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)     

Net Ca2+ influx and sarcoplasmic reticulum Ca2+ uptake in resting single myocytes of the rat heart: comparison with guinea-pig

We tested Shattock and Bers' (1989) hypothesis according to which in rat cardiac myocytes net Ca2+ influx during diastole via Na/Ca exchange provides the main route of entry of Ca2+ available for activation of contractions. We used injections of caffeine into the close vicinity of the single, isolated rat or guinea-pig ventricular myocytes in order to release Ca2+ from sarcoplasmic reticulum (SR). The cells responded to caffeine with a transient contracture, the amplitude of which was regarded as a relative index of SR Ca2+ content. Application of caffeine deprived the SR of Ca2+. This was manifested by a very small (rat) or absent (guinea-pig) contractile response to the second application of caffeine and by a decrease of the amplitude of the first post caffeine contraction to 8 +/- 3% (rat) or to 16 +/- 6% (guinea-pig) of control. In the rat myocytes SR deprived of Ca2+ was able to recover its Ca2+ store even in the resting cell. This was indicated by the time dependent recovery of contractile response to the second application of caffeine and of the amplitude of the post-caffeine electrically evoked contractions. The recovery of post-caffeine contractile responses was completely inhibited by Ca2+ free solution, by 5.0 mM Ni+ and by low K+ (1.0 mM) hyperpolarising solution superfused from the first application of caffeine or during rest. The recovery was enhanced by superfusion of the cells with low Na+ (50%) solution. These results show that there is a considerable net Ca2+ influx by means of Na/Ca exchange and then the SR Ca2+ uptake in the resting rat myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

'Calcium paradox' in the heart is modulated by cell sodium during the calcium-free period

We hypothesized that after a Ca2+-free period the magnitude of the Na+ gradient at the onset of Ca2+ reperfusion would grade the ensuing cell Ca2+ gain. Rabbit interventricular septa perfused with Hepes buffered solution (pH 7.4, [Ca2+] = 1.0 mM) and stimulated to contract isometrically at 60 min-1 at 30 degrees C were exposed to a 30-min Ca2+-free period followed by 30-min of Ca2+ re-introduction. Cell Na without Ca2+-free perfusion was 137 +/- 5 mumol/g dry wt. During the Ca2+-free period, the perfusate was manipulated to result in three groups of septa in which cell Na just prior to Ca2+ re-introduction was 64 +/- 9 (perfusate [Na+] reduced to 47 mM), 170 +/- 12 (perfusate unaltered), and 293 +/- 16 mumol/g dry wt (addition of 5 X 10(-5) M ouabain). Following Ca2+ re-introduction, cell Ca2+ content was 3.4 +/- 0.5, 6.5 +/- 1.0, and 10.6 +/- 0.7 mumol/g dry wt in the low, intermediate, and high cell Na+ groups, respectively. Similar marked and highly significant gradations among the three groups were observed in the extent of cell K+ loss and recovery of contractile function during Ca2+ reintroduction. These results indicate that (1) myocardial cell Na+ increases during Ca2+ free perfusion and (2) the magnitude of the Na+ gradient at the end of the Ca2+ free period is an important determinant of the extent of cell Ca2+ gain, cell K+ loss, and reduction of contractile function with Ca2+ re-introduction, which collectively have been referred to as the 'calcium paradox' in the heart.     

Dual actions of 1,25-dihydroxycholecalciferol on intracellular Ca2+ in GH4C1 cells: evidence for effects on voltage-operated Ca2+ channels and Na+/Ca2+ exchange

In GH4C1 rat pituitary cells, 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] amplifies the TRH-induced spike phase of increase in cytosolic free calcium ([Ca2+]i). In the present report we describe the results of investigations on the mechanisms of action of 1,25-(OH)2D3 on Ca2+ homeostasis in these cells. Pretreatment with 1 nM 1,25-(OH)2D3 for at least 24 h caused no change in basal uptake of 45Ca2+ compared with that in control cells or in 45Ca2+ uptake induced by the calcium channel agonist Bay K 8644. However, when the cells were depolarized with 50 mM K+, 1,25-(OH)2D3-treated cells showed an up to 90% enhancement of uptake (3-120 min) of 45Ca2+. An enhanced increase in [Ca2+]i was also observed in fura-2-loaded cells. The effect was specific and dose dependent for 1,25-(OH)2D3. The calcium channel antagonists nimodipine and verapamil inhibited completely the enhancing action of 1,25-(OH)2D3 as did the protein synthesis inhibitor cycloheximide. No enhanced uptake of 45Ca2+ into intracellular stores was detected when cells were incubated with 1,25-(OH)2D3. Na+/Ca2+ exchange was determined by measuring exchange of extracellular 45Ca2+ for intracellular Na+. Na+/Ca2+ exchange was dependent on intracellular Na+, was inactive when Li+ replaced Na+, was insensitive to calcium channel antagonists, and showed electrogenic properties. In cells incubated with 1,25-(OH)2D3 for at least 24 h, Na+/Ca2+ exchange was enhanced up to 54% compared with that in control cells. Enhanced exchange was dose dependent and specific for 1,25-(OH)2D3. Ca2+ channel antagonists were without effect while dichlorobenzamil inhibited partially the 1,25-(OH)2D3 enhancement of Na+/Ca2+ exchange. Cycloheximide abolished completely the action of 1,25-(OH)2D3 on Na+/Ca2+ exchange. We conclude that in GH4C1 cells, 1,25-(OH)2D3 enhances membrane calcium transport by modulating voltage-operated Ca2+ channels and activating Na+/Ca2+ exchange by mechanisms requiring new protein synthesis.     

Mobilization of extracellularly bound Ca2+ during high K+ and norepinephrine stimulation of the rabbit aorta

The effects of alpha-adrenergic stimulation and high K+-induced membrane depolarization on 45Ca2+ uptake and tension generation in the rabbit aorta were investigated. Tension and unidirectional 45Ca2+ uptake were increased by both stimulants. Moreover, this 'steady-state' uptake remained elevated for as long as the stimulants were present. When tissues were preincubated in 45Ca2+-containing PSS prior to the Ne or high K+ challenge the resulting Ca2+ uptake showed an 'initial burst' of uptake which was not observed in the 'steady-state' experiments. The magnitude of the 'initial burst' increased with time displaying a t1/2 for exchange of 1.25 min for both high K+ and Ne, suggesting that this Ca2+ source is shared by both stimulants. The 'initial burst' became Ca2+ saturated when [Ca2+]o was between 0.5 and 1.5 mM, was enhanced by 45Ca2+ preincubation in a solution of lowered ionic strength and was inhibited (approximately 70%) by D600 (5 X 10(-6) M). In contrast, the 'steady-state' uptake was linearly dependent on [Ca2+]o up to 6.4 mM, was 90% inhibited by 5 X 10(-6) M D600 and was unaffected by lowered ionic strength. It is concluded that the properties displayed by the Ca2+ source responsible for the 'initial burst' and 'steady-state' uptake suggest that they are of distinct origin; the 'initial burst' being derived from a bound extracellular Ca2+ pool and the 'steady-state' uptake resulting from the uptake of free Ca2+ dissolved in the extracellular space.     

Role of changes in [Ca2+]i in energy deprivation contracture

Mechanisms of energy deprivation contracture were investigated in cultured chick embryo ventricular cells. In the presence of zero-extracellular-Na+, (choline chloride substitution)-nominal-zero-Ca2+ [( Ca2+] approximately 5 microM), exposure of ventricular cells to 1 mM cyanide (CN) and 20 mM 2-deoxyglucose (2-DG)-zero-glucose solution resulted in the development of a contracture (video motion detector) in 5.9 +/- 0.5 minutes. Early after contracture development, the resupply of extracellular Na+, in the continued presence of CN + 2-DG, resulted in a rapid partial relaxation (t1/2 = 1.9 +/- 0.3 seconds), associated with an increase in 45Ca efflux, presumably due to transsarcolemmal Ca2+ extrusion due to Na+-Ca2+ exchange. Resupply of glucose and removal of CN + 2-DG, in the continued absence of Na+, resulted in an initially slower (t1/2 = 11.6 +/- 2.5 seconds), but more complete relaxation of contracture, which was not associated with increased Ca2+ efflux. Pretreatment with 20 mM caffeine delayed the onset of contracture (9.2 +/- 1.1 minutes) and resulted in a contracture that could not be relaxed by resupply of external Na+ only. Studies using the fluorescent Ca2+ probe indo 1 demonstrated that in zero-Na+-zero-Ca2+ solutions, contracture due to CN + 2-DG was associated with an initial rise in [Ca2+]i but that this did not account for all of contracture force development. In cells exposed to CN + 2-DG in the presence of normal extracellular Na+ and Ca2+ concentrations, a small rise in [Ca2+]i was associated with initial contracture development, consistently preceding the development of a larger accelerated contracture presumably due to ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible evidence for transmembrane K(+)-H+ exchange system in guinea pig myocardium.

The aim of this study was to obtain evidence for a transmembrane K(+)-H+ exchange system in Langendorff-perfused whole hearts and isolated ventricular myocytes of guinea pig. Effluent relation between K+ and pH in the whole hearts perfused with HEPES-buffered Tyrode's solution indicated a significant (p < 0.05) functional coupling of K+ uptake and H+ extrusion that was energy-dependent and omeprazole (OPZ)-sensitive. Administration of OPZ (0.3 mM) or dimethylamiloride (0.1 mM), an inhibitor of Na(+)-H+ antiport, to whole hearts subjected to the repetitive NH4Cl applications implied that both Na(+)-H+ and putative K(+)-H+ countertransports contribute to the regulation of intracellular pH. In isolated myocytes, voltage-dependent L-type Ca current (ICa) was inhibited by OPZ (0.3 mM) under K(-)- and Na(+)-free condition by 11 to 14%, and was inhibited to a greater extent (i.e., by 36 to 40%) by this agent in the presence of K+. OPZ-induced inhibition of the putative K(+)-H+ exchanger likely resulted in subsarcolemmal acidification which was responsible for the rate-independent suppression of ICa. In conclusion, these data provide functional evidence for a myocardial transmembrane K(+)-H+ exchanger.     

Role of cyclic ADP-ribose in Ca2+-induced Ca2+ release and vasoconstriction in small renal arteries

Cyclic-ADP-ribose (cADPR) has been reported to serve as a second messenger to mobilize intracellular Ca2+ independent of IP3 in a variety of mammalian cells. This cADPR-mediated Ca2+ signaling pathway importantly participates in the regulation of various cell functions. The present study determined the role of endogenous cADPR in mediating ryanodine-sensitive Ca2+-induced Ca2+ release (CICR) in vascular myocytes from small renal arteries and vasomotor response of these arteries. In freshly-isolated renal arterial myocytes, addition of CaCl2 (0.01, 0.1, and 1 mM) into the Ca2+-free bath solution produced a rapid Ca2+ release response from the sarcoplasmic reticulum (SR), with a maximal increase of 237+/-25 nM at 1 mM CaCl2. This CaCl2 response was significantly blocked by a cell-membrane permeant cADPR antagonist, 8-bromo-cADP-ribose (8-br-cADPR) (30 microM) or ryanodine (50 microM). Caffeine, a classical CICR or ryanodine receptor activator was found to stimulate the SR Ca2+ release (Delta[Ca2+]i: 253+/-35 nM), which was also attenuated by 8-br-cADPR or ryanodine. Using isolated and pressurized small renal arteries bathed with Ca2+-free solution, both CaCl2 and caffeine-induced vasoconstrictions were significantly attenuated by either 8-br-cADPR or ryanodine. Biochemical analyses demonstrated that CaCl2 and caffeine did not increase cADPR production in these renal arterial myocytes, but confocal microscopy showed that a dissociation of the accessory protein, FK506 binding protein 12.6 (FKBP12.6) from ryanodine receptors was induced by CaCl2. We conclude that cADPR importantly contributes to CICR and vasomotor responses of small renal arteries through enhanced dissociation of ryanodine receptors from their accessory protein.     

H+ disposal by rabbit gastric mucosal surface cells

Exposure of isolated gastric mucosal surface cells to NH4+ results in acidification of cells as determined by a fluorescent dye technique using acridine orange. The resulting intracellular pH gradient is maintained when cells are suspended in either buffered HCO3- -free Ringer's or choline chloride solution. Cells suspended in a Na+-containing but K+-free solution exhibit dissipation of the proton gradient. When Na+ is added to cells suspended in Na+, K+-free solution, the gradient rapidly dissipates with a half-maximal response occurring at 56 mM Na+. The effect of Na+ is amiloride sensitive with half-maximal inhibition occurring at 38 microM at a Na+ concentration of 50 mM. The K+ does not cause dissipation of the gradient and neither ouabain nor valinomycin have an effect. Yet, K+ has a modulating influence on Na+/H+ exchange by the isolated surface cells. The addition of K+ to acid-loaded cells resuspended in Na+-free solution decreases the ability of subsequent Na+ addition to evoke gradient dissipation. The data suggest that Na+/H+ exchange appears to be at least one mechanism whereby gastric mucosal surface cells could protect themselves against diffusing acid. This ion exchange mechanism is amiloride sensitive and appears to be unrelated to Na+, K+ adenosine triphosphatase activity, but is affected by the external K+ concentration.     

Reexpression of T-type Ca2+ channel gene and current in post-infarction remodeled rat left ventricle

OBJECTIVE: T-type Ca2+ currents (I(Ca-T)) are present in neonatal rat myocytes but is not detected in adult ventricular myocytes. The present study was designed to investigate the expression of the T-type Ca2+ channel gene and current in post-infarction remodeled hypertrophied rat left ventricle (LV). METHODS: We compared the expression of T-type Ca2+ channel gene alpha-1G in neonatal rat LV, in adult sham-operated LV and remodeled hypertrophied LV 3 to 4 weeks post-myocardial infarction (MI) using RNase protection assay (RPA). The cDNA fragment of alpha-1G used in RPA was obtained from poorly conserved region of recently published T-type Ca2+ channel coding sequence of rat by RT-PCR. The fragment was verified by restriction enzyme digestion and sequencing. The presence of I(Ca-T) in LV of sham and post-MI rats was examined using patch-clamp techniques. In the presence of K+-free, Na+-free external solution, I(Ca-T) was separated from I(Ca-L) by different holding potentials (HP). I(Ca-T) was also recorded during depolarization to -40 mV from a HP of -80 mV with NaCl in external solution and I(Na) suppressed by 100 microM tetrodotoxin (TTX). RESULTS: The T-type Ca2+ channel gene alpha-1G was expressed in neonatal heart, the expression level decreased by 80%, in adult sham heart and was reexpressed in MI (158% increases compared to sham; P<0.01). I(Ca-T) was recorded in 11 of 31 MI cells in presence of K+-free, Na+-free external solution and in 9 of 14 cells when I(Na) was suppressed by TTX. I(Ca-T) was not detected in any of 21 sham cells. I(Ca-T) density was 1.1+/-0.4 pA/pF. I(Ca-T) was more sensitive to Ni2+ and less sensitive to nisoldipine. CONCLUSIONS: T-type Ca2+ channel gene and current are reexpressed in rat post-MI remodeled LV myocytes. Its functional significance in the post-MI remodeling process remains to be defined.     

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.     

K+-induced dilation of hamster cremasteric arterioles involves both the Na+/K+-ATPase and inward-rectifier K+ channels

OBJECTIVE: The mechanism by which elevated extracellular potassium ion concentration ([K+]o) causes dilation of skeletal muscle arterioles was evaluated. METHODS: Arterioles (n = 111) were hand-dissected from hamster cremaster muscles, cannulated with glass micropipettes and pressurized to 80 cm H2O for in vitro study. The vessels were superfused with physiological salt solution containing 5 mM KCl, which could be rapidly switched to test solutions containing elevated [K+]o and/or inhibitors. The authors measured arteriolar diameter with a computer-based diameter tracking system, vascular smooth muscle cell membrane potential with sharp micropipettes filled with 200 mM KCl, and changes in intracellular Ca2+ concentration ([Ca2+]i) with Fura 2. Membrane currents and potentials also were measured in enzymatically isolated arteriolar muscle cells using patch clamp techniques. The role played by inward rectifier K+ (KIR) channels was tested using Ba2+ as an inhibitor. Ouabain and substitution of extracellular Na+ with Li+ were used to examine the function of the Na+/K+ ATPase. RESULTS: Elevation of [K+]o from 5 mM up to 20 mM caused transient dilation of isolated arterioles (27 +/- 1 microm peak dilation when [K+]o was elevated from 5 to 20 mM, n = 105, p <.05). This dilation was preceded by transient membrane hyperpolarization (10 +/-1 mV, n = 23, p <.05) and by a fall in [Ca2+]i as indexed by a decrease in the Fura 2 fluorescence ratio of 22 +/- 5% (n = 4, p <.05). Ba(2+) (50 or 100 microM) attenuated the peak dilation (40 +/- 8% inhibition, n = 22) and hyperpolarization (31 +/- 12% inhibition, n = 7, p <.05) and decreased the duration of responses by 37 +/-11% (n = 20, p < 0.05). Both ouabain (1 mM or 100 microM) and replacement of Na+ with Li+ essentially abolished both the hyperpolarization and vasodilation. CONCLUSIONS: Elevated [K+]o causes transient vasodilation of skeletal muscle arterioles that appears to be an intrinsic property of the arterioles. The results suggest that K+-induced dilation involves activation of both the Na+/K+ ATPase and KIR channels, leading to membrane hyperpolarization, a fall in [Ca2+]i, and culminating in vasodilation. The Na+/K+ ATPase appears to play the major role and is largely responsible for the transient nature of the response to elevated [K+]o, whereas KIR channels primarily affect the duration and kinetics of the response.     

Muscarinic acetylcholine receptor activation induces Ca2+ mobilization and Na+/K+-ATPase activity inhibition in eel enterocytes

The effect of carbachol (Cch) on intracellular calcium concentration ([Ca2+]i) in eel enterocytes was examined using the fluorescent Ca2+ indicator fura-2. Cch caused a biphasic increase in [Ca2+]i, with an initial spike followed by a progressively decreasing level (over 6 min) to the initial, pre-stimulated, level. The effect of Cch was dose-dependent with a 7.5-fold increase in [Ca2+]i over basal level induced by the maximal dose of Cch (100 microM). In Ca2+-free/EGTA buffer the effect of Cch was less pronounced and the [Ca2+]i returned rapidly to basal levels. The increment of [Ca2+]i was dose-dependently attenuated in cells pre-treated with U73122, a specific inhibitor of phospholipase C, suggesting that the Cch-stimulated increment of [Ca2+]i required inositol triphosphate formation. In the presence of extracellular Ca2+, thapsigargin (TG), a specific microsomal Ca2+-ATPase inhibitor, caused a sustained rise in [Ca2+]i whereas in Ca2+-free medium the increase in [Ca2+]i was transient; in both cases, subsequent addition of Cch was without effect. When 2 mM CaCl2 were added to the cells stimulated with TG or with Cch in Ca2+-free medium, a rapid increase in [Ca2+]i was detected, corresponding to the capacitative Ca2+ entry. Thus, both TG and Cch depleted intracellular Ca2+ stores and stimulated influx of extracellular Ca2+ consistent with capacitative Ca2+ entry. K+ depolarization obtained with increasing concentrations of KCl in the extracellular medium induced a dose-related increase in [Ca2+]i which was blocked by 2 microM nifedipine, a non-specific L-type Ca2+ channel blocker. Nifedipine also changed significantly the height of the Ca2+ transient, and the rate of decrement to the pre-stimulated [Ca2+]i level, indicating that Ca2+ entry into enterocytes also occurs through an L-type voltage-dependent calcium channel pathway. We also show that isolated enterocytes stimulated with increasing Cch concentrations (0.1-1000 microM) showed a dose-dependent inhibition of the Na+/K+-ATPase activity. The threshold decrease was at 1 microM Cch; it reached a maximum at 100 microM (50.5% inhibition) and did not decrease further with the use of higher dose. The effect of Cch on Na+/K+-ATPase activity was dependent on both protein kinase C (PKC) and protein phosphatase calcineurin activation since the PKC inhibitor calphostin C abolished Cch effects, while the calcineurin inhibitor FK506 augmented Cch effect. Collectively, these data establish a functional pathway by which Cch can modulate the activity of the Na+/K+-ATPase through a PKC-dependent (calphostin C-sensitive) pathway and a calcineurin-dependent (FK506-sensitive) pathway.     

Effects of active oxygen generated by DTT/Fe2+ on cardiac Na+/Ca2+ exchange and membrane permeability to Ca2+

Sarcolemmal vesicles isolated from bovine heart were preincubated at 37 degrees C with an oxygen radical generating system consisting of 1 mM dithiothreitol (DTT) and 50 microM FeSO4. Exposure of the vesicles for 1 to 40 mins stimulated Na+/Ca2+ exchange about 2.5-fold. The DTT/Fe2+ treatment decreased the apparent Km for Ca2+ of Nai+-dependent Ca2+ uptake by 80% (from 63 to 13 microM). The effect on Vmax was much smaller however. The resulting stimulation of exchange activity was diminished by the presence of desferrioxamine (95%) or catalase (60%). In contrast, superoxide dismutase and sodium formate did not prevent the effects of DTT/Fe2+ on the exchanger. Neither Zn2+ nor Ga3+ could replace Fe2+ in the stimulation of Na+/Ca2+ exchange. Passive Ca2+ efflux was determined by first allowing Na+/Ca2+ exchange to continue to plateau values and then diluting the loaded vesicles in the presence of EGTA. Ca2+ leakage from the vesicles was slightly but significantly (P less than 0.05) increased by the action of DTT/Fe2+, the rate constants for the passive Ca2+ efflux being 0.22 and 0.26/min in control and treated groups, respectively. The calcium loading observed in myocytes in ischemia/reperfusion injury suggests that the stimulation of Na+/Ca2+ exchange by active oxygen may moderate the myocardial response to oxygen mediated injuries including ischemia/reperfusion injury. However, the clinical relevance of these phenomena is far from clear as the stimulation depends in part on the Km for Ca2+ prior to treatment.     

Independence of the positive inotropic effect of ouabain from the inhibition of the heart Na+/K+ pump.

Isolated left atria from guinea pigs were stimulated at 3.3 Hz and bathed at 30 degrees C in Tyrode's solution containing 6 mM KCl. After equilibration, this solution was replaced by a low-K solution or by Tyrode's solution containing ouabain or dihydroouabain. These treatments evoked an increase in the contractility of the atria. The time to peak increase was about 30 min, and the inotropic effect was sustained for at least 40 min. After 30 min, 42K was added to the bathing solution in order to estimate the activity of the Na+/K+ pump. A linear relationship was observed between the degree of inhibition of the Na+/K+ pump and the increase in systolic tension. The regression line was the same for low-K solutions and dihydroouabain but not for ouabain. For a given degree of inhibition of the pump, ouabain evoked a higher increase in contractility. These findings indicate that inhibition of the Na+/K+ pump can be the only mechanism responsible for the positive inotropic effect of dihydroouabain but cannot be the sole mechanism for that of ouabain.     

Calcium and magnesium transport by in situ mitochondria: electron probe analysis of vascular smooth muscle

The extent, time course, and reversibility of mitochondrial Ca2+ uptake secondary to cellular Ca2+ influx stimulated by massive Na+ efflux were evaluated by electron probe microanalysis of rabbit portal vein smooth muscle. Strips of portal vein were Na+ loaded for 3 hours at 37 degrees C in a K+-free 1 mM ouabain solution, after which rapid Na+ efflux was induced by washing with a Na+-free K+-Li+ solution (1 mM ouabain). Li+ washing Na+-loaded portal vein produced a large transient contraction accompanied by an increase (over 100-fold) in mitochondrial Ca2+ and also significant (p less than 0.05) increases in phosphorus and Mg2+. The Ca2+ loading of the mitochondria was reversed during prolonged Li+ wash, and by 2 hours, mitochondrial Ca2+, Mg2+, and phosphorus had returned to control levels. The maximal contractile response to stimulation remained normal, demonstrating that pathologic Ca2+ loading of mitochondria is reversible in situ and compatible with normal maximal force developed by the smooth muscle. Mitochondrial Ca2+ and phosphorus uptake were reduced but still significant when the Li+ wash contained 0.2 mM Ca2+ or when ouabain was omitted. The fact that mitochondrial Ca2+ loading accompanied submaximal contractions during 0.2 mM Ca2+-Li wash suggests "supranormal" affinity of mitochondria for Ca2+ and may be due, in part, to reverse operation of the mitochondrial Na+-Ca2+ exchanger. Mitochondrial Ca2+, Mg2+, and phosphorus uptake were eliminated when the Li+ wash was performed at 2 degrees C or when the wash contained no Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiac cell action potential duration is dependent upon induced changes in free Ca2+ activity during pH changes in vitro

We examined how changes in solution pH alter myocardial cell action potentials (AP) with and without changes in free [Ca2+] caused by pH induced effects on calcium binding. Guinea pig ventricular tissue was isolated, superfused either with Krebs-Ringer (K-R) bicarbonate, phosphate buffered solution, or with Hepes buffered solution, and electrically paced during control (5% CO2 in O2), acidic (12% CO2), and alkalotic (0% CO2) conditions. Action potentials were recorded with intracellular microelectrodes. Extracellular free [Ca2+] was measured with a calcium ion selective electrode and total soluble calcium was measured by ultrafiltration and spectrophotometry. With a total [CaCl2] of 2.5 mM in the K-R solution, we found a free [Ca2+] of 2.14 mM at pH 7.44 (control), 2.48 mM at pH 6.97 and 1.60 mM at pH 8.19; total soluble calcium concentration was 2.00 mM at pH 8.19. In the Hepes solution, free [Ca2+] was only slightly altered (2.42 to 2.55 mM) within this pH range. Equivalent acidosis of either K-R or Hepes suffusate significantly, and similarly, prolonged the AP and its refractory period. Alkalosis of the Hepes suffusate shortened the AP; but equivalent alkalosis of the K-R suffusate prolonged the AP as did a reduction of [CaCl2] in Hepes suffusate from 3.0 to 1.5 mM at pH 7.43. Our study demonstrates that a paradoxical increase in APD occurs because free Ca2+ ion activity falls in K-R solution and overrides the effect of alkalosis alone to decrease APD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of maitotoxin with voltage-sensitive calcium channels in cultured neuronal cells.

The dinoflagellate toxin maitotoxin (MTX) stimulated 45Ca2+ uptake in cultured NG108-15 neuroblastoma X glioma cells. Depolarizing stimuli (e.g., 50 mM K+) produced an immediate stimulation in Ca2+ uptake, whereas that produced by MTX occurred only after a lag period of about 2 min. MTX did not stimulate Ca2+ uptake into fibroblasts. Both 50 mM K+- and MTX-stimulated Ca2+ uptake was blocked by organic calcium channel antagonists (nitrendipine, D-600, diltiazem) at very low concentrations. Cd2+ was also a potent blocker. The novel dihydropyridine BAY K8644 enhanced Ca2+ uptake in the presence of 50 mM K+ but had no effect in 5 mM Ca2+. However, in the presence of MTX, BAY K8644 stimulated Ca2+ uptake in 5 mM K+. The effects of MTX were not blocked by tetrodotoxin but were decreased in Na+-free medium. MTX did not stimulate Na+ uptake into NG108-15 cells and did not alter [3H]nitrendipine binding to rat brain cortical synaptosomes. It is concluded that MTX may alter the voltage dependence of calcium-channel activation.     

Ca2+/calmodulin inhibition and phospholipase C-linked Ca2+ Signaling in clonal beta-cells

Neurotransmitters and hormones, such as arginine vasopressin (AVP) and bombesin, evoke frequency-modulated repetitive Ca2+ transients in insulin-secreting HIT-T15 cells by binding to receptors linked to phospholipase C (PLC). The role of calmodulin (CaM)-dependent mechanisms in the generation of PLC-linked Ca2+ transients was investigated by use of the naphthalenesulfonamide CaM antagonists W-7 and W-13 and their dechlorinated control analogs W-5 and W-12. W-7 (10-30 microM) and W-13 (30-100 microM), but not W-5 (100 microM) and W-12 (300 microM), reversibly inhibited the AVP- and bombesin-induced Ca2+ transients. As the generation of PLC-linked Ca2+ transients requires mobilization of internal Ca2+ and Ca2+ influx through voltage-sensitive (VSCC) and -insensitive (VICC) Ca2+ channels, the effects of the W compounds on these processes were further investigated. First, W-7 dose dependently diminished K+ (45 mM)-induced Ca2+ signals (IC50, approximately 25 microM), and W-13 (100 microM) reduced the K+ (45 mM)-induced [Ca2+]i rise by about 40-60%, whereas W-5 (100 microM) and W-12 (300 microM) had no effect. In addition, W-7 (100 microM) inhibited whole cell Ca2+ currents in mouse beta-cells by about 60%. Second, pretreatment of cells (5 min) with W-7 (30 microM), but not W-5 (30 microM), inhibited agonist-induced internal Ca2+ mobilization by about 75% in Ca2+-free medium. Neither W-7 (30 microM) nor W-5 (30 microM) affected AVP (100 nM)-stimulated formation of IP3. Third, capacitative Ca2+ influx through VICC activated by thapsigargin (2 microM) in the presence of verapamil (50 microM) was inhibited by W-7 (30 microM) but not by W-5 (30 microM). As all of the W compound effects corresponded well to their reported anticalmodulin activity, a specific anticalmodulin action can be assumed. Thus, Ca2+ via activation of CaM-dependent processes could provide positive feedback on the generation of PLC-linked Ca2+ transients in HIT-T15 cells. This appears to involve CaM-dependent regulation of both mobilization of internal Ca2+ and Ca2+ influx through VSCC and VICC.     

Effect of perfusate [Ca2+] on cardiac sarcoplasmic reticulum Ca2+ release channel in isolated rat hearts.

The effect of perfusate [Ca2+] on the function of cardiac sarcoplasmic reticulum (CSR) was assessed by the oxalate-supported Ca2+ uptake rate of ventricular homogenates of isolated rat hearts maintained in a modified Langendorff preparation. The total Ca2+ pumping activity of the CSR was determined by using 20 microM ruthenium red or 625 microM ryanodine to close the CSR Ca2+ release channel. The homogenate Ca2+ uptake rate in the absence of ruthenium red or ryanodine decreased progressively with increasing perfusate [Ca2+] (25.7 +/- 1.2, 21.4 +/- 1.5, 17.2 +/- 1.1, and 16.3 +/- 1.2 [mean +/- SEM] nmol Ca2+.min-1.mg-1 for hearts perfused for 5 minutes with 0.2, 1.4, 2.8, and 5.6 mM Ca2+, respectively; p = 0.0001; n = 8). This depression was not observed when Ca2+ uptake was assayed in the presence of ryanodine or ruthenium red. Since the Ca2+ uptake in the presence of ryanodine or ruthenium red is determined by the Ca(2+)-ATPase, this result suggests that perfusion with varying [Ca2+] did not affect the Ca(2+)-ATPase. The observed decrease in Ca2+ uptake in the absence of ryanodine or ruthenium red is caused by an increased efflux through the ryanodine-sensitive Ca2+ release channel. When hearts perfused for 5 minutes with 0.2 or 5.6 mM Ca2+ were reperfused for 10 minutes with 1.4 mM Ca2+, homogenate Ca2+ uptake rates were restored to near control levels. These effects of perfusate Ca2+ were not direct effects, because changes in the [Ca2+] of the homogenization medium did not alter the homogenate Ca2+ uptake activity in either the presence or absence of ryanodine. The homogenate Ca2+ uptake rates were unaffected by prior active loading of the CSR with Ca2+. These results suggest a regulatory role of perfusate Ca2+ in increasing the open state of the ryanodine-sensitive Ca2+ release channel that is distinct from the beat-to-beat regulation of Ca2+ release from the CSR by Ca2+ (Ca(2+)-induced Ca2+ release).