ATP-dependent Na+ transport in cardiac sarcolemmal vesicles

Although the enzyme (Na⁺ + K⁺)-ATPase has been extensively characterized, few studies of its major role, ATP-dependent Na⁺ pumping, have been reported in vesicular preparations. This is because it is extremely difficult to determine fluxes of isotopic Na⁺ accurately in most isolated membrane systems. Using highly purified cardiac sarcolemmal vesicles, we have developed a new technique to detect relative rates of ATP-dependent Na⁺ transport sensitively. This technique relies on the presence of Na⁺-Ca²⁺ exchange and ATP-driven Na⁺ pump activities on the same inside-out sarcolemmal vesicles. ATP-dependent Na⁺ uptake is monitored by a subsequent Na_i⁺-dependent Ca²⁺ uptake reaction (Na⁺-Ca²⁺ exchange) using ⁴⁵Ca²⁺. We present evidence that the Na⁺-Ca²⁺ exchange will be linearly related to the prior active Na⁺ uptake. Although this method is indirect, it is much more sensitive than a direct approach using Na⁺ isotopes. Applying this method, we measure cardiac ATP-dependent Na⁺ transport and (Na⁺ + K⁺)-ATPase activities in identical ionic media. We find that the (Na⁺ + K⁺)-ATPase and the Na⁺ pump have identical dependencies on both Na⁺ and ATP. The dependence on [Na⁺] is sigmoidal, with a Hill coefficient of 2.8. Na⁺ pumping is half-maximal at [Na⁺] = 9 mM. The K_m for ATP is 0.21 mM. ADP competitively inhibits ATP-dependent Na⁺ pumping. This approach should allow other new investigations on on ATP-dependent Na⁺ transport across cardiac sarcolemma.     

ATP-dependent calcium transport in cardiac sarcolemmal membrane vesicles

Cardiac sarcolemmal (SL) membrane vesicles accumulated Ca in the presence of ATP. The accumulated Ca was released by osmotic shock and by the Ca ionophore A23187, indicating that the Ca had been transported into the vesicle interior. Ca uptake by the SL vesicles was not inhibited by ruthenium red, 2,4-dinitrophenol, carbonyl cyanide $\text{m}$-chlorophenyl hydrazone, of NaN₃, agents that are known to inhibit mitochondrial Ca transport activity. In contrast to the behavior of cardiac sarcoplasmic reticulum, Ca accumulation by the SL vesicles was not stimulated by oxalate and could not driven by p-nitrophenylphosphate hydrolysis. NaCl inhibited ATP-dependent Ca uptake by the SL vesicles. This effect was shown to be due to a stimulation of Ca efflux by Na, mediated by the sarcolemmal NaCa exchange system. The results provide conclusive evidence for the presence of an ATP-dependent Ca “pump” in the cardiac SL membrane.     

Characterization of Na+-dependent phosphate transport in cardiac sarcolemmal vesicles

Pi uptake by purified bovine cardiac sarcolemmal vesicles was stimulated by an inwardly directed Na+ gradient, but not by such gradients of K+, Rb+, Li+, and choline. When Na+ was present both inside and outside the vesicles, or when Na+ gradient was dissipated by monensin, the Na+-dependent Pi uptake increased with time, reached a peak, and then declined approaching a steady state. The initial rate of Na+-dependent Pi uptake was a saturable function of Pi concentration (Km = 0.5 mM). These findings indicate the existence of a Na+,Pi-cotransporter in the sarcolemma. The Na+-activation curve of the Pi uptake exhibited positive cooperativity, suggesting the requirement for multiple Na+ binding to the functional unit of the carrier. The initial rate of Na+-dependent Pi uptake decreased as extra-vesicular pH increased in the range of 5.5-8.7. The uptake rate increased under conditions that are known or expected to generate an inside-negative membrane potential, indicating that Pi uptake is accompanied by the uptake of positive charge. These results suggest the electrogenic cotransports of two Na+ and one H2PO4-. We conclude that this cotransporter catalyzes the secondary active transport of Pi across the cardiac plasma membrane and regulates myocardial energy metabolism. We also suggest that the cotransporter may control intracellular Na+ and thus be involved in the regulation of trans-sarcolemmal Ca2+ movement and cardiac contractility.     

Na+-H+ exchange in cardiac sarcolemmal vesicles

The transport of Na+ by a purified sarcolemmal vesicular preparation from canine ventricular tissue was studied as a function of both internal and external pH. The uptake of Na+ into sarcolemmal vesicles increased upon raising the extravesicular pH of the reaction medium. Half-maximal uptake of Na+ was observed at a pHo of about 8.1 and maximal uptake occurred at pH 8.6. The uptake of Na+ by sarcolemma was also dependent upon the intravesicular pH. Na+ uptake into sarcolemmal vesicles was greatly attenuated in the absence of a H+ gradient across the membrane. Transport of Na+ was potently inhibited by amiloride, a known blocker of Na+-H+ exchange. LiCl was also an effective inhibitor of Na+ transport. In the presence of optimal H+ gradients, Na+ uptake was linear for the first 5 seconds of the reaction and exhibited a Vmax of 290 nmol Na+/mg per min and a KNa of 3.5 mM. These experiments strongly indicate the presence of a Na+-H+ exchange system in cardiac sarcolemma. This activity appeared to be relatively specific for this membrane fraction. The identification of Na+-H+ exchange activity in a sarcolemmal vesicular fraction from the heart will permit extensive characterization of the regulation and kinetics of this antiporter in future investigations.     

Na+-dependent alkaline earth metal uptake in cardiac sarcolemmal vesicles

The ability of alkaline earth metals (M2+) to substitute for Ca2+ in Na+-Ca2+ exchange was examined in sarcolemmal vesicles isolated from the canine heart. 85Sr2+ and 133Ba2+, in addition to 45Ca2+, were used to determine the characteristics of Na+-M2+ exchange. The Na+i-dependent M2+ uptake was measured as a function of time, with t ranging from 0.5 to 360 s, [Na+]i = 140 mM and [M2+]o = 40 microM. This function was linear for Ca2+ and Sr2+ uptake for approx. 6 s and for Ba2+ for about 60 s. Plateau levels were achieved within 120 s for Ca2+ and Sr2+ but Ba2+ took considerably longer. The Km values for Na+-M2+ exchange, derived from Eadie-Hofstee plots, were 30, 58, and 73 microM for Ca2+, Sr2+ and Ba2+, respectively. The Na+i-dependent uptake of all three ions was stimulated in the presence of 0.36 microM valinomycin. Na+-Ca2+ exchange was also measured in the presence of either 20 microM Sr2+ or 100 microM Ba2+. Both of these ions behaved (at these concentrations) as competitive inhibitors of Na+-Ca2+ exchange with the KI being 32 microM for Sr2+ and 92 microM for Ba2+. Passive efflux was determined by first allowing Na+-M2+ exchange to continue to plateau values and then diluting the loaded vesicles in the presence of EGTA. The rate constants for the passive efflux were 8.4, 6.3 and 4.4 min-1 for Ca2+, Sr2+ and Ba2+, respectively.     

Cationic interactions with Na+-H+ exchange and passive Na+ flux 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 stumulation 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.     

Protein methylation inhibits Na+-Ca2+ exchange activity in cardiac sarcolemmal vesicles

We have examined the effect of membrane methylation on the Na+-Ca2+ exchange activity of canine cardiac sarcolemmal vesicles using S-adenosyl-L-methionine as methyl donor. Methylation leads to approximately 40% inhibition of the initial rate of Nai+-dependent Ca2+ uptake. The inhibition is due to a lowering of the Vmax for the reaction. The inhibition is not due to an effect on membrane permeability and is blocked by S-adenosyl-L-homocysteine, an inhibitor of methylation reactions. The following experiments indicated that inhibition of Na+-Ca2+ exchange was due to methylation of membrane protein and not due to methylated phosphatidylethanolamine (PE) compounds (i.e., phosphatidyl-N-monomethylethanolamine (PMME) or phosphatidyl-N,N'-dimethylethanolamine (PDME]: (1) We solubilized sarcolemma and reconstituted activity into vesicles containing no PE. The inhibition by S-adenosyl-L-methionine was not diminished in this environment. (2) We reconstituted sarcolemma into vesicles containing PMME or PDME. These methylated lipid components had no effect on Na+-Ca2+ exchange activity. (3) We verified that many membrane proteins, probably including the exchanger, become methylated.     

Enrichment of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by alkaline extraction

Exposure of canine cardiac sarcolemmal vesicles to alkaline media (greater than or equal to pH 12) results in the extraction of 33% of the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that specific proteins are being solubilized. Most of the phospholipid and sialic acid remains with the pellet after centrifugation. Electron microscopy reveals that alkaline treatment does not cause gross morphological damage to the vesicles, although freeze-fracture demonstrates some aggregation of intramembrane particles. The data indicate that high pH probably removes peripheral proteins and leaves the integral proteins in place. We find complete recovery of Na+-Ca2+ exchange activity in alkaline-extracted membranes after solubilization and reconstitution. These vesicles contain only 50% of the protein of vesicles reconstituted from control sarcolemma. Thus, the specific activity of Na+-Ca2+ exchange is doubled. Alkaline extraction is a useful and reproducible procedure for enrichment of the Na+-Ca2+ exchange protein. (Na+ + K+)-ATPase is completely inactivated by exposure to pH 12 medium though immunodetection shows that the (Na+ + K+)-ATPase proteins are not extracted. We detect both alpha and alpha + forms of (Na+ + K+)-ATPase and deduce that the Na+ pump proteins do not comprise a major fraction of sarcolemmal protein.     

Inhibition of Na+-Ca2+ exchange mechanism in cardiac sarcolemmal vesicles by harmaline

1. Harmaline was found to inhibit the Na+-Ca2+ exchange mechanism present in cardiac sarcolemmal vesicles. 2. The inhibition was dose-dependent and was observed in the range 10(-5) M-10(-2) M harmaline. 3. The effect was demonstrated on both 45Ca2+-uptake and 45Ca2+-efflux. 4. The observed Ki value for harmaline inhibition of 45Ca2+-uptake was found to be 2.5 X 10(-4) M.     

Modulation of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by Ca2+ antagonists

Summary The purpose of this study was to examine the effect of three classes of Ca²⁺ antagonists, diltiazem, verapamil and nifedipine on Na⁺-Ca²⁺ exchange mechanism in the sarcolemmal vesicles isolated from canine heart. Na⁺-Ca²⁺ exchange and Ca²⁺ pump (ATP-dependent Ca²⁺ uptake) activities were assessed using the Millipore filtration technique. sarcolemmal vesicles used in this study are estimated to consist of several subpopulations wherein 23% are inside-out and 55% are right side-out sealed vesicles in orientation. The affect of each Ca²⁺ antagonist on the Na⁺-dependent Ca²⁺ uptake was studied in the total population of sarcolemmal vesicles, in which none of the agents depressed the initial rate of Ca²⁺ uptake until concentrations of 10 M were incubated in the incubation medium. However, when sarcolemmal vesicles were preloaded with Ca²⁺ via ATP-dependent Ca²⁺ uptake, cellular Ca²⁺ influx was depressed only by verapamil (28%) at 1 M in the efflux medium with 8 mM Na⁺. Furthermore, inhibition of Ca²⁺ efflux by verapamil was more pronounced in the presence of 16 mM Na⁺ in the efflux medium. The order of inhibition was; verapamil > diltiazem > nifedipine. These results indicate that same forms of Ca²⁺-antagonist drugs may affect the Na⁺-Ca²⁺ exchange mechanism in the cardiac sarcolemmal vesicles and therefore we suggest this site of action may contribute to their effects on the myocardium.     

Demonstration of a Na+/H+ exchange activity in purified canine cardiac sarcolemmal vesicles

Purified canine cardiac sarcolemmal membrane vesicles exhibit a sodium ion for proton exchange activity (Na+/H+ exchange). Na+/H+ exchange was demonstrated both by measuring rapid 22Na uptake into sarcolemmal vesicles in response to a transmembrane H+ gradient and by following H+ transport in response to a transmembrane Na+ gradient with use of the probe acridine orange. Maximal 22Na uptake into the sarcolemmal vesicles (with starting intravesicular pH = 6 and extravesicular pH = 8) was approximately 20 nmol/mg protein. The extravesicular Km of the Na+/H+ exchange activity for Na+ was determined to be between 2 and 4 mM (intravesicular pH = 5.9, extravesicular pH = 7.9), as assessed by measuring the concentration dependence of the 22Na uptake rate and the ability of extravesicular Na+ to collapse an imposed H+ gradient. All results suggested that Na+/H+ exchange was reversible and tightly coupled. The Na+/H+ exchange activity was assayed in membrane subfractions and found most concentrated in highly purified cardiac sarcolemmal vesicles and was absent from free and junctional sarcoplasmic reticulum vesicles. 22Na uptake into sarcolemmal vesicles mediated by Na+/H+ exchange was dependent on extravesicular pH, having an optimum around pH 9 (initial internal pH = 6). Although the Na+/H+ exchange activity was not inhibited by tetrodotoxin or digitoxin, it was inhibited by quinidine, quinacrine, amiloride, and several amiloride derivatives. The relative potencies of the various inhibitors tested were found to be: quinacrine greater than quinidine = ethylisopropylamiloride greater than methylisopropylamiloride greater than dimethylamiloride greater than amiloride. The Na+/H+ exchange activity identified in purified cardiac sarcolemmal vesicles appears to be qualitatively similar to Na+/H+ exchange activities recently described in intact cell systems. Isolated cardiac sarcolemmal vesicles should prove a useful model system for the study of Na+/H+ exchange regulation in myocardial tissue.     

Modulation of Na+-Ca2+ exchange and Ca2+ permeability in cardiac sarcolemmal vesicles by doxylstearic acids

We examine the effects of 5-, 12- and 16-doxylstearic acids on the Na+-Ca2+ exchange and passive Ca2+ permeability of cardiac sarcolemmal vesicles. Stearic acid is a weak stimulator of Na+-Ca2+ exchange. A doxyl moiety potentiates stimulation with the order of increasing potency being 5-, 12- and then 16-doxylstearic acid. Stearic acid has little effect on vesicle Ca2+ permeability but again the doxylstearates are more effective. The sequence of potency is reversed, however, from that for increasing Na+-Ca2+ exchange. 5-Doxylstearic acid most markedly exchanges passive Ca2+ flux followed by the 12-, and then 16-doxylstearic acids. Methyl esters of the doxylstearates have no effect on either Na+-Ca2+ exchange or Ca2+ permeability. We model the results as follows. For a fatty acid to stimulate Na+-Ca2+ exchange activity, an anionic charge is required to interact with the exchanger protein at the membrane surface. Stimulation is potentiated by a perturbation (such as provided by a doxyl group) within the lipid bilayer. The perturbation is most effective at a location towards the center of the bilayer. To increase passive Ca2+ permeability an anionic charge is again essential. Disorder within the bilayer is also important, but now the most important site is near the membrane surface. Results of experiments with linolenic and gamma-linolenic acid and previous studies with other fatty acids also support this model.     

Altered cardiac Na(+)/H(+) exchange in phospholipase D-treated sarcolemmal vesicles

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.     

Effects of fatty acids on Na+-Ca2+ exchange and Ca2+ permeability of cardiac sarcolemmal vesicles

We have previously reported that anionic phospholipids (Philipson, K.D., and Nishimoto, A.Y. (1984) J. Biol. Chem. 259, 16-19) and other anionic amphiphiles (Philipson, K.D. (1984) J. Biol. Chem. 259, 13999-14002) stimulate Na+-Ca2+ exchange in cardiac sarcolemmal vesicles. To further these studies, we have now investigated the effects of a variety of fatty acids on both Na+-Ca2+ exchange and passive Ca2+ permeability. Na+-Ca2+ exchange was stimulated by fatty acids by up to 150%. Unsaturated fatty acids were more potent than saturated fatty acids, and the stimulation was primarily due to a decrease in the apparent KM (Ca2+). There was a positive correlation between the ability of a fatty acid to stimulate Na+-Ca2+ exchange and to increase passive Ca2+ permeability. The methyl esters of fatty acids had no effects on either exchange or permeability indicating the importance of anionic charge. We conclude that the combination of local lipid disorder and anionic charge regulate Na+-Ca2+ exchange. Perturbations of the bilayer hydrophobic region and increased negative surface charge are both required for fatty acids to increase passive Ca2+ flux. Na+-Ca2+ exchange is stimulated when the ratio of membrane free fatty acid to phospholipid is about 5%. This level of fatty acid is achieved during 1 h of myocardial ischemia (Chien, K. R., Han, A., Sen, A., Buja, L. M., and Willerson, J. T. (1984) Circ. Res. 54, 313-322), indicating that ischemia could induce altered sarcolemmal Ca2+ transport due to fatty acid accumulation.     

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.     

Ion specificity of cardiac sarcolemmal Na+/H+ antiporter

In bovine cardiac sarcolemmal vesicles, an outward H+ gradient stimulated the initial rate of amiloride-sensitive uptake of 22Na+, 42K+, or 86Rb+. Release of H+ from the vesicles was stimulated by extravesicular Na+, K+, Rb+, or Li+ but not by choline or N-methylglucamine. Uptakes of Na+ and Rb+ were half-saturated at 3 mM Na+ and 3 mM Rb+, but the maximal velocity of Na+ uptake was 1.5 times that of Rb+ uptake. Na+ uptake was inhibited by extravesicular K+, Rb+, or Li+, and Rb+ uptake was inhibited by extravesicular Na+ or Li+. Amiloride-sensitive uptake of Na+ or Rb+ increased with increase in extravesicular pH and decrease in intravesicular pH. In the absence of pH gradient, there were stimulations of Na+ uptake by intravesicular Na+ and K+ and of Rb+ uptake by intravesicular Rb+ and Na+. Similarly, there were trans stimulations of Na+ and Rb+ efflux by extravesicular alkali cations. The data suggest the existence of a nonselective antiporter catalyzing either alkali cation/H+ exchange or alkali cation/alkali cation exchange. Since increasing Na+ caused complete inhibition of Rb+/H+ exchange, but saturating K+ caused partial inhibitions of Na+/H+ exchange and Na+/Na+ exchange, the presence of a Na(+)-selective antiporter is also indicated. Although both antiporters may be involved in pH homeostasis, a role of the nonselective antiporter may be in the control of Na+/K+ exchange across the cardiac sarcolemma.     

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.     

Identification of dystrophin in cardiac sarcolemmal vesicles

We have identified dystrophin in highly purified sarcolemmal vesicles isolated from canine and bovine hearts using specific antibodies against the COOH-terminal region of the protein. Bovine cardiac sarcolemma contained a single immunoreactive protein band (Mr. approximately 400,000) whereas the canine cardiac membrane contained a doublet (Mr. approximately 420,000 and approximately 380,000). The higher molecular weight form of canine cardiac dystrophin was more abundant than the lower molecular weight form. These highly purified preparations of the sarcolemmal vesicles should provide a useful tool for structural and functional analysis of the interaction of dystrophin with the plasma membrane.     

Alterations by saponins of passive Ca2+ permeability and Na+-Ca2+ exchange activity of canine cardiac sarcolemmal vesicles

Saponins can both permeabilize cell plasma membranes and cause positive inotropic effects in isolated cardiac muscles. Different saponins vary in their relative abilities to cause each effect suggesting that different mechanisms of action may be involved. To investigate this possibility, we have compared the effects of seven different saponins on the passive Ca2+ permeability and Na+-Ca2+ exchange activity of isolated canine cardiac sarcolemmal membranes. Saponins having hemolytic activity reversibly increased the passive efflux of Ca2+ from sarcolemmal vesicles preloaded with 45Ca2+ with the following order of potency: echinoside-A greater than echinoside-B greater than holothurin-A greater than holothurin-B greater than sakuraso-saponin. Ginsenoside-Rd and desacyl-jego-saponin, which lack hemolytic activity, had no significant effect on this variable. The saponins also stimulated Na+-Ca2+ exchange activity measured as Na+-dependent Ca2+ uptake by sarcolemmal vesicles. Ginsenoside-Rd and desacyl-jego-seponin, which did not affect passive Ca2+ permeability, stimulated the uptake, while in contrast, echinoside-A and -B only slightly increased or decreased this latter variable. Thus, the abilities of these compounds to enhance Na+-Ca2+ exchange activity seem to be inversely related to their abilities to increase the Ca2+ permeability. Effects by the echinosides on Na+-Ca2+ exchange may be masked by the loss of Ca2+ from the vesicles due to the increased permeability. These results suggest that the saponins interact with membrane constituent(s) that can influence the passive Ca2+ permeability and the Na+-Ca2+ exchange activity of cardiac sarcolemmal membranes.     

ATP-dependent H+ transport in synaptosomal membrane vesicles of the rat brain

H+ transport into synaptosomal membrane vesicles of the rat brain was stimulated by ATP and to a lesser extent by GTP, but not by ITP, CTP, UTP, ADP, AMP or beta, gamma-methylene ATP. ATP at concentrations up to 200 mM concentration-dependently stimulated the rate of H+ transport with a Km value of 0.6 mM, but at higher concentrations of this nucleotide the rate decreased. Other nucleotides such as CTP, UTP, GTP and AMP, or products of ATP hydrolysis i.e. ADP and Pi also reduced the ATP-stimulated H+ transport. The inhibition by GTP and ADP was not affected by the ATP concentration. These findings suggest that plasma membranes of nerve endings transport H+ from inside to outside of the cells utilizing energy from ATP hydrolysis, and that this transport is regulated by the intracellular concentration of nucleotides and Pi on sites other than those involved in substrate binding.