Sarcoplasmic Reticulum Calcium Pump Research Articles

The sarcoplasmic reticulum calcium pump is functionally altered in dystrophic muscle

In Duchenne muscular dystrophy, muscle cells, which lack the protein dystrophin, have been reported to have elevated resting intracellular calcium levels. It has also been noted that, compared to normal muscle, intracellular [Ca 2+] in dystrophic muscle returns more slowly to its resting level following contractile stimulation. Consistent with this, it has been suggested that dystrophin is directly involved in the regulation of Ca 2+ influx. A secondary alteration in the sarcoplasmic reticulum Ca 2+ pump, however, could also contribute to, or be responsible for, the abnormal Ca 2+ handling seen. To determine whether the Ca 2+ pump is functionally altered in dystrophic muscle, we examined Ca 2+ uptake by vesicles derived from skeletal muscle sarcoplasmic reticulum of normal and dystrophic (mdx) mice. The Hill coefficient and the Ca 2+ sensitivity of the Ca 2+-ATPase were the same in both cases. The maximum velocity of Ca 2+ uptake, however, normalized to the ATPase content of the vesicles, was less for mdx muscle.

Molecular dynamics in mouse atrial tumor sarcoplasmic reticulum

We have determined directly the effects of the inhibitory peptide phospholamban (PLB) on the rotational dynamics of the calcium pump (Ca-ATPase) of cardiac sarcoplasmic reticulum (SR). This was accomplished by comparing mouse ventricular SR, which has PLB levels similar to those found in other mammals, with mouse atrial SR, which is effectively devoid of PLB and thus has much higher (unregulated) calcium pump activity. To obtain sufficient quantities of atrial SR, we isolated the membranes from atrial tumor cells. We used time-resolved phosphorescence anisotropy of an erythrosin isothiocyanate label attached selectively and rigidly to the Ca-ATPase, to detect the microsecond rotational motion of the Ca-ATPase in the two preparations. The time-resolved phosphorescence anisotropy decays of both preparations at 25 degrees C were multi-exponential, because of the presence of different oligomeric species. The rotational correlation times for the different oligomers were similar for the two preparations, but the total decay amplitude was substantially greater for atrial tumor SR, indicating that a smaller fraction of the Ca-ATPase molecules exists as large aggregates. Phosphorylation of PLB in ventricular SR decreased the population of large-scale Ca-ATPase aggregates to a level similar to that of atrial tumor SR. Lipid chain mobility (fluidity), detected by electron paramagnetic resonance of stearic acid spin labels, was very similar in the two preparations, indicating that the higher protein mobility in atrial tumor SR is not due to higher lipid fluidity. We conclude that PLB inhibits by inducing Ca-ATPase lateral aggregation, which can be relieved either by phosphorylating or removing PLB.

Activation of protein kinase C inhibits potassium currents in cultured endothelial cells.

The effect of protein kinase C on potassium channels in cultured endothelial cells was investigated by using whole-cell patch-clamp techniques. Activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), but not phorbol 12-monomyristate (PMM), an inactive analogue of phorbol esters, depressed an outward calcium-dependent potassium current. The inhibitory actions of PMA and PDBu could be reversed by the kinase inhibitor H-7. Cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum calcium pump, and LP-805, a novel vasodilator which also releases endothelium-derived relaxing factors, activated the outward calcium-dependent potassium conductance. PMA and PDBu, but not PMM, reduced the outward conductance induced by cyclopiazonic acid and LP-805. These effects of PMA and PDBu on potassium currents may be mediated either by phosphorylation of ion channels, or by decreasing intracellular calcium concentration.

Sarcoplasmic reticulum calcium pump in cardiac and slow twitch skeletal muscle but not fast twitch skeletal muscle undergoes phosphorylation by endogenous and exogenous Ca2+/calmodulin-dependent protein kinase. Characterization of optimal conditions for calcium pump phosphorylation.

We have demonstrated recently that in cardiac sarcoplasmic reticulum (SR), a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase) phosphorylates and activates the Ca(2+)-pumping ATPase (Ca(2+)-ATPase) in addition to phosphorylating the previously characterized substrates, phospholamban, and Ca2+ release channel (ryanodine receptor) (Xu, A., Hawkins, C., and Narayanan, N. (1993) J. Biol. Chem. 268, 8394-8397). The present study shows that a CaM kinase regulatory system capable of modulating SR Ca2+ pump activity through direct phosphorylation of the Ca(2+)-ATPase is functional in slow twitch but not fast twitch skeletal muscle. Incubation of SR vesicles isolated from rabbit slow twitch (soleus) and fast twitch (adductor magnus) skeletal muscles in the presence of Ca2+ and calmodulin resulted in phosphorylation of the Ca(2+)-ATPase in slow twitch muscle SR but not in fast twitch muscle SR. Exogenous CaM kinase II, which stimulated phosphorylation of the cardiac and slow twitch muscle SR Ca(2+)-ATPase, failed to phosphorylate fast twitch muscle SR Ca(2+)-ATPase. These observations demonstrate that CaM kinase-catalyzed phosphorylation of the Ca2+ pump is isoform-specific since heart and slow twitch muscle express the same Ca(2+)-ATPase isoform (SERCA2a), which is distinct from that of fast twitch muscle (SERCA1). As in the case of cardiac SR Ca(2+)-ATPase, phosphorylation of the slow twitch muscle SR Ca(2+)-ATPase (occurring at a serine residue) resulted in a 2-fold increase in catalytic activity of the enzyme without alteration in its Ca2+ sensitivity. In addition, Ca2+/calmodulin-dependent prephosphorylation of slow twitch muscle SR resulted in a greater than 2-fold increase in its Ca2+ transport activity. In both cardiac and slow twitch muscle SR, phosphorylation of the Ca(2+)-ATPase by the endogenous CaM kinase occurred rapidly (maximum within 2 min at 37 degrees C), had similar pH optimum (8.5-9.0), temperature optimum (30 degrees C), and calmodulin concentration-dependence (k0.5 50-60 nM). cAMP-dependent protein kinase did not phosphorylate the Ca(2+)-ATPase appreciably in either cardiac or slow twitch muscle SR. These findings suggest a muscle-specific role for the membrane-associated CaM kinase in the modulation of Ca2+ uptake and release functions of the SR. In cardiac and slow twitch muscle, phosphorylation of the SR Ca(2+)-ATPase by CaM kinase might provide a novel mechanism for the modulation of the enzymatic and Ca2+ transport functions of this enzyme.

Effects of a nonionic detergent on calcium uptake by cardiac microsomes.

We investigated the effects of the nonionic detergent octaethylene glycol monododecyl ether (C12E8) on the sarcoplasmic reticulum calcium pump in cardiac microsomes in view of its specific effects on different ATP-accelerated steps in the catalytic cycle of the Ca-ATPase in leaky fast skeletal muscle microsomes. At low concentrations of MgATP2- (< 2.5 microM), a nonsolubilizing concentration of added C12E8 (15 microM) increased apparent Vmax(MgATP) of oxalate-facilitated calcium uptake associated with MgATP2- binding to the high affinity catalytic site. An ATP induced acceleration of calcium uptake, attributable to regulatory nucleotide binding, was seen between 2 and 3 microM MgATP2- in both C12E8-treated and control microsomes. These effects of C12E8 are similar to those seen previously with trypsin treatment of microsomes [Lu, Y.-Z., Xu, Z.-C., & Kirchberger, M.A. (1993) Biochemistry 32, 3105-3111]. However, at a saturating Ca2+ between 3 and 10 microM MgATP2-, C12E8 produced a greater reduction in the magnitude of the ATP-induced acceleration of calcium uptake seen with trypsin. At 1 mM MgATP2-, C12E8 and trypsin as well as protein kinase A-catalyzed microsomal phosphorylation all increased the Ca2+ affinity of the pump, but only the latter two treatments significantly increased apparent Vmax(Ca). In fact in trypsin-treated and phosphorylated microsomes, C12E8 reduced Vmax(Ca) to close to the control values; it reduced Vmax(Ca) only slightly in control microsomes. Under our experimental conditions, comparable effects of 15 microM C12E8 on calcium uptake were absent in fast skeletal muscle microsomes, which lack phospholamban.(ABSTRACT TRUNCATED AT 250 WORDS)

CIDNP study of the aromatic side chain interactions in myotoxin alpha.

CIDNP and COSY measurements were applied to study aromatic side chain interactions and conformations in myotoxin alpha, a Crotalus venom toxin which acts as blocker of the Ca2+ influx in the sarcoplasmic reticulum calcium pump. New evidence for the existence of a hydrophobic aromatic cluster at the amino terminus was obtained. This cluster consists of Tyr1, His5, His10, and (possibly) F12. The CIDNP data clearly establish that the usual order of the tyrosine 2, 6 and 3, 5 proton signals of Tyr1 is inverted, because of the large diamagnetic shielding effects of one ring on the other. The lines of the 2, 6 ring protons of Tyr1 and proton 4 in each of His5 and His10 are significantly broadened, an outcome of the side-chain hydrophobic interaction. The aromatic cluster could possibly present a hydrophobic sticky patch for binding of toxin by Ca2+ ATPase.

Control of the calcium pump of cardiac sarcoplasmic reticulum. A specific role for the pentameric structure of phospholamban?

Intracellular sources of calcium provide most of that required for cardiac muscle contraction. Regulation of the filling of these calcium stores by control of the enzyme responsible for calcium uptake, the sarcoplasmic reticular calcium pump, provides a mechanism by which the contractile properties of the heart can be adapted. In fact, this single biochemical event accounts for most of the changes in calcium handling associated with beta adrenergic stimulation. The calcium pump of cardiac sarcoplasmic reticulum is regulated by the phosphorylation status of a second sarcoplasmic reticular component, phospholamban. This is a low molecular weight membrane protein which assembles as an oligomer (homopentamer) and inhibits pump function by physical interaction with a "regulatory" motif on the pump. This interaction (and the state of inhibition) is broken following phosphorylation of phospholamban by cAMP-, cGMP, or Ca2+/calmodulin dependent protein kinases. This model of pump control shares a number of features associated with control of the plasma membrane calcium pump; however, the oligomeric arrangement of the regulator is specific for the sarcoplasmic reticular ATPase. The functional significance of the oligomer is currently unknown, but this format may provide functional advantages such as (1) offering an effective substrate structure for kinase/phosphatase, promoting rapid changes in phosphate status; (2) facilitating intrapentamer cooperation, translating low stoichiometric phosphorylation into large changes in pump function; or (3) coordinating multiple pump units around a single pentamer thus restricting conformational freedom and suppressing enzyme activity.

Coexistence of high- and low-affinity calcium binding sites of the sarcoplasmic reticulum calcium pump

We have recently shown [Mészáros, L. G., & Bak, J. (1992) Biochemistry 31, 1195-1200] that, during the rapid phase of Ca2+ uptake into sarcoplasmic reticulum (SR), internalization and binding of Ca2+ to the cytoplasmic high-affinity binding sites of the Ca2+ ATPase occur simultaneously, resulting in a transient supernumerary Ca/ATP stoichiometry. Here we address the question of whether the cytoplasmic high-affinity and the luminal low-affinity Ca2+ binding sites of the SR Ca2+ ATPase also coexist. SR vesicles were loaded with Ca2+ (0-10 mM), and then the kinetics of EP formation and decomposition as well as the maximum level of EP formed from radiolabeled ATP were determined at conditions which only allow single-cycle reactions to occur: empty or Ca-loaded SR vesicles (in micromolar extravesicular Ca2+) were either mixed with ATP plus millimolar EGTA or added in amounts that set a Ca2+ ATPase/ATP ratio of 80-85 at the initiation of the reaction. The rates of EP formation and decomposition were both significantly reduced in Ca-loaded, compared to empty (ionomycin-treated), vesicles. However, the value of EPmax was unaltered by Ca-loading, suggesting the existence of the enzyme intermediate, E.Ca2(cyt).Ca2(lum), i.e., the coexistence of the cytoplasmic and the luminal Ca2+ binding sites of the Ca-pump. These results suggest that the uphill transport of Ca2+ might not be based on an alternating relocation and conversion of the Ca2+ binding sites of the Ca2+ ATPase.

Effect of pH on stability of sarcoplasmic reticulum calcium pump in rabbit heart.

The sarcoplasmic reticulum (SR) membranes isolated from rabbit heart were preincubated at pH 6.8 or 7.8 and their Ca2+ pump properties were compared at pH 6.8. The ATP-dependent azide insensitive oxalate-stimulated Ca2+ uptake was reduced more rapidly from the membranes preincubated at 37 degrees C at pH 7.8 than from those preincubated at pH 6.8. The Ca(2+)-Mg(2+)-ATPase, and the Ca(2+)-dependent formation of 110 kDa acylphosphate were also inhibited by the preincubation at the higher pH. Including 1 mM DTT in the preincubation medium reduced the inactivation. The preincubation at 37 degrees C in the presence or absence of DTT caused membranes to become more leaky as the loss of Ca2+ uptake was more rapid than that of ATPase or the acylphosphate formation. The loss of these activities was not accompanied by a breakdown of the protein as monitored in Western blots. It is hypothesized that the SR Ca2+ pump inactivation involves a key-SH group and that the lower pH provides a compensatory protective mechanism for the SR during acidosis.

Regulation of sarcoplasmic reticulum calcium pump gene expression by hindlimb unweighting.

Hindlimb unweighting (HU) causes upregulation of several muscle-specific genes responsible for the slow-to-fast transition in soleus skeletal muscle properties despite the profound muscle atrophy. The purpose of this study was to examine the expression of the fast and slow isoforms of the sarcoplasmic reticulum Ca(2+)-ATPase at the mRNA and protein level in the soleus muscle over a time course of HU and relate them to Ca(2+)-dependent ATPase activity and selected contractile properties. mRNA levels of the acetylcholine receptor (AChR) were measured to compare the signal of unweighting with denervation. Atrophy of the soleus muscles from tail-suspended rats was observed at all time points with muscle mass decreased by 52% at 28 days of HU (P < 0.05). Northern blot analysis showed the relative expression of the fast Ca2+ pump mRNA increased by 0, 250, 910, 1,340, and 4,050% over control levels at 1, 4, 7, 14, and 28 days of HU, respectively, whereas changes in slow mRNA were variable and modest in comparison. For the same time points, Western blot analysis showed relative expression of the fast Ca2+ pump protein increased by 30, 110, 320, 280, and 300% over control levels, whereas the slow-pump protein expression was unchanged except for a 75% decrease at 28 days of HU. Specific Ca(2+)-dependent ATPase activity was increased (P < 0.05) by 170% at 28 days of HU. Contractile properties measured in vitro at 14 and 28 days revealed time to peak tension and one-half relaxation time were shortened (P < 0.05) and a rightward shift in the tension-frequency curves in unloaded soleus muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Sarcoplasmic reticulum calcium transport and Ca(2+)-ATPase gene expression in thoracic and abdominal aortas of normotensive and spontaneously hypertensive rats.

Increased intracellular Ca2+ concentration has been associated with the elevation of vascular tone in hypertensive animals. The increase in free cytosolic Ca2+ may partially result from a reduced activity of the sarcoplasmic reticulum (SR) calcium pump. Accordingly we investigated the Ca2+ transport function and the expression of the Ca(2+)-ATPase gene in thoracic and abdominal aortas of normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Total SR Ca2+ pump activity was estimated by measuring the oxalate-stimulated Ca2+ transport rate on crude homogenates. Ca2+ transport was also measured on highly active microsomal fractions. Our data indicate that the Ca2+ uptake rate, expressed per mg of protein or per g of muscle, is greater in homogenates from aortas of SHR when compared with that of WKY rats. In microsomal fractions isolated from thoracic aortas of SHR compared with WKY rats, the activity and density of SR Ca2+ pump were only slightly increased. The SR Ca2+ transport rate and the amount of each SR Ca(2+)-ATPase mRNA isoform, i.e. SERCA 2a and SERCA 2b, normalized to 18 S ribosomal RNA, were greater in thoracic than in abdominal aorta in both strains. When compared with WKY rats, the level of each SERCA mRNA isoform is higher in the abdominal aorta of SHR but appears similar in the thoracic aorta. Thus, in contrast to previously published data that documented a depressed SR Ca2+ transport activity in the aorta of SHR, the present data indicate that the SR function is increased. These changes in SR activity are accompanied by quantitative changes in expression of the SR Ca(2+)-ATPase gene without alterations in the SR Ca(2+)-ATPase mRNA isoforms pattern.

Evidence for an effect of phospholamban on the regulatory role of ATP in calcium uptake by the calcium pump of the cardiac sarcoplasmic reticulum.

The purpose of this study was to investigate the functional relationship between phospholamban and the nucleotide site of the calcium pump protein of the cardiac sarcoplasmic reticulum. We used control and trypsin-treated cardiac microsomes in which cleavage of the inhibitory cytoplasmic domain of phospholamban is associated with an activation of the calcium pump similar to that produced by protein kinase A catalyzed phospholamban phosphorylation. Phenylglyoxal was shown to inactivate the calcium pump in a pseudo-first-order reaction by binding to a single Arg at the nucleotide binding site. No differences upon trypsin treatment of microsomes were observed in the kinetics of phenylglyoxal inactivation or the ability of millimolar ATP to protect against inactivation. In subsequent kinetic studies, Ca-uptake rates measured at saturating Ca2+ and 5 microM-1 mM MgATP2- were increased 15-32% by trypsin treatment in each of three different microsome preparations. Double-reciprocal plots of the data showed marked downward curvature indicating an acceleratory effect associated with ligand binding to a lower affinity site. At 0.32 microM Ca2+, Ca-uptake rates were lower than at 11 microM Ca2+ but were stimulated to a greater extent by trypsin treatment; control microsomes showed reduced evidence of apparent negative cooperativity. At 0-2 microM MgATP2- and saturating Ca2+, there was a 50% increase in Vmax(app) when the Hill coefficient (N) was 1. At 0-10 microM MgATP2-, second-site binding was evident. At both 0-10 microM and 5 microM-1 mM MgATP2-, trypsin-treated microsomes showed greater activation of Ca uptake attributable to second-site binding than did control microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

The MgATPase activity of rat cardiac sarcoplasmic reticulum is a function of the calcium ATPase protein

Magnesium-dependent ATPase (MgATPase) activity is associated with many E 1-E 2 or P-type transport ATPases including the sarcoplasmic reticulum (SR) calcium ATPase. The SR isolated from rat heart has a MgATPase activity which is 6–12 times faster than the MgATPase activity of the SR isolated from dog heart. To determine the origin of the high MgATPase activity of rat heart SR, we compared and contrasted cardiac SR isolated from both species. The preparations were similar in the following ways: (i) contamination by other organelles; (ii) the comigration of MgATPase activity with calcium-dependent ATPase (CaATPase) activity through a sucrose gradient; (iii) a similar ATPase activity sensitivity to pH and ATP concentration; (iv) the high and similar of sensitivity of ATPase activity to detergent; and (v) a similar protein profile. In both preparations, a single protein in the 105,000-Da region of polyacrylamide gels was phosphorylated by ATP, and the phosphorylated species was an acylphosphate formed in the presence and absence of calcium. Dimethyl sulfoxide, which slows acylphosphoenzyme breakdown, markedly inhibited both CaATPase and MgATPase activities of both preparations but not other enzyme activities. Importantly, the specific inhibitor of the SR calcium pump, thapsigargin, completely inhibited the CaATPase activity with an I 50 of 6–7 n m; however, a higher concentration (I 50 of 2 μ m) was required to inhibit the MgATPase activity of the rat cardiac SR. These results provide evidence that the Mg-ATPase activity of rat cardiac SR is part of the enzyme cycle of the calcium ATPase protein.

Effects of low myoplasmic Mg2+ on calcium binding by parvalbumin and calcium uptake by the sarcoplasmic reticulum in frog skeletal muscle.

The effects of low intracellular free Mg2+ on the myoplasmic calcium removal properties of skeletal muscle were studied in voltage-clamped frog skeletal muscle fibers by analyzing the changes in intracellular calcium and magnesium due to membrane depolarization under various conditions of internal free [Mg2+]. Batches of fibers were internally equilibrated with cut end solutions containing two calcium indicators, antipyrylazo III (AP III) and fura-2, and different concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor [Ca2+] and [Mg2+] transients, whereas fura-2 fluorescence was mostly used to monitor resting [Ca2+]. Shortly after applying an internal solution containing less than 60 microM free Mg2+ to the cut ends of depolarized fibers most of the fibers exhibited spontaneous repetitive movements, suggesting that free internal Mg2+ might affect the activity of the sarcoplasmic reticulum (SR) calcium channels at rest. The spontaneous contractions generally subsided. In polarized fibers the maximal amplitude of the calcium transient elicited by a depolarizing pulse was about the same whatever the internal [Mg2+], but its decay after the end of the pulse slower in low [Mg2+]. In low [Mg2+] (less than 0.14 mM), the mean rate constant of decay obtained from fitting a single exponential plus a constant to the decay of the calcium transients was approximately 30% of its value in the control fibers (1 mM internal [Mg2+]). A model characterizing the main calcium removal properties of a frog skeletal muscle fiber, including the SR pump and the Ca-Mg sites on parvalbumin, was fitted to the decay of the calcium transients. Results of the fits show that in low internal [Mg2+] the slowing of the decay of the calcium transient can be well predicted by both a decreased rate of SR calcium uptake and an expected decreased resting magnesium occupancy of parvalbumin leading to a reduced contribution of parvalbumin to the overall rate of calcium removal. These results are thus consistent with the known properties of parvalbumin as a Ca-Mg buffer and furthermore suggest that in an intact portion of a muscle fiber, the activity of the SR calcium pump can be affected by the level of free Mg2+.

Molecular mechanism of sarcoplasmic reticulum calcium pump

Molecular mechanism of sarcoplasmic reticulum calcium pump

Developmental regulation of the sarcoplasmic reticulum calcium pump in the rabbit heart.

Previous studies have demonstrated that myocardial function changes during mammalian perinatal development. The purpose of this study was to evaluate the subcellular basis underlying the slower relaxation in the developing heart by examining perinatal changes in sarcoplasmic reticulum (SR) function, and in SR Ca2+ pump protein and mRNA abundance. We measured Ca2+ uptake and ATPase rates in isolated fetal, newborn, and adult rabbit cardiac SR membranes. In fetal and adult SR membranes, we estimated the active Ca2+ pump protein content by measuring the steady state Ca(2+)-dependent phosphoenzyme content; the total Ca2+ pump protein content was estimated by Western analysis of the immunoreactive Ca2+ pumps. We isolated RNA from fetal and adult hearts and estimated the SR Ca2+ pump mRNA content by Northern analysis. Ca2+ uptake and ATPase rates were significantly lower in the fetal and newborn SR membranes compared with the adult. The contents of active and total Ca2+ pump protein and of Ca2+ pump mRNA were 52-63% lower in the fetus than in the adult. These results indicate that a great deal of the slower sarcoplasmic reticulum Ca2+ uptake and ATPase rates in the fetal rabbit heart can be related to lower Ca2+ pump mRNA and protein contents. It is evident that transcriptional and/or posttranscriptional regulation of the SR Ca2+ pump may form an important part of the subcellular basis of the perinatal change in mammalian cardiac relaxation.

Mechanism of inhibition of the calcium pump of sarcoplasmic reticulum by thapsigargin.

The steady-state ATPase activity of sarcoplasmic-reticulum (Ca(2+)-Mg2+)-ATPase is inhibited by thapsigargin at a molar ratio of 1:1, with a dissociation constant for thapsigargin estimated to be in the sub-nanomolar range. In the presence of thapsigargin, only a single Ca2+ ion binds to the ATPase. Similarly, addition of thapsigargin to the ATPase incubated in the presence of Ca2+ results in the release of one of the two originally bound Ca2+ ions. As monitored by the fluorescence of nitrobenzo-2-oxa-1,3-diazole-labelled ATPase, thapsigargin appears to shift the transition between E1 and E2 conformations towards E2. Addition of thapsigargin prevents phosphorylation of the ATPase by P(i) and results in a very low steady-state level of phosphorylation of the ATPase by ATP, as observed previously for nonylphenol.

Simultaneous internalization and binding of calcium during the initial phase of calcium uptake by the sarcoplasmic reticulum Ca pump.

The kinetics of Ca2+ transport mediated by the sarcoplasmic reticulum (SR) Ca-ATPase were investigated by rapid kinetic techniques that either measure the disappearance of Ca2+ from the medium [stopped-flow photometry of Ca2+ indicators or rapid filtration (method 1)] or directly detect the changes in the accessibility of Ca2+ to the exterior of the membrane, i.e., occlusion of Ca2+ within the Ca pump and Ca2+ transport into the lumen of SR vesicles [EGTA quench (method 2)]. SR vesicles were preincubated in micromolar Ca2+ to form the E.2Cacyt intermediate of the Ca-ATPase, and then Ca2+ transport was initiated by addition of ATP. It was found that Ca2+ uptake measured by method 1 began with no lag phase, in spite of the prediction of kinetic models of the Ca-ATPase. Instead, the time course of Ca2+ uptake was found to have two components: a fast and a slow phase, similar to that obtained using method 2, although the rate constant of the fast phase determined by method 1 was considerably lower than that measured by method 2. The fast phase of Ca2+ uptake measured by method 1 was not influenced by either Ca2+ ionophore or detergent treatment, whereas the slow phase was diminished.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of rat cardiac calcium pump activity by cationic amphiphilic drugs

Cationic amphiphilic drugs such as imipramine, chlorimipramine, chlorpromazine, chlorphentermine, and chloroquine have been shown to cause myocardial toxicity including arrhythmia, cardiomyopathy, and myocarditis. Since calcium transport by sarcoplasmic reticulum (SR) plays a critical role in contraction-relaxation coupling of myocardium, the toxicity exhibited by these drugs could be due to their effects on this phenomenon. Hence, we have studied the effects in vitro and in vivo of imipramine, chlorimipramine, chlorpromazine, chloroquine, and chlorphentermine on cardiac SR 45Ca-uptake and Ca2+-ATPase in male Fisher-344 rats. For in vitro studies, cardiac SR was prepared from normal rat hearts. For in vivo studies, rats were treated with drugs by oral gavage (p.o) at a dose of 20 pair-fed. All these drugs in vitro inhibited the cardiac SR 45Ca-uptake to a varying extent. The order of potency is chlorimipramine > chlorpromazine > imipramine > chlorphentermine > chloroquine. The inhibitory pattern of these drugs on cardiac SR Ca2+-ATPase also followed the same trend. The chlorinated analogs of these drugs are more potent in inhibiting 45Ca-uptake as well as Ca2+-ATPase as compared to their nonchlorinated con-geners. In vivo, all the drugs except chloroquine inhibited the 45Ca-uptake and Ca2+-ATPase activity to a different extent. The inhibition of cardiac SR calcium pump activity was seen as early as 7 days of treatment with chlorimipramine whereas all the other drugs (imipramine, chlorpromazine, and chlorphentermine) inhibited this calcium pump only at 21 days of treatment. Although in vivo chloroquine-induced inhibition of calcium pump activity did not reach significant level, it seems that there may be an effect at higher concentrations. In summary, the present results indicate that these cationic amphiphilic drugs might be exerting myocardial toxicity due to changes in cellular Ca2+ homeostasis because of their effects on cardiac SR calcium transport phenomenon. © 1992 Wiley-Liss, Inc.

Kinetic characterization of the normal and procaine-perturbed reaction cycles of the sarcoplasmic reticulum calcium pump.

We investigated the effect of the local anesthetic procaine on the activity of the calcium pump protein of sarcoplasmic reticulum (SR) vesicles. Procaine slowed down the rate of calcium uptake by SR vesicles without enhancing the vesicles' passive permeability. This slowing of the unidirectional pumping rate was reflected by the inhibition of the maximal rate of the transport-coupled Ca(2+)-ATPase activity. The inhibition was dependent on Mg2+ concentration; at optimal (i.e. low) concentrations of magnesium, half-maximal inhibition occurred with procaine concentrations close to 15-20 mM. Inhibition of ATPase was not mediated by a change in the properties of the bulk lipid phase. Procaine moderately reduced the true affinity of ATPase for ATP, whereas equilibrium binding of calcium to ATPase in the absence of ATP was virtually not modified by procaine. In fast-kinetics studies, we explored the various intermediate steps in the ATPase catalytic cycle, in order to determine which of them were targets for inhibition by procaine. We found that procaine slowed down ATPase dephosphorylation, an effect which is at least partly responsible for the observed inhibition of overall ATPase activity. In contrast, procaine accelerated the calcium-induced transconformation of unphosphorylated ATPase in the absence of ATP, and altered neither the rate of the Ca(2+)-dependent phosphorylation of ATPase, nor the rate of the dissociation of Ca2+ from phosphorylated ATPase towards the SR lumen, a critical step, the rate of which was measured by a novel fast-filtration method. These results are discussed with respect to the possible site(s) of binding of this amphiphile on the ATPase, and in relation to the contribution of individual steps in the catalytic cycle to the rate limitation of unperturbed SR ATPase activity.