Sodium-dependent calcium release from vascular smooth muscle mitochondria.

Interest in mitochondrial calcium (Ca2+) uptake and release waned as it became apparent that sarcoplasmic reticulum calcium stores dominate the control of cytoplasmic calcium concentration. Our recent demonstration of a very large rise in vascular smooth muscle (VSM) cytoplasmic sodium (Na+) concentration after inhibition of the sodium, potassium-ATPase (sodium pump) led us to several questions. Do VSM mitochondria show Na(+)-dependent Ca2+ release? Are the documented changes in cytoplasmic Na+ concentration sufficient to cause Ca2+ release? Do features of the cardiac mitochondrial exchange system, including differential sensitivity to a number of calcium antagonists and cation specificity, apply to VSM? We isolated mitochondria from bovine aorta and mesenteric arteries and employed arsenazo III as the Ca2+ indicator. Mitochondria from arterial vessels accumulated added calcium (up to 50 nmol Ca2+/mg protein) and released Ca2+ on exposure to Na+. This concentration-dependent relationship was linear from 0 to 10 mM of Na+, and it plateaued between 20 mM and 40 mM of Na+. VSM mitochondria exposed to 20 mM Na+ released 118 +/- 25 nmol Ca2+ per mg mitochondrial protein in 20 min, when a new equilibrium was reached. Lithium (Li+), in contrast to Na+, produced much smaller amounts of Ca2+ release from the VSM mitochondria. Na+-dependent Ca2+ release was antagonized in a concentration-dependent manner by diltiazem (0-320 microM) with a Ki of 10.2 microM. Nifedipine had a lesser effect, and verapamil produced almost no inhibition. VSM mitochondria responses resemble those from heart mitochondria in that Na+-dependent Ca2+ release is present with a similar range of sensitivity to Na+ and a similar pattern of influence of diltiazem, nifedipine and verapamil. However, the influence of Li+ on Ca2+ release was much smaller and the amount of the Ca2+ released was much greater for VSM mitochondria compared with that reported for heart mitochondria. The large amount of Ca2+ released and the range of Na+ concentration that provoked Ca2+ release being within the physiologically achievable range raise the interesting possibility that these mechanisms may modify intramitochondrial cytosolic Ca2+ concentration, and hence could potentially contribute to the contractile response that follows inhibition of the sodium pump.     

Sodium-lithium exchange and sodium-proton exchange are mediated by the same transport system in sarcolemmal vesicles from bovine superior mesenteric artery

Several laboratories have reported that Na+-Li+ countertransport activities are increased in red blood cells from patients with essential hypertension. It has been proposed that the activity of this red blood cell transport system might reflect the activity of a similar system in vascular smooth muscle. We previously demonstrated Na+-Li+ exchange in sarcolemmal vesicles from canine artery and proposed that this transport function might be mediated by the Na+-H+ exchanger. In the present studies, however, we were unable to demonstrate Na+-Li+ countertransport in canine red blood cells. Since bovine red blood cells have a vigorous Na+-Li+ exchanger and we previously demonstrated Na+-H+ exchange in sarcolemmal vesicles from bovine artery, we wished to determine whether bovine sarcolemmal vesicles mediate Na+-Li+ exchange and whether this transport function is mediated via the Na+-H+ exchanger. We found that an outwardly directed proton or Li+ gradient stimulated 22Na+ uptake in sarcolemmal vesicles from bovine superior mesenteric artery. Li+ gradient-stimulated Na+ uptake was not due to electrical coupling between the two ions, was not affected by a change in membrane potential, and could not be explained by the parallel operation of Li+-H+ and Na+-H+ exchange. External Li+ inhibited proton gradient-stimulated Na+ uptake, and external protons inhibited Li+ gradient-stimulated Na+ uptake. Na+ efflux from vesicles was stimulated by inwardly directed gradients for Li+ or protons, and these effects were not additive. Proton efflux from vesicles was stimulated by inwardly directed gradients for Na+ or Li+, and these effects were not additive. Finally, Na+-H+ exchange and Na+-Li+ exchange in sarcolemmal vesicles were inhibited by 5-(N-ethyl-N-isopropyl)amiloride in an identical dose-dependent manner. In conclusion, Na+-Li+ countertransport could not be demonstrated in canine red blood cells, but as is the case with bovine red blood cells, sarcolemmal vesicles from bovine artery mediate Na+-Li+ countertransport. This transport function and sarcolemmal Na+-H+ exchange are mediated via a single 5-(N-ethyl-N-isopropyl)amiloride-sensitive cation exchanger with affinity for Na+, Li+, and protons. The cow, as opposed to the dog, may be a good animal model to test whether the activity of red blood cell Na+-Li+ countertransport is predictive of the activity of Na+-Li+ (and Na+-H+) exchange in vascular smooth muscle.     

Role of the Na+-Ca2+ exchange in the calcium paradox in frog auricular trabeculae

After a Ca2+-free ouabain perfusion of 8-10 min duration, reperfusion of isolated stimulated frog auricular trabeculae with Ca2+ ouabain containing medium resulted in a large and transient contracture. The contracture was weaker in a quiescent preparation or in the absence of ouabain. In sodium-free (Li+ substitute) ouabain containing medium, the amplitude of the contracture was largely decreased while it disappeared completely in Na+-free medium without ouabain. Moreover this contracture was suppressed by 15 mM Mn2+. Although the approach was only indirect, these results suggest that the contracture is due to an entry of Ca2+ ions through the Na+-Ca2+ exchange mechanism and that this could be the unique route of calcium entry during calcium repletion.     

Influence of race, sex, and blood pressure on erythrocyte sodium transport in humans

Sodium transport of erythrocytes from normotensive and essential hypertensive subjects was evaluated by determining ouabain-sensitive and ouabain-insensitive sodium efflux rates, Na+-Li+ countertransport rates, Li+-K+ cotransport rate constants (lithium replacing sodium), intracellular sodium concentrations, and the number of Na+,K+-adenosine triphosphatase (ATPase) sites per erythrocyte. Subjects included men and women, blacks and whites. Hypertensive subjects had significantly higher sodium transport than did normotensive subjects for ouabain-sensitive sodium efflux (p less than 0.025) and Na+-Li+ countertransport (p less than 0.001). Sexual differences were noted for ouabain-sensitive (p less than 0.001) and ouabain-insensitive (p less than 0.001) sodium efflux, for intracellular sodium concentration (p less than 0.025), and for the Li+-K+ cotransport rate constant (p less than 0.005), all with higher values for men than for women. Racial differences were noted for ouabain-insensitive sodium efflux (p less than 0.005), Na+-Li+ countertransport (p less than 0.001), and the Li+-K+ cotransport rate constant (p less than 0.001); values were higher in whites than blacks for all three measurements. The number of [3H]ouabain binding sites was lower for blacks (p less than 0.001) and the intracellular sodium concentration was higher for blacks (p less than 0.001). Among all subjects, significant (p less than 0.001) correlations were found between intracellular sodium concentration and the number of Na+,K+-ATPase sites per erythrocyte (r = -0.78) and between the ouabain-sensitive sodium efflux per site and intracellular sodium concentration (r = 0.85, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell membrane transport of magnesium

In order to maintain low intracellular Mg(2+) concentration ( [Mg(2+)] (i)), Mg(2+) has to be extruded from cell interior by active transport. In this article, properties of the active Mg(2+) transport in cardiac myocytes are reviewed. After [Mg(2+)] (i) was increased in the solution containing high Mg(2+) concentration, the reduction of extracellular Mg(2+) concentration ( [Mg(2+)] (o)) down to 1 mM caused a rapid decrease in [Mg(2+)] (i) only in the presence of extracellular Na(+);extracellular Na(+) stimulates the Mg(2+) efflux in a concentration dependent fashion with half maximal activation at 53 mM. When the rate of Mg(2+) efflux was compared under different levels of intracellular Na(+) concentration ( [Na(+)] (i)), intracellular Na(+) loading by ouabain decreased the rate of Mg(2+) efflux with 50% inhibition at - 40 mM [Na(+)] (i). In the experiments where the myocytes were voltage clamped at - 80 mV using the perforated patch-clamp technique with amphotericin B, the increase in pipette Na(+) concentration from 0 mM to 130 mM significantly decreased the rate of Mg(2+) efflux. The rate of Mg(2+) efflux was greater at the higher initial levels of [Mg(2 + )] (o), and was nearly zero at the basal levels;the efflux was half activated at 1.9 mM [Mg(2+)] (i). The Mg(2+) efflux was significantly slower at higher [Mg(2+)] (o) (50% inhibition at 10 mM). These results are consistent with the Mg(2+) efflux activity driven by the [Na(+)] gradient across cell membrane, or the Na(+)-Mg(2+) exchange.     

Characterization of H+ efflux pathways in rat hepatocytes

A pH-stat method was used to characterize H+ efflux pathways in hepatocytes in order to determine if Na+/H+ and Ca++/H+ exchange are involved in H+ efflux from hepatocytes under basal conditions and if cyclic AMP analogs affect Na+/H+ exchange. Total H+ efflux of freshly prepared hepatocytes ranged from 10 to 15 nmoles per min per mg protein. A part of total H+ efflux (35 to 50%) was dependent on extracellular Na+. This Na+-dependent H+ efflux was (i) inhibited by amiloride with a half-maximal effect at 0.3 mM, (ii) inhibited by ouabain, (iii) dependent on extracellular pH and (iv) characterized by a Km of 15 +/- 3 mM Na+ and a Vmax of 9 +/- 0.07 nmoles per min per mg protein. Amiloride, ouabain and replacement of Na+ by choline also decreased intracellular pH determined from equilibrium distribution of dimethyloxazolidinedione. Li+ could partially substitute for Na+ in Na+-dependent H+ efflux and in maintaining intracellular pH. Efflux of CO2 and lactic acid from hepatocytes represented 80% of Na+-independent H+ efflux. Efflux of H+ in the presence and absence of Na+ was not significantly altered by extracellular Ca++ (less than 10 microM and 1.0 mM). Thus, Ca++/H+ exchange is unlikely to contribute significantly to total H+ efflux from hepatocytes. Cyclic AMP analogs, dibutyryl cyclic AMP and 8-bromo cyclic AMP, inhibited amiloride-sensitive Na+-dependent H+ efflux, and dibutyryl cyclic AMP decreased intracellular pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+-Ca2+ exchange in cultured vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) contract as intracellular free calcium ([Ca2+]i) rises. While Na+-Ca2+ exchange has been proposed to contribute to transmembrane Ca2+ flux, its role in cultured VSMC is unknown. Accordingly, we have investigated the role of Na+-Ca2+ exchange in unidirectional and net transmembrane Ca2+ fluxes in cultured rat aortic VSMC under basal conditions and following agonist-mediated stimulation. Transmembrane Ca2+ uptake was significantly increased in response to a low external Na+ concentration ([Na+]o) compared with 140 mM [Na+]o. Na+-dependent Ca2+ uptake in response to low [Na+]o was further increased by intracellular Na+ loading by preincubation of the VSMC with 1 mM ouabain. Under steady-state conditions, Ca2+ content varied inversely with [Na+]o, increasing from 1.0 nmol Ca2+/mg protein at 140 mM [Na+]o to 4.0 nmol Ca2+/mg protein at 20 mM [Na+]o. Increasing [K+]o to 55 mM also enhanced Na+-dependent Ca2+ influx. Augmentation of Ca2+ uptake with K+ depolarization was not significantly inhibited by the calcium channel antagonist verapamil. Transmembrane Ca2+ efflux was increased in response to 130 mM [Na+]o compared with zero [Na+]o (iso-osmotic substitution with choline+), and was further stimulated by the vasoconstrictor angiotensin II, which is known to elevate [Ca2+]i. These changes in [Ca2+]i were studied directly using fura-2 fluorescence measurements. Elevated [Ca2+]i levels returned to baseline more rapidly in the presence of normal (130 mM) [Na+]o compared with zero [Na+]o (iso-osmotic substitution with choline+). These findings suggest that a bidirectional Na+-Ca2+ exchange mechanism is present in cultured rat aortic VSMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of high extracellular Ca2+ and Mg2+ concentrations on the levels of inositol 1,3,4,5-tetrakisphosphate in bovine parathyroid cells

We examined the effects of calcium (Ca2+), magnesium (Mg2+), and fluoride ion (F-) on inositol phosphate accumulation in bovine parathyroid cells prelabeled with [3H] inositol to determine whether these agents might modulate cytosolic Ca2+ through accumulation of intracellular inositol 1,3,4,5-tetrakisphosphate (IP4), which has been postulated to be a mediator of the influx of extracellular Ca2+. Both Ca2+ and Mg2+ produced dose-dependent increases in IP4 in the presence of Li+, with maximal 19- and 4-fold increases with 5.0 mM Ca2+ and 20 mM Mg2+, respectively. A 50% rise in IP4 was evident within 1 min in response to 5.0 mM Ca2+. In the absence of Li+, a 2-fold increase in IP4 was seen with 5.0 mM Ca2+ only after 15 min. Fluoride ion generated a dose-dependent increase in IP4, with a 48% rise at 10 mM F-, presumably by activating phospholipase-C through a guanine nucleotide regulatory (G) protein-dependent process. We conclude that inositol 1,4,5-trisphosphate generated in response to high extracellular Ca2+ and Mg2+ concentrations can be converted to IP4 in parathyroid cells. The slow kinetics for the increase in IP4 with high Ca2+ in the absence of Li+, however, do not support a role for IP4 in the early phase of the sustained increase in cytosolic Ca2+ produced by high extracellular Ca2+ concentrations.     

Evidence for synergism between copper and prostaglandin E2 in stimulating the release of gonadotropin-releasing hormone from median eminence explants: Na+/Cl- requirements

Copper (Cu) and PGE2 are known to stimulate LHRH release from explants of the median eminence area (MEA) by two mechanisms distinguishable by their Ca2+ dependence. Moreover, exposure to Cu and PGE2 results in an amplified release of LHRH which is partially Ca2+ dependent, thus, resembling the release process stimulated by PGE2 alone. We have shown that LHRH release stimulated by Cu alone is Na+/Cl- dependent. By defining the Na+/Cl- dependence of PGE2- and Cu/PGE2-stimulated release of LHRH, we wished to ascertain if there is synergism between Cu and PGE2 actions. MEA of adult male rats were incubated for 5 min with 150 microM Cu and then for 15 min with 10 microM PGE2 (Cu/PGE2). Controls were incubated with Cu or PGE2. LHRH release into the medium was evaluated by RIA. Substituting Cl- in the incubation buffer with the non-permeant anion, isethionate, did not alter PGE2 stimulation of LHRH release, but it drastically inhibited Cu/PGE2 stimulation of LHRH release, indicating that this process requires a permeant monovalent anion. PGE2 and Cu/PGE2 stimulation of LHRH release were both inhibited when Na+ was substituted with Li+, or when 0.5 mM ouabain was included in the Na+-containing buffer; neither 10 microM tetrodotoxin (TTX) nor 100 microM amiloride were inhibitory. To ascertain if Na+ is required for Cu uptake, we evaluated the uptake of 67Cu by MEA explants and found that neither ouabain nor Li+ inhibited uptake, indicating that the extracellular Na+ and the activity of Na+/K+ ATPase are required for the process of LHRH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Erythrocyte membrane lipids and cationic transport systems in men.

OBJECTIVE: The relationship between erythrocyte membrane and plasma lipids and various transmembrane erythrocyte cationic fluxes was examined in 53 normal men. DESIGN: Different measurements of erythrocyte transport systems were obtained: Na(+)-Li+ countertransport activity; Na+, K+ cotransport activity; Na+, K(+)-ATPase pump activity and the ground membrane permeability for Na+ and K+ as well as the intra-erythrocyte Na+, K+ and Mg2+ concentrations. Plasma cholesterol, triglycerides, phospholipids, free fatty acids, low- and high-density lipoprotein cholesterol levels and the erythrocyte membrane contents of cholesterol, phospholipids and free fatty acids were obtained from fasting subjects. RESULTS: In single regression analysis the erythrocyte Na(+)-Li+ countertransport and Na+, K+ cotransport activities were negatively related to the erythrocyte membrane cholesterol, phospholipids and free fatty acids contents. The Na+, K(+)-ATPase pump activity as assessed by the ouabain-sensitive Na+ efflux was also inversely related to the membrane cholesterol and phospholipids contents. In multiple regression analysis the red blood cell Na(+)-Li+ countertransport activity was independently and negatively related to the membrane cholesterol and free fatty acids contents. CONCLUSION: Our data show that an elevated level of erythrocyte membrane lipids in normal men is accompanied by lower Na(+)-Li+ countertransport, Na+, K+ cotransport and Na+, K(+)-ATPase pump activities.     

Effect of ouabain on cellular free calcium and cellular cyclic AMP production in response to arginine vasopressin in rat renal papillary collecting tubule cells in culture

The effect of extracellular calcium (Ca2+) on the cellular action of arginine vasopressin (AVP) was examined using an Na+, K+-ATPase inhibitor in rat renal papillary collecting tubule cells in culture. The pretreatment of cells with ouabain enhanced basal and AVP-induced cAMP production in a dose-dependent manner. The augmentation by ouabain of cellular cAMP production in response to AVP was totally abolished by co-treatment with cobalt, lanthanum, verapamil or Ca2+-free medium containing 1 mmol EGTA/l, each blocking cellular Ca2+ uptake by different mechanisms. Two other findings indicated that ouabain directly stimulated cellular Ca2+ mobilization; namely, that ouabain significantly increased 45Ca2+ influx and cellular free Ca2+ concentration [( Ca2+]i) determined by Fura-2 fluorescence. The ouabain-induced increase in [Ca2+]i was completely blocked by either cobalt or Ca2+-free medium containing 1 mmol EGTA/l. AVP at 0.1 mumol/l increased [Ca2+]i to 177.1 +/- 26.2 nmol/l from 92.2 +/- 8.0 nmol/l (P less than 0.01) in renal papillary collecting tubule cells, and ouabain significantly enhanced the AVP-induced increase in [Ca2+]i. The increase of cellular free Ca2+ induced by ouabain probably binds to calmodulin to form an active complex of Ca2+-calmodulin in the cell, since two chemically dissimilar antagonists of calmodulin attenuated the enhancement by ouabain of cAMP production in response to AVP. These results therefore indicate that ouabain increases cellular Ca2+ uptake and enhances AVP-induced cellular free Ca2+ mobilization and its own second messenger cAMP production in renal papillary collecting tubule cells, and that extracellular Ca2+ is an important source for ouabain-mobilized cellular Ca2+.     

Na+-H+ exchange in gastric glands as measured with a cytoplasmic-trapped, fluorescent pH indicator.

We have used the pH-sensitive, fluorescent, cytoplasmic-trapped dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to identify Na+-H+ exchange in gastric glands isolated from rabbit stomachs by high-pressure perfusion and collagenase digestion. The fluorescence of BCECF-loaded glands was calibrated in terms of cytosolic pH (pHc) by permeabilizing the cell membranes and titrating the extracellular solution to different pH values. In one set of experiments in Cl--free solutions, glands were treated with 0.1 mM ouabain for 45 min to increase cellular cytosolic molar sodium ion concentration [( Na+]c) to high levels. Subsequent suspension of these cells in a Na+-free Ringer's solution (to generate [Na+]c greater than [Na+]o) caused cells to acidify rapidly (t1/2 approximately equal to 60 sec) from pHc approximately equal to 7.15 to pHc approximately equal to 6.55. Subsequent addition of 100 mM Na+ or Li+, but not K+, caused cells rapidly to increase pHc (t1/2 approximately equal to 30 sec) toward the control value. These changes of pHc were blocked when ouabain-treated glands had been preequilibrated for 10 min with 1 mM amiloride, and this block was overcome by adding 10 microM monensin (an ionophore that artificially exchanges Na+ for H+). In another set of experiments in Cl--containing Ringer's solution, glands were acid-loaded by treatment with 30 mM NH4Cl for 4 min, followed by washing the NH4Cl from the solutions. Under these conditions, pHc decreased from 7.02 to approximately equal to 6.5; subsequent alkalinization of cells back to control pHc was stimulated by Na+ (t1/2 approximately equal to 60 sec), but not K+, and was inhibited by 1 mM amiloride. This amiloride block also was overcome by further addition of 10 microM monensin. We conclude that gastric glands contain a Na+-H+ exchanger that appears independent of Cl-, not activated by K+, and blocked by 1 mM amiloride. This exchanger is likely localized to the serosal membrane of gland cells. Na+-H+ exchange may play an important role in regulation of pHc in oxyntic and chief cells exposed to high luminal acidity, where back diffusion of H+ into cells may occur at rapid rates.     

Canine cardiac sarcolemmal vesicles demonstrate rapid initial Na(+)-Ca2+ exchange activity

To identify a rapid, uninhibited rate of exchange activity, we investigated in canine sarcolemmal vesicles the rapid kinetics of Na(+)-Ca2+ exchange. Sarcolemmal vesicles were incubated in 160 mM NaCl and 20 mM HEPES at 25 degrees C (pH 7.4) and actively loaded with 45Ca2+ for 2 minutes by Na(+)-Ca2+ exchange. After further uptake was inhibited by dilution into 0.15 mM Na(+)-free EGTA, sarcolemmal vesicles were immobilized on a rapid filtration apparatus that allowed millisecond resolution of 45Ca2+ fluxes. In the presence of external NaCl (Na+o) but not other monovalent cations (i.e., K+, Li+), a biphasic pattern of Ca2+ release was observed--an initial brief and rapid rate of Ca2+ release followed by a second slower, prolonged phase of Ca2+ release. Semilogarithmic plots of sarcolemmal Ca2+ content versus time were not linear but were consistent with a biexponential rate of Na+o-induced Ca2+ release during the first several seconds of the exchange reaction. The fast phase of Na+o-stimulated Ca2+ release was several thousand-fold more rapid than that in the absence of Na+o. Both phases of Ca2+ release showed a similar Na+o dependence (Km, approximately 12 mM) with evidence of a positive cooperative effect of Na+. Vmax of the fast and slow phases were approximately 37.0 and approximately 0.76 nmol/mg/sec, respectively. Using rapid-reaction techniques, we demonstrated in the present study that the initial velocity of sarcolemmal Na(+)-Ca2+ exchange activity is greater than previously reported in sarcolemmal vesicles and that this exchange process exhibits complex rate behavior with a biphasic pre-steady state kinetic pattern.     

Ca2+ transport systems mediating the high K+/low Na+-induced uptake of Ca2+ into rat atrium

The effect of high K+/low Na+-Tyrode's solution on Ca2+ uptake into neonatal rat atrium was studied using 45Ca2+. Substitution of 60-129 mM Na+ in Tyrode's solution by equimolar concentrations of K+ or choline, significantly (with the exception of 60 mM choline substitution) increased Ca2+ uptake above control. Furthermore, the Ca2+ uptake stimulated by K+ substitution was significantly greater than that stimulated by choline substitution at the corresponding concentrations. The choline/low Na+-induced Ca2+ uptake (i.e. that above the Ca2+ uptake measured in normal Tyrode's solution) was increased by pre-exposure to either ice-cold Tyrode's solution for 1 h (approximately 36% increase) or to K+-free Tyrode's solution for 3 h (approximately 100% increase). The choline/low Na+-induced Ca2+ uptake was abolished by the hypertonic addition of NaCl (returning the bathing Na+ concentration to normal), increased (approximately 140%) by the addition of 1.8 mM PO4(3-)-free Hepes buffered choline/low Na+ media, but unaffected by 0.2 mM cadmium. The high K+/low Na+-induced Ca2+ uptake (i.e. that above the Ca2+ uptake measured in normal Tyrode's solution) was relatively insensitive to pre-exposure to cold (0% change) or K+-free media (11% increase) and only 50% inhibited by the hypertonic addition of NaCl (returning the bathing Na+ concentration to normal). However, the high K+/low Na+-induced Ca2+ uptake was 57% inhibited by 0.2 mM cadmium and approximately 30% inhibited by the addition of 1.8 mM PO4(3-) to HCO3-/PO4(3-)-free Hepes buffered high K+/low Na+ media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium transport by plasma membranes from a glucose-responsive rat insulinoma

Inside-out plasma membrane vesicles from a glucose-responsive rat insulinoma showed an ATP- and Mg2(+)-dependent uptake of Ca2+. The Km (concentration giving half-maximal activity) for Ca2+ was 60 nM. In the presence of 0.4 microM free Ca2+, the Km for ATP was 15 microM, and the Km for Mg2+ was 4 microM. Glucose (30 mM) decreased Ca2+ uptake by 50%, while other insulin secretagogues had no effect, except for glyceraldehyde, which stimulated Ca2+ uptake. Calmodulin increased the uptake of Ca2+, while trifluoperazine and vanadate inhibited the uptake. The Ca2(+)- and Mg2(+)-dependent ATPase from this tumor has a 10- to 20-fold higher requirement for Ca2+, which suggests that this enzyme is not responsible for Ca2+ transport, rather, Ca2+ transport activity represents only a small fraction of the total Ca2(+)-ATPase activity. The physiological importance of Ca2+ transport in insulin secretion is evident from the inhibition of Ca2+ uptake by glucose, which leads to a decrease in Ca2+ efflux from the cell. This inhibition would lead to an increase in intracellular free Ca2+ and insulin release.     

Na(+)-Ca2+ exchange, myoplasmic Ca2+ concentration, and contraction of arterial smooth muscle.

Na(+)-Ca2+ exchange is proposed to be an important regulator of myoplasmic intracellular Ca2+ concentration ([Ca2+]i) and contraction in vascular smooth muscle. We investigated the role of Na(+)-Ca2+ exchange in regulating [Ca2+]i in swine carotid arterial tissues that were loaded with aequorin to allow simultaneous measurement of [Ca2+]i and force. Reversal of Na(+)-Ca2+ exchange, by reduction of extracellular Na+ concentration ([Na+]o) to 1.2 mM, induced a large increase in aequorin-estimated [Ca2+]i and a low [Ca2+]i sensitivity. The contraction induced by 1.2 mM [Na+]o was partially caused by depolarization and opening of L-type Ca2+ channels because 10 microM diltiazem partially attenuated the 1.2 mM [Na+]o-induced increases in [Ca2+]i. High dose ouabain (10 microM), a putative endogenous Na+,K(+)-ATPase inhibitor, increased both [Ca2+]i and force. However, the increases in [Ca2+]i and force were mostly blocked by 10 microM phentolamine, suggesting the predominant effect of ouabain was to increase norepinephrine release from nerve terminals. In the presence of 10 microM phentolamine, 10 microM ouabain slightly accentuated 1 microM histamine-induced increases in [Ca2+]i and force. The ouabain dose necessary to induce contraction in the absence of phentolamine was significantly less than the ouabain dose necessary to accentuate histamine-induced contractions in the presence of phentolamine. These results suggest that Na(+)-Ca2+ exchange exists in swine arterial smooth muscle. These data also suggest that ouabain (which should increase [Na+]i and inhibit Na(+)-Ca2+ exchange) primarily enhances contractile function in the swine carotid artery by releasing catecholamines from nerve terminals; direct action of Na+,K(+)-ATPase inhibitors on smooth muscle appears to occur only with very high doses.     

Effects of simulated ischemia and reperfusion on the sarcoplasmic reticulum of digitonin-lysed cardiomyocytes.

ATP-dependent, inorganic phosphate-supported 45Ca2+ uptake by digitonin-lysed adult rat ventricular cardiomyocytes was used to evaluate the effects of simulated ischemia and reperfusion on the physically intact sarcoplasmic reticulum. Mitochondrial reactions were inhibited with rotenone and oligomycin. 45Ca2+ accumulation in the presence of the calcium efflux inhibitors, procaine (10 mM) and ruthenium red (30 microM), was used to characterize unidirectional uptake kinetics. A decrease in pH from 7.2 to 6.6 increased the [Ca2+] K0.5 from 0.5 to 2.0 microM and reduced the apparent Vmax by 28%. In the absence of procaine and ruthenium red, at a free [Mg2+] of 0.5 mM, maximum net uptake occurred at pCa 6.2 when pH was 7.2 and at pCa 6.0 when pH was 6.6. At lower pCa, net Ca2+ accumulation declined. Increasing free [Mg2+] from 0.5 to 1 mM at pH 6.6 or to 2.5 mM at pH 7.2 increased net 45Ca2+ accumulation in the absence of procaine and ruthenium and shifted maximum uptake to pCa 5.6 and 6.0, respectively. Increases in cytosolic free [Mg2+] thought to occur during myocardial ischemia are therefore capable of inhibiting calcium efflux from the sarcoplasmic reticulum. Reducing [ATP] from 10 to 1 mM reduced maximum net 45Ca2+ uptake by 30% both in the presence and absence of efflux inhibitors. Preincubation of intact myocytes under conditions designed to simulate ischemia and reperfusion decreased 45Ca2+ uptake greater than or equal to 50%. The data indicate that myocardial ischemia and reperfusion can alter both Ca2+ accumulation and calcium release by the sarcoplasmic reticulum.     

Elevated cytosolic Na+ decreases mitochondrial Ca2+ uptake during excitation-contraction coupling and impairs energetic adaptation in cardiac myocytes

Mitochondrial Ca2+ ([Ca2+]m) regulates oxidative phosphorylation and thus contributes to energy supply and demand matching in cardiac myocytes. Mitochondria take up Ca2+ via the Ca2+ uniporter (MCU) and extrude it through the mitochondrial Na+/Ca2+ exchanger (mNCE). It is controversial whether mitochondria take up Ca2+ rapidly, on a beat-to-beat basis, or slowly, by temporally integrating cytosolic Ca2+ ([Ca2+]c) transients. Furthermore, although mitochondrial Ca2+ efflux is governed by mNCE, it is unknown whether elevated intracellular Na+ ([Na+]i) affects mitochondrial Ca2+ uptake and bioenergetics. To monitor [Ca2+]m, mitochondria of guinea pig cardiac myocytes were loaded with rhod-2-acetoxymethyl ester (rhod-2 AM), and [Ca2+]c was monitored with indo-1 after dialyzing rhod-2 out of the cytoplasm. [Ca2+]c transients, elicited by voltage-clamp depolarizations, were accompanied by fast [Ca2+]m transients, whose amplitude (delta) correlated linearly with delta[Ca2+]c. Under beta-adrenergic stimulation, [Ca2+]m decay was approximately 2.5-fold slower than that of [Ca2+]c, leading to diastolic accumulation of [Ca2+]m when amplitude or frequency of delta[Ca2+]c increased. The MCU blocker Ru360 reduced delta[Ca2+]m and increased delta[Ca2+]c, whereas the mNCE inhibitor CGP-37157 potentiated diastolic [Ca2+]m accumulation. Elevating [Na+]i from 5 to 15 mmol/L accelerated mitochondrial Ca2+ decay, thus decreasing systolic and diastolic [Ca2+]m. In response to gradual or abrupt changes of workload, reduced nicotinamide-adenine dinucleotide (NADH) levels were maintained at 5 mmol/L [Na+]i, but at 15 mmol/L, the NADH pool was partially oxidized. The results indicate that (1) mitochondria take up Ca2+ rapidly and contribute to fast buffering during a [Ca2+]c transient; and (2) elevated [Na+]i impairs mitochondrial Ca2+ uptake, with consequent effects on energy supply and demand matching. The latter effect may have implications for cardiac diseases with elevated [Na+]i.     

Na+-H+ exchange and other ion-transport systems in erythrocytes of essential hypertensives and spontaneously hypertensive rats: a comparative analysis

The activity of ion-transport systems and Ca2+-induced erythrocyte haemolysis were compared between patients with essential hypertension and two strains of spontaneously hypertensive rats. Previous data on the increased rate of Na+-Li+ countertransport in erythrocytes of essential hypertensives were confirmed in this study. However, identification of Na+-Li+ countertransport in rat erythrocytes remained a complicated person because of the high rate of sodium-independent efflux of Li+. The rate of Na+-H+ exchange increased by 50-80% both in spontaneously hypertensive Wistar-Kyoto rats (SHR) and in patients with essential hypertension. No difference between Milan hypertensive strain rats (MHS) and Milan normotensive strain rats (MNS) was found. The rate of Na+,K+ cotransport increased in SHR and MHS erythrocytes compared with rats of the control strains [normotensive Wistar-Kyoto rats (WKY) and MNS; 30-50 and 90-110%, respectively]. No difference in this parameter was found between patients with essential hypertension and healthy subjects. Erythrocytes of patients with essential hypertension and of SHR were characterized by a higher sensitivity of their K+ channels to the increased concentration of intracellular Ca2+. This parameter did not change in MHS erythrocytes. Ca2+-induced haemolysis increased four- to fivefold in MHS erythrocytes compared with MNS and did not change in erythrocytes of SHR and patients with essential hypertension. The conclusion from these data is that the SHR strain is a more adequate model of human essential hypertension than the MHS.     

Influence of ruthenium red on rat heart subcellular calcium transport

Ruthenium red inhibited Ca2+-ATPase and ATP-independent Ca2+ binding with rat heart sarcolemma in a concentration dependent manner; significant effects were evident at 0.25 microM and higher concentrations. The apparent Ka for Ca2+-ATPase was 1.02 +/- 0.02 mM Ca2+ and 1.47 +/- 0.12 mM Ca2+ in the absence and presence of 2.5 microM ruthenium red, respectively; however, no change in the Vmax (41.2 +/- 1.6 mumol Pi/mg/h) was observed. Likewise, the affinity of Ca2+ for both low and high affinity Ca2+ binding sites in sarcolemma was decreased by ruthenium red. Sarcolemmal Na+-dependent Ca2+ uptake, ATP-dependent Ca2+ accumulation, Mg2+-ATPase and Na+,K+-ATPase activities were not affected by ruthenium red. In sarcoplasmic reticulum preparations, ruthenium red (0.25 to 25 microM) enhanced Ca2+ uptake without altering the Ca2+-stimulated ATPase activity. The observed increase in Ca2+ uptake appears to be due to the depressant effect of the dye on Ca2+ release from the sarcoplasmic reticulum. In mitochondrial preparations, ruthenium red (0.025 to 25 microM) showed a marked inhibitory effect on Ca2+ uptake activity whereas the Mg2+-ATPase activity was unaltered. In isolated rat hearts, 0.025 microM ruthenium red produced a slight negative inotropic effect, whereas 0.25 to 2.5 microM ruthenium red elicited a biphasic response both in terms of developed tension and resting tension. High concentrations of ruthenium red (12.5 to 25 microM) resulted in the development of contracture. Electron microscopic studies revealed the presence of ruthenium red in the myoplasm of hearts perfused for 15 to 30 mins with 2.5 to 5 microM dye.(ABSTRACT TRUNCATED AT 250 WORDS)