6 microM) did not require any divalent cation and showed comparatively lower affinities for AdoMet. Developmental-related differences were found to exist among these three catalytic sites when phospholipid methylation was carried out in 1- to 12-week-old rats. The results suggest that three distinct methyltransferase sites are involved in cardiac sarcolemmal phospholipid methylation.

Animals↗