The effect of oxytocin and sigetin on Ca2+ transport in the fraction of plasma membranes of myometrial cells

Oxytocin and sigetin were studied for their effect on the active and passive transport of Ca2+ in the fraction of myometrium sarcolemma in women. Oxytocin (5.10(-7) M) introduced into the sarcolemma vesicles and sigetin (5.10(-3) M) added into the incubation medium inhibit Mg2+, ATP-dependent accumulation of Ca2+ in these structures. The both agents in the mentioned concentration do not affect the passive release of cation from vesicles. A conclusion is drawn that inhibition of the calcium pump of myometrium cell plasma membranes underlies the physiological action of oxytocin and sigetin as stimulators of the contractile activity of the myometrium.     

Passive transport of calcium into plasma membrane fractions of myometrium cells

The apparent values of intravesicular volume (45 microliter/mg of protein), maximal capacity of adsorbed calcium binding on the inner surface of the vesicles (4.5 nmol/mg of protein) and dissociation constants for the Ca2+-binding site complexes (36 microM) were determined from the analysis of peculiarities of passive transport of 45Ca2+ into cow myometrium sarcolemmal vesicles. The kinetics of passive efflux of ionized Ca2+ from the vesicles is described by a two-phase exponential curve. Dilution of the vesicles with a dilution medium is associated with a rapid efflux of ionized Ca2+ from the intravesicular space resulting in dissociation of the Ca2+-binding site complexes on the inner surface of the vesicles and, correspondingly, in the passage from a rapid to the slow phase of Ca2+ efflux from the vesicles which is limited by the dissociation of the Ca2+-binding site complexes. The values of the apparent rate constants for the transmembrane transfer of Ca2+ and dissociation of the Ca2+-binding site complexes (0.73 and 0.02 min-1, respectively) and the permeability of sarcolemmal vesicles for the cation (10(-15) mol of Ca2+/cm2.s) were determined. Alkalinization of the dilution medium stimulates 45Ca2+ release from the vesicles. The blockers of passive Co2+ and Mn2+ transport injected into the vesicles inhibit the efflux of 45Ca2+ from the vesicles. The data obtained were used to analyze the role of sarcolemma in the Ca2+ control of myometrium contraction.     

Contribution of the Mg2+-, ATP- AND Na+-dependent Ca2+-transport systems to the regulation of Ca2+ concentration in myometrial cells

Studies with sarcolemma from cattle myometrium containing inside-out cytoplasmic vesicles, using Ca2+-EGTA buffer, showed that the affinity of ionized Ca2+ for the Mg2+- or ATP-dependent transport is higher than that for the Na+-Ca2+ exchange system (Kd = 3,2 X 10(-6) and (4.3-5.3) X 10(-5) M), respectively. The Km values for MgATP are 2.15 mM. Oxytocin added to the homogenization medium containing rabbit and cattle myometrium cells, i.e. during the formation of closed sarcolemmal fragments, resulted in inhibition of Mg2+, ATP-dependent accumulation of 45Ca2+ by plasma membranes. However, an addition of oxytocin to the incubation medium did not affect the kinetics of active accumulation of Ca2+. It was assumed that the system of non-electrogenic Na+-Ca2+ exchange in the myometrium possessing a low affinity for Ca2+ provides for the maintenance of ionized Ca2+ concentration in the myocytes at 10(-5) M. Therefore, this system cannot induce relaxation of mechanical tension of the uterus. Further decrease of Ca2+ in the myoplasm from 10(-5) to 10(-7) M and, correspondingly, the relaxation of myometrium is provided for by the Mg2+, ATP-dependent efflux of Ca2+ from the myocytes having a high affinity for this cation. The decrease of the activity of ATP-dependent Ca2+-pump by oxytocin is the cause of Ca2+ elevation in the myoplasm and, consequently, of myometrium contraction.     

Effect of the membrane potential on the passive transport of Ca2+ in fractions of vesicles of the myometrium sarcolemma

Using a potential-sensitive fluorescent probe diS-C3-(5), the formation of the membrane (K+-diffusion) potential, delta psi, in the myometrium sarcolemmal vesicular fraction was demonstrated. The magnitude of this potential corresponds to that calculated according to the Nernst equation, is time-stable (characteristic dissociation time--3-5 min) and temperature-dependent and is generated upon the substitution of the anion (Cl- for gluconate-) and the compensating cation (Na+ for Tris+, choline+). The change in delta psi from -61 to 0 mV leads to the activation of passive Ca2+ efflux from the vesicles (with choline+ as the compensating cation in the dilution medium). At the same value of the potential, i. e., -61 mV, the substitution of choline in the dilution medium for Na+ or Li+ stimulates the passive release of Ca2+. Co2+, Mn2+ and D-600 suppress this process by 15-20% in depolarized vesicles which points to the inhibition of Ca2+ release with an alteration of the membrane potential value from 0 to -61 mV (20%). The potential-dependent component of passive Ca2+ transport is characterized by saturation with the substrate (Km = 0.5 mM). The dependence of Ca2+ flux release from the sarcolemmal vesicles on the membrane potential value (-60-+27 mV) is bell-shaped and qualitatively relative to the volt-amper characteristics of the steady state Ca2+ flux in single smooth muscle cells. Analysis of experimental results revealed that the potential-dependent component of passive Ca2+ transport in myometrium sarcolemmal vesicles is determined by the non-activated Ca2+ conductivity of plasma membrane.     

Isolation and characteristics of the plasma membrane fraction from the swine myometrium

An accelerated method is developed for isolating a fraction of plasma membranes of pig myometrium using ultracentrifugation within the sucrose density gradient (15% and 30%). The membranes possessed the high activity of 5'-nucleotidase and Na+, K+-ATPase and the low activity of rhotenon-insensitive NADH-cytochrome c reductase. The vesicularized preparations of plasma membranes are able of ATP-dependent accumulation of Ca2+ (7.5 +/- 0.3 nmol. 45Ca2+ per 1 mg of protein for 15 min). Phosphate increases the calcium accumulation in the presence of ATP and Mg2+. Ionophore A 23187 promotes a complete and rapid release of the previously active-accumulated calcium. The release of 45Ca2+ accumulated by the membrane fraction may be reached by introduction of 1 mM EGTA or DS-Na into the incubation medium, that evidences for the cation accumulation inside closed structures. Using concanavalin-A-sepharose 4B it is shown that 60% of membrane vesicles are turned inside out. The low saponine concentrations (0.0005%) which inhibit Ca2+-accumulation by plasma membranes but not by the endoplasmic reticulum inhibit this process by 60-70% in preparations of the isolated membrane fraction. The method has certain advantages over the previously applied methods used for isolating of plasma membrane fragments from smooth muscles.     

Role of Na+/H+ exchange in thrombin- and arachidonic acid-induced Ca2+ influx in platelets

Platelet activation is accompanied by an increase of cytosolic free Ca2+ concentration, [Ca2+]i, (due to both extracellular Ca2+ influx and Ca2+ movements from the dense tubular system) and an Na+ influx associated with H+ extrusion. The latter event is attributable to the activation of Na+/H+ exchange, which requires Na+ in the extracellular medium and is inhibited by amiloride and its analogs. The present study was carried out to determine whether a link exists between Ca2+ transients (measured by the quin2 method and the 45CaCl2 technique) and Na+/H+ exchange activation (studied with the pH-sensitive intracellular probe, 6-carboxyfluorescein) during platelet stimulation. Washed human platelets, stimulated with thrombin and arachidonic acid, showed: (1) a large and rapid [Ca2+]i rise, mostly due to a Ca2+ influx through the plasma membrane; (2) a marked intracellular alkalinization. Both phenomena were markedly inhibited in the absence of extracellular Na+ or in the presence of an amiloride analog (EIPA). Monensin, a cation exchanger which elicits Na+ influx and alkalinization, and NH4Cl, which induces alkalinization only, were able to evoke an increase in [Ca2+]i, mostly as an influx from the extracellular medium. Our results suggest that Ca2+ influx induced by thrombin and arachidonic acid in human platelets is strictly dependent on Na+/H+-exchange activation.     

Extracellular pH modifies mitochondrial control of capacitative calcium entry in Jurkat cells

It was found that a collapse of the mitochondrial calcium buffering caused by the protonophoric uncoupler CCCP, antimycin A plus oligomycin, or the inhibitor of the mitochondrial Ca2+/Na+ exchanger led to a strong inhibition of thapsigargin-induced capacitative Ca2+ entry (CCE) into Jurkat cells suspended in a medium at pH 7.2. The effect of these inhibitors was markedly less significant at higher extracellular pH. Moreover, dysfunction of the mitochondrial calcium handling greatly decreased CCE sensitivity to extracellular Ca2+ when the pH of extracellular solution was 7.2 (apparent Kd toward extracellular Ca2+ rose from 2.3 +/- 0.6 mm in control cells to 11.0 +/- 1.7 mM in CCCP-treated cells) as compared with pH 7.8 (apparent Kd toward extracellular Ca2+ increased from 1.3 +/- 0.4 mM in control cells to 2.4 +/- 0.4 mM in uncoupler-treated cells). Changes in intracellular pH triggered by methylamine did not influence Ca2+ influx. This suggests that, in Jurkat cells, store-operated calcium channels sense extracellular pH change as a parameter that modifies their sensitivity to intracellular Ca2+. In contrast, in human osteosarcoma cells, changes in extracellular pH as well as mitochondrial uncoupling did not exert any inhibitory effects on CCE.     

Na+/Ca2+ exchange in isolated smooth muscle cells demonstrated by the fluorescent calcium indicator fura-2

Fura-2, a novel fluorescent indicator of cytoplasmic calcium concentrations ([Ca2+i]), was 'loaded' into smooth muscle cells isolated from guinea pig taenia coli. Resting cells maintained a stable [Ca2+i] of 107 +/- 26 nM (n = 13), which could be perturbed with ionomycin. [Ca2+i] was elevated by stimulation of the cells with carbachol or 50 mM KCl. Reduction of the plasmalemmal Na+ concentration gradient by inhibition of the Na+/K+-ATPase with ouabain markedly elevated [Ca2+i]; this elevation was dependent on extracellular Ca2+. [Ca2+i] was also increased by replacement of the extracellular Na+ with an organic cation.     

Transmembrane Ca2+ exchange in depolarized rat myometrium mitochondria

Polarization of the inner membrane is the key factor in maintenance of the physiologically significant cations accumulation, in particular Ca2+, in the mitochondria. It has been well established that mitochondria accumulate calcium through the uniporter, driven by the mitochondrial membrane potential. Nevertheless, it has been shown that depolarized mitochondria also accumulate Ca2+. The aim of this paper is to investigate free Ca level in depolarized myometrium mitochondria. As we have shown previously Ca2+ addition to the incubation medium, that did not contain K-phosphate, ATP and Mg2+, led to inner mitochondrial membrane depolarization. Nevertheless Ca2+ addition to such medium led to the concentration-dependent accumulation of this cation in the matrix. RuR or Mg addition to the incubation medium led to the higher elevation of mitochondrial Ca2+ level in depolarized mitochondria. Mitochondrial Ca2+ level was not affected by 5 microM cyclosporine A. It was suggested that H+/Ca2+ exchanger could provide calcium accumulation in depolarized mitochondria. The elevation of mitochondrial Ca2+ level after addition of Mg2+ and RuR may be due to inhibition of Ca2+- efflux through Ca2+ uniporter.     

Properties of adenylate cyclase from plasma membranes of the rabbit myometrium in different functional states

Adenylate cyclase of plasma membranes from the nonpregnant rabbit myometrium shows the maximum activity at pH 7.7-7.9, is characterized by apparent Km for ATP amounting to 0.38 +/- 0.09 mM, V--125 +/- 34.4 pmol min/mg protein, is activated at most by 15-20 mM Mg2+ and F-. Adenylate cyclase of plasma membranes from the pregnant rabbit myometrium is characterized by apparent Km for ATP amounting to 0.74 +/- 0.06 mM, V--77.3 +/- 6.0 pmol/min/mg protein, is activated at most by 5-10 mM Mg2+ and 10-15 mM F-; the pH optimum for the adenylate cyclase in this functional state is 7.3. Adenylate cyclase in the state of labour is characterized by apparent Km for ATP amounting to 0.46 +/- 0.11 mM, V--34.8 +/- 4.6 pmol/min/mg protein, is activated at most by 10-15 mM Mg2+ and F-, shows the same activity at pH 7.3-8.5. Adenylate cyclase of myometrium in three investigated states is activated by 2 mM EGTA; 10(-7) M Ca2+ decreases activation caused by EGTA; higher concentrations of Ca2+ decrease the basal activity of the enzyme.     

Manganese Uptake and Efflux in Cultured Rat Astrocytes

Astrocytes play a central role in manganese (Mn) regulation in the CNS. Using primary astrocyte cultures from neonatal rat brains, these studies demonstrate a specific high-affinity transport system for Mn2+. Saturation kinetics are clearly indicated by both 1/v versus 1/s plots (Km = 0.30 +/- 0.03 microM; Vmax = 0.30 +/- 0.02 nmol/mg of protein/min) and plots of v versus [s]. Several divalent cations (Co2+, Zn2+, and Pb2+) failed to inhibit the initial rate of 54Mn2+ uptake. In contrast, extracellular Ca2+ at 10 microM decreased 54Mn2+ uptake. Exchange with extracellular Mn2+ was not obligatory for the efflux of 54Mn2+ into extracellular medium because efflux occurred into Mn(2+)-free extracellular medium, but efflux of 54Mn2+ was enhanced when astrocytes were equilibrated in the presence of unlabeled Mn2+. Efflux of 54Mn2+ was biphasic with both a rapid and a slow component. Efflux was most rapid during the first 10 min of incubation, with 27.5 +/- 2.2% of 54Mn2+ transported extracellularly, and 37.2 +/- 1.2% of preloaded 54Mn2+ was retained by the astrocytes at 120 min. These studies show, for the first time, that mammalian astrocytes can transport Mn via a specific transport system.     

Oxytocin regulates the plasma membrane Ca2+ transport in rat myometrium.

Development of myometrium in young female rats was stimulated by administration of diethylstilboestrol. Plasma membrane and sarcoplasmic reticulum from rat myometrium were separated by a new and rapid method using a Percoll gradient. Calcium uptake was inhibited in plasma membrane vesicles isolated from oxytocin-treated myometrium, while no consistent effect of oxytocin was found on the Ca2+ uptake in the sarcoplasmic reticulum. Oxytocin regulated the plasma membrane Ca2+ pump by decreasing its apparent affinity for Ca2+ without affecting its maximal velocity. The K1/2 for Ca2+ in the absence of calmodulin was 0.41 +/- 0.04 microM in normal membranes; this was increased to 0.93 +/- 0.12 microM in oxytocin-treated membranes. Calmodulin decreased the K1/2 for Ca2+ to 0.27 +/- 0.027 microM and oxytocin also increased this, to 0.46 +/- 0.061 microM. The effect of oxytocin on the plasma membrane Ca2+ pump was highly dependent on the hormonal status of the animals. When the diethylstilboestrol was administered together with progesterone, the inhibitory action of oxytocin was totally suppressed, consistent with the expected action of this agent. The results suggest that regulation of the plasma membrane Ca2+ pump may be important in the prolonged elevation of intracellular Ca2+ caused by oxytocin.     

Effect of ethanol on accumulation of Ca ions in intracellular structures of rat myometrium under conditions of first creating ethanol-dependent models

In the experiments which have been conducted on digitonin-treated myometrium cell suspensions of nonpregnant rats the direct influence of ethanol in concentration 0-10% on the Ca2+ accumulation in mitochondria and endoplasmic reticulum have been studied. Studies have been conducted on the different models, such as, control (model I), subacute (model II), acute (model III) and chronic ethanol consumption (model IV). It has been shown for all models that the dependence of Ca2+ accumulation by mitochondria on the concentration of ethanol in incubation medium is bell-shaped. Acute and chronic ethanol consumption resulted in statistically reliable decrease in the amount of accumulated cations. Nevertheless the I50 values were the same for the models I-III and were 8-9%, although in the case of model IV this one was only 4.0 +/- 0.6%. The increase of ethanol concentration in the incubation medium caused of Ca2+ accumulation decreasing in the endoplasmic reticulum for all studied models, the values of I50 also decreased for models II-IV (2.8 +/- 0.2; 2.5 +/- 0.2 and 2.3 +/- 0.3% respectively) relative to the control (3.8 +/- 0.2%). At the level of model I oxytocin-inhibited component of the endoplasmic reticulum Ca2+ uptake was more stable to the ethanol effects than oxytocin-independent one. Although the sensitivity of the first one to the ethanol effects at the level of models II-IV rose, that parameter for the oxytocin-independent component was not changed. The mechanisms of ethanol effects on Ca2+ accumulation in the myometrium intracellular structures have been discussed.     

Calcium uptake in isolated brush-border vesicles from rat small intestine.

Ca2+ uptake in brush-border vesicles isolated from rat duodena was studied by a rapid-filtration technique. Ca2+ uptake showed saturation kinetics, was dependent on the pH and ionic strength of the medium and was independent of metabolic energy. Uptake activity was readily inhibited by Ruthenium Red, La3+, tetracaine, EGTA, choline chloride and Na+ or K+. The effect of variations in medium osmolarity on Ca2+ uptake and the ionophore A23187-induced efflux of the cation from preloaded vesicles indicated that the Ca2+-uptake process involved binding to membrane components, as well as transport into an osmotically active space. Scatchard-plot analyses of the binding data suggested at least two classes of Ca2+-binding sites. The high-affinity sites, Ka = (2.7 +/- 1.1) x 10(4) M-1 (mean +/- S.D.) bound 3.2 +/- 0.8 nmol of Ca2+/mg of protein, whereas the low-affinity sites (Ka = 60 +/- 6 M-1) bound 110 +/- 17 nmol of Ca2+/mg of protein. In the presence of 100 mM-NaCl, 1.7 and 53 nmol of Ca2+/mg of protein were bound to the high- and low-affinity sites respectively. Decreased Ca2+-uptake activity was observed in vesicles isolated from vitamin D-deficient as compared with vitamin D-replete animals and intraperitoneal administration of 1,25-dihydroxycholecalciferol to vitamin D-deficient rats 16 h before membrane isolation stimulated the initial rate of Ca2+ uptake significantly. The data indicated that Ca2+ entry and/or binding was passive and may involve a carrier-mediated Ca2+-uptake component that is associated with the brush-border membrane. Altering the electrochemical potential difference across the membrane by using anions of various permeability and selected ionophores appeared to increase primarily binding to the membrane rather than transport into the intravesicular space. Since there is considerable binding of Ca2+ to the vesicle interior, a comprehensive analysis of the transport properties of the brush-border membrane remains difficult at present.     

The role of sarcolemma and mitochondria in calcium-dependent control of myometrium relaxation

Using atomic absorption spectroscopy, it was shown that the amount of firmly bound Ca2+ in cattle mitochondria and myometrium sarcolemma is 160 +/- 10 and 30 +/- 10 mumol/kg of wet tissue, respectively. The Ca2+ 1 accumulating capacity of mitochondria (350 nmol per mg of protein) markedly exceeds that of sarcolemmal vesicles (30 nmol per mg of protein). Using a Ca2+-EGTA buffer, it was found that the affinity of ionized Ca for the mitochondrial transport system (Km = 5.69 microM) is higher than that for the Na+-Ca2+ system of sarcolemma exchange (Km = 30 microM), but is markedly lower than that for the Mg2+, ATP-dependent Ca2+ efflux (Km = 0.35 microM). A kinetic analysis demonstrated that the sarcolemmal Ca2+ pump is incapable of causing complete relaxation of the smooth muscle within the physiologically significant time, whereas the Ca2+ transport system of mitochondria evokes this process within 21 s. However, the contribution of the Ca2+ pump to the regulation of the Ca2+ content in myocytes is paralleled with the accumulation of Ca2+ in mitochondria and is realized at low concentrations of this cation in the myoplasm, i.e., at late steps of relaxation. A mechanism of Ca2+ control over myometrium relaxation is proposed. The system of non-electrogenic Na+-Ca2+ exchange maintains Ca2+ concentration in the myoplasm as high as 10(-5) M. Mitochondria which accumulate the bulk of Ca2+ rapidly decrease its concentration in the cytoplasm down to 10(-6)-10(-7) M; at these values, the activity of the sarcolemmal Ca2+ pump with a high affinity for the transfer substrate is manifested. In this way, the Ca2+ pump accomplishes fine regulation of Ca2+ concentration in the myocytes.     

Calcium influx is not required for thyrotropin-releasing hormone stimulation of prolactin release from GH3 cells

TRH stimulation of prolactin release from GH3 cells is dependent on Ca2+; however, whether TRH-induced influx of extracellular Ca2+ is required for stimulated secretion remains controversial. We studied prolactin release from cells incubated in medium containing 110 mM K+ and 2 mM EGTA which abolished the electrical and Ca2+ concentration gradients that usually promote Ca2+ influx. TRH caused prolactin release and 45Ca2+ efflux from cells incubated under these conditions. In static incubations, TRH stimulated prolactin secretion from 11.4 +/- 1.2 to 19 +/- 1.8 ng/ml in control incubations and from 3.2 +/- 0.6 to 6.2 +/- 0.8 ng/ml from cells incubated in medium with 120 mM K+ and 2 mM EGTA. We conclude that Ca2+ influx is not required for TRH stimulation of prolactin release from GH3 cells.     

Sodium-dependent regulation of steroidogenesis in rat granulosa cells: possible involvement of a Na+/H+ antiport

The possible role of Na+/H+ antiport in the gonadotropic regulation of steroidogenesis was examined in rat granulosa cells incubated for up to 6 h in a chemically defined medium in the absence or presence of Na+ (128 mM), gonadotropin (FSH or LH; 0-500 ng/ml), dibutyryl cyclic AMP [Bu)2cAMP; 2 mM) and amiloride (0-1 mM). Replacement of Na+ (Na+0) in the incubation medium with choline chloride resulted in a marked decrease in basal and LH-, FSH- and (Bu)2cAMP-stimulated progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) synthesis in vitro. The Na+/H+ exchange inhibitor, amiloride significantly suppressed basal and hormone-stimulated progestin production dose-dependently in the presence of Na+0. However, it was without effect in Na+-deficient medium. The effect of the inhibitor on progestin production appeared to be directed at specific step(s) involved in the synthesis of pregnenolone, as concentrations of amiloride which inhibited progesterone production failed to influence the metabolism of exogenous pregnenolone to progestins. Cell viability and the incorporation of [3H]leucine into acid-precipitable material were not affected by amiloride. Our findings support the contention that extracellular sodium is important for steroidogenesis in rat granulosa cells. The inhibition by amilordie indicates an involvement of the Na+/H+ exchange in the regulation of this granulosa cell function.     

Anion-dependent Mg2+ influx and a role for a vacuolar H+-ATPase in sheep ruminal epithelial cells

The K+-insensitive component of Mg2+ influx in primary culture of ruminal epithelial cells (REC) was examined by means of fluorescence techniques. The effects of extracellular anions, ruminal fermentation products, and transport inhibitors on the intracellular free Mg2+ concentration ([Mg2+]i), Mg2+ uptake, and intracellular pH were determined. Under control conditions (HEPES-buffered high-NaCl medium), the [Mg2+]i of REC increased from 0.56 +/- 0.14 to 0.76 +/- 0.06 mM, corresponding to a Mg2+ uptake rate of 15 microM/min. Exposure to butyrate did not affect Mg2+ uptake, but it was stimulated (by 84 +/- 19%) in the presence of CO2/HCO(-)3. In contrast, Mg2+ uptake was strongly diminished if REC were suspended in HCO(-)3-buffered high-KCl medium (22.3 +/- 4 microM/min) rather than in HEPES-buffered KCl medium (37.5 +/- 6 microM/min). After switching from high- to low-Cl- solution, [Mg2+]i was reduced from 0.64 +/- 0.09 to 0.32 +/- 0.16 mM and the CO2/HCO(-)3-stimulated Mg2+ uptake was completely inhibited. Bumetanide and furosemide blocked the rate of Mg2+ uptake by 64 and 40%, respectively. Specific blockers of vacuolar H+-ATPase reduced the [Mg2+]i (36%) and Mg2+ influx (38%) into REC. We interpret this data to mean that the K+-insensitive Mg2+ influx into REC is mediated by a cotransport of Mg2+ and Cl- and is energized by an H+-ATPase. The stimulation of Mg2+ transport by ruminal fermentation products may result from a modulation of the H+-ATPase activity.     

Extracellular ATP increases cation fluxes in human erythrocytes by activation of the P2X7 receptor

Canine erythrocytes are known to undergo a reversible increase in cation permeability when incubated with extracellular ATP. We have examined the expression and function of P2X receptors on human erythrocytes using confocal microscopy and a panel of anti-P2X(1-7) antibodies and have measured monovalent cation fluxes in the presence of various nucleotide agonists. Human erythrocytes expressed P2X7 receptors on all cells examined from eight of eight subjects, as well as P2X2 at a far lower staining intensity in six of eight subjects. ATP stimulated the efflux of 86Rb+ (K+) from human erythrocytes in a dose-dependent fashion with an EC50 of approximately 95 microM. Other nucleotides also induced an efflux of 86Rb+ from erythrocytes with an order of agonist potency of 2'- and 3'-O(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP (2MeSATP) > adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), whereas ADP or UTP had no effect. ATP-induced efflux of 86Rb+ from erythrocytes was inhibited by extracellular Na+ and oxidized ATP, as well as by KN-62, an antagonist specific for the human P2X7 receptor. When erythrocytes were incubated in isotonic KCl medium, the addition of ATP stimulated an 86Rb+ influx approximately equal in magnitude to ATP-stimulated 86Rb+ efflux from the same cells. BzATP also stimulated the influx of 22Na+ into erythrocytes incubated in isotonic NaCl medium. Both ATP-induced efflux and influx of 86Rb+ and 22Na+ were impaired in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X7 receptor. These results suggest that the reversible permeabilization of erythrocytes by extracellular ATP is mediated by the P2X7 receptor.     

Mechanism of the change in erythrocyte osmotic resistance in rats exposed to valinomycin: the features seen in spontaneous hypertension

Introduction of valinomycin into erythrocyte incubation medium increased the cell stability to water-induced hemolysis. In these conditions the erythrocytes of spontaneously hypertensive and normotensive (control) rats release 63.2 +/- 1.5% and 80.9 +/- 1.6%, respectively, of the total hemoglobin content. Valinomycin effect is completely abolished with K+ substitution for Na+ and is independent of extracellular Ca2+ concentration. Valinomycin had no effect on human erythrocyte osmotic stability. It has been shown that valinomycin-induced kinetics of Na+ and K+ redistribution was different in human and rat erythrocytes. The distinctions are thought to be related to specific anion transport mediated by the third band protein--the main component of membrane cytoskeleton.