Biomedical subjects

N S Dhalla

Publications and source records attributed to N S Dhalla.

At least 73 records · Page 4Linked to original sources

PubMed1998

Modification of ischemia-reperfusion-induced changes in cardiac sarcoplasmic reticulum by preconditioning.

To examine the effects of ischemic preconditioning on ischemia-reperfusion-induced changes in the sarcoplasmic reticulum (SR) function, isolated rat hearts were either perfused with a control medium for 30 min or preconditioned with three episodes of 5-min ischemia and 5-min reperfusion before sustained ischemia for 30 min followed by reperfusion for 30 min was induced. Preconditioning itself depressed cardiac function (left ventricular developed pressure, peak rate of contraction, and peak rate of relaxation) and SR Ca2+-release and -uptake activities as well as protein content and Ca2+/calmodulin-dependent protein kinase (CaMK) phosphorylation of Ca2+-release channels by 25-60%. Global ischemia for 30 min produced marked depressions in SR Ca2+-release and -uptake activities as well as SR Ca2+-pump protein content in control hearts; these changes were significantly attenuated by preconditioning. Compared with the control preparations, preconditioning improved the recovery of cardiac function and SR Ca2+-release and -uptake activities as well as Ca2+-release channel and Ca2+-pump protein contents in the ischemic-reperfused hearts. Unlike the protein kinase A-mediated phosphorylation in SR membranes, the CaMK-mediated phosphorylations at Ca2+-release channels, Ca2+ pump, and phospholamban were depressed in the ischemic hearts; these changes were prevented by preconditioning. These results indicate that ischemic preconditioning may exert beneficial effects on ischemia-reperfusion-induced alterations in SR function by preventing changes in Ca2+-release channel and Ca2+-pump protein contents in the SR membrane.

Animals↗

PubMed1998

Mechanisms of lysophosphatidylcholine-induced increase in intracellular calcium in rat cardiomyocytes.

Previous reports have demonstrated that lysophosphatidylcholine (LPC) increases the intracellular concentration of calcium ([Ca++]i) in the heart; however, the mechanisms responsible for this increase are not clear. We examined the effect of exogenous LPC on [Ca++]i in freshly isolated cardiomyocytes from adult rats. Our results showed that LPC elevated the [Ca++]i in a dose-dependent (2.5-10 microM) manner. The LPC (10 microM)-induced increase in [Ca++]i was augmented upon increasing the concentration of extracellular Ca++ and was abolished by the removal of Ca++ from the medium. Preincubation of cardiomyocytes with sarcolemmal L-type Ca++ channel blocker, verapamil, did not affect the LPC-evoked increase in [Ca++]i significantly. On the other hand, ouabain, a Na(+)-K+ ATPase inhibitor, and low concentrations of extracellular Na+ enhanced the LPC response. The LPC-induced increase in [Ca++]i was attenuated significantly by the inhibitors of Na(+)-Ca++ exchanger such as Ni++ and amiloride. Depletion of the sarcoplasmic reticulum (SR) Ca++ stores by low micromolar concentrations of ryanodine (a SR Ca(++)-release channel activator) or by thapsigargin (a SR Ca(++)-pump ATPase inhibitor) depressed the LPC-mediated increase in [Ca++]i. Combined blockade of Na(+)-Ca++ exchanger and inhibition of SR Ca(++)-pump or ryanodine receptor had an additive effect on the LPC response. These observations suggest that the increase in [Ca++]i induced by LPC depends on both Ca(++)-influx from the extracellular space and Ca(++)-release from the SR stores. Furthermore, Na(+)-Ca++ exchange plays a critical role in the LPC-mediated entry of Ca++ into cardiomyocytes.

Amiloride↗

PubMed1998

Role of H2O2 in changing beta-adrenoceptor and adenylyl cyclase in ischemia-reperfused hearts.

In view of the accumulation of H2O2 in the myocardium due to ischemia-reperfusion and changes in beta-adrenoceptor mechanisms in the ischemic-reperfused heart, we investigated the effects of H2O2 on the beta-adrenoceptor, G-protein and adenylyl cyclase complex. Rat hearts were perfused with 1 mM H2O2 for 10 min before isolating membranes for measuring the biochemical activities. The stimulation of adenylyl cyclase by different concentrations of isoproterenol was depressed upon perfusing hearts with H2O2. Both the affinity and density of beta1-adrenoceptors as well as the density of the beta2-adrenoceptors were decreased whereas the affinity of beta2-adrenoceptors was increased by H2O2 perfusion. Competition curves did not reveal any effect of H2O2 on the proportion of coupled receptors in the high affinity state. The basal as well as forskolin-, NaF- and Gpp(NH)p-stimulated adenylyl cyclase activities were depressed by perfusing the heart with H2O2. Catalase alone or in combination with mannitol was able to significantly decrease the magnitude of alterations due to H2O2. The positive inotropic effect of 1 microM isoproterenol was markedly attenuated upon perfusing hearts with 200-500 microM H2O2 for 10 min. These results suggest that H2O2 may depress the beta1-adrenoceptor, Gs-proteins and catalytic subunit of the adenylyl cyclase enzyme and thus may play an important role in attenuating the beta-adrenoceptor linked signal transduction due to ischemia-reperfusion injury.

Adenylyl Cyclases↗

PubMed1998

Cardiac sarcolemmal Na(+)-Ca2+ exchange and Na(+)-K+ ATPase activities and gene expression in alloxan-induced diabetes in rats.

To determine the sequence of alterations in cardiac sarcolemmal (SL) Na(+)-Ca2+ exchange, Na(+)-K+ ATPase and Ca(2+)-transport activities during the development of diabetes, rats were made diabetic by an intravenous injection of 65 mg/kg alloxan. SL membranes were prepared from control and experimental hearts 1-12 weeks after induction of diabetes. A separate group of 4 week diabetic animals were injected with insulin (3 U/day) for an additional 4 weeks. Both Na(+)-K+ ATPase and Ca(2+)-stimulated ATPase activities were depressed as early as 10 days after alloxan administration; Mg2+ ATPase activity was not depressed throughout the experimental periods. Both Na(+)-Ca2+ exchange and ATP-dependent Ca(2+)-uptake activities were depressed in diabetic hearts 2 weeks after diabetes induction. These defects in SL Na(+)-K+ ATPase and Ca-transport activities were normalized upon treatment of diabetic animals with insulin. Northern blot analysis was employed to compare the relative mRNA abundances of alpha 1-subunit of Na(+)-K+ ATPase and Na(+)-Ca2+ exchanger in diabetic ventricular tissue vs. control samples. At 6 weeks after alloxan administration, a significant depression of the Na(+)-K+ ATPase alpha 1-subunit mRNA was noted in diabetic heart. A significant increase in the Na(+)-Ca2+ exchanger mRNA abundance was observed at 3 weeks which returned to control by 5 weeks. The results from the alloxan-rat model of diabetes support the view that SL membrane abnormalities in Na(+)-K+ ATPase, Na+Ca2+ exchange and Ca(2+)-pump activities may lead to the occurrence of intracellular Ca2+ overload during the development of diabetic cardiomyopathy but these defects may not be the consequence of depressed expression of genes specific for those SL proteins.

Alloxan↗

PubMed1998

Differential influence of fasting and BM13.907 treatment on growth and phenotype of pressure overloaded rat heart.

To examine metabolic influences on markers of the fetal phenotype of pressure overloaded rat heart, rats with stenosis of the abdominal aorta were intermittently fasted for 10-12 weeks. Although intermittent fasting, which reduces insulin mediated glucose uptake in the heart and moderate pressure overload of the left ventricle increased the proportion of myosin beta-heavy chains (beta-MHC) and reduced the Ca(2+)-stimulated ATPase activity of sarcoplasmic reticulum (SR) to a similar extent, these interventions had no additive effects when combined. Furthermore, addition of sucrose (0.8%) to the drinking water prevented the changes in both the beta-MHC proportion and SR Ca(2+)-stimulated ATPase activity due to pressure overload or fasting. To assess the effects of a drug which stimulates glucose-carrier translocation, rats with stenosis of the abdominal aorta were treated with BM13.907 (50 and 100 mg/kg daily for 10-12 weeks). This treatment amplified the left ventricular hypertrophy (+43 vs. 21% of untreated rats) and increased the beta-MHC proportion. The SR Ca(2+)-stimulated ATPase activity of pressure overloaded rats treated with BM13.907 (100 mg/kg/day) was, however, not reduced compared with sham operated control rats. Thus, an intervention which is known to stimulate insulin-mediated glucose-carrier translocation, but not glucose-carrier activation, partially prevented the characteristic phenotype of pressure overloaded hearts. These data provide further evidence in favor of metabolic influences linked to glucose uptake on growth and phenotype of the pressure overloaded heart.

Administration, Oral↗

PubMed1997

Modification of heart sarcolemmal phosphoinositide pathway by lysophosphatidylcholine.

Although lysophosphatidylcholine (lyso-PtdCho) accumulates in the sarcolemmal (SL) membrane and alters its function during myocardial ischemia and diabetic cardiomyopathy, the effects of lyso-PtdCho on SL signalling processes have not yet been investigated. The present study was carried out to examine the actions of lyso-PtdCho on the rat heart SL membrane enzymes involved in the phosphoinositide pathway. Different lyso-PtdCho species (10 to 200 microM) inhibited the activities of both phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase in the SL membrane in a concentration-dependent manner. The inhibitory potency of lyso-PtdCho compounds for phosphatidylinositol kinase was lyso-PtdCho plasmalogen > 1-oleoyl-lyso-PtdCho > 1-stearoyl-lyso-PtdCho > 1-palmitoyl-lyso-PtdCho, and that for phosphatidylinositol-4-phosphate kinase was lyso-PtdCho plasmalogen > 1-oleoyl-lyso-PtdCho > 1-palmitoyl-lyso-PtdCho > 1-stearoyl-lyso-PtdCho. The inhibitory effect of lyso-PtdCho on phosphatidylinositol-4-phosphate kinase was greater than that on phosphatidylinositol kinase. Lyso-PtdCho structural analogues, such as phosphatidylcholine, lysophosphatidic acid, lysophosphatidylethanolamine, L-alpha-glycerophosphate, oleate and phosphorylcholine, did not affect the phosphoinositide kinases, suggesting that the intact structure of lyso-PtdCho was required for the inhibition of the kinases. The detrimental action of lyso-PtdCho on PtdIns kinase was potentiated by acidosis. Unlike Ca2+, ATP (0.1 and 4 mM) increased lyso-PtdCho-induced deactivation of the kinases. Both enzyme activities were found to be depressed in the ischemic-reperfused or diabetic hearts. None of the tested lyso-PtdCho species altered phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis by SL phospholipase C. These results indicate that accumulation of lyso-PtdCho in the SL membrane under pathological conditions may diminish the availability of the PtdIns(4,5)P2 substrate for the production of second messengers by receptor-linked phospholipase C.

1-Phosphatidylinositol 4-Kinase↗

PubMed1997

Phosphorylation of cardiac Na+-K+ ATPase by Ca2+/calmodulin dependent protein kinase.

Na+-K+ ATPase is known to be involved in the transport of sodium and potassium across the cell membrane. We describe here a novel mechanism for the regulation of cardiac Na+-K+ ATPase through phosphorylation by a Ca2+/calmodulin-dependent protein kinase (CaM kinase) present in the sarcolemmal membrane. Incubation of cardiac sarcolemma in the presence of Ca2+ and calmodulin resulted in phosphorylation of a 110 kDa protein, identified as the alpha-subunit of Na+-K+ ATPase. The compound W-7, a potent inhibitor of calmodulin, caused significant inhibition of the CaM kinase-mediated phosphorylation while ouabain, a potent inhibitor of Na+-K+ ATPase, had no effect. Furthermore, phosphorylation of the sarcolemmal membrane with Ca2+/calmodulin caused significant reduction in the activity of Na+-K+ ATPase. These results suggest that phosphorylation of the alpha-subunit of Na+-K+ ATPase by an endogenous CaM kinase may lead to an inhibition of its catalytic activity.

Animals↗

PubMed1997

Identification of changes in cardiac phospholipase C activity in congestive heart failure.

Although phosphoinositide-specific phospholipase C (PLC) is involved in signal transduction mechanisms of the myocardial cell. very little is known about its status in congestive heart failure (CHF). We have examined the PLC activity in sarcolemmal and cytosolic fractions isolated from the viable left ventricle of rats at 8 weeks (moderate stage of CHF) and 16 weeks (severe stage of CHF) after occlusion of the left anterior descending coronary artery; the hypertrophied right ventricle was used for comparison. At 8 weeks, the hydrolysis of phosphatidylinositol 4,5-bisphosphate by sarcolemmal PLC was reduced by 37% of sham control values only in the left ventricle, whereas at 16 weeks, PLC-mediated hydrolysis was depressed in both left and right ventricles by 25% and 30%, respectively. The hydrolysis of phosphatidylinositol 4-monophosphate (PIP) was reduced by 25% of control value only in the severely failing left ventricle, while the phosphatidylinositol (PI) hydrolysis remained unaltered. Kinetic studies of PLC activity in the left ventricle showed a depression of V(max) at moderate and severe failure stages, whereas the affinity for the substrate was increased in the left ventricle at 8 weeks and decreased in the right ventricle at 16 weeks. The only difference observed between experimental and control groups at the cytosolic level, was a significant enhancement of PLC activity in the severely failing left ventricle when PIP was given as a substrate, and in the corresponding right ventricle when PI was the substrate. The results of this study identify time-related defects in sarcolemmal PLC in right and left ventricles during the development of CHF due to myocardial infarction.

Analysis of Variance↗

PubMed1997

Beta-adrenoceptor-linked signal transduction in ischemic-reperfused heart and scavenging of oxyradicals.

In order to examine the mechanisms of ischemia-reperfusion induced changes in beta-adrenoceptor-linked signal transduction pathway, isolated rat hearts perfused in the absence or presence of superoxide dismutase (SOD) plus catalase (CAT) were made ischemic for 30 min and then reperfused for 60 min. The left ventricular developed pressure as well as the rare of contraction and rate of relaxation were markedly decreased, whereas the left ventricular end-diastolic pressure increased in the ischemic hearts. A significant increase in the density and affinity of beta 1-adrenoceptors without any changes in the characteristics of beta 2-adrenoceptors was evident in cardiac membranes obtained from the ischemic hearts. The recovery of contractile abnormalities in the ischemic heart was depressed upon reperfusion; the ischemic-reperfused hearts also showed attenuated inotropic responses to isoproterenol. The affinities and densities of beta- and beta-adrenoceptors were decreased in the ischemic-reperfused hearts; the magnitude of changes in beta 1-adrenoceptors was greater than that in beta 2-adrenoceptors. The isoproterenol-stimulated adenylyl cyclase activity was depressed in both ischemic hearts and ischemic-reperfused hearts. The basal and forskolin-stimulated adenylyl cyclase activities were unaltered due to ischemia but were increased upon reperfusion. The NaF- and 5'-Guanylyl-imidodiphosphate[Gpp(NH)p]-stimulated adenylyl cyclase activities were depressed in the ischemic hearts and increased in the ischemic reperfused hearts. Cholera toxin (CT)-stimulated adenylyl cyclase as well as the CT-catalysed ADP-ribosylation activity and stimulatory G protein (Gs protein) immunoreactivity were decreased in the ischemic hearts and increased in the reperfused hearts. Pertussis toxin (PT)-stimulated adenylyl cyclase activity was unaltered in both ischemic and ischemic-reperfused hearts, whereas the PT-catalysed ribosylation and inhibitory G protein (Gi protein) immunoactivity were slightly increased in the reperfused myocardium. Thus the inability of isoproterenol to stimulate adenylyl cyclase in the ischemic-reperfused hearts may be due to alterations mainly in the characteristics of beta 1-adrenoceptors including density, affinity and coupling with the adenylyl cyclase. Scavenging of oxyradicals by the addition of SOD plus CAT in the perfusion medium prevented the reperfusion-induced changes in contractile function, inotropic responses of the heart to isoproterenol, activation of adenylyl cyclase by isoproterenol, as well as densities and affinities of beta-adrenoceptors in cardiac membranes. These results suggest that the depressed contractile activity and the attenuated inotropic responses of ischemic-reperfused hearts to isoproterenol as well as the defects in beta-adrenoceptor-linked signal transduction may be due to the formation of oxyradicals in the myocardium.

Adenylate Cyclase Toxin↗

PubMed1997

Phosphatidic acid: a potential signal transducer for cardiac hypertrophy.

Phosphatidic acid (PA) is mainly formed by the hydrolysis of phosphatidylcholine due to the activation of phospholipase D (PLD). PA is also generated by phosphorylation of diacylglycerol (DAG) due to the action of DAG kinase and is converted to DAG under the action of PA phosphohydrolase. Most of the positive inotropic agents which are known to stimulate cardiac hypertrophy, have been shown to increase the level of PA in cardiac sarcolemma. Although the growth factor-like effect of PA has been recognized in a wide variety of tissues, there is a lack of similar information in adult cardiomyocytes. By using single cardiomyocytes, we have now shown that PA increased the basal [Ca2+]i level without significant effect on the amplitude of Ca2+ transients. PA (10-50 mu M) also increased the [Ca2+]i in cardiac cell suspension. PA has also been shown to stimulate protein synthesis in cardiomyocytes, which is inhibited by a PKC inhibitor as well as a Ca2+ chelator. PA at the concentration of 1-50 mu M was observed to stimulate the activity of PLC in cardiac sarcolemma; this effect was attenuated by a PLC inhibitor. Since DAG, formed due to the activation of PLC, is considered to play a crucial role in regulating the activity of protein kinase C (PKC), the positive feedback effect of PA on this pathway may be essential for maintaining the sustained elevation in the activity of PKC during the development of cardiac hypertrophy. In view of these observations and other facts available in the literature, it is suggested that PA may be a potential signal transducer for the development of cardiac hypertrophy.

Animals↗

PubMed1997

Differential changes in sympathetic activity in left and right ventricles in congestive heart failure after myocardial infarction.

Although congestive heart failure subsequent to myocardial infarction is known to be associated with increased sympathetic activity, very little information regarding changes in the sympathetic nerves in the left and right ventricles at various stages after infarction is available. Male Sprague-Dawley rats were subjected to coronary artery ligation and studied 4 and 8 weeks later; these animals had mild and moderate stages of congestive heart failure. A sham group, without coronary ligation, was used as control. Four weeks after myocardial infarction, plasma and ventricular (left and right) epinephrine (EPI), unlike norepinephrine (NE), were markedly increased. Whereas plasma catecholamine (EPI and NE) levels were increased 8 weeks after infarction, NE concentration in the left ventricle was unchanged but EPI concentration was increased in comparison with sham control. The right ventricle showed an increased level of both NE and EPI 8 weeks after infarction. Measurement of the rate of change in the specific activity of NE (NE turnover) in the left and right ventricles 8 weeks after infarction revealed an increase in NE turnover in the left ventricle, without any changes in the right ventricle. The concentration of EPI, unlike NE, was increased in the kidney, spleen, and brain 8 weeks after coronary occlusion. These results are interpreted to mean that congestive heart failure caused by myocardial infarction is associated with differential changes in the status of sympathetic nerves in the left and right ventricles; sympathetic activity is increased only in the left ventricle, whereas the right ventricle may play an adaptive role by increasing catecholamine stores during the development of heart failure.

Animals↗

PubMed1997

Myosin light-chain phosphorylation in diabetic cardiomyopathy in rats.

The regulatory myosin light chain (MLC) is phosphorylated in cardiac muscle by Ca2+/calmodulin-dependent MLC kinase (MLCK) and is considered to play a modulatory role in the activation of myofibrillar adenosine triphosphatase (ATPase) and the process of force generation. Since the depression in cardiac contractile function in chronic diabetes is associated with a decrease in myofibrillar ATPase activity, we investigated changes in MLC phosphorylation in diabetic heart. Rats were made diabetic by injecting streptozotocin (65 mg/kg intravenously), and the hearts were removed 8 weeks later; some 6-week diabetic animals were injected with insulin (3 U/d) for 2 weeks. Changes in the relative MLC and MLCK protein contents were measured by electrophoresis and immunoblot assay, whereas phosphorylated and unphosphorylated MLCs were separated on 10% acrylamide/urea gel and identified by Western blot. MLC and MLCK contents were decreased markedly (40% to 45%) and MLC phosphorylation was decreased significantly (30% to 45%) in the diabetic rat heart homogenate in comparison to control values. The changes in MLC and MLCK content in diabetic heart were partially reversible, whereas changes in MLC phosphorylation were normalized upon treatment with insulin. These results suggest that decreased protein contents of MLC and MLCK and phosphorylation of MLC may contribute to the depression of cardiac myofibriliar ATPase activity and heart dysfunction in diabetic cardiomyopathy.

Adenosine Triphosphatases↗

PubMed1997

Subcellular alterations in cardiac phospholipase D activity in chronic diabetes.

Several studies have suggested that myocardial phospholipase D (PLD) and its hydrolytic product, phosphatidic acid (PtdOH), may regulate Ca2+ movements and contractile performance of the heart. Since abnormal intracellular Ca2+ handling is a major factor of myocardial dysfunction in chronic diabetes, we examined subcellular changes in PLD activity in myocardium from insulin-dependent diabetic rats. Diabetes in rats was induced by a single i.v. injection of streptozotocin (65 mg/kg body wt) and 8 weeks later the ventricular tissue was processed for the isolation of sarcolemma, sarcoplasmic reticulum and mitochondria. Compared to age-matched controls, the sarcolemmal, sarcoplasmic reticular and mitochondrial PLD activities were significantly depressed in the diabetic animals. The depressed sarcolemmal PLD activity was normalized, whereas the sarcoplasmic reticular and mitochondrial enzyme activities were partially reversed upon treating the 6-week diabetic rats with insulin for a period of 2 weeks. These data suggest that the reduction of PLD-derived PtdOH may lead to an impairment in this phospholipid signal transduction pathway and subsequent cardiac dysfunction in chronic diabetes.

Animals↗

PubMed1997

Differential changes in left and right ventricular adenylyl cyclase activities in congestive heart failure.

The status of beta-adrenergic receptors and adenylyl cyclase in crude membranes from both left and right ventricles was examined when the left coronary artery in rats was occluded for 4, 8, and 16 wk. The adenylyl cyclase activity in the presence of isoproterenol was decreased in the uninfarcted (viable) left ventricle and increased in the right ventricle subsequent to myocardial infarction. The density of beta1-adrenergic receptors, unlike beta2-receptors, was reduced in the left ventricle, whereas no change in the characteristics of beta1- and beta2-adrenergic receptors was seen in the right ventricle. The catalytic activity of adenylyl cyclase was depressed in the viable left ventricle but was unchanged in the right ventricle. In comparison to sham controls, the basal, as well as NaF-, forskolin-, and 5'-guanylyl imidodiphosphate [Gpp(NH)p]-stimulated adenylyl cyclase activities were decreased in the left ventricle and increased in the right ventricle of the experimental animals. Opposite alterations in the adenylyl cyclase activities in left and right ventricles from infarcted animals were also seen when two types of purified sarcolemmal preparations were employed. These changes in adenylyl cyclase activities in the left and right ventricles were dependent on the degree of heart failure. Furthermore, adenosine 3',5'-cyclic monophosphate contents were higher in the right ventricle and lower in the left ventricle from infarcted animals injected with saline, isoproterenol, or forskolin in comparison to the controls. The results suggest differential changes in the viable left and right ventricles with respect to adenylyl cyclase activities during the development of congestive heart failure due to myocardial infarction.

Adenylyl Cyclases↗

PubMed1997

Fura-2 fluorescent technique for the assessment of Ca2+ homeostasis in cardiomyocytes.

Ca2+ homeostasis plays a pivotal role in maintaining cell growth and function. Many heart diseases are related to the abnormalities in Ca2+ mobilization and extrusion. Ca2+-sensitive fluorescent dyes have been used successfully to estimate intracellular free Ca2+ ([Ca2+]i) level and the mechanisms of Ca2+ movements in living cells. This article is focused on the methodology involving the use of Fura-2/AM or free Fura-2 to measure agonist-induced Ca2+ mobilization as well as the mechanisms of changes in [Ca2+]i in cardiomyocytes. Methods involving Fura-2 technique for the measurement of Ca2+ extrusion from the cells and Ca2+ reuptake by sarcoplasmic reticulum (SR) are also described. The prevention of KCl-induced increase in the intracellular Ca2+ is shown by chelating the extracellular Ca2+ with EGTA or by the presence of Ca2+-channel inhibitors such as verapamil and diltiazem. The involvement of SR in the ATP-induced increase in intracellular Ca2+ is illustrated by the use of Ca2+-pump inhibitors, thapsigargin and cyclopiazonic acid as well as ryanodine which deplete the SR Ca2+ storage. The use of 2-nitro-4-carboxyphenyl N,N-diphenyl carbamate (NCDC), an inhibitor of inositol 1,4,5-trisphosphate (IP3) production, is described for the attenuation of phosphatidic acid (PA) induced increase in Ca2+-mobilization. The increase in intracellular Ca2+ in cardiomyocytes by PA, unlike that by KCl or ATP, was observed in diabetic myocardium. Thus, it appears that the Fura-2 method for the measurement of Ca2+ homeostasis in cardiomyocytes is useful in studying the pathophysiology and pharmacology of Ca2+ movements.

Animals↗

PubMed1997

Biphasic alterations in cardiac beta-adrenoceptor signal transduction mechanism due to oxyradicals.

To assess the effects of oxyradicals on cardiac beta-adrenoceptors, G-proteins and adenylyl cyclase, rat heart membranes were incubated with xanthine (X) plus xanthine oxidase (XO) for different intervals. The basal as well as forskolin-, NaF-, 5'-guanylylimidodiphosphate and isoproterenol-stimulated adenylyl cyclase activities showed an increase at 10 min and a decrease at 30 min of incubation with X plus XO. Treatment of membranes with H2O2 also produced biphasic changes in adenylyl cyclase activities. The density of beta1-adrenoceptors was decreased when cardiac membranes were treated with X plus XO for 10 and 30 min whereas the affinity of beta1-adrenoceptors was increased after 10 min and reduced after 30 min of incubation. The beta2-adrenoceptors were not modified at 10 min whereas incubation of cardiac membranes with X plus XO for 30 min increased the affinity and decreased the density. Cholera toxin-stimulated adenylyl cyclase activity, cholera toxin-catalyzed ADP-ribosylation and stimulatory guanine nucleotide binding protein immunoreactivity in cardiac membranes were increased at 10 min and decreased at 30 min of incubation with X plus XO. However, the pertussis toxin-stimulated adenylyl cyclase activity, pertussis toxin-catalyzed ADP ribosylation and inhibitory guanine nucleotide binding protein immunoreactivity were not affected on treatment of membranes with X plus XO. Addition of superoxide dismutase plus catalase in the incubation medium prevented the X plus XO-induced alterations in adenylyl cyclase activities, stimulatory guanine nucleotide binding protein-related ADP-ribosylation and changes in the characteristics of beta-adrenoceptors except the increased affinity of beta1-adrenoceptors at 10 min of incubation. These data suggest that alterations in the beta1-adrenoceptor-linked stimulatory guanine nucleotide binding protein-adenylyl cyclase pathway due to X plus XO are biphasic in nature and these changes may likely be due to the formation of H2O2.

Adenylyl Cyclases↗

PubMed1996

Stimulation of protein synthesis by phosphatidic acid in rat cardiomyocytes.

Phosphatidic acid (PA) was observed to stimulate protein synthesis in adult cardiomyocytes in a time- and concentration-dependent manner. The maximal stimulation in protein synthesis (142 +/- 12% vs 100% as the control) was achieved at 10 microM PA within 60 min and was inhibited by actinomycin D (107 +/- 4% of the control) or cycloheximide (105 +/- 6% of the control). The increase in protein synthesis due to PA was attenuated or abolished by preincubation of cardiomyocytes with a tyrosine kinase inhibitor, genistein (94 +/- 9% of the control), phospholipase C inhibitors 2-nitro-4-carboxyphenyl N,N-diphenyl carbamate or carbon-odithioic acid O-(octahydro-4,7-methanol-1H-inden-5-yl (101 +/- 6 and 95 +/- 5% of the control, respectively), protein kinase C inhibitors staurosporine or polymyxin B (109 +/- 3 and 93 +/- 3% of the control), and chelators of extracellular and intracellular free Ca2+ EGTA or BAPTA/AM (103 +/- 6 and 95 +/- 6% of the control, respectively). PA at different concentrations (0.1 to 100 microM) also caused phosphorylation of a cell surface protein of approximately 24 kDa. In addition, mitogen-activated protein kinase was stimulated by PA in a concentration-dependent manner; maximal stimulation (217 +/- 6% of the control) was seen at 10 microM PA. These data suggest that PA increases protein synthesis in adult rat cardiomyocytes and thus may play an important role in the development of cardiac hypertrophy.

Animals↗

PubMed1996

Alteration in cardiac sarcolemmal ATP receptors by oxyradicals.

We previously demonstrated that cardiac sarcolemmal membranes bind [35S]ATP gamma S at both low and high affinity binding sites. In this study we examined the effects of some P2-purinoceptor antagonists as well as of two oxidants (H2O2 and HOCl) on the high affinity ATP-binding sites under in vitro conditions. It was found that putative P2-purinoceptor antagonists such as Cibacron blue, suramin, and 4,4'-diisothiocyanatostilbene 2-2 acid markedly inhibited specific ATP-binding with sarcolemmal membrane. H2O2 produced a biphasic effect (first increase and then decrease) on the specific ATP-binding with cardiac sarcolemma in a time- and concentration-dependent manner; these effects were prevented by catalase. On the other hand, HOCl markedly inhibited ATP-binding; this inhibition was prevented by l-methionine. These results suggest that the high affinity ATP-binding sites in cardiac sarcolemma may represent the P2-purinoceptors, which are susceptible to modification by oxidative stress under pathophysiological conditions including myocardial ischemia-reperfusion injury.

Adenosine Triphosphate↗