Expression of the Na(+)/Ca(2+) exchanger in skeletal muscle.

The expression of the Na(+)/Ca(2+) exchanger was studied in differentiating muscle fibers in rats. NCX1 and NCX3 isoform (Na(+)/Ca(2+) exchanger isoform) expression was found to be developmentally regulated. NCX1 mRNA and protein levels peaked shortly after birth. Conversely, NCX3 isoform expression was very low in muscles of newborn rats but increased dramatically during the first 2 wk of postnatal life. Immunocytochemical analysis showed that NCX1 was uniformly distributed along the sarcolemmal membrane of undifferentiated rat muscle fibers but formed clusters in T-tubular membranes and sarcolemma of adult muscle. NCX3 appeared to be more uniformly distributed along the sarcolemma and inside myoplasm. In the adult, NCX1 was predominantly expressed in oxidative (type 1 and 2A) fibers of both slow- and fast-twitch muscles, whereas NCX3 was highly expressed in fast glycolytic (2B) fibers. NCX2 was expressed in rat brain but not in skeletal muscle. Developmental changes in NCX1 and NCX3 as well as the distribution of these isoforms at the cellular level and in different fiber types suggest that they may have different physiological roles.

Left ventricular alterations in a model of fetal left ventricular overload.

Congenital aortic coarctation is well tolerated by the fetus because the foramen ovale and ductus arteriosus equalize intracardiac and great arteries pressures and shunts. The pathologic consequences only emerge after birth with closure of the foramen ovale and ductus arteriosus. There is, however, no documentation of myocardial effects in utero of the left ventricular (LV) pressure overload induced by aortic banding. We investigated whether prenatal aortic banding could be detrimental at the structural and/or functional level. The goal of the present study was to investigate the cardiac effects of LV pressure overload in a fetal lamb model. Nine fetal lambs underwent preductal banding of the aortic arch in utero at midgestation (CoA group), whereas their twins underwent sham surgery. All fetuses were studied between 27 and 37 d after surgery for LV pressure, anatomic and histologic anomalies, and steady state sarcoendoplasmic reticulum calcium ATPase (SERCA 2a) mRNA and protein levels and pump activity. Surgery resulted in severe aortic coarctation in all the animals in the CoA group and was associated with a 65% increase in the LV weight to body weight ratio relative to the sham-operated group (p < 0.001). Hemodynamic and histologic studies showed an evolutionary pattern depending on duration of the experimental coarctation with a shift occurring at 30 d of coarctation. The initial response of cardiomyocytes to ventricular overload was hypertrophy of the myocytes, followed by myocyte hyperplasia. Compared with sham, there was an apparent decrease in the percentage of binucleated cells in the CoA group after 30 d of coarctation. The earliest response to LV pressure overload appears to occur at the molecular level. Indeed, sarcoendoplasmic reticulum calcium ATPase (SERCA 2a) mRNA levels fell significantly to only 28.6% of the sham group value (p = 0.023), independently of the duration of coarctation. In the fetal lamb, the pressure overload-induced hypertrophy resulting from progressive aortic coarctation leads to hemodynamic and lesional abnormalities and slows ontogenic maturation.

Expression of the sodium/calcium exchanger in mammalian skeletal muscle cells in primary culture.

Previous investigations have demonstrated molecular and functional expression, at early phases of development of skeletal muscle cells in primary culture, of cardiac isoforms of proteins involved in calcium transport and regulation, like the L-type calcium channel. Here the expression of the cardiac isoform of the Na(+)/Ca(2+) exchanger (NCX1) was studied in skeletal muscle cells developing in vitro, by using biochemical, immunological, and electrophysiological techniques. Northern and Western blot experiments revealed the presence of this cardiac exchanger and its increasing expression during the early phases of development. Confocal imaging of myotubes showed an NCX1 distribution that was predominantly sarcolemmal. The whole-cell patch-clamp technique allowed us to record ionic currents, the direction and the amplitude of which depended on extracellular sodium and calcium concentrations. The developmental changes of this functional expression could be correlated with the molecular NCX1 expression changes. Taken together these data demonstrate the presence of the NCX1 isoform of the Na(+)/Ca(2+) exchanger during in vitro myogenesis and reinforce the theory that significant levels of cardiac-type proteins are transiently expressed during the early phases of the skeletal muscle cell development.

Synthesis of some hydroxynaphthazarins and their cardioprotective effects under ischemia-reperfusion in vivo.

A series of hydroxynaphthazarins has been synthesized. Some of them were found in in vivo experiments to be protectors of myocardium under ischemia-reperfusion and to reduce the infarction zone by 50% without any adverse effect. All compounds exhibit a moderate or small toxicity and are active in low doses.

Cooperative interaction between Ca2+ binding sites in the hydrophilic loop of the Na(+)-Ca2+ exchanger.

A high affinity Ca(2+)-binding domain which is located in a middle portion of the large intracellular loop of the Na(+)-Ca2+ exchanger contains two highly acidic sequences, each characterized by three consecutive aspartic acid residues (Levitsky DO, Nicoll DA, and Philipson KD (1994) J Biol Chem 269: 22847-22852). This portion of the protein provides secondary Ca2+ regulation of the exchanger activity. To determine number of Ca2+ binding sites participating in formation of the high affinity domain, we isolated polypeptides of different lengths encompassing the domain and measured 45Ca2+ binding. The fusion proteins containing the high affinity domain were obtained in a Ca(2+)-bound form and as evidenced by shifts in there mobility in SDS-polyacrylamide gels after EGTA treatment. The Ca2+ binding curves obtained after equilibrium dialysis reached saturation at 1 microM free Ca2+, Kd value being approx. 0.4 microM. The Ca2+ binding occurred in a highly cooperative manner. Upon saturation, the amount of Ca2+ ion bound varied from 1.3-2.1 mol per mol protein. Proteins with an aspartate in each acidic sequence mutated lacked the positive cooperativity, had lower Ca2+ affinity and bound two to three times less Ca2+. Na(+)-Ca2+ exchangers of tissues other than heart though different from the cardiac exchanger by molecular weight most likely possess a similar Ca2+ binding site. It is concluded that, by analogy with Ca2+ binding proteins of EF-type, the high Ca(2+)-affinity domain of the Na(+)-Ca2+ exchanger is comprised of at least two binding sites interacting cooperatively.

Regulation of the cardiac Na(+)-Ca2+ exchanger by Ca2+. Mutational analysis of the Ca(2+)-binding domain.

The sarcolemmal Na(+)-Ca2+ exchanger is regulated by intracellular Ca2+ at a high affinity Ca2+ binding site separate from the Ca2+ transport site. Previous data have suggested that the Ca2+ regulatory site is located on the large intracellular loop of the Na(+)-Ca2+ exchange protein, and we have identified a high-affinity 45Ca2+ binding domain on this loop (Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. 1994. Journal of Biological Chemistry. 269:22847-22852). We now use electrophysiological and mutational analyses to further define the Ca2+ regulatory site. Wild-type and mutant exchangers were expressed in Xenopus oocytes, and the exchange current was measured using the inside-out giant membrane patch technique. Ca2+ regulation was measured as the stimulation of reverse Na(+)-Ca2+ exchange (intracellular Na+ exchanging for extracellular Ca2+) by intracellular Ca2+. Single-site mutations within two acidic clusters of the Ca2+ binding domain lowered the apparent Ca2+ affinity at the regulatory site from 0.4 to 1.1-1.8 microM. Mutations had parallel effects on the affinity of the exchanger loop for 45Ca2+ binding (Levitsky et al., 1994) and for functional Ca2+ regulation. We conclude that we have identified the functionally important Ca2+ binding domain. All mutant exchangers with decreased apparent affinities at the regulatory Ca2+ binding site also have a complex pattern of altered kinetic properties. The outward current of the wild-type Na(+)-Ca2+ exchanger declines with a half time (th) of 10.8 +/- 3.2 s upon Ca2+ removal, whereas the exchange currents of several mutants decline with th values of 0.7-4.3 s. Likewise, Ca2+ regulation mutants respond more rapidly to Ca2+ application. Study of Ca2+ regulation has previously been possible only with the exchanger operating in the reverse mode as the regulatory Ca2+ and the transported Ca2+ are then on opposite sides of the membrane. The use of exchange mutants with low affinity for Ca2+ at regulatory sites also allows demonstration of secondary Ca2+ regulation with the exchanger in the forward or Ca2+ efflux mode. In addition, we find that the affinity of wild-type and mutant Na(+)-Ca2+ exchangers for intracellular Na+ decreases at low regulatory Ca2+. This suggests that Ca2+ regulation modifies transport properties and does not only control the fraction of exchangers in an active state.

Nonlinear relationship between erythrocyte sodium-lithium countertransport and blood pressure in man.

A relationship between erythrocyte Na+/Li(+)-countertransport activity and blood pressure was studied in a randomly selected sample (95 subjects) with full range of blood pressure, from a representative group of inhabitants of one of Moscow's districts. The mean rate of erythrocyte Na+/Li(+)- countertransport activity was higher (p < 0.01 or less) in the groups of subjects with both borderline (BH) and moderate essential hypertension (EH) as compared with the group of normotensives (NT). A positive correlation was found between the erythrocyte Na+/Li(+)- countertransport rate and age and body weight in the entire selected group. The total contribution of these confounding parameters is responsible for 20.4% of the interindividual variability of the Na+/Li(+)- countertransport activity. The individual Na+/Li(+)- countertransport values remained unchanged during at least 2 years of follow-up. A nonlinear relationship between erythrocyte Na+/Li(+)- countertransport activity and blood pressure was established in the entire group. No significant association between blood pressure and Na+/Li(+)- countertransport was seen at high and low values of these two parameters. A pronounced change in the erythrocyte Na+/Li(+)- countertransport values occurred within a narrow borderline blood pressure range.

Identification of the high affinity Ca(2+)-binding domain of the cardiac Na(+)-Ca2+ exchanger.

The cardiac sarcolemmal Na(+)-Ca2+ exchanger transports Na+ and Ca2+ but is also regulated by Ca2+ at a high affinity binding site. A large intracellular, hydrophilic loop of the exchanger has been suggested to contain the Ca(2+)-binding regulatory domain (Matsuoka S., Nicoll, D. A., Reilly, R. F., Hilgemann, D. W., and Philipson, K. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3870-3874). To localize the Ca(2+)-binding site(s), we expressed portions of the exchanger loop as fusion proteins and measured binding by 45Ca2+ overlay. The high affinity binding domain is located near the center of the loop and binds Ca2+ in a cooperative manner. K0.5 ranges from 0.3 to 3 microM in the presence of 0.2-5 mM Mg2+. The binding region (amino acids 371-508) has two highly acidic sequences, each characterized by 3 consecutive aspartic acid residues. Ca2+ affinity markedly decreases when these aspartates are mutated. The Ca(2+)-binding region does not contain an EF-hand motif. The mobilities during SDS-polyacrylamide gel electrophoresis of fusion proteins with high Ca2+ affinity differ depending on the presence or absence of Ca2+ in the gel loading buffer. The high affinity Ca(2+)-binding domain is probably responsible for the secondary Ca2+ regulation of the Na(+)-Ca2+ exchanger.

Red blood cell sodium-lithium countertransport in patients with essential and renal hypertension.

The erythrocyte Na+/Li(+)-countertransport activity was studied in patients with essential hypertension (n = 59), chronic glomerulonephritis (n = 30), chronic pyelonephritis (n = 26), renovascular hypertension (n = 35) and pheochromocytoma (n = 3). The erythrocyte Na+/Li(+)-countertransport (SLC) activity was on average higher (p < 0.02) in the patients with essential hypertension as compared to those with secondary hypertension, although a clear distinction between both groups was not possible. After surgical treatment of the patients with atherosclerotic renal artery stenosis, fibromuscular dysplasia or pheochromocytoma, no change in erythrocyte SLC activity was observed. However, blood pressure was significantly reduced.

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.

Age-related changes in sarcoplasmic reticulum Ca(2+)-ATPase and alpha-smooth muscle actin gene expression in aortas of normotensive and spontaneously hypertensive rats.

The expression of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase gene and the SR Ca2+ pump function were investigated in thoracic aortas of 5- and 17-week-old normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). The relative level of the two isoforms of SR Ca(2+)-ATPase mRNA expressed in the aorta (i.e., SERCA 2a and SERCA 2b) was determined by quantitative S1 nuclease protection analysis and normalized to the level of alpha-smooth muscle (alpha-Sm) actin mRNA. The level of alpha-Sm actin mRNA itself was normalized to the level of 18S ribosomal RNA using slot-blot hybridization assays. Total SR Ca2+ pump activity was estimated by measuring the rate of oxalate-supported Ca2+ uptake in homogenates. At 5 weeks, the amount of SERCA 2a and SERCA 2b mRNA, normalized to 18S ribosomal RNA, and the ratio of alpha-Sm actin mRNA to 18S RNA were identical in SHR and WKY rats. The Ca2+ pump activity was similar in the two strains of rats at 5 weeks. From 5 to 17 weeks, the amount of SERCA 2a mRNA increased in both strains while the level of SERCA 2b mRNA remained constant. The Ca2+ pump activity was unchanged in SHRs and tended to decrease in WKY rats. Accordingly, the change in the ratio of the SR Ca(2+)-ATPase mRNA isoforms does not appear to influence SR function. The level of alpha-Sm actin mRNA and SERCA 2a mRNA increased in parallel from 5 to 17 weeks in both strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of alkaline metal ions with Ca(2+)-binding sites of Ca(2+)-ATPase of sarcoplasmic reticulum: 23Na-NMR studies.

The analysis of the 23Na-NMR signal shape variations in the presence of vesicles of light sarcoplasmic reticulum (SR) shows the existence of sodium sites on the membranes with Kd values of about 10 mM. Other monovalent cations displace Na+ from SR fragments in a competitive manner according to the row K+ greater than Rb+ greater than Cs+ greater than Li+. Calcium ions also reduce Na+ binding, the Na+ desorption curve being of a two-stage nature, which, as suggested, indicates the existence of two types of Ca(2+)-sensitive Na+ binding sites (I and II). Sites of type I and II are modified by Ca2+ in submicromolar and millimolar concentrations, respectively. Analysis of sodium (calcium) desorption produced by calcium (sodium) allowed us to postulate the competition of these two cations for sites I and identity of these sites to high-affinity Ca(2+)-binding ones on the Ca(2+)-ATPase. Sites I weakly interact with Mg2+ (KappMg approximately 30 mM). Reciprocal effects of sodium and calcium on binding of each other to sites II cannot be described by a simple competition model, which indicates nonhomogeneity of these sites. A portion of sites I (approximately 70%) interacts with Mg2+ (KappMg = 3-4 mM). The pKa value of sites II is nearly 6.0. The number of sites II is three times greater than that of sites I. In addition, sites with intermediate affinity for Ca2+ were found with Kd values of 2-5 microM. These sites were revealed due to the reducing of the sites II affinity for Na+ upon Ca2+ binding to SR membranes. It can thus be concluded that in nonenergized SR there are binding sites for monovalent cations of at least three types: (1) sites I (which also bind Ca2+ at low concentrations), (2) magnesium-sensitive sites II and (3) magnesium-insensitive sites II.

Uptake of calcium by pancreatic islet cell microsomes: inhibition by a monoclonal antibody to heart sarcoplasmic reticulum.

The uptake of Ca2+ by microsomes is thought to participate in the control of cytosolic Ca2+ activity in the insulin-producing pancreatic B-cell. In order to study such a phenomenon methods were developed to isolate microsomes from rat parotid cells, pancreatic islets and tumoral islet cells of the RINm5F line. In the latter case, a subcellular microsomal fraction was prepared in which the ratio of microsomal/mitochondrial enzyme markers, as well as that of ruthenium red-resistant/sensitive 45Ca2+ uptake was 20 times higher than in the corresponding mitochondrial subcellular fraction. The ATP-dependent net uptake of 45Ca2+ by RINm5F cell microsomes was inhibited at low temperature and by either vanadate or a monoclonal antibody to dog heart sarcoplasmic reticulum. Although the uptake of Ca2+ by microsomes may account for only a minor fraction of ATP consumption, its synarchistic regulation by ATP and Ca2+, at close-to-physiological concentrations, appeared well suited to play a major regulatory role in the control of cytosolic Ca2+ activity in intact islet cells.

Monoclonal antibodies to dog heart sarcoplasmic reticulum as markers of endoplasmic reticulum.

A monoclonal antibody (mAb 4B4) was raised against purified sarcoplasmic reticulum vesicles from canine myocardium, and shown to inhibit Ca2+ uptake by microsomes isolated from cardiac, skeletal, and smooth muscle. The amount of mAb 4B4 needed to inhibit the Ca2+ uptake 50% at a given membrane concentration correlated with the amount of Ca2+ pump protein in the microsomal preparation. This is consistent with the observation the mAb 4B4 binds specifically to the sarcoplasmic/endoplasmic reticulum Ca2+ pump (Mr 100 kDa), but has no effect on the T-tubule Mg2+-ATPase. Changes in the binding of mAb 4B4 to crude microsomes isolated from dog heart after various durations of global ischemia showed that the decrease in microsomal Ca2+ transport during the first 15 min of ischemia correlated with a loss of active Ca2+ pump molecules. The monoclonal antibody mAb 4B4 may therefore serve as a specific marker for the sarcoplasmic/endoplasmic reticulum Ca2+ pump system in various cells, and can provide quantitative information about the loss of active Ca2+ pump proteins under pathological conditions.

Free radical membrane scavengers in myocardium of rats of different age exposed to chronic hypoxia.

Age-dependent changes in myocardial content of vitamin E and total hydrophobic free radical scavengers were estimated in rats of different age. Comparatively high values of vitamin E (0.10-0.12 mg/g fresh tissue) were found in 2-day-old rats. The content of vitamin E was lower in 15- and 60-day-old and 40% higher in 120-day-old rats as compared with new-born animals. Total scavengers were monotonously falling down with age. Vitamin E contribution to total scavengers activity gradually increased in postnatal developmental period of cardiac muscle (from 10% for 2-day-old rats to 50% for 120-day-old rats). Chronic hypoxia (intermittent high altitude hypoxia - 7000 m) had no effect on the levels of cardiac total scavengers and vitamin E when the animals were acclimatized from the 5th postnatal day. Acclimatization of adult animals induced a drop in concentration of vitamin E and did not affect the level of total scavengers.

Monoclonal antibodies to dog heart sarcoplasmic reticulum. Antibodies that inhibit Ca2+-pump systems of cardiac and skeletal muscles.

Purified sarcoplasmic reticulum (SR) vesicles from dog heart were used as an antigen to produce monoclonal antibodies (mAbs) to the Ca2+-ATPase. Nine of twelve clones of hybridoma cells produce mAbs which cross-react with seven SR preparation isolated from cardiac and skeletal muscles of various species. Three mAbs of IgM type interact with the 45-kDa tryptic fragment of rabbit skeletal muscle Ca2+-ATPase and markedly inhibit Ca2+ uptake (by 95%) and ATPase activity (by 80%) and decrease (by 30-50%) the steady-state level of the Ca2+-ATPase phosphoenzyme. The ATPase activity could be completely blocked by one of these mAbs if the incubation medium was supplemented with 2 microM orthovanadate. On the other hand, when SR vesicles were treated with increasing concentrations of a nonionic detergent C12E8, the inhibiting effect of mAb 4B4 is diminished. It is concluded that the mAbs inhibit the Ca2+-ATPase only if the enzyme exists in an oligomeric form. The inhibition of the SR activities is due to an effect of the mAbs on the whole active center of the enzyme, rather than on a single partial reaction.

Characterization of Ca2+ release from the sarcoplasmic reticulum of myocardium and vascular smooth muscle.

The effects of Ca2+ and inositol-1,4,5-trisphosphate (IP3) as putative inducers of Ca2+ release from sarcoplasmic reticulum (SR) vesicles were studied. Addition of Ca2+ (5-50 microM) or caffeine (5 mM) to calcium loaded SR vesicles from canine ventricular myocardium caused immediate release of Ca2+; whereas, no such Ca2+ release was observed in canine aortic SR vesicles. Fractionation of the cardiac SR by zonal centrifugation of calcium/oxalate-loaded SR vesicles showed that Ca2+-induced Ca2+ release was present in a caffeine-sensitive, ryanodine-sensitive, calsequestrin-enriched fraction, probably derived from the junctional SR. By contrast, IP3 (greater than 0.1 microM) stimulated Ca2+ release from aortic SR, but had no such effect on cardiac SR (even with 50 microM IP3. These data indicate a difference in Ca2+ release mechanisms in SR from the heart and vascular smooth muscle.

Monoclonal antibodies to dog heart sarcoplasmic reticulum. Application of the mAbs for studies of the structure and function of Ca2+-pumps.

One of monoclonal antibodies (mAbs) raised against purified dog heart sarcoplasmic reticulum (SR) efficiently decreases Ca2+-pump and Ca2+-ATPase activities of various SR preparations. The ATPase activity that is insensitive to the mAb (10-20% of the initial value) is present both in light and heavy fractions of rabbit skeletal muscle SR. The residual activity is completely blocked by 2 microM vanadate. The inhibition of the ATPase by the mAb is prevented in the presence of a nonionic detergent C12E8. It is concluded that the inhibiting effect of the mAb takes place when the Ca2+-ATPase exists in an oligomeric form. Another mAb does not affect SR functions and is specific only for Ca2+-ATPase from cardiac and slow muscle cells. Decrease in the Ca2+-pump activity of SR fractions from ischemic myocardium is accompanied by a diminished binding of both mAbs with the antigen. The mAbs described could be employed for differentiating endoplasmic reticulum and plasma-lemmal calcium pump systems, visualization of SR in the cells and estimating its amount in membrane preparations and tissue homogenates.

Effects of changing Ca2+-to-H+ ratio on Ca2+ uptake by cardiac sarcoplasmic reticulum.

Ca uptake by fragments of cardiac sarcoplasmic reticulum (SR) in the absence of oxalate depends on the ratios between concentrations of free Ca2+ and H+ and on the level of Ca loading. Using a rapid quenching technique, we showed that at [Ca2+]f less than 1 microM or in short time intervals after ATP addition (less than or equal to 1 s), pH decrease led to a significant inhibition of Ca accumulation. If Ca transport was quenched in 1 s, [Ca2+]f for half-maximal stimulation of the process would be 0.3, 1.0, and 4.0 microM at pH 7.2, 6.8, and 6.2, respectively. At higher [Ca2+]f and at longer intervals, low pH increases the amount of Ca taken up by SR. These phenomena could not be explained by an interaction of ethyleneglycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) with the Ca2+-pump system since they were also demonstrated without application of Ca-EGTA buffers. Scatchard plot analysis of ATP-independent Ca binding to SR shows that apparent Kd for Ca increases from 0.3 microM at pH 7.2 to 7.4 microM at pH 6.2. pH shifts do not change the number of high-affinity Ca-binding sites. The results suggest a competition between Ca2+ and H+ for high-affinity binding sites on Ca2+-ATPase molecules. This may cause a delay of myocardial relaxation during ischemia when a decrease in intracellular pH takes place.