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.     

ATP stimulation of Na+/Ca2+ exchange in cardiac sarcolemmal vesicles

In cardiacsarcolemmal vesicles, MgATP stimulatesNa⁺/Ca²⁺exchange with the following characteristics:1) increases 10-fold the apparentaffinity for cytosolic Ca²⁺;2) a Michaelis constant for ATP of~500 µM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory;4) not observed in the presence of20 µM eosin alone but reinstated when vanadate is added;5) mimicked by adenosine5'-O-(3-thiotriphosphate), without the need for vanadate, but not by ,-methyleneadenosine 5'-triphosphate; and 6) notaffected by unspecific protein alkaline phosphatase but abolished by aphosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLCeffect is counteracted by phosphatidylinositol. In addition, in theabsence of ATP,L--phosphatidylinositol4,5-bisphosphate (PIP₂) was ableto stimulate the exchanger activity in vesicles pretreated with PI-PLC.This MgATP stimulation is not related to phosphorylation of thecarrier, whereas phosphorylation appeared in the phosphoinositides,mainly PIP₂, thatcoimmunoprecipitate with the exchanger. Vesicles incubated with MgATPand no Ca²⁺ show a markedsynthesis ofL--phosphatidylinositol4-monophosphate (PIP) with little production ofPIP₂; in the presence of 1 µM Ca²⁺, the net synthesis of PIP issmaller, whereas that of PIP₂increases ninefold. These results indicate thatPIP₂ is involved in the MgATPstimulation of the cardiacNa⁺/Ca²⁺exchanger through a fast phosphorylation chain: aCa²⁺-independent PIP formationfollowed by a Ca²⁺-dependentsynthesis of PIP₂.

     

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.     

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.     

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.     

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.     

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.     

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.