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.     

Stimulation of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by phospholipase D

Treatment of canine cardiac sarcolemmal vesicles with phospholipase D resulted in a large stimulation (up to 400%) of Na+-Ca2+ exchange activity. The phospholipase D treatment decreased the apparent Km (Ca2+) for the initial rate of Nai+-dependent Ca2+ uptake from 18.2 +/- 2.6 to 6.3 +/- 0.3 microM. The Vmax increased from 18.0 +/- 3.6 to 31.5 +/- 3.6 nmol of Ca2+/mg of protein/s. The effect was specific for Na+-Ca2+ exchange; other sarcolemmal transport enzymes ((Na+, K+)-ATPase; ATP-dependent Ca2+ transport) are inhibited by incubation with phospholipase D. Phospholipase D had little effect on the passive Ca2+ permeability of the sarcolemmal vesicles. After treatment with 0.4 unit/ml of phospholipase D (20 min, 37 degrees C), the sarcolemmal content of phosphatidic acid rose from 0.9 +/- 0.2 to 8.9 +/- 0.4%; simultaneously, Na+-Ca2+ exchange activity increased 327 +/- 87%. It is probable that the elevated phosphatidic acid level is responsible for the enhanced Na+-Ca2+ exchange activity. In a previous study (Philipson, K. D., Frank, J. S., and Nishimoto, A. Y. (1983) J. Biol. Chem. 258, 5905-5910), we hypothesized that negatively charged phospholipids were important in Na+-Ca2+ exchange, and the present results are consistent with this hypothesis. Stimulation of Na+-Ca2+ exchange by phosphatidic acid may be important in explaining the Ca2+ influx which accompanies the phosphatidylinositol turnover response which occurs in a wide variety of tissues.     

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.     

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.     

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.     

Symmetry properties of the Na+-Ca2+ exchange mechanism in cardiac sarcolemmal vesicles

The Na+-Ca2+ exchange mechanism in cardiac sarcolemmal vesicles can catalyze the exchange of Ca2+ on either side of the sarcolemmal membrane for Na+ on the opposing side. Little is known regarding the relative affinities of Na+ and Ca2+ for exchanger binding sites on the intra- and extracellular membrane surfaces. We have previously reported (Philipson, K.D. and Nishimoto, A.Y. (1982) J. Biol. Chem. 257, 5111-5117) a method for measuring the Na+-Ca2+ exchange of only the inside-out vesicles in a mixed population of sarcolemmal vesicles (predominantly right-side-out). We concluded that the apparent Km(Ca2+) for Na+i-dependent Ca2+ uptake was similar for inside-out and right-side-out vesicles. In the present study, we examine in detail Na+o-dependent Ca2+ efflux from both the inside-out and the total population of vesicles. To load vesicles with Ca2+ prior to measurement of Ca2+ efflux, four methods are used: 1, Na+-Ca2+ exchange; 2, passive Ca2+ diffusion; 3, ATP-dependent Ca2+ uptake; 4, exchange of Ca2+ for Na+ which has been actively transported into vesicles by the Na+ pump. The first two methods load all sarcolemmal vesicles with Ca2+, while the latter two methods selectively load inside-out vesicles with Ca2+. We are able to conclude that the dependence of Ca2+ efflux on the external Na+ concentration is similar in inside-out and right-side-out vesicles. Thus the apparent Km(Na+) values (approximately equal to 30 mM) of the Na+-Ca2+ exchanger are similar on the two surfaces of the sarcolemmal membrane. In other experiments, external Na+ inhibited the Na+i-dependent Ca2+ uptake of the total population of vesicles much more potently than that of the inside-out vesicles. Apparently Na+ can compete for the Ca2+ binding site more effectively on the external surface of right-side-out than on the external surface of inside-out vesicles. Thus, although affinities for Na+ or Ca2+ (in the absence of the other ion) appear symmetrical, the interactions between Na+ and Ca2+ at the two sides of the exchanger are not the same. The Na+-Ca2+ exchanger is not a completely symmetrical transport protein.     

Inhibition by taurine of Na(+)-Ca2+ exchange in sarcolemmal membrane vesicles from bovine and guinea pig hearts

1. Taurine, but not GABA, beta-alanine and glycine, inhibited Na(+)-dependent Ca2+ uptake in bovine cardiac sarcolemmal membrane vesicles in a dose-dependent manner. 2. The inhibition of Na(+)-dependent Ca2+ uptake was noncompetitive with respect to Ca2+ concentration. 3. The inhibitory effect of taurine on the exchange was also observed in cardiac sarcolemmal vesicles prepared from guinea pig, but not from rat. 4. Taurine did not affect Na(+)-dependent Ca2+ efflux nor ATP-dependent Ca2+ uptake in the bovine cardiac membranes.     

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.     

Inhibition of the Na+/Ca2+ exchange in cardiac sarcolemmal vesicles by amiloride

The pyrazine diuretic amiloride inhibits the Na+/Ca2+ exchange activity of cardiac sarcolemmal vesicles in a concentration-dependent way. A good relationship between the uptake of amiloride by the vesicles and the inhibition of the exchanger has been found. Kinetic analyses indicate that the inhibition of Na+/Ca2+ exchange activity by amiloride is non-competitively removed by Ca2+ and competitively overcome by an outwardly directed Na+ gradient.     

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.     

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.     

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.     

Stimulation of Na+-Ca2+ exchange in heart sarcolemma by insulin

Insulin was found to stimulate Na+-dependent Ca2+ uptake in dog heart sarcolemma in a concentration dependent manner (0.001 to 1 milliunits/ml). Maximal stimulation (160 to 170%) was seen at 0.1 to 1 milliunits/ml of insulin. Unlike Na+-dependent Ca2+ uptake, ATP-dependent Ca2+ uptake was unaltered by 1 microunit/ml of insulin. However, high concentrations of insulin (0.01 to 1 milliunits/ml) significantly increased the ATP-dependent Ca2+ uptake activity of heart sarcolemma; maximal increase (60%) was observed at 1 milliunit/ml of insulin. The Na+ K+-ATPase activity did not change upon incubating sarcolemma with insulin. The membrane preparation exhibited specific insulin binding characteristics. The Scatchard plot analysis of the data indicated two binding sites for insulin; the association constants for the high and low affinity sites were 2 X 10(9) M-1 and 4.4 X 10(8) M-1, respectively. These results support the view regarding the presence of insulin receptors in the heart cell membrane and indicate a dramatic effect of insulin on the sarcolemmal Ca2+ transport systems.