Extracellular nucleotide signaling in the inner ear

Extracellular nucleotides, particularly adenosine 5'-triphosphate (ATP), act as signaling molecules in the inner ear. Roles as neurotransmitters, neuromodulators, and as autocrine or paracrine humoral factors are evident. The diversity of the signaling pathways for nucleotides, which include a variety of ATP-gated ion channels (assembled from different subtypes of P2X-receptor subunit) and also different subtypes of G protein-coupled nucleotide receptors (P2Y receptors) supports a major physiological role for ATP in the regulation of hearing and balance. Almost invariably both P2X and P2Y receptor expression is apparent in the complex tissue structures associated with the inner-ear labyrinth. However P2X-receptor expression, commonly associated with fast neurotransmission, is apparent not only with the cochlear and vestibular primary afferent neurons, but also appears to mediate humoral signaling via ATP-gated ion channel localization to the endolymphatic surface of the cochlear sensory epithelium (organ of Corti). This is the site of the sound-transduction process and recent data, including both electrophysiological, imaging, and immunocytochemistry, has shown that the ATP-gated ion channels are colocalized here with the mechano-electrical transduction channels of the cochlear hair cells. In contrast to this direct action of extracellular ATP on the sound-transduction process, an indirect effect is apparent via P2Y-receptor expression, prevalent on the marginal cells of the stria vascularis, a tissue that generates the standing ionic and electrical gradients across the cochlear partition. The site of generation of these gradients, including the dark-cell epithelium of the vestibular labyrinth, may be under autocrine or paracrine regulation mediated by P2Y receptors sensitive to both purines (ATP) and pyrimidines such as UTP. There is also emerging evidence that the nucleoside adenosine, formed as a breakdown product of ATP by the action of ectonucleotidases and acting via P1 receptors, is also physiologically significant in the inner ear. P1-receptor expression (including A1, A2, and A3 subtypes) appear to have roles associated with stress, acting alongside P2Y receptors to enhance cochlear blood flow and to protect against the action of free radicals and to modulate the activity of membrane conductances. Given the positioning of a diverse range of purinergic-signaling pathways within the inner ear, elevations of nucleotides and nucleosides are clearly positioned to affect hearing and balance. Recent data clearly supports endogenous ATP- and adenosine-mediated changes in sensory transduction via a regulation of the electrochemical gradients in the cochlea, alterations in the active and passive mechanical properties of the cells of the sensory epithelium, effects on primary afferent neurons, and control of the blood supply. The field now awaits conclusive evidence linking a physiologically-induced modulation of extracellular nucleotide and nucleoside levels to altered inner ear function.     

Neural systems affected in developmental dyslexia revealed by functional neuroimaging

Spiral ganglion neurones in rat cochlea express three different isoforms of the P2X2 receptor subunit which assemble into ATP-gated ion channels. Two of these P2X2R subunit isoforms have previously been detected in other auditory tissues. The third isoform (designated P2X2-3R) has not been described. This isoform lacks 39 bp immediately prior to the stop codon, corresponding to a 13 amino acid deletion of the extreme C-terminus domain. Using direct in situ RT-PCR, expression of P2X2R mRNA was confined to a subpopulation of type I spiral ganglion neurones. This study supports a role for extracellular ATP as a neurotransmitter for a discrete population of auditory neurones where variation in P2X2R isoform assembly may confer functional heterogeneity, including enhanced desensitization.     

Detection of polymorphic triplet repeats in the genomes of patients suffering from bipolar affective disorder

P2X receptors are a family of ion channels gated by extracellular ATP. Each member of the family can form functional homomeric channels, but only P2X2 and P2X3 have been shown to combine to form a unique heteromeric channel. Data from in situ hybridization studies suggest that P2X1 and P2X5 may also co-assemble. In this study, we tested this hypothesis by expressing recombinant P2X1 and P2X5 receptor subunits either individually or together in human embryonic kidney 293 cells. In cells expressing the homomeric P2X1 receptor, 30 microM alpha,beta-methylene ATP (alpha,beta-me-ATP) evoked robust currents that completely desensitized in less than 1 sec, whereas alpha,beta-me-ATP failed to evoke current in cells expressing the homomeric P2X5 receptor. By contrast, alpha, beta-me-ATP evoked biphasic currents with a pronounced nondesensitizing plateau phase in cells that co-expressed both subunits. Further, the EC50 for alpha,beta-me-ATP was greater in cells expressing both P2X1 and P2X5 than in cells expressing P2X1 alone (5 and 1.6 microM, respectively). Heteromeric assembly was confirmed using a co-immunoprecipitation assay of epitope-tagged P2X1 and P2X5 subunits. In summary, this study provides biochemical and functional evidence of a novel channel formed by P2X subunit heteropolymerization. This finding suggests that heteromeric subunit assembly constitutes an important mechanism for generating functional diversity of ATP-mediated responses.     

N-Linked glycosylation is essential for the functional expression of the recombinant P2X2 receptor

P2X receptors are integral membrane proteins that belong to the growing family of transmitter-gated ion channels. The extracellular domain of these receptors contains several consensus sequences for N-linked glycosylation that may contribute to the functional expression of the channel. We have previously reported the extracellular orientation of asparagine residues 182, 239, and 298 of the P2X2 receptor subunit by showing that the protein is glycosylated at each site [Torres, G. E., et al. (1998) FEBS Lett. 425, 19-23 (1)]. In this study, we focused on the consequences of removing N-linked glycosylation from the P2X2 receptor by using two different approaches, tunicamycin treatment or site-directed mutagenesis. HEK-293 cells stably transfected with the P2X2 receptor subunit showed little or no response to ATP after tunicamycin treatment. In addition, loss of function was observed with the elimination of all three N-linked glycosylation sites from P2X2. Cell surface labeling with biotin or indirect immunofluorescence revealed that the expression of the nonglycosylated receptors produced by either tunicamycin or site-directed mutagenesis is greatly reduced at the cell surface, indicating that the nonglycosylated P2X2 receptors are retained inside the cell. These data provide the first direct evidence for a critical role of N-linked glycosylation in the cell surface expression of a P2X receptor subunit.     

Identification of amino acid residues contributing to the pore of a P2X receptor

P2X receptors are ion channels opened by extracellular ATP. The seven subunits currently known are encoded by different genes. It is thought that each subunit has two transmembrane domains, a large extracellular loop, and intracellular N- and C-termini, a topology which is fundamentally different from that of other ligand-gated channels such as nicotinic acetylcholine or glutamate receptors. We used the substituted cysteine accessibility method to identify parts of the molecule that form the ionic pore of the P2X2 receptor. Amino acids preceding and throughout the second hydrophobic domain (316-354) were mutated individually to cysteine, and the DNAs were expressed in HEK293 cells. For three of the 38 residues (I328C, N333C, T336C), currents evoked by ATP were inhibited by extracellular application of methanethiosulfonates of either charge (ethyltrimethylammonium, ethylsulfonate) suggesting that they lie in the outer vestibule of the pore. For two further substitutions (L338C, D349C) only the smaller ethylamine derivative inhibited the current. L338C was accessible to cysteine modification whether or not the channel was opened by ATP, but D349C was inhibited only when ATP was concurrently applied. The results indicate that part of the pore of the P2X receptor is formed by the second hydrophobic domain, and that L338 and D349 are on either side of the channel 'gate'.     

Proton potentiation of ATP-gated ion channel responses to ATP and Zn2+ in rat nodose ganglion neurons

1. The modulation by protons of ATP-gated ion channel responses to ATP and Zn2+ was studied in freshly isolated rat nodose ganglion neurons using the whole cell patch-clamp technique. 2. Reduced external pH enhanced, whereas elevated external pH suppressed, current activated by 10 microM ATP. The pH producing the half-maximal effect (EC50) at this ATP concentration was 7.1. 3. Acidification shifted the ATP concentration-response curve to the left, decreasing the EC50 for ATP, and alkalinization shifted the ATP concentration-response curve to the right, increasing the EC50 for ATP. Fitting the data to a single-site pH model yielded an apparent pKa of the site on the ATP-gated ion channel of 7.6. Between pH 6.8 and 7.8, a change of 0.1 pH unit was calculated to change the ATP EC50 by 4.03 microM. Changing pH did not alter the maximal response to ATP. 4. The potentiating effect of protons appeared to be due to a direct action on the ATP-gated channel, as it could not be explained by an increase in the concentration of one or more species of ATP. 5. Lowering pH also increased the potency of Zn2+ for enhancement of ATP-activated current without altering its maximal response. Changing the pH from 7.3 to 6.8 changed the Zn2+ EC50 from 12 to 1.7 microM. 6. The potentiation of ATP-activated current by protons could not be attributed solely to an increase in the affinity of the receptor for Zn2+, as the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine did not alter the effect of protons. 7. Protons and Zn2+ do not appear to act at the same site on ATP-gated channels, as responses to maximally effective concentrations of Zn2+ were enhanced further by protons and vice versa. 8. These results suggest that protons regulate the function of P2X purinoceptors in rat nodose ganglion neurons by modulating the affinity of the binding sites for ATP and Zn2+ on these receptor channels.     

Acid pH augments excitatory action of ATP on a dissociated mammalian sensory neuron

ATP stimulates nociceptive neurons via an action on ligand-gated ion channels. Since tissue injury and inflammation result in both localized acidosis and release of ATP, we studied the effect of acid pH on ATP-gated ion channels in rat nodose ganglion neurons. Lowering pH dramatically increased membrane depolarization and action potential firing elicited by ATP. ATP-activated current was enhanced by acid pH and suppressed by alkaline pH. A pH of 7.2 produced the half-maximal effect. Acidification increased the apparent affinity of the receptor for ATP, as evidenced by a parallel shift of the ATP concentration-response curve to the left. The observations suggest that the localized acidosis associated with tissue injury may enhance pain perception via an action on ATP-gated ion channels on mammalian sensory neurons.     

The P2X1 receptor, an adenosine triphosphate-gated cation channel, is expressed in human platelets but not in human blood leukocytes

Extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) activate multiple types of P2-nucleotide receptors expressed in platelets or leukocytes. Electrophysiological and biochemical studies have indicated expression of the P2X1 receptor, an ATP-gated cation channel, in human and rat platelets, rat basophilic leukemia (RBL) cells, and phorbol myristate acetate (PMA)-differentiated HL-60 myeloid cells. Although these findings suggest that P2X1 receptors are present in both blood leukocytes and blood platelets, the relative levels of P2X1 receptor expression and function in human blood leukocytes and platelets have not been directly characterized. On the basis of both immunoblot analysis and functional assays of P2X1 receptor-mediated ionic fluxes, we report that there is significant expression of P2X1 receptors in human platelets, but not in neutrophils, monocytes, or blood lymphocytes. Thus, unlike platelets and myeloid progenitor cell lines, fully differentiated human blood leukocytes do not express functionally significant numbers of P2X1 receptors, suggesting the downregulation of P2X1 receptor gene expression during the differentiation of phagocytic leukocytes. By contrast, P2X1 receptor expression is strongly maintained during megakaryocytic differentiation and platelet release. Immunoblot analysis indicated that the platelet P2X1 receptor migrates as an approximately 60-kD protein during SDS-electrophoresis under reducing or nonreducing conditions. Treatment of platelet membranes with endoglycosidase-F causes the P2X1 receptor band to migrate as a 46-kD protein, verifying the highly glycosylated nature of the mature receptor protein. Additional studies of nucleotide-induced changes in Ca2+ influx/mobilization demonstrated that the platelet P2X1 receptors are pharmacologically distinct from the well-characterized ADP receptors of these cells. This finding suggests a unique role for these ATP-gated ion channels during hemostasis or thrombosis.     

A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP

Extracellular ATP exerts its effects through P2 purinoceptors: these are ligand-gated ion channels (P2x) or G-protein-coupled receptors (P2Y, P2U). ATP at P2x receptors mediates synaptic transmission between neurons and from neurons to smooth muscle, being responsible, for example, for sympathetic vasoconstriction in small arteries and arterioles. We have now cloned a complementary DNA encoding the P2x receptor from rat vas deferens and expressed it in Xenopus oocytes and mammalian cells. ATP activates a cation-selective ion channel with relatively high calcium permeability. Structural predictions suggest that the protein (399 amino acids long) is mostly extracellular and contains only two transmembrane domains plus a pore-forming motif which resembles that of potassium channels. The P2x receptor thus defines a new family of ligand-gated ion channels.     

Membrane topology of an ATP-gated ion channel (P2X receptor)

Western blots of Xenopus oocyte membrane preparations showed that the apparent molecular mass of the wild type P2X2 receptor (about 65 kDa) was reduced by pretreatment with endoglycosidase H. Mutagenesis of one or more of three potential asparagines (N182S, N239S, and N298S) followed by Western blots showed that each of the sites was glycosylated in the wild type receptor. Functional channels were formed by receptors lacking any single asparagine, but not by channels mutated in two or three positions. Artificial consensus sequences (N-X-S/T) introduced into the N-terminal region (asparagine at position 9, 16, or 26) were not glycosylated. Asparagines were glycosylated when introduced at the C-terminal end of the first hydrophobic domain (positions 62 and 66) and at the N-terminal end of the second hydrophobic domain (position 324). A protein in which the C terminus of one P2X2 subunit was joined to the N terminus of a second P2X2 subunit (from a concatenated cDNA) had twice the molecular mass of the P2X2 receptor subunit, and formed fully functional channels. The experiments provide direct evidence for the topology originally proposed for the P2X receptor, with intracellular N and C termini, two membrane-spanning domains, and a large extracellular loop.     

ATP stimulates sympathetic transmitter release via presynaptic P2X purinoceptors

ATP is a fast transmitter in sympathetic ganglia and at the sympathoeffector junction. In primary cultures of dissociated rat superior cervical ganglion neurons, ATP elicits noradrenaline release in an entirely Ca2+-dependent manner. Nevertheless, ATP-evoked noradrenaline release was only partially reduced (by approximately 50%) when either Na+ or Ca2+ channels were blocked, which indicates that ATP receptors themselves mediated transmembrane Ca2+ entry. An "axonal" preparation was obtained by removing ganglia from explant cultures, which left a network of neurites behind; immunostaining for axonal and dendritic markers revealed that all of these neurites were axons. In this preparation, ATP raised intraaxonal Ca2+ and triggered noradrenaline release, and these actions were not altered when Ca2+ channels were blocked by Cd2+. Hence, Ca2+-permeable ATP-gated ion channels, i.e., P2X purinoceptors, are located at presynaptic sites and directly mediate Ca2+-dependent transmitter release. These presynaptic P2X receptors displayed a rank order of agonist potency of ATP >/= 2-methylthio-ATP > ATPgammaS > alpha,beta-methylene-ATP approximately beta,gamma-methylene-L-ATP and were blocked by suramin or PPADS. ATP, 2-methylthio-ATP, and ATPgammaS also evoked inward currents measured at neuronal somata, but there these agonists were equipotent. Hence, presynaptic P2X receptors resemble the cloned P2X2 subtype, but they appear to differ from somatodendritic P2X receptors in terms of agonist sensitivity. Suramin reduced depolarization-evoked noradrenaline release by up to 20%, when autoinhibitory mechanisms were inactivated by pertussis toxin. These results indicate that presynaptic P2X purinoceptors mediate a positive, whereas G-protein-coupled P2Y purinoceptors mediate a negative, feedback modulation of sympathetic transmitter release.     

An aspartic acid residue near the second transmembrane segment of ATP receptor/channel regulates agonist sensitivity

Charged or polarized amino acid residues near or within the second transmembrane (M2) segment of neuronal ATP receptor/channels (P2X2 receptors) were neutralized by site-directed mutagenesis, and the properties of the mutants were electrophysiologically characterized using Xenopus oocytes. When Asp315 was substituted with Val (D315V), the sensitivity to ATP was reduced by about 60-fold. The sensitivity to ATP was not affected by the neutralization of Lys324, which is involved in a Walker type A ATP-binding sequence, Lys366, Tyr330, or Asn333. With D315V channels, the sensitivities to other agonists (ADP, ATP gamma S, and 2-methylthio ATP) were also reduced. The sensitivities to antagonists (suramin and Cibacron Blue F3GA) were, however, not affected by this neutralization. The results suggest that Asp315, which is assumed to be present in the extracellular region near the M2 segment of P2X2 receptor/channels, serves to maintain agonist sensitivity.     

Functional modulation of P2X2 receptors by cyclic AMP-dependent protein kinase

It is generally believed that protein phosphorylation is an important mechanism through which the functions of voltage- and ligand-gated channels are modulated. The intracellular carboxyl terminus of P2X2 receptor contains several consensus phosphorylation sites for cyclic AMP (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC), suggesting that the function of the P2X2 purinoceptor could be regulated by the protein phosphorylation. Whole-cell voltage-clamp recording was used to record ATP-evoked cationic currents from human embryonic kidney (HEK) 293 cells stably transfected with the cDNA encoding the rat P2X2 receptor. Dialyzing HEK 293 cells with phorbol 12-myristate 13-acetate, a PKC activator, failed to affect the amplitude and kinetics of the ATP-induced cationic current. The role of PKA phosphorylation in modulating the function of the P2X2 receptor was investigated by internally perfusing HEK 293 cells with 8-bromo-cAMP or the purified catalytic subunit of PKA. Both 8-bromo-cAMP and PKA catalytic subunit caused a reduction in the magnitude of the ATP-activated current without affecting the inactivation kinetics and the value of reversal potential. Site-directed mutagenesis was also performed to replace the intracellular PKA consensus phosphorylation site (Ser431) with a cysteine residue. In HEK 293 cells expressing (S431C) mutant P2X2 receptors, intracellular perfusion of 8-bromo-cAMP or purified PKA catalytic subunit did not affect the amplitude of the ATP-evoked current. These results suggest that as with other ligand-gated ion channels, protein phosphorylation by PKA could play an important role in regulating the function of the P2X2 receptor and ATP-mediated physiological effects in the nervous system.     

P2X3 is expressed by DRG neurons that terminate in inner lamina II

The P2X3 receptor subunit, a member of the P2X family of ATP-gated ion channels, is almost exclusively localized in sensory neurons. In the present study, we sought to gain insight into the role of P2X3 and P2X3-containing neurons in sensory transmission, using immunohistochemical approaches. In rat dorsal root ganglia (DRG), P2X3-immunoreactivity (-ir) was observed in small- and medium-sized neurons. Approximately 40% of DRG neuronal profiles in normal rats contained P2X3-ir. In rats that had received neonatal capsaicin treatment, the number of P2X3-positive neurons was decreased by approximately 70%. Analysis of the colocalization of P2X3-ir with cytochemical markers of DRG neurons indicated that approximately 94% of the P2X3-positive neuronal profiles were labelled by isolectin B4 from Bandeiraea simplicifolia, while only 3% contained substance P-ir, and 7% contained somatostatin-ir. In dorsal horn of rat spinal cord, P2X3-ir was observed in the inner portion of lamina II and was reduced subsequent to dorsal rhizotomy, as well as subsequent to neonatal capsaicin treatment. Finally, P2X3-ir accumulated proximal to the site of sciatic nerve ligation, and was seen in nerve fibres in skin and corneal epithelium. In summary, our results suggest that P2X3 is expressed by a functionally heterogeneous population of BSI-B4-binding sensory neurons, and is transported into both central and peripheral processes of these neurons.     

Cystic fibrosis transmembrane regulator-independent release of ATP. Its implications for the regulation of P2Y2 receptors in airway epithelia

The cystic fibrosis (CF) transmembrane regulator (CFTR) is a cyclic AMP-dependent Cl- channel that is defective in CF cells. It has been hypothesized that CFTR exhibits an ATP release function that controls the airway surface ATP concentrations. In airway epithelial cells, CFTR-independent Ca2+-activated Cl- conductance is regulated by the P2Y2 receptor. Thus, ATP may function as an autocrine signaling factor promoting Cl- secretion in normal but not CF epithelia if ATP release is defective. We have tested for CFTR-dependent ATP release using four independent detection systems. First, a luciferase assay detected no differences in ATP concentrations in the medium from control versus cyclic AMP-stimulated primary normal human nasal epithelial (HNE) cells. A marked accumulation of extracellular ATP resulted from mechanical stimulation effected by a medium displacement. Second, high pressure liquid chromatography analysis of 3H-labeled species released from [3H]adenine-loaded HNE cells revealed no differences between basal and cyclic AMP-stimulated cells. Mechanical stimulation of HNE cells again resulted in enhanced accumulation of extracellular [3H]ATP and [3H]ADP. Third, when measuring ATP concentrations via nucleoside diphosphokinase-catalyzed phosphorylation of [alpha-33P]dADP, equivalent formation of [33P]dATP was observed in the media of control and cyclic AMP-stimulated HNE cells and nasal epithelial cells from wild-type and CF mice. Mechanically stimulated [33P]dATP formation was similar in both cell types. Fourth, 1321N1 cells stably expressing the human P2Y2 receptor were used as a reporter system for detection of ATP via P2Y2 receptor-promoted formation of [3H]inositol phosphates. Basal [3H]inositol phosphate accumulation was of the same magnitude in control and CFTR-transduced cells, and no change was observed following addition of forskolin and isoproterenol. In both cell types, mechanical stimulation resulted in hexokinase-attenuable [3H]inositol phosphate formation. In summary, our data suggest that ATP release may be triggered by mechanical stimulation of cell surfaces. No evidence was found supporting a role for CFTR in the release of ATP.     

An open multicentre study of tropisetron for cisplatin-induced nausea and vomiting

1. P2X-receptors are ligand-gated ion channels which activate within milliseconds of agonist binding, causing rapid cellular depolarization and excitation. This makes them ideally suited to mediate the rapid neurotransmitter functions of adenosine 5'-triphosphate (ATP). 2. The initial postjunctional response of the vas deferens and most blood vessels to sympathetic nerve stimulation is a rapid, transient excitatory junction potential (EJP). With sufficient stimulation EJPs summate and the membrane depolarizes sufficiently to open voltage-dependent calcium channels, initiating a calcium action potential and contraction. 3. EJPs are inhibited by desensitization of the P2X-receptor by the stable agonist alpha, beta-methyleneATP (alpha, beta-meATP) and by the P2X-receptor antagonists ANAPP3, suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, indicating that they are consequent upon activation of the P2X-receptor. 4. The P2X-receptor was originally defined by contractile studies in smooth muscle preparations, where a rank order of agonist potency of alpha, beta-meATP > > 2-methylthioATP (2-meSATP) > or = ATP was found. However, recent results show that the potency of ATP and 2-meSATP, but not alpha, beta-meATP, is decreased by 100-to 1000-fold by breakdown and when this is prevented, ATP and 2-meSATP are more potent than alpha, beta-meATP as agonists at the P2X-receptor. 5. This conclusion was supported by the cloning and functional expression of the P2X1-receptor from the rat bladder. A total of seven P2X-subunits have since been cloned and the P2X1-subunit is thought to be the predominant subunit expressed in vascular smooth muscle cells.     

Nucleotide receptors

Adenosine 5'-triphosphate (ATP) and/or related nucleotides act at both ionotropic (P2X) and metabotropic (P2Y) receptors. P2X receptor subunits (P2X1-P2X7) form ligand-gated cation channels, as homomultimers or heteromultimers. Recent work indicates that P2X3 subunits participate in channels expressed by nociceptive sensory neurons, and that the second of the two transmembrane domains of each subunit contributes to the ion permeation pathway. P2X7 subunits form large cytolytic pores in addition to cation channels; they have been found in macrophages and brain microglia. P2Y receptors form a distinct subset of G-protein-coupled receptors; most couple through G proteins to phospholipase C, but inhibition of adenylate cyclase and N-type Ca2+ channels, and activation of K+ channels also occurs. Expressed P2Y receptors have generally been distinguished pharmacologically by the rank order of effectiveness of agonists; some prefer pyrimidines to purines. Recent studies suggest that it is important to use purified nucleotides in such classifications. Several P2Y receptors have a very widespread tissue distribution.     

Mechanical stress induces release of ATP from Ehrlich ascites tumor cells

The supernatant from a suspension of Ehrlich cells exposed to centrifugation at 700xg for 45 s induced a transient increase in the intracellular concentration of free, cytosolic Ca2+, [Ca2+]i, as well as activation of an outwardly rectifying whole-cell current when added to a suspension of non-stimulated cells. These effects were inhibited by suramin, a non-specific P2 receptor antagonist, and mimicked by ATP. Reversed phase HPLC analysis revealed that the supernatant from Ehrlich cells exposed to centrifugation contained 2. 6+/-0.2 microM ATP, and that the mechanical stress-induced release of ATP was inhibited by glibenclamide and verapamil, non-specific inhibitors of the cystic fibrosis transmembrane conductance regulator and P-glycoprotein, respectively. After trypan blue staining, less than 0.5% of the cells were unable to extrude the dye. Addition of extracellular ATP induced a suramin-sensitive, transient, concentration-dependent increase in [Ca2+]i, activation of an outwardly rectifying whole-cell current and a hyperpolarization of the plasma membrane. The ATP-induced hyperpolarization of the plasma membrane was strongly inhibited in the presence of charybdotoxin (ChTX), an inhibitor of several Ca2+-activated K+ channels, suggesting that stimulation of P2 receptors in Ehrlich cells evokes a Ca2+-activated K+ current. The relative potencies of several nucleotides (ATP, UTP, ADP, 2-MeSATP, alpha,beta-MeATP, bzATP) in eliciting an increase in [Ca2+]i, as well as the effect of repetitive addition of nucleotides were investigated. The results lead us to conclude that mechanical stimulation of Ehrlich cells leads to release of ATP, which in turn stimulates both P2Y1 and P2Y2 receptors, resulting in Ca2+ influx as well as release and activation of an outwardly rectifying whole-cell current.