Membrane transport of potassium ions in erythrocytes of the American black bear, Ursus americanus

1. Membrane transport of K ions was investigated in red blood cells of bears by methods of measurement of unidirectional isotopic fluxes. 2. Unlike red cells of dogs, red cells of bears exhibited a significant, though small, component of ouabain-sensitive K influx. 3. Ouabain-insensitive K influx, as in other carnivore cells, was activated by swelling and inhibited by shrinkage. Swelling-induced K influx was dependent upon presence of chloride ions but was not inhibited by furosemide or bumetanide. 4. Ouabain-sensitive K influx was largest with ATP and with high concentration of Na in the cell, but it persisted in the absence of cytoplasmic Na or ATP. It was also resistant to the drug, harmaline, at a concentration that in other cells fully inhibits ouabain-sensitive K influx. 5. It was concluded that under such adverse conditions ouabain-sensitive K influx represents another mode of the Na/K pump not fully described elsewhere. 6. Also, as in low K red cells of sheep and goat, apparent absence of Na/K pump activity in carnivore red cells may represent suppression rather than elimination of activity. 7. Ouabain-insensitive K influx showed a seasonal pattern with minima occurring in early winter, earlier than for the minimum observed in Na influx. 8. Ouabain-sensitive K influx tended to be lower in the hibernation season of the bear, but the seasonal pattern was not consistent.     

Lithium transport pathways in human red blood cells

In human red cells, Li is extruded against its own concentration gradient if the external medium contains Na as a dominant cation. This uphill net Li extrusion occurs in the presence of external Na but not K, Rb, Cs, choline, Mg, or Ca, is ouabain-insensitive, inhibited by phloretin, and does not require the presence of cellular ATP. Li influx into human red cells has a ouabain-sensitive and a ouabain-insensitive but phloretin-sensitive component. Ouabain-sensitive Li influx is competitively inhibited by external K and Na and probably involves the site on which the Na-K pump normally transports K into red cells. Ouabain does not inhibit Li efflux from red cells containing Li concentrations below 10 mM in the presence of high internal Na or K, whereas a ouabain-sensitive Li efflux can be measured in cells loaded to contain 140 mM Li in the presence of little or no internal Na or K. Ouabain-insensitive Li efflux is stimulated by external Na and not by K, Rb, Cs, choline, Mg, or Ca ions. Na-dependent Li efflux does not require the presence of cellular ATP and is inhibited by phloretin, furosemide, quinine, and quinidine. Experiments carried out in cells loaded in the presence of nystatin to contain either only K or only Na show that the ouabain-insensitive, phloretin-inhibited Li movements into or out of human red cells are stimulated by Na on the trans side and inhibited by Na on the cis side of the red cell membrane. The characteristics of the Na-dependent unidirectional Li fluxes and uphill Li extrusion are similar, suggesting that they are mediated by the same Na-Li countertransport system.     

Hypoxia-induced increase in intracellular calcium concentration in endothelial cells: role of the Na(+)-glucose cotransporter.

Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet-activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca(2+)](i)) which then activates the phospholipase A(2) (PLA(2)). The link between the decrease in ATP and the increase in [Ca(2+)](i) was not known and is investigated in this work. We first showed that the presence of extracellular Na(+) was necessary to observe the hypoxia-induced increase in [Ca(2+)](i) and the activation of PLA(2). This increase was not due to the release of Ca(2+) from intracellular stores, since thapsigargin did not inhibit this process. The Na(+)/Ca(2+) exchanger was involved since dichlorobenzamil inhibited the [Ca(2+)](i) and the PLA(2) activation. The glycolysis was activated, but the intracellular pH (pH(i)) in hypoxic cells did not differ from control cells. Finally, the hypoxia-induced increase in [Ca(2+)](i) and PLA(2) activation were inhibited by phlorizin, an inhibitor of the Na(+)-glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na(+) through the activated Na(+)-glucose cotransport followed by the activation of the Na(+)/Ca(2+) exchanger, resulting in a net influx of Ca(2+).     

Extracellular ATP induces ion fluxes and inhibits growth of Friend erythroleukemia cells

Extracellular ATP (1 mM) inhibited the growth of Friend virus-infected murine erythroleukemia cells (MEL cells) but had no effect on dimethyl sulfoxide-induced differentiation. ATP (1 mM) also caused changes in the permeability of MEL cells to ions. There was an increased influx of 45Ca2+ from a basal level of 5 pmol/min to 18 pmol/min/10(6) cells to achieve a 2-fold increase in steady-state Ca2+ as measured at isotopic equilibration. Ca2+ influx was blocked by diisothiocyanostilbene disulfonate (DIDS), an inhibitor of anion transport. ATP also stimulated Cl- uptake, and this flux was inhibited by DIDS. The ratio of ATP stimulated Cl- to Ca2+ uptake was 1.6:1. K+ and Na+ influx were also stimulated by ATP, but phosphate uptake was inhibited; the Na+ influx dissipated the Na+ gradient and thus inhibited nutrient uptake. ATP-stimulated K+ influx was ouabain inhibitable; however, the total cellular K+ decreased due to an ATP-stimulated ouabain-resistant K+ efflux. Na+ influx and Ca2+ influx occurred by separate independent routes, since Na+ influx was not inhibited by DIDS. The effects observed were specific for ATP *K1/2 MgATP = 0.7 mM) since AMP, GTP, adenosine, and the slowly hydrolyzable ATP analogue adenyl-5'-yl imidodiphosphate were without effect. The major ionic changes in the cell were a decrease in K+ and increase in Na+; cytoplasmic pH and free Ca2+ did not change appreciably. These ATP-induced changes in ion flux are considered to be responsible for growth inhibition.     

Calcium transport mechanisms in dog red blood cells studied from measurements of initial flux rates

Ca2+ efflux from dog red blood cells loaded with Ca2+ using the A23187 ionophore could be separated into two main components: (1) Mg- and ATP-dependent (active transport) and (2) dependent on external Na (K1/2 around 15 mM); at 80 microM internal free Ca the relative magnitudes of these fluxes were 70% and 30% respectively. The Na-dependent Ca2+ efflux had the following additional properties: (i) it was partially inhibited by ATP depletion or preincubation with vanadate, but it was not affected by Mg2+ depletion; (ii) it failed to be stimulated by external monovalent cations other than Na: (iii) it was stimulated by reduction in the internal Na+ concentration. Both active and Na-dependent Ca2+ efflux remained unchanged in hypotonic solutions or in solutions with alkaline pH (8.5). In cells containing ATP and Mg2+, external Ca2+ inhibited Ca2+ efflux (K1/2 around 1 mM); on the other hand, in Mg-free dog red cells external Ca2+ stimulated Ca2+ efflux (K1/2 about 30 microM). In Mg-depleted red cells incubated in the absence of external Na2+, Ca2+ influx as a function of external Ca2+ followed a monotonically saturable function (K1/2 around 20 microM): addition of Na resulted in (i) inhibition of Ca2+ influx and (ii) a sigmoid relationship between flux and external Ca2+. Intracellular Ca2+ stimulated the external Na-dependent Ca2+ efflux along a sigmoid curve (K1/2 around 30 microM); on the other hand the Ca pump had a biphasic response to internal Ca2+: stimulation at low internal Ca2+ (K1/2 between 1 and 10 microM), followed by a decline at internal Ca2+ concentrations higher than 50 microM.     

Ca2+-activated Na+ fluxes in human red cells. Amiloride sensitivity

The effect of Ca2+ on the ouabain- and bumetanide-resistant Na+ fluxes in intact red cells was studied at relatively constant internal Ca2+, membrane potential, and cell volume. The red cell calcium concentration was modified using the ionophore A23187. In fresh red cells, the Na+ influx and efflux (1.2 +/- 0.13 and 0.26 +/- 0.07 mmol/liter cells x h, respectively) were not affected by amiloride (1 mM). When external Ca2+ was raised from 0 to 150 microM, in the presence of A23187, both the Na+ influx and efflux were stimulated (about 3.5-fold). The Ca2+-activated Na+ efflux and influx had an apparent Km for activation by Ca2+o of about 25 microM. The Ca2+-dependent Na+ transport was inhibited 30-60% by amiloride (ID50 = 17.3 +/- 8 microM). Amiloride, however, had no effect on the Ca2+-dependent K+ influx. The amiloride-sensitive (AS) transport pathway was a linear function of the Na+o concentration in the range from 0 to 75 mM. The Ca2+i activation seems to depend on the metabolic integrity of red cells. 1) It does not take place in ATP-depleted red cells; 2) ATP-repletion of ATP-depleted red cells fully restored AS Na influx; and 3) ATP-enrichment (ATP-red cells) enhanced the AS Na influx by about 100%. The Ca2+-activated AS Na+ influx was not affected by either DIDS or trifluoperazine. The present results indicate that in human erythrocytes an increase in internal Ca2+ activates on otherwise silent AS Na+-transport system, which is dependent on the metabolic integrity of the red cells.     

Effect of cell ageing on Ca2+ influx into human red cells

The effect of cell ageing on Ca2+ entry was studied in this work, using sub-populations of young and old human red cells, separated by stringent percoll density gradients. Additionally, the influence of an osmotic gradient was investigated as a model for shear stress. Ca2+ entry was assessed at 37 degrees C, under conditions where the Ca2+ pump was either inhibited by NaVO3 (0.5-10 mM) or inactivated by ATP depletion. The entry was linear with time up to 1 h. No differences in Ca2+ influx between the two sub-populations were detected in isotonic Na(+)-medium. In contrast, after incubation in anisosmotic media, Ca2+ entry into old cells was significantly higher than into younger cells. In hypotonic Na(+)-medium, the entry into old cells was not affected by La3+ (10 microM) whilst it was partially blocked by Gd3+ at a similar level (half-maximal effect attained with about 1 microM Gd3+). The entry into young cells was only slightly stimulated by these lanthanides at low concentrations (10 microM), regardless of the tonicity of incubation medium. Further increasing Gd3+ levels above 10 microM markedly enhanced Ca2+ entry into both cell types. The selective blockade of Ca2+ influx by low Gd3+ concentrations suggests presence of mechano-sensitive channels, that become preferentially activated in old cells. Activation of these channels during in-vivo microcirculation may help to explain the increased Ca2+ content of senescent cells.     

Sodium and calcium movements in dog red blood cells

Determinants of 45Ca influx, 45Ca efflux, and 22Na efflux were examined in dog red blood cells. 45Ca influx is strongly influenced by the Na concentration on either side of the membrane, being stimulated by intracellular Na and inhibited by extracellular Na. A saturation curve is obtained when Ca influx is plotted as a function of medium Ca concentration. The maximum Ca influx is a function of pH (increasing with greater alkalinity) and cell volume (increasing with cell swelling). Quinidine strongly inhibits Ca influx. Efflux of 45Ca is stimulated by increasing concentrations of extracellular Na. 22Na efflux is stimulated by either Ca or Na in the medium, and the effects of the two ions are mutually exclusive rather than additive. Quinidine inhibits Ca-activated 22Na efflux. The results are considered in terms of a model for Ca-Na exchange, and it is concluded that the system shows many features of such a coupled ion transport system. However, the stoichiometric ratio between Ca influx and Ca-dependent Na efflux is highly variable under different experimental conditions. Because the Ca fluxes may reflect a combination of ATP-dependent, outward transport and Na-linked passive movements, the true stoichiometry of an exchanger may not be ascertainable in the absence of a specific Ca pump inhibitor. The meaning of these observations for Ca-dependent volume regulation by dog red blood cells is discussed.     

The ATP4- receptor-operated ion channel of human lymphocytes: inhibition of ion fluxes by amiloride analogs and by extracellular sodium ions.

Extracellular ATP is known to increase the membrane permeability of a variety of cells. Addition of ATP to human leukemic lymphocytes loaded with the Ca2+ indicator, fura-2, induced a rise in cytosolic Ca2+ concentration which was attenuated or absent in NaCl media compared with KCl, choline Cl, or NMG Cl media. In contrast, anti-immunoglobulin antibody gave similar Ca2+ transients in NaCl and KCl media. A half-maximal inhibition of peak ATP-induced Ca2+ response was observed at 10-16 mM extracellular Na+. Basal 45Ca2+ influx into lymphocytes was stimulated 9.6-fold by ATP added to cells in KCl media, but the effect of ATP was greatly reduced for cells in NaCl media. Hexamethylene amiloride blocked 74% of the ATP-stimulated Ca45 uptake of cells in KCl media. Flow cytometry measurements of fluo-3-loaded cells confirmed that the ATP-induced rise in cytosolic Ca2+ was inhibited either by extracellular Na+ or by addition of hexamethylene amiloride. Extracellular ATP stimulated 86Rb efflux from lymphocytes 10-fold and this increment was inhibited by the amiloride analogs in a rank order of potency 5-(N-methyl-N-isobutyl)amiloride greater than 5-(N,N-hexamethylene)amiloride greater than 5-(N-ethyl-N-isopropyl)amiloride greater than amiloride. ATP-induced 86Rb efflux showed a sigmoid dependence on the concentration of ATP and Hill analysis gave K1/2 of 90 and 130 microM and n values of 2.5 and 2.5 for KCl and NaCl media, respectively. However, the maximal ATP-induced 86Rb efflux was 3-fold greater in KCl than in NaCl media. Raising extracellular Na+ from 10 to 100 mM increased ATP-induced Na+ influx from a mean of 2.0 to 3.7 nEq/10(7) cells/min, suggesting either saturability or self-inhibition by Na+ of its own influx. These data suggest that ATP opens a receptor-operated ion channel which allows increased Ca2+ and Na+ influx and Rb+ efflux and these fluxes are inhibited by extracellular Na+ ions as well as by the amiloride analogs.     

Delivery of ion pumps from exogenous membrane-rich sources into mammalian red blood cells.

Using polyethylene glycol-mediated fusion of ATP-ase-enriched (native) microsomes with red blood cells, we have delivered sarcoplasmic reticulum (SR) Ca-ATPase and kidney Na,K-ATPase into the mammalian erythrocyte membrane. Experiments involving delivery of the SR Ca-ATPase into human red cells were first carried out to assess the feasibility of the fusion protocol. Whereas there was little detectable 45Ca2+ uptake into control cells in either the absence or presence of extracellular ATP, a marked time-dependent uptake of 45Ca2+ was observed in the presence of ATP in cells fused with SR Ca-ATPase. Comparison of the kinetics of uptake into microsome-fused cells versus native SR vesicles supports the conclusion of true delivery of pumps into the red cell membrane. Thus, the time to reach steady state was more than two orders of magnitude longer in the (large) cells versus the native SR vesicles. Na,K-ATPase from dog and rat kidney microsomes were fused with red cells of humans, sheep, and dogs. Using dog kidney microsomes fused with dog red cells which are practically devoid of Na,K-ATPase, functional incorporation of sodium pumps was evidenced in ouabain-sensitive Rb+ uptake and Na+ efflux energized by intracellular ATP, as well as in ATP-stimulated Na+ influx and Rb+ efflux from inside-out membrane vesicles prepared from the fusion-treated cells. From analysis of the biphasic kinetics of ouabain-sensitive Na+ efflux under conditions of limited intracellular Na+ concentration, it is concluded that the kidney pumps are incorporated into a relatively small fraction (approximately 15%) of the red cells. This system provides a uniquely useful system for studying the behavior of native sodium pumps in a compartment (red cell) of small surface/volume ratio. The newly incorporated native kidney pumps, while of the same isoform as the endogenous red cell pump, behave differently from the endogenous red cell sodium pump with respect to their very low "uncoupled" Na+/O flux activity.     

Instrumental role of Na+ in NMDA excitotoxicity in glucose-deprived and depolarized cerebellar granule cells

In glucose-deprived cerebellar granule cells, substitution of extracellular Na+ with Li+ or Cs+ prevented N-methyl-D-aspartate (NMDA)-induced excitotoxicity. NMDA stimulated 45Ca2+ accumulation and ATP depletion in a Na-dependent manner, and caused neuronal death, even if applied while Na,K-ATPase was inhibited by 1 mM ouabain. The cells treated with NMDA in the presence of ouabain accumulated sizable 45Ca2+ load but most of them failed to elevate cytosolic [Ca2+] upon mitochondrial depolarization. Na/Ca exchange inhibitor, KB-R7943, inhibited Na-dependent and NMDA-induced 45Ca2+ accumulation but only if Na,K-ATPase activity was compromised by ouabain. In cells energized by glucose and exposed to NMDA without ouabain, KB-R7943 reduced NMDA-elicited ionic currents by 19% but failed to inhibit 45Ca2+ accumulation. It appears that a large part of NMDA-induced Ca2+ influx in depolarized and glucose-deprived cells is mediated by reverse Na/Ca exchange. A high level of reverse Na/Ca exchange operation is maintained by a sustained Na+ influx via NMDA channels and depolarization of the plasma membrane. In cells energized by glucose, however, most Ca2+ enters directly via NMDA channels because Na,K-ATPase regenerating Na+ and K+ concentration gradients prevents Na/Ca exchange reversal. Since under these conditions Na/Ca exchange extrudes Ca2+, its inhibition destabilizes Ca2+ homeostasis.     

Kinetics and stoichiometry of coupled Na efflux and Ca influx (Na/Ca exchange) in barnacle muscle cells

Coupled Na+ exit/Ca2+ entry (Na/Ca exchange operating in the Ca2+ influx mode) was studied in giant barnacle muscle cells by measuring 22Na+ efflux and 45Ca2+ influx in internally perfused, ATP-fueled cells in which the Na+ pump was poisoned by 0.1 mM ouabain. Internal free Ca2+, [Ca2+]i, was controlled with a Ca-EGTA buffering system containing 8 mM EGTA and varying amounts of Ca2+. Ca2+ sequestration in internal stores was inhibited with caffeine and a mitochondrial uncoupler (FCCP). To maximize conditions for Ca2+ influx mode Na/Ca exchange, and to eliminate tracer Na/Na exchange, all of the external Na+ in the standard Na+ sea water (NaSW) was replaced by Tris or Li+ (Tris-SW or LiSW, respectively). In both Na-free solutions an external Ca2+ (Cao)-dependent Na+ efflux was observed when [Ca2+]i was increased above 10(-8) M; this efflux was half-maximally activated by [Ca2+]i = 0.3 microM (LiSW) to 0.7 microM (Tris-SW). The Cao-dependent Na+ efflux was half-maximally activated by [Ca2+]o = 2.0 mM in LiSW and 7.2 mM in Tris-SW; at saturating [Ca2+]o, [Ca2+]i, and [Na+]i the maximal (calculated) Cao-dependent Na+ efflux was approximately 75 pmol#cm2.s. This efflux was inhibited by external Na+ and La3+ with IC50's of approximately 125 and 0.4 mM, respectively. A Nai-dependent Ca2+ influx was also observed in Tris-SW. This Ca2+ influx also required [Ca2+]i greater than 10(-8) M. Internal Ca2+ activated a Nai-independent Ca2+ influx from LiSW (tracer Ca/Ca exchange), but in Tris-SW virtually all of the Cai-activated Ca2+ influx was Nai-dependent (Na/Ca exchange). Half-maximal activation was observed with [Na+]i = 30 mM. The fact that internal Ca2+ activates both a Cao-dependent Na+ efflux and a Nai-dependent Ca2+ influx in Tris-SW implies that these two fluxes are coupled; the activating (intracellular) Ca2+ does not appear to be transported by the exchanger. The maximal (calculated) Nai-dependent Ca2+ influx was -25 pmol/cm2.s. At various [Na+]i between 6 and 106 mM, the ratio of the Cao-dependent Na+ efflux to the Nai-dependent Ca2+ influx was 2.8-3.2:1 (mean = 3.1:1); this directly demonstrates that the stoichiometry (coupling ratio) of the Na/Ca exchange is 3:1. These observations on the coupling ratio and kinetics of the Na/Ca exchanger imply that in resting cells the exchanger turns over at a low rate because of the low [Ca2+]i; much of the Ca2+ extrusion at rest (approximately 1 pmol/cm2.s) is thus mediated by an ATP-driven Ca2+ pump.(ABSTRACT TRUNCATED AT 400 WORDS)     

Non-selective cation channels and oxidative stress-induced cell swelling

Necrosis is considered as a non-specific form of cell death that induces tissue inflammation and is preceded by cell swelling. This increase in cell volume has been ascribed mainly to defective outward pumping of Na+ caused by metabolic depletion and/or to increased Na+ influx via membrane transporters. A specific mechanism of swelling and necrosis driven by the influx of Na+ through nonselective cation channels has been recently proposed (Barros et al., 2001a). We have characterized further the properties of the nonselective cation channel (NSCC) in HTC cells. The NSCC shows a conductance of approximately 18 pS, is equally permeable to Na+ and K+, impermeant to Ca2+, requires high intracellular Ca2+ as well as low intracellular ATP for activation and is inhibited by flufenamic acid. Hydrogen peroxide induced a significant increase in cell volume that was dependent on external Na+. We propose that the NSCC, which is ubiquitous though largely inactive in healthy cells, becomes activated under severe oxidative stress. The ensuing Na+ influx initiates via positive feedback a series of metabolic and electrolytic disturbances, resulting in cell death by necrosis.     

ATP modulation of mitogen activated protein kinases and intracellular Ca2+ in breast cancer (MCF-7) cells

In the breast tumor cell line MCF-7, extracellular nucleotides induce transient elevations in intracellular calcium concentration ([Ca(2+)](i)). In this study we show that stimulation with ATP or UTP sensitizes MCF-7 cells to mechanical stress leading to an additional transient Ca(2+) influx. ATP> or =ATPgamma-S> or =UTP>ADP=ADPbeta-S elevate [Ca(2+)](i), proving the presence of P2Y(2)/P2Y(4) purinergic receptor subtypes. In addition, cell stimulation with ATP, ATPgamma-S or UTP but not ADPbeta-S induced the phosphorylation of ERK1/2, p38 and JNK1/2 mitogen activated protein kinases (MAPKs). The use of Gd(3+), La(3+) or a Ca(2+)-free medium, inhibited ATP-dependent stress activated Ca(2+) (SAC) influx, but had no effect on MAPK phosphorylation. ATP-induced activation of MAPKs was diminished by two PI-PLC inhibitors and an IP(3) receptor antagonist. These results evidence an ATP-sensitive SAC influx in MCF-7 cells and indicate that phosphorylation of MAPKs by ATP is dependent on PI-PLC/IP(3)/Ca(2+)(i) release but independent of SAC influx in these cells, differently to other cell types.     

The sodium and potassium activated ATPase. II. Comparative study of intestinal epithelium and red cells

The Na-K ATPase found in sedimentable fractions of intestinal epithelium of rats hydrolyzed cytidine triphosphate nearly as well as ATP (25% to 50%); was active only in presence of divalent cations, with specificity for Mg (100%), Mn (50%) and Ca (10%); showed a plateau of activation when Mg concentrations were in excess of substrate; and was inhibited by a second divalent cation (Zn > Mn > Ca), and by 3 × 10^?4 M ouabain (50%). Parallel assays of rat red cell ghosts showed differences in substrate specificity (CTP was not utilized), in activation kinetics (activation peak with Mg) and in greater specificity to Mg (Mn was a weaker activator and Zn was a weaker inhibitor). Stabilities also differed in the two preparations: Na? K ATPase of intestinal epithelium was activated by sucrose extraction and denatured during cytolysis at room temperature, while that of red cell fragments was denatured during sucrose extraction and preserved by hemolysis at room temperature. Other properties of Na? K ATPase studied in the two tissues included activation by monovalent cations (optimum at 160 mM Na, 15 mM K), specificity to monovalent cations, and sensitivity to lipid solvents and to some drugs. The data were discussed in terms of comparative properties of Na? K ATPases of various cells. Residual ATPase activities of intestinal epithelium and red cell ghosts were shown to differ in substrate specificity, inhibition and activation. “Residual ATPase” from intestinal epithelium was a zinc-activated nucleoside polyphosphate phosphohydrolase, while ghosts contained Mg? ATPase. Only the latter enzyme was specific to ATP and Mg, activated by Ca in presence of Mg, and sensitive to inhibition by PCMB and Zn.     

Evidence for receptor-mediated calcium entry and refilling of intracellular calcium stores in FRTL-5 rat thyroid cells.

The aim of the present study was to investigate the relationship between agonist-induced changes in intracellular free Ca2+ ([Ca2+]i) and the refilling of intracellular Ca2+ stores in Fura 2-loaded thyroid FRTL-5 cells. Stimulating the cells with ATP induced a dose-dependent increase in ([Ca2+]i). The ATP-induced increase in [Ca2+]i was dependent on both release of sequestered intracellular Ca2+ as well as influx of extracellular Ca2+. Addition of Ni2+ prior to ATP blunted the component of the ATP-induced increase in [Ca2+]i dependent on influx of Ca2+. In cells stimulated with ATP in a Ca(2+)-free buffer, readdition of Ca2+ induced a rapid increase in [Ca2+]i; this increase was inhibited by Ni2+. In addition, the ATP-induced influx of 45Ca2+ was blocked by Ni2+. Stimulating the cells with noradrenaline (NA) also induced release of sequestered Ca2+ and an influx of extracellular Ca2+. When cells were stimulated first with NA, a subsequent addition of ATP induced a blunted increase in [Ca2+]i. If the action of NA was terminated by addition of prazosin, and ATP was then added, the increase in [Ca2+]i was restored to control levels. Addition of Ni2+ prior to prazosin inhibited the restoration of the ATP response. In the presence of extracellular Mn2+, ATP stimulated quenching of Fura 2 fluorescence. The quenching was probably due to influx of Mn2+, as it was blocked by Ni2+. The results thus suggested that stimulating release of sequestered Ca2+ in FRTL-5 cells was followed by influx of extracellular Ca2+ and rapid refilling of intracellular Ca2+ stores.     

Activation of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange by protein phosphatase inhibitors in red blood cells of the frog<Emphasis Type="Italic"> Rana ridibunda</Emphasis>

The present study was designed to evaluate the role of protein phosphatases in regulation of sodium transport in the marsh frog erythrocytes using 22Na as a tracer. For this purpose the cells were treated with several known inhibitors of protein phosphatases. In standard isotonic medium, exposure of the cells to 10 mmol l(-1) NaF, 20 nmol l(-1) calyculin A or 0.1 mmol l(-1) cantharidin resulted in a significant (1.7-fold) increase in unidirectional ouabain-insensitive Na+ influx. The Na+ influx in frog red cells was progressively activated as the medium osmolality was increased by addition of 100, 200 or 300 mmol l(-1) sucrose to standard isotonic medium. The stimulatory effect of protein phosphatase blockers on Na+ influx was much higher in hypertonic medium containing 100 or 200 mmol l(-1) sucrose than that in isotonic medium. Stimulation of Na+ transport enhanced with increasing concentrations of calyculin A, and half-maximal activation (EC50) was obtained at 16 nmol l(-1). However, Na+ influx induced by strong hypertonic treatment (+300 mmol l(-1) sucrose) was not altered further in the presence of protein phosphatase inhibitors. The changes in Na+ influx evoked by protein phosphatase inhibitors and hypertonic treatment were associated with a rise in the intracellular Na+, but not K+, content. Enhancement in Na+ influx after addition of protein phosphatase blockers to cell suspension in isotonic or hypertonic media was almost completely inhibited by Na+/H+ exchange inhibitors, amiloride and ethyl-isopropyl-amiloride. The basal Na+ influx in frog erythrocytes in isotonic medium was relatively low (1.7 mmol/l cells/h) and not affected by 1 mmol l(-1) amiloride. Thus, the data obtained clearly indicate that Na+/H+ exchanger in the marsh frog red blood cells is under tight regulatory control, in all likelihood via protein phosphatases of types PP-1 and PP-2A.     

Activation of sodium transport in rat erythrocytes by inhibition of protein phosphatases 1 and 2A

Four structurally different protein phosphatases (PPs) inhibitors - fluoride, calyculin A, okadaic acid and cantharidin--were tested for their ability to modulate unidirectional Na(+) influx in rat red blood cells. Erythrocytes were incubated at 37 degrees C in isotonic and hypertonic media containing 1 mM ouabain and (22)Na in the absence or presence of PP inhibitors. Exposure of the cells to 20 mM fluoride or 50 nM calyculin A for 1 h under isosmotic conditions caused a significant stimulation of Na(+) influx, whereas addition of 200 microM cantharidin or 100 nM okadaic acid had no effect. After 2 h of treatment, however, all these PPs blockers significantly enhanced Na(+) transport in rat erythrocytes. Selective inhibitors of PP-1 and PP-2A types, calyculin A, cantharidin and okadaic acid, produced similar ( approximately 1.2-1.4-fold) stimulatory effects on Na(+) influx in the cells. Activation of Na(+) influx was unchanged with increasing calyculin A concentration from 50 to 200 nM. No additive stimulation of Na(+) influx was observed when the cells were treated with combination of 20 mM fluoride and 50 nM calyculin A. Na(+) influx induced by PPs blockers was inhibited by 1 mM amiloride and 200 muM bumetanide approximately in the equal extent, indicating the involvement of Na(+)/H(+) exchange and Na-K-2Cl cotransport in sodium transport through rat erythrocytes membrane. Activation of Na(+) transport in the cells induced by calyculin A and fluoride was associated with increase of intracellular Na(+) content. Shrinkage of the rat erythrocytes resulted in 2-fold activation of Na(+) influx. All tested PPs inhibitors additionally activated the Na(+) influx by 70-100% above basal shrinkage-induced level. Amiloride and bumetanide have diminished both the shrinkage-induced and PPs-inhibitors-induced Na(+) influxes. Thus, our observations clearly indicate that activities of Na(+)/H(+) exchanger and Na-K-2Cl cotransporter in rat erythrocytes are regulated by protein phosphatases and stimulated when protein dephosphorylation is inhibited.     

Ca-induced K transport in human red blood cell ghosts containing arsenazo III. Transmembrane interactions of Na, K, and Ca and the relationship to the functioning Na-K pump

Increasing free intracellular Ca (Cai) from less than 0.1 microM to 10 microM by means of A23187 activated Ca-stimulated K transport and inhibited the Na-K pump in resealed human red cell ghosts. These ghosts contained 2 mM ATP, which was maintained by a regenerating system, and arsenazo III to measure Cai. Ca-stimulated K transport was activated 50% at 2-3 microM free Cai and the Na-K pump was inhibited 50% by 5-10 microM free Cai. Free Cai from 1 to 8 microM stimulated K efflux before it inhibited the Na-K pump, dissociating the effect of Ca on the two systems. 3 microM trifluoperazine inhibited Ca-stimulated K efflux and 0.5 mM quinidine reduced Na-K pumping by 50%. In other studies, incubating fresh intact cells in solutions containing Ca and 0.5 microM A23187 caused the cells to lose K heterogeneously. Under the same conditions, increasing A23187 to 10 microM initiated a homogeneous loss of K. In ATP-deficient ghosts containing Cai equilibrated with A23187, K transport was activated at the same free Cai as in the ghosts containing 2 mM ATP. Neither Cao nor the presence of an inward Ca gradient altered the effect of free Cai on the permeability to K. In these ghosts, transmembrane interactions of Na and K influenced the rate of Ca-stimulated K efflux independent of Na- and K-induced changes in free Cai or sensitivity to Cai. At constant free Cai, increasing Ko from 0.1 to 3 mM stimulated K efflux, whereas further increasing Ko inhibited it. Increasing Nai at constant Ki and free Cai markedly decreased the rate of efflux at 2 mM Ko, but had no effect when Ko was greater than or equal to 20 mM. These transmembrane interactions indicate that the mechanism underlying Ca-stimulated K transport is mediated. Since these interactions from either side of the membrane are independent of free Cai, activation of the transport mechanism by Cai must be at a site that is independent of those responsible for the interaction of Na and K. In the presence of A23187, this activating site is half-maximally stimulated by approximately 2 microM free Ca and is not influenced by the concentration of ATP. The partial inhibition of Ca-stimulated K efflux by trifluoperazine in ghosts containing ATP suggests that calmodulin could be involved in the activation of K transport by Cai.(ABSTRACT TRUNCATED AT 400 WORDS)     

Caffeine activates a mechanosensitive Ca(2+) channel in human red cells

Caffeine is known to activate influx of both mono- and divalent cations in various cell types, suggesting that this xanthine opens non-selective cation channels at the plasma membrane. This possibility was investigated in human erythrocytes, studying the caffeine action on net Ca(2+), Na(+) and K(+) movements in ATP-depleted cells. Whole populations and subpopulations of young and old erythrocytes were employed. Caffeine was tested in the presence of known mechanosensitive channel blockers (Gd(3+), neomycin and amiloride) and ruthenium red as a possible inhibitor. Caffeine enhanced net cation fluxes in a concentration-dependent way. In whole populations, the Ca(2+) entry elicited by 20 mM caffeine was fully suppressed by Gd(3+) (5 microM), amiloride (250 microM) and ruthenium red (100 microM) and partially blocked by neomycin (100 microM). The above blockers also inhibited caffeine-dependent Na(+) entry whilst showing antagonistic effects on the corresponding K(+) efflux. These compounds fully suppressed hypotonically-induced (-35 mOsm/kg) Ca(2+) influx at nearly the same concentrations completely blocking caffeine-stimulated Ca(2+) entry. The effect of inhibitors on Ca(2+) influx in young cells exceeded that in old cells at similar concentrations. The results clearly show that caffeine stimulates a stretch-activated Ca(2+) channel in human red cells and that aged cells are less susceptible to mechanosensitive channel blockers.