Premethylation of purified porcine cardiac sarcolemma (SL) in the presence of 0.15, 10 and 150 microM S-adenosyl-L-methionine (AdoMet) did not change the phosphorylation of SL proteins catalyzed either by intrinsic cyclic AMP-dependent protein kinase (cAK) or by added catalytic (C) subunit of this enzyme. On the other hand, membrane exhibited increased lipid methyltransferase activity after preincubation with MgATP and C subunit. Prephosphorylation of membranes stimulated the total [3H]-methyl incorporation into SL lipids assayed at 0.15 microM [3H]AdoMet due to an enhancement of Vmax and without changes in the Km value for AdoMet. Analysis of the methylated lipid products revealed an increased methyl group incorporation into a nonpolar lipid fraction whereas phosphatidylethanolamine-N-methylation was not affected by phosphorylation. The results suggest that the cyclic AMP-mediated signal transduction at the level of cardiac SL is not affected by methylation-induced modifications of the membrane lipid microdomains. On the other hand, an intrinsic SL lipid methyltransferase activity is apparently not related to the N-methylation of phospholipids, is modulated by cyclic AMP-dependent protein phosphorylation.

Adenosine Triphosphate↗

PubMedDate not supplied

Characteristics of Ca2(+)-stimulated ATPase in rat heart sarcolemma in the presence of dithiothreitol and alamethicin.

We have studied the activities of Ca2(+)-stimulated ATPase in rat heart sarcolemma upon modulating the redox state of membrane thiol groups with dithiothreitol (DTT). The suitability of alamethicin to unmask the latent activity of this enzyme was also investigated. The Ca2(+)-stimulated ATPase in sarcolemma exhibited two activation sites--one with low affinity (Km = 0.70 +/- 0.2 mM; Vmax = 10.0 +/- 2.2 mumol Pi/mg/h) and the other with high affinity (Km = 0.16 +/- 0.7 mM; Vmax = 4.6 +/- 0.8 mumol Pi/mg/h) for Mg2+ ATP. Alamethicin at a ratio of 1:1 with the sarcolemmal protein caused a 3-fold activation of Ca2(+)-stimulated ATPase without affecting its sensitivity to Ca2+ or Mg2+ ATP. Treatment of sarcolemma with deoxycholate or sodium dodecyl sulfate resulted in a total loss of the enzyme activity; high concentrations of alamethicin also showed a detergent-like action on the sarcolemmal vesicles. DTT at 5-10 mM concentrations caused a 4-5 fold activation of Ca2(+)-stimulated ATPase in sarcolemma and this effect was observed to be dependent on the concentration of Mg2+ ATP. DTT increased the affinity of the enzyme to Mg2+ ATP at the high affinit site and enhanced the Vmax at the low affinity site in addition to increasing the sensitivity of Ca2(+)-stimulated ATPase to Ca2+. DTT protected the Ca2(+)-stimulated ATPase against deterioration by detergents and restored the enzyme activity after treatment with N-ethylmaleimide. The mechanism of action of DTT on Ca2(+)-stimulated ATPase may involve the reduction of essential thiols at the active site of the enzyme or its interaction with specific DTT-dependent inhibitor protein. No changes in the sensitivity of sarcolemmal Ca2(+)-stimulated ATPase to orthovanadate was evident in the absence or presence of DTT and alamethicin. The results suggest the use of both DTT and alamethicin for the determination of Ca2(+)-stimulated ATPase activity in sarcolemmal preparations.

Alamethicin↗

PubMedDate not supplied

Relationship between mechanical dysfunction and depression of sarcolemmal Ca(2+)-pump activity in hearts perfused with oxygen free radicals.

Although in vitro studies have shown that oxygen free radicals depress the sarcolemmal Ca(2+)-pump activity and thereby may cause the occurrence of intracellular Ca2+ overload for the genesis of contractile failure, the exact relationship between changes in sarcolemmal Ca(2+)-pump activity and cardiac function due to these radicals is not clear. In this study we examined the effects of oxygen radicals on sarcolemmal Ca2+ uptake and Ca(2+)-stimulated ATPase activities as well as contractile force development by employing isolated rat heart preparations. When hearts were perfused with medium containing xanthine plus xanthine oxidase, the sarcolemmal Ca(2+)-stimulated ATPase activity and ATP-dependent Ca2+ accumulation were depressed within 1 min whereas the developed contractile force, rate of contraction and rate of relaxation were increased at 1 min and decreased over 3-20 min of perfusion. The resting tension started increasing at 2 min of perfusion with xanthine plus xanthine oxidase. Catalase showed protective effects against these alterations in heart function and sarcolemmal Ca(2+)-pump activities upon perfusion with xanthine plus xanthine oxidase whereas superoxide dismutase did not exert such effects. The combination of catalase and superoxide dismutase did not produce greater effects in comparison to catalase alone. These results are consistent with the view that the depression of heart sarcolemmal Ca2+ pump activities may result in myocardial dysfunction due to the formation of hydrogen peroxide and/or hydroxyl radicals upon perfusing the hearts with xanthine plus xanthine oxidase.

Animals↗

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Sarcolemmal Na+-Ca2+ exchange and Ca2+-pump activities in cardiomyopathies due to intracellular Ca2+-overload.

In order to identify defects in Na+-Ca2+ exchange and Ca2+-pump systems in cardiomyopathic hearts, the activities of sarcolemmal Na+-dependent Ca2+ uptake, Na+-induced Ca2+ release, ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase were examined by employing cardiomyopathic hamsters (UM-X7.1) and catecholamine-induced cardiomyopathy produced by injecting isoproterenol into rats. The rates of Na+-dependent Ca2+ uptake, ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase activities of sarcolemmal vesicles from genetically-linked cardiomyopathic as well as catecholamine-induced cardiomyopathic hearts were decreased without any changes in Na+-induced Ca2+-release. Similar results were obtained in Ca2+-paradox when isolated rat hearts were perfused for 5 min with a medium containing 1.25 mM Ca2+ following a 5 min perfusion with Ca2+-free medium. Although a 2 min reperfusion of the Ca2+-free perfused hearts depressed sarcolemmal Ca2+-pump activities without any changes in Na+-induced Ca2+-release, Na+-dependent Ca2+ uptake was increased. These results indicate that alterations in the sarcolemmal Ca2+-efflux mechanisms may play an important role in cardiomyopathies associated with the development of intracellular Ca2+ overload.

Adenosine Triphosphatases↗

PubMedDate not supplied

Decreased expression of cardiac sarcoplasmic reticulum Ca(2+)-pump ATPase in congestive heart failure due to myocardial infarction.

Myocardial infarction in rats induced by occluding the left coronary artery for 4, 8 and 16 weeks has been shown to result in congestive heart failure (CHF) characterized by hypertrophy of the viable ventricular myocardial tissue. We have previously demonstrated a decreased calcium transport activity in the sarcoplasmic reticulum (SR) of post-myocardial infarction failing rat hearts. In this study we have measured the steady state levels of the cardiac SR Ca(2+)-pump ATPase (SERCA2) mRNA using Northern blot and slot blot analyses. The relative amounts of SERCA2 mRNA were decreased with respect to GAPDH mRNA and 28 S rRNA in experimental failing hearts at 4 and 8 weeks post myocardial infarction by about 20% whereas those at 16 weeks declined by about 35% of control values. The results obtained by Western blot analysis, revealed that the immunodetectable levels of SERCA2 protein in 8 and 16 weeks postinfarcted animals were decreased by about 20% and 30%, respectively. The left ventricular SR Ca(2+)-pump ATPase specific activity was depressed in the SR preparations of failing hearts as early as 4 weeks post myocardial infarction and declined by about 65% at 16 weeks compared to control. These results indicate that the depressed SR Ca(2+)-pump ATPase activity in CHF may partly be due to decreased steady state amounts of SERCA2 mRNA and SERCA2 protein in the failing myocardium.

Animals↗

PubMedDate not supplied

Inhibition of cardiac sarcolemma Na(+)-K+ ATPase by oxyradical generating systems.

The Na(+)-K+ ATPase activity and SH group content were decreased whereas malondialdehyde (MDA) content was increased upon treating the porcine cardiac sarcolemma with xanthine plus xanthine oxidase, which is known to generate superoxide and other oxyradicals. Superoxide dismutase either alone or in combination with catalase and mannitol fully prevented changes in SH group content but the xanthine plus xanthine oxidase-induced depression in Na(+)-K+ ATPase activity as well as increase in MDA content were prevented partially. The Lineweaver-Burk plot analysis of the data for Na(+)-K+ ATPase activity in the presence of different concentrations of MgATP or Na+ revealed that the xanthine plus xanthine oxidase-induced depression in the enzyme activity was associated with a decrease in Vmax and an increase in Km for MgATP; however, Ka value for Na+ was decreased. Treatment of sarcolemma with H2O2 plus Fe2+, an hydroxyl and other radical generating system, increased MDA content but decreased both Na(+)-K+ ATPase activity and SH group content; mannitol alone or in combination with catalase prevented changes in SH group content fully but the depression in Na(+)-K+ ATPase activity and increase in MDA content were prevented partially. The depression in the enzyme activity by H2O2 plus Fe2+ was associated with a decrease in Vmax and an increase in Km for MgATP. These results indicate that the depressant effect of xanthine plus xanthine oxidase on sarcolemmal Na(+)-K+ ATPase may be due to the formation of superoxide, hydroxyl and other radicals. Furthermore, the oxyradical-induced depression in Na(+)-K+ ATPase may be due to the formation of superoxide, hydroxyl and other radicals.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMedDate not supplied

Renaissance of cytochemical localization of membrane ATPases in the myocardium.

ATPases of cardiac cells are known to be among the most important enzymes to maintain the fluxes of vital cations by hydrolysis of the terminal high-energy phosphate of ATP. Biochemically the activities of Ca(2+)-pump ATPase, Ca2+/Mg(2+)-ecto ATPase, Na+,K(+)-ATPase and Mg(2+)-ATPase are determined in homogenates and isolated membranes as well as in myofibrillar and mitochondrial fractions of various purities. Such techniques permit estimation of enzyme activities in vitro under optimal conditions without precise enzyme topography. On the other hand, cytochemical methods demonstrate enzyme activity in situ, but not under optimal conditions. Until recently several cytochemical methods have been employed for each enzyme in order to protect its specific activity and precise localization but the results are difficult to interpret. To obtain more consistent data from biochemical and cytochemical point of view, we modified cytochemical methods in which unified conditions for each ATPase were used. The fixative solution (1% paraformaldehyde-0.2% glutaraldehyde in 0.1 M Tris Base buffer, pH 7.4), the same cationic concentrations of basic components in the incubation medium (0.1 M Tris Base, 2 mM Pb(NO2)3, 5 mM MgSO4, 5 mM ATP) and selective stimulators or inhibitors were employed. The results reveal improved localization of Ca(2+)-pump ATPase, Na(+)-K+ ATPase and Ca2+/Mg(2+)-ecto ATPase in the cardiac membrane.

Adenosine Triphosphatases↗

PubMedDate not supplied

Protective effect of vanadate on oxyradical-induced changes in isolated perfused heart.

In order to examine the mechanisms of the beneficial effects of vanadate on cardiac dysfunction in chronic diabetes, rat hearts were perfused with xanthine plus xanthine oxidase, an oxyradical generating system in the absence or presence of vanadate. The heart failed to generate contractile force and increased the resting tension markedly within 5 min of perfusion with xanthine plus xanthine oxidase. These changes were prevented by the addition of 4 microM vanadate in the perfusion medium. The protective effects of vanadate on the loss of developed tension and increased resting tension due to xanthine plus xanthine oxidase were dose-dependent (0.1-5 microM). Perfusion of the hearts with glucose-free medium did not abolish the protective actions of vanadate. The sarcolemmal Ca(2+)-pump (ATP-dependent Ca2+ uptake and Ca(2+)-stimulated ATPase) and Na(+)-dependent Ca2+ uptake activities were decreased upon perfusing the hearts with a medium containing xanthine plus xanthine oxidase for 5 min; these effects were prevented by the addition of 2-4 microM vanadate in the perfusion medium. The signals for superoxide radicals produced by xanthine plus xanthine oxidase, as detected by electron paramagnetic resonance spectroscopic technique, were inhibited by 5-100 microM vanadate. These results suggest that vanadate is an oxyradical scavenger and thus may prevent heart dysfunction under some pathological conditions by its antioxidant action.

Animals↗

PubMedDate not supplied

Alterations in heart and serum lysosomal activities in streptozotocin-induced diabetes.

Alterations in the cardiac tissue and serum acid hydrolase activities were studied in chronic streptozotocin-induced diabetes in rats. No changes were observed in total cardiac tissue homogenate lysosomal enzyme activities at 4 weeks of diabetes but there were significant alterations in the distribution of selected enzymes. Significant decreases in nonsedimentable beta-N-acetylglucosaminidase (NAG) and beta-galactosidase (Gal) activities were observed at 4 weeks of diabetes. At 8 weeks of the disease, decreased activities of NAG and Gal were observed in heart homogenates but no changes were apparent in alpha-mannosidase (Man) or acid phosphatase activities. Nonsedimentable activities of NAG and both sedimentable and nonsedimentable activities of Gal were decreased at 8 weeks. At 16 weeks of the diabetic condition, increased activities of NAG, Gal and acid phosphatase were observed. This increase at 16 weeks of the disease was due to an increase in sedimentable enzyme activity. At all times of diabetes, serum enzyme activities were significantly increased. Insulin treatment reversed all of the observed changes in tissue homogenates, but serum levels were not completely reversed. These results suggest that cardiac lysosomal hydrolases are probably only involved in the later stages of the diabetic cardiomyopathy when extensive ultrastructural derangements are evident. The present evidence also suggests that the heart may be a source of serum hydrolase activities.

Acetylglucosaminidase↗

PubMedDate not supplied

Alterations in heart sarcolemmal Ca2(+)-ATPase and Ca2(+)-binding activities due to oxygen free radicals.

Effects of oxygen free radicals on Ca2+/Mg2+ ATPase and ATP-independent Ca2(+)-binding activities were examined in rat heart sarcolemma. Membranes were incubated with different oxygen radical generating media such as xanthine + xanthine oxidase, hydrogen peroxide, and hydrogen peroxide + Fe2+. In the presence of xanthine + xanthine oxidase, Ca2+ ATPase activity was stimulated and this effect was prevented by the addition of superoxide dismutase. Hydrogen peroxide also showed a significant increase in Ca2(+)-ATPase activity in a dose-dependent manner and this effect was blocked by catalase. On the other hand, a combination of hydrogen peroxide + Fe2+ decreased Ca2(+)-ATPase activity; this depression was prevented by the addition of D-mannitol. The observed change in Ca2(+)-ATPase activity due to oxygen free radicals was associated with changes in Vmax, whereas Ka remained unaffected. Both xanthine + xanthine oxidase and hydrogen peroxide increased whereas, hydrogen peroxide + Fe2+ inhibited the ATP-independent Ca2(+)-binding activities. It is suggested that oxygen free radicals may influence Ca2+ movements in the cell by altering the Ca2+/Mg2+ ATPase and Ca2(+)-binding activities of the membrane and these effects may be oxygen-radical species specific.

Animals↗

PubMedDate not supplied

A biphasic change in contractile proteins during the development of cardiac hypertrophy in pigs.

A non-failing hypertrophy of the left ventricle was produced in the pig heart by supravalvular banding of aorta for 4, 8 and 12 weeks and the myosin and myofibrillar adenosine triphosphatase activities were measured. A significant increase in myosin Ca2+-ATPase activity was seen at 4 weeks of hypertrophy, but at 8 and 12 weeks this activity was significantly decreased compared to sham control. Similar changes were also seen in actin-activated myosin ATPase activities at 4, 8 and 12 weeks of hypertrophy. There were no changes in the K+- and NH4+-EDTA-stimulated ATPAse activities of myosin. Basal ATPase activities of myofibrils were decreased at 4 and 8 weeks of hypertrophy and there was no change in this activity at 12 weeks of hypertrophy. Ca2+ stimulated ATPase activity of myofibrils was significantly increased at 4 weeks, normal at 8 weeks and significantly reduced at 12 weeks of hypertrophy. The changes in ATPase activities were not due to any alterations of proteins by high concentrations of salts during the purification of myosin. The non-hypertrophied right ventricle from the banded animals did not show any change in the basal or Ca2+ stimulated myofibrillar ATPase activities. It is suggested that hypertrophy of the myocardium is accompanied by specific changes in the enzyme activities of the contractile proteins and the biphasic responses may correlate with the functional state of the myocardium subjected to a chronic increase in pressure.

Adenosine Triphosphatases↗

PubMedDate not supplied

Negatively charged sites and calcium binding in the isolated rat heart sarcolemma.

Colloidal iron staining, calcium binding and enzyme activities were studied in the isolated rat heart sarcolemma. Colloidal iron staining of the sarcolemma revealed a high density of negatively charged sites associated with the cell surface. This membrane fraction was found to have calcium binding activity at both low (0.1 mM) and high (1.25 mM) concentrations of calcium. Pretreatment of the sarcolemma with either trypsin, phospholipase C or neuraminidase, was associated with a reduction in colloidal iron staining as well as decreased calcium-binding activity at high concentrations of calcium. Calcium binding at low concentrations was decreased by both trypsin and neuraminidase. Mg2+ ATPase, Ca2+ ATPase, and Na+-K+ ATPase activities were altered by neuraminidase and trypsin treatments, whereas phospholipase C treatment altered Na+-K+ ATPase only. It is concluded that both surface negative charge and calcium-binding sites associated with the isolated rat heart sarcolemma are contributed by a mosaic of biomolecules including proteins, phospholipids and glycoproteins, and alterations in the surface charge may influence the activities of membrane-bound enzymes.

Animals↗

PubMedDate not supplied

Calcium movements in relation to heart function.

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Animals↗

PubMedDate not supplied

Cardiac alpha- and beta-adrenergic receptor alterations in diabetic cardiomyopathy.

The effect of chronic experimental diabetes on the adrenergic receptors in the rat heart was investigated. Diabetes was induced by streptozotocin (65 mg/kg; i.v.) administration, animals were sacrificed 8 weeks later, and positive as well as negative dF/dt values were determined in isolated papillary muscle preparations. Stimulation of the contractile force generation by isoproterenol and methoxamine was attenuated in diabetic preparations. Beta- and alpha-adrenergic receptor bindings were determined in cardiac membranes by employing 3H-dihydroalprenolol and 3H-dihydroergocryptine respectively. Reduced number of beta- and alpha-receptor binding sites without changes in the affinity constants were observed in diabetic myocardium. Such a decrease in alpha- and beta-receptor density in the heart may account for the depressed contractile responsiveness to adrenergic stimuli in diabetic cardiomyopathy.

Animals↗