Modification of heart sarcolemmal Na+/K+-ATPase activity during development of the calcium paradox

This study examined the status of sarcolemmal Na⁺/K⁺-ATPase activity in rat heart under conditions of Ca²⁺-paradox to explore the existence of a relationship between changes in Na⁺/K⁺-pump function and myocardial Na⁺ as well as K⁺ content. One min of reperfusion with Ca²⁺ after 5 min of Ca²⁺-free perfusion reduced Na⁺/K⁺-ATPase activity in the isolated heart by 53% while Mg²⁺-ATPase, another sarcolemmal bound enzyme, retained 74% of its control activity. These changes in sarcolemmal ATPase activities were dependent on the duration and Ca²⁺ concentration of the initial perfusion and subsequent reperfusion periods; however, the Na⁺/K⁺-ATPase activity was consistently more depressed than Mg²⁺-ATPase activity under all conditions. The depression in both enzyme activities was associated with a reduction in V_max without any changes in K_m values. Low Na⁺ perfusion and hypothermia, which protect the isolated heart from the Ca²⁺-paradox, also prevented reperfusion-induced enzyme alterations. A significant relationship emerged upon comparison of the changes in myocardial Na⁺ and K⁺ content to Na⁺/K⁺-ATPase activity under identical conditions. At least 60% of the control enzyme activity was necessary to maintain normal cation gradients. Depression of the Na⁺/K⁺-ATPase activity by 60-65% resulted in a marked increase and decrease in intracellular Na⁺ and K⁺ content, respectively. These results suggest that changes in myocardial Na⁺ and K⁺ content during Ca²⁺-paradox are related to activity of the Na⁺/K⁺-pump; the impaired Na⁺/K⁺-ATPase activity may lead to augmentation of Ca²⁺-overload via an enhancement of the Na⁺/Ca²⁺-exchange system.     

Mechanisms that may be involved in calcium tolerance of the diabetic heart

In diabetes the hearts exhibit impaired membrane functions, but also increased tolerance to Ca²⁺ (iCaT) However, neither the true meaning nor the molecular mechanisms of these changes are fully understood. The present study is devoted to elucidation of molecular alterations, particularly those induced by non-enzymatic glycation of proteins, that may be responsible for iCaT of the rat hearts in the stage of fully developed, but still compensated diabetic cardiomyopathy (DH). Insulin-dependent diabetes (DIA) was induced by a single i.v. dose of streptozotocin (45 mg.kg^-1). Beginning with the subsequent day, animals obtained 6 U insulin daily. Glucose, triglycerides, cholesterol and glycohemoglobin were investigated in blood. ATPase activities, the kinetics of activation of (Na,K)-ATPase by Na⁺ and K⁺, further the fluorescence anisotropy of diphenyl-hexatriene as well as the order parameters of membranes in isolated heart sarcolemma (SL) were also investigated. In addition, the degree of glycation and glycation-related potency for radical generation in SL proteins were determined by investigating their fructosamine content. In order to study calcium tolerance of DH in a 'transparent' model, hearts were subjected to calcium paradox (Ca-Pa, 3 min of Ca²⁺ depletion; 10 min of Ca²⁺ repletion). In this model of Ca²⁺-overload, Ca²⁺ ions enter the cardiac cells in a way that is not mediated by receptors. Results revealed that more than 83% of the isolated perfused DH recovered, while the non-DIA control hearts all failed after Ca-Pa. DH exhibited well preserved SL ATPase activities and kinetics of (Na,K)-ATPase activation by Na⁺, even after the Ca-Pa. This was considered as a reason for their iCaT. Pretreatment and administration of resorcylidene aminoguanidine (RAG 4 or 8 mg.kg^-1) during the disease prevented partially the pathobiochemical effects of DIA-induced glycation of SL proteins. DIAinduced perturbations in anisotropy and order parameters of SL were completely prevented by administration of RAG (4 mg.kg^-1). Although, the latter treatment exerted little influence on the (Na,K)-ATPase activity, it decreased the calcium tolerance of the DH. Results are supporting our hypothesis that the glycation-induced enhancement in free radical formation and protein crosslinking in SL may participate in adaptive mechanisms that may be also considered as 'positive' and are responsible for iCaT of the DH.     

Decreased Ca2+-binding and Ca2+-ATPase activities in heart sarcolemma upon phospholipid methylation

Heart sarcolemma has been shown to possess three catalytic sites (I, II and III) for methyl transferase activity (Panagia V, Ganguly PK and Dhalla NS. Biochim Biophys Acta 792: 245–253, 1984). In this study we examined the effect of phosphatidylethanolamine N-methylation on ATP-independent Ca²⁺ binding and ATPase activities in isolated rat heart sarcolemma. Both low affinity (1.25 mM Ca²⁺) and high affinity (50 µM Ca²⁺) Ca²⁺ binding activities were decreased following incubation of sarcolemmal membranes with AdoMet under optimal conditions for site II and III. Similarly, Ca²⁺ ATPase activities measured at 1.25 mM and 4 mM Ca²⁺ were depressed by phospholipid N-methylation. S-adenosyl homocysteine, a specific inhibitor of phospholipid N-methylation, prevented the depression of low affinity Ca²⁺ binding and Ca²⁺ ATPase activities, whereas the methylation-induced effect on the high affinity Ca²⁺ binding was not influenced by this agent. Pretreatment of sarcolemma with methyl acetimidate hydrochloride, an amino group blocking agent, also prevented the methylation-induced inhibition of both Ca²⁺ binding and Ca²⁺ ATPase. A further decrease in Ca²⁺ binding and Ca²⁺ ATPase activities together with a marked increase in the intramembranal level of PC was seen when membranes were methylated under the site III conditions in the presence of phosphatidyldimethylethanolamine as exogenous substrate. There was no effect of phospholipid methylation on sarcolemmal Na⁺-K⁺ ATPase and Mg²⁺ ATPase activities. These results indicate a role of phospholipid N-methylation in the regulation of sarcolemmal Ca²⁺ ATPase and low affinity ATP-independent Ca²⁺ binding.     

Effect of calcium on the structure-function relationship of (Na+ + K+)-ATPase in cardiac sarcolemma

Calcium-induced changes in (Na+ + K+)-ATPase activity and structural changes of membrane bound proteins in rat heart sarcolemma were investigated. Increasing concentrations of Ca2+ (0.1-8.0 mmol.l-1) gradually inhibited the (Na+ + K+)-ATPase activity and decreased the alpha-helix content of sarcolemmal proteins. Mathematical and graphical analysis of observed data yielded a quantitative relationship between Ca2+-induced changes in (Na+ + K+)-ATPase activity and the secondary structure of membrane proteins in cardiac sarcolemma.     

A comparative study of the rat heart sarcolemmal Ca2+-dependent ATPase and myosin ATPase

In order to gain some information regarding Ca²⁺-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca²⁺-ATPase activity of myosin purified from rat heart. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by Ca²⁺ but the maximal activation of Ca²⁺-dependent ATPase required 4 mM Ca²⁺ whereas that of myosin ATPase required 10 mM Ca²⁺. These ATPases were also activated by other divalent cations in the order of Ca²⁺ > Mn²⁺ > Sr²⁺ > Br²⁺ > Mg²⁺; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca²⁺-dependent ATPase was not activated by actin. The pH optima of Ca²⁺-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na⁺ markedly inhibited Ca²⁺-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca²⁺-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca²⁺-dependent ATPase. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH₄-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca²⁺-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3–4 mM Ca²⁺ and 3 to 4 mM ATP like sarcolemmal Ca²⁺-dependent ATPase. K⁺ stimulated both ATPase activities in the absence of Ca²⁺ and inhibited in the presence of Ca²⁺. Both enzymes were inhibited by Na⁺, Mg²⁺, La³⁺, and azide similarly. However, Ca²⁺ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca²⁺ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca²⁺-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca²⁺-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca²⁺-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca²⁺-ATPase. These observations suggest that Ca²⁺-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils.     

Selective and reversible inhibition of heart sarcolemmal (Na+ + K+)-ATPase by p-bromophenyl isothiocyanate. Evidence for a sulfhydryl group in the ATP-binding site of the enzyme

Isothiocyanates are potent modifiers of thiol groups, and they have been successfully applied in studying the active site structure of renal (Na+ + K+)-ATPase. However, very little has been known on interactions of isothiocyanates with myocardial sarcolemmal ATPases. In the present study the mode of interaction and inhibitory effect of p-bromophenyl isothiocyanate (BPITC) on isolated rat heart sarcolemmal preparation ATPase activities not exhibiting (Mg-Ca)-ATPase activity was investigated. BPITC in concentrations of 10(-7)-10(-4) mol . l-1 inhibited selectively and non-competitively the (Na+ + K+)-ATPase activity in the sarcolemma with an ID50 around 2.10(-7) mol . l-1. The non-specific interaction of BPITC with bivalent cations, namely with Mg2+ and Ca2+, in the reaction system was eliminated by preincubation of membranes with BPITC keeping the ratio of inhibitor to membrane protein concentration constant. Under these conditions no considerable inhibitory effects were observed on Mg2+-ATPase or the low-affinity Ca2+-ATPase of sarcolemma. Preincubation of membranes with 2 mmol . l-1 ATP protected (Na+ + K+)-ATPase activity against inhibition by BPITC. The interaction of BIPTC with the sarcolemma proved to be reversible in the presence of beta-mercaptoethanol or dithiothreitol.     

Ethanol protects the heart against the calcium paradox injury

Rat hearts were depleted of Ca2+ (less than 10(-9) M) for 10 min, followed by 15 min of Ca2+-repletion. The calcium paradox injury occurs during Ca2+-repletion, after a period of calcium depletion. The calcium paradox injury was assessed by percent recovery (hemodynamics, [Ca2+]i, and energy levels) during Ca2+-repletion. A decrease in Na+ concentration during Ca2(+)-depletion did not allow for recovery during Ca2(+)-repletion, however 2.5% and 5% ethanol during Ca2(+)-depletion allowed for an approximate 50% recovery during Ca2(+)-repletion. A combination of ethanol (2.5% or 5%) with a low extracellular Na+ concentration (88 mM) allowed for complete recovery. Ethanol prevented a depletion of diastolic [Ca2+]i during Ca2(+)-depletion, and allowed for a return of normal diastolic [Ca2+]i during Ca2(+)-repletion. Ethanol modulates the activity of the Na+/Ca2+ exchanger and protects against the Ca2(+)-paradox injury.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: I. Impairment of ATP-dependent Ca2+ transport

Effects of endotoxin administration on the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca²⁺ transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the V_max for ATP and for Ca²⁺ were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca²⁺ were not significantly affected after endotoxin administration. Magnesium (1–5 mM) stimulated while vanadate (0.25–3.0 M) inhibited the ATP-dependent Ca²⁺ transport, but the Mg²⁺-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca²⁺. Additional experiments show that the Ca²⁺ sensitivity of the Ca²⁺-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca²⁺ pumping without affecting Ca²⁺-ATPase activity. Since sarcolemmal ATP-dependent Ca²⁺ transport plays an important role in the regulation of cytosolic Ca²⁺ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca²⁺ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.     

Isolation and characterization of cardiac sarcolemma

A procedure was developed for the isolation of cardiac sarcolemmal vesicles. These vesicles are enriched about ten-fold (with respect to the tissue homogenate) in K⁺-stimulated p-nitrophenylphosphatase, (Na⁺ + K⁺)-ATPase, 5'-nucleotidase activities and sialic acid content, all of which are believed to be components of the sarcolemma. The sarcolemma of tissue culture cardiac cells were radioiodinated and the distribution of this radioiodine paralleled the distribution of the other membrane markers above. There was very little contamination of the sarcolemmal fraction by sarcoplasmic reticulum (as judged by Ca²⁺-ATPase and glucose-6-phosphatase activities) or inner mitochondrial membranes (as judged by succinate dehydrogenase activity). There may, however, be some contamination by outer mitochondrial membranes (as judged by monoamine oxidase and rotenone-insensitive NADH cytochrome c reductase activities) which have rarely been monitored in cardiac sarcolemmal preparations. The purity of this preparation is good when compared with other cardiac sarcolemmal preparations. This preparation should be very useful in studying the roles of the cardiac sarcolemma (e.g. in excitation contraction coupling and Ca²⁺ binding).     

Regulation of cardiac sarcolemmal Ca2+ channels and Ca2+ transporters by thyroid hormone

In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca²⁺-channels, Na⁺–Ca²⁺ exchange and Ca²⁺-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca²⁺-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development+(dP/dt)max and pressure fall–(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and –(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca²⁺ and Ca²⁺ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca²⁺ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [³H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (B_max) without any change in the dissociation constant for receptor-ligand complex (K_d) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake and ouabain-sensitive Na⁺–K⁺ ATPase were decreased whereas the Mg²⁺-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na⁺–K⁺ ATPase activity, whereas Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake, and Mg²⁺-ATPase activities were unchanged. The V_max and K_a for Ca²⁺ of cardiac sarcolemmal Na⁺–Ca²⁺ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca²⁺-transport processes is regulated by thyroid hormones and the modification of Ca²⁺-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart.     

Effects of lanthanum on the heart sarcolemmal ATPase and calcium binding activities.

Effects of lanthanum on Ca2+-ATPase, Mg2+-ATPase, Na+-K+-ATPase, and calcium binding activities were studied in rat heart sarcolemma. Ten to 100 micrometers lanthanum depressed significantly the Ca2+-ATPase activity and 50--200 micrometers lanthanum inhibited the calcium binding activity. Lineweaver-Burk plots of the Ca2+-ATPase activity showed that the inhibition by lanthanum was competitive with calcium concentration. Neither Mg2+-ATPase nor Na+-K+-ATPase activities were affected by lanthanum when the assay medium contained 1 mM EDTA; however, in the absence of EDTA, these enzyme activities were significantly decreased by 10--100 micrometers lanthanum. Rat hearts perfused with HEPES buffer containing 0.5 mM lanthanum showed electron-dense deposits restricted to the outer cell surface and the sarcolemma obtained from these hearts also had the deposits, indicating that the membrane fraction isolated by the hypotonic shock--LiBr treatment method is of sarcolemmal origin. The Ca2+-ATPase activity of the sarcolemma isolated from lanthanum-perfused hearts, unlike the Mg2+-ATPase, Na+-K+-ATPase, and calcium binding activities, was significantly less than the control value. From these observations it is suggested that lanthanum may influence calcium movement across the sarcolemma by affecting sarcolemmal ATPase and calcium binding activities.     

Activation of Ca2+/Mg2+ ATPase in heart sarcolemma upon electrical stimulation

Electrical stimulation of the rat heart sarcolemmal membranes with a square wave current was found to increase Ca²⁺-ATPase activity. This activation of the enzyme was dependent upon the voltage of the electric current, frequency of stimulation and duration of stimulation of the sarcolemmal membranes. The increase in ca²⁺-ATPase was reversible upon terminating the electrical stimulation. The activation of sarcolemmal Ca²⁺-ATPase due to electrical stimulation was markedly depressed when the reaction was carried out at high pH (7.8 to 8.2), low pH (6.6 to 7.0), high temperatures (45 to 50°C) and low temperatures (17 to 25°C) of the incubation medium. Ca²⁺-antagonists, verapamil and D-600, unlike other types of inhibitors such as propranolol and ouabain, were found to reduce the activation of sarcolemmal Ca²⁺-ATPase by electrical stimulation. These results support the view that Ca²⁺/Mg²⁺ ATPase may be involved in the gating mechanism for opening Ca²⁺-channels in the sarcolemmal membrane upon excitation of the cardiac muscle.     

Localization of (Ca2+ + Mg2+)-ATPase,Ca2+ pump and other ATPase activities in cardiac sarcolemma

N-Ethylmaleimide was employed as a surface label for sarcolemmal proteins after demonstrating that it does not penetrate to the intracellular space at concentrations below 1·10^?4 M. The sarcolemmal markers, ouabain-sensitive (Na⁺ + K⁺)-ATPase and Na⁺/Ca²⁺-exchange activities, were inhibited in N-ethylmaleimide perfused hearts. Intracellular activities such as creatine phosphokinase, glutamate-oxaloacetate transaminase and the internal phosphatase site of the Na⁺ pump (K⁺-p-nitrophosphatase) were not affected. Almost 20% of the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump were inhibited indicating the localization of a portion of this activity in the sarcolemma. Sarcolemma purified by a recent method (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 27–39) from N-ethylmaleimide-perfused hearts showed loss of approx. 85% of its (Ca²⁺ + Mg²⁺-ATPase and Ca²⁺ pump compared to control hearts. (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump activities showed two classes of sensitivity to vanadate ion inhibition. The high vanadate affinity class ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for inhibition approx. 1.5 μM) may be localized in the sarcolemma and represented approx. 20% of the total inhibitable activity in agreement with estimates from N-ethylmaleimide studies. Sucrose density fractionation indicated that only a small portion of Mg²⁺-ATPase and Ca²⁺-ATPase may be associated with the sarcolemma. The major portion of these activities seems to be associated with high density particles.     

Cellular damage in the rat heart caused by caffeine or dinitrophenol

1. Langendorff-perfusion of rat hearts with either 10 mM caffeine or 1 mM 2,4-dinitrophenol (DNP) caused severe ultrastructural damage to the myofilaments and mitochondria that was similar to that found in a standard Ca²⁺-paradox.2. This damage occurred in the presence and absence of extracellular Ca²⁺3. Creatine kinase (CK) release (indicative of sarcolemma breakdown) was not recorded unless the caffeine- or DNP-perfusion was preceded by Ca²⁺₀-depletion.4. It is concluded that: (i) the pathways leading to damage to the myofilaments and sarcolemma are independent; (ii) the CK release mechanism requires dual activation of Ca²⁺₀-depletion plus a rise in [Ca²⁺]_i; and (iii) current theories concerning the mechanisms underlying the genesis of the Ca²⁺-paradox are incorrect or incomplete.     

Nitric oxide protects cardiac sarcolemmal membrane enzyme function and ion active transport against ischemia-induced inactivation

Nitric oxide (NO.) generated from nitric oxide synthase (NOS) isoforms bound to cellular membranes may serve to modulate oxidative stresses in cardiac muscle and thereby regulate the function of key membrane-associated enzymes. Ischemia is known to inhibit the function of sarcolemmal enzymes, including the (Na+ + K+)-ATPase, but it is unknown whether concomitant injury to sarcolemma (SL)-associated NOS isoforms may contribute to this process by reducing the availability of locally generated NO. Here we report that nNOS, as well as eNOS (SL NOSs), are tightly associated with cardiac SL membranes in several different species. In isolated perfused rat hearts, global ischemia caused a time-dependent irreversible injury to cardiac SL NOSs and a disruption of SL NO. generation. Pretreatment with low concentrations of the NO. donor 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7) markedly protected both SL NOS and (Na+ + K+)-ATPase functions against ischemia-induced inactivation. Moreover, ischemia impaired SL Na+/K+ binding, and NOC-7 significantly prevented ischemic injury to the ion binding sites on (Na+ + K+)-ATPase. These novel findings indicate that NO. can protect cardiac SL NOSs and (Na+ + K+)-ATPase against ischemia-induced inactivation and suggest that locally generated NO. may serve to regulate SL Na+/K+ ion active transport in the heart.     

Characteristics of Ca2+-stimulated ATPase in rat heart sarcolemma in the presence of dithiothreitol and alamethicin

We have studied the activities of Ca²⁺-stimulated ATPase in rat heart sarcolemma upon modulating the redox state of membrane thiol groups with dithiothreitol (DTT). The suitability of alamethicin to unmask the latent activity of this enzyme was also investigated. The Ca²⁺-stimulated ATPase in sarcolemma exhibited two activation sites — one with low affinity (Km = 0.70 ± 0.2 mM; Vmax = 10.0 ± 2.2 mol Pi/mg/h) and the other with high affinity (Km = 0.16 ± 0.7 mM; Vmax = 4.6 ± 0.8 mol Pi/mg/h) for Mg²⁺ATP. Alamethicin at a ratio of 1:1 with the sarcolemmal protein caused a 3-fold activation of Ca²⁺-stimulated ATPase without affecting its sensitivity to Ca²⁺ or Mg²⁺ATP. Treatment of sarcolemma with deoxycholate or sodium dodecyl sulfate resulted in a total loss of the enzyme activity; high concentrations of alamethicin also showed a detergent-like action on the sarcolemmal vesicles. DTT at 5–10 mM concentrations caused a 4–5 fold activation of Ca²⁺-stimulated ATPase in sarcolemma and this effect was observed to be dependent on the concentration of Mg²⁺ATP. DTT increased the affinity of the enzyme to Mg²⁺ATP at the high affinity site and enhanced the Vmax at the low affinity site in addition to increasing the sensitivity of Ca²⁺-stimulated ATPase to Ca²⁺. DTT protected the Ca²⁺-stimulated ATPase against deterioration by detergents and restored the enzyme activity after treatment with N-ethylmaleimide. The mechanism of action of DTT on Ca²⁺-stimulated ATPase may involve the reduction of essential thiols at the active site of the enzyme or its interaction with specific DTT-dependent inhibitor protein. No changes in the sensitivity of sarcolemmal Ca²⁺-stimulated ATPase to orthovanadate was evident in the absence or presence of DTT and alamethicin. The results suggest the use of both DTT and alamethicin for the determination of Ca²⁺-stimulated ATPase activity in sarcolemmal preparations.     

A protein activator of the plasma membrane Ca++-ATPase of heart sarcolemma

A detergent extract of dog or beef heart sarcolemmal vesicles was prepared and found to have a stimulatory effect on the Ca⁺⁺-ATPase of plasma membranes from human erythrocyte and cardiac sarcolemma. A procedure is described which enriches the activating fraction. The protein nature of the preparation is illustrated by its sensitivity to boiling and to the proteolytic enzyme(s) trypsin and chymotrypsin. SDS polyacrylamide gels indicate that the protein(s) involved have a molecular weight of 56 and 60 kDa. The sarcolemmal activator can stimulate the Ca⁺⁺-ATPase activity of the isolated enzyme more than 100% in the presence of saturating amounts of calmodulin. The activation is calcium dependent, being greatest at approximately 10µm Ca⁺⁺, free, but does not change theK _m for Ca⁺⁺. A possible physiological role for the activator is discussed.     

Inhibition of Na+-Ca2+ exchange in heart sarcolemmal vesicles by phosphatidylethanolamine N-methylation

The effect of phosphatidylethanolamine N-methylation on Na+-Ca2+ exchange was studied in sarcolemmal vesicles isolated from rat heart. Phosphatidylethanolamine N-methylation following incubation of membranes with S-adenosyl-L-methionine, a methyl donor for the enzymatic N-methylation, inhibited Nai+-dependent Ca2+ uptake by about 50%. The N-methylation reaction did not alter the passive permeability of the sarcolemmal vesicles to Na+ and Ca2+ and did not modify the electrogenic characteristics of the exchanger. The depressant effect of phosphatidylethanolamine N-methylation on Nai+-dependent Ca2+ uptake was prevented by S-adenosyl-L-homocysteine, an inhibitor of the N-methylation. Pretreatment of sarcolemma with methyl acetimidate hydrochloride, an amino-group-blocking agent, also prevented methylation-induced inhibition of Ca2+ uptake. In the presence of exogenous phospholipid substrate, the phospholipid N-methylation process in methyl-acetimidate-treated sarcolemmal vesicles was restored and the inhibitory effect on Ca2+ uptake was evident. These results suggest that phosphatidylethanolamine N-methylation influences the heart sarcolemmal Na+-Ca2+ exchange system.     

Localization of (Ca2+ + Mg2+)-ATPase, Ca2+ pump and other ATPase activities in cardiac sarcolemma

N-Ethylmaleimide was employed as a surface label for sarcolemmal proteins after demonstrating that it does not penetrate to the intracellular space at concentrations below 1.10(-4) M. The sarcolemmal markers, ouabain-sensitive (Na+ +K+)-ATPase and Na+/Ca2+-exchange activities, were inhibited in N-ethylmaleimide perfused hearts. Intracellular activities such as creatine phosphokinase, glutamate-oxaloacetate transaminase and the internal phosphatase site of the Na+ pump (K+-p-nitrophosphatase) were not affected. Almost 20% of the (Ca2+ +Mg2+)-ATPase and Ca2+ pump were inhibited indicating the localization of a portion of this activity in the sarcolemma. Sarcolemma purified by a recent method (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 27-39) from N-ethylmaleimide-perfused hearts showed loss of approx. 85% of its (Ca2+ +Mg2+-ATPase and Ca2+ pump compared to control hearts. (Ca2+ +Mg2+)-ATPase and Ca2+ pump activities showed two classes of sensitivity to vanadate ion inhibition. The high vanadate affinity class (K1/2 for inhibition approx. 1.5 microM) may be localized in the sarcolemma and represented approx. 20% of the total inhibitable activity in agreement with estimates from N-ethylmaleimide studies. Sucrose density fractionation indicated that only a small portion of Mg2+-ATPase and Ca2+-ATPase may be associated with the sarcolemma. The major portion of these activities seems to be associated with high density particles.     

Exaprolol as a modulator of heart sarcolemmal (Na+ + K+)-ATPase. Evidence for interaction with an essential sulfhydryl group in the catalytic centre of the enzyme

Beta-adrenoceptor blocking agents may have, in addition to their primary action, also ancillary effects on the cell membrane. In the present paper the non-specific interaction of exaprolol with the ATPase systems in isolated rat heart sarcolemmal membranes was investigated. When preincubated with sarcolemmal membranes in vitro, exaprolol in concentrations below 10(-4) mol.l-1 had no significant effect on sarcolemmal Mg2+-, Ca2+- and (Na+ + K+)-ATPase activities. At exaprolol concentration of 10(-4) mol.l-1 the Mg2+- and Ca2+-ATPase activities became inhibited whereas the (Na+ + K+)-ATPase activity was markedly stimulated. A kinetic analysis of these interactions revealed a non-competitive inhibition of Mg2+- and Ca2+-ATPase. In the case of (Na+ + K+)-ATPase a synergistic type of stimulation characterized by an exaprolol-induced conversion of an essential sulfhydryl group in the active site of the enzyme to the more reactive [S-] form has been observed thus increasing the affinity of the enzyme to ATP. Exaprolol concentrations exceeding 5 X 10(-4) mol.l-1 induced an overall depression of the investigated enzyme activities.