Effects of monoclonal antibody against phospholamban on calcium pump ATPase of cardiac sarcoplasmic reticulum.

A monoclonal antibody against phospholamban has been reported to increase Ca2+ uptake by cardiac sarcoplasmic reticulum. We compared the effect of this antibody on Ca2+ pump ATPase activity of cardiac sarcoplasmic reticulum vesicles to the effect of cAMP-dependent phosphorylation of phospholamban. The antibody markedly stimulated the Ca(2+)-dependent ATPase activity in parallel to the increase in Ca2+ uptake by cardiac sarcoplasmic reticulum. When the Ca(2+)-dependent profile of the ATPase activity was compared, the KCa was shifted from 1.24 to 0.62 microM by the antibody, whereas cAMP-dependent phosphorylation of phospholamban shifted the KCa to 0.84 microM. When cardiac sarcoplasmic reticulum vesicles were treated with both cAMP-dependent protein kinase and the antibody, the stimulation was the same as that with the antibody alone. Thus, the Ca2+ pump ATPase seems to be fully activated by the antibody. The stoichiometry between Ca2+ uptake and ATPase rate was around 1 and no significant change was observed by the treatment with the antibody. Therefore, the stimulation of Ca2+ uptake of cardiac sarcoplasmic reticulum by the antibody occurred by the stimulation of Ca2+ pump ATPase, not by other mechanisms such as channel activity of phospholamban. These results indicate that the binding of the antibody to phospholamban produces essentially the same mode of action on Ca2+ pump ATPase as that of phospholamban phosphorylation. The antibody and phospholamban phosphorylation appear to release the inhibitory action of phospholamban on Ca2+ pump ATPase, resulting in the stimulation of Ca2+ pump.     

Ca2+-transport ATPases of vascular smooth muscle

To characterize the Ca2+-transport properties of the plasma membrane and of the endoplasmic reticulum of bovine pulmonary artery, membrane vesicles are subfractionated by a procedure of density-gradient centrifugation that takes advantage of the selective effect of digitonin on the density of plasma-membrane vesicles. The obtained endoplasmic-reticulum fraction contains hardly any plasma-membrane vesicles, whereas the plasma-membrane fraction is still contaminated by a substantial amount of endoplasmic-reticulum vesicles. An adenosine 5'-triphosphate (ATP) energized Ca2+-transport system and a Ca2+-stimulated ATPase activity are present in both subcellular fractions. The Ca2+ transport by the plasma membrane is catalyzed by a (Ca2+,Mg2+)-ATPase of Mr 130,000. It binds calmodulin and it has a low steady-state phosphoprotein intermediate level. The endoplasmic-reticulum vesicles contain a Ca2+-transport ATPase of Mr 100,000 that is characterized by a high steady-state phosphointermediate level. It is antigenically related to the Ca2+-pump protein of cardiac sarcoplasmic reticulum. Phospholamban, the regulatory protein of the Ca2+-transport enzyme of cardiac sarcoplasmic reticulum, is also present in the endoplasmic reticulum of the pulmonary artery. A comparison of these fractions with the previously characterized fractions from porcine gastric smooth muscle reveals important differences in the basal Mg2-ATPase activity, in the ratio of the (Ca2+,Mg2+)-ATPase of the plasmalemma to that of the endoplasmic reticulum, and in the ratio of the (Na+,K+)-ATPase activity to the plasmalemmal (Ca2+,Mg2+)-ATPase activity. These differences can be ascribed in part to the species and in part to the tissue. These data suggest that in the bovine pulmonary artery the Ca2+ extrusion via the ATP-dependent Ca2+ pump may have a less predominant role, and that the Ca2+ uptake by the endoplasmic reticulum, and possibly also the Ca2+ extrusion via the Na+-Ca2+ exchanger could be more important in this tissue than in the porcine stomach.     

Identification and characterization of proteins in sarcoplasmic reticulum from normal and failing human left ventricles

Monoclonal and polyclonal antibodies to the major sarcoplasmic reticulum proteins of rabbit skeletal and canine cardiac muscle have been used to identify and characterize the corresponding components of human cardiac sarcoplasmic reticulum. The Ca2(+)-transporting ATPase of human cardiac sarcoplasmic reticulum was identified as a 105,000-Da protein antigenically distinct from its rabbit skeletal muscle counterpart. Human cardiac sarcoplasmic reticulum also contained 53,000- 155,000- and 165,000-Da glycoproteins antigenically related to the low and high molecular weight glycoproteins of canine cardiac and rabbit skeletal muscle sarcoplasmic reticulum. The ryanodine-sensitive Ca2+ channel of human cardiac sarcoplasmic reticulum was identified as a 400,000-Da protein antigenically related to its counterparts in canine cardiac and rabbit skeletal muscle. Human cardiac calsequestrin was identified as a 52,000-Da protein. Human phospholamban was identified as a 29,000-Da substrate for phosphorylation by cAMP-dependent protein kinase. Immunoblots of sarcoplasmic reticulum from the normal left ventricles of four unmatched organ donors and the excised failing left ventricles of nine patients with idiopathic dilated cardiomyopathy were compared in search of qualitative differences in the protein patterns of the failing hearts. No such differences were found with respect to the Ca2+ ATPase, the 53,000-Da glycoprotein, the ryanodine-sensitive Ca2+ channel, calsequestrin or phospholamban. In contrast, the 165,000-Da glycoprotein band, present in all four preparations from nonfailing hearts, was absent from three of nine preparations from failing hearts, and staining of the 155,000-Da glycoprotein in these three preparations appeared to be relatively increased. The absence of the 165,000-Da glycoprotein band may identify or reflect a pathogenetic mechanism in a subset of patients with idiopathic dilated cardiomyopathy.     

The effect of pH on the transient-state kinetics of Ca2+-Mg2+-ATPase of cardiac sarcoplasmic reticulum. A comparison with skeletal sarcoplasmic reticulum

The effect of pH on the Ca2+-Mg2+-dependent ATPase of sarcoplasmic reticulum (SR) was investigated with a rapid mixing quench-flow apparatus capable of measuring phosphorylation and dephosphorylation at times as rapid as 4 msec. The rates of formation and decomposition of the phosphorylated intermediate (E approximately P) of the Ca2+-Mg2+-ATPase were studied in the pH range between 7.6 and 6.0. At pH 6.8, the rates of formation of the phosphorylated intermediate of the Ca2+-Mg2+-ATPase of sarcoplasmic reticulum are the same (t1/2 = 10 msec) for cardiac and skeletal sarcoplasmic reticulum preloaded with calcium, but decrease as the pH is lowered. The effect of acid pH (6.0) is more pronounced for cardiac sarcoplasmic reticulum (t 1/2 = 47 msec) than for skeletal sarcoplasmic reticulum (t 1/2 = 17 msec), in agreement with studies showing that acidosis has a more pronounced effect on cardiac muscle than on skeletal muscle. In addition, a decrease in pH results in a decrease in the rate of the E approximately P decomposition step (the slowest step in the SR reaction sequence). The E approximately P decomposition half-lives were observed to be 97 and 77 msec, respectively for cardiac and skeletal SR at pH 6.8. At pH 6.0, the half-lives were increased to 136 and 178 msec for cardiac and skeletal SR, respectively.     

Calreticulin and calsequestrin are differentially distributed in canine heart

Cardiac sarcoplasmic reticulum (SR) consists of three continuous yet distinct regions: longitudinal, corbular, and junctional. Ca(2+)uptake, catalyzed by the Ca(2+)-dependent ATPase, is thought to occur throughout the SR whereas Ca(2+)release occurs in the region of the junctional SR. In the SR, Ca(2+)is stored in a complex with Ca(2+)-binding proteins such as calsequestrin. In the present study, the distribution of the high-affinity calcium-binding protein, calreticulin, in canine cardiac SR was determined and compared with the distribution of other SR proteins. Three types of distribution were observed. Many proteins, including the Ca(2+)-ATPase and two mannose-containing glycoproteins (52 and 131 kDa), were present in all subfractions. These proteins were absent or depleted from the sarcolemma-enriched fraction. The ryanodine receptor/Ca(2+)release channel and calsequestrin were localized to the junctional SR. Calreticulin immunoreactivity was detected in fractions containing longitudinal SR but not in fractions enriched in sarcolemma or junctional SR. Hence calreticulin is present in a unique vesicular fraction and the Ca(2+)-binding proteins calreticulin and calsequestrin display different patterns of distribution in canine heart.     

A proton gradient controls a calcium-release channel in sarcoplasmic reticulum

Sarcoplasmic reticulum vesicles from mammalian skeletal muscle have previously been shown to develop a proton gradient (alkaline inside) of 0.15-0.5 pH units during active Ca2+ uptake. We found that dissipation of this gradient by the proton ionophores gramicidin, nigericin, and carbonyl cyanide p-trichloromethoxyphenylhydrazone caused a rapid transient tension in skinned rabbit psoas muscle fibers. Increases, but not decreases, in medium pH of approximately 0.2 units over the range from pH 6.5 to pH 7.5 also elicited transient tensions. In isolated vesicles, physiological levels of Ca2+ (3.3 microM), inhibited pH-induced Ca2+ release. Dicyclohexylcarbodiimide blocked pH- and ionophore-induced Ca2+ release under conditions in which it could bind to sarcoplasmic reticulum proteins but did not inhibit Ca2+ uptake. We propose that a proton gradient generated across sarcoplasmic reticulum membranes during Ca2+ uptake maintains a Ca2+ release channel in a closed conformation and that dissipation of this gradient permits the Ca2+ release channel to open. We further propose that elevated myoplasmic Ca2+ also causes the Ca2+ channel to close, permitting Ca2+ uptake through Ca2+/Mg2+-ATPase to function effectively. As the proteolipids of sarcoplasmic reticulum bind dicyclohexylcarbodiimide under conditions in which Ca2+ release is blocked and as they have previously been shown to have Ca2+ ionophoric activity, we propose that the Ca2+-release channel either resides in the proteolipids or is controlled by H+ fluxes through the proteolipids.     

Remodeling of Cardiac Membranes During the Development of Congestive Heart Failure

Various proteins such as Ca2+channels, Ca2+-pump ATPase, Na+–Ca2+exchanger, and Na+-K+ATPase in the sarcolemmal (SL) membrane are considered to be intimately involved in Ca2+-influx and Ca2+-efflux processes in the cardiomyocyte. On the other hand, Ca2+-pump ATPase, Ca2+-release channels, Ca2+-regulatory protein (phospholamban), and Ca2+-binding protein (calsequestrin) in the sarcoplasmic reticulum (SR) are known to participate in raising and lowering the intracellular concentration of Ca2+for the occurrence of cardiac contraction and relaxation processes. Therefore, a defect in any of the SL and SR proteins can be seen to result in Ca2+-handling abnormalities in cardiomyocytes and subsequently in cardiac dysfunction during the development of heart failure. In this review, evidence is presented to show that changes in the expression of genes specific for cardiac membrane proteins may lead to remodeling of both SR and SL membranes during the development of heart failure. Although a great deal of work on changes in gene expression for the SR membrane proteins has been carried out in the failing heart, relatively little information regarding changes in gene expression for SL proteins has appeared in the literature. Prevention of remodeling of cardiac membranes by modification of changes in the gene expression is suggested to serve as an important target for the treatment of heart failure.     

Effect of thyroid hormone on the expression of mRNA encoding sarcoplasmic reticulum proteins.

The purpose of this study was to determine the expression of genes encoding various sarcoplasmic reticulum components that are functionally coupled with calcium release, uptake, and storage function during cardiac hypertrophy induced by thyroid hormone. Hyperthyroidism was induced in two groups of rabbits by the injection of 200 micrograms/kg L-thyroxine (T4) daily for 4 days (T4-4-day group) and 8 days (T4-8-day group). Hypothyroidism was induced in another group of rabbits by adding 0.8 mg/ml propylthiouracil to the drinking water for 4 weeks. The relative expression level of mRNA encoding different sarcoplasmic reticulum proteins was determined by RNA slot blot and Northern blot analysis. In hyperthyroid hearts, the steady-state level of cardiac ryanodine receptor mRNA and sarcoplasmic reticulum cardiac/slow-twitch Ca(2+)-ATPase mRNA were both increased to 147% (T4-4-day group) and 186% (T4-8-day group) of control, respectively, but decreased to 71% and 75%, respectively, in hypothyroid ventricles. The mRNA level for phospholamban was decreased in both hyperthyroidism (T4-8-day group, 72%) and hypothyroidism (77%) in these hearts. On the other hand, calsequestrin mRNA levels did not change in hyperthyroid and hypothyroid ventricles. In accord with the changes in Ca(2+)-ATPase mRNA levels, the Ca(2+)-ATPase protein was increased to 199% (T4-8-day group) in hyperthyroid ventricles and decreased to 86% of control in hypothyroid ventricles. The expression levels of ryanodine receptor, Ca(2+)-ATPase, phospholamban, and calsequestrin mRNAs were similarly altered in skeletal muscle tissues from hyperthyroid and hypothyroid rabbits. These results indicate that the mRNA levels of sarcoplasmic reticulum proteins responsible for calcium release and calcium uptake are coordinately regulated in response to changes in thyroid hormone level in both heart and skeletal muscle. These changes in mRNA level should lead to changes in protein levels and thus to altered calcium release and uptake in the chronic stages of hyperthyroidism and hypothyroidism.     

Function and regulation of sarcoplasmic reticulum Ca2+-ATPase: advances during the past decade and prospects for the coming decade

In cardiac muscle, the contraction-relaxation cycle is tightly controlled by the regulated release and uptake of intracellular Ca2+ between sarcoplasmic reticulum and cytoplasm. A major protein controlling Ca2+ cycling is Ca2+-ATPase (SERCA2a) located in the sarcoplasmic reticulum membrane. The function of SERCA2a protein is regulated by the phosphorylatable protein, phospholamban. Phosphorylation of phospholamban releases its inhibitory effect on SERCA2a through direct molecular interaction. Recently, mice whose SERCA2a function is increased (overexpression of the gene) or lost (knock out) were developed. These mice demonstrated that SERCA2a pump levels are a major determinant of cardiac muscle contractility and relaxation. These studies open the prospect that the overexpression of SERCA2a can correct cardiac dysfunction seen in heart failure. Advances in knowledge concerning the function and gene regulation of SERCA2a are discussed in this review.     

Quercetin inhibits Ca2+ uptake but not Ca2+ release by sarcoplasmic reticulum in skinned muscle fibers.

Quercetin inhibited Ca2+-dependent ATP hydrolysis, ATP-dependent Ca2+ uptake, chelator-induced [ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] Ca2+ release, and ATP synthesis coupled to Ca2+ release in isolated vesicles of sarcoplasmic reticulum. Use of this inhibitor permitted evaluation of whether Ca2+ release from sarcoplasmic reticulum in situ occurs through a reversal of the uptake pathway. Release of Ca2+ from the sarcoplasmic reticulum of skinned muscle fibers can be detected by the measurement of tension in the fiber. If the sarcoplasmic reticulum of these preparations is first allowed to accumulate Ca2+, tension development may be induced by the addition of Ca2+ itself or of caffeine to the bathing medium or by depolarization with Cl-. The presence of quercetin during the loading phase inhibited Ca2+ uptake by sarcoplasmic reticulum in situ. When quercetin was added together with initiators of tension development, however, the rate of tension development was enhanced 4- to 7-fold and the relaxation rate of the fibers was greatly inhibited. These results suggest that quercetin had no effect on Ca2+ release in skinned fiber; its effect on Ca2+ reuptake could account for the apparent enhancement of the release rate and for the prolonged relaxation time. These observations rule out reversal of the Ca2+ pump as the mechanism of Ca2+ release in situ.     

Effects of phospholamban phosphorylation catalyzed by adenosine 3':5'-monophosphate- and calmodulin-dependent protein kinases on calcium transport ATPase of cardiac sarcoplasmic reticulum

To elucidate the role of 22000-dalton protein phospholamban, a putative regulator of Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum, we examined the relationship between cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban and their effects on ATPase activity and calcium transport of cardiac sarcoplasmic reticulum. Cardiac microsomes were incubated with [gamma-32P]ATP or unlabeled ATP, catalytic subunit of cyclic AMP-dependent protein kinase and/or exogenous calmodulin, and subsequently assayed for ATPase activity and calcium uptake by cardiac sarcoplasmic reticulum. Cyclic AMP-dependent phosphorylation of phospholamban was independent of Ca2+, whereas calmodulin-dependent phosphorylation of phospholamban was dependent on Ca2+ within a range between 0.2 and 50 microM. Cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban occurred independently; when both kinases were operative, the amounts of phosphorylation were additive. Under these conditions, the phosphoproteins formed by cyclic AMP- and calmodulin-dependent protein kinases electrophoretically migrated as 11000-dalton components when sodium dodecyl sulfate-solubilized phosphoproteins were boiled prior to polyacrylamide gel electrophoresis. The ATPase activity was stimulated by either cyclic AMP- or calmodulin-dependent phosphorylation of phospholamban at Ca2+ concentrations up to 2 microM. The extents of stimulation of ATPase activity were additive when both types of phosphorylation were functional. Calcium uptake was similarly augmented by cyclic AMP- and/or calmodulin-dependent phosphorylation of phospholamban. These results indicate that Ca2+-dependent ATPase and calcium transport of cardiac sarcoplasmic reticulum are regulated by phospholamban phosphorylation catalyzed by cyclic AMP- and calmodulin-dependent protein kinases, thus suggesting a dual role of phospholamban in active calcium transport.     

Digestion of cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles with calpain II. Effects on the Ca2+ release channel

The Ca2+ release channel and ryanodine receptor are activities copurifying with the 400,000-450,000 Da high molecular weight protein of cardiac and skeletal junctional sarcoplasmic reticulum. Calpain II, an endogenous cytosolic protease, was used to selectively degrade the high molecular weight protein in cardiac and skeletal muscle sarcoplasmic reticulum vesicles, and its effects on the activity of the Ca2+ release channel and [3H]ryanodine binding sites were analyzed. Degradation of the high molecular weight protein was associated with appearance of 315,000 and 150,000 Da proteolytic fragments and with a change in the ultrastructure of the "feet," extravesicular projections that protrude from the junctional sarcoplasmic reticulum membrane. The maximal number of [3H]ryanodine binding sites and the affinities of the sites for ryanodine were not remarkably affected by calpain II. Ca2+ release channels recorded from nondegraded cardiac and skeletal membrane vesicle preparations had slope conductances of 85 and 110 pS, respectively, measured with 1 microM cis-Ca2+ and 50 mM trans-Ba2+. Proteolysis did not alter the unitary channel conductances but did increase the percentage of channel open times from 36% to more than 90%. After proteolysis, channel opening remained dependent on micromolar cis-Ca2+, and high concentrations of ryanodine (300 microM) still blocked the channel. Our results suggest that proteolysis of the Ca2+ release channel with calpain II selectively impairs its inactivation, leaving its unitary conductance and the requirement for micromolar Ca2+ intact.     

Junctin and calsequestrin overexpression in cardiac muscle: the role of junctin and the synthetic and delivery pathways for the two proteins

Ca(2+) storage and release in muscle cells are controlled by a complex of junctional sarcoplasmic reticulum (jSR) proteins, that includes the calcium-binding protein calsequestrin (CSQ), the Ca(2+)-release channel (ryanodine receptor or RyR) and two transmembrane proteins that bind to RyR: junctin (JNC) and triadin (Tr). The relationship between CSQ and JNC, and their contributions to the architecture of the jSR vesicle was studied in transgenic mice with combined overexpression of CSQ and JNC. We find that CSQ, on its own, has a diffuse disposition in the sarcoplasmic reticulum (SR) lumen. Overexpression of JNC results in a tighter packing of CSQ in proximity of the SR membrane, presumably due to the binding of CSQ to the membrane by JNC. Quantitative and qualitative analysis of structural changes in the overexpressing as well as in the normally differentiating myocardium illustrate the synthetic pathways and the events in the targeting and delivery of CSQ and JNC to the jSR of the differentiating cardiac myocyte. CSQ is delivered from the Golgi to the SR, where it buds out into precursors of the jSR vesicles. JNC reaches the jSR vesicles directly, but its arrival is delayed relative to CSQ.     

Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase.

Thapsigargin, a tumor-promoting sesquiterpene lactone, discharges intracellular Ca2+ in rat hepatocytes, as it does in many vertebrate cell types. It appears to act intracellularly, as incubation of isolated rat liver microsomes with thapsigargin induces a rapid, dose-dependent release of stored Ca2+. The thapsigargin-releasable pool of microsomal Ca2+ includes the pools sensitive to inositol 1,4,5-trisphosphate and GTP. Thapsigargin pretreatment of microsomes blocks subsequent loading with 45Ca2+, suggesting that its target is the ATP-dependent Ca2+ pump of endoplasmic reticulum. This hypothesis is strongly supported by the demonstration that thapsigargin causes a rapid inhibition of the Ca2(+)-activated ATPase activity of rat liver microsomes, with an identical dose dependence to that seen in whole cell or isolated microsome Ca2+ discharge. The inhibition of the endoplasmic reticulum isoform of the Ca2(+)-ATPase is highly selective, as thapsigargin has little or no effect on the Ca2(+)-ATPases of hepatocyte or erythrocyte plasma membrane or of cardiac or skeletal muscle sarcoplasmic reticulum. These results suggest that thapsigargin increases the concentration of cytosolic free Ca2+ in sensitive cells by an acute and highly specific arrest of the endoplasmic reticulum Ca2+ pump, followed by a rapid Ca2+ leak from at least two pharmacologically distinct Ca2+ stores. The implications of this mechanism of action for the application of thapsigargin in the analysis of Ca2+ homeostasis and possible forms of Ca2+ control are discussed.     

Influence of protein kinase phosphorylation on isolated sarcolemmal membranes

A cardiac muscle sarcolemmal preparation, enriched in adenylate cyclase, Na+, K+ -ATPase, beta, muscarinic and ouabain receptors, also contained endogenous protein kinase activity. Phosphorylation of sarcolemmal membrane proteins by the endogenous protein kinase occurred mainly on 22 000 and 12 000 Mr proteins. To determine the effect of this phosphorylation on sarcolemmal properties, sarcolemmal vesicles were preincubated under conditions for optimal phosphorylation while control vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both control and phosphorylated vesicles were centrifuged, resuspended in 10 mM Tris-Cl (pH 7.4) and subsequently assayed for ATPase activities and for binding of ouabain, dihydroalprenolol and quinuclidinyl benzilate to the membranes. Sarcolemmal phosphorylation was associated with an increase in Ca2+ -ATPase activity but had no effect on Mg2+ ATPase or Na+, K+ -ATPase activity or on ouabain binding. Muscarinic receptor and beta-adrenoreceptor binding also appeared to be unaffected.     

Characterization and pharmacological relevance of high affinity binding sites for [3H]LY186126, a cardiotonic phosphodiesterase inhibitor, in canine cardiac membranes

[3H]LY186126, an analogue of the cardiotonic agent indolidan, was shown to bind reversibly and with high affinity (Kd = 4 nM) to a single class of binding sites within canine myocardial vesicles. Binding site density measured in various cardiac membrane fractions correlated well with Ca2+-ATPase activity (r = 0.94; p less than 0.01), but not with Na+,K+-ATPase or azide sensitive ATPase, indicating a localization of these sites within sarcoplasmic reticulum membranes. Divalent cations were required for binding and displayed the following order of activation: Zn2+ greater than Mn2+ greater than Mg2+ greater than Ca2+. Differential activation of [3H]LY186126 binding by various divalent cations was due to alterations in binding site density, rather than affinity. cGMP and selective inhibitors of type IV membrane-bound phosphodiesterase (SR-PDE), for example, indolidan, milrinone, imazodan, and enoximone, selectively displaced bound [3H]LY186126 caffeine, theophylline, and rolipram were relatively impotent as inhibitors of radiolabel binding. Kd values from displacement curves were highly correlated with IC50 values for inhibition of SR-PDE (r = 0.92; p less than 0.001). In addition, Kd values correlated well with published ED50 values for increases in cardiac contractility in pentobarbital-anesthetized dogs (r = 0.94; p less than 0.001). The results support the hypothesis that [3H]LY186126 labels the pharmacological receptor for the class of positive inotropic agents characterized as isozyme-selective phosphodiesterase inhibitors. Furthermore, the data suggest that the identity of the site labeled by [3H]LY186126 is SR-PDE, the type IV isozyme of cardiac phosphodiesterase located in the sarcoplasmic reticulum.     

Compensatory adaptation of the heart to chronic rate overload: increase in calcium transport ATPase activity of myocardial sarcoplasmic reticulum

This study was undertaken to test the hypothesis that a compensatory response of the heart to a chronic and continuous, metabolic and heart rate overload was an increase in the calcium sequestering activity of the myocardial sarcoplasmic reticulum. Calcium sequestering activity was estimated by determination of the calcium-dependent ATPase (Ca2+-ATPase) activity of isolated microsomes. Chronic rate overload was modelled by comparing: dysthyroid and control rats; control swine and swine with implanted cardiac pacemakers set at 180 beats/min; and different species of mammals with widely different heart rates. The myocardial sarcoplasmic reticulum Ca2+-ATPase pump activity was significantly increased by 39% for hyperthyroid rats compared to control rats and by 87% for control rats compared to thyroidectomized rats; by 63% for paced swine compared to control swine; and by 43% for rats compared to guinea pigs, by 140% for guinea pigs compared to dogs and by 120% for dogs compared to cows. These data indicate that calcium sequestering activity of myocardial sarcoplasmic reticulum increases in equivalent proportion to the chronotropic demand and that heart rate is a hemodynamic correlate of the sarcoplasmic reticulum Ca2+-ATPase activity.     

Inhibition of Na+/Ca2+ exchange in membrane vesicle and papillary muscle preparations from guinea pig heart by analogs of amiloride.

Na+/Ca2+ exchange is inhibited in both guinea pig cardiac membrane vesicles and papillary muscles in a concentration-dependent fashion by several analogs of the pyrazine diuretic amiloride. Structure/activity studies based on transport measurements in vesicles prepared from guinea pig left ventricle indicate that hydrophobic substitutions at the terminal nitrogen atom of the guanidinium moiety of amiloride improved the inhibitory potency almost 100-fold over that of the parent compound. 3',4'- Dichlorobenzamil ( DCB ) is one of the most active inhibitors (IC50 = 17 microM). In electrically stimulated papillary muscles isolated from guinea pig heart, 10-40 microM DCB decreases contractile force. At 100 microM inhibitor, diastolic tension is significantly increased. The positive inotropic responses to veratridine and ouabain are inhibited by 20 and 40 microM DCB . Since the responses to these interventions were a consequence of increased intracellular Na+ concentration, these data indicate that DCB is an inhibitor of Na+-dependent Ca2+ influx in the intact tissue. Interpretation of mechanical responses elicited by paired pulses suggests that 40 microM but not 100 microM DCB decreases release of Ca2+ from the sarcoplasmic reticulum. The mechanical data obtained with concentrations of DCB that inhibited Na+/Ca2+ exchange in vesicles suggest that a significant amount of Ca2+ can enter the cardiac cell via Na+/Ca2+ exchange under normal conditions and that this transport system may be an important source of Ca2+ supplying the sarcoplasmic reticulum in guinea pig heart. Moreover, these amiloride analogs function as potent inhibitors of the positive inotropic effect caused by increased intracellular Na+ concentration.     

Isolation and characterization of purified sarcoplasmic reticulum membranes from isolated adult rat ventricular myocytes.

We have demonstrated for the first time the isolation of sarcoplasmic reticulum (SR) membranes from adult rat ventricular myocytes obtained from a single rat heart. The myocyte SR preparation exhibits similar Ca(2+)-transport and Ca2+/K(+)-ATPase activity as well as a similar protein profile to SR membranes isolated from intact rat heart tissue. This SR preparation exhibited a Ca2+/K(+)-ATPase activity of 371 +/- 55 nmol/min/mg protein (mean +/- S.E.M.; n = 5) and an oxalate-stimulated Ca(2+)-uptake activity of 103 +/- 4 nmol/min/mg protein (mean +/- S.E.M.; n = 6). Pretreatment of the SR vesicles with 5 microM ruthenium red increased the oxalate-stimulated Ca(2+)-uptake to 204 +/- 12 nmol/min/mg protein demonstrating the presence of junctional SR membranes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis shows that the isolated SR membranes contained protein bands at 430 (Ca(2+)-release channel), 100 (Ca2+/K(+)-ATPase), 55 (calsequestrin and/or calreticulin) and 53 kDa (glycoprotein). Western blots of myocyte SR membranes stained with ruthenium red detected 2 major Ca(2+)-binding protein bands in this preparation at 53-55 kDa (calsequestrin and/or calreticulin) and 97-100 kDa (Ca2+/K(+)-ATPase). The presence of phospholamban, a regulatory protein of the Ca2+/K(+)-ATPase of cardiac SR, was confirmed in the myocyte SR membranes by western blots probed with a monoclonal antibody to phospholamban. Isoproterenol stimulation of intact [32P]orthophosphate equilibriated myocytes was associated with an increase in the phosphorylation of 3 distinct proteins (27, 31 and 152 kDa) in myocyte homogenates. The 27 kDa phosphorylated protein was identified in purified SR membranes as phospholamban my migration on electrophoretic gels and by immunoblotting. The ability to prepare SR membranes from intact isolated adult rat ventricular myocytes makes this system a potentially useful model for the study of SR regulation by protein phosphorylation.     

Influence of ruthenium red on rat heart subcellular calcium transport

Ruthenium red inhibited Ca2+-ATPase and ATP-independent Ca2+ binding with rat heart sarcolemma in a concentration dependent manner; significant effects were evident at 0.25 microM and higher concentrations. The apparent Ka for Ca2+-ATPase was 1.02 +/- 0.02 mM Ca2+ and 1.47 +/- 0.12 mM Ca2+ in the absence and presence of 2.5 microM ruthenium red, respectively; however, no change in the Vmax (41.2 +/- 1.6 mumol Pi/mg/h) was observed. Likewise, the affinity of Ca2+ for both low and high affinity Ca2+ binding sites in sarcolemma was decreased by ruthenium red. Sarcolemmal Na+-dependent Ca2+ uptake, ATP-dependent Ca2+ accumulation, Mg2+-ATPase and Na+,K+-ATPase activities were not affected by ruthenium red. In sarcoplasmic reticulum preparations, ruthenium red (0.25 to 25 microM) enhanced Ca2+ uptake without altering the Ca2+-stimulated ATPase activity. The observed increase in Ca2+ uptake appears to be due to the depressant effect of the dye on Ca2+ release from the sarcoplasmic reticulum. In mitochondrial preparations, ruthenium red (0.025 to 25 microM) showed a marked inhibitory effect on Ca2+ uptake activity whereas the Mg2+-ATPase activity was unaltered. In isolated rat hearts, 0.025 microM ruthenium red produced a slight negative inotropic effect, whereas 0.25 to 2.5 microM ruthenium red elicited a biphasic response both in terms of developed tension and resting tension. High concentrations of ruthenium red (12.5 to 25 microM) resulted in the development of contracture. Electron microscopic studies revealed the presence of ruthenium red in the myoplasm of hearts perfused for 15 to 30 mins with 2.5 to 5 microM dye.(ABSTRACT TRUNCATED AT 250 WORDS)