Effect of anoxia on intracellular ATP, Na+i, Ca2+i, Mg2+i, and cytotoxicity in rat hepatocytes.

The effects of anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with SBFI, intracellular pH (pHi) with BCECF, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, ATP by 31P NMR spectroscopy in real time, and intracellular free Mg2+ (Mg2+i) from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. After 1 h of anoxia, beta-ATP fell 66%, and 85% after 2 h, while the Pi/ATP ratio increased 10-fold from 2.75 to 28.3. Under control conditions, the resting cytosolic free calcium was 127 +/- 6 nM. Anoxia increased Ca2+i in two distinct phases: a first rise occurred within 15 min and reached a mean value of 389 +/- 35 nM (p less than 0.001). A second peak reached a maximum value of 1.45 +/- 0.12 microM (p less than 0.001) after 1 h. During the first hour of anoxia, Na+i increased from 15.9 +/- 2.4 mM to 32.2 +/- 1.2 mM (p less than 0.001), Mg2+i doubled from 0.51 +/- 0.05 to 1.12 +/- 0.01 mM (p less than 0.001), and pHi decreased from 7.41 +/- 0.03 to 7.06 +/- 0.1 (p less than 0.001). LDH release doubled during the first hour and increased 6-fold during the second hour of anoxia. Upon reoxygenation, ATP, Ca2+i, Mg2+i, Na+i, and LDH returned near the control levels within 45 min. To determine whether the increased LDH release was related to the rise in Ca2+i, and whether the increased Ca2+i was caused by Ca2+ influx, the cells were perfused with Ca(2+)-free KHB (+ 0.1 mM EGTA) during the anoxic period. After 2 h of anoxia in Ca(2+)-free medium, beta-ATP again fell 90%, but Ca2+i, after the first initial peak, fell below control levels, and LDH release increased only 2.7-fold. During reoxygenation, Ca2+i, ATP, Na+i, and LDH returned near the control levels within 45 min. These results suggest that the rise in Ca2+i induced by anoxia is caused by an influx of Ca2+ from the extracellular fluid, and that LDH release and cell injury may be related to the resulting rise in Ca2+i.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.     

The effects of anoxia on cytosolic free calcium, calcium fluxes, and cellular ATP levels in cultured kidney cells

The effect of anoxia and substrate removal on cytosolic free calcium (Ca2+i), cell calcium, ATP content, and calcium efflux was determined in cultured monkey kidney cells (LLC-MK2) exposed to 95% N2, 5% CO2 for 60 min. In the control period, the basal Ca2+i level was 70.8 +/- 9.4 nM. During 1 h of anoxia without substrate, ATP content decreased 70%, Ca2+i and calcium efflux increased 2.5-fold, while the total cell calcium did not change. When the cells were perfused again with O2 and 5 mM glucose, the ATP concentration, Ca2+i, and calcium efflux returned to control levels within 15-20 min. In the presence of 20 mM glucose, anoxia did not produce any change in ATP, in Ca2+i or in calcium efflux. An important source of calcium contributing to the rise in Ca2+i induced by anoxia appears to be extracellular because the rate of rise in Ca2+i is proportional to the extracellular calcium concentration, and because La3+ which blocks calcium influx greatly reduces the rise in Ca2+i. Mitochondria appear to control Ca2+i as well since the early rise in Ca2+i cannot be blocked by La3+ during the initial phase of anoxia, and since the mitochondrial inhibitor carbonyl cyanide p-trifluoromethoxyphenylhydrazone increases Ca2+i further during reoxygenation and slows the return of Ca2+i to control levels.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

Effect of Quin-2 on 45Ca2+ uptake mediated by Na+i/Ca2+o exchange and 45Ca2+ efflux in rat brain synaptosomes: a requirement for [Ca2+]i

The Na+/Ca2+ exchanger of squid axons, barnacle muscle and sarcolemma requires micromolar intracellular calcium for activation in the Na+i/Ca2+o exchange mode ('reverse' Na+/Ca2+ exchange). The requirement for [Ca2+]i has been demonstrated with the use of intracellular calcium buffers, such as Quin-2, to inhibit Na+i/Ca2+o exchange. However, the inhibition of Na+i/Ca2+o exchange in mammalian nerve terminals loaded with Quin-2 has not been observed [7], suggesting a lower sensitivity to low [Ca2+]i for this system. In contrast, the results reported herein indicate that 45Ca2+ uptake in synaptosomes through Na+i/Ca2+o exchange is inhibited by Quin-2 much in the same way as it is in the squid, provided that synaptosomes are preincubated in low Ca2+ medium to avoid saturation of Quin-2. Under these conditions, 45Ca2+ efflux via Ca2+i/Ca2+o exchange is also inhibited. Our results indicate that the Na+i/Ca2+o and Ca2+i/Ca2+o modes of the Na+/Ca2+ exchanger from rat brain synaptosomes require intracellular calcium for activation. However, because no clear relationship between the observed [Ca2+]i values and the inhibition of Na+i/Ca2+o exchange has been found, it is suggested that localised submembrane calcium concentrations not detected by the [Ca2+]i probe might regulate the exchanger.     

In dialyzed squid axons oxidative stress inhibits the Na+/Ca2+ exchanger by impairing the Cai2+-regulatory site

The Na(+)/Ca(2+) exchanger, a major mechanism by which cells extrude calcium, is involved in several physiological and physiopathological interactions. In this work we have used the dialyzed squid giant axon to study the effects of two oxidants, SIN-1-buffered peroxynitrite and hydrogen peroxide (H(2)O(2)), on the Na(+)/Ca(2+) exchanger in the absence and presence of MgATP upregulation. The results show that oxidative stress induced by peroxynitrite and hydrogen peroxide inhibits the Na(+)/Ca(2+) exchanger by impairing the intracellular Ca(2+) (Ca(i)(2+))-regulatory sites, leaving unharmed the intracellular Na(+)- and Ca(2+)-transporting sites. This effect is efficiently counteracted by the presence of MgATP and by intracellular alkalinization, conditions that also protect H(i)(+) and (H(i)(+) + Na(i)(+)) inhibition of Ca(i)(2+)-regulatory sites. In addition, 1 mM intracellular EGTA reduces oxidant inhibition. However, once the effects of oxidants are installed they cannot be reversed by either MgATP or EGTA. These results have significant implications regarding the role of the Na(+)/Ca(2+) exchanger in response to pathological conditions leading to tissue ischemia-reperfusion and anoxia/reoxygenation; they concur with a marked reduction in ATP concentration, an increase in oxidant production, and a rise in intracellular Ca(2+) concentration that seems to be the main factor responsible for cell damage.     

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes.     

Characterization of Na+/H+ exchange in FRTL-5 thyroid cells. Evidence for dependence on activation of protein kinase C.

Na+/H+ exchange activity was investigated in cultured rat thyroid follicular FRTL-5 cells using the pH sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Basal intracellular pH (pHi) was 7.13 +/- 0.10 in cells incubated in Hepes-buffered saline solution. The intracellular buffering capacity beta i was determined using the NH4Cl-pulse method, yielding a beta i value of 85 +/- 12 mM/pH unit. The relationship between extracellular Na+ and the initial rate of alkalinization of acid-loaded cells showed simple saturation kinetics, with an apparent Km value of 44 +/- 26 mM, and an Vmax value of 0.3 +/- 0.01 pH unit/min. The agonist-induced activation of Na+/H+ exchange was investigated in cells acidified with nigericin. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) or ATP induced rapid cytosolic alkalinization in acid-loaded cells. The action of both TPA and ATP was abolished by preincubating the cells with 100 microM amiloride, by substituting extracellular Na+ with equimolar concentrations of choline+, and by pretreating the cells with TPA for 24 h. Chelating extracellular Ca2+, or depleating intracellular Ca2+ pools did not affect the ATP-induced alkalinization. The results indicate, that FRTL-5 cells have a functional Na+/H+ exchange mechanism. Furthermore, stimulation of protein kinase C activity is of importance in activating the antiport.     

Antisense inhibition of Na+/Ca2+ exchange during anoxia/reoxygenation in ventricular myocytes

This study investigated the role of the Na+/Ca2+ exchanger (NCX) in regulating cytosolic intracellular Ca2+ concentration ([Ca2+]i) during anoxia/reoxygenation in guinea pig ventricular myocytes. The hypothesis that the NCX is the predominant mechanism mediating [Ca2+]i overload in this model was tested through inhibition of NCX expression by an antisense oligonucleotide. Immunocytochemistry revealed that this antisense oligonucleotide, directed at the area around the start site of the guinea pig NCX1, specifically reduced NCX expression in cultured adult myocytes by 90 +/- 4%. Antisense treatment inhibited evoked NCX activity by 94 +/- 3% and decreased the rise in [Ca2+]i during anoxia/reoxygenation by 95 +/- 3%. These data suggest that NCX is the predominant mechanism mediating Ca2+ overload during anoxia/reoxygenation in guinea-pig ventricular myocytes.     

Intracellular Ca2+ requirement for activation of the Na+/H+ exchanger in vascular smooth muscle cells

The role for intracellular Ca2+ in modulating activity of the Na+/H+ exchanger was studied in cultured vascular smooth muscle cells. Na+/H+ exchange was activated by four distinct stimuli: 1) phorbol 12-myristate 13-acetate, 2) thrombin, 3) cell shrinkage, and 4) intracellular acid loading. [Ca2+]i was independently varied between 40 and 200 nM by varying the bathing Ca2+ from 10 nM to 5.0 mM. Thrombin-induced intracellular Ca2+ transients were blocked with bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM). In the absence of stimulators of Na+/H+ exchange, varying [Ca2+]i above or below the basal level of 140 nM did not activate Na+/H+ exchange spontaneously. However, varying [Ca2+]i did affect stimulus-induced activation of Na+/H+ exchange. Activation of the exchanger by phorbol 12-myristate 13-acetate was blunted by reduced intracellular Ca2+ (half-maximal activity at 50-90 nM [Ca2+]i), consistent with a Ca2+ requirement for protein kinase C (Ca2+/phospholipid-dependent enzyme). Activation of the exchanger by thrombin in protein kinase C-depleted cells was also sensitive to reduced intracellular Ca2+ (half-maximal activity at 90-140 nM [Ca2+]i) and was increased 40% by raising [Ca2+]i to 200 nM. Activation of the exchanger by cell shrinkage or intracellular acid loads was not significantly affected over the range of [Ca2+]i tested. Thus, altered [Ca2+]i does not itself affect Na+/H+ exchange activity in vascular smooth muscle but instead modulates activation of the transporter by particular stimuli.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

Ca2+ translocation and catalytic activity of the sarcoplasmic reticulum ATPase. Modulation by ATP, Ca2+, and Pi

The ratio between Ca2+ uptake and Ca(2+)-dependent ATP hydrolysis measured in sarcoplasmic reticulum vesicles of rabbit skeletal muscle was found to vary greatly depending on the concentrations of oxalate or Pi used. In the presence of 5 mM oxalate, 20 mM Pi, and 1 mM Pi, the ratios found were in the range of 1.4-2.3, 0.6-0.8, and 0.01-0.10, respectively. The rates of Ca2+ exchange and ATP synthesis were measured at the steady state by adding trace amounts of 45Ca and 32Pi, after the vesicles had been loaded with Ca2+. In the presence of 1 mM Pi, 10 mM MgCl2, and 0.2 mM CaCl2, the ratio between Ca2+ exchange and ATP synthesis varied from 9 to 14. This ratio approached two when Ca2+ in the medium was reduced to a very low level, or when in the presence of Ca2+, dimethyl sulfoxide was added to the assay medium, or when the Pi concentration was raised from 1 to 20 mM. A ratio of two was also measured when the steady state was attained using ITP instead of ATP. In all the conditions that led to a ratio close to two, there was an increase in the fraction of enzyme phosphorylated by Pi. It is proposed that the coupling between Ca2+ translocation and ATP hydrolysis or synthesis is modulated by the phosphorylation of the ATPase by Pi.     

Multisite control of the Crabtree effect in ascites hepatoma cells.

AS-30D hepatoma cells, a highly oxidative and fast-growing tumor line, showed glucose-induced and fructose-induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively. To advance the understanding of the underlying mechanism of this process, the effect of 5 mM glucose or 10 mM fructose on the intracellular concentration of several metabolites was determined. The addition of glucose or fructose lowered intracellular Pi (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8). Glucose and fructose increased the content of AMP (30%), glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate (15, 13 and 50 times, respectively). The cytosolic concentrations of Ca2+ and Mg2+ were not modified. The addition of galactose or glycerol did not modify the concentrations of the metabolites. Mitochondria isolated from AS-30D cells, incubated in media with low Pi (0.6 mM) at pH 6.8, exhibited a 40% inhibition of oxidative phosphorylation. The data suggest that the Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose.     

Video imaging of cytosolic Ca2+ in pancreatic beta-cells stimulated by glucose, carbachol, and ATP.

In order to define the differences in the distribution of cytosolic free Ca2+ ([Ca2+]i) in pancreatic beta-cells stimulated with the fuel secretagogue glucose or the Ca(2+)-mobilizing agents carbachol and ATP, we applied digital video imaging to beta-cells loaded with fura-2.83% of the cells responded to glucose with an increase in [Ca2+]i after a latency of 117 +/- 24 s (mean +/- S.E., 85 cells). Of these cells, 16% showed slow wave oscillations (frequency 0.35/min). In order to assess the relationship between membrane potential and the distribution of the [Ca2+]i rise, digital image analysis and perforated patch-clamp methods were applied simultaneously. The system used allowed sufficient temporal resolution to visualize a subplasmalemmal Ca2+ transient due to a single glucose-induced action potential. Glucose could also elicit a slow depolarization which did not cause Ca2+ influx until the appearance of the first of a train of action potentials. [Ca2+]i rose progressively during spike firing. Inhibition of Ca2+ influx by EGTA abolished the glucose-induced rise in [Ca2+]i. In contrast, the peak amplitude of the [Ca2+]i response to carbachol was not significantly different in normal or in Ca(2+)-deprived medium. Occasionally, the increase of the [Ca2+]i rise was polarized to one area of the cell different from the subplasmalemmal rise caused by glucose. The amplitude of the response and the number of responding cells were significantly increased when carbachol was applied after the addition of high glucose (11.2 mM). ATP also raised [Ca2+]i and promoted both Ca2+ mobilization and Ca2+ influx. The intracellular distribution of [Ca2+]i was homogeneous during the onset of the response. A polarity in the [Ca2+]i distribution could be detected either in the descending phase of the peak or in subsequent peaks during [Ca2+]i oscillations caused by ATP. In the absence of extracellular Ca2+, the sequential application of ATP and carbachol revealed that carbachol was still able to raise [Ca2+]i after exhaustion of the ATP response. This may be due to desensitization to the former agonist, since the response occurred in the same area of the cell. These results reveal subtle differences in [Ca2+]i distribution following membrane depolarization with glucose or the application of Ca(2+)-mobilizing agonists.     

In dialyzed squid axons Ca2+i activates Ca2+o-Na+i and Na+o-Na+i exchanges in the absence of Ca chelating agents

We used internally dialyzed squid axons to explore whether the reported activatory effect of Ca2+i on the partial reactions of the Na+-Ca2+ exchange (essential activator) is secondary to the presence of Ca2+ chelating agents in the internal medium. The effect of Ca2+i pulses on both the reverse (Ca2+o-dependent Na+ efflux) and Na+-Na+ exchange (Na+o-dependent Na+ efflux) modes of the Na+-Ca2+ exchange was studied in axons dialyzed without EGTA. For these experiments a substantial inhibition of the Ca2+ buffer capacity of the axoplasm was achieved by the use of Ruthenium red (10-20 microM), cyanide (1 mM) and vanadate (1 mM) in the dialysis solution. Our results indicate that the Ca2+i requirement of the reverse and Na+-Na+ exchange can not be explained by a direct inhibition of the Na+-Ca2+ exchanger by EGTA. In fact, both modes of operation of the exchanger can be activated by internal Ca2+ ions in the complete absence of Ca2+ chelating agents thus indicating that the 'catalytic' effect of Ca2+i on the Na+-Ca2+ exchanger is a real phenomenon.     

Ouabain-insensitive Na+ stimulation of a microsomal Mg2+ -ATPase in gills of sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain a Mg2+-dependent Na+-stimulated ATPase activity in the absence of K+, whose characteristics are compared with those of the (Na+ + K+)-ATPase of the same preparations. The activity at 30 degrees C is 11.3 mumol Pi X mg-1 protein X hr-1 under optimal conditions (5 mM MgATP, 75 mM Na+, 75 mM HEPES, pH 6.0) and exhibits a lower pH optimum than the (Na+ + K+)-ATPase. The Na+ stimulation of ATPase is only 17% inhibited by 10-3M ouabain and completely abolished by 2.5 mM ethacrinic acid which on the contrary cause, respectively, 100% and 34% inhibition of the (Na+ + K+)-ATPase. Both Na+-and (Na+ + K+)-stimulated activities can hydrolyze nucleotides other than ATP in the efficiency order ATP greater than CTP greater than UTP greater than GTP and ATP greater than CTP greater than GPT greater than UTP, respectively. In the presence of 10(-3)M ouabain millimolar concentrations of K+ ion lower the Na+ activation (90% inhibition at 40 mM K+). The Na+-ATPase is less sensitive than (Na+ + K+)-ATPase to the Ca2+ induced inhibition as the former is only 57.5% inhibited by a concentration of 1 X 10(-2)M which completely suppresses the latter. The thermosensitivity follows the order Mg2+--greater than (Na+ + K+)--greater than Na+-ATPase. A similar break of the Arrhenius plot of the three enzymes is found. Only some of these characteristics do coincide with those of a Na+-ATPase described elsewhere. A presumptive physiological role of Na+-ATPase activity in seawater adapted teleost gills is suggested.     

Parathyroid hormone inhibition of Na+/phosphate cotransport in OK cells: intracellular [Ca2+] as a second messenger

Parathyroid hormone increases cellular cAMP, 1,2-diacylglycerol, inositol 1,4,5-trisphosphate and cytosolic Ca2+ concentration ([Ca2+]i) in OK cells. In the present study, we determined the importance of the PTH-dependent increase in [Ca2+]i in the control of sodium-dependent phosphate (Na+/Pi) cotransport. PTH (10(-7) M) results in a transient increase in [Ca2+]i from basal levels of 67 +/- 4 nM to maximal concentrations of 190 +/- 9 nM. The increase in [Ca2+]i was dose-dependent with half-maximal increases at about 5.10(-8) M PTH. These hormone levels were 10(3)-fold higher than that required for half-maximal inhibition of Na+/Pi cotransport. Clamping [Ca2+]i with either intracellular Ca2+ chelators or by ionomycin in the presence of high concentrations of extracellular Ca2+ did not alter PTH-dependent inhibition of Na/Pi cotransport. Nor did indomethacin, an inhibitor of the cyclooxygenase pathway, influence the hormonal inhibition of cotransport. Accordingly, these data suggest that changes in [Ca2+]i and/or activation of the phospholipase A2 and the cyclooxygenase pathways are not involved in signal induction of the PTH-mediated control of Na+/Pi cotransport.     

(Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in kidney of sea bass (Dicentrarchus labrax L.)

1. Sea bass kidney microsomal preparations contain two Mg2+ dependent ATPase activities: the ouabain-sensitive (Na+ + K+)-ATPase and an ouabain-insensitive Na+-ATPase, requiring different assay conditions. The (Na+ + K+)-ATPase under the optimal conditions of pH 7.0, 100 mM Na+, 25 mM K+, 10 mM Mg2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 mumol Pi/mg protein per hr whereas the Na+-ATPase under the conditions of pH 6.0, 40 mM Na+, 1.5 mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 mumol Pi/mg protein per hr. 2. The (Na+ + K+)-ATPase is specifically inhibited by ouabain and vanadate; the Na+-ATPase specifically by ethacrynic acid and preferentially by frusemide; both activities are similarly inhibited by Ca2+. 3. The (Na+ + K+)-ATPase is specific for ATP and Na+, whereas the Na+-ATPase hydrolyzes other substrates in the efficiency order ATP greater than GTP greater than CTP greater than UTP and can be activated also by K+, NH4+ or Li+. 4. Minor differences between the two activities lie in the affinity for Na+, Mg2+, ATP and in the thermosensitivity. 5. The comparison between the two activities and with what has been reported in the literature only partly agree with our findings. It tentatively suggests that on the one hand two separate enzymes exist which are related to Na+ transport and, on the other, a distinct modulation in vivo in different tissues.     

Mechanisms of regulation of intracellular distribution of Ca2+ in adipose tissue

The kinetics of influx and efflux of 45Ca and its accumulation by the subcellular membranes of adipose tissue have been studied. The initial rate of Ca2+ efflux does not depend on the intracellular concentration of Na+ and K+. The rate of exchange between intracellular 45Ca and 40Ca of the incubation medium is independent on concentration of Na+ and K+ in the incubation mixture. This suggests the absence of Na,Ca-transmembrane exchange in the adipocytes. The changes in the ratio of intracellular concentration of Na+ and K+ by the factors inhibiting the activity ofNa,K-ATPase cause redistribution of Ca in the intracellular pools of the adipocytes. The lypolytic agents (adrenalin, adrenocorticotropic hormone, caffeine) but not dibutytyl-3' : 5'-AMP, accelerate Ca2+ efflux from the adipocytes. At physiological concentrations of ATP, succinate and Pi the highest Ca-accumulating activity is observed in adipose tissue mitochondria. The highest initial rate of Ca uptake, as in the case of contractile tissues, is detected in the endoplasmic reticulum membranes. In contrast to the plasma membranes and reticulum, in which the Ca-accumulating capacity is independent of ATP concentration up to 0.5 mM, the Ca-accumulating capacity of mitochondria decreases 8--9-fold with a reduction in ATP concentration from 4 down to 1 mM. The physiological significance of this phenomenon in the action mechanism of lipolytic agents, which reduce the ATP content in the adipocytes, is discussed.     

Secretagogue-induced mobilization of an intracellular Mg2+ pool in rat sublingual mucous acini.

The regulation of the intracellular free Mg2+ concentration ([Mg2+]i) was monitored in rat sublingual mucous acini using dual wavelength microfluorometry of the Mg(2+)-sensitive dye mag-fura-2. Acini attached to coverslips and superfused continuously with a Mg(2+)-containing medium (0.8 mM) have a steady-state [Mg2+]i of 0.35 +/- 0.01 mM. Adjusting the extracellular Mg2+ concentration to 0 and 10 mM or removing extracellular Na+ did not alter the resting [Mg2+]i. Stimulation with the Ca(2+)-mobilizing, muscarinic agonist, carbachol, induced a sustained increase in [Mg2+]i (approximately 50%; t1/2 < 20 s; Kd approximately 1.5 microM), the magnitude and the duration of which were unchanged in Mg(2+)-depleted medium indicating that the rise in [Mg2+]i was generated by Mg2+ release from an intracellular Mg2+ pool. Forskolin, which increases the intracellular cAMP content, produced a small, transient increase in the [Mg2+]i (< 10%). Muscarinic stimulation in a Ca(2+)-free medium blunted the initial increase in [Mg2+]i by approximately 50%, whereas the sustained increase in [Mg2+]i was lost. When the muscarinic-induced increase in [Ca2+]i was blocked by 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate, an inhibitor of the agonist-sensitive intracellular Ca2+ release pathway, both the initial and the sustained phases of the increase in [Mg2+]i were virtually eliminated. Thapsigargin and 2,5-di-(terbutyl)-1,4-benzohydroquinone, which increase [Ca2+]i by inhibiting microsomal Ca(2+)-ATPase, caused a dramatic increase in [Mg2+]i. Stimulation in a Na(+)-free medium or in the presence of bumetanide, an inhibitor of Na+/K+/2Cl- cotransport, blunted the agonist-induced rise in [Mg2+]i (approximately 50%), whereas ouabain, a Na+,K(+)-ATPase inhibitor, had no significant effect. FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), a mitochondrial uncoupler, mobilized an intracellular Mg2+ pool as well. The carbachol-induced increase in [Mg2+]i was markedly inhibited by FCCP (approximately 80%), suggesting that the same pool(s) of Mg2+ were primarily involved. The above results provide strong evidence that Ca(2+)-mobilizing agonists increase cytoplasmic free [Mg2+] by releasing an intracellular pool of Mg2+ that is associated with a rise in the [Na+]i.