Effect of reserpine pretreatment on calcium transport and Ca++-Mg++ adenosine triphosphatase activity of guinea-pig cardiac microsomes

A previous report from this laboratory showed that reserpine pretreatment, in appropriate doses and under restricted conditions, increased the inotropic responsiveness of guinea-pig hearts to calcium. The enhanced responsiveness was characterized by a selective increase in the rate of ventricular relaxation (-dP/dt). We therefore hypothesized that reserpine might alter calcium uptake or (Ca++-Mg++) adenosine triphosphatase (ATPase) activity of guinea-pig ventricular sarcoplasmic reticulum (SR). Pretreatment of guinea pigs with reserpine (2.5 mg/kg/day, 2 days) significantly elevated ATP-dependent, Tris oxalate-facilitated SR Ca++ uptake and increased the calcium-sensitive component of the SR (Ca++) ATPase activity. These changes appeared to be functionally related to a reserpine-induced potentiation of ventricular relaxation rate, as estimated by the relationship between negative and positive left ventricular dP/dt of isolated working guinea-pig hearts. An alternative dose of reserpine (5 mg/kg, -24 hr), which had been demonstrated to produce an equivalent degree of catecholamine depletion, had no effect on either the inotropic responsiveness to calcium or on the SR calcium uptake or ATPase activities. The exact mechanism for these reserpine-induced alterations in calcium homeostasis remains to be elucidated.     

Stimulation of cardiac slow Ca++ current by high concentration of cimetidine

Electrophysiological studies on the cardiac effects of a H2-antagonist, cimetidine, were examined in three kinds of preparations: guinea-pig papillary muscles, rat left atria and perfused chick hearts. Cimetidine (10(-5) to 10(-4) M) depressed or abolished the slow action potentials (APs) induced by histamine (10(-5) to 10(-4) M) in hearts whose fast Na+ channels had been inactivated by 25 mM K+. Higher concentrations of cimetidine (10(-3) to 5 X 10(-3) M) increased myocardial cyclic AMP level and allowed the generation of slow APs in such inexcitable tissues. These cimetidine-induced slow APs were not prevented by propranolol (10(-6) to 10(-5) M) or pretreatment with 6-hydroxydopamine (50 mg/kg). These results suggest that cimetidine, in doses higher than that required to block cardiac H2-receptors, may have a cardio-stimulating action mediated through increase of inward Ca++ current.     

Effects of 2-nicotinamidoethyl nitrate on agonist-sensitive Ca++ release and Ca++ entry in rabbit aorta

The effects of 2-nicotinamidoethyl nitrate (SG-75) on norepinephrine (NE)- and KCI-induced responses in rabbit aorta were quantitated, correlated with 45Ca studies and compared with the effects of nifedipine (NIF) on similar parameters. NE- and KCI-induced dose-response relationships were differentially depressed by SG-75 (NE much greater than KCI) and NIF (KCI much greater than NE). Responses to KCI were relatively insensitive to prior SG-75, yet moderately relaxed by subsequent SG-75. Conversely, NIF markedly inhibited and completely relaxed similar responses. Responses to NE were relaxed and inhibited with SG-75, but unaffected by NIF. Responses to NE in La or O-Ca++ + ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid plus D600 (with and without KCI) solutions were phasic, reduced by SG-75 and insensitive to NIF. NE-dependent, Ca++-induced responses in a O-Ca++ + ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid plus D600 solution (with and without KCI) were attenuated by SG-75. Equilibrated (60 min) La -resistant (residual), high apparent affinity Ca++ binding was increased 26% with SG-75 and decreased 34% with NIF, yet neither altered the rate of exchange (10 min). Rate of exchange at low apparent affinity, residual sites was increased 21% by SG-75 without altering equilibrated values, whereas NIF reduced equilibrated values 11%, without affecting rate. NE reduced, SG-75 + NE augmented and NIF + NE decreased, in an additive fashion, high apparent affinity, residual bound Ca++. Residual Ca++ binding at low apparent affinity sites was increased with 160 mM substituted KCI (380%). This increase was only partially inhibited with SG-75, and eliminated by NIF. Net Ca++ efflux was persistently slowed by SG-75 and unaltered by NIF. The primary effects of SG-75 appear to be depression of Ca++ release and inhibition of receptor-operated (potential-independent) Ca++ entry, with limited attenuation of voltage-dependent Ca++ entry. NIF primarily inhibits voltage-dependent Ca++ entry.     

Comparative interactions of organic Ca++ channel antagonists with myocardial Ca++ and K+ channels

Dose-dependent inhibition by three organic calcium channel antagonists, D-600, nisoldipine and diltiazem, of the inward calcium current (iCa) and the delayed, outward potassium current (iK) in single frog atrial cells was examined using a voltage clamp technique. At holding potentials of -60 mV, low concentrations of these antagonists produced considerable inhibition of iCa without significant alterations in iK, suggesting that iK in single frog atrial cells is not a calcium-activated K+ conductance. Higher concentrations of each of these antagonists, however, inhibited iK. The estimated Kd values for inhibition of iCa and iK, respectively, were 3.7 X 10(-7) M and 8.2 X 10(-4) M for D-600, 1.6 X 10(-8) M and 1.6 X 10(-5) M for nisoldipine and 4.4 X 10(-6) M and 3.3 X 10(-4) M for diltiazem. Under these experimental conditions, D-600 and nisoldipine interact more selectively with myocardial Ca++ channels than K+ channels compared to diltiazem, which is less selective. In addition, the inhibition of iK by each of these antagonists was found to exhibit an apparent voltage dependence; block was enhanced at more negative membrane potentials and relieved at more positive membrane potentials. This voltage-dependent block of iK is, therefore, opposite to the voltage-dependent inhibition of iCa produced by these compounds, where block of iCa is accentuated at positive membrane potentials.     

Influence of Mg++ on the effect of diltiazem to increase dihydropyridine binding to receptors on Ca++-channels in chick cardiac and skeletal muscle membranes

The influence of Mg++ on the effect of diltiazem to increase ligand binding to a high-affinity state of dihydropyridine receptors on voltage-dependent Ca-channels has been studied in chick cardiac and skeletal muscle membranes at 25 degrees C. The high-affinity binding of the Ca-channel inhibitors (+)-[3H]PN 200-110 and [3H]nitrendipine to cardiac membranes was depressed markedly by EDTA and restored fully by the addition of free Mg++ (Ptasienski et al., Biochem. Biophys. Res. Commun. 129: 910-917, 1985). Similar results have now been obtained with skeletal muscle membranes. In the presence of EDTA alone, diltiazem, which binds to another receptor on the Ca-channel, increased the high-affinity binding of both ligands to cardiac and skeletal muscle membranes. However, in the presence of added Mg++, diltiazem had smaller or no effects on the binding of these dihydropyridines. Analyses of the data indicated that both Mg++ and diltiazem could increase the maximum binding (Bmax) for these ligands, but the effect of diltiazem was smaller than, and not additive to, that of Mg++. Specific binding of the Ca-channel activator [3H]Bay k 8644 was only observed in assays containing Mg++ in excess of EDTA. The Bmax for [3H]Bay k 8644 in skeletal muscle membranes was less than that for [3H]PN 200-110 and [3H]nitrendipine, whereas with cardiac membranes equal Bmax values were obtained for all ligands. Diltiazem increased the Bmax for [3H]Bay k 8644 in skeletal muscle, but not in cardiac membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ouabain on the concentration of free cytosolic Ca++ and on contractility in cultured rat cardiac myocytes

Effects of ouabain on [Ca++]i and on contractility was measured in quin2 and fura2 loaded cultured neonatal rat cardiac myocytes. Addition of ouabain (5 x 10(-8) to 5 x 10(-6) M) to cultured myocytes exposed to balanced buffered salt solution (BSS) caused a transient increase in [Ca++]i, followed by slow oscillations for about 10 min, and by an elevated steady state level of [Ca++]i thereafter. Concentrations of ouabain between 10(-7) and 5 x 10(-7) M caused an increase in the amplitude of systolic motion (ASM) whereas concentrations above 10(-6) caused a decrease in the ASM, an increase in the beating frequency and an upward shift of the base line, indicating impaired relaxation. When ouabain was added to cardiac myocytes exposed to Ca++-free BSS the increase in [Ca++]i was not observed, but only a transient decrease. To investigate the effect of [K+]o on the ouabain-induced changes in [Ca++]i, ouabain was added to cells exposed to BSS containing low K+ concentration (1 mM instead of 5 mM in balanced BSS). In this medium the increase in ASM by ouabain was similar to that in balanced BSS. Addition of ouabain caused a transient decrease in [Ca++]i. There was no initial increase in [Ca++]i and the steady state level of [Ca++]i was not elevated as compared with the same cells before the addition of ouabain. Similar results were observed in cells loaded with quin2 or with fura2. In view of these results the mechanism of action of ouabain on cardiac myocytes is discussed.     

Effect of morphine on synaptosomal Ca++ uptake

The effect of morphine on the uptake of 45Ca++ was studied in synaptosomes from mouse brain using two procedures, centrifugation and filtration. The addition of morphine (1.7 x 10(-7) or 3.4 x 10(-7) M) reduced 45CA++ uptake by either technique, although the basal 45Ca++ uptake by the filtration method was approximately 7-fold higher than that by the centrifugation procedure. Similar effects were obtained after acute morphine treatment with 10 mg/kg s.c. Previous naloxone in vitro treatment (1.9 x 10(-8) M) or in vivo administration (2 mg/kg s.c.) reversed the morphine inhibition of the 45Ca++ uptake. On the other hand, after the animal was rendered tolerant and dependent by morphine pellet implantation, an enhancement of the synaptosomal 45Ca++ uptake was observed. It is concluded that changes in Ca++ fluxes in synaptosomes observed after acute and chronic morphine treatment may be involved with morphine pharmacological action related with analgesia, tolerance and physical dependence.