Rat Cardiac Sarcolemma Research Articles

CO2 permeability and carbonic anhydrase activity of rat cardiomyocytes.

To determine the CO2 permeability (PCO2 ) of plasma membranes of cardiomyocytes. These cells were chosen because heart possesses the highest rate of O2 consumption/CO2 production in the body. Cardiomyocytes were isolated from rat hearts using the Langendorff technique. Cardiomyocyte suspensions exhibited a vitality of 2-14% and were studied by the previously described mass spectrometric 18 O-exchange technique deriving PCO2 . We showed by mass spectrometry and by carbonic anhydrase (CA) staining that non-vital cardiomyocytes are free of CA and thus do not contribute to the mass spectrometric signal, which is determined exclusively by the fully functional vital cardiomyocytes. Lysed cardiomyocytes yielded an intracellular CA activity for vital cells of 5070; that is, the rate of CO2 hydration inside the cell is accelerated 5071-fold. Using this number, analyses of the mass spectrometric recordings from cardiomyocyte suspensions yield a PCO2 of 0.10 cm s-1 (SD ± 0.06, n = 15) at 37 °C. In comparison with the PCO2 of other cells, this value is quite high and about identical to that of the human red cell membrane. As no major protein CO2 channels such as aquaporins 1 and 4 are present in rat cardiac sarcolemma, the high PCO2 of this membrane is likely due to its low cholesterol content of about 0.2 (mol cholesterol)·(mol total membrane lipids)-1 . Previous work predicted a PCO2 of ≥0.1 cm s-1 from this level of cholesterol. We conclude that the low cholesterol establishes a PCO2 high enough to render the membrane resistance to CO2 diffusion almost negligible, even under conditions of maximal O2 consumption of the heart.

Inhibition of [3H]-U69593 binding and the cardiac effects of U50, 488H by calcium channel blockers in the rat heart.

1. The calcium channel blockers (CCBs), nifedipine, nicardipine, diltiazem and verapamil, were used to displace the binding of [3H]-U69593 ((5a, 7a,8b)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4,5] dec-8-yl)-benzeneacetamide), a specific kappa-opioid agonist, in the rat cardiac sarcolemma. The CCBs competed with the binding of [3H]-U69593 (4 nM) in a dose-dependent manner. The displacing potency of verapamil was 55 times greater than that of nifedipine. 2. The effects of two CCBs, verapamil and nifedipine, on the arrhythmogenic action of kappa-receptor stimulation by a specific kappa-receptor agonist, U50,488H (trans-(+/-(-3),4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeacetamide methanesulphonate), were also studied in the rat isolated perfused heart. U50,488H 80-800 nmol dose-dependently induced arrhythmias, which were completely abolished by a selective kappa-receptor antagonist, nor-BNI (nor-binaltorphimine, 17,17'-(dicyclopropylmethyl)-6,6',7,7'-6,6'-imino-7,7'-binorphinan -3,4',14, 14'-tetrol), at 100 nmol. The arrhythmogenic effect was also attenuated by both verapamil and nifedipine in a dose-dependent manner. The ED50 values for verapamil and nifedipine were 2.75 and 63.7 nmol, respectively. The antiarrhythmic potencies of these two CCBs were correlated to their displacing potencies and inversely related to their well known potencies in inhibiting transmembrane Ca2+ influx in the cardiac muscle. 3. Measurement of [Ca2+]i in the absence of free extracellular Ca2+ by a spectrofluorometric method, with fura-2 as Ca2+ indicator, showed that U50,488H 5 x 10(-5) M slowly increased [Ca2+]i in single ventricular myocytes and this effect was abolished by pretreatment with nor-BNI (5 microM), or ryanodine (5 microM). Verapamil 1 and 10 microM abolished the effect of U50,488H in 37.5% (3 out of 8) and 100% (12 out of 12) of the cells studied, respectively. On the other hand, nifedipine 10 and 100 microM had no effect at all. Neither verapamil nor nifedipine exerted any significant effect on the caffeine-induced Ca2+ transient. 4. The observations suggest that CCBs may inhibit the actions of kappa-receptor stimulation at the level of the kappa-receptor.

Characterization and localization of an ATP diphosphohydrolase activity (EC 3.6.1.5) in sarcolemmal membrane from rat heart.

In the present report we describe an ATP diphosphohydrolase (apyrase EC 3.6.1.5) in rat cardiac sarcolemma. It is Ca2+ dependent and is insensitive to ouabain, orthovanadate, N-ethylmaleimide (NEM), lanthanum, and oligomycin that are classical ATPase inhibitors. Sodium azide that is a mitochondrial inhibitor at low concentrations, did not affect the enzyme activity at 5.0 mM or below. In contrast, at high concentrations (> 10 mM) sodium azide inhibited the enzyme. Levamisole, a specific inhibitor of alkaline phosphatase and P1, P5-di(adenosine 5'-)pentaphosphate (Ap5A), a specific inhibitor of adenylate kinase did not inhibit the enzyme. Mercury chloride showed a parallel inhibition of the hydrolysis of both substrates of apyrase. Similar inhibition profiles are powerful evidence for a common catalytic site for the hydrolysis of both substrates. The enzyme has an optimum pH range of 7.5-8.0 and catalyzes the hydrolysis of triphospho- and diphosphonucleosides other than ATP or ADP. The apparent Km (Michaelis constant) and Vmax (maximal velocity) are 62.1 +/- 5.2 microM and 1255.7 +/- 178 micromol inorganic phosphate liberated/min/mg with ATP and 59.4 +/- 4.3 microM and 269.2 +/- 39 micromol inorganic phosphate liberated/min/mg with ADP. Enzyme markers indicated that this apyrase is associated with the plasma membrane. A deposition of lead phosphate granules on the outer surface of the sarcolemmal vesicles was observed by electron microscopy in the presence of either ATP or ADP as substrate. It is suggested that the ATP diphosphohydrolase could regulate the concentration of extracellular adenosine, and thus is important in the control of vascular tone and coronary flow.

Differential Sensitivity of Rat Cardiac Sarcolemma and Mitochondria to Damage Induced by Lipid Peroxidation

It is well known that the sarcolemma is the organelle most susceptible to lipid peroxidative attack in the isolated membrane preparations. To determine whether this also occurs in the intact heart, we studied the effect of cumene hydroperoxide, an agent capable of initiating lipid peroxidation, on the ultrastructure and lanthanum (La) staining of isolated rat hearts perfused with HEPES buffer (pH 7.4) containing: 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 3 mM HEPES, 1.5 mM CaCl2 and 11 mM glucose. No ultrastructural alterations or intracellular deposits of La were observed in myocytes of rats perfused with HEPES buffer. Perfusion with cumene hydroperoxide (0.5 mM) for 30 min induced a release of malondialdehyde-like substance in the perfusate and a spectrum of myocardial ultrastructural alterations. La was always observed only outside the sarcolemma in myocytes with moderate damage consisting of clearing of the mitochondrial matrix and slight margination of chromatin in the nuclei. Intracellular La was found in myocytes with severe and irreversible damages consisting of fragmentation of cristae and electron-dense amorphous particles in mitochondria. La was deposited on the outer surface of the mitochondrial membranes, lipid droplets and myofilaments. These data suggest that mitochondria are more susceptible than is the sarcolemma to lipid peroxidation induced by cumene hydroperoxide in the beating rat heart.

Effect of dietary fats on some membrane-bound enzyme activities, membrane lipid composition and fatty acid profiles of rat heart sarcolemma.

The effect of various dietary fats on membrane lipid composition, fatty acid profiles and membrane-bound enzyme activities of rat cardiac sarcolemma was assessed. Four groups of male weanling Charles Foster Young rats were fed diets containing 20% of groundnut, coconut, safflower or mustard oil for 16 weeks. Cardiac sarcolemma was prepared from each group and the activities of Na+, K(+)-ATPase, 5'-nucleotidase, Ca(2+)-ATPase and acetylcholinesterase were examined. ATPase activities were similar in all groups except the one fed coconut oil, which had the highest activities. Acetylcholinesterase activity was also similar in all the groups, however, it was significantly higher in the group fed mustard oil. No significant changes were observed among the groups in 5'-nucleotidase activity, in the cholesterol-to-phospholipid molar ratio and in sialic acid content. The coconut, safflower and mustard oil diets significantly increased cholesterol and phospholipid contents and the lipid-to-protein ratio of cardiac sarcolemma as compared to feeding the groundnut oil diet. The fatty acid composition of membrane lipids was quite different among the various groups, reflecting the type of dietary fat given. The total unsaturated-to-saturated fatty acid ratio was not different among the various groups; however, the levels of some major fatty acids such as palmitic (16:0), oleic (18:1) and linoleic (18:2) acids were significantly different. Cardiac sarcolemma of the group fed safflower oil had the highest polyunsaturated fatty acid content. The results suggest that dietary fats induce changes not only in the fatty acid composition of the component lipids but also in the activities of sarcolemmal enzymes involved in the regulation of cardiac function.

Endogenous substrates of rat heart protein kinase C type I, II, and III isozymic forms in cardiac sarcolemma.

The endogenous substrate proteins of rat cardiac protein kinase C type I, II, and III isozymic forms were studied in rat cardiac sarcolemma. The 19-, 21-, 29-, 35-, and 95-kDa proteins were phosphorylated by both types II and III, but not type I. The extent of phosphorylation by individual protein kinase C isozymic forms was additive and equal to the extent of phosphorylation observed when a mixture of isozymic forms was employed. The extent of phosphorylation of the 21-kDa protein by type III was much higher than that by type II. These results suggest that the protein kinase C isozymes have preferences for specific endogenous substrate proteins. The phosphorylation of these endogenous substrate proteins by protein kinase C isozymes probably plays a role in cardiac cell functions.

Congestive heart failure secondary to myocardial infarction: Abnormalities in rat cardiac sarcolemma

Congestive heart failure secondary to myocardial infarction: Abnormalities in rat cardiac sarcolemma

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.

Opioid receptors in rat cardiac sarcolemma: effect of phenylephrine and isoproterenol

The present study demonstrates the presence of opioid receptors in the rat cardiac sarcolemma isolated by the hypotonic LiBr-shock procedure. Opioid binding was measured by using [3H]U69 593, [3H](2-D-penicillamine,5-D-penicillamine)enkephalin ([3H]DPDPE) or [3H][D-Ala2,MePhe4,Gly-(ol)5]enkephalin ([3H]DAGO) as selective radioligands for K, delta and mu opioid receptors, respectively. Both the K- and delta-selective ligands exhibited highly specific (75-86%) binding, saturable at a concentration of about 20 nM. No specific binding for the selective agonist DAGO was observed. A marked increase in both [3H]U69 593 and [3H]DPDPE binding was observed after incubation of the sarcolemma with the alpha-adrenoceptor agonist phenylephrine or with the beta-adrenoceptor agonist isoproterenol. These stimulatory effects were associated with an increase in the Bmax values, a decrease in the Kd values, and were completely antagonized by the respective antagonists phentolamine and propranolol.

Separation and characteristics of inside-out and right side-out vesicles from a rat cardiac sarcolemma preparation

1. 1. Purified cardiac sarcolemma (SL) vesicles are highly suitable to study various Ca 2+-transport systems present in the SL. 2. 2. We describe in this paper the separation of the Inside-Out (IO) and Right side-Out (RO) oriented vesicle subpopulations from a purified rat heart SL preparation. 3. 3. The isolated subfractions were characterized with respect to the number of beta-adrenergic binding sites and the Ca 2+-uptake and (Ca 2+-Mg 2+)-ATPase activities. 4. 4. It was found that the Ca 2+-uptake and the (Ca 2+-Mg 2+)-ATPase activities reside in the IO fraction and are virtually absent in the RO fraction, confirming that the active Ca 2+-uptake represents the outward directed sarcolemmal Ca 2+-flux.

Respective involvements of high- and low- affinity digitalis receptors in the inotropic response of isolated rat heart to ouabain

High- and low-affinity digitalis receptors coexist in rat cardiac sarcolemma. In this study, their relative involvement in the inotropic effect of ouabain was evaluated on an isolated Langendorff rat heart preparation working under isovolumic conditions at a low external calcium concentration (0.25 mM). This involvement was estimated according to both the development of the inotropic response to ouabain (10 −8–10 −4 M) and the time course of the washing out the biological effect. In each phenomenon considered, and whatever the index of inotropy chosen, the high-affinity digitalis receptor ( ec 50: 1–2 × 10 −8 M) contributed to 25–40% of the maximal inotropy (evoked by 10 −4 m ouabain). Low-affinity receptors ( ec 50: 1–2 × 10 −5 M) acounted for 60–75%. These apparent affinities were identical to those previously determined in sarcolemma isolated from rat heart perfused with 0.25 mM Ca 2+. The biphasic effect of ouabain was related to both the inhibition of high- and low-sensitivity Na +, K +-ATPase forms and the corresponding number of ouabain-binding sites occupied. These results support the concept that the Na +, K +-ATPase highly sensitive to ouabain as revealed by lowering calcium is in the in vivo manifestation of the high-sensitivity inotropic component.

Modulation of ATP-dependent calcium extrusion and Na +/Ca 2+ exchange across rat cardiac sarcolemma by calcium antagonists

The calcium antagonists verapamil, bepridil, nifedipine and nimodipine inhibited ATP-dependent 45Ca 2+ uptake in purified rat ventricular sarcolemma vesicles dose dependently. This inhibition was preceded by a slight stimulation in the case of the two dihydropyridines, but not with bepridil and verapamil. In contrast, Na +/Ca 2+ exchange was only inhibited by verapamil and bepridil and not affected by the dihydropyridines. The steepness of the inhibition curves was significantly different for the two processes. No stereoselectivity was found with either process for inhibition by the verapamil enantiomers. Inhibition of the exchange was not due to a decrease of the exchange velocity but to a decrease in exchange capacity. Variation of the antagonist preincubation time did not modify the inhibition of the uptake. The results indicate that two different sites, located at the inner surface of the sarcolemma are involved in the modulation of the ATP-dependent uptake and the Na +/Ca 2+ exchange. However, the possibility cannot be ruled out that inhibition of the exchange process is also mediated by an extracellularly located site.

Comparison of (Ca 2+-Mg 2+-ATPase and Ca 2+-ATPase in rat cardiac sarcolemma

1. 1. The calcium dependency of the Ca 2+-pump ATPase of rat cardiac sarcolemma was investigated in the presence and absence of EGTA and EDTA in combination with two free Mg 2+-ion concentrations. 2. 2. The results showed: (1) that Mg 2+-ions are not essential for the turnover of the Ca 2+-pump ATPase; (2) that the Ca 2+-affinity is regulated by the concentration of the calcium-chelator complex present in the medium; (3) that (Ca 2+-Mg 2+)ATPase and Ca 2+-ATPase are probably expressions of the same Ca 2+-pump ATPase in the plasma membrane of the cell.

Effect of Dietary Fats on the Lipid Composition and Enzyme Activities of Rat Cardiac Sarcolemma

The effect of dietary lipids on the lipid composition and the activites of some enzymes of cardiac sarcolemma were studied. Feeding rats coconut oil-rich diet for 4 weeks resulted in a significant decrease in 5′-nucleotidase, phosphodiesterase I and p-nitrophenylphosphatase activity of cardiac sarcolemma as compared with feeding rats safflower oil. Sarcolemma from animals fed coconut oil diet contained a significantly lower concentration of total polyunsaturated fatty acids and a higher concentration of total monounsaturated fatty acids than that from rats fed safflower oil. Most of the alterations in polyunsaturated fatty acids were found in 20:4, whereas those of the monounsaturates were found in 18:1. Among all the phosphoglycerides, the fatty acid composition of the phosphatidylcholine exhibited the largest alterations as a result of coconut oil feeding. No dietary effect was observed in the sarcolemma content of cholesterol and phospholipid. These studies clearly indicate that manipulation of dietary lipids influences both the fatty acid composition and some functional properties of the sarcolemma membranes.

Cyclic AMP induced stimulation and inhibition of Ca 2+-uptake in rat cardiac sarcolemma vesicles

Cyclic AMP induced stimulation and inhibition of Ca 2+-uptake in rat cardiac sarcolemma vesicles

Comparison of cyclic amp-dependent phosphorylation of sarcolemma and sarcoplasmic reticulum from rat cardiac ventricle muscle

1. 1. The phosphorylation by cAMP and protein kinase I of rat cardiac sarcolemma (SL) and sarcoplasmic reticulum (SR) isolated from the same homogenate, was compared. 2. 2. In both fractions, the phosphate incorporation is strongly dependent on the ATP and the membrane protein concentration. 3. 3. SDS-gel electrophoresis reveals that in the SL preparation a protein of M r = 24,500 and a glycoprotein of M r = 17,500 are mainly phosphorylated, while in the SR fraction the main phosphate incorporation is found in a protein having a M r = 37,000. 4. 4. Isoprenaline stimulates the phosphorylation of SL but not of SR. Propranolol abolished that stimulatory action of isoprenaline completely, suggesting that the β-adrenoceptor is involved.

CA2+-free perfusion of rat heart reveals a (Na+ + K+)ATPase form highly sensitive to ouabain.

The sensitivities of the cardiac (Na+ + K+)ATPases to inhibition by ouabain have been shown to correlate with the pharmacologically active digitalis concentrations in the various source species1. The rat heart enzyme is generally considered to be insensitive to ouabain with half-maximal inhibition at ∼6 × 10−5 M ouabain2,3 and a KD for binding3–7 of 1–6 × 10−5 M. However, recent data5–7 showed that rat cardiac sarcolemma preparations can bind ouabain with high affinity (KD 1–3 × 10−7M) with no detectable concomitant enzyme inhibition in vitro but producing a sustained positive inotropic effect in vivo. In the present study, we measured the (Na+ + K+)ATPase inhibition by ouabain in sarcolemma-enriched fractions obtained from rat hearts relaxed by perfusion with Ca2+-free solutions. In these conditions, two enzyme forms having high and low sensitivities to ouabain were found (half-maximal inhibitions with 1.2×10−8 and 6×10−5M, respectively). Their relative proportion is close to 1. Confirming previous data2–7, the highly sensitive form was not detected in preparations from hearts maintained at a physiological calcium level. This heterogeneous population of functional (Na+ + K+)ATPases suggests that, in rat heart, the positive inotropic effect of low doses of ouabain in vivo5–9 would act through the inhibition9 of the enzyme activity highly sensitive to ouabain as detected in vitro.