Selective activation of Ca2+ influx by extracellular ATP in a pancreatic beta-cell line (HIT)

The action of exogenous ATP on cytoplasmic free Ca2+ ([Ca2+]i) was studied in insulin secreting cells using fura-2. Stimulation of clonal pancreatic beta-cells (HIT) with ATP (range 2-20 microM) evoked a sustained elevation in [Ca2+]i. ATP selectively promoted Ca2+ influx and not Ca2+ mobilization since (1) the effect required external Ca1+ and (2) was observed in cells in which internal stores were depleted with ionomycin (3) the rate of Mn2+ influx, measured as the quenching of the fura-2 signal, was accelerated by ATP. The action of ATP was unaffected by the voltage-sensitive Ca2+ channel blockers nifedipine and verapamil as well as by a depolarizing concentration of K+. The effect on [Ca2+]i was highly specific for ATP since AMP, ADP, adenosine 5'-[gamma-thio]triphosphate, adenosine 5'-[beta, gamma-methylene]triphosphate, GTP and adenosine were ineffective. In normal pancreatic islet cells, both exogenous ATP (range 0.2-2 microM) and ADP induced a transient Ca2+ elevation that did not require external Ca2+. The nucleotide specificity of the effect on [Ca2+]i suggests that ATP activates P2 gamma purinergic receptors in normal beta-cells. Thus, ATP evokes a Ca2+ signal in clonal HIT cells and normal islet cells by different transducing systems involving distinct purinoreceptors. A novel mechanism for increasing [Ca2+]i by extracellular ATP is reported in HIT cells, since the nucleotide specificity and the selective activation of Ca2+ influx without mobilization of internal Ca2+ stores cannot be explained by mechanisms already described in other cell systems.     

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.     

Extracellular nucleotides mediate Ca2+ fluxes in J774 macrophages by two distinct mechanisms

We have studied the effects of extracellular nucleotides on the cytosolic free calcium concentration [( Ca2+]i) in J774 macrophages using quin2 and indo-1 as indicator dyes. Micromolar quantities of ATP induced a biphasic increase in [Ca2+]i: a rapid and transient increase (peak I) which was due to mobilization of Ca2+ from intracellular stores and a second more sustained elevation (peak II) due to influx of extracellular Ca2+. The sustained peak II elevation had two components, a "low threshold" (1 microM ATP) response which saturated at 10-50 microM ATP and a "high threshold" response, apparent at [ATP] greater than 100 microM. The latter component was not seen with nucleotides other than ATP and correlated with an ATP-induced generalized increase in plasma membrane permeability. A variant J774 cell line was isolated which does not demonstrate this ATP-induced increase in plasma membrane permeability; nevertheless, it demonstrated both the release of Ca2+ from intracellular stores and the low threshold component of the Ca2+ influx across the plasma membrane in response to nucleoside di- and triphosphates. Several lines of evidence indicate that the fully ionized (i.e. free acid) forms of nucleoside di- and triphosphates were the ligands that mediated these increases in [Ca2+]i. These data show that extracellular nucleotides mediate Ca2+ fluxes by two distinct mechanisms in J774 cells. In one, the rise in [Ca2+]i is due to release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane. This response is elicited preferentially by the free acid forms of purine and pyrimidine nucleoside di- and triphosphates. In the other, the rise in [Ca2+]i reflects a more generalized increase in plasma membrane permeability and is elicited by ATP4- only.     

Extracellular ATP triggers superoxide production in human neutrophils

The effects of ATP on the concentration of cytosolic calcium [( Ca2+]i) were examined with respect to early events associated with activation of the superoxide (O2-)-generating system in human neutrophils. Addition of ATP to cytochalasin B-treated neutrophils resulted in two sequential increase in [Ca2+]i: an initial phase presumably related to the mobilization of Ca2+ from intracellular stores and a second phase dependent upon the presence of extracellular Ca2+. The second phase was associated with an increase in the rate of O2- production, which also required the presence of extracellular Ca2+. The results suggest that increased Ca2+ influx may act to trigger a cascade of Ca2+-sensitive events, leading to stimulated O2-production.     

ATP-Stimulated Ca2+ Influx and Phospholipase D Activities of a Rat Brain-Derived Type-2 Astrocyte Cell Line, RBA-2, Are Mediated Through P2X7 Receptors

This study characterizes and examines the P2 receptor-mediated signal transduction pathway of a rat brain-derived type 2 astrocyte cell line, RBA-2. ATP induced Ca2+ influx and activated phospholipase D (PLD). The ATP-stimulated Ca2+ influx was inhibited by pretreating cells with P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), in a concentration-dependent manner. The agonist 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) stimulated the largest increases in intracellular Ca2+ concentrations ([Ca2+]i); ATP, 2-methylthioadenosine triphosphate tetrasodium, and ATPgammaS were much less effective, whereas UTP, ADP, alpha,beta-methylene-ATP, and beta,gamma-methylene-ATP were ineffective. Furthermore, removal of extracellular Mg2+ enhanced the ATP- and BzATP-stimulated increases in [Ca2+]i. BzATP stimulated PLD in a concentration- and time-dependent manner that could be abolished by removal of extracellular Ca2+ and was inhibited by suramin, PPADS, and oxidized ATP. In addition, PLD activities were activated by the Ca2+ mobilization agent, ionomycin, in an extracellular Ca2+ concentration-dependent manner. Both staurosporine and prolonged phorbol ester treatment inhibited BzATP-stimulated PLD activity. Taken together, these data indicate that activation of the P2X7 receptors induces Ca2+ influx and stimulates a Ca2+-dependent PLD in RBA-2 astrocytes. Furthermore, protein kinase C regulates this PLD.     

ATP-induced calcium mobilization in human neutrophils

Extracellular ATP at 10 microM increased the concentration of cytoplasmic free Ca2+ ( [Ca2+]i) 3-fold in human neutrophils. The [Ca2+]i was measured by fura-2 fluorescence. The effect was rapid but transient: [Ca2+]i reached a maximum within 10 s and then returned to its resting value after 2-3 min. The rise in [Ca2+]i elicited by ATP was unaffected by the removal of extracellular Ca2+, indicating that the primary source of Ca2+ is from intracellular stores. In contrast to ATP, neither ADP nor AMP, at concentrations as high as 100 microM, caused any detectable changes in [Ca2+]i. Among other nucleotide triphosphates tested, UTP was as effective as ATP in causing a transient rise in [Ca2+]i, and prevented a subsequent response to ATP. Similarly, ATP prevented a subsequent response to UTP but the second response could be obtained when the initially added ATP was removed by the addition of hexokinase. Phorbol myristate acetate, the activator of Ca2+, phospholipid-dependent protein kinase, completely inhibited the ATP-induced increases in [Ca2+]i without affecting the basal [Ca2+]i level. The results suggest that extracellular ATP stimulates human neutrophils by causing the release of calcium from intracellular storage pools by mechanisms which can be inhibited by phorbol myristate acetate.     

Identification of P2Y purinoceptors associated with voltage-activated cation channels in cardiac ventricular myocytes of the rat

Extracellular ATP has vasodilatory and inotropic effects in the heart. We have demonstrated that extracellular ATP, in a concentration-dependent manner (10 nM-0.1 mM), increased [Ca2+]i in suspensions of isolated fura-2-loaded rat cardiac ventricular myocytes (maximum 96 +/- 10% increase over basal levels, SEM, n = 12, P less than 0.01). The increase in [Ca2+]i was often biphasic, with an initial fast phase (less than 1 s) of low amplitude, followed by a slower phase of higher amplitude. A second application of ATP had little effect, and ATP abolished the effect of subsequent electrical stimulations, even through the cells were still able to respond with an increase in [Ca2+]i to KCl-induced depolarization or stimulation by caffeine. Pretreatment of cells with nifedipine, verapamil, caffeine, ryanodine, or 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride attenuated the effect of extracellular ATP on [Ca2+]i, and binding of extracellular free calcium by excess EGTA completely abolished the effects of extracellular ATP and electrical stimulation. Extracellular ATP increased bisoxonol fluorescence in ventricular myocytes, indicating depolarization of the sarcolemma. Pretreatment of the myocytes with tetrodotoxin (50 microM), or replacement of NaCl in the incubation buffer with the impermeant cation N-methyl-D-glucamine, suppressed the extracellular ATP effect on [Ca2+]i. ADP and AMP had smaller effects on [Ca2+]i than ATP; adenosine had no effect. ATP analogues showed the following rank order of potency in increasing [Ca2+]i or bisoxonol fluorescence: ATP greater than or equal to 2-methylthioATP much greater than adenosine 5'-O-[3-thio]triphosphate greater than adenosine 5'-[alpha, beta-methylene]triphosphate approximately adenosine 5'-[beta, gamma-methylene]triphosphate approximately adenosine 5'-[beta, gamma-imino]triphosphate greater than adenosine. These data are consistent with the presence of purinoceptors (P2Y subtype) on the sarcolemma of cardiac ventricular myocytes of the rat, which upon activation lead to depolarization and activation of cation channels of the sarcolemma and flux of extracellular Ca2+ into the cells. This may result in further flux of Ca2+ into the cytosol from intracellular stores. The effects of extracellular ATP on [Ca2+]i in rat cardiac ventricular myocytes may, in part, explain the direct inotropic effects of extracellular ATP on the mammalian heart.     

Basolateral store-operated Ca(2+)-entry in polarized human bronchial and colonic epithelial cells

Bronchial epithelial cells respond to extracellular nucleotides from the luminal and basolateral side activating Cl- secretion via [Ca2+]i increase. In this study we investigated the differences of apically (ap) and basolaterally (bl) stimulated [Ca2+]i signals in polarized human bronchial epithelial cells (16HBE14o-). Specifically we investigated the localization of 'capacitative Ca2+ entry' (CCE). 16HBE14o- cells grown on permeable filters were mounted into an Ussing chamber built for the simultaneous measurement of Fura-2 fluorescence and electrical properties. Application of ATP from both sides induced a rapid [Ca2+]i increase and subsequent sustained [Ca2+]i plateau due to transmembraneous Ca(2+)-influx. The use of different nucleotides revealed the following rank order or potency which was very similar for addition from the apical or basolateral side: UTP (EC50 ap: 4 microM, bl: 5 microM) > ATP (EC50 ap: 4 microM, bl: 10 microM) > ADP (n = 4-7 from both sides). 2-MeS-ATP, AMP, adenosine and beta gamma-methylene ATP were ineffective (n = 3 from both sides). The ATP- (ap and bl) induced Ca2+ influx was only abolished by removal of basolateral Ca2+. This was also true for receptor-independent activation of Ca(2+)-influx by intracellular Ca(2+)-store depletion with 2,5 Di-(tert-butyl)-1,4-benzohydroquinone (BHQ) (10 microM). Also in polarized T84 cells the basolateral carbachol and BHQ activated Ca2+ plateau was exclusively sensitive to removal of basolateral Ca2+. We propose that in all polarized epithelial cells the CCE entry pathway is located in the basolateral membrane. We furthermore suggest that Ca2+[i elevating agonists acting from the apical side of the epithelium lead to the opening of a basolateral CCE pathway.     

Characterization of ATP receptor which mediates norepinephrine release in PC12 cells.

PC12 cells, a rat pheochromocytoma cell line, has been reported to release norepinephrine in response to extracellular ATP in the presence of extracellular Ca2+. The potency order of ATP analogues was adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than adenosine 5'-O-(1-thiotriphosphate) = 2-methylthioadenosine 5'-triphosphate (MeSATP) greater than 2'- and 3'-O-(4-benzoyl-benzoyl)ATP (BzATP) greater than ADP greater than 5-adenylylimidodiphosphate. Adenosine 5'-O-(2-thiodiphosphate), beta, gamma-methyleneadenosine 5'-triphosphate, AMP and adenosine were inactive. The ATP action in the absence of extracellular Ca2+, suggests a small but appreciable contribution of intracellular Ca2+ mobilization, for norepinephrine release. However, for some ATP derivatives, like BzATP, almost no contribution of the phospholipase C-Ca2+ pathway is suggested, based on their low activity in inositol phosphates production. To identify the ATP-receptor protein, PC12 cell membranes were photoaffinity-labeled with [32P]BzATP. SDS-PAGE analysis showed that a 53-kDa protein labeling was inhibited by ATP and its derivatives, as well as by P2-antagonists, suramin and reactive blue 2, which inhibit the nucleotide-induced norepinephrine release. The inhibitory activity of the nucleotides was, in parallel with their potency, to induce norepinephrine release. Despite their inability to release norepinephrine, GTP and GTP gamma S inhibited the BzATP labeling, suggesting the participation of a putative G protein in the ATP-receptor-mediated actions. We suggest that the 53-kDa protein on the PC12 cell surface is an ATP receptor, which mediates the norepinephrine release, depending, mainly, on extracellular Ca2+ gating.     

Simultaneous exposure to ATP and phenylephrine induces a sustained elevation in the intracellular calcium concentration in supraoptic neurons

Vasopressin (VP) release from the hypothalamo-neurohypophyseal system (HNS) is stimulated by ATP activation of P2X purinergic receptors and by activation of 1-adrenergic receptors by phenylephrine (PE). These responses are potentiated by simultaneous exposure to ATP+PE. Potentiation was blocked by depleting intracellular calcium stores with thapsigargin. To test the hypothesis that the synergistic response to ATP+PE reflects alterations in the intracellular calcium concentration ([Ca2+]i), [Ca2+]i was monitored in supraoptic neurons in HNS explants loaded with fura 2-AM. Both ATP and PE induced rapid, but transient, elevations in [Ca2+]i. In 0.3 mM Ca2+, the peak response to ATP was greater than to PE but did not differ from the peak response to ATP+PE. A sustained elevation in [Ca2+]i was induced by ATP+PE, that was greater than ATP or PE alone. In 2 mM Ca2+, the peak response to ATP+PE was significantly greater than to either ATP or PE alone, and the sustained response to ATP+PE was greater than to either agent alone. Responses were comparable in the presence of TTX. The sustained elevation in [Ca2+]i was also observed when ATP+PE was removed after 1 min, but it was eliminated by either thapsigargin or removing external calcium, indicating that both calcium influx and calcium release from internal stores are required. Some cells were vasopressinergic based on a VP-induced increase in [Ca2+]i. These observations support the hypothesis that simultaneous exposure to ATP+PE induces a different pattern of [Ca2+]i than either agent alone that may initiate events leading to synergistic stimulation of VP release.     

Regulation of Ca2+ influx during mitosis: Ca2+ influx and depletion of intracellular Ca2+ stores are coupled in interphase but not mitosis.

Activation of a wide variety of membrane receptors leads to a sustained elevation of intracellular Ca2+ ([Ca2+]i) that is pivotal to subsequent cell responses. In general, in nonexcitable cells this elevation of [Ca2+]i results from two sources: an initial release of Ca2+ from intracellular stores followed by an influx of extracellular Ca2+. These two phases, release from intracellular stores and Ca2+ influx, are generally coupled: stimulation of influx is coordinated with depletion of Ca2+ from stores, although the mechanism of coupling is unclear. We have previously shown that histamine effects a typical [Ca2+]i response in interphase HeLa cells: a rapid rise in [Ca2+]i followed by a sustained elevation, the latter dependent entirely on extracellular Ca2+. In mitotic cells only the initial elevation, derived by Ca2+ release from intracellular stores, occurs. Thus, in mitotic cells the coupling of stores to influx may be specifically broken. In this report we first provide additional evidence that histamine-stimulated Ca2+ influx is strongly inhibited in mitotic cells. We show that efflux is also strongly stimulated by histamine in interphase cells but not in mitotics. It is possible, thus, that in mitotics intracellular stores are only very briefly depleted of Ca2+, being replenished by reuptake of Ca2+ that is retained within the cell. To ensure the depletion of Ca2+ stores in mitotic cells, we employed the sesquiterpenelactone, thapsigargin, that is known to affect the selective release of Ca2+ from intracellular stores by inhibition of a specific Ca(2+)-ATPase; reuptake is inhibited. In most cells, and in accord with Putney's capacitative model (1990), thapsigargin, presumably by depleting intracellular Ca2+ stores, stimulates Ca2+ influx. This is the case for interphase HeLa cells. Thapsigargin induces an increase in [Ca2+]i that is dependent on extracellular Ca2+ and is associated with a strong stimulation of 45Ca2+ influx. In mitotic cells thapsigargin also induces a [Ca2+]i elevation that is initially comparable in magnitude and largely independent of extracellular Ca2+. However, unlike interphase cells, in mitotic cells the elevation of [Ca2+]i is not sustained and 45Ca2+ influx is not stimulated by thapsigargin. Thus, the coupling between depletion of intracellular stores and Ca2+ influx is specifically broken in mitotic cells. Uncoupling could account for the failure of histamine to stimulate Ca2+ influx during mitosis and would effectively block all stimuli whose effects are mediated by Ca2+ influx and sustained elevations of [Ca2+]i.     

Extracellular ATP-induced inward current in isolated epithelial cells of the endolymphatic sac.

Using whole-cell patch-clamp technique and Fura-2 fluorescence measurement, the presence of ATP-activated ion channels and its dependence on intracellular Ca2+ concentration ([Ca2+]i) in the epithelial cells of the endolymphatic sac were investigated. In zero current-clamp configuration, the average resting membrane potential was -66.8+/-1.3 mV (n=18). Application of 30 microM ATP to the bath induced a rapid membrane depolarization by 43.1+/-2.4 mV (n=18). In voltage-clamp configuration, ATP-induced inward current at holding potential (VH) of -60 mV was 169.7+/-6.3 pA (n=18). The amplitude of ATP-induced currents increased in sigmoidal fashion over the concentration range between 0.3 and 300 microM with a Hill coefficient (n) of 1.2 and a dissociation constant (Kd) of 11.7 microM. The potency order of purinergic analogues in ATP-induced current, which was 2MeSATP>ATPgammas>/=ATP>alpha, beta-ATP>ADP=AMP>/=adenosine=UTP, was consistent with the properties of the P2Y receptor. The independence of the reversal potential of the ATP-induced current from Cl- concentration suggests that the current is carried by a cation channel. The relative ionic permeability ratio of the channel modulated by ATP for cations was Ca2+>Na+>Li+>Ba2+>Cs+=K+. ATP (10 microM) increased [Ca2+]i in an external Ca2+-free solution to a lesser degree than that in the external solution containing 1.13 mM CaCl2. ATP-induced increase in [Ca2+]i can be mimicked by application of ionomycin in a Ca2+-free solution. These results indicate that ATP increases [Ca2+]i through the P2Y receptor with a subsequent activation of the non-selective cation channel, and that these effects of ATP are dependent on [Ca2+]i and extracellular Ca2+.     

Activation of purinergic P2X receptors inhibits P2Y-mediated Ca2+ influx in human microglia

Purinoceptor (P2X and P2Y) mediated Ca2+ signaling in cultured human microglia was studied using Ca2+ sensitive fluorescence microscopy. ATP (at 100 microM) induced a transient increase in [Ca2+]i in both normal and Ca(2+)-free solution suggesting a primary contribution by release from intracellular stores. This conclusion was further supported by the failure of ATP to cause a divalent cationic influx in Mn2+ quenching experiments. However, when fluorescence quenching was repeated after removal of extracellular Na+, ATP induced a large influx of Mn2+, indicating that inward Na+ current through a non-selective P2X-coupled channel may normally suppress divalent cation influx. Inhibition of Mn2+ entry was also found when microglia were depolarized using elevated external K+ in Na(+)-free solutions. The possibility of P2X inhibition of Ca2+ influx was then investigated by minimizing P2X contributions of purinergic responses using either the specific P2Y agonist, ADP-beta-S in the absence of ATP or using ATP combined with PPADS, a specific inhibitor of P2X receptors. In quenching studies both procedures resulted in large increases in Mn2+ influx in contrast to the lack of effect observed with ATP. In addition, perfusion of either ATP plus PPADS or ADP-beta-S alone caused a significantly enhanced duration (about 200%) of the [Ca2+]i response relative to that induced by ATP. These results show that depolarization induced by P2X-mediated Na+ influx inhibits store-operated Ca2+ entry resulting from P2Y activation, thereby modulating purinergic signaling in human microglia.     

Auranofin modulated cytoplasmic free calcium in neutrophils by mobilizing intracellular calcium and inhibiting protein kinase

The effect of the lipophilic gold compound, auranofin (AUR) on the calcium homeostasis of human neutrophils treated with or without n-formyl-methionyl-leucyl-phenylalanine (FMLP) was investigated. In agreement with previous reports, FMLP induced a rapid release of intracellular Ca2+ stores followed by a smaller influx of extracellular Ca2+. AUR and staurosporine enhanced while phorbol 12-myristate 13-acetate suppressed the secondary influx of Ca2+. Mn2(+)-quenching-of-fluorescence studies indicate that phorbol 12-myristate 13-acetate incubation blocked cation entry. AUR or staurosporine potentiation of FMLP effects on cytoplasmic free Ca2+ [( Ca2+]i) was attributed to suppression of negative feedback effects of protein kinase C. AUR (5-45 microM) per se induced a slow release of internal Ca2+ stores followed by a delayed influx of extracellular Ca2+. Control studies showed that AUR did not induce the formation of inositol 1,4,5-trisphosphate, lyse cells, or promote dye leakage. Dithiothreitol suppressed the AUR effect. AUR triggered biphasic but smaller increases in [Ca2+]i of neutrophil cytoplasts. Studies with permeabilized neutrophils showed that AUR directly released Ca2+ from internal stores. By comparison, gold sodium thiomalate, which had no effect on intact cells, also released Ca2+ from permeabilized cells. Present results indicate that AUR modulated [Ca2+]i directly by mobilized Ca2+ from multiple storage sites and indirectly by inhibiting protein kinase C.     

P2X and P2Y purinoreceptors mediate ATP-evoked calcium signalling in optic nerve glia in situ

It is known that ATP acts as an extracellular messenger mediating Ca2+ signalling in glial cells. Here, the mechanisms involved in the ATP-evoked increase in glial [Ca2+]i were studied in situ, in the acutely isolated rat optic nerve. ATP and agonists for P2X (a,b-metATP) and P2Y (2MeSATP) purinoreceptors triggered raised glial [Ca2+]i, and there was no significant difference between cells identified morphologically as astrocytes and oligodendrocytes. Dose-response curves indicated that P2Y receptors were activated at nanomolar concentrations, whereas P2X purinoreceptors were only activated above 10 microM. The rank order of potency for several agonists indicated optic nerve glia expressed heterogeneous purinoreceptors, with P2Y1< or = P2Y2/4< or = P2X. The ATP evoked increase in [Ca2+]i was reversibly blocked by the P2X/Y purinoreceptor antagonist suramin (100 microM) and markedly reduced by thapsigargin (10 microM), which blocks IP3-dependent release of Ca2+ from intracellular stores. Removal of extracellular Ca2+ reduced the ATP evoked increase in [Ca2+]i and completely blocked its recovery, indicating that refilling of intracellular stores was ultimately dependent on Ca2+ influx from the extracellular milieu. The results implicate ATP as an important signal in CNS white matter astrocytes and oligodendrocytes in situ, and indicate that metabotropic P2Y purinoreceptors mobilize intracellular Ca2+ at physiological concentrations of ATP, whereas ionotropic P2X purinoreceptors induce Ca2+ influx across the plasmalemma only at high concentrations of ATP, such as occur following CNS injury.     

Effect of p-chloroamphetamine on calcium movement and viability in renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, the effect of p-chloroamphetamine, a neurotoxin that depletes intracellular serotonin, on intracellular Ca2+ concentration ([Ca2+]i) and viability was measured by using the Ca2+-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium. p-Chloroamphetamine (> or = 10 microM) caused a rapid rise of [Ca2+]i in a concentration-dependent manner. p-Chloroamphetamine-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+. p-Chloroamphetamine-induced extracellular Ca2+ influx was also suggested by Mn2+ influx-induced fura-2 fluorescence quench. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which p-chloroamphetamine failed to increase [Ca2+]i; also, pretreatment with p-chloroamphetamine reduced 50% of thapsigargin-sensitive Ca2+ stores. U73122, an inhibitor of phospholipase C, abolished ATP (but not p-chloroamphetamine)-induced [Ca2+]i rise. Overnight incubation with 1-500 microM p-chloroamphetamine decreased cell viability. These findings suggest that p-chloroamphetamine evokes a rapid increase in [Ca2+]i in renal tubular cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release, and is cytotoxic.     

Depolarization of the membrane potential decreases the ATP-induced influx of extracellular Ca2+ and the refilling of intracellular Ca2+ stores in rat thyroid FRTL-5 cells.

The aim of the present study was to investigate the effect of membrane depolarization on ATP-induced changes in intracellular Ca2+ ([Ca2+]i) and the refilling of intracellular Ca2+ stores in thyroid follicular FRTL-5 cells. Depolarizing the cells with 50 mM K+, an amount sufficient to almost totally depolarize the cells as determined by bisoxonal, significantly reduced the ATP-induced uptake of 45Ca2+. This effect was not dependent on an enhanced efflux of Ca2+, as no difference in the ATP-induced efflux of 45Ca2+ was obtained between control cells and depolarized cells. The ATP-induced transient increase in [Ca2+]i in Fura-2 loaded cells was not altered by depolarization, whereas the ATP-induced plateau in [Ca2+]i was decreased compared with control cells. Furthermore, in cells stimulated with ATP in a Ca(2+)-free buffer, readdition of Ca2+ after the termination of the ATP response induced a decreased response in [Ca2+]i in depolarized cells. Refilling of intracellular Ca2+ stores was investigated by first stimulating the cells with noradrenaline (NA). The effect of NA was then terminated with prazosin, and the cells restimulated with ATP. In cells depolarized with high K+, the response to ATP was decreased compared with that seen in control cells. The results thus suggest that both the ATP-induced influx of extracellular Ca2+ and the refilling of intracellular Ca2+ stores is decreased in depolarized FRTL-5 cells.     

Adenosine triphosphate induces a low [Ca2+]i sensitivity of phosphorylation and an unusual form of receptor desensitization in smooth muscle.

The contractile sensitivity of smooth muscle to changes in myoplasmic [Ca2+] is dependent on the form of stimulation. Both myosin phosphorylation and force are less sensitive to increases in [Ca2+]i derived from Ca2+ entry through L-type Ca2+ channels than to increases in [Ca2+] induced by agents which release internal Ca2+ stores. We hypothesized that activation of receptor-operated channels should produce a [Ca2+]i sensitivity similar to that induced by opening L channels. Aequorin-estimated myoplasmic [Ca2+] and myosin light chain phosphorylation were measured in swine carotid media tissues stimulated with ATP, an activator of the only known receptor-operated cation channel in smooth muscle. ATP, via activation of a P2x purinergic receptor, induced large, transient increases in [Ca2+]i, yet only small transient elevations in phosphorylation or force. Rapid desensitization to ATP was partially, but not completely, caused by hydrolysis of ATP into adenosine since 1) alpha-beta-methylene ATP (a poorly hydrolyzable analog of ATP) produced larger, yet still transient increases in [Ca2+]i, phosphorylation, and force; 2) BW A1433U, a P1 (adenosine) receptor antagonist, enhanced ATP-induced contractions; and 3) ATP, but not alpha-beta-methylene ATP increased bath [adenosine]. The [Ca2+]i sensitivity of phosphorylation during P2x receptor activation was similar to that observed with KCl-depolarization-induced opening of L channels, supporting the hypothesis that transplasmalemmal Ca2+ influx produces less phosphorylation and force than mobilization of intracellular Ca2+ stores. Cumulative additions of higher alpha-beta-methylene ATP concentrations induced repeated transient contractions, indicative of an unusual form of receptor desensitization which could be explained if the affinity of the P2x receptor for ATP, but not the receptor number were rapidly reduced.     

Extracellular ATP induces a nonspecific permeability of thymocyte plasma membranes

We have previously demonstrated that extracellular ATP can give medullary thymocytes the calcium message required for the induction of their blastogenesis, without mobilization of intracellular calcium. We describe here the effects of extracellular nucleotides on membrane permeability to monovalent and divalent cations in mouse thymocytes. Among all nucleotides tested, under physiological conditions, only ATP and, to a lesser extent, 2-methylthio-ATP, adenosine 5'-O-(3-thio-triphosphate), and ADP were able to depolarize thymocyte plasma membranes and to induce Na+ and Ca2+ influxes into thymocytes; other nonhydrolysable ATP analogs were only effective in the absence of Mg2+. The ATP-induced effects were inhibited in a dose-dependent manner by Mg2+ and greatly potentiated in its absence, which suggests that the tetrabasic ATP4 is probably the active species and that a phosphotransferase activity is not involved in its effects. There ATP-mediated changes in ion fluxes result from an increase in nonspecific permeability of thymocyte membranes, probably by pore formation. These ion flux changes might be responsible for the mitogenic induction of phorbol 12-myristate 13-acetate treated medullary thymocytes. The potency order for the adenine derivatives to affect these fluxes (ATP greater than ADP much greater than AMP greater than adenosine) suggests the presence of ATP specific receptors (P2 purinergic receptors) on thymocyte plasma membranes.     

Increased plasma membrane permeability to Ca2+ in anti-Ig-stimulated B lymphocytes is dependent on activation of phosphoinositide hydrolysis

The biochemical basis of Ca2+ mobilization after anti-Ig binding to B cell Ag-R has been further characterized by flow cytometric analysis of indo-1-loaded B cells. The ability to distinguish intracellular Ca2+ release from extracellular Ca2+ influx by using an extracellular calcium depletion-repletion approach has allowed us to study the relationship between the mobilization of Ca2+ from these sources. Studies involving manipulation of the Ca2+ gradient across the plasma membrane indicate that a significant portion of the Ca2+ mobilization response is preserved even when the normal inwardly directed Ca2+ gradient is reversed. In the presence of an extracellular calcium concentration ([Ca2+]o) of 10 microM, the response to anti-Ig is not blocked by the organic Ca2+ channel blockers. This response is not reduced by further depletion of [Ca2+]o by EGTA Ca2+-binding buffers. Thus, the Ca2+ response that occurs when [Ca2+]o less than or equal to 10 microM represents intracellular calcium release. Analysis of B cells stimulated with anti-Ig in low Ca2+ medium ([Ca2+]o = less than 10 microM) followed by repletion of [Ca2+]o to 1 to 5 mM reveals that a significant increase in permeability of the plasma membrane to Ca2+ develops in the stimulated cells. The resultant Ca2+ influx is nimodipine (20 microM) sensitive. Both intracellular Ca2+ release and Ca2+ influx are reduced in parallel as the concentration of anti-Ig stimulus is decreased, suggesting that Ca2+ influx may be coupled to the release of intracellular stores. Neomycin blocks anti-Ig-stimulated formation of inositol trisphosphate, which mediates release of Ca2+ from the endoplasmic reticulum. It also blocks the anti-Ig-induced release of intracellular Ca2+ stores as well as Ca2+ influx, indicating that both responses may be dependent upon phosphatidylinositol 4,5-bisphosphate hydrolysis.