Ca2+, K+ redistributions and alpha-adrenergic activation of glycogenolysis in perfused rat livers

1. The alpha-adrenergic activation of glycogenolysis was investigated in isolated rat livers perfused in a non-recirculating system. Net uptake and/or release of Ca2+, K+ and H+ by the liver (measured by ion-selective electrodes) were correlated with the glycogenolytic effects of phenylephrine. Uptake and retention of 45Ca by the mitochondria of perfused livers were studied to obtain information on the role played by exchangeable mitochondrial calcium in alpha-adrenergic activation of glycogenolysis. 2. Between 1 and 5 min after starting the addition of phenylephrine a net release of Ca2+ was observed, this was paralleled by an uptake of K+. Production rates of glucose and lactate from endogenous glycogen started to increase at the same time. During the following minutes K+ was released. 2 mM EGTA and a high concentration of Mg2+ strongly diminished the ionic and metabolic responses to phenylephrine, 0.2 mM EGTA was less effective. 3. High concentrations of K+ prevented the metabolic response to phenylephrine but had no effect on the release of Ca2+ into the extracellular medium. Tetracaine activated glycogenolysis and suppressed all the effects of the alpha-adrenergic agonist. 4. Experiments with 45Ca provided no evidence for an alpha-adrenergic release of Ca2+ from the exchangeable mitochondrial pool. Incorporation of 45Ca into the mitochondria of perfused livers was enhanced by phenylephrine. 5. We propose that the alpha-adrenergic release of Ca2+ from a pool located close to the surface of the cell is capable of triggering the glycogenolytic response.     

Regulation of intracellular magnesium by Mg2+ efflux

Chicken erythrocytes were loaded with Mg2+ by incubation with the cation ionophore A 23187 in the presence of Mg2+. After removing A 23187 by intensive washing with serum albumin and reincubating the Mg2+-loaded cells, Mg2+ was transported out of the cells until the original Mg2+ content was achieved. The net Mg2+ efflux followed Michaelis-Menten-kinetics and was independent of extracellular and intracellular Ca2+ and calmodulin. The net Mg2+ efflux was not affected by adrenalin, isoproterenol, p-chloromercuribenzenesulfonate, ouabain and tetrodotoxin, but was inhibited by dicyclohexylcarbodiimide, KCN, iodoacetate, high extracellular concentrations of Mg2+, Mn2+ and when extracellular Na+ was substituted by choline or K+. The efflux of 1 Mg2+ was coupled with the uptake of 2 Na+. It is concluded that there exists an additional gating process at the inner cell surface becoming active only at increased concentrations of intracellular free Mg2+ regulating the exit of Mg2+ by the efflux system.     

Alpha 1-adrenergic stimulation causes Mg2+ release from perfused rat liver

The possibility that Mg2+ mobilization is stimulated in perfused liver by alpha 1-adrenergic agonists was studied by measuring Mg2+ release in response to 0.5 and 20 microM phenylephrine. During preperfusion exogenous Mg2+ was added to the medium to give 1.2 mM. 5 min before starting the addition of phenylephrine the infusion of exogenous Mg2+ was stopped. Mg2+ in the perfusate leaving the liver was measured by atomic absorption spectroscopy. Analysis of the Mg2+ decay curves with two exponential models indicated that phenylephrine caused dose-dependent Mg2+ release from perfused rat livers.     

Tetracaine stimulates insulin secretion through the mobilization of Ca2+ from thapsigargin- and IP3-insensitive Ca2+ reservoir in pancreatic beta-cells

The effect of tetracaine on 45Ca efflux, cytoplasmic Ca2+ concentration [Ca2+]i, and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the 45Ca efflux from isolated islets in a dose-dependentOFF efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]i in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 microM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 microM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in 45Ca efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.     

Effect of thyroid hormone on intracellular Ca2+ mobilization by noradrenaline and vasopressin in relation to glycogenolysis in rat liver

The relation between Ca2+ efflux, Ca2+ mobilization from mitochondria and glycogenolysis was studied in perfused euthyroid and hypothyroid rat livers stimulated by Ca2+-mobilizing hormones. Ca2+ efflux, induced by noradrenaline (1 microM) in the absence or presence of DL-propranolol (10 microM) from livers perfused with medium containing a low concentration of Ca2+ (approx. 24 microM), was decreased by more than 50% in hypothyroidism. This correlated with an equal decrease of the fractional mobilization of mitochondrial Ca2+, which could account for 65% of the difference between the net amounts of Ca2+ expelled from the euthyroid and hypothyroid livers. With vasopressin (10 nM) similar results were found, suggesting that hypothyroidism has a general effect on mobilization of internal Ca2+. In normal Ca2+ medium (1300 microM), however, the effect of vasopressin on net Ca2+ fluxes and phosphorylase activation was not impaired in hypothyroidism, indicating that Ca2+ mobilization from the mitochondria in this case plays a minor role in phosphorylase activation. The alpha 1-adrenergic responses of Ca2+ efflux, phosphorylase activation and glucose output, glucose-6-phosphatase activity and oxygen consumption in hypothyroid rat liver were completely restored by in vivo T3 injections (0.5 micrograms per 100 g body weight, daily during 3 days). Perfusion with T3 (100 pM) during 19 min did not influence hypothyroid rat liver oxygen consumption and alpha 1-receptor-mediated Ca2+ efflux. However, this in vitro T3 treatment showed a completely recovered alpha 1-adrenergic response of phosphorylase and a partly restored glucose-6-phosphatase activity and glucose output. The results indicate that thyroid hormones may control alpha 1-adrenergic stimulation of glycogenolysis by at least two mechanisms, i.e., a long-term action on Ca2+ mobilization, and a short-term action on separate stages of the glycogenolytic process.     

The relationship between extracellular K+ and Ca2+ on aminopyrine accumulation in rat parietal cells.

The effects of extracellular K+ in relation to extracellular Ca2+ on acid production were studied. Studies were performed in vitro using isolated cells from rat stomachs, and acid production was indirectly determined by 14C-aminopyrine (AP) accumulation. In the absence of K+ in the incubation medium histamine-stimulated AP accumulation ratios were significantly decreased independently in the presence or absence of extracellular Ca2+. Under basal conditions, in the absence of extracellular Ca2+, increasing concentrations of extracellular K+ enhanced AP accumulation ratios to significantly higher than those found in the presence of Ca2+. In histamine-, cAMP-, and carbachol-stimulated parietal cells, high K+ concentrations increased AP accumulation significantly less in Ca(2+)-free than in Ca(2+)-containing media. High K+ also induced significantly both an increase in cytosolic free Ca2+ concentration and 45Ca2+ uptake. The present results confirmed the importance of K+ in gastric acid production and suggested a role for Ca2+ as a modulator of mechanisms of parietal cell stimulation.     

alpha(1)-Adrenoceptor-induced Mg2+ extrusion from rat hepatocytes occurs via Na(+)-dependent transport mechanism

The stimulation of the alpha(1)-adrenergic receptor by phenylephrine results in a sizable extrusion of Mg2+ from liver cells. Phenylephrine-induced Mg2+ extrusion is almost completely abolished by the removal of extracellular Ca2+ or in the presence of SKF-96365, an inhibitor of capacitative Ca2+ entry. In contrast, Mg2+ extrusion is only partially inhibited by the Ca2+-channel blockers verapamil, nifedipine, or (+)BAY-K8644. Furthermore, Mg2+ extrusion is almost completely prevented by TMB-8 (a cell-permeant inhibitor of the inositol trisphosphate receptor), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (an intracellular Ca2+-chelating agent), or W-7 (a calmodulin inhibitor) Thapsigargin can mimic the effect of phenylephrine, and the coaddition of thapsigargin and phenylephrine does not result in an enlarged extrusion of Mg2+ from the hepatocytes. Regardless of the agonist used, Mg2+ extrusion is inhibited by >90% when hepatocytes are incubated in the presence of physiological Ca(2+) but in the absence of extracellular Na(+). Together, these data suggest that the stimulation of the hepatic alpha(1)-adrenergic receptor by phenylephrine results in an extrusion of Mg2+ through a Na(+)-dependent pathway and a Na(+)-independent pathway, both activated by changes in cellular Ca2+.     

Compartmental analysis of 45Ca2+ efflux in perfused rat liver: effects of hormonal stimulation.

The kinetics of calcium movements in the isolated perfused rat liver were examined using compartmental analysis of the efflux profiles of 45Ca2+ from 45Ca(2+)-equilibrated livers under a variety of calcium concentrations and hormonal treatments. From the 45Ca2+ efflux profiles, we determined that a three compartment model was appropriate to describe the movements of calcium in the liver on the time scale of the experiments. Hormonal treatment with the alpha-adrenergic agonist, phenylephrine, or the vasoactive peptide, vasopressin, during the efflux period lowered significantly the rate of transfer of Ca2+ between the internal compartments at all of the calcium concentrations employed. Also, phenylephrine treatment leads to increased transfer of Ca2+ into the liver from the perfusate. The temporal characteristics of the phenylephrine and vasopressin sensitive Ca2+ pools were examined by pulsing livers, loaded for variable periods of time with 45Ca2+, with the two hormones during the efflux of 45Ca2+ to measure the kinetics of Ca2+ exchange in the hormone-sensitive pools. Results from these experiments indicate that the rate of unstimulated Ca2+ efflux, k2, for the phenylephrine and vasopressin sensitive Ca2+ pools, modeled as a one compartment system, are the same, 0.074 and 0.078 min-1 for phenylephrine and vasopressin respectively, corresponding to half times for turnover of the pool(s) of 9.3 and 8.9 min, respectively.     

Detection of ligand-activated conductive Ca2+ channels in human B lymphocytes

It has been assumed that uptake of extracellular Ca2+ occurs through ligand-activated Ca2+ channels in anti-IgM stimulated human B cells. If so, then uptake should be associated with a depolarizing inward current. Instead, a hyperpolarization due to Ca2+-sensitive K+ conductance is observed. To demonstrate conductive Ca2+ channels in human B lymphocytes, we loaded the cells with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetate (BAPTA), an intracellular Ca2+ chelating agent. This increased the magnitude of the Ca2+ current and delayed the Ca2+-dependent K+ conductance. In BAPTA-loaded B cells suspended in Ca2+-free medium and activated with anti-IgM, reintroduction of Ca2+ resulted in a depolarization that was inhibited by high (microM) concentrations of verapamil and was observed when Ca2+ was replaced by Ba2+ but not Mg2+. These data demonstrate the opening of selective, Ca2+ conductive channels in human B cells following cross-linking of surface immunoglobulins.     

Effects of Ca2+ on the sodium pump observed in cardiac myocytes isolated from guinea pigs

It is presently unknown whether Ca2+ plays a role in the physiological control of Na+/K+-ATPase or sodium pump activity. Because the enzyme is exposed to markedly different intra- and extracellular Ca2+ concentrations, tissue homogenates or purified enzyme preparations may not provide pertinent information regarding this question. Therefore, the effects of Ca2+ on the sodium pump were examined with studies of [3H]ouabain binding and 86Rb+ uptake using viable myocytes isolated from guinea-pig heart and apparently maintaining ion gradients. In the presence of K+, a reduction of the extracellular Ca2+ increased specific [3H]ouabain binding observed at apparent binding equilibria: a half-maximal stimulation was observed when extracellular Ca2+ was lowered to about 50 microM. The change in [3H]ouabain binding was caused by a change in the number of binding sites accessible by ouabain instead of a change in their affinity for the glycoside. Ouabain-sensitive 86Rb+ uptake was increased by a reduction of extracellular Ca2+ concentration. Benzocaine in concentrations reported to reduce the rate of Na+ influx failed to influence the inhibitory effect of Ca2+ on glycoside binding. When [3H]ouabain binding was at equilibrium, the addition of Ca2+ decreased and that of EGTA increased the glycoside binding. Mn2+, which does not penetrate the cell membrane, had effects similar to Ca2+. In the absence of K+, cells lose their tolerance to Ca2+. Reducing Ca2+ concentration prevented the loss of rod-shaped cells but failed to affect specific [3H]ouabain binding observed in the absence of K+. These results indicate that a large change in extracellular Ca2+ directly affects the sodium pump in cardiac myocytes isolated from guinea pigs.     

Distinct effects of various amino acids on 45Ca2+ fluxes in rat pancreatic islets.

The effects of three types of amino acids on 45Ca2+ fluxes in rat pancreatic islets have been compared. Alanine, a non-insulinotropic neutral amino acid, transported with Na+, increased 45Ca2+ efflux in the presence or in the absence of extracellular Ca2+, but not in the absence of Na+. Its effects in Na+-solutions were practically abolished by 7 mM-glucose. Alanine slightly stimulated 45Ca2+ influx (5 min uptake) only when Na+ was present. Two insulinotropic cationic amino acids (arginine and lysine) triggered similar changes in 45Ca2+ efflux. They accelerated the efflux in the presence of Ca2+ and inhibited the efflux in a Ca2+-free medium, whether glucose was present or not. In an Na+-free Ca2+-medium, arginine and lysine markedly accelerated 45Ca2+ efflux, but this effect was suppressed by 7 mM-glucose. Arginine stimulated 45Ca2+ influx irrespective of the presence or absence of glucose and Na+. Leucine, a neutral insulinotropic amino acid well metabolized by islet cells, inhibited 45Ca2+ efflux from the islets in a Ca2+-free medium; this effect was potentiated by glutamine. In the presence of Ca2+ and Na+, leucine was ineffective alone, but triggered a marked increase in 45Ca2+ efflux when combined with glutamine. In an Na+-free Ca2+-medium, leucine accelerated 45Ca2+ efflux to the same extent with or without glutamine. Leucine also stimulated 45Ca2+ influx in the presence or in the absence of Na+, but its effects were potentiated by glutamine only in the presence of Na+. The results show that amino acids of various types cause distinct changes in 45Ca2+ fluxes in pancreatic islets. Certain of these changes involve an Na+-mediated mobilization of cellular Ca2+ from sequestering sites where glucose appears to exert an opposite effect.     

The Ca2+-mobilizing actions of vasopressin and angiotensin differ from those of the alpha-adrenergic agonist phenylephrine in the perfused rat liver.

A Ca2+-sensitive electrode was used to study net Ca2+-flux changes induced by the administration of phenylephrine, vasopressin and angiotensin to the perfused rat liver. The studies reveal that, although the Ca2+ responses induced by vasopressin and angiotensin are similar, they are quite different from the Ca2+ fluxes induced by phenylephrine. The administration of phenylephrine is accompanied by a stimulation of a net amount of Ca2+ efflux (140 nmol/g of liver). A re-uptake of a similar amount of Ca2+ occurs only after the hormone is removed. In contrast, the administration of vasopressin or angiotensin to livers perfused with 1.3 mM-Ca2+ induces the release of a relatively small amount of Ca2+ (approx. 40 nmol/g of liver) during the first 60 s. This is followed by a much larger amount of Ca2+ uptake (70-140 nmol/g of liver) after 1-2.5 min of hormone administration, and a slow efflux or loss of a similar amount of Ca2+ over a period of 6-8 min. At lower concentrations of perfusate Ca2+ (less than 600 microM) these hormones induce only a net efflux of the ion. These results suggest that at physiological concentrations of extracellular Ca2+ the mechanism by which alpha-adrenergic agonists mobilize cellular Ca2+ is different from that involving vasopressin and angiotensin. It seems that the hormones may have quite diverse effects on Ca2+ transport across the plasma membrane and perhaps organellar membranes in liver.     

Respiration-dependent uptake and extrusion of Mg2+ by isolated heart mitochondria

It has been known for some time that isolated heart mitochondria can both take up and extrude Mg2+ by respiration-dependent, uncoupler-sensitive processes. A re-examination of these reactions reveals that the respiration-dependent uptake of Mg2+ can be quite rapid and efficient and is apparently preceded by a passive binding to the inner membrane. The rate of Mg2+ uptake can exceed 30 ng ion/min/mg protein at an efficiency of about 1 ng ion Mg2+ accumulated per ng atom O2 consumed. Passive binding and respiration-dependent accumulation of Mg2+ are strongly inhibited by K+ and other monovalent cations and the uptake reaction is further decreased by the presence of ATP or ADP. Under conditions approaching those faced by mitochondria in situ (state 3 respiration in a KCl medium) the rate of Mg2+ uptake, as estimated from 28Mg2+ distribution, is no more than 0.25 ng ion/min/mg. When heart mitochondria are suspended in a Mg2+-free medium, a slow, respiration-dependent Mg2+ efflux is seen. This reaction is quite insensitive to external K+ and otherwise shows an inhibitor profile markedly different from that of the Mg2+ accumulation reaction. Neither the uptake nor the loss of Mg2+ is inhibited by ruthenium red or diltiazem. These reactions therefore appear unrelated to those involved in the uptake and release of Ca2+. It is concluded that heart mitochondria have separate pathways available for Mg2+ uptake and release.     

alpha-Adrenergic stimulation of trans-sarcolemma electron efflux in perfused rat heart. Possible regulation of Ca2+-channels by a sarcolemma redox system

The role of trans-sarcolemma membrane electron efflux in the alpha-adrenergic control of Ca2+ influx in perfused rat heart was examined. Electron efflux was measured by monitoring the rate of reduction of extracellular ferricyanide and compared with changes in contractility, as an indirect assessment of changes in cytoplasmic Ca2+ concentration. Methoxamine and phenylephrine each increased the rate of ferricyanide reduction from 80 to approx. 114 nmol/min per g wet wt. of heart, with half-maximal activation occurring at 10 microM for each agonist. Activation of the rate of ferricyanide reduction by both 10 microM methoxamine and 10 microM phenylephrine was blocked by the alpha-adrenergic antagonist, phenoxybenzamine, but not by the beta-antagonist, propranolol. Stimulation of the rate of ferricyanide reduction by the alpha-agonist coincided with the increase in contractility, each reaching maximum values at approx. 80 s. Removal of the alpha-agonists led to parallel decreases in contractility and the rate of reduction, each returning to pre-stimulation values in approx. 400 s. In addition, the relationship between Ca2+ and ferricyanide reduction was examined. Perfusion of the heart with medium containing 6 mM CaCl2 significantly increased contractility and the rate of ferricyanide reduction. Perfusion of the heart with low Ca2+ diminished contractility, did not affect the rate of ferricyanide reduction, but amplified the stimulatory effect of methoxamine on this rate. The increase in ferricyanide reduction by alpha-adrenergic agonists resulted from a change in the apparent Vmax, indicative of an increase in electron efflux sites in the plasma membrane. It is concluded that alpha-adrenergic control of electron efflux closely parallels changes in contractility and therefore changes in the cytoplasmic concentration of Ca2+. The data suggest that alpha-agonist-mediated changes in electron efflux may lead to Ca2+ influx.     

The Ca2+-dependent actions of the alpha-adrenergic agonist phenylephrine on hepatic glycogenolysis differ from those of vasopressin and angiotensin

The stimulation of hepatic glycogenolysis by the Ca2+-dependent hormones phenylephrine, vasopressin and angiotensin II was studied as a function of intracellular and extracellular Ca2+. In the isolated perfused rat liver the decline in glucose formation was monophasic ('half-life' approximately equal to 3 min) with vasopressin (1 nM) or angiotensin II (0.05 microM), but biphasic (half-life of 4.8 min and 17.6 min) in the presence of the alpha-agonist phenylephrine (0.01 mM), indicating either a different mode of mobilization or the mobilization of additional intracellular calcium stores. Under comparable conditions an elevated [Ca2+] level was maintained in the cytosol of hepatocytes for at least 10 min in the presence of phenylephrine, but not vasopressin. Titration experiments performed in the isolated perfused liver to restore cellular calcium revealed differences in the hormone-mediated uptake of Ca2+. The onset in glucose formation above that seen in the absence of exogenous calcium occurred at approximately 30 microM or 70-80 microM Ca2+ in the presence of phenylephrine or vasopressin respectively. The shape of the response curve was sigmoidal for vasopressin and angiotensin II, but showed a distinct plateau between 0.09 mM and 0.18 mM in the presence of phenylephrine. The plateau was also observed at phenylephrine concentrations as low as 0.5 microM. The formation of plateaus observed after treatment of the liver with A 23187, but not after EGTA, is taken as an indication that intracellular calcium stores are replenished. A participation of the mitochondrial compartment could be excluded by pretreatment of the liver with the uncoupler 2,4-dinitrophenol. Differences in the Ca2+ dependence of the glycogenolytic effects of these hormones were also revealed by kinetic analysis. It is concluded that phenylephrine differs from vasopressin and angiotensin II in that, in addition to a more common, non-mitochondrial pool, which is also responsive to the vasoactive peptides, the agonist mobilizes Ca2+ from a second, non-mitochondrial pool. The results are consistent with the proposal that Ca2+ transport across subcellular membranes may be subject to different hormonal control.     

Stimulation of glycogenolysis by adenine nucleotides in the perfused rat liver.

Infusion of adenine nucleotides and adenosine into perfused rat livers resulted in stimulation of hepatic glycogenolysis, transient increases in the effluent perfusate [3-hydroxybutyrate]/[acetoacetate] ratio, and increased portal vein pressure. In livers perfused with buffer containing 50 microM-Ca2+, transient efflux of Ca2+ was seen on stimulation of the liver with adenine nucleotides or adenosine. ADP was the most potent of the nucleotides, stimulating glucose output at concentrations as low as 0.15 microM, with half-maximal stimulation at approx. 1 microM, and ATP was slightly less potent, half-maximal stimulation requiring 4 microM-ATP. AMP and adenosine were much less effective, doses giving half-maximal stimulation being 40 and 20 microM respectively. Non-hydrolysed ATP analogues were much less effective than ATP in promoting changes in hepatic metabolism. ITP, GTP and GDP caused similar changes in hepatic metabolism to ATP, but were 10-20 times less potent than ATP. In livers perfused at low (7 microM) Ca2+, infusion of phenylephrine before ATP desensitized hepatic responses to ATP. Repeated infusions of ATP in such low-Ca2+-perfused livers caused homologous desensitization of ATP responses, and also desensitized subsequent Ca2+-dependent responses to phenylephrine. A short infusion of Ca2+ (1.25 mM) after phenylephrine infusion restored subsequent responses to ATP, indicating that, during perfusion with buffer containing 7 microM-Ca2+, ATP and phenylephrine deplete the same pool of intracellular Ca2+, which can be rapidly replenished in the presence of extracellular Ca2+. Measurement of cyclic AMP in freeze-clamped liver tissue demonstrated that adenosine (150 microM) significantly increased hepatic cyclic AMP, whereas ATP (15 microM) was without effect. It is concluded that ATP and ADP stimulate hepatic glycogenolysis via P2-purinergic receptors, through a Ca2+-dependent mechanism similar to that in alpha-adrenergic stimulation of hepatic tissue. However, adenosine stimulates glycogenolysis via P1-purinoreceptors and/or uptake into the cell, at least partially through a mechanism involving increase in cyclic AMP. Further, the hepatic response to adenine nucleotides may be significant in regulating hepatic glucose output in physiological and pathophysiological states.     

Effects of low extracellular sodium on cytosolic ionized calcium. Na+-Ca2+ exchange as a major calcium influx pathway in kidney cells

The effects of extracellular Na+ (Na+o) on cytosolic ionized calcium (Ca2+i) and on calcium and sodium fluxes were measured in monkey kidney cells (LLC-MK2). Ca2+i was measured with aequorin and the ion fluxes with 45Ca and 22Na. Na+-free media rapidly increased Ca2+i from 60 to a maximum of about 700 nM in 2-3 min. After the peak, Ca2+i declined and reached a plateau of about twice the resting Ca2+i. The peak Ca2+i was inversely proportional to Na+o and directly proportional to the extracellular calcium concentration (Ca2+o). On the other hand, a pH of 6.8 reduced and Ca2+o substitution with Sr2+ completely blocked the Ca2+i response to low Na+o. A Na+-free medium stimulated calcium efflux from the cells 4-5-fold, a response which was abolished in the absence of extracellular Ca2+. Na+-free media also stimulated calcium influx and sodium efflux. The cell calcium content, however, was not increased. These results indicate that removal of extracellular Na+ increases Ca2+i by stimulating calcium influx and not by inhibiting calcium efflux; the increased calcium influx takes place on the Na+-Ca2+ antiporter operating in the reverse mode in exchange for sodium efflux. The increased calcium efflux occurs as a consequence of the rise in Ca2+i and presumably takes place on the (Ca2+-Mg2+) ATPase-dependent calcium pump.     

Intracellular and extracellular concentrations of Na+ modulate Mg2+ transport in rat ventricular myocytes

Apparent free cytoplasmic concentrations of Mg2+ ([Mg2+]i) and Na+ ([Na+]i) were estimated in rat ventricular myocytes using fluorescent indicators, furaptra (mag-fura-2) for Mg2+ and sodium-binding benzofuran isophthalate for Na+, at 25 degrees C in Ca2+-free conditions. Analysis included corrections for the influence of Na+ on furaptra fluorescence found in vitro and in vivo. The myocytes were loaded with Mg2+ in a solution containing 24 mM Mg2+ either in the presence of 106 mM Na+ plus 1 mM ouabain (Na+ loading) or in the presence of only 1.6 mM Na+ to deplete the cells of Na+ (Na+ depletion). The initial rate of decrease in [Mg2+]i from the Mg2+-loaded cells was estimated in the presence of 140 mM Na+ and 1 mM Mg2+ as an index of the rate of extracellular Na+-dependent Mg2+ efflux. Average [Na+]i, when estimated from sodium-binding benzofuran isophthalate fluorescence in separate experiments, increased from 12 to 31 mM and 47 mM after Na+ loading for 1 and 3 h, respectively, and decreased to approximately 0 mM after 3 h of Na+ depletion. The intracellular Na+ loading significantly reduced the initial rate of decrease in [Mg2+]i, on average, by 40% at 1 h and by 64% at 3 h, suggesting that the Mg2+ efflux was inhibited by intracellular Na+ with 50% inhibition at approximately 40 mM. A reduction of the rate of Mg2+ efflux was also observed when Na+ was introduced into the cells through the amphotericin B-perforated cell membrane (perforated patch-clamp technique) via a patch pipette that contained 130 mM Na+. When the cells were heavily loaded with Na+ with ouabain in combination with intracellular perfusion from the patch pipette containing 130 mM Na+, removal of extracellular Na+ caused an increase in [Mg2+]i, albeit at a very limited rate, which could be interpreted as reversal of the Mg2+ transport, i.e., Mg2+ influx driven by reversed Na+ gradient. Extracellular Na+ dependence of the rate of Mg2+ efflux revealed that the Mg2+ efflux was activated by extracellular Na+ with half-maximal activation at 55 mM. These results contribute to a quantitative characterization of the Na+-Mg2+ exchange in cardiac myocytes.     

Magnesium permeability of sarcoplasmic reticulum. Mg2+ is not countertransported during ATP-dependent Ca2+ uptake by sarcoplasmic reticulum

Magnesium transport across sarcoplasmic reticulum (SR) vesicles was investigated in reaction mixtures of various composition using antipyrylazo III or arsenazo I to monitor extravesicular free Mg2+. The half-time of passive Mg2+ efflux from Mg2+-loaded SR was 100 s in 100 mM KCl, 150 S in 100 mM K gluconate, and 370 S in either 100 mM Tris methanesulfonate or 200 mM sucrose solutions. The concentration and time course of Mg2+ released into the medium was also measured during ATP-dependent Ca2+ uptake by SR. In reaction mixtures containing up to 3 mM Mg2+, small changes in free magnesium of 10 microM or less were accurately detected without interference from changes in free Ca2+ of up to 100 microM. Three experimental protocols were used to determine whether the increase of free [Mg2+] in the medium after an addition of ATP was due to Mg2+ dissociated from ATP following ATP hydrolysis or to Mg2+ translocation from inside to outside of the vesicles. 1) In the presence of ATP-regenerating systems which maintained constant ATP to ADP ratios and normal rates of active Ca2+ uptake, the increase of Mg2+ in the medium was negligible. 2) Mg2+ released during ATP-dependent Ca2+ uptake by SR was similar to that observed during ATP hydrolysis catalyzed by apyrase, in the absence of SR. 3) In SR lysed with Triton X-100 such that Ca2+ transport was uncoupled from ATPase activity, the rate and amount of Mg2+ release was greater than that observed during ATP-dependent Ca2+ uptake by intact vesicles. Taken together, the results indicate that passive fluxes of Mg2+ across SR membranes are 10 times faster than those of Ca2+ and that Mg2+ is not counter-transported during active Ca2+ accumulation by SR even in reaction mixtures containing minimal concentrations of membrane permeable ions that could be rapidly exchanged or cotransported with Ca2+ (e.g. K+ or Cl-).     

Assay of muscarinic acetylcholine receptor function in cultured cardiac cells by stimulation of 86Rb+ efflux

An assay for the increase in potassium permeability mediated by muscarinic acetylcholine receptors (mAChR) in cultured cardiac cells is described, using the K+ ion substitute 86Rb+ as the tracer ion. Cardiac cells accumulate 86Rb+ from the extracellular medium in a Na+/K+ ATPase-dependent manner. Subsequent efflux of 86Rb+ in the absence and presence of muscarinic agonists follows kinetics similar to those previously reported for 42K+. The mAChR agonist carbamylcholine (carbachol) stimulated 86Rb+ efflux with an EC50 of 50 nM. The half-time for efflux is reduced by greater than 40% at maximally effective concentrations of agonist. Stimulation of 86Rb+ efflux by carbachol is blocked by the mAChR antagonist atropine with an IC50 of 15 nM. The stimulation of 86Rb+ efflux by carbachol is not affected by the presence of the Na+/K+ ATPase inhibitor ouabain. This assay provides a method for quantitating the mAChR-mediated increase in K+ permeability in cardiac cells without the use of 42K+.