ATPase Activity Of Sarcolemma Research Articles

Sarcolemmal Ca2+ transport activities in cardiac hypertrophy caused by pressure overload

To examine Ca2+ transport activities in sarcolemmal membrane in cardiac hypertrophy caused by pressure overload, rats were subjected to aortic banding for 28 days. Heart-to-body weight ratio was increased by 46% in aortic-banded animals in comparison with the sham-operated rats. Ouabain-sensitive Na+-K+-ATPase activity in sarcolemma (SL) from hypertrophied hearts was not different from that in the control preparation. The initial rate of Na+-dependent Ca2+ uptake in SL vesicles from the hypertrophied hearts was stimulated by 53% compared with the control vesicles. ATP-dependent Ca2+ uptake and Ca2+-stimulated adenosinetriphosphatase (ATPase) activities in SL from hypertrophied hearts were increased by 35%. The number of Ca2+ channels estimated by [5-methyl-3H]nitrendipine binding was decreased by 33% in SL from hypertrophied hearts. Total and individual phospholipid contents in the hypertrophied heart preparations were not different from those in the control, except that phosphatidylcholine and phosphatidylethanolamine contents were significantly increased. Sarcolemmal preparations from hypertrophied hearts from the 22-wk-old spontaneously hypertensive rats exhibited changes in Na+-Ca2+ exchange and Ca2+-pump activities (similar to those observed in banded hearts), whereas the Na+-K+-ATPase activity decreased, [3H]nitrendipine binding increased, and phospholipid contents were not different. Thus, although differences were defined between two types of hypertrophy, these results suggest alterations in the sarcolemmal Ca2+ transport activities that may serve as an adaptive mechanism for the removal of Ca2+ from the myocardial cells during the development of cardiac hypertrophy.

Specific stimulation of heart sarcolemmal [formula omitted] ATPase by concanavalin A

The effects of concanavalin A (Con A) on membrane Ca 2+ Mg 2+ ATPase activities as well as the characteristics of Con A binding were examined by employing rat heart sarcolemmal preparations. Con A stimulated the Ca 2+ ATPase and Mg 2+ ATPase activities in sarcolemma; maximal stimulation in these parameters was seen at a concentration of 10 μg/ml. The observed effects of Con A were blocked by α-methylmannoside. Sarcolemmal Na +-K + ATPase and Ca 2+-stimulated ATPase were not affected by Con A. Likewise, Con A did not alter the mitochondrial, sarcoplasmic reticular, and myofibrillar ATPase activities. Con A was found to bind to sarcolemma; α-methylmannoside prevented this binding. The Scatchard plot analysis of the data on specific Con A binding showed a straight line with a K d of about 530 n m and a B max of 235 pmol/mg protein, thus indicating that there was only one kind of binding site for Con A in sarcolemma. These results suggest that Con A is a specific activator of the low affinity Ca 2+ Mg 2+ ATPase system in the heart sarcolemmal membrane.

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.

Biochemical properties of membranes isolated from calcium-depleted rabbit hearts.

The purpose of this study was to define the biochemical properties of sarcolemma from the calcium-depleted rabbit heart. Calcium repletion after calcium-free perfusion results in irreversible damage to the heart (calcium paradox). No difference was found in specific activity of the Na+ -Ca++ antiporter in a crude preparation of sarcolemmal vesicles that was isolated from calcium-depleted hearts, compared with control perfused hearts. Likewise, the passive calcium efflux from sarcolemmal vesicles, preloaded with calcium via the Na+ -Ca++ antiporter, showed rates that were identical with control values. This indicates that the sarcolemma calcium permeability is not affected by calcium-free perfusion of the heart. Na+,K+ -ATPase activity in sarcolemma isolated from calcium-depleted hearts was reduced by 75% (P less than 0.005) compared with the control activity. Sarcolemmal phosphoproteins, whether produced by endogenous cyclic AMP- or calcium-calmodulin-dependent protein kinase, were not altered by calcium-free perfusion of the heart. The content of an important calcium-binding site in the myocardial cell, the sialic acid residues, was also estimated. Only a long period (60 minutes) of calcium-free perfusion resulted in a significant decrease (by 68%, P less than 0.025) of sialic acid content in the homogenate but not in the sarcolemma preparation. In hearts that were reperfused for 15 minutes with a normal calcium concentration (1.3 mM), sarcolemmal Na+,K+ -ATPase remained depressed and calcium permeability was still unchanged. It is possible that the sarcolemma isolation method selected a distinct part of the sarcolemma from the calcium-depleted and repleted heart that had no modified glycocalyx and permeability barriers to calcium ions, and that another part of the sarcolemma with altered properties was lost during the isolation procedure. Another possibility is that reconstitution processes during isolation affected membrane permeability properties. The results of the Na+,K+ -ATPase measurements provide evidence that the net calcium gain of the cells after calcium repletion may be associated, in part, with a loss in ability of the sarcolemma to remove calcium from the cytosol.

Membrane localization of myocardial type II cyclic amp-dependent protein kinase activity

Crude cardiac membrane vesicles were separated into subfractions of sarcolemma and sarcoplasmic reticulum. The subfractions were used to determine the origin and type of cyclic AMP-dependent protein kinase activity present in myocardial membranes. A cyclic AMP-binding protein of molecular weight 55 000 was covalently labeled with the photoaffinity probe 8-azido adenosine 3′,5′-mono[ 32P]phosphate, and found to copurify with the (Na + + K +)-ATPase activity of sarcolemma, and away from the (Ca 2+ + K +)-ATPase activity of sarcoplasmic reticulum. Endogenous cyclic AMP-dependent protein kinase activity also copurified with sarcolemma. Protein substrates phosphorylated by cyclic AMP-dependent protein kinase activity had apparent molecular weights of 21 000 and 8000 and were present in both sarcolemma and sarcoplasmic reticulum. However, while addition of cyclic AMP alone resulted in phosphorylation of sarcolemma proteins, both cyclic AMP and exogenous, soluble cyclic AMP-dependent kinase were required for phosphorylation of sarcoplasmic reticulum proteins. Addition of the calcium-binding protein, calmodulin, to either sarcolemma or sarcoplasmic reticulum resulted in phosphorylation of the 21 000 and 8000-dalton proteins, as well. The results suggest that cardiac sarcolemma contains an intrinsic type II cyclic AMP-dependent protein kinase activity that is not present in sarcoplasmic reticulum. On the other hand, Ca 2+- and calmodulin-dependent protein kinase activity is present in both sarcolemma and sarcoplasmic reticulum.

Changes in subcellular membranes in rat heart perfused with diethyl ether

Alterations in contractile force as well as heart sarcolemmal, microsomal, and mitochondrial activities were investigated by perfusing rat hearts with different concentrations (0.5–1.5% (v/v)) of ether for 2 min. Contractile force of the perfused heart was depressed by 0.5–1.0% ether and completely abolished by 1.25–1.5% ether. The contractile force recovered fully upon reperfusion when the hearts were preperfused with 0.5–0.75% ether but the recovery was partial in hearts preperfused with 1.0–1.25% ether. The hearts preperfused with 1.5% ether did not recover their ability to generate contractile force upon reperfusion. The Na+–K+-ATPase activity of sarcolemma obtained from hearts perfused with 0.75–1.0% ether was higher than the control value. The sarcolemmal Na+–K+-ATPase, Mg2+-ATPase, Ca2+-ATPase, calcium binding, and adenylate cyclase activities were decreased in hearts perfused with 1.5% ether and these activities were further decreased upon reperfusion. The microsomal calcium binding and uptake activities were depressed in hearts perfused with 1.0–1.5% ether and these changes were irreversible. The mitochondrial calcium binding and uptake activities as well as oxidative phosphorylation activity were also depressed in hearts perfused with 1.0–1.5% ether; however, irreversible damage was seen in hearts perfused with 1.5% ether. Electron microscopic examination showed occasional vacuolization of mitochondria in hearts perfused with 1% ether and swelling of sarcoplasmic reticulum and mitochondria as well as damage to myofilaments were seen in hearts perfused with 1.5% ether. These results suggest the involvement of alterations in subcellular membrane system in the cardiotoxic actions of ether.