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Myocardial ischemia-reperfusion activates the Na(+)/H(+) exchanger, which induces arrhythmias, cell damage, and eventually cell death. Inhibition of the exchanger reduces cell damage and lowers the incidence of arrhythmias after ischemia-reperfusion. The omega-3 polyunsaturated fatty acids (PUFAs) are also known to be cardioprotective and antiarrhythmic during ischemia-reperfusion challenge. Some of the action of PUFAs may occur via inhibition of the Na(+)/H(+) exchanger. The purpose of our study was to determine the capacity for selected PUFAs to alter cardiac sarcolemmal (SL) Na(+)/H(+) exchange. Cardiac membranes highly enriched in SL vesicles were exposed to 10-100 microM eicosapentanoic acid (EPA) or docosahexanoic acid (DHA). H(+)-dependent (22)Na(+) uptake was inhibited by 30-50% after treatment with > or =50 microM EPA or > or =25 microM DHA. This was a specific effect of these PUFAs, because 50 microM linoleic acid or linolenic acid had no significant effect on Na(+)/H(+) exchange. The SL vesicles did not exhibit an increase in passive Na(+) efflux after PUFA treatment. In conclusion, EPA and DHA can potently inhibit cardiac SL Na(+)/H(+) exchange at physiologically relevant concentrations. This may explain, in part, their known cardioprotective effects and antiarrhythmic actions during ischemia-reperfusion.

Cardiac sarcolemmal Na(+)/H(+) exchange is critical for the regulation of intracellular pH, and its activity contributes to ischemia-reperfusion injury. It has been suggested that the membrane phospholipid environment does not modulate Na(+)/H(+) exchange. The present study was carried out to determine the effects on Na(+)/H(+) exchange of modifying the endogenous membrane phospholipids through the addition of exogenous phospholipase D. Incubation of 0.825 U of phospholipase D with 1 mg of porcine cardiac sarcolemmal vesicles hydrolyzed 34 +/- 2% of the sarcolemmal phosphatidylcholine and increased phosphatidic acid 10.2 +/- 0.5-fold. Treatment of vesicles with phospholipase D resulted in a 46 +/- 2% inhibition of Na(+)/H(+) exchange. Na(+)/H(+) exchange was measured as a function of reaction time, extravesicular pH, and extravesicular Na(+). All of these parameters of Na(+)/H(+) exchange were inhibited following phospholipase D treatment compared with untreated controls. Passive efflux of Na(+) was unaffected. Treatment of sarcolemmal vesicles with phospholipase C had no effect on Na(+)/H(+) exchange. We conclude that phospholipase D-induced changes in the cardiac sarcolemmal membrane phospholipid environment alter Na(+)/H(+) exchange.

Fibroblast growth factor-2 (FGF-2), administered to the isolated rat heart by perfusion and under constant pressure, is protective against ischemia-reperfusion (I-R). Here we have investigated whether FGF-2 cardioprotection: (a) is dependent on flow modulation; (b) is linked to effects on contractility; (c) is mediated by protein kinase C (PKC); and (d) is linked to PKC and/or mitogen activated protein kinase (MAPK) associated with the sarcolemma. The isolated rat heart was used as a model. Under conditions of constant flow FGF-2 induced significant improvement in recovery of contractile function during I-R. Under constant perfusion pressure, FGF-2 induced a negative inotropic effect (15% decrease in developed pressure). Chelerythrine, a specific PKC inhibitor, prevented both the FGF-2-induced negative inotropic effect before ischemia, and cardioprotection during I-R. FGF-2 induced a chelerythrine-preventable, five-fold increase in sarcolemmal calcium-independent PKC activity. It also increased the association of PKC subtypes -epsilon and -delta with sarcolemmal membranes, detected by Western blotting, as well as, for PKC delta, by immunolocalization. FGF-2 increased the association of PKC epsilon with the membrane fraction of adult cardiomyocyte in culture, confirming that it can affect PKC signaling in cardiomyocytes directly and in a manner similar to its effects in situ. Finally, FGF-2 induced increased active MAPK at sarcolemmal as well as cytosolic sites. Active sarcolemmal MAPK remained elevated when the FGF-2-induced protection was prevented by chelerythrine. In conclusion, we have provided evidence that cardioprotection by FGF-2 is independent of flow modulation. PKC activation mediates both the FGF-2-induced negative inotropic effect before ischemia and the cardioprotective effect assessed during reperfusion, suggesting a cause and effect relationship. Furthermore, FGF-2 cardioprotection is linked to targeting of sarcolemmal sites by calcium-independent PKC.

Tension generation and Ca2+ flux in smooth muscle varies depending upon the diameter of a vessel and its location. The purpose of the present investigation was to determine if the biochemical characteristics of the Na(+)-Ca2+ exchanger and the Ca2+ channel differ in sarcolemmal membrane preparations isolated from a large conduit vessel (thoracic aorta) or from large and small coronary arteries. We also investigated the possibility of differences between sarcolemmal membranes isolated from coronary arteries dissected from the right and left ventricles. The purification of the sarcolemmal membranes was of a similar magnitude amongst the different groups. Contamination of the sarcolemmal membranes with other membranous organelles was negligible and similar amongst the groups. The Km and Vmax of Na(+)-dependent Ca2+ uptake in sarcolemmal vesicles was similar amongst the groups. Calcium channel characteristics were examined by measuring [3H] PN200-110 binding to sarcolemmal vesicles. The right coronary artery membranes from both large and small caliber vessels exhibited a higher Kd and the small right coronary artery sarcolemmal preparation had a lower maximal binding density for [3H] PN200-110. The results suggest that the right coronary artery, and in particular the small diameter right coronary artery, possesses altered Ca2+ channel characteristics in isolated sarcolemmal membranes.

The effect of R 56865 (N-[1-[4-(4-fluorophenoxy)butyl]-piperidinyl]-N-methyl-2- benzothiazolamine, CAS 104606-13-5) on Na+/H+ exchange and Na+/Ca2+ exchange was studied in isolated cardiac sarcolemmal vesicles. R 56865 inhibited Na+/H+ exchange with an ED50 of 180 mumol/l at a concentration of 0.05 mmol/l Na+. The potency of R 56865 decreased with increasing concentrations of Na+. Na+/Ca2+ exchange was also inhibited. The ED50 amounted to 45 mumol/l. This ED50 was close to that of the reference compound dichlorobenzamil (ED50 = 27 mumol/l). The inhibitory potency of R 56865 on Na+/H+ and Na+/Ca2+ exchange is far too low to explain the already reported beneficial effects of this inhibitor of Na+ and Ca+ overload during ouabain intoxication and ischemia and reperfusion.

Ca2+ binding has been studied in isolated heart sarcolemmal membranes using the 45Ca overlay technique. 45Ca bound to two sarcolemmal polypeptides of 125 kDa and 97 kDa in preparations from dog, rabbit, cow and pig. During fractionation on DEAE ion-exchange and wheat-germ lectin affinity columns, the two Ca2(+)-binding polypeptides copurified with the dihydropyridine receptor associated with the voltage gated Ca2+ channel. These polypeptides were the major proteins in the isolated fraction as judged by silver staining in SDS-PAGE. Antisera raised against purified dog heart, sarcolemma indicated that the 125 and 97 kDa polypeptides were highly antigenic components of this membrane. The antisera cross-reacted with similar polypeptides in cardiac sarcolemmal preparations from rabbit, cow and pig, but not sarcoplasmic reticulum membranes. Purified antibodies against the 125 kDa polypeptide did not cross-react with the 97 kDa polypeptide, while antibodies against the 97 kDa polypeptide did not cross-react with the 125 kDa polypeptide. Both the 125 kDa and 97 kDa polypeptides bound wheat-germ lectin, suggesting both were glycoproteins. It is unlikely that these Ca2+ binding glycoproteins represent subunits of the dihydropyridine receptor-Ca2+ channel in this membrane.

Oxygen free radicals have the ability to oxidize cholesterol. However, nothing is known about the effects of cholesterol oxidation on ion transport in isolated myocardial membranes. The purpose of the present study was to investigate the effects of in situ oxidative modification of sarcolemmal cholesterol on Ca2+ flux. Cholesterol oxidase was used to oxidatively modify membrane cholesterol. After incubation of cardiac sarcolemmal vesicles with cholesterol oxidase, cholest-4-en-3-one (cholestenone) was the predominant species of oxidated cholesterol produced. Cholesterol oxidase inhibited sarcolemmal Na(+)-Ca2+ exchange in a concentration-dependent manner. Both the Vmax and Km of the reaction were altered after cholesterol oxidase treatment. Extensive treatment of the sarcolemmal membranes with cholesterol oxidase increased the passive permeability characteristics of the membrane. Passive Ca2+ efflux from the sarcolemmal vesicles was stimulated by increasing the concentration of cholesterol oxidase. ATP-dependent Ca2+ uptake was also inhibited after cholesterol oxidase treatment, but it was not as sensitive as the Na(+)-Ca2+ exchange. Conversely, passive Ca2+ binding to sarcolemmal vesicles was strikingly stimulated by cholesterol oxidase treatment. The results demonstrate that oxidative modification of sarcolemmal membrane cholesterol can directly affect ionic interactions with the sarcolemmal vesicle and provide potentially important mechanistic information for the molecular basis of the effects of free radicals on ion flux and function in the heart.

The effects of platelet-activating factor (PAF) on Na(+)-dependent calcium uptake in myocardial sarcolemmal vesicles were examined in order to clarify its mechanism of inotropic action on the heart. PAF (40 and 20 microM) significantly inhibited Na(+)-Ca2+ exchange by 61% and 37%, respectively. Both initial rate of exchange and maximal exchange were inhibited. The Km for the reaction was not altered but Vmax was lowered 55% by PAF. Lyso-PAF inhibited Na(+)-Ca2+ exchange to a similar degree as PAF. CV-3988, a specific PAF receptor antagonist, failed to diminish the inhibitory effect of PAF on Na(+)-Ca2+ exchange, suggesting that the effect of PAF on Na(+)-Ca2+ exchange is not via a receptor mechanism. The passive permeability of sarcolemmal vesicles to Ca2+ was markedly elevated after PAF treatment. However, this effect could not account for the decrease in Na(+)-Ca2+ exchange. Interestingly, passive Ca2+ binding to cardiac sarcolemma was increased by 40 microM PAF. This study indicates that a depression of Na(+)-Ca2+ exchange probably does not play a role in the negative inotropic effect of PAF on the myocardium under physiological conditions. Its mechanism of action on Na(+)-Ca2+ exchange is discussed.

Intracellular pH and [Na+] in the heart are regulated by the sarcolemmal membrane Na(+)-H+ exchange pathway. No data are currently available regarding the adaptation of this system to pathological conditions in the heart. Because ionic interactions with the heart are altered in cardiomyopathy during chronic experimental diabetes, it was hypothesized that Na(+)-H+ exchange may become abnormal. In addition, the effects of treating diabetic rats with daily injection of L-propionylcarnitine were investigated to determine whether alterations in lipid metabolism may be involved in any potential changes in ion transport. Rats were injected with streptozotocin (65 mg/kg) and killed 8-10 wk later, and sarcolemmal membrane vesicles were isolated from pooled ventricles. Significant depressions in Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity and Na(+)-Ca2+ exchange were observed in the diabetic preparations in comparison to control. L-Propionylcarnitine treatment of the diabetic rats partially normalized these activities. A striking depression in cardiac sarcolemmal Na(+)-H+ exchange was observed in the diabetic animals in comparison to control, and this was not a result of a nonspecific increase in membrane permeability. L-Propionylcarnitine treatment of the diabetic rats did not improve sarcolemmal Na(+)-H+ exchange.

Heart sarcolemmal membranes were isolated by the hypotonic shock-LiBr treatment from rats with chronic diabetes induced by a streptozotocin (65 mg/kg, iv) injection. Sarcolemmal Mg2+-dependent ATPase activity was elevated, whereas 5'-nucleotidase and K+-p-nitrophenylphosphatase activities in diabetic heart were depressed in comparison to control preparations. Although patent Na+-K+-ATPase and patent ouabain-sensitive Na+-K+-ATPase activities were unaltered, latent Na+-K+-ATPase activities, as determined in membranes after alamethicin or deoxycholate treatments, were found to be significantly depressed in diabetic animals. A depression in the latent Na+-K+-ATPase activity in diabetic preparations was also observed in membranes prepared by the sucrose density gradient method. Insulin-treated diabetic rats were observed to have normalized latent Na+-K+-ATPase activities. Total phospholipid content did not differ, but cholesterol content of the sarcolemmal membranes was significantly increased in diabetic heart preparations. Sarcolemmal Na+-K+-ATPase activity in diabetic heart was more resistant to treatments with filipin, an agent known to bind with cholesterol residues. These results suggest that chronic experimental diabetes is associated with some defects in sarcolemmal enzymatic activities and composition.

The purpose of this study was to identify alterations in specific enzyme and Ca2+ binding activities in cardiac sarcolemmal fractions from UM-X7.1 myopathic Syrian hamsters during the development of cardiomyopathy. Experimental and healthy control animals were examined from 25 to 200 days of age. Sarcolemmal Na+, K+-ATPase activity was depressed in the myopathic hamsters throughout the time course of this study. Sarcolemmal ATP-independent Ca2+ binding was found to be depressed in experimental animals as early as 55 days of age. Ca2+ -stimulated, Mg2+ -dependent ATPase activity was depressed in the experimental animals by 90 days of age and this decrease in enzyme activity was accompanied by a decrease in ATP-dependent Ca2+ binding capacity of the sarcolemmal membranes. Mg2+ -ATPase and Ca2+ -ATPase activities were only affected in the latter stages of the disease (155 to 200 days old). NaF, epinephrine and Gpp(NH)p stimulation of the sarcolemmal adenylate cyclase activity was also observed to be attenuated during the latter stages of the disease. These defects in adenylate cyclase system of the sarcolemmal fraction appeared specific since basal adenylate cyclase activity was not altered at any age studied. The results demonstrate that the earliest lesions in sarcolemmal activity in myopathic hamster heart occur in Na+, K+-ATPase and ATP-independent Ca2+ binding capacity. These defects correspond temporally to the initial stages of cardiac necrotic development in this strain of myopathic hamster.

Chronic streptozotocin-induced diabetes in rats was associated with a significant loss in the ability of isolated cardiac sarcolemmal membranes to bind Ca2+. Administration of insulin to the diabetic rats normalized the sarcolemmal Ca2+ binding capacity. The content of sialic acid residues, which are considered to represent a superficial Ca2+ pool in sarcolemma, was decreased in preparations from diabetic rats, and this change also was reversible upon insulin treatment of the diabetic rats. Treatment of sarcolemma with neuraminidase decreased Ca2+ binding by 37% in control preparations but had no effect on diabetic preparations. Diphosphatidylglycerol content was decreased but other acidic phospholipids such as phosphatidylinositol and phosphatidylserine, which also bind Ca2+, were not altered during diabetes. An increase in lysophosphatidylcholine and a decrease in phosphatidylethanolamine contents were observed in membranes isolated from diabetic rats. These results suggest that some alterations occur in Ca2+ binding and composition of heart sarcolemma in chronically diabetic rats and may provide further insight into the pathogenesis of diabetic cardiomyopathy.

The purpose of this study was to examine the movements of Ca2+ into the myocardium from the extracellular space during the course of muscle contraction. Equilibration of the rabbit right ventricular wall with perfusate containing 45Ca was measured by collecting effluent droplets over time. This protocol was carried out in a quiescent muscle and then repeated 15-20 minutes later in an identical fashion except that halfway through the collection period the muscle was stimulated to contract. We were thus able to quantitate the contraction-dependent changes in 45Ca movements. In control experiments using [58Co]EDTA as an extracellular space marker, we observed that contraction altered the volume of this space. This alteration in extracellular space was relatively small, and the quantitation of 45Ca movements was corrected for this change. The addition of 1 microM Bay K 8644 to the perfusate stimulated tension and augmented Ca2+ depletion from the extracellular space. The addition of 15 microM nifedipine to the perfusate significantly reduced both tension development and Ca2+ depletion from the extracellular space of the muscle. Net contraction-dependent movement of Ca2+ from the extracellular space into the myocardium under control conditions at 1 mM [Ca2+] was 10-14 mumol Ca2+/kg tissue wet wt/beat. This value indicates either a large contribution of Ca2+-induced Ca2+ release from the sarcoplasmic reticulum and/or significant contribution of sarcolemmal bound Ca2+ to excitation-contraction coupling in the rabbit ventricle.

Modification of the cholesterol content of highly purified cardiac sarcolemma from dog ventricles was accomplished by incubation with phosphatidylcholine liposomes containing various amounts of cholesterol. The degree of cholesterol enrichment could be varied by changing the liposomal cholesterol/phospholipid ratio or varying the liposome-membrane incubation time. Na+-Ca2+ exchange measured in cholesterol-enriched sarcolemmal vesicles was increased up to 48% over control values. The stimulation of Na+-Ca2+ exchange was associated with an increased affinity of the exchanger for Ca2+ (Km = 17 microM compared with Km = 22 microM for control preparations). Na+-Ca2+ exchange measured in cholesterol-depleted membrane preparations was decreased by 15%. This depressed activity was associated with a decreased affinity of the exchanger for Ca2+ (Km = 27 microM). These changes were not due to either a change in membrane permeability to Ca2+ or an increase in the amount of Ca2+ bound to sarcolemmal vesicles. The stimulating effect of cholesterol enrichment was specific to the Na+-Ca2+ exchange process since sarcolemmal Ca2+-Mg2+ ATPase activity was depressed 40% by cholesterol enrichment. Further, K+-p-nitrophenylphosphatase and Na+-K+ ATPase activities were depressed in both cholesterol-depleted and cholesterol-enriched sarcolemmal vesicles. In situ oxidation of membrane cholesterol completely eliminated Na+-Ca2+ exchange. These results suggest that cholesterol is intimately associated with Na+-Ca2+ exchange and may interact with the exchange protein and modulate its activity.

Cardiac contractile function is dependent on the integrity and function of the sarcolemmal membrane. Swimming exercise training is known to increase cardiac contractile performance. The purpose of the present study was to examine whether a swimming exercise program would alter sarcolemmal enzyme activity, ion flux, and composition in rat hearts. After approximately 11 wk of exercise training, cardiac myosin and actomyosin Ca2+-adenosinetriphosphatase (ATPase) activity was significantly higher in exercised rat hearts than in sedentary control rat hearts. Glycogen content was increased in plantaris and gastrocnemius muscles from exercised animals as was succinic dehydrogenase activity in gastrocnemius muscle of exercised rats in comparison to sedentary rat preparations. Sarcolemmal vesicles were isolated from hearts of exercise-trained and control rats. Sarcolemmal Na+-K+-ATPase and K+-p-nitrophenylphosphatase activities, Na+-Ca2+ exchange, and passive Ca2+ binding did not differ between the two groups. ATP-dependent Ca2+ uptake and 5'-nucleotidase activity were elevated in the cardiac sarcolemmal vesicles isolated from exercised animals compared with sedentary control rats. Sarcolemmal phospholipid composition was not altered by the exercise training. Our results demonstrate that swimming training in rats does not affect most parameters of cardiac sarcolemmal function or composition. However, the elevated sarcolemmal Ca2+ pump activity in exercised rats may help to reduce intracellular Ca2+ and augment cardiac relaxation rates. The enhanced 5'-nucleotidase activity may stimulate adenosine production, which could affect myocardial blood flow. The present results further our knowledge on the subcellular response of the heart to swimming training in the rat.

The purpose of this investigation was to study the effects of a distinct type of phospholipase C on sarcolemmal Na+-Ca2+ exchange. With this phospholipase C (Staphylococcus aureus), treatment of cardiac sarcolemmal vesicles resulted in a specific hydrolysis of membrane phosphatidylinositol. This hydrolysis of phosphatidylinositol also released two proteins (110 and 36 kDa) from the sarcolemmal membrane. Phospholipase C pretreatment of the sarcolemma resulted in an unexpected stimulation of Na+-Ca2+ exchange. The Vmax of Na+-Ca2+ exchange was increased but the Km for Ca2+ was not altered. This stimulation was specific to the Na+-Ca2+ exchange pathway. ATP-dependent Ca2+ uptake was depressed after phospholipase C treatment, but passive membrane permeability to Ca2+ was unaffected. Sarcolemmal Na+,K+-ATPase activity was not altered, whereas passive Ca2+ binding was modestly decreased after phospholipase C pretreatment. The stimulation of Na+-Ca2+ exchange after phosphatidylinositol hydrolysis was greater in inside-out vesicles than in a total population of vesicles of mixed orientation. This finding suggests that the cardiac sarcolemmal Na+-Ca2+ exchanger is functionally asymmetrical. The results also suggest that membrane phosphatidylinositol is inhibitory to the Na+-Ca2+ exchanger or, alternatively, this phospholipid may anchor an endogenous inhibitory protein in the sarcolemmal membrane. The observation that a transsarcolemmal Ca2+ flux pathway may be stimulated solely by phosphatidylinositol hydrolysis independently of phosphoinositide metabolic products like inositol triphosphate is novel.

Different amino acid residues in cardiac sarcolemmal vesicles were modified by incubation with various chemical reagents. The effects of these modifications on sarcolemmal Na+-Ca2+ exchange were examined. Dithiothreitol, an agent that maintains sulfur-containing residues in a reduced state, caused a time- and concentration-dependent decrease in Na+-Ca2+ exchange. The treatment with dithiothreitol resulted in a decrease in Vmax values but did not alter the Km for Ca2+ for the Na2+-Ca2+ exchange reaction. If Na+ replaced K+ as the ion present during the modification of sarcolemmal membranes with dithiothreitol, there was substantially less of an inhibitor effect on Na+-Ca2+ exchange. Similar results were obtained with reduced glutathione, a reagent that also maintains sulfur-containing residues in a reduced state. Two sulfhydryl modifying reagents, methylmethanethiosulfonate and N'-ethylmaleimide, were capable of altering Na+-Ca2+ exchange, and the type of ion present during modification significantly affected the extent of this alteration. Almost all of the chemical reagents investigated that modified other amino acid resides (carboxyl, lysyl, histidyl, tyrosyl, tryptophanyl, arginyl and hydroxyl) had the capacity to alter Na+-Ca2+ exchange after preincubation with the sarcolemmal membrane vesicles. However, the sulfur residue-modifying reagents were the only compounds to exhibit significant differences in their action on Na+-Ca2+ exchange, depending on whether Na+ or K+ was present in the preincubation modification medium. The tryptophan modifier, N-bromosuccinimide, was the sole reagent that elicited a substantial increase in membrane permeability. The evidence is consistent with the hypothesis that sulfur-containing residues interact with a Na+-binding site for Na+-Ca2+ exchange in cardiac sarcolemmal vesicles.

Na+-H+ exchange and passive Na+ flux were investigated in cardiac sarcolemmal vesicles as a function of changing the ionic composition of the reaction media. The inclusion of EGTA in the reaction medium resulted in a potent stimulation of Na+ uptake by Na+-H+ exchange. It was found that millimolar concentrations of Mg2+ and Li+ were capable of inhibiting Na+-H+ exchange by 80%. One mechanism by which these ions may inhibit intravesicular Na+ accumulation by Na+-H+ exchange is via an increase in Na+ efflux. An examination of Na+ efflux kinetics from vesicles pre-loaded with Na+ revealed that Na+, Ca2+, Mg2+ and Li+ could stimulate Na+ efflux. Na+-H+ exchange was potently inhibited by an organic divalent cation, dimenthonium, which screens membrane surface charge. This would suggest that Na+-H+ exchange occurs in the diffuse double layer region of cardiac sarcolemma and this phenomenon is distinctly different from other Na+ transport processes. The results in this study indicate that in addition to a stimulation of Na+ efflux, the inhibitory effects of Mg2+, Ca2+ and Li+ on Na+-H+ exchange may also involve a charge dependent screening of Na+ interactions with the membrane.