Mechanisms underlying delayed afterdepolarizations in hypertrophied left ventricular myocytes of rats.

Cardiac hypertrophy was induced in rats by daily injection of isoproterenol (5 mg/kg ip) for 7 days. Membrane voltage and currents were recorded using the whole cell patch-clamp technique in left ventricular myocytes from control and hypertrophied hearts. Ryanodine-sensitive delayed afterdepolarizations (DADs) and transient inward current (I(ti)) appeared in hypertrophied cells more often and were of larger amplitude than in control cells. DADs and I(ti) are carried principally by Na/Ca exchange with smaller contributions from a nonselective cation channel and from a Cl- channel. The latter is expressed only in hypertrophied myocytes. In hypertrophy, the density of caffeine-induced Na/Ca exchange current (I(Na/Ca)) was increased by 26%, sarcoplasmic reticulum (SR) Ca2+ content as assessed from the integral of I(Na/Ca) was increased by 30%, the density of Na-pump current (I(pump)) was reduced by 40%, and the intracellular Na+ content, measured by Na+-selective microelectrodes was increased by 55%. The results indicate that DADs and I(ti) are generated by spontaneous Ca2+ release from an overloaded SR caused by a downregulated Na pump and an upregulated Na/Ca exchange. These findings may explain the propensity for arrhythmias seen in this model of hypertrophy.

An unknown endogenous inhibitor of Na/Ca exchange can enhance the cardiac muscle contractility.

The cardiac sarcolemma Na/Ca exchanger is a key system for controlling the intracellular calcium levels during the excitation-contraction coupling. Here, we test the hypothesis that the heart tissue contains a putative endogenous factor having a capacity to modulate the Na/Ca exchanger and muscle contractility. The concentrated cardiac extracts inhibit the Na(i)- or Ca(i)-dependent (45)Ca uptakes in isolated cardiac sarcolemma vesicles as well as the Na(o)-dependent Ca efflux, monitored by extravesicular Ca probe fluo-3. The inhibitory activity has been purified approximately 2000-fold by normal and reversed-phase HPLC procedures. The inhibitory activity is eluted from the Sephadex G-10 in the range of 350-550 Da, suggesting that the inhibitory factor is a low-molecular-weight substance. The mass spectra analysis shows a number of signals within m/z 380-560; however, it is not clear at this moment whether these recordings represent the mass of putative inhibitory factor or irrelevant impurities. The endogenous inhibitory factor of Na/Ca exchange does not resemble the properties (HPLC retention time, mass spectra, amino acid analysis, etc.) of autoinhibitory XIP peptide. The addition of inhibitory factor to muscle strip of guinea pig ventricles induces 2- to 5-fold enhancement of isometric contractions, thereby exhibiting a strong positive inotropic effect. This effect is a dose-dependent phenomenon, which can be reversed by washing the inhibitory factor from the organ bath. Assuming a molecular weight of 350-550 Da, the effective concentrations of putative inhibitor must be <10(-6) M. Therefore, the present findings demonstrate that the mammalian heart contains a low-molecular-weight factor that can inhibit Na/Ca exchange and enhance the cardiac contractility.

Time-resolved monitoring of electrogenic Na+-Ca2+ exchange in the isolated cardiac sarcolemma vesicles by using a rapid-response fluorescent probe.

As a major Ca exit system in myocytes, the electrogenic Na+-Ca2+ exchange is exposed to rapid changes of regulatory factors (e.g., cytosolic Ca) during the excitation-contraction coupling. The dynamic aspects of the exchanger response to regulatory factors have not been resolved in the past due to technical limitations. Here, we describe stopped-flow protocols for monitoring the electrogenic activity of Na+-Ca2+ exchange in cardiac sarcolemma vesicles by using a rapid-response voltage-sensitive dye Merocyanine-540 (M540). The M540 signal of Nao-dependent Ca efflux is generated by mixing the Ca-loaded vesicles with Na buffer, yielding 160 mM extravesicular Na and 6 microM Cafree. This signal is inhibited by a cyclic peptide blocker (FRCRCFa), by a Ca ionophore (ionomycin), or by an electrogenic uncoupler (valinomycin or FCCP). The M540 signal of Nao-dependent Ca efflux shows a rapid pre-steady-state burst (210 s-1), followed by slow steady-state phase (

The role of sarcoplasmic reticulum in the protective effect of class III drugs against Ca2+ overload.

UNLABELLED: Various studies on humans and experimental mammals showed that d-sotalol and tedisamil (class III antiarrhythmic drugs with positive inotropic effect) facilitate spontaneous ventricular defibrillation. Following our previous results, we summarized that spontaneous ventricular defibrillation requires high level of intercellular coupling and synchronization, both of which depends on intracellular free Ca2+ concentration. We hypothesized that any antiarrhythmic compound that facilitates spontaneous defibrillation, including d-sotalol and tedisamil, should prevent intracellular free Ca2+ overload most likely by elevating cAMP level and enhancing cAMP-related Ca2+ uptake of the sarcoplasmic reticulum (SR). The aim of the present study was to examine the role of the SR uptake function in their effect against Ca2+ overload. METHODS: The effect of d-sotalol, tedisamil and dbcAMP on increased intracellular Ca2+ level were examined in cultured rat cardiomyocytes during blockade of SR Ca2+ uptake by administration of thapsigargin (TG), a selective inhibitor of Ca2+-ATPase. RESULTS: Administration of 3 x 10(-6) mol/l TG, prior to d-sotalol, tedisamil and dbcAMP, significantly increased intracellular free Ca2+ concentration and prevented the effect of d-sotalol, tedisamil or dbcAMP to decrease intracellular Ca2+ level to its beseline, while 10(-6) mol/l TG prevented it only partially. Administration of either d-sotalol or tedisamil (at concentration of 10(-5) mol/l) before the administration of 10(-6) mol/l TG prevent the TG induced elevation of [Ca2+]i. CONCLUSION: These results support our hypothesis that d-sotalol and tedisamil prevent Ca2+ overload by the cAMP dependent SR Ca2+ uptake.

Actions of myristyl-FRCRCFa, a cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchanger, tested in rabbit ventricular myocytes.

In cardiac muscle, the electrogenic Na-Ca exchanger plays important roles in determining action potential shape and in the beat-to-beat homeostasis of intracellular calcium. In this study we tested the actions of a putative cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchange, "Myristyl- (Myr-) FRCRCFa". Experiments were performed using isolated rabbit right ventricular myocytes and whole-cell patch-clamp at 35-37 degreesC. The Na-Ca exchange current (INa-Ca), L-type calcium current (ICa,L), inward rectifier potassium current (IK1) and delayed rectifier potassium current (IK) were compared in untreated cells and cells incubated in a solution containing N-myristylated FRCRCFa. With other major currents blocked, INa-Ca was measured as the Ni-sensitive component of current during a voltage ramp applied from the holding potential of -40 mV, between +80 and -120 mV (ramp velocity 0.1 V s-1). In untreated cells, INa-Ca at +60 mV was 7.1+/-0.6 pA/pF and at -100 mV was -2.7+/-0.3 pA/pF (n=9). After a 15-min pre-incubation with 20 microM Myr-FRCRCFa, INa-Ca was reduced to 4.2+/-0.3 pA/pF at +60 mV and -1. 5+/-0.2 pA/pF at -100 mV (P<0.02; n=7). After incubation with 20 microM Myr-FRCRCFa for 1 h, INa-Ca at both potentials was further reduced (2.3+/-0.8 pA/pF at +60 mV; -0.9+/-0.3 pA/pF at -100 mV; P<0. 008 compared with control; n=4). Under selective recording conditions for ICa,L, there was little difference in ICa,L density between untreated and cells incubated with Myr-FRCRCFa. A Boltzmann fit to the ICa,L/V relation showed no significant alteration of half-maximal activation potential or slope factor of activation. IK1 was also largely unaffected by pre-incubation of cells with Myr-FRCRCFa. IK, measured as deactivating tail current following 1-s test depolarisations to a range of test potentials, was also not significantly altered by Myr-FRCRCFa. The suppression of INa-Ca in cells incubated in Myr-FRCRCFa suggests that addition of the myristyl group to FRCRCFa peptide conveys cell permeancy to the peptide and that Myr-FRCRCFa applied externally to rabbit ventricular myocytes is moderately effective as an INa-Ca blocker. ICa,L, IK1 and IK were largely unaffected by Myr-FRCRCFa. N-Myristylation of such conformationally constrained hexapeptides may, therefore, provide a means of producing cell-permeant inhibitors of the cardiac Na-Ca exchanger.

The peptide "FRCRCFa", dialysed intracellularly, inhibits the Na/Ca exchange in rabbit ventricular myocytes with high affinity.

We investigated the effect in rabbit ventricular myocytes of "FRCRCFa", a newly developed peptide inhibitor of the Na/Ca exchange. Myocytes were whole-cell patch clamped and experiments were carried out at 36 degrees C. The Na/Ca exchange was measured selectively, by blocking interfering ion channel currents and the Na/K pump, as the membrane current which could be inhibited by 5 mM nickel (Ni; a known blocker of the Na/Ca exchange). Increasing concentrations of FRCRCFa dialysed into the cell from the patch-pipette inhibited the Na/Ca exchange current. The dose/response curve could be fitted by a function for co-operative ligand binding, which predicted a KD for FRCRCFa-mediated inhibition of 22.7 +/- 3.7 nM, with a Hill coefficient of 0.61 +/- 0.06. Pipette FRCRCFa concentrations of 1 micro;M and above were sufficient to cause complete inhibition of Na/Ca exchange current. The inhibitory effect of FRCRCFa was independent of membrane potential and relatively selective: 10 micro;M FRCRCFa dialysed into the cell had no effect on the L-type Ca current and delayed rectifier and inward rectifier K currents. Thus FRCRCFa appears to be a potent and relatively selective inhibitor of the Na/Ca exchange in intact cardiac myocytes, and may be of value for studies of the Na/Ca exchange.

Inhibition of the cardiac sarcolemma Na+/Ca2+ exchanger by conformationally constrained small cyclic peptides.

Positively charged cyclic peptides (three to seven amino acids) have been tested for their inhibitory effects on Na+/Ca2+ exchange in the cardiac sarcolemma vesicles. The lead structure of Phe-Arg-Cys-Arg-Cys-Phe-CONH2 (FRCRCFa) has been systematically modified for identification of important pharmacophores. In cyclic peptides (intramolecular S-S bond, the carboxyl terminal is locked with amide (CONH2), and positive charge is retained by one or two arginines, ornithines, or lysines. Thirty-five different cyclic peptides show IC50 values in the range of 2-800 microM, suggesting that some specific structure-activity relationships may determine the inhibitory effects. Shortening of the FRCRCFa length to four amino acids decreases the inhibitory potency by 10-80-fold. The substitution of Arg2 or Arg4 in FRCRCFa with lysine or ornithine decreases the inhibitory potency by 5-12-fold, suggesting that both arginines are beneficial for inhibition. The substitution of Phe1 in FRCRCFa by 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid produces a potent inhibitor (IC50 = 2-4 microM). The N-myristoylated FRCRCFa exhibits an inhibitory potency (IC50 = 8-10 microM) similar to that of the parent FRCRCFa peptide, thereby arousing a new possibility for the development of a cell-permeable blocker of the Na+/Ca2+ exchanger, D-Arg4 or D-Cys5 substitutions in FRCRCFa do not alter the inhibitory effect, whereas the L-to-D substitutions of other amino acids in FRCRCFa reduce the inhibitory potency by 4-5-fold. Thus, the L-to-D substitutions of Arg4 and/or Cys5 have a potential to increase the peptide stability to proteolytic degradation. The insertion of proline outside of the ring of FRCRCFa diminishes the inhibitory potency by 3-6-fold, whereas proline introduction into the ring decreases the inhibitory potency by 16-20-fold. The replacement of Cys3 and Cys5 in FRCRCFa with beta, beta-dimethylcystein has no significant effect on the inhibitory potency, suggesting that the S-S bond is not exposed to the interface of the peptide/receptor interaction. In conclusion, the current data support a proposal that the conformationally constrained Arg-Cys-Arg-Cys structure is obligatory for inhibition of Na+/Ca2+ exchange, whereas hydrophobic additions at the carboxyl and amino ends have limited effects in increasing the inhibitory potency.

Rapid interaction of FRCRCFa with the cytosolic side of the cardiac sarcolemma Na(+)-Ca2+ exchanger blocks the ion transport without preventing the binding of either sodium or calcium.

Positively charged cyclic hexapeptide Phe-Arg-Cys-Arg-Cys-Phe-CONH2 (FRCRCFa) represents a group of conformationally constrained peptides that block the cardiac sarcolemma Na(+)-Ca2+ exchanger [Khananshvili et al. (1995b) J. Biol. Chem. 270, 16182-16188]. Here, we study the kinetic mechanisms of FRCRCFa-induced inhibition of Na(+)-Ca2+ exchange and its partial reaction, the Ca(2+)-Ca2+ exchange. The Nai-dependent 45Ca uptake and Cai-dependent 45Ca uptake were measured by adding the EGTA quench in the semirapid mixer. The reverse mode of Na(+)-Ca2+ exchange (Nao-dependent Ca efflux) was monitored (t = 10-5000 ms) in the stopped-flow machine by measuring extravesicular free calcium with a fluorescence probe fluo-3. Saturating concentrations of FRCRCFa inhibit completely the forward and reverse modes of exchange, suggesting that the inside-out vesicles contribute to most (if not all) of the exchange activity. A short time exposure (t = 10-20 ms) of FRCRCFa with the vesicles is enough to reach a rapid equilibrium between FRCRCFa and a putative inhibitory site at the extravesicular (cytosolic) side of the membrane. A lower limit for the second-order rate constant of FRCRCFa binding can be estimated as a kon of > 10(6) M-1 s-1. A possible competition between FRCRCFa and either Na+ or Ca2+ has been tested at the extravesicular (cytosolic) side of the membrane. At different extravesicular Cao concentrations of 13-250 microM, FRCRCFa inhibits the Na(+)-Ca2+ and Ca(2+)-Ca2+ exchanges with an IC50 of 11-16 microM, suggesting no competition between FRCRCFa and Ca2+. At different extravesicular Nao concentrations of 40-160 mM, FRCRCFa inhibits Nao-dependent Ca efflux with an IC50 of 12-18 microM, suggesting that FRCRCFa and Na+ do not compete for binding at the extravesicular side. A mild proteolytic treatment of vesicles activates the Nai-dependent 45Ca uptake, but has a little effect on the FRCRCFa-induced inhibition. Thus, the "inhibitory site" is still functional after the proteolytic treatment of the inside-out vesicles. In conclusion, a rapid (< 20 ms) interaction of extravesicular (cytosolic) FRCRCFa with the exchanger prevents the ion translocation through the exchanger, while the inhibitory peptide does not interact with the ion transport sites of the exchanger at the cytosolic side of the membrane.

Rate-limiting mechanisms of exchange reactions in the cardiac sarcolemma Na(+)-Ca2+ exchanger.

The effects of temperature, pH, voltage and K+ were tested on Na(+)-Ca2+ and Ca(2+)-Ca2+ exchanges with a goal to elucidate the rate-limiting mechanisms. The initial rates (t = 1 s) of Nai- and Cai-dependent 45Ca uptakes were measured in the sarcolemma vesicles. At pH 7.4 the Ca(2+)-Ca2+ exchange shows a bell-shaped temperature curve with a maximum at 27-29 degrees C. This effect is not caused by irreversible inactivation of the exchanger. The increase of pH from pH 6.0 to 7.4 in the K(+)-free medium decelerates the Ca(2+)-Ca2+ exchange 1.5-2.0-fold, while the addition of K+ accelerates the Ca(2+)-Ca2+ exchange 2.0-3.0-fold. Therefore, the accelerating effect of K+ opposes the decelerating effect of deprotonation. Temperatures increase (6-45 degrees C) in the K(+)-free medium (pH 7.4) elevates the Na(+)-Ca2+/Ca(2+)-Ca2+ exchange ratio from 0.8 to 5.0. With varying temperatures (6-37 degrees C) and pH 5.0-9.7, K+ has no considerable effect on Na(+)-Ca2+ exchange but accelerates the Ca(2+)-Ca2+ exchange 2-3-fold. At 6-45 degrees C and fixed pH 7.4, the inside-positive potential (delta psi > or = +200 mV) accelerates the Na(+)-Ca2+ exchange 1.7-2.0-fold, suggesting that the same rate-limiting reaction controls the Na(+)-Ca2+ exchange at various temperatures. It is concluded that (a) At pH > 6.5 (6-45 degrees C and 0-100 mM K+) the voltage-sensitive Na+ efflux limits the Na(+)-Ca2+ exchange, while the Ca2+ efflux limits the Ca(2+)-Ca2+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Positively charged cyclic hexapeptides, novel blockers for the cardiac sarcolemma Na(+)-Ca2+ exchanger.

Positively charged cyclic hexapeptides have been synthesized and tested for their effects on the cardiac sarcolemma Na(+)-Ca2+ exchange activities with a goal to identify a potent blocker. The cyclic hexapeptides, having the different amino acid sequence, contain two arginines (to retain a positive charge), two phenylalanines (to control hydrophobicity), and two cysteines (to form an intramolecular S-S bond). The effect of cyclic hexapeptides were tested on Na(+)-Ca2+ exchange and its partial reaction, the Ca(2+)-Ca2+ exchange, by measuring the 45Ca fluxes in the semi-rapid mixer or monitoring the calcium-sensitive dye Arsenazo III and voltage-sensitive dyes (Oxanol-V or Merocyanine-540). Seven cyclic hexapeptides inhibit Na(+)-Ca2+ exchange with a different potency (IC50 = 2-300 microM). Phe-Arg-Cys-Arg-Cys-Phe-CONH2 (FRCRCFa) inhibits the Na+i-dependent 45Ca uptake (Na(+)-Ca2+ exchange) and Ca2+i-dependent 45Ca uptake (Ca(2+)-Ca2+ exchange) in the isolated cardiac sarcolemma vesicles with IC50 = 10 +/- 2 microM and IC50 = 7 +/- 3 microM, respectively. Interaction of FRCRCFa with a putative inhibitory site does not involve a "slow" binding (a maximal inhibitory effect is already observed after t = 1 s of mixing). The inside positive potential, generated by Na+o-dependent Ca2+ efflux, was monitored by Oxanol-V (A635-A612) or Merocyanine-540 (A570-A500). In both assay systems, FRCRCFa inhibits the Na(+)-Ca2+ exchange with IC50 = 2-3 microM, while a complete inhibition occurs at 20 microM FRCRCFa. The forward (Na+i-dependent Ca2+ influx) and reverse (Na+o-dependent Ca2+ efflux) modes of Na(+)-Ca2+ exchange, monitored by Arsenazo III (A600-A785), are also inhibited by FRCRCFa. The L-Arg4-->D-Arg4 substitution in FRCRCFa does not alter the IC50, meaning that this structural change may increase a proteolytic resistance without a loss of inhibitory potency. At fixed [Na+]i (160 mM) or [Ca2+]i (250 microM) and varying 45Cao (2-200 microM), FRCRCFa decreases Vmax without altering the Km. Therefore, FRCRCFa is a noncompetitive inhibitor in regard to extravesicular Ca2+ either for Na(+)-Ca2+ or Ca(2+)-Ca2+ exchange. It is suggested that FRCRCFa prevents the ion movements through the exchanger rather than the ion binding.

The cardiac Na(+)-Ca2+ exchanger: relative rates of calcium and sodium movements and their modulation by protonation-deprotonation of the carrier.

The exchange cycle of the cardiac Na(+)-Ca2+ exchanger can be described as separate steps of Ca2+ and Na+ transport [Khananshvili, D. (1990) Biochemistry 29, 2437-2442]. In order to determine the relative rates of Na+ and Ca2+ movement during the Na(+)-Ca2+ and Ca(2+)-Ca2+ exchange modes, the ratios (R) of Na(+)-Ca2+/Ca(2+)-Ca2+ exchanges were estimated with saturating concentrations of ions at both sides of the membrane. The effect of extravesicular pH and voltage (potassium valinomycin) on the initial rates (t = 1 s) of Na(+)-Ca2+ and Ca(2+)-Ca2+ exchange were investigated by assuming that, under the conditions tested, the intravesicular pH (pH 7.4) is not affected. Na(+)- or Ca(2+)-preloaded sarcolemma vesicles were diluted rapidly in assay medium containing 45Ca and buffer (pH 5.0-10.9), and the reaction of 45Ca uptake was quenched by using a semi-rapid-mixing device. Under conditions in which [45Ca]o = [Ca]i = 250 microM, the pH-dependent curve of Ca(2+)-Ca2+ exchange shows a bell shape in the acidic range (pKa1 = 5.1 +/- 0.1 and pKa2 = 6.5 +/- 0.2) followed by activation of the exchange in the alkaline range (pKa3 = 10.0 +/- 0.2). With [45Ca]o = 250 microM and [Na]i = 160 mM, the Na(+)-Ca2+ exchange increases monotonically from pH 5.0 to 9.5 (pKa1 = 5.1 +/- 0.1, pKa2 = 7.2 +/- 0.2, and pKa3 = 9.1 +/- 0.2). At pH < 6.1, the ratio of Na(+)-Ca2+/Ca(2+)-Ca2+ exchange is close to unity (R approximately 1), while it increases to R = 3-4 in the range of pH 7.1-9.3.(ABSTRACT TRUNCATED AT 250 WORDS)

Phe-Met-Arg-Phe-NH2 (FMRFa)-related peptides inhibit Na(+)-Ca2+ exchange in cardiac sarcolemma vesicles.

The molluscan cardioexcitatory tetrapeptide FMRF-amide (Phe-Met-Arg-Phe-NH2) and related peptides inhibit Na(+)-Ca2+ exchange in calf cardiac sarcolemma vesicles. FMRFa itself has a low inhibitory potency (IC50 = 750 microM) which completely resides in its COOH-terminal RFa portion. The physiologically active analog FLRFa is 10-fold more potent (IC50 = 60 microM). Two other substitutions of the Met2 in FMRFa, by either Ile or Lys increase inhibitory potency 7- and 50-fold, respectively. The inhibitory potency increases 300-500-fold if the NH2-terminal Phe1 in FMRFa is substituted by either Val or His (IC50 = 1-2 microM). The inhibitory activity of WnLRFa (IC50 = 40 microM) is lost when either the NH2-terminal amino group is acylated or the NH2-terminal Trp1 is deleted. These data suggest that the COOH-terminal portion is essential for the basic low potency inhibition of Na(+)-Ca2+ exchange, whereas the NH2-terminal portion is important for the potentiation of the inhibitory activity. Although the IC50 values of various peptides range widely (10(-6)-10(-3) M), all of them induce a complete inhibition. The dose-response pattern of the peptide-induced inhibition is identical for the Na(+)-Ca2+ exchange and its partial reaction, the Ca(2+)-Ca2+ exchange. The inhibitory effect is reversible and affects both Nai(or Cai)-dependent 45Ca uptake and Nao-dependent 45Ca efflux, suggesting that the bidirectional movements of ions are altered. A mild pretreatment of vesicles with trypsin augments the Na(+)-Ca2+ exchange 1.5-fold but diminishes the inhibitory potency 3-4-fold, suggesting that the inhibition is mediated by an extravesicular membrane protein. The characteristics of the peptide-induced inhibition resemble the effect of opiates on Na(+)-Ca2+ exchange. FLRFa and dextrorphan (a non-opioid stereoisomer of an opiate agonist) are mutually exclusive inhibitors, suggesting that they may bind to the same site. This putative site lacks the pharmacological properties of opiate receptors and may be located either on the Na(+)-Ca2+ exchanger or at its vicinity. Endogenous analogs of FMRFa may regulate intracellular calcium via Na(+)-Ca2+ exchange.

The effect of opiate agonists and antagonists on Na(+)-Ca2+ exchange in cardiac sarcolemma vesicles.

Opiate agonists and antagonists inhibit Na(+)-Ca2+ exchange in the isolated cardiac sarcolemma vesicles. Non-opioid stereoisomers (dextrorphan, Mr 1542MS, WIN 44,441-3) display effects similar to their opioid isomers (levorphanol, Mr 1543MS, WIN 44,441-2) suggesting that inhibition is not mediated by opiate receptors. Naloxone (permeable) and methylnaloxone (impermeable) inhibit the Na(+)-Ca2+ exchange similarly, suggesting an extravesicular location of inhibitory site. The inhibitory potency of naloxone is pH-independent in the range of 7.4-9.1, suggesting that the charge-carrying properties of drug-protein interactions are not altered under the tested conditions. Opiates display similar dose-response relationships for Na(+)-Ca2+ exchange and its partial reaction, the Ca(2+)-Ca2+ exchange. The opiate-induced inhibition is complete and noncompetitive in regard to extravesicular calcium. These data suggest that opiates do not bind to the Ca(2+)-binding domain (A-site), but they may interest either with the Na(+)-binding site (B-site) or with a putative opiate-binding site, presumably located outside of the ion-binding vicinity. Further studies on structure-activity relationship might lead to the discovery of potent and more specific inhibitors of cardiac Na(+)-Ca2+ exchanger. A possible relevance of these findings to some non-opioid pharmacological effects of naloxone on the cardiac muscle is suggested.

Voltage-dependent modulation of ion binding and translocation in the cardiac Na(+)-Ca2+ exchange system.

The transport of Na+ and Ca2+ ions in the cardiac Na(+)-Ca2+ exchanger can be described as separate events (Khananshvili, D. (1990) Biochemistry 29, 2437-2442). Thus, the Na(+)-Na+ and Ca(2+)-Ca2+ exchange reactions reflect reversible partial reactions of the transport cycle. The effect of diffusion potentials (K(+)-valinomycin) on different modes of the Na(+)-Ca2+ exchanger (Na(+)-Ca2+, Ca(2+)-Ca2+, and Na(+)-Na+ exchanges) were tested in reconstituted proteoliposomes, obtained from the Triton X-100 extracts of the cardiac sarcolemmal membranes. The initial rates of the Nai-dependent 45Ca-uptake (t = 1 s) were measured in EGTA-entrapped proteoliposomes at different voltages. At the fixed values of voltage [45 Ca]o was varied from 4 to 122 microM, and [Na]i was saturating (150 mM). Upon varying delta psi from -94 to +91 mV, the Vmax values were increased from 9.5 +/- 0.5 to 26.5 +/- 1.5 nmol.mg-1.s-1 and the Km from 17.8 +/- 2.5 to 39.1 +/- 5.2 microM, while the Vmax/Km values ranged from only 0.53 +/- 0.08 to 0.73 +/- 0.17 nmol.mg-1.s-1.microM-1. The equilibrium Ca(2+)-Ca2+ exchange was voltage sensitive at very low [Ca]o = [Ca]i = 2 microM, while at saturating [Ca]o = [Ca]i = 200 microM the Ca(2+)-Ca2+ exchange became voltage-insensitive. The rates of the equilibrium Na(+)-Na+ exchange appears to be voltage insensitive at saturating [Na]o = [Na]i = 160 mM. Under the saturating ionic conditions, the rates of the Na(+)-Na+ exchange were at least 2-3-fold slower than the Ca(2+)-Ca2+ exchange. The following conclusions can be drawn. (a) The near constancy of the Vmax/Km for Na(+)-Ca2+ exchange at different voltages is compatible with the ping-pong model proposed previously. (b) The effects of voltage on Vmax of Na(+)-Ca2+ exchange are consistent with the existence of a single charge carrying transport step. (c) It is not yet possible to clearly assign this step to the Na+ or Ca2+ transport half of the cycle although it is more likely that 3Na(+)-transport is a charge carrying step. Thus, the unloaded ion-binding domain contains either -2 or -3 charges (presumably carboxyl groups). (d) The binding of Na+ and Ca2+ appears to be weakly voltage-sensitive. The Ca(2+)-binding site may form a small ion-well (less than 2-3 A).

Distinction between the two basic mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system.

In order to distinguish between the Ping-Pong and sequential mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system, the initial rates of the Nai-dependent 45Ca uptake (t = 1 s) were measured in reconstituted proteoliposomes, loaded with a Ca chelator. Under "zero-trans" conditions ([Na]o = [Ca]i = 0) at a fixed [Na]i = 10-160 mM with varying [45Ca]o = 2.5-122 microM for each [Na]i, the Km and Vmax values increased from 7.7 to 33.5 microM and from 2.3 to 9.0 nmol.mg-1.s-1, respectively. The Vmax/Km values show a +/- 2-10% deviation from the average value of 0.274 nmol.mg-1.s-1.microM-1 over the whole range of [Na]i. These deviations are within the standard error of Vmax (+/- 3-7%), Km (+/- 11-17%), and Vmax/Km (+/- 11-19%). This suggests that, under conditions in which Vmax and Km are [Na]i dependent and vary 4-5-fold, the Vmax/Km values are constant within the experimental error. In the presence of K(+)-valinomycin the Vmax/Km values are 0.85 +/- 0.17 and 1.08 +/- 0.18 nmol.mg-1.s-1.microM-1 at [Na]i = 20 and 160 mM, respectively, suggesting that under conditions of "short circuit" of the membrane potential the Vmax/Km values still exhibit the [Na]i independence. At a very low fixed [45Ca]o = 1.1 microM with varying [Na]i = 10-160 mM, the initial rates were found to be [Na]i independent. At a high fixed [45Ca]o = 92 microM the initial rates show a sigmoidal dependence on the [Na]i with Vmax = 13.8 nmol.mg-1.s-1, KmNa = 21 mM, and Hill coefficient nH = 1.5. The presented data support a Ping-Pong (consecutive) mechanism of cation transport in the Na(+)-Ca2+ exchanger.

The calcium ATPase of sarcoplasmic reticulum is inhibited by one Ca2+ ion.

Inhibition by calcium of the steady-state turnover of the calcium ATPase from sarcoplasmic reticulum of rabbit muscle follows a Hill slope of 0.8 +/- 0.2 (pH 7.0, 0.1 M KCl, varying [Mg2+] and 2 microM A23187 ionophore). It is concluded that dissociation of the two Ca2+ ions from E-P.Ca2 is sequential and that the inhibition arises from the binding of one Ca2+ to A-P.Ca1.

Two-step internalization of Ca2+ from a single E approximately P.Ca2 species by the Ca2+-ATPase.

Phosphorylation by ATP of E.*Ca2 (sarcoplasmic reticulum vesicles (SRV) with bound 45Ca2+) during 5-10 ms leads to the occlusion of 2 *Ca2+/EPtot [quench by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) alone] in both "empty" (10 microM free Ca2+in) or "loaded" SRV (20-40 mM free Ca2+in). The rate of Ca2+ "internalization" from the occluded E approximately P.*Ca2 was measured by using an ADP + EGTA quench; a *Ca2+ ion that is not removed by this quench is defined as internalized. In the presence of 20-40 mM unlabeled Ca2+ inside SRV, 1 *Ca2+/EPtot is internalized from 45Ca-labeled E approximately P.*Ca2 with a first-order rate constant of kl = 34 s-1. Empty SRV take up 2 *Ca2+/EPtot with the same initial rate, but the overall rate constant is kobsd = 17 s-1. The apparent rate constant (kb = 17 s-1) for internalization of the second *Ca2+ is inhibited by [Ca]in, with K0.5 approximately 1.3 mM and a Hill coefficient of n = 1.1. These data show that the two Ca2+ ions are internalized sequentially, presumably from separate sequential sites in the channel. [32P]EP.Ca2 obtained by rapid mixing of E.Ca2 with [gamma-32P]ATP and EGTA disappears in a biphasic time course with a lag corresponding to approximately 34 s-1, followed by EP* decay with a rate constant of approximately 17 s-1. This shows that both Ca2+ ions must be internalized before the enzyme changes its specificity for catalysis of phosphoryl transfer to water instead of to ADP. Increasing the concentration of ATP from 0.25 to 3 mM accelerates the rate of 45Ca2+ internalization from 34 to 69 s-1 for the first Ca2+ and from 17 to 34 s-1 for the second Ca2+. High [ATP] also accelerates both phases of [32P]EP.Ca2 disappearance by the same factor. The data are consistent with a single form of ADP-sensitive E approximately P.Ca2 that sequentially internalizes two ions. The intravesicular volume was estimated to be 2.0 microL/mg, so that one turnover of the enzyme gives 4 mM internal [Ca2+].