Differential effect of global ischemia on the ryanodine-sensitive and ryanodine-insensitive calcium uptake of cardiac sarcoplasmic reticulum

The effect of ischemia on the function of cardiac sarcoplasmic reticulum (SR) was assessed by the calcium uptake rate of rat whole-heart homogenates in the presence of 10 mM oxalate. Previous studies have shown that this uptake is restricted to the SR. The contribution of the ryanodine-sensitive fractions of the SR to the total homogenate uptake was assessed by using 20 microM ruthenium red and 625 microM ryanodine to close the SR calcium release channel under previously established optimal conditions. Global ischemia of 10, 15, 30, and 60 minutes depressed homogenate calcium uptake rate 19 +/- 2%, 50 +/- 6%, 65 +/- 3%, and 81 +/- 5%, respectively. This decrease was not observed when the uptake rates were measured after closure of the calcium channel with ryanodine or ruthenium red. Similar results were obtained with a Langendorff in vitro perfusion preparation, in which calcium uptake was decreased 35 +/- 5%, 37 +/- 8%, 58 +/- 7%, and 64 +/- 4% after 10, 15, 30, and 60 minutes of ischemia, but no significant decrease was observed when homogenate uptake rates were measured in the presence of ryanodine. Thus, ischemia caused a depression in the calcium uptake rate of cardiac SR only when this activity was measured in the absence of SR calcium channel blockers. Reperfusion of ischemic hearts in a Langendorff preparation resulted in recovery of homogenate calcium uptake activity that correlated well with the return to sinus rhythm of the reperfused hearts. These reperfused hearts showed no change in the calcium uptake rate measured in the presence of ryanodine. These results suggest that the decrease in homogenate calcium uptake caused by ischemia is not due to a defect in calcium pumping capabilities but is due to an increased efflux through the ryanodine-sensitive calcium release channel of cardiac SR.     

Effect of ischemia and reperfusion on sarcoplasmic reticulum calcium uptake.

To investigate the mechanism underlying postischemic cardiac dysfunction (myocardial stunning), contractility and adenine nucleotide metabolism were studied in three groups of isolated perfused rabbit hearts (control, ischemic, and reperfused), whereas Ca2+ uptake by the sarcoplasmic reticulum (SR) was measured in homogenates obtained from them. The hearts were Langendorff-perfused under constant pressure with Krebs-Henseleit solution at 37 degrees C. Global normothermic ischemia was produced by closing the perfusion line. In the reperfused group, after 15 minutes of ischemia, Krebs-Henseleit solution was perfused for 10 minutes. Developed left ventricular pressure (control, 104 +/- 6.3 mm Hg) and left ventricular dP/dt (2,063 +/- 256.6 mm Hg.sec-1) were significantly decreased in reperfused hearts (left ventricular pressure, 78 +/- 5.9 mm Hg; left ventricular dP/dt, 1,339 +/- 216.3 mm Hg.sec-1). Myocardial ATP content (control, 13.6 +/- 0.98 mumol/g dry wt) decreased during ischemia (4.5 +/- 1.23 mumol/g) but was restored to control level on reperfusion (11.8 +/- 0.68 mumol/g). Maximum velocity of Ca2+ uptake by the SR (Vmax) (control, 49.3 +/- 2.54 nmol.min-1 x mg-1) was significantly depressed by ischemia (36.3 +/- 1.94 nmol.min-1 x mg-1) but was restored to the control value after a 10-minute reperfusion (45.3 +/- 0.79 nmol.min-1 x mg-1). Apparent dissociation constant KCa and the Hill coefficient for Ca2+ uptake were not different between control, ischemia, and reperfusion. To test for the possible role of the SR Ca(2+)-release channel in the effect of ischemia and reperfusion, we measured Ca2+ uptake after incubation of homogenates with 610 microM ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of regional ischemia on the ryanodine-sensitive Ca2+ release channel of canine cardiac sarcoplasmic reticulum.

In mammalian myocardium, muscle contraction is regulated by the rapid release of Ca2+ ions through ryanodine-sensitive Ca2+ release channels present in the intracellular membrane compartment, sarcoplasmic reticulum (SR). In this study, the effects of regional ischemia on intrinsic SR Ca2+ release channel function were determined by studying the Ca2+ transport and release, and [3H]ryanodine binding properties of whole muscle homogenates and SR-enriched membrane fractions from normal and ischemic myocardium. Measurement of oxalate-supported 45Ca(2+)-uptake rates before and after pretreatment with 1 mM ryanodine, indicated that the SR Ca2+ release channel retained its ability to be effectively closed by the channel-specific probe ryanodine after 15 and 60 min of ischemia. 45Ca2+ efflux from, and high-affinity [3H]ryanodine binding to SR-enriched vesicle fractions indicated retention of regulation of Ca2+ release channel activity by Ca2+, Mg2+ and adenine nucleotide in 15 and 60 min ischemic samples. Further, sodium dodecylsulfate polyacrylamide gel and immunoblot analysis revealed no proteolytic degradation of the M(r) 565,000 SR Ca2+ release channel polypeptide after 15 and 60 min of ischemia. These results suggested a minimal, if any, loss of intrinsic SR Ca2+ release channel function in ischemic hearts.     

Molecular mechanism of calcium uptake and release by cardiac sarcoplasmic reticulum

Cardiac sarcoplasmic reticulum (SR) plays a special role in controlling free calcium ions (Ca) in heart muscle cells. Ca stored in the SR is released through the Ca release channels when the sarcolemmal membrane is depolarized, thereby inducing contraction, while Ca is reaccumulated by the Ca pump to induce relaxation. In the latter process, the Ca pump of cardiac SR has a regulatory system by cAMP-dependent phosphorylation of a SR protein, phospholamban. Recently, significant progress has been achieved in understanding the molecular mechanisms of Ca release and uptake and its regulation. The structures of the Ca pump and phospholamban have been defined at molecular levels. A direct interaction between these two proteins was demonstrated. The Ca release channel was identified, and turned out to be the foot structure which in situ connects the SR to the sarcolemma/transverse tubule.     

High molecular weight proteins purified from cardiac junctional sarcoplasmic reticulum vesicles are ryanodine-sensitive calcium channels

The cardiac high molecular weight proteins/ryanodine receptors were purified to homogeneity from junctional sarcoplasmic reticulum membranes and shown to exhibit large conductance calcium channel activity. High molecular weight proteins were solubilized from junctional sarcoplasmic reticulum in zwitterionic detergent and purified by size-exclusion chromatography followed by sucrose density gradient centrifugation. The purified proteins exhibited an apparent Mr = 400,000-350,000, and bound [3H]ryanodine with a Kd of 4.6 nM and a Bmax of 140-280 pmol/mg protein. High molecular weight proteins demonstrated divalent cation channel activity after incorporation into planar lipid bilayers. Two channel types were identified. Large conductance channels had a slope conductance of 96 +/- 13 pS and a Erev of 42 +/- 9 mV (n = 5); small conductance channels had a slope conductance of 5.5 +/- 1 pS [1.0 microM cis CaCl2; 50 mM trans Ba(OH)2]. Reducing cis calcium from 1 microM to 1 nM reduced the large conductance channel open time from 7 +/- 1% to 0.1% (holding potential, -100 mV). Adding ATP (1 mM) to the cis chamber increased channel open time from 6 +/- 1% to 52 +/- 4% (holding potential, -100 mV); 10 nM ryanodine increased and 100 microM ryanodine decreased percent of open time of the 96 pS channel, without altering unitary channel conductance. The large conductance channel was similar to the calcium release channel detected in native canine cardiac junctional sarcoplasmic reticulum vesicles. Our data suggest that the ryanodine receptor, the calcium-release channel, and the high molecular weight proteins are all identical proteins containing allosteric regulatory sites for calcium, ATP, and ryanodine.     

Altered calcium uptake by the sarcoplasmic reticulum following cardiac transplantation in humans

Abnormalities in the diastolic properties of the heart have been described following human cardiac transplantation and may reflect, at least in part, decreased Ca2+ uptake by the sarcoplasmic reticulum. This possibility was evaluated by obtaining serial myocardial biopsies in 13 patients who underwent cardiac transplantation for severe heart failure. Oxalate-supported Ca2+ uptake by the sarcoplasmic reticulum was measured in homogenates of 83 ventricular biopsies from transplanted hearts. Biopsies from seven subjects with normal cardiac function and morphology served as controls. In the transplanted hearts, there was a tendency for Ca2+ uptake rate to decline with time so that 4-5 months postoperatively, it was significantly lower (4.5 +/- 0.5 nmoles Ca2+/mg/min) compared to controls (5.6 +/- 0.5 nmoles Ca2+/mg/min, p less than 0.01). Plasma norepinephrine levels fell from the high preoperative values (689 +/- 50 pg/mL) towards normal (215 +/- 7 pg/mL) within 30 days after transplantation. Subsequently, however, there was a tendency for norepinephrine levels to increase (369 +/- 55 pg/mL at 4 months). In four patients for which serial observations were available, there was an inverse relationship between myocardial Ca2+ uptake and plasma norepinephrine levels. These results indicate the feasibility of obtaining reproducible serial measurements of Ca2+ uptake in human cardiac biopsies. The decline in sarcoplasmic reticulum function following cardiac transplantation may be, in part, the biochemical basis for the reported impairment in diastolic relaxation.     

Calcium efflux from cardiac sarcoplasmic reticulum: effects of calcium and magnesium

The dependence of the rate of calcium efflux from cardiac sarcoplasmic reticulum on the concentration of ionized calcium in the medium has been investigated. A high concentration of ionized calcium outside the sarcoplasmic reticulum stimulates calcium efflux. Stimulation of calcium efflux from cardiac sarcoplasmic reticulum by high levels of ionized calcium outside the membranes is inhibited by magnesium. This inhibition can be overcome at very high concentrations of calcium in the medium. This resembles the magnesium inhibition of calcium triggered calcium release from skinned cardiac muscle fibres, which can also be overcome by increasing the concentration of calcium in the medium. Ouabain at 10^?6m and verapamil at 10^?5m have no effet on the stimulation of calcium efflux by calcium.     

Biphasic effects of doxorubicin on the calcium release channel from sarcoplasmic reticulum of cardiac muscle

To define the mechanism of doxorubicin cardiotoxicity, the effects of doxorubicin and caffeine were examined on calcium release channels from cardiac sarcoplasmic reticulum. We found that calcium release from cardiac sarcoplasmic reticulum vesicles was induced by both compounds. When sarcoplasmic reticulum vesicles were incorporated into planar lipid bilayers, calcium-permeable channels were observed. Addition of caffeine (2.5-10 mM) increased channel open probability from less than 0.1% to 40%, and this effect persisted for a mean of 44 minutes. In contrast, doxorubicin (2.5-10 microM) had a biphasic effect; initially, doxorubicin activated the channel, whereas after a mean of 8 minutes, the channel became irreversibly inhibited. Although the degree of channel activation by doxorubicin was concentration dependent, the time needed to inactivate the channel was concentration independent. Pretreatment with dithiothreitol (0.2 mM) prevented doxorubicin-induced channel inactivation, and channel activity persisted for an average of 58 minutes. Dithiothreitol alone did not alter channel open probability. Our results support the hypotheses that 1) the integrity of sulfhydryl groups is important for some aspects of calcium release channel function and 2) activation and inactivation of the channel are separable processes. The biphasic effect of doxorubicin on channel function may also correspond to the clinically observed adverse effects of doxorubicin, a widely used chemotherapeutic agent that, after prolonged usage, causes a dilated cardiomyopathy.     

Degradation of the cardiac sarcoplasmic reticulum in acute myocardial ischemia

The degradation of the sarcoplasmic reticulum (SR) in acute myocardial ischemia was studied with references to the regional irreversibility and to the mechanism of ischemic degradation by the measurements of Ca++-stimulated ATPase activity and composition of the major ATPase protein of the SR and activity of cathepsin B of the SR and lysosome (Ly) fractions. Ca++-stimulated ATPase activity decreased to 66% of that of the nonischemic portion at 20 min after coronary ligation in the subendocardium (Endo) and to 44% at 30 min in the subepicardium (Epi). Composition of the major ATPase protein decreased to 55% and 73% at 30 min in Endo and Epi, respectively. In both SR and Ly fractions cathepsin B exhibited the maximal activity at 6.0-6.5, and pH dependent. And incubation of the SR at pH 6.0 induced the degradation of the ATPase protein quite similarly to that in vivo ischemia. These results suggest that the degradation of the SR membrane of ischemic myocardial cells begins earlier in Endo 20 to 30 min after the cease of the coronary blood flow, and extends to Epi later. Cathepsin B is strongly conceivable to play an initial role of necrotic process of the ischemic myocardial cells by activation inside of the SR in ischemic acidic state.     

The reversal of the calcium pump of cardiac sarcoplasmic reticulum

Summary The reversal of the calcium pump of cardiac sarcoplasmic reticulum (SR) prepared from dogs was investigated. Phosphorylation of the calcium transport ATPase by orthophosphate and ATP synthesis from ADP and orthophosphate by SR passively preloaded with calcium are demonstrated. The ADP-dependent calcium efflux from SR loaded with calcium in the presence of acetylphosphate is stoichiometrically coupled to ATP synthesis from ADP and orthophosphate.

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Die Umkehr der Calcium-Pumpe des myokardialen sarkoplasmatischen Retikulums

Zusammenfassung Die Umkehr der Calcium-Pumpe des myokardialen sarkoplasmatischen Retikulums (SR) wurde untersucht. SR-Vesikel, die passiv durch Präinkubation mit hohen Calciumkonzentrationen mit Calcium beladen wurden, synthetisieren ATP aus Orthophosphat und ADP. Die ATP-Synthese erfolgt über die Phosphoryllerung der Calcium-Transport-ATPase des SR durch Orthophosphat und eine Dephosphorylierung des EP durch ADP. Die Rate der Calcium-Freisetzung aus SR-Vesikel — die Calcium-Beladung erfolgte aktiv in Gegenwart von Acetylphosphat — wird durch ADP erhöht. Der ADP-abhängige Calcium-Efflux ist stöchiometrisch mit der ATP-Synthese aus Orthophosphat und ADP gekoppelt.

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Paper, presented at the Erwin Riesch Symposium, Tübingen, September 26–29, 1976

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With 3 figures and 1 table     

Effects of ischemia on the isolation and function of canine cardiac sarcoplasmic reticulum

Normothermic global ischemia of 7, 10, 15 and 60 min was found to depress oxalate supported calcium uptake rate measured either in unfractionated homogenates or isolated sarcoplasmic reticulum. The degree of depression increased with the duration of ischemia. Comparison of the isolated sarcoplasmic reticulum with unfractionated homogenates showed that the isolated sarcoplasmic reticulum was more damaged by ischemia than the unfractionated homogenate. The cause of this discrepancy was not due to inactivation of sarcoplasmic reticulum during isolation but was due to the discard of greater portions of undamaged sarcoplasmic reticulum as the ischemic period increased. Ischemia preferentially affected that sarcoplasmic reticulum most easily fragmented by homogenization. To determine if the depression of sarcoplasmic reticulum function is uniform throughout the isolated fraction, we compared several properties of the isolated fractions. After 10 min of ischemia, extensive properties such as calcium oxalate uptake rate, calcium ATPase rate, calcium oxalate capacity and steady-state calcium loading were depressed 50, 41, 48 and 24% respectively. In contrast, intensive properties such as permeability, calcium-ATPase turnover rate, and ratio of forward nucleotide flux to reverse nucleotide flux were unaffected by ischemia. However, one intensive property, the coupling ratio, was depressed 20%. We conclude from this difference in the effects of ischemia on extensive and intensive properties that the major effect of ischemia is to inactivate the Ca-ATPase.     

Phosphodiesterase inhibitors and the cardiac sarcoplasmic reticulum calcium release channel: differential effects of milrinone and enoximone.

STUDY OBJECTIVE--The aim was to investigate possible interactions between milrinone or enoximone and the calcium release channel from cardiac sarcoplasmic reticulum. DESIGN--A membrane preparation enriched with "heavy" sarcoplasmic reticulum vesicles containing the calcium release channel was prepared from sheep myocardium. The incorporation of these vesicles into artificial lipid bilayers permitted investigation of the effects of the drugs on single calcium release channels under voltage clamp conditions. The effects of the drugs on radiolabelled ryanodine binding were also investigated as a functional probe for the activity of large populations of channels. MEASUREMENTS AND MAIN RESULTS--Milrinone (100 microM-2 mM) caused a reversible activation of channel opening when added at the cytoplasmic face of the channel. Lifetime analysis suggests this activation is synergistic with the effects of calcium on the channel. Milrinone also stimulated [3H]ryanodine binding, consistent with the proposition that it is an activating ligand of the calcium release channel. Enoximone (100 microM-1 mM) was without effect on both single channel activity and [3H]ryanodine binding. CONCLUSIONS--Activation of the calcium release channel probably contributes to the positive inotropic action in vivo of milrinone but not enoximone. Other drugs which activate the calcium release channel have been shown to be cardiotoxic, but it is not known whether this is a specific effect of channel activation or a more general result of raising cytoplasmic calcium concentration within the myocyte. Further research is required to determine accurately the mechanism of action of drugs with phosphodiesterase inhibitory activity.     

Effect of hypothermic ischemia and reperfusion on calcium transport by myocardial sarcolemma and sarcoplasmic reticulum.

The effects of hypothermic ischemia and reperfusion on sarcolemma and sarcoplasmic reticulum Ca2+ transport were studied in vesicles isolated from rabbit hearts. Hypothermic global ischemia was produced by immersing hearts in saline at 4 degrees C for 3 h. Following hypothermic ischemia, reperfusion was carried out for 40 min using a Langendorff perfusion system for the working heart. Na+,K(+)-ATPase activity of sarcolemmal vesicles (SL), was not depressed by hypothermic ischemia nor by ischemia and reperfusion. The initial rate of Na(+)-Ca2+ exchange in SL vesicles was not depressed, but the maximum amount of Ca2+ uptake was increased both after hypothermic ischemia and after reperfusion. Ca2+ uptake activity of sarcoplasmic reticulum vesicles (SR) isolated from hearts subjected to hypothermic ischemia was slightly lower than that of control, and was further reduced following reperfusion. Ca(2+)-ATPase activity of SR was unaffected by hypothermic ischemia, while it was markedly lowered after reperfusion. Although the phosphoenzyme level in SR vesicles was slightly decreased, the turnover rate was reduced after reperfusion. Reperfusion injury thus took place mainly in SR while SL appeared to be tolerant to ischemia and reperfusion.     

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.     

Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes

Cardiac mitochondria can take up Ca²⁺, competing with Ca²⁺ transporters like the sarcoplasmic reticulum (SR) Ca²⁺-ATPase. Rapid mitochondrial [Ca²⁺] transients have been reported to be synchronized with normal cytosolic [Ca²⁺]_i transients. However, most intra-mitochondrial free [Ca²⁺] ([Ca²⁺]_mito) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca²⁺]_mito in single rat myocytes using the ratiometric Ca²⁺ indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca²⁺]_mito dependence on [Ca²⁺]_i (with 5 mM EGTA) was sigmoid with [Ca²⁺]_mito < [Ca²⁺]_i for [Ca²⁺]_i below 475 nM. With low [EGTA] (50 μM) and 150 nM [Ca²⁺]_i (± 15 mM Na⁺) cyclical spontaneous SR Ca²⁺ release occurred (5–15/min). Changes in [Ca²⁺]_mito during individual [Ca²⁺]_i transients were small ( 2–10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca²⁺ handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca²⁺]_mito and spontaneous Ca²⁺ transients (confirming that SR Ca²⁺ releases caused the [Ca²⁺]_mito rise). Confocal imaging of local [Ca²⁺]_mito (using rhod-2) showed that [Ca²⁺]_mito rose rapidly with a delay after SR Ca²⁺ release (with amplitude up to 10 nM), but declined much more slowly than [Ca²⁺]_i (time constant 2.8 ± 0.7 s vs. 0.19 ± 0.06 s). Total Ca²⁺ uptake for larger [Ca²⁺]_mito transients was  0.5 μmol/L cytosol (assuming 100:1 mitochondrial Ca²⁺ buffering), consistent with prior indirect estimates from [Ca²⁺]_i measurements, and corresponds to  1% of the SR Ca²⁺ uptake during a normal Ca²⁺ transient. Thus small phasic [Ca²⁺]_mito transients and gradually integrating [Ca²⁺]_mito signals occur during repeating [Ca²⁺]_i transients.     

The effect of pH on the calcium dependence of calcium accumulation in dog cardiac muscle sarcoplasmic reticulum

Net Ca2+ accumulation in vesicles of dog cardiac sarcoplasmic reticulum (CSR) was evaluated at three different pHs: 6.0, 6.8 and 7.6. The Ca2+ sequestration by CSR depends on Ca2+ concentration and on pH values. The curves that show the relationship between Ca2+ accumulated by CSR and external Ca2+ concentrations were shifted with pH changes, both in the absence and in the presence of potassium oxalate. Considering the curve at pH 6.8 as reference, a lower Ca concentration was needed to obtain the half-maximal value in Ca sequestration under pH 7.6 (0.04 +/- 0.006 and 0.79 +/- 0.09 microM at pH 7.6 and 6.8, respectively). Opposite results were obtained under pH 6.0 (13.66 +/- 1.29 microM). Net calcium release during active accumulation of Ca2+ and Ca2+ efflux from passively 45Ca2+ loaded CSR microsomes were significantly higher at alkaline pH than at acidic pH. The results suggest that in CSR alkaline pH would promote the increase in the rates of both, Ca2+ release and active Ca2+ accumulation, while opposite effects would be expected under acidic pH. Therefore, pH changes may regulate both, the Ca2+ level upon which the SR Ca2+ pump works (permeability effect) and the sequestration rate of the Ca2+ pump (variation in the affinity for calcium).     

Effect of antihypertensive therapy on calcium transport by cardiac sarcoplasmic reticulum of SHRs

Cardiac hypertrophy develops during the course of blood pressure elevation in spontaneously hypertensive rats (SHRs) and is associated with defective calcium transport by cardiac sarcoplasmic reticulum (SR). AT 20 weeks of age, calcium uptake is reduced in SHRs (42 +/- 1.3 vs 64 +/- 1.6 nmol X mg-1 X min-1 in age-matched normotensive Wistar-Kyoto rats, P less than 0.01), while Ca2+ ATPase activity is enhanced (44 +/- 1.1 vs 35 +/- 0.7 nmol X mg-1 X min-1 in WKYs, P = 0.02); this results in low stoichiometry between calcium uptake and ATP hydrolysis in SHRs. The steady-state levels of the phosphoprotein intermediate [EP] of the transport ATPase are higher in normotensive rats (0.97 +/- 0.1 vs 0.67 +/- 0.08 nmol X mg-1 in SHRs, P less than 0.01) but the Ca2+- and ATP-dependency are similar in the two groups. In order to study the relative roles of hypertension and cardiac hypertrophy in the depression of SHR function, 20-week old SHRs and normotensive rats were treated for 10 weeks with either hydralazine (100 mg X litre-1) or alpha-methyldopa (8 g X litre-1). Both therapeutic regimens resulted in near normalisation of blood pressure of SHRs (hydralazine: 18.1 +/- 0.5 kPa [136 +/- 4 mmHg]; alpha-methyldopa 17.6 +/- kPa [132 +/- 3 mmHg]). Regression of cardiac hypertrophy, however, was seen only in the alpha-methyldopa-treated group, as judged by changes in left ventricular weight, RNA/DNA ratio, and hydroxyproline content. Furthermore, improvement in calcium transport capacity by the SHR, as reflected in higher calcium uptake and stoichiometric ratio between uptake and ATP hydrolysis, was found after alpha-methyldopa, but not hydralazine treatment. These results indicate that reversal of cardiac hypertrophy is required for improvement in calcium transport by cardiac SR after antihypertensive therapy of SHRs.