ATPase Activity Of Sarcoplasmic Reticulum Research Articles

O-201 - The effect of a Molsidomine metabolite (SIN-1A) on (Ca2+-Mg2+ )ATPase activity in sarcoplasmic reticulum from guinea-pig aortic smooth muscle

O-201 - The effect of a Molsidomine metabolite (SIN-1A) on (Ca2+-Mg2+ )ATPase activity in sarcoplasmic reticulum from guinea-pig aortic smooth muscle

The major metabolite of doxorubicin is a potent inhibitor of membrane-associated ion pumps. A correlative study of cardiac muscle with isolated membrane fractions.

Doxorubicin (adriamycin) is a highly effective cancer chemotherapeutic drug but its clinical utility is limited by its cardiotoxicity. Doxorubicinol, the major metabolite of doxorubicin, is up to 10 times more potent than doxorubicin at inhibiting isometric contraction of the papillary muscle isolated from the right ventricle of rabbit heart. Doxorubicinol also increases resting tension of isolated cardiac muscle indicative of incomplete relaxation between contractions, a characteristic of doxorubicinol but not of doxorubicin. This study assesses the effect(s) of doxorubicinol on a variety of ion pumps which may explain, in part, the action of the metabolite in the intact muscle. We find the doxorubicinol is a potent inhibitor (IC50 less than 5 micrograms/ml) of calcium-stimulated ATPase activity of sarcoplasmic reticulum from canine heart and rabbit skeletal muscle. At comparable levels, doxorubicinol is also a potent inhibitor of (Na + K)-ATPase of cardiac sarcolemma and the Mg-dependent ATPase activity referable to the F0F1 proton pump of mitochondria. For each of these ion pumps, doxorubicinol is at least 80 times more potent an inhibitor than doxorubicin. Doxorubicinol, between 10 and 50 micrograms/ml, increases resting tension up to 4-fold in isolated papillary muscles cyclically contracting at 30 times/min. Resting stress is relatively insensitive to doxorubicin. Thus, doxorubicinol is a potent inhibitor of several key cationic pumps that directly or indirectly regulate cell calcium and inhibits relaxation in the isolated fiber preparation. These observations add a new dimension to understanding the cardiotoxicity of doxorubicin.

Preparation and characterization of longitudinal tubules of sarcoplasmic reticulum from fast skeletal muscle

Sarcoplasmic reticulum (SR) serves a central role in calcium uptake and release, thereby regulating muscle relaxation and contraction, respectively. Recently, we have isolated fractions referable to longitudinal tubules (R2) and terminal cisternae (R4), the two major types of Sarcoplasmic reticulum (A. Saito et al. (1984) J. Cell Biol. 99, 875–885). The terminal cisternae contain two types of membranes, the calcium pump membrane and the junctional face membrane. The terminal cisternae are filled with electronopaque contents which serve as a Ca 2+ reservoir. The longitudinal tubules consist mainly of the calcium pump membrane. In this study, we describe a new longitudinal tubule fraction (F2) and characterize it together with the R2 and R4 SR fractions. The calcium pump membrane of the longitudinal tubules is a highly specialized membrane consisting of about 90% calcium pump protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extensive changes in morphology can be observed in the SR fractions referable to osmotic differences during the fixation conditions using either glutaraldehyde-tannic acid or osmium tetroxide fixatives. The changes include swelling or shrinkage and aggregation of the compartmental contents when the fixative contains calcium ions. The two types of SR have different osmotic permeability to the same medium, as indicated by differential swelling or shrinkage. Both longitudinal tubule and terminal cisternae vesicles of SR appear larger and are spherical vesicles when the glutaraldehyde-tannic acid fixative is isotonic as compared with the “standard” fixation method. We have previously reported that the ruthenium red-sensitive calcium release channels are localized to the terminal cisternae. The terminal cisternae as isolated are leaky to Ca 2+ since these channels are in the “open state” (S. Fleischer et al. (1985) Proc. Natl. Acad. Sci USA 82, 7256–7259). Thus, the Ca 2+,Mg 2+-dependent ATPase (Ca 2+ ATPase) rate is only slightly enhanced in the presence of a Ca 2+ ionophore, which dissipates the Ca 2+ gradient across the SR membrane. We now find that preincubation with ruthenium red restores the tight coupling of the Ca 2+ ATPase activity to Ca 2+ transport. That is to say, ATPase activity is reduced and the addition of ionophore stimulates the Ca 2+ ATPase activity 4– to 7-fold. The Ca 2+ ATPase activity in longitudinal tubules is already tightly coupled. It is minimal after a Ca 2+ gradient has been generated, but can be stimulated 9– to 20-fold when the Ca 2+ gradient is dissipated with ionophore. This finding suggests that the Ca 2+ ATPase activity in SR is tightly coupled to Ca 2+ transport in situ.

Gingerol, a novel cardiotonic agent, activates the Ca 2+-pumping ATPase in skeletal and cardiac sarcoplasmic reticulum

Gingerol, isolated as a potent cardiotonic agent from the rhizome of ginger, stimulated the Ca 2+-pumping activity of fragmented sarcoplasmic reticulum (SR) prepared from rabbit skeletal and dog cardiac muscles. The extravesicular Ca 2+ concentrations of the heavy fraction of the fragmented SR (HSR) were measured directly with a Ca 2+ electrode to examine the effect of gingerol on the SR. Gingerol (3–30 μM) accelerated the Ca 2+-pumping rate of skeletal and cardiac SR in a concentration-dependent manner. The rate of 45Ca 2+ uptake of HSR was also increased markedly by 30 μM gingerol without affecting the 45Ca 2+ efflux from HSR. Furthermore, gingerol activated Ca 2+-ATPase activities of skeletal and cardiac SR (EC 50, 4 μM). The activation of SR Ca 2+-ATPase activity by gingerol (30 μM) was completely reversed by 100-fold dilution with the fresh saline solution. Kinetic analysis of activating effects of gingerol suggests that the activation of SR Ca 2+-ATPase is uncompetitive and competitive with respect to Mg · ATP at concentrations of 0.2–0.5 mM and above 1 mM, respectively. Kinetic analysis also suggests that the activation by gingerol is mixed-type with respect to free Ca 2+ and this enzyme is activated probably due to the acceleration of enzyme-substrate complex breakdown. Gingerol had no significant effect on sarcolemmal Ca 2+-ATPase, myosin Ca 2+-ATPase, actin-activated myosin ATPase and cAMP-phosphodiesterase activities, indicating that the effect of gingerol is rather specific to SR Ca 2+-ATPase activity. Gingerol may provide a valuable chemical tool for studies aimed at clarifying the regulatory mechanisms of SR Ca 2+-pumping systems and the causal relationship between the Ca 2+-pumping activity of SR and muscle contractility.

Mechanistic origin of the kinetic cooperativity for the ATPase activity of sarcoplasmic reticulum.

The (Ca2+-Mg2+)-ATPase from sarcoplasmic reticulum presents negative cooperativity for the hydrolysis of Mg2+-ATP at different concentration ranges of this substrate. A kinetic model is proposed according to which Mg2+-ATP may bind to three different enzymatic species present during the catalytic cycle. E (K1 = 1 microM). E' approximately P.Ca2 (K9 = 500 microM) and EP (K7 = 20 microM), accelerating the release of Pi. The fact that each of these species has a different affinity for Mg2+-ATP allows a significant enhancement of the rate of Pi release to the medium at the different ranges of Mg2+-ATP concentration where the enzyme shows a kinetic cooperativity. The kinetic analysis of this mechanism yields an equation which is a ratio of two cubic polynomials (3:3 rate equations) with respect to Mg2+-ATP and which may explain the negative cooperativity of the enzyme at different concentration ranges of Mg2+-ATP.

Comparison of ATP-dependent calcium transport and calcium-activated ATPase activities of cardiac sarcoplasmic reticulum and sarcolemma from rats of various ages

Age-associated decline in the Ca 2+ pump function of cardiac sarcoplasmic reticulum (SR), and increase in the Ca 2+ pump activity of sarcolemma (SL) were suggested by my previous study which compared the ATP-energized in vitro Ca 2+ transport activities of these membranes from young (3–4-month-old) and aged (24–25-month-old) rat myocardium ( Biochim. Biophys. Acta, 678 (1981) 442–459). In the present study, ATP-dependent Ca 2+ transport and Ca 2 sensitive ATPase activities of SR and SL derived from the myocardium of rats aged 3 (young), 6 (young adult), 12 (adult), 18 (aging) and 24 (aged) months were determined so as to further characterize age-related changes in the Ca 2+ transport function of these membranes. The rates of ATP-dependent Ca 2+ accumulation by SR from 3- and 6-month-old rats were virtually similar whereas the rates of Ca 2+ accumulation by this membrane from 12-, 18- and 24-month-old rats were significantly lower when compared to 3- or 6-month-old rats; the magnitude of this age-related decline amounted to approx. 18, 45 and 50%, respectively, for SR from 12-, 18- and 24-month-old animals. In contrast to the above findings with SR, SL from 18- and 24-month-old rats displayed substantially higher rates (approx. 45 and 80% increase, respectively, at 18 and 24 months of age) of ATP-dependent Ca 2+ accumulation than SL preparations from 3-, 6- and 12-month-old rats; no significant age-related difference was evident between the latter three age groups. The divergent age-related changes in the Ca 2+ accumulating activities of SR and SL were seen at varying Ca 2+ concentrations (0.54–25.2 μM). With either membrane, kinetic analysis showed that the velocity of Ca 2+ transport, but not the apparent affinity of the transport system for Ca 2+, underwent age-related changes. The Ca 2+-stimulated ATPase activities of SR and SL were not altered significantly with increasing age from 3 to 24 months. Comparison of the ‘combined Ca 2+ transport activity’ of SR and SL from rats of various ages showed a significant overall age-related decline in the rates of Ca 2+ transport via the ATP-driven membrane Ca 2+ pumps; this decrement in membrane function was moderate at 12 months of age (approx. 16%) and became pronounced with advancing age thereafter (approx. 35 and 38%, respectively, at 18 and 24 months of age). Similar progressive age-related decline was observed in the ATP-dependent Ca 2+ sequestering activity of cardiac homogenates. No significant age-related difference was evident in the ATP- and respiration-supported Ca 2+ uptake activity of mitochondria isolated from 6- and 24-month-old rats. The above findings suggest that, barring compensatory age-related increment in Ca 2+ efflux via Na +-Ca + exchange across the SL, the overall ability of the myocardial cell to sequester Ca 2+ from the sarcoplasm declines with aging, mainly due to deterioration in the Ca 2+ pump activity of SR. This impairment in SR function is likely the major factor underlying the prolongation of cardiac relaxation seen with aging. The age-associated decline in the Ca 2+ transport activity of SR may involve uncoupling of the energy transduction and ion translocation functions of this membrane Ca 2+ pump.

Coronary artery ligation and reperfusion in rabbits made diabetic with alloxan.

The biochemical and functional changes associated with ligation (40 min) of the left circumflex coronary artery and subsequent reperfusion (60 min) in the rabbit made diabetic with alloxan were studied and compared with those of control animals. Measurement of haemodynamic parameters revealed that both left ventricular pressure and mean arterial pressure were significantly (P less than 0.05) decreased after ligation and reperfusion in the diabetic animals compared with controls. Analysis of subcellular organelle enzyme markers from the ischaemic tissue revealed that sarcolemmal Na+,K+-ATPase, mitochondrial ATPase and sarcoplasmic reticulum ATPase activities were decreased after ligation to the same extent in the diabetic and control animals. However, upon reperfusion, the recovery of mitochondrial ATPase activity was significantly (P less than 0.05) less in the diabetic animals than in the controls. Ion measurements revealed a significant (P less than 0.05) depletion of Mg in diabetic hearts before ligation, and this was augmented during reperfusion. In contrast, a significantly (P less than 0.05) higher calcium accumulation was observed upon reperfusion in the hearts of diabetic animals. Similarly, both tissue ATP levels and the ability of the mitochondria to generate ATP were depressed to a greater degree in the diabetic animals. Our results indicate, therefore, a greater susceptibility of the diabetic myocardium to ischaemic/reperfusion injury which in the clinical situation would exacerbate the problems associated with atherosclerosis and possibly contribute to the high mortality from cardiovascular complications in diabetic patients.

Inhibition of sarcoplasmic reticulum Ca 2+-ATPase by 2-mercaptoethanol

The Ca 2+-dependent ATPase activity of sarcoplasmic reticulum was inhibited when membrane vesicles were incubated at 0°C in presence of thiols. 2-mercaptoethanol was the most effective inhibitor from the thiols tested. The effect of 2-mercaptoethanol on the ATPase activity was biphasic; enzyme inhibition originally increased and then decreased with increasing thiol concentration. The inhibitory action of this thiol was significantly higher at low membrane concentrations and the rate of inactivation at 22°C was considerably lower than that at 0°C. Ca 2+-ATPase previously inhibited by 2-mercaptoethanol was partially reactivated by incubation with periodate.

Effect of superoxide generation on rat heart mitochondrial pyruvate utilization

Previous research has shown that heart mitochondria are able to produce reactive species of oxygen such as superoxide radicals, hydrogen peroxide and hydroxyl radicals [ 10, 11]. When these compounds are formed beyond a certain level they are not completely removed by the enzymatic and metabolic processes which neutralize their toxicity, and as a result they are able to produce structural and functional damages that impair mitochondrial function [ 5, 10]. In order to study the molecular mechanism/s by which the oxygen radicals may function as mediators of cellular injury a flow of these radicals by chemical, enzymatic or photochemical methods has been generated in vitro in the presence of cellular preparations. For example, the exposure of isolated subcellular particles to the enzymatic flow of oxygen radicals produced by the reaction of xanthine oxidase upon xanthine reduced both calcium uptake velocity and Ca 2+-ATPase activity in sarcoplasmic reticulum [ 7], while it reduced Ca 2+-stimulated ATPase activity in myofibrillar preparations [ 4]. In addition, incubation with the xanthine oxidase reaction produced an impairment of the respiratory functions associated with an increased lipid peroxidation in the isolated mitochondria [ 5, 10]. These negative effects were augmented in α-tocopherol-deficient mitochondria [ 3], but were opposed by the exogenous addition of superoxide dismutase [ 10]. This report shows that the superoxide radicals generated by the xanthine oxidase reaction reduced rat heart mitochondrial respiration induced by pyruvate. This negative effect was partially prevented by superoxide dismutase and catalase and by thiol protecting agents. Moreover, the generation of free radicals caused a significant reduction in the rate of (1- 14C)-pyruvate decarboxylation, while it did not change the transport of pyruvate into mitochondria.

Ischaemia-induced changes in canine cardiac sarcoplasmic reticulum.

Effects of myocardial ischaemia on sarcoplasmic reticulum (SR) of dog hearts were investigated. Regional ischaemia was produced by occlusion of the left circumflex artery, and a microsomal fraction enriched in vesicles of SR was isolated from subendocardium (Endo) and subepicardium (Epi) of control and ischaemic areas of the heart. No significant changes occurred in ischaemic Epi. A loss of in vitro activities (ie calcium transport and ATPase) was found for SR from ischaemic Endo which paralleled the changes in the histology of the tissue. At 5 min of coronary occlusion, Ca2+ binding and Ca2+-ATPase activities of SR from ischaemic Endo were normal. A decrease in the activities of SR was first evident at 15 min after the occlusion, decreased further at about 30 min and remained at that level at 60 min of ischaemia. The maximal rate of Ca2+ uptake did not parallel the Ca2+-binding and Ca2+-ATPase activities. The degree of cAMP-dependent phosphorylation by endogenous and exogenous protein kinase was not different between SR from control and ischaemic areas. A participatory role of SR in the ischaemic impairment of left ventricular systolic and diastolic performance is discussed.

Inhibition of sarcoplasmic reticulum calcium pump by cytosolic protein(s) endogenous to heart and slow skeletal muscle but not fast skeletal muscle

Cytosol from rabbit heart and slow and fast skeletal muscles was fractionated using (NH 4) 2SO 4 to yield three cytosolic protein fractions, viz., CPF-I (protein precipitated at 30% saturation), CPF-II (protein precipitated between 30 and 60% saturation), and cytosol supernatant (protein soluble at 60% saturation). The protein fractions were dialysed and tested for their effects on ATP-dependent, oxalate-supported Ca 2+ uptake by sarcoplasmic reticulum from heart and slow and fast skeletal muscles. CPF-I from heart and slow muscle, but not from fast muscle, caused marked inhibition (up to 95%) of Ca 2+ uptake by sarcoplasmic reticulum from heart and from slow and fast muscles. Neither unfractionated cytosol nor CPF-II or cytosol supernatant from any of the muscles altered the Ca 2+ uptake activity of sarcoplasmic reticulum. Studies on the characteristics of inhibition of sarcoplasmic reticulum Ca 2+ uptake by CPF-I (from heart and slow muscle) revealed the following: (a) Inhibition was concentration- and temperature-dependent (50% inhibition with approx. 80 to 100 μg CPF-I; seen only at temperatures above 20°C). (b) The inhibitor reduced the velocity of Ca 2+ uptake without appreciably influencing the apparent affinity of the transport system for Ca 2+. (c) Inhibition was uncompetitive with respect to ATP. (d) Sarcoplasmic reticulum washed following exposure to CPF-I showed reduced rates of Ca 2+ uptake, indicating that inhibition results from an interaction of the inhibitor with the sarcoplasmic reticulum membrane. (e) Concomitant with the inhibition of Ca 2+ uptake, CPF-I also inhibited the Ca 2+-ATPase activity of sarcoplasmic reticulum. (f) Heat-treatment of CPF-I led to loss of inhibitor activity, whereas exposure to trypsin appeared to enhance its inhibitory effect. (g) Addition of CPF-I to Ca 2+-preloaded sarcoplasmic reticulum vesicles did not promote Ca 2+ release from the vesicles. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum Ca 2+ pump in heart and slow skeletal muscle but not in fast skeletal muscle. The characteristics of the inhibitor and its apparently selective distribution suggest a potentially important role for it in the in vivo regulation of sarcoplasmic reticulum Ca 2+ pump, and therefore in determining the duration of Ca 2+ signal in slow-contracting muscle fibers.

Tb3+ binding to Ca2+ and Mg2+ binding sites on sarcoplasmic reticulum ATPase.

The interactions of Tb3+ and sarcoplasmic reticulum (SR) were investigated by inhibition of Ca2+-activated ATPase activity and enhancement of Tb3+ fluorescence. Ca2+ protected against Tb3+ inhibition of SR ATPase activity. The apparent association constant for Ca2+, determined from the protection, was about 6 x 10(6) M-1, suggesting that Tb3+ inhibits the ATPase activity by binding to the high affinity Ca2+ binding sites. Mg2+ did not protect in the 2-20 mM range. The association constant for Tb3+ binding to this Ca2+ site was estimated to be about 1 x 10(9) M-1. No cooperativity was observed for Tb3+ binding. No enhancement of Tb3+ fluorescence was detected. A second group of binding sites, with weaker affinity for Tb3+, was observed by monitoring the enhancement of Tb3+ fluorescence (lambda ex 285 nm, lambda em 545 nm). The fluorescence intensity increased 950-fold due to binding. Ca2+ did not complete for binding at these sites, but Mg2+ did. The association constant for Mg2+ binding was 94 M-1, suggesting that this may be the site that catalyzes phosphorylation of the ATPase by inorganic phosphate. For vesicles, Tb3+ binding to these Mg2+ sites was best described as binding to two classes of binding sites with negative cooperativity. If the SR ATPase was solubilized in the nonionic detergent C12E9 (dodecyl nonaoxyethylene ether alcohol), in the absence of Ca2+, only one class of Tb3+ binding sites was observed. The total number of sites appeared to remain constant. If Ca2+ was included in the solubilization step, Tb3+ binding to these Mg2+ binding sites displayed positive cooperativity (Hill coefficient, 2.1). In all cases, the apparent association constant for Tb3+, in the presence of 5 mM MgCl2, was in the range of 1-5 x 10(4) M-1.

Alteration of sarcoplasmic reticulum after denervation of chicken pectoralis muscle.

To determine the neural influence on the function of the sarcoplasmic reticulum (SR) of fast-twitch skeletal muscle, the superior pectoralis muscle of adult chicken was denervated, and the SR was isolated at 20 days post-denervation. The isolated SR was probably derived from the longitudinal SR and was relatively free of contaminants. The protein profile of the SR was quantitatively changed after denervation with an increase in the M55 and 30000-mol.wt. proteins relative to the Ca2+-ATPase. Ca2+-dependent ATPase activity and phosphoenzyme formation were lower in the denervated-muscle SR; however, the enzyme catalytic-centre activity was similar to the control value. The decrease in Ca2+-ATPase activity in denervated-muscle SR was accompanied by a lower Ca2+ accumulation so that the relationship between Ca2+ accumulation and Ca2+-dependent ATPase activity was well maintained in the SR from denervated muscle. The data imply that denervation may result in a diminution of functional Ca2+ pump sites. Evidence is presented, though, which suggests that denervation affects a single class of Ca2+-binding sites of the Ca2+-ATPase, resulting in a lower affinity for Ca2+.

Effects of adrenalectomy and in vivo administration of dexamethasone on ATP-dependent calcium accumulation by sarcoplasmic reticulum from rat heart

ATP-dependent Ca 2+ accumulation and (Mg 2+ + Ca 2+)ATPase activities were determined in cardiac sarcoplasmic reticulum (SR) from control, adrenalectomized and adrenalectomized-dexamethasone-treated rats. SR from adrenalectomized rats displayed significantly diminished rates (∼40 to 50%, P < 0.001) of Ca 2+ accumulation compared to SR from control rats. The decreased Ca 2+ accumulating activity of the membranes from adrenalectomized compared to control rats could be observed at varying Ca 2+ concentrations (0.5 to 11.9 μ m); kinetic analysis showed that the velocity of Ca 2+ transport but not the apparent affinity of the transport system for Ca 2+ was altered by adrenalectomy. ATPase activities (with Mg 2+ or with Mg 2+ + Ca 2+) of SR were not altered significantly following adrenalectomy. In vivo administration of dexamethasone to adrenalectomized rats led to a partial (75 to 80%) yet significant ( P < 0.01 to 0.05) restoration of Ca 2+ accumulating activity of SR towards normal; dexamethasone-treatment caused decrease in basal (Mg 2+)ATPase activity and enhancement in (Mg 2+ + Ca 2+)ATPase activity. When added in vitro, dexamethasone had no effect on Ca 2+ accumulating and ATPase activities of SR from control or adrenalectomized rats. These findings imply an important role for glucocorticoids in the maintenance of membrane Ca 2+ transport function, and therefore, normal myocardial contractility.

Glucose, insulin, potassium protection during the course of hypothermic global ischemia and reperfusion: a new proposed mechanism by the scavenging of free radicals.

Glucose, insulin, potassium (GIK: 300 g glucose + 50 U insulin + 80 mEq KC1/L) was administered to anesthetized dogs as a 30-ml bolus followed by 1.5 ml/kg/h for 2 h. Five populations were studied: control (C, n = 6); 60 min hypothermic arrest both without (I, n = 6) and with pretreatment (I + GIK, n = 6); 60 min hypothermic arrest followed by reperfusion without (R, n = 6) and with pretreatment (R + GIK, n = 6). Glycogen content declined during the ischemic and reperfusion periods whether or not GIK pretreatment was utilized. Glycogen values did not differ significantly among the four groups. GIK pretreatment significantly protected sarcoplasmic reticulum (SR) calcium uptake rates. SR Ca2+ + Mg2+ adenosine triphosphatase (ATPase) activity was unaffected in the I group, depressed in the R group, but protected by GIK pretreatment. Myofibrillar pCa-ATPase activity was significantly depressed in the I group and unaffected by GIK pretreatment. In the R + GIK group, myofibrillar pCa-ATPase activity was identical to controls at all calcium concentrations except for Vmax. In vitro, generation of the superoxide anion by a xanthine-xanthine oxidase system at pH 7.0 significantly depressed both SR calcium uptake and ATPase activity, and this depression was partially reversible by glucose. Generation of the hydroxyl free radical and pH 6.4 significantly depressed calcium uptake but not ATPase activity, and this depression was reversible with glucose + superoxide dismutase. GIK pretreatment exerts a protective effect on the excitation-contraction coupling system during hypothermic global ischemia and reperfusion. Glycogen augmentation after short-term GIK infusion was not significantly different. It is hypothesized that an additional mechanism by which GIK may protect subcellular function is by serving as a scavenger of free radicals generated during the ischemic/reperfusion process.

Calcium transport and ATPase activities of sarcoplasmic reticulum with adenosine 5'-O-(2-thiotriphosphate) diastereomers as substrates.

Calcium transport and ATPase activities of sarcoplasmic reticulum with adenosine 5'-O-(2-thiotriphosphate) diastereomers as substrates.

Effect of disuse on sarcoplasmic reticulum in fast and slow skeletal muscle

The effect of 6 wk of hindlimb immobilization on rat skeletal muscle sarcoplasmic reticulum (SR) was determined in the slow-twitch, type I soleus (SOL), the fast-twitch, type IIA deep region of the vastus lateralis (DVL), and the fast-twitch, type IIB superficial region of the vastus lateralis (SVL). Immobilization produced a significant decline in the Ca2+ uptake rate (Vmax) of SR vesicles from the slow SOL (0.930 +/- 0.116 to 0.365 +/- 0.071 mumol Ca2+ . mg-1 . min-1), while the SR Vmax increased in the fast SVL (2.763 +/- 0.133 to 5.209 +/- 0.687) and was unaltered in the DVL. Vesicles from the fast SVL and DVL also exhibited a higher total Ca2+ uptake capacity following immobilization. An evaluation of the time course of the immobilization-mediated effect revealed an increased Ca2+ uptake capacity in all three samples after 1 wk. In the SOL total Ca2+ uptake returned to control level after 2 wk, while in the fast-twitch muscles the higher capacities were maintained. The Ca2+-stimulated SR ATPase activity was not altered in any of the muscles studies, although the total SR ATPase activity increased twofold in the slow SOL.

Equilibrium and kinetic studies of calcium transport and ATPase activity in sarcoplasmic reticulum.

A number of equilibrium and kinetic measurements are presented to characterize the partial reactions of the ATPase and transport cycle in sarcoplasmic reticulum vesicles. The cycle begins with calcium and nucleotide binding on sites available on the outer surface of the vesicles. A phosphorylated enzyme intermediate is then formed, and the calcium sites are subjected to a change in their orientation and their affinity for calcium. It is shown that steps involved in calcium release on the inner side of the vesicles are rate limiting for the cycle, and are followed by hydrolytic cleavage of the intermediate with release of inorganic phosphate and recycling of the enzyme.

Uncoupling of Ca2+ transport in sarcoplasmic reticulum as a result of labeling lipid amino groups and inhibition of Ca2+-ATPase activity by modification of lysine residues of the Ca2+-ATPase polypeptide.

Limited labeling of amino groups with fluorescamine in fragmented sarcoplasmic reticulum vesicles inhibits Ca2+-ATPase activity and Ca2+ transport. Under the labeling conditions used, 80% of the label reacts with phosphatidylethanolamine and 20% with the Ca2+-ATPase polypeptide. This degree of labeling does not result in vesicular disruption or in loss of vesicular proteins and does not increase the membrane permeability to Ca2+. Fluorescamine labeling of a purified Ca2+-ATPase devoid of aminophospholipids also inhibits Ca2+-ATPase activity, suggesting that labeling of lysine residues of the enzyme polypeptide is responsible for the inhibition of Ca2+-ATPase activity in sarcoplasmic reticulum. Fluorescamine labeling interferes with phosphoenzyme formation and decomposition in both the native vesicles and the purified enzyme; addition of ATP during labeling, and with less effectiveness ADP or AMP, protects both partial reaction steps. Addition of a nonhydrolyzable ATP analog protects phosphoenzyme formation but not decomposition. The inhibition of Ca2+ transport but not of Ca2+-ATPase occurs in sarcoplasmic reticulum vesicles labeled in the presence of ATP, indicating that the transport reaction is uncoupled from the Ca2+-ATPase reaction. The inhibition of Ca2+ transport but not of Ca2+-ATPase activity is also found in sarcoplasmic reticulum vesicles in which only phosphatidylethanolamine has reacted with fluorescamine. Furthermore, the extent of labeling of phosphatidylethanolamine is correlated with the inhibition of Ca2+ transport rates. The inhibition of Ca2+ transport is a reflection of the inhibition of Ca2+ translocation and is not due to an increase in Ca2+ efflux. We propose that labeling of phosphatidylethanolamine perturbs the lipid environment around the enzyme, producing a specific defect in the Ca2+ translocation reaction.

Muscle fatigue with prolonged exercise: contractile and biochemical alterations.

Alterations in the contractile and biochemical properties of fast and slow skeletal muscle were studied in rats following a prolonged swim to exhaustion. The exercise produced glycogen depletion (less than 1 mg/g tissue) in muscles representative of all three fiber types; the isometric contractile properties were altered in the 84% type I soleus (SOL) and the 60% type IIa extensor digitorium longus (EDL) but not in the 100% type IIb superficial region of the vastus lateralis (SVL). Peak tetanic tension (Po) and the rate of tension development and decline all decreased after prolonged exercise in both the SOL and the EDL. The maximal isotonic shortening velocity was highly correlated with the myofibrillar ATPase activity, and both were relatively resistant to fatigue. Furthermore, the Ca2+ sensitivity of the myofibrils was unaffected by exercise in both fast and slow muscle. The Ca2+ uptake capacity of the sarcoplasmic reticulum (SR) was reduced in both the SOL and the fast-twitch type IIa deep region of the vastus lateralis, whereas the SR ATPase activity was unchanged. Our findings provide evidence that prolonged exercise produces alterations in contractile and biochemical properties of type I and IIa but not type IIb fibers and that muscle fatigue as measured by a decline in Po is not necessarily correlated with glycogen depletion.