Comparison of the effect of ATP on intracellular calcium ion dynamics between rat testicular and cerebral arteriole smooth muscle cells

Adenosine 5'-triphosphate (ATP), when released from neuronal and non-neuronal tissues, interacts with cell surface receptors produces a broad range of physiological responses. The goal of the present study was to examine the issue of whether vascular smooth muscle cells respond to ATP. To this end, the dynamics of the intracellular concentration of calcium ions ([Ca(2+)](i)) in smooth muscle cells in testicular and cerebral arterioles was examined by laser scanning confocal microscopy. ATP produced an increase in [Ca(2+)](i) in arteriole smooth muscle cells. While P1 purinoceptor agonists had no effect on this process, P2 purinoceptor agonists induced a [Ca(2+)](i) increase and a P2 purinoceptor antagonist, suramin, completely inhibited ATP-induced [Ca(2+)](i) dynamics in both arteriole smooth muscle cells.In testicular arterioles, Ca(2+) channel blockers and the removal of extracellular Ca(2+), but not thapsigargin pretreatment, abolished the ATP-induced [Ca(2+)](i) dynamics. In contrast, Ca(2+) channel blockers and the removal of extracellular Ca(2+) did not completely inhibit ATP-induced [Ca(2+)](i) dynamics in cerebral arterioles. Uridine 5'-triphosphate caused an increase in [Ca(2+)](i) only in cerebral arterioles and alpha,beta-methylene ATP caused an increase in [Ca(2+)](i) in both testicular and cerebral arterioles.We conclude that testicular arteriole smooth muscle cells respond to extracellular ATP via P2X purinoceptors and that cerebral arteriole smooth muscle cells respond via P2X and P2Y purinoceptors.     

Nongenomic inhibition of catecholamine secretion by 17beta-estradiol in PC12 cells

We investigated the effects of 17beta-estradiol, an estrogen, on [(3)H]norepinephrine ([(3)H]NE) secretion in PC12 cells. Pretreatment with 17beta-estradiol reduced 70 mM K(+)-induced [(3)H]NE secretion in a concentration-dependent manner with a half-maximal inhibitory concentration (IC(50)) of 2 +/- 1 microM. The 70 mM K(+)-induced cytosolic free Ca(2+) concentration ([Ca(2+)](i)) rise was also reduced when the cells were treated with 17beta-estradiol (IC(50) = 15 +/- 2 microM). Studies with voltage-sensitive calcium channel (VSCC) antagonists such as nifedipine and omega-conotoxin GVIA revealed that both L- and N-type VSCCs were affected by 17beta-estradiol treatment. The 17beta-estradiol effect was not changed by pretreatment of the cells with actinomycin D and cycloheximide for 5 h. In addition, treatment with pertussis or cholera toxin did not affect the inhibitory effect of 17beta-estradiol. 17beta-Estradiol also inhibited the ATP-induced [(3)H]NE secretion and [Ca(2+)](i) rise. In PC12 cells, the ATP-induced [Ca(2+)](i) rise is known to occur through P2X(2) receptors, the P2Y(2)-mediated phospholipase C (PLC) pathway, and VSCCs. 17beta-Estradiol pretreatment during complete inhibition of the PLC pathway and VSCCs inhibited the ATP-induced [Ca(2+)](i) rise. Our results suggest that 17beta-estradiol inhibits catecholamine secretion by inhibiting L- and N-type Ca(2+) channels and P2X(2) receptors in a nongenomic manner.     

Effects of ATP on intracellular calcium dynamics of neurons and satellite cells in rat superior cervical ganglia

Adenosine 5'-triphosphate (ATP) which is released from neuronal and non-neuronal tissues interacts with cell surface receptors to produce a broad range of physiological responses. The present study addressed the issue of whether the cells of the superior cervical ganglia (SCG) respond to ATP. To this end, the dynamics of the intracellular calcium ion concentration ([Ca2+]i) of neurons and satellite cells in intact SCG was analyzed by laser scanning confocal microscopy. ATP produced an increase of [Ca2+]i in both neurons and satellite cells; initially, ATP elicited [Ca2+]i increase in satellite cells and, subsequently, a [Ca2+]i change in neurons was observed. P1 purinoceptor agonists had no effect on this process, but P2 purinoceptor agonists induced [Ca2+]i increase and suramin totally inhibited ATP-induced [Ca2+]i dynamics in both neurons and satellite cells. In satellite cells, Ca2+ channel blockers and the removal of extracellular Ca2+, but not thapsigargin pretreatment, abolished ATP-induced [Ca2+]i dynamics. In contrast, thapsigargin pretreatment abolished ATP-induced [Ca2+]i dynamics in neurons. Reactive blue-2 inhibited the ATP-induced reaction on neurons alone. Uridine 5'-triphosphate caused a [Ca2+]i increase in neurons and alpha,beta-methylene ATP caused a [Ca2+]i increase in satellite cells. We concluded that neurons respond to extracellular ATP mainly via P2Y purinoceptors and that satellite cells respond via P2X purinoceptors.     

Activation of P2Y but not P2X(4) nucleotide receptors causes elevation of [Ca2+]i in mammalian osteoclasts

Extracellular nucleotides cause elevation of cytosolic free Ca2+ concentration ([Ca2+](i)) in osteoclasts, although the sources of Ca2+ are uncertain. Activation of P2Y receptors causes Ca2+ release from stores, whereas P2X receptors are ligand-gated channels that mediate Ca2+ influx in some cell types. To examine the sources of Ca2+, we studied osteoclasts from rat and rabbit using fura 2 fluorescence and patch clamp. Nucleotide-induced rise of ([Ca2+](i)) persisted on removal of extracellular Ca2+ (Ca), indicating involvement of stores. Inhibition of phospholipase C (PLC) with U-73122 or inhibition of endoplasmic reticulum Ca(2+)-ATPase with cyclopiazonic acid or thapsigargin abolished the rise of ([Ca2+](i)). After store depletion in the absence of Ca, addition of Ca led to a rise of ([Ca2+](i)) consistent with store-operated Ca2+ influx. Store-operated Ca2+ influx was greater at negative potentials and was blocked by La(3+). In patch-clamp studies where PLC was blocked, ATP induced inward current indicating activation of P2X(4) nucleotide receptors, but with no rise of ([Ca2+](i)). We conclude that nucleotide-induced elevation of [Ca(2+)](i) in osteoclasts arises primarily through activation of P2Y nucleotide receptors, leading to release of Ca2+ from intracellular stores.     

ATP increases intracellular calcium in supraoptic neurons by activation of both P2X and P2Y purinergic receptors

ATP increases intracellular calcium concentration ([Ca(2+)](i)) in supraoptic nucleus (SON) neurons in hypothalamo-neurohypophyseal system explants loaded with the Ca(2+)-sensitive dye, fura 2-AM. Involvement of P2X purinergic receptors (P2XR) in this response was anticipated, because ATP stimulation of vasopressin release from hypothalamo-neurohypophyseal system explants required activation of P2XRs, and activation of P2XRs induced an increase in [Ca(2+)](i) in dissociated SON neurons. However, the ATP-induced increase in [Ca(2+)](i) persisted after removal of Ca(2+) from the perifusate ([Ca(2+)](o)). This suggested involvement of P2Y purinergic receptors (P2YR), because P2YRs induce Ca(2+) release from intracellular stores, whereas P2XRs are Ca(2+)-permeable ion channels. Depletion of [Ca(2+)](i) stores with thapsigargin (TG) prevented the ATP-induced increase in [Ca(2+)](i) in zero, but not in 2 mM [Ca(2+)](o), indicating that both Ca(2+) influx and release of intracellular Ca(2+) contribute to the ATP response. Ca(2+) influx was partially blocked by cadmium, indicating a contribution of voltage-gated Ca(2+) channels. PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid), and iso-PPADS, P2XR antagonists, attenuated, but did not abolish, the ATP-induced increase in [Ca(2+)](i). Combined treatment with PPADS or iso-PPADS and TG prevented the response. A cocktail of P2YR agonists consisting of UTP, UDP, and 2-methylthio-ADP increased [Ca(2+)](i) (with or without tetrodotoxin) that was markedly attenuated by TG. 2-Methylthio-ADP alone induced consistent and larger increases in [Ca(2+)](i) than UTP or UDP. MRS2179, a specific P2Y(1)R antagonist, eliminated the response to ATP in zero [Ca(2+)](o). Thus, both P2XR and P2YR participate in the ATP-induced increase in [Ca(2+)](i), and the P2Y(1)R subtype is more prominent than P2Y(2)R, P2Y(4)R, or P2Y(6)R in SON.     

Regulation by P2 agonists of the intracellular calcium concentration in epithelial cells freshly isolated from rat trachea.

Epithelial cells were isolated from rat trachea by incubation of the organ in a calcium-free medium. The intracellular concentration of calcium ([Ca(2+)](i)) was measured with the calcium-sensitive fluorescent dye fura2. In resting conditions, the cells maintained a low [Ca(2+)](i) in spite of the presence of millimolar concentration of calcium in the incubation medium. These cells had retained intracellular stores of calcium which were emptied after exposure of the cells to thapsigargin, an inhibitor of intracellular calcium ATPases. Substance P (125 nM) transiently increased 2.5-fold the [Ca(2+)](i). ATP (1 mM) doubled the [Ca(2+)](i) after a few seconds and further induced a sustained increase of the [Ca(2+)](i). Coomassie blue fully blocked the response to ATP and extracellular magnesium only inhibited the delayed response to ATP. Among purinergic analogs, only benzoyl-ATP (Bz-ATP), an agonist on P2X ionotropic purinergic receptors, reproduced the response to ATP. UTP and 2-methylthioATP (two agonists on P2Y metabotropic purinergic receptors) transiently increased the [Ca(2+)](i). Thapsigargin, ATP and Bz-ATP increased the uptake of extracellular calcium. RT-PCR analysis revealed that two metabotropic receptors (P2Y(1) and P2Y(2)) and two ionotropic receptors (P2X(4) and P2X(7)) were expressed by the cells present in the suspension. It is concluded that purinergic agonists can modulate the response of rat tracheal epithelial cells by several mechanisms. The activation of metabotropic receptors should mobilize intracellular IP(3)-sensitive calcium pools. The activation of the ionotropic receptors should not only open a non-specific cation channel leading to the entry of calcium but should also induce the formation of pores in cells expressing the P2X(7) receptors, which could be deleterious to these cells.     

Activation of P2Y2 purinoceptor inhibits the activity of the Na+/K+-ATPase in HeLa cells

The role of ATP on regulation of the Na(+)/K(+)-ATPase activity in the human cancerous HeLa cells was investigated. HeLa cells stimulated with increasing ATP concentrations showed a dose-dependent inhibition of the Na(+)/K(+)-ATPase activity. These effects were also obtained by UTP. ATP and UTP provoked a rise in intracellular calcium concentration ([Ca(2+)](i)) persisting for at least 4 min. The inhibitor of phospholipase C, U73122, blocked the elevation of [Ca(2+)](i) provoked by ATP/UTP. The expression of mRNA for P2Y2 and P2Y6 receptors was demonstrated by RT-PCR. ATP/UTP activated PKC-alpha, -betaI and -epsilon isoforms, but not PKC-delta and -zeta. The inhibition of the Na(+)/K(+)-ATPase activity by ATP/UTP was blocked by G?6976, a specific inhibitor of the calcium-dependent PKCs. In conclusion, our results suggest that ATP/UTP modulate Na(+)/K(+)-ATPase activity in HeLa cells through the P2Y2 purinoceptor via calcium mobilisation and activation of calcium-dependent PKCs.     

Bcl2 mitigates Ca2+ entry and mitochondrial Ca2+ overload through downregulation of L-type Ca2+ channels in PC12 cells

Altered calcium homeostasis and increased cytosolic calcium concentrations ([Ca(2+)](c)) are linked to neuronal apoptosis in epilepsy and in cerebral ischemia, respectively. Apoptotic programmed cell death is regulated by the antiapoptotic Bcl2 family of proteins. Here, we investigated the role of Bcl2 on calcium (Ca(2+)) homeostasis in PC12 cells, focusing on L-type voltage-dependent calcium channels (VDCC). Cytosolic Ca(2+) transients ([Ca(2+)](c)) and changes of mitochondrial Ca(2+) concentrations ([Ca(2+)](m)) were monitored using cytosolic and mitochondrially targeted aequorins of control PC12 cells and PC12 cells stably overexpressing Bcl2. We found that: (i) the [Ca(2+)](c) and [Ca(2+)](m) elevations elicited by K(+) pulses were markedly depressed in Bcl2 cells, with respect to control cells; (ii) such depression of [Ca(2+)](m) was not seen either in digitonin-permeabilized cells or in intact cells treated with ionomycin; (iii) the [Ca(2+)](c) transient depression seen in Bcl2 cells was reversed by shRNA transfection, as well as by the Bcl2 inhibitor HA14-1; (iv) the L-type Ca(2+) channel agonist Bay K 8644 enhanced K(+)-evoked [Ca(2+)](m) peak fourfold in Bcl2, and twofold in control cells; (v) in current-clamped cells the depolarization evoked by K(+) generated a more hyperpolarized voltage step in Bcl2, as compared to control cells. Taken together, our experiments suggest that the reduction of the [Ca(2+)](c) and [Ca(2+)](m) transients elicited by K(+), in PC12 cells overexpressing Bcl2, is related to the reduction of Ca(2+) entry through L-type Ca(2+) channels. This may be due to the fact that Bcl2 mitigates cell depolarization, thus diminishing the recruitment of L-type Ca(2+) channels, the subsequent Ca(2+) entry, and mitochondrial Ca(2+) overload.     

Effect of erythromycin on ATP-induced intracellular calcium response in A549 cells

ATP induced a biphasic increase in the intracellular Ca(2+)concentration ([Ca(2+)](i)), an initial spike, and a subsequent plateau in A549 cells. Erythromycin (EM) suppressed the ATP-induced [Ca(2+)](i) spike but only in the presence of extracellular calcium (Ca(2+)(o)). It was ineffective against ATP- and UTP-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] formation and UTP-induced [Ca(2+)](i) spike, implying that EM perturbs Ca(2+) influx from the extracellular space rather than Ca(2+)release from intracellular Ca(2+) stores via the G protein-phospholipase C-Ins(1,4,5)P(3) pathway. A verapamil-sensitive, KCl-induced increase in [Ca(2+)](i) and the Ca(2+) influx activated by Ca(2+) store depletion were insensitive to EM. 3'-O-(4-benzoylbenzoyl)-ATP evoked an Ca(2+)(o)-dependent [Ca(2+)](i) response even in the presence of verapamil or the absence of extracellular Na(+), and this response was almost completely abolished by EM pretreatment. RT-PCR analyses revealed that P2X(4) as well as P2Y(2), P2Y(4), and P2Y(6) are coexpressed in this cell line. These results suggest that in A549 cells 1) the coexpressed P2X(4) and P2Y(2)/P2Y(4) subtypes contribute to the ATP-induced [Ca(2+)](i) spike and 2) EM selectively inhibits Ca(2+) influx through the P2X channel. This action of EM may underlie its clinical efficacy in the treatment of airway inflammation.     

ATP induces intracellular calcium increases and actin cytoskeleton disaggregation via P2x receptors

The consequences of purinoceptor activation on calcium signalling, inositol phosphate metabolism, protein secretion and the actin cytoskeleton were demonstrated in the WRK-1 cell line. Extracellular ATP was used as a secretagogue to induce a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)), acting via P2x purinergic receptors, which causes actin skeleton disaggregation and protein secretion. ATP bound specifically to purinergic receptors, with Ki of 0.8 microM. The magnitude order for binding of different nucleotides was alpha beta-Met-ATP >or= dATPalphaS > ATP >or= ADP > UTP > AMP > suramin. No increase in inositol phosphates (IPs) was observed after ATP application suggesting that the purinergic sites in WRK-1 cells are not of a P2y type. ATP (1-100 microM) caused a concentration-dependent increase in [Ca(2+)](i)(EC(50)= 30 microM). The responses were reproducible without any desensitization over several applications. The response to ATP was abolished when extracellular calcium ([Ca(2+)](e)) was reduced to 100 nM. A non-specific purinergic antagonist, suramin, reversibly inhibited the ATP-response suggesting that ATP is able to bind to P2x purinergic sites to trigger Ca(2+) entry and increase of [Ca(2+)](i). ATP induced a concentration-dependent disaggregation of actin and exocytotic release of proteins both, which were dependent upon [Ca(2+)](e). Similarly, alpha,beta-Met-ATP, a potent P2x agonist also stimulated Ca(2+) mobilization, actin network destructuration, and protein release. In the isolated rat neurohypophysial nerve terminals, ATP was shown to act as a physiological stimulus for vasopressin release via Ca(2+) entry through a P2x receptor [6]. Here, we show that in these nerve terminals, ATP is also able to induce actin disaggregation by a Ca(2+) dependent mechanism. Thus, actin cytoskeleton alterations induced by ATP through activation of P2x receptors could be a prelude to exocytosis.     

Sustained calcium entry through P2X nucleotide receptor channels in human airway epithelial cells

Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing [Ca(2+)](i) in human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Cytosolic Ca(2+) was measured by fluorospectrometry using the fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in [Ca(2+)](i) derived from intracellular stores in a Na(+)-rich environment. In contrast, ATP induced an increase in [Ca(2+)](i) that had transient and sustained components in a Na(+)-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased [Ca(2+)](i) only in Na(+)-free medium, suggesting competition between Na(+) and Ca(2+) through P2XRs. Biochemical evidence showed that the P2X(4) receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca(2+) channels, voltage-dependent Ca(2+) channels, or Na(+)/Ca(2+) exchanger in the ATP-induced sustained Ca(2+) signal was ruled out. In conclusion, these data show that epithelial P2X(4) receptors serve as ATP-gated calcium entry channels that induce a sustained increase in [Ca(2+)](i). In airway epithelia, a P2XR-mediated Ca(2+) signal may have therapeutic benefit for CF.     

P2X2 receptors are essential for [Ca2+]i increases in response to ATP in cultured rat myenteric neurons

We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+]i responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+]i that was greatly decreased by the removal of extracellular Ca2+ concentration ([Ca2+]o). The amplitude of the [Ca2+]i response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+]o-free solution, ATP produced a small transient rise in [Ca2+]i similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+]i was ATP > or = adenosine 5'-O-3-triphosphate > or = CTP > or = 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X2 receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X2 receptors, resulting in a [Ca2+]i increase dependent on [Ca2+]o in rat myenteric neurons. A small part of the ATP-induced [Ca2+]i increase may be also mediated via a P2Y receptor-related mechanism.     

Contribution of two ionotropic purinergic receptors to ATP responses in submandibular gland ductal cells

The effect of extracellular ATP on salivary gland function was compared in wild-type (WT) and P2X(7) knockout (KO) mice. The increase in the intracellular concentration of calcium ([Ca(2+)](i)) in response to carbachol was similar in submandibular ductal cells of WT and KO mice. ATP and its analog, benzoyl-ATP, induced a sustained increase in the [Ca(2+)](i) in WT animals. In KO mice, ATP slightly and transiently increased the [Ca(2+)](i) and benzoyl-ATP had no effect. The response to ATP of WT but not KO mice was blocked by KN-62, Coomassie blue and magnesium. The small response of ATP observed in KO mice was completely blocked in the absence of extracellular calcium, unchanged by U73122 and potentiated by ivermectin indicating the probable involvement of a P2X(4) receptor. A RT-PCR and a Western blot confirmed the presence of these receptors in ducts of both WT and KO mice. ATP increased the permeability of the cells to ethidium bromide and stimulated a phospholipase A(2) activity in WT but not KO mice. Mice submandibular gland cells secreted IL-1beta but this secretion was not modified by ATP and was similar in both groups of animals. The volume of saliva provoked by pilocarpine and the concentration of proteins, sodium and chloride in this saliva was similar in both groups of animals. The concentration of potassium was higher in KO mice. We can conclude that the major purinergic receptors expressed in mice submandibular ductal cells are P2X(7) receptors but that P2X(4) receptors are also involved in some ATP effects.     

Potentiation by propofol of the response of rat submandibular acinar cells to purinergic agonists

The effect of propofol (2,6-diisopropylphenol) on the intracellular concentration of calcium ([Ca(2+)](i)) and on the response of rat submandibular acini to purinergic agonists was studied. By itself, propofol (60 to 200 microM) slowly increased the [Ca(2+)](i) without affecting the production of inositol phosphates. The increase of the [Ca(2+)](i) involved for about 50% the mobilization of thapsigargin-sensitive intracellular calcium pools. The rest of the calcium originated from a pool distinct from mitochondria. Propofol also increased the uptake of extracellular calcium but not manganese by a mechanism inhibited by nickel. The variation of the [Ca(2+)](i) by propofol provoked a decrease of cell volume measured by light scattering. Propofol increased the effect of a maximal concentration of extracellular ATP on the [Ca(2+)](i). This interaction could be observed when propofol and ATP were added simultaneously to the medium but not when propofol had been removed from the medium before adding ATP. Among ATP analogs, propofol only increased the response to benzoyl-ATP (Bz-ATP). The blockade of P2X(7) receptors with oxidized ATP or Coomassie blue did not prevent the interaction between propofol and ATP. The effect of propofol could also be observed even when the concentration of ATP(4-) was decreased by extracellular magnesium to such a level that only P2X(4) receptors could possibly be activated by the nucleotide. Propofol had no effect on the uptake of manganese, the formation of pores and the activation of phospholipase D in response to a P2X(7) agonist. These results exclude an interaction with this receptor.It is concluded that, in rat submandibular acini, propofol can increase the [Ca(2+)](i) and decrease the cell volume. Propofol can also modulate the activation of P2X(4) receptors by extracellular nucleotides. These effects are observed at concentrations of propofol reached during the induction of anesthesia and might explain why hypersalivation has been reported as one of the side-effects of propofol.     

Effect of development on [Ca2+]i transients to ATP in petrosal ganglion neurons: a pharmacological approach using optical recording

ATP, acting through P2X(2)/P2X(3) receptor-channel complexes, plays an important role in carotid body chemoexcitation in response to natural stimuli in the rat. Since the channels are permeable to calcium, P2X activation by ATP should induce changes in intracellular calcium ([Ca(2+)](i)). Here, we describe a novel ex vivo approach using fluorescence [Ca(2+)](i) imaging that allows screening of retrogradely labeled chemoafferent neurons in the petrosal ganglion of the rat. ATP-induced [Ca(2+)](i) responses were characterized at postnatal days (P) 5-8 and P19-25. While all labeled cells showed a brisk increase in [Ca(2+)](i) in response to depolarization by high KCl (60 mM), only a subpopulation exhibited [Ca(2+)](i) responses to ATP. ATP (250-1,000 μM) elicited one of three temporal response patterns: fast (R1), slow (R2), and intermediate (R3). At P5-8, R2 predominated and its magnitude was attenuated 44% by the P2X(1) antagonist, NF449 (10 μM), and 95% by the P2X(1)/P2X(3)/P2X(2/3) antagonist, TNP-ATP (10 μM). At P19-25, R1 and R3 predominated and their magnitudes were attenuated 15% by NF449, 66% by TNP-ATP, and 100% by suramin (100 μM), a nonspecific P2 purinergic receptor antagonist. P2X(1) and P2X(2) protein levels in the petrosal ganglion decreased with development, while P2X(3) protein levels did not change significantly. We conclude that the profile of ATP-induced P2X-mediated [Ca(2+)](i) responses changes in the postnatal period, corresponding with changes in receptor isoform expression. We speculate that these changes may participate in the postnatal maturation of chemosensitivity.     

Effect of diindolylmethane on Ca(2+) homeostasis and viability in PC3 human prostate cancer cells

The effect of the natural product diindolylmethane on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in PC3 human prostate cancer cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Diindolylmethane at concentrations of 20-50 μM induced [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). Diindolylmethane-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca(2+)](i) rise. At concentrations of 50-100 μM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/PI staining data implicate that diindolylmethane (50 and 100 μM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca(2+)](i) rise in PC3 cells by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via phospholipase A(2)-sensitive store-operated Ca(2+) channels. Diindolylmethane caused cell death in which apoptosis may participate.     

Evidence on the operation of ATP-induced capacitative calcium entry in breast cancer cells and its blockade by 17beta-estradiol

Little is known about the regulation of cytosolic calcium Ca(2+) levels ([Ca(2+)](i)) in breast cancer cells. We investigated the existence of capacitative calcium entry (CCE) in the tumorigenic cell line MCF-7 and its responsiveness to ATP. MCF-7 cells express purinergic receptors as well as estrogen receptors (ER). Depletion of calcium stores with thapsigargin (TG, 500 nM) or ATP (10 microM) in the absence of extracellular Ca(2+), resulted in a rapid and transient elevation in [Ca(2+)](i). After recovery of basal levels, Ca(2+) readmission (1.5 mM) to the medium increased Ca(2+) influx (twofold over basal), reflecting pre-activation of a CCE pathway. Cells pretreated with TG were unable to respond to ATP, thus indicating that the same Ca(2+) store is involved in their response. Moreover, IP(3)-dependent ATP-induced calcium mobilization and CCE were completely blocked using compound U-73122, an inhibitor of phospholipase C. Compound 2-APB (75 microM) and Gd(3+) (10 microM), antagonists of the CCE pathway, completely prevented ATP-stimulated capacitative Ca(2+) entry. CCE in MCF-7 cells was highly permeable to Mn(2+) and to the Ca(2+) surrogate Sr(2+). Mn(2+) entry sensitivity to Gd(3+) matched that of the Ca(2+) entry pathway. 17Beta-estradiol blocked ATP-induced CCE, but was without effect on TG-induced CCE. Besides, the estrogen blockade of the ATP-induced CCE was completely abolished by preincubation of the cells with an ER monoclonal antibody. ER alpha immunoreactivity could also be detected in a purified plasma membrane fraction of MCF-7 cells. These results represent the first evidence on the operation of a ATP-responsive CCE pathway in MCF-7 cells and also indicate that 17beta-estradiol interferes with this mechanism by acting at the cell surface level.     

NO upregulation of a cyclic nucleotide-gated channel contributes to calcium elevation in endothelial cells

We investigated whether nitric oxide (NO) upregulates a cyclic nucleotide-gated (CNG) channel and whether this contributes to sustained elevation of intracellular calcium levels ([Ca(2+)](i)) in porcine pulmonary artery endothelial cells (PAEC). Exposure of PAEC to an NO donor, NOC-18 (1 mM), for 18 h increased the protein and mRNA levels of CNGA2 40 and 50%, respectively (P < 0.05). [Ca(2+)](i) in NO-treated cells was increased 50%, and this increase was maintained for up to 12 h after removal of NOC-18 from medium. Extracellular calcium is required for the increase in [Ca(2+)](i) in NO-treated cells. Thapsigargin induced a rapid cytosolic calcium rise, whereas both a CNG and a nonselective cation channel blocker caused a faster decline in [Ca(2+)](i), suggesting that capacitive calcium entry contributes to the elevated calcium levels. Antisense inhibition of CNGA2 expression attenuated the NO-induced increases in CNGA2 expression and [Ca(2+)](i) and in capacitive calcium entry. Our results demonstrate that exogenous NO upregulates CNGA2 expression and that this is associated with elevated [Ca(2+)](i) and capacitive calcium entry in porcine PAEC.     

Ionotropic NMDA and P2X1/5 receptors mediate synaptically induced Ca2+ signalling in cortical astrocytes

Local, global and propagating calcium (Ca(2+)) signals provide the substrate for glial excitability. Here we analyse Ca(2+) permeability of NMDA and P2X(1/5) receptors expressed in cortical astrocytes and provide evidence that activation of these receptors trigger astroglial Ca(2+) signals when stimulated by either endogenous agonists or by synaptic release of neurotransmitters. The Ca(2+) permeability of the ionotropic receptors was determined by reversal potential shift analysis; the permeability ratio P(Ca)/P(K) was 3.1 for NMDA receptors and 2.2 for P2X(1/5) receptors. Selective stimulation of ionotropic receptors (with NMDA and α,β-methyleneATP) in freshly isolated cortical astrocytes induced ion currents associated with transient increases in cytosolic Ca(2+) concentration ([Ca(2+)](i)). Stimulation of neuronal afferents in cortical slices triggered glial synaptic currents and [Ca(2+)](i) responses, which were partially blocked by selective antagonists of NMDA (D-AP5 and UBP141) and P2X(1/5) (NF449) receptors. We conclude that ionotropic receptors contribute to astroglial Ca(2+) signalling and may provide a specific mechanism for fast neuronal-glial signalling at the synaptic level.