P2Y-receptor regulates size of endothelial cells in an intracellular Ca2+ dependent manner

The effects of P2 receptor agonists on cell size and intracellular calcium levels, [Ca(2+)](i), was investigated using cultured endothelial cells isolated from the caudal artery of male Wistar rats. Cell size and [Ca(2+)](i) were measured using a phase-contrast and fluorescent confocal microscopic image analyzer and a Calcium Green fluorescence probe. P2Y receptor agonists, 2-methylthio ATP (2meS-ATP), ADP, UTP and ATP decreased the cell size and increased [Ca(2+)](i) in endothelial cells from rat caudal artery. However, alpha,beta-methylene ATP, a P2X receptor agonist, did not induce these responses. The decrease in size and the increase in [Ca(2+)](i), by 2meS-ATP were blocked by PPADS (P2-antagonist), suramin (P2-antagonist), thapsigargin (Ca(2+) pump inhibitor) and U-73122 (phospholipase C inhibitor). The present results show that activation of P2Y receptors, not P2X receptors, induces a decrease in cell size and an increase in [Ca(2+)](i), and the pharmacological properties of these two responses are the same. We concluded that the size of endothelial cells is regulated by P2Y receptors via intracelluar Ca(2+) derived from Ca(2+) stores.     

Mitochondrial calcium transport is regulated by P2Y1- and P2Y2-like mitochondrial receptors

Ischemia-reperfusion injury remains a major clinical problem in liver transplantation. One contributing factor is mitochondrial calcium (mCa(2+)) overload, which triggers apoptosis; calcium also regulates mitochondrial respiration and adenosine 5'-triphosphate (ATP) production. Recently, we reported the presence of purinergic P2Y(1)- and P2Y(2)-like receptor proteins in mitochondrial membranes. Herein, we present an evaluation of the functional characteristics of these receptors. In experiments with isolated mitochondria, specific P2Y(1) and P2Y(2) receptors ligands: 2-methylthio-adenosine 5'-diphosphate (2meSADP) and uridine 5'-triphosphate (UTP), respectively, were used, and mitochondrial calcium uptake was measured. 2meSADP and UTP had a maximum effect at concentrations in the range of the known P2Y(1) and P2Y(2) receptors. The P2Y inhibitor phosphate-6-azophenyl-2',4'-disulfonate (PPADS) blocked the effects of both ligands. The phospholipase C (PLC) antagonist U73122 inhibited the effect of both ligands while its inactive analog U73343 had no effect. These data strongly support the hypothesis that mitochondrial Ca(2+) uptake is regulated in part by adenine nucleotides via a P2Y-like receptor mechanism that involves mitochondrial PLC activation.     

Purine and pyrimidine nucleotide-sensitive phospholipase A(2) in ampulla from frog semicircular canal

This study was attempted to characterize pharmacologically the P2Y receptors triggering phospholipase A(2) (PLA(2)) activation in ampulla from frog semicircular canal. A microassay was developed to screen the abilities of UTP analogs to stimulate [(3)H]arachidonic acid release by labeled ampullas. At 26 degrees C UTP induced a dose-dependent and saturable increase of PLA(2) activity (apparent activation constant 1.3 +/- 0.4 microM, Hill coefficient 0.9 +/- 0.2, maximal stimulating factor 2.0 +/- 0.1). The rank order of potency of agonists for PLA(2) activation was UTP > or = UDP > adenosine 5'-O-(2-thiodiphosphate) = adenosine 5'-O-(3-thiotriphosphate) > or = ATP = 2-methylthio-ATP > or = ADP = diadenosine tetraphosphate > or = alpha,beta-methylene-ATP = CTP > 2' and 3'-O-(4-benzoylbenzoyl)-ATP > or = AMP = UMP > uridine and adenosine. UTP- and 2-methylthio-ATP-induced PLA(2) activations were inhibited by U-73122, GF-109203X, and methyl arachidonyl fluorophosphate. Basal activity was stimulated by phorbol ester and epinephrine and reduced by vasotocin, isoproterenol, prostaglandin E(2), cAMP, and forskolin. H-89 restored the cAMP- and forskolin-inhibited PLA(2) activities. Results indicate that P2Y receptor-mediated PLA(2) stimulation requires phopholipase C and protein kinase C activations and basal activity is inhibited by agonist-stimulated cAMP-dependent mechanisms.     

Coexpression of several types of metabotropic nucleotide receptors in single cerebellar astrocytes

We have examined the expression of mRNA for several P2Y nucleotide receptors by northern blot analysis in purified type 1 cerebellar astrocyte cultures. These results suggest that different P2Y subtypes could be responsible for ATP metabotropic calcium responses in single type 1 astrocytes. To identify these subtypes we have studied the pharmacological profile of ATP calcium responses using fura-2 microfluorimetry. All tested astrocytes responded to ATP and UTP stimulations evoking similar calcium transients. Most astrocytes also responded to 2-methylthioATP and ADP challenges. The agonist potency order was 2-methylthioATP > ADP > ATP = UTP. Cross-desensitization experiments carried out with ATP, UTP, and 2-methylthioATP showed that 2-methylthioATP and UTP interact with different receptors, P2Y(1) and P2Y(2) or P2Y(4). In a subpopulation of type 1 astrocytes, ATP prestimulation did not block UTP responses, and UDP elicited clear intracellular Ca(2+) concentration responses at very low concentrations. 2-MethylthioATP and UTP calcium responses exhibited different sensitivity to pertussis toxin and different inhibition patterns in response to P2 antagonists. The P2Y(1)-specific antagonist N:(6)-methyl-2'-deoxyadenosine 3', 5'-bisphosphate (MRS 2179) specifically blocked the 2-methylthio-ATP responses. We can conclude that all single astrocytes coexpressed at least two types of P2Y metabotropic receptors: P2Y(1) and either P2Y(2) or P2Y(4) receptors. Moreover, 30-40% of astrocytes also coexpressed specific pyrimidine receptors of the P2Y(6) subtype, highly selective for UDP coupled to pertussis-toxin insensitive G protein.     

Activation of P2Y2 receptor induces c-FOS protein through a pathway involving mitogen-activated protein kinases and phosphoinositide 3-kinases in HeLa cells

The effects of P2Y2 purinoceptor activation on c-Fos expression and the signaling pathways evoked by extracellular ATP/UTP in HeLa cells were investigated. We found that P2Y2 activation induced c-Fos protein and phosphorylated the extracellular signal-regulated kinases 1 and 2 (ERK1/2). The P2Y2-stimulated c-Fos induction was partly blocked (a) by U73122, a phospholipase C inhibitor, (b) by G?6976, a conventional PKC inhibitor, (c) by PD098059, a mitogen-activated protein kinase kinase inhibitor, and, moreover, (d) by the inhibitors of phosphoinositide 3-kinases (PI3K), LY294002 and wortmannin. When G?6976 and PD098059, or G?6976 and wortmannin, were combined there was a totally inhibition of P2Y2-induced c-Fos increase. Either U73122 or G?6976 did not inhibit ERK1/2 phosphorylation induced by ATP/UTP, while it was inhibited by LY294002 (or wortmannin) and by staurosporine. Additionally, wortmannin inhibited the cytosol-to-membrane translocation of PKC- epsilon induced by ATP/UTP. These data indicated that agonist-induced PI3K and downstream PKC- epsilon activation mediated the effect of ATP/UTP on ERK1/2 activation. To test the biological consequences of ERK1/2 activation, the effect of P2Y2 on cell functions were examined. P2Y2 stimulation increased cell proliferation and this effect was attenuated by PD098059 in a dose-dependent manner, thereby indicating that the ERK pathway mediates mitogenic signaling by P2Y2. In conclusion, the activation of conventional PKCs through P2Y2 receptor acts in concert with ERK and PI3K/PKC- epsilon pathways to induce c-Fos protein and HeLa cell proliferation.     

Analysis of purine- and pyrimidine-induced vascular responses in the isolated rat cerebral arteriole

Effects of extraluminal UTP were studied and compared with vascular responses to ATP and its analogs in rat cerebral-penetrating arterioles. UTP, UDP, 2-methylthio-ATP, and alpha,beta-methylene-ATP dilated arterioles at the lowest concentration and constricted them at high concentrations. Low concentrations of ATP dilated the vessels; high concentrations caused a biphasic response, with transient constriction followed by dilation. Endothelial impairment inhibited ATP- and UTP-mediated dilation and potentiated constriction to UTP but not to ATP. ATP- and 2-methylthio-ATP- but not UTP-mediated constrictions were inhibited by desensitization with 10(-6) M alpha,beta-methylene-ATP or 3 x 10(-6) M pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). PPADS at 10(-4) M abolished the UTP-mediated constriction and induced vasodilation in a dose-dependent manner but did not affect the dilation to ATP. These results suggest that in rat cerebral microvessels 1) ATP and 2-methylthio-ATP induce transient constriction via smooth muscle P(2X1) receptors in the cerebral arteriole, 2) UTP stimulates two different classes of P(2Y) receptors, resulting in constriction (smooth muscle P(2Y4)) and dilation (possibly endothelial P(2Y2)), and 3) ATP and UTP produce dilation by stimulation of a single receptor (P(2Y2)).     

P2Y(11), a purinergic receptor acting via cAMP, mediates secretion by pancreatic duct epithelial cells

Pancreatic duct epithelial cells (PDEC) mediate the exocrine secretion of fluid and electrolytes. We previously reported that ATP and UTP interact with P2Y(2) receptors on nontransformed canine PDEC to increase intracellular free Ca2+ concentration ([Ca2+](i)) and stimulate Ca2+-activated Cl- and K+ channels. We now report that ATP interacts with additional purinergic receptors to increase cAMP and activate Cl- channels. ATP, 2-methylthio-ATP, and ATP-gamma-S stimulated a 4- to 10-fold cAMP increase with EC(50) of 10-100 microM. Neither UTP nor adenosine stimulated a cAMP increase, excluding a role for P2Y(2) or P1 receptors. Although UTP stimulated an (125)I(-) efflux that was fully inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), ATP stimulated a partially resistant efflux, suggesting activation of additional Cl- conductances through P2Y(2)-independent and Ca2+-independent pathways. In Ussing chambers, increased cAMP stimulated a much larger short-circuit current (I(sc)) increase from basolaterally permeabilized PDEC monolayers than increased [Ca2+](i). Luminal ATP and UTP and serosal UTP stimulated a small Ca2+-type I(sc) increase, whereas serosal ATP stimulated a large cAMP-type I(sc) response. Serosal ATP effect was inhibited by P2 receptor blockers and unaffected by BAPTA-AM, supporting ATP activation of Cl- conductances through P2 receptors and a Ca2+-independent pathway. RT-PCR confirmed the presence of P2Y(11) receptor mRNA, the only P2Y receptor acting via cAMP.     

Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium

The loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial HCO(3)(-) secretion contributes to the pathogenesis of pancreatic and biliary disease in cystic fibrosis (CF) patients. Recent studies have investigated P2Y(2) nucleotide receptor agonists, e.g., UTP, as a means to bypass the CFTR defect by stimulating Ca(2+)-activated Cl(-) secretion. However, the value of this treatment in facilitating transepithelial HCO(3)(-) secretion is unknown. Gallbladder mucosae from CFTR knockout mice were used to isolate the Ca(2+)-dependent anion conductance during activation of luminal P2Y(2) receptors. In Ussing chamber studies, UTP stimulated a transient peak in short-circuit current (I(sc)) that declined to a stable plateau phase lasting 30-60 min. The plateau I(sc) after UTP was Cl(-) independent, HCO(3)(-) dependent, insensitive to bumetanide, and blocked by luminal DIDS. In pH stat studies, luminal UTP increased both I(sc) and serosal-to-mucosal HCO(3)(-) flux (J(s-->m)) during a 30-min period. Substitution of Cl(-) with gluconate in the luminal bath to inhibit Cl(-)/HCO(3)(-) exchange did not prevent the increase in J(s-->m) and I(sc) during UTP. In contrast, luminal DIDS completely inhibited UTP-stimulated increases in J(s-->m) and I(sc). We conclude that P2Y(2) receptor activation results in a sustained (30-60 min) increase in electrogenic HCO(3)(-) secretion that is mediated via an intracellular Ca(2+)-dependent anion conductance in CF gallbladder.     

P2Y(2) receptor-stimulated phosphoinositide hydrolysis and Ca(2+) mobilization in tracheal epithelial cells

Extracellular nucleotides have been implicated in the regulation of secretory function through the activation of P2 receptors in the epithelial tissues, including tracheal epithelial cells (TECs). In this study, experiments were conducted to characterize the P2 receptor subtype on canine TECs responsible for stimulating inositol phosphate (InsP(x)) accumulation and Ca(2+) mobilization using a range of nucleotides. The nucleotides ATP and UTP caused a concentration-dependent increase in [(3)H]InsP(x) accumulation and Ca(2+) mobilization with comparable kinetics and similar potency. The selective agonists for P1, P2X, and P2Y(1) receptors, N(6)-cyclopentyladenosine and AMP, alpha,beta-methylene-ATP and beta, gamma-methylene-ATP, and 2-methylthio-ATP, respectively, had little effect on these responses. Stimulation of TECs with maximally effective concentrations of ATP and UTP showed no additive effect on [(3)H]InsP(x) accumulation. The response of a maximally effective concentration of either ATP or UTP was additive to the response evoked by bradykinin. Furthermore, ATP and UTP induced a cross-desensitization in [(3)H]InsP(x) accumulation and Ca(2+) mobilization. These results suggest that ATP and UTP directly stimulate phospholipase C-mediated [(3)H]InsP(x) accumulation and Ca(2+) mobilization in canine TECs. P2Y(2) receptors may be predominantly mediating [(3)H]InsP(x) accumulation, and, subsequently, inositol 1,4,5-trisphosphate-induced Ca(2+) mobilization may function as the transducing mechanism for ATP-modulated secretory function of tracheal epithelium.     

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.     

ATP-mediated killing of Mycobacterium bovis bacille Calmette-Guérin within human macrophages is calcium dependent and associated with the acidification of mycobacteria-containing phagosomes

We previously demonstrated that extracellular ATP stimulated macrophage death and mycobacterial killing within Mycobacterium bovis Bacille Calmette-Guérin (BCG)-infected human macrophages. ATP increases the cytosolic Ca(2+) concentration in macrophages by mobilizing intracellular Ca(2+) via G protein-coupled P2Y receptors, or promoting the influx of extracellular Ca(2+) via P2X purinoceptors. The relative contribution of these receptors and Ca(2+) sources to ATP-stimulated macrophage death and mycobacterial killing was investigated. We demonstrate that 1) ATP mobilizes Ca(2+) in UTP-desensitized macrophages (in Ca(2+)-free medium) and 2) UTP but not ATP fails to deplete the intracellular Ca(2+) store, suggesting that the pharmacological properties of ATP and UTP differ, and that a Ca(2+)-mobilizing P2Y purinoceptor in addition to the P2Y(2) subtype is expressed on human macrophages. ATP and the Ca(2+) ionophore, ionomycin, promoted macrophage death and BCG killing, but ionomycin-mediated macrophage death was inhibited whereas BCG killing was largely retained in Ca(2+)-free medium. Pretreatment of cells with thapsigargin (which depletes inositol (1,4,5)-trisphosphate-mobilizable intracellular stores) or 1,2-bis-(2-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid acetoxymethyl ester (an intracellular Ca(2+) chelator) failed to inhibit ATP-stimulated macrophage death but blocked mycobacterial killing. Using the acidotropic molecular probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine, it was revealed that ATP stimulation promoted the acidification of BCG-containing phagosomes within human macrophages, and this effect was similarly dependent upon Ca(2+) mobilization from intracellular stores. We conclude that the cytotoxic and bactericidal effects of ATP can be uncoupled and that BCG killing is not the inevitable consequence of death of the host macrophage.     

ATP stimulates Ca2+ oscillations and contraction in airway smooth muscle cells of mouse lung slices

In airway smooth muscle cells (SMCs) from mouse lung slices, > or =10 microM ATP induced Ca2+ oscillations that were accompanied by airway contraction. After approximately 1 min, the Ca2+ oscillations subsided and the airway relaxed. By contrast, > or =0.5 microM adenosine 5'-O-(3-thiotriphosphate) (nonhydrolyzable) induced Ca2+ oscillations in the SMCs and an associated airway contraction that persisted for >2 min. Adenosine 5'-O-(3-thiotriphosphate)-induced Ca2+ oscillations occurred in the absence of external Ca2+ but were abolished by the phospholipase C inhibitor U-73122 and the inositol 1,4,5-trisphosphate receptor inhibitor xestospongin. Adenosine, AMP, and alpha,beta-methylene ATP had no effect on airway caliber, and the magnitude of the contractile response induced by a variety of nucleotides could be ranked in the following order: ATP = UTP > ADP. These results suggest that the SMC response to ATP is impaired by ATP hydrolysis and mediated via P2Y(2) or P2Y(4) receptors, activating phospholipase C to release Ca2+ via the inositol 1,4,5-trisphosphate receptor. We conclude that ATP can serve as a spasmogen of airway SMCs and that Ca2+ oscillations in SMCs are required to sustain airway contraction.     

Purinergic P2Y2 receptors mediate rapid Ca(2+) mobilization, membrane hyperpolarization and nitric oxide production in human vascular endothelial cells

In blood vessels, stimulation of the vascular endothelium by the Ca(2+)-mobilizing agonist ATP initiates a number of cellular events that cause relaxation of the adjacent smooth muscle layer. Although vascular endothelial cells are reported to express several subtypes of purinergic P2Y and P2X receptors, the major isoform(s) responsible for the ATP-induced generation of vasorelaxant signals in human endothelium has not been well characterized. To address this issue, ATP-evoked changes in cytosolic Ca(2+), membrane potential and acute nitric oxide production were measured in isolated human umbilical vein endothelial cells (HUVECs) and profiled using established P2X and P2Y receptor probes. Whereas selective P2X agonist (i.e. α,β-methyl ATP) and antagonists (i.e. TNP-ATP and PPADS) could neither mimic nor block the observed ATP-evoked cellular responses, the specific P2Y receptor agonist UTP functionally reproduced all the ATP-stimulated effects. Furthermore, both ATP and UTP induced intracellular Ca(2+) mobilization with comparable EC(50) values (i.e. 1-3μM). Collectively, these functional and pharmacological profiles strongly suggest that ATP acts primarily via a P2Y2 receptor sub-type in human endothelial cells. In support, P2Y2 receptor mRNA and protein were readily detected in isolated HUVECs, and siRNA-mediated knockdown of endogenous P2Y2 receptor protein significantly blunted the cytosolic Ca(2+) elevations in response to ATP and UTP, but did not affect the histamine-evoked response. In summary, these results identify the P2Y2 isoform as the major purinergic receptor in human vascular endothelial cells that mediates the cellular actions of ATP linked to vasorelaxation.     

Expression of functional receptor-coupled TRPC3 channels in DT40 triple receptor InsP3 knockout cells

The TRPC3 channel, an intensively studied member of the widely expressed transient receptor potential (TRP) family, is a Ca(2+)-conducting channel activated in response to phospholipase C-coupled receptors. Despite scrutiny, the receptor-induced mechanism to activate TRPC3 channels remains unclear. Evidence indicates TRPC3 channels interact directly with intracellular inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) and that channel activation is mediated through coupling to InsP(3)Rs. TRPC3 channels were expressed in DT40 chicken B lymphocytes in which all three InsP(3)R genes were deleted (DT40InsP(3)R-k/o). Endogenous B-cell receptors (BCR) coupled through Syk kinase to phospholipase C-gamma (PLC-gamma) activated the expressed TRPC3 channels in both DT40w/t and DT40InsP(3)R-k/o cells. The diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) also activated TRPC3 channels independently of InsP(3)Rs. BCR-induced TRPC3 activation was blocked by the PLC enzymic inhibitor, U-73122, and also blocked by wortmannin-induced PLC substrate depletion. Neither U-73122 nor wortmannin modified either OAG-induced TRPC3 activation or store-operated channel activation in DT40 cells. Cotransfection of cells with both G protein-coupled M5 muscarinic receptors and TRPC3 channels resulted in successful M5 coupling to open TRPC3 channels mediated by PLC-beta. We conclude that TRPC3 channels are activated independently of InsP(3)Rs through DAG production resulting from receptor-mediated activation of either PLC-gamma or PLC-beta.     

Increase of [Ca(2+)](i) via activation of ATP receptors in PC-Cl3 rat thyroid cell line.

In PC-Cl3 rat thyroid cell line, ATP and UTP provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Thapsigargin (TG) caused a rapid rise in [Ca(2+)](i) and subsequent addition of ATP was without effect. The transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with the specific inhibitor of phospholipase C (PLC), U73122. These data suggest that the ATP-stimulated increment of [Ca(2+)](i) required InsP(3) formation and binding to its specific receptors in Ca(2+) stores. Desensitisation was demonstrated with respect to the calcium response to ATP and UTP in Fura 2-loaded cells. Further studies were performed to investigate whether the effect of ATP on Ca(2+) entry into PC-Cl3 cells was via L-type voltage-dependent Ca(2+) channels (L-VDCC) and/or by the capacitative pathway. Nifedipine decreased ATP-induced increase on [Ca(2+)](i). Addition of 2 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment of the cells with TG or with 100 microM ATP in Ca(2+)-free medium. These data indicate that Ca(2+) entry into PC-Cl3 stimulated with ATP occurs through both an L-VDCC and through a capacitative pathway. Using buffers with differing Na(+) concentrations, we found that the effects of ATP were dependent of extracellular Na(+), suggesting that a Na(+)/Ca(2+) exchange mechanism is also operative. These data suggest the existence, in PC-Cl3 cell line, of a P2Y purinergic receptor able to increase the [Ca(2+)](i) via PLC activation, Ca(2+) store depletion, capacitative Ca(2+) entry and L-VDCC activation.     

ATP-dependent activation of K(Ca) and ROMK-type K(ATP) channels in human submandibular gland ductal cells.

[Ca(2+)](i) and membrane current were measured in human submandibular gland ductal (HSG) cells to determine the regulation of salivary cell function by ATP. 1-10 microM ATP activated internal Ca(2+) release, outward Ca(2+)-dependent K(+) channel (K(Ca)), and inward store-operated Ca(2+) current (I(SOC)). The subsequent addition of 100 microM ATP activated an inwardly rectifying K(+) current, without increasing [Ca(2+)](i). The K(+) current was also stimulated by ATP in cells treated with thapsigargin in a Ca(2+)-free medium and was blocked by glibenclamide and tolbutamide, but not by charybdotoxin. This suggests the involvement of a Ca(2+)-independent, sulfonylurea-sensitive K(+) channel (K(ATP)). UTP mimicked the low [ATP] effects, while benzoyl-ATP activated internal Ca(2+) release, a Ca(2+) influx pathway, and K(Ca). Thus, ATP acts via P(2U) (P2Y(2)) and P(2Z) (P2X(7)) receptors to increase [Ca(2+)](i) and activate K(Ca), but not K(ATP). Importantly, (i) ROMK1 and the cystic fibrosis transmembrane regulator protein (but not SUR1, SUR2A, or SUR2B) and (ii) cAMP-stimulated Cl(-) and K(+) currents were detected in HSG cells. These data demonstrate for the first time that a ROMK-type K(ATP) channel is present in salivary gland duct cells that is regulated by extracellular ATP and possibly by the cystic fibrosis transmembrane regulator. This reveals a potentially novel mechanism for K(+) secretion in these cells.