Glutamate release and synapsin-I phosphorylation induced by P2X7 receptors activation in cerebellar granule neurons

The present work reports that activation of P2X7 receptor induces synaptic vesicle release in granule neurons and phosphorylation of synapsin-I by calcium-calmodulin-dependent protein kinase II (CaMKII), which in turn modulates secretory event. ATP, in absence of magnesium, induced a concentration-dependent glutamate release with an EC50 value of 1.95 microM. The involvement of P2X7 receptor was suggested when maximal secretory response was significantly reduced by the selective P2X7 antagonist Brilliant Blue G (BBG; 100 nM) and abolished by removing extracellular Ca2+. The involvement of P2X7 receptor on synaptic vesicle release was confirmed by measuring the release of FM 1-43 dye. In this case, pharmacological activation of P2X7 was achieved with the more selective agonist 2'-3'-o-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP; 100 microM) showing a significant FM 1-43 release that was blocked by BBG (100 nM), by Zn2+ ions (100 microM), both P2X7 blockers, but not by suramin (100 microM), antagonist of P2X1, P2X2, P2X3 and P2X5. In addition, BzATP, through P2X7 receptor activation, significantly increased the phosphorylation of synapsin-I, the main presynaptic target of CaMKII. Both effects mediated by BzATP were inhibited by the CaMKII inhibitors KN-62 (10 microM) and KN-93 (10 microM). These results suggest, therefore, that Ca2+ entrance mediated by P2X7 receptor induces glutamate release and in parallel synapsin-I phosphorylation.     

Extracellular ATP facilitates flow-induced vasodilatation in rat small mesenteric arteries

ATP can be released from endothelial cells, and this release is increased by intraluminal flow in blood vessels. In the present study, the effect of extracellular ATP (1 microM) on flow-induced vasodilatation was investigated in isolated and pressurized rat small mesenteric arteries. In the absence of extracellular ATP, only 46% of arteries developed dilatation in response to flow, and this response was both transient and unstable. In marked contrast, with ATP present, all vessels developed a prolonged and stable dilatation in response to flow. Even in the vessels that failed to respond to flow in the absence of ATP, dilatation could be stimulated once ATP was present. The ability of ATP to facilitate flow-induced vasodilatation was mimicked by UTP (1 microM), a P2Y agonist, or 3'-O-(4-benzoyl)benzoyl ATP (BzATP; 10 microM), an agonist for P2X1, P2X7, and P2Y11 purinoceptors. The involvement of P2X7 purinoceptors was further supported by the inhibitory effect of KN-62 (1 microM), a P2X7 antagonist, on the action of BzATP. P2X1 and P2X3 purinoceptors were not involved because their receptor agonist alpha,beta-methylene ATP had no effect. The facilitating effect of ATP on flow dilatation was also attenuated by the combined application of reactive blue 2 (100 microM), a P2Y antagonist, and suramin (100 microM), a nonselective P2X and P2Y antagonist. Furthermore, flow-induced dilatation obtained in the presence of ATP was reproducible. In contrast, in the additional presence of the ectonucleotidase inhibitor ARL-67156 (10 microM), although the first dilatation was normal, the responses to the second and later exposures to flow were greatly attenuated. The nonhydrolyzable ATP analogs adenosine-5'-(3-thiotriphosphate)trilithium salt (1 microM) and adenosine 5'-(beta,gamma-imido) triphosphate tetralithium salt hydrate (10 microM) had similar effects to those of ARL-67156. These data suggest that ATP acts through both P2X and P2Y purinoceptors to facilitate flow-induced vasodilatation and that ectonucleotidases prevent this effect by degrading ATP on the endothelial cell surface.     

P2X7 nucleotide receptor activation enhances IFN gamma-induced type II nitric oxide synthase activity in BV-2 microglial cells

Under normal and pathological conditions, brain cells release nucleotides that regulate a wide range of cellular responses due to activation of P2 nucleotide receptors. In this study, the effect of extracellular nucleotides on IFN gamma-induced NO release in murine BV-2 microglial cells was investigated. BV-2 cells expressed mRNA for metabotropic P2Y and ionotropic P2X receptors. Among the P2 receptor agonists tested, ATP, ADP, 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP), and 2-methylthio-ATP (2-MeSATP), but not UTP, enhanced IFN gamma-induced iNOS expression and NO production, suggesting that the uridine nucleotide receptors P2Y2 and P2Y6 are not involved in this response. U0126, an antagonist for MEK1/2, a kinase that phosphorylates the extracellular signal-regulated kinases ERK1/2, decreased IFN gamma-induced NO production. BzATP, a potent P2X7 receptor agonist, was more effective than ATP, ADP, or 2-MeSATP at enhancing IFN gamma-induced ERK1/2 phosphorylation. Consistent with activation of the P2X7 receptor, periodate-oxidized ATP, a P2X7 receptor antagonist, and suramin, a non-specific P2 receptor antagonist, inhibited the effect of ATP or BzATP on IFN gamma-induced NO production, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), an antagonist of several P2X receptor subtypes, was ineffective. These results suggest that activation of P2X7 receptors may contribute to inflammatory responses in microglial cells seen in neurodegenerative diseases.     

Canine erythrocytes express the P2X7 receptor: greatly increased function compared with human erythrocytes

Over three decades ago, Parker and Snow (Am J Physiol 223: 888-893, 1972) demonstrated that canine erythrocytes undergo an increase in cation permeability when incubated with extracellular ATP. In this study we examined the expression and function of the channel/pore-forming P2X(7) receptor on canine erythrocytes. P2X(7) receptors were detected on canine erythrocytes by immunocytochemistry and immunoblotting. Extracellular ATP induced (86)Rb(+) (K(+)) efflux from canine erythrocytes that was 20 times greater than that from human erythrocytes. The P2X(7) agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-trisphosphate (BzATP) was more potent than ATP, and both stimulated (86)Rb(+) efflux from erythrocytes in a dose-dependent fashion with EC(50) values of approximately 7 and approximately 309 microM, respectively. 2-Methylthioadenosine 5'-triphosphate and adenosine 5'-O-(3-thiotriphosphate) induced a smaller (86)Rb(+) efflux from erythrocytes, whereas ADP, AMP, UTP, or adenosine had no effect. ATP-induced (86)Rb(+) efflux from erythrocytes was inhibited by oxidized ATP, KN-62, and Brilliant blue G, known P2X(7) antagonists. ATP also induced uptake of choline(+) into canine erythrocytes that was 60 times greater than that into human erythrocytes. Overnight incubation of canine erythrocytes with ATP and BzATP induced phosphatidylserine exposure in >80% of cells and caused up to 20% hemolysis. In contrast, <30% of human erythrocytes showed phosphatidylserine exposure after overnight incubation with ATP and BzATP, and hemolysis was negligible. Flow cytometric measurements of ATP-induced ethidium(+) uptake showed that P2X(7) function was three times lower in canine monocytes than in human monocytes. These data show that the massive cation permeability increase induced by extracellular ATP in canine erythrocytes results from activation and opening of the P2X(7) receptor channel/pore.     

Activity-dependent development of P2X7 current and Ca2+ entry in rabbit osteoclasts

Bone remodeling is regulated by local factors and modulated by mechanical stimuli. Mechanical stimulation can cause release of ATP, an agent that stimulates osteoclastic resorption at low concentrations and inhibits at high concentrations. We examined whether osteoclasts express P2X(7) receptors, which are activated by high concentrations of ATP and can behave as ion channels or cause the formation of membrane pores. Rabbit osteoclasts were studied using patch clamp techniques. Successive or prolonged applications of 2'- & 3'-O-(4-benzoylbenzoyl)-ATP (BzATP, a relatively potent P2X(7) agonist) or high concentrations of ATP caused the development of a slowly deactivating inward current. The underlying channel was permeable only to small cations, ruling out pore formation. Divalent cations reduced current magnitude, consistent with the presence of P2X(7) receptors, a finding confirmed in rat osteoclasts by immunocytochemistry. Successive applications of BzATP also elicited [Ca(2+)](i) elevations that required extracellular Ca(2+). The BzATP-induced current and the rise of [Ca(2+)](i) were temporally associated, and both were inhibited by PPADS, a P2X(7) antagonist. This study demonstrates that high concentrations of ATP activate P2X(7) receptors and provides the first functional evidence for an extracellular ligand-gated Ca(2+) influx pathway in osteoclasts. ATP released in response to mechanical stimuli may act through P2X(7) receptors to inhibit osteoclastic resorption.     

Modeling P2Y receptor-Ca2+ response coupling in taste cells

Here we elaborated an analytical approach for the simulation of dose-response curves mediated by cellular receptors coupled to PLC and Ca(2+) mobilization. Based on a mathematical model of purinergic Ca(2+) signaling in taste cells, the analysis of taste cells responsiveness to nucleotides was carried out. Consistently with the expression of P2Y(2) and P2Y(4) receptors in taste cells, saturating ATP and UTP equipotently mobilized intracellular Ca(2+). Cellular responses versus concentration of BzATP, a P2Y(2) agonist and a P2Y(4) antagonist, implicated high and low affinity BzATP receptors. Suramin modified the BzATP dose-response curve in a manner that suggested the low affinity receptor to be weakly sensitive to this P2Y antagonist. Given that solely P2Y(2) and P2Y(11) are BzATP receptors, their high sensitivity to suramin is poorly consistent with the suramin effects on BzATP responses. We simulated a variety of dose-response curves for different P2Y receptor sets and found that the appropriate fit of the overall pharmacological data was achievable only with dimeric receptors modeled as P2Y(2)/P2Y(4) homo- and heterodimers. Our computations and analytical analysis of experimental dose-response curves raise the possibility that ATP responsiveness of mouse taste cells is mediated by P2Y(2) and P2Y(4) receptors operative mostly in the dimeric form.     

P2X₇-mediated calcium influx triggers a sustained, PI3K-dependent increase in metabolic acid production by osteoblast-like cells

The P2X? receptor is an ATP-gated cation channel expressed by a number of cell types, including osteoblasts. Genetically modified mice with loss of P2X? function exhibit altered bone formation. Moreover, activation of P2X? in vitro stimulates osteoblast differentiation and matrix mineralization, although the underlying mechanisms remain unclear. Because osteogenesis is associated with enhanced cellular metabolism, our goal was to characterize the effects of nucleotides on metabolic acid production (proton efflux) by osteoblasts. The P2X? agonist 2',3'-O-(4-benzoylbenzoyl)ATP (BzATP; 300 μM) induced dynamic membrane blebbing in MC3T3-E1 osteoblast-like cells (consistent with activation of P2X? receptors) but did not induce cell death. Using a Cytosensor microphysiometer, we found that 9-min exposure to BzATP (300 μM) caused a dramatic increase in proton efflux from MC3T3-E1 cells (～2-fold), which was sustained for at least 1 h. In contrast, ATP or UTP (100 μM), which activate P2 receptors other than P2X?, failed to elicit a sustained increase in proton efflux. Specific P2X? receptor antagonists A 438079 and A 740003 inhibited the sustained phase of the BzATP-induced response. Extracellular Ca2? was required during P2X? receptor stimulation for initiation of sustained proton efflux, and removal of extracellular glucose within the sustained phase abolished the elevation elicited by BzATP. In addition, inhibition of phosphatidylinositol 3-kinase blocked the maintenance but not initiation of the sustained phase. Taken together, we conclude that brief activation of P2X? receptors on osteoblast-like cells triggers a dramatic, Ca2?-dependent stimulation of metabolic acid production. This increase in proton efflux is sustained and dependent on glucose and phosphatidylinositol 3-kinase activity.     

Pharmacological Properties and Physiological Function of a P2X-Like Current in Single Proximal Tubule Cells Isolated from Frog Kidney

Although previous studies have provided evidence for the expression of P2X receptors in renal proximal tubule, only one cell line study has provided functional evidence. The current study investigated the pharmacological properties and physiological role of native P2X-like currents in single frog proximal tubule cells using the whole-cell patch-clamp technique. Extracellular ATP activated a cation conductance (P2X(f)) that was also Ca2+-permeable. The agonist sequence for activation was ATP = αβ-MeATP > BzATP = 2-MeSATP, and P2X(f) was inhibited by suramin, PPADS and TNP-ATP. Activation of P2X(f) attenuated the rundown of a quinidine-sensitive K+ conductance, suggesting that P2X(f) plays a role in K+ channel regulation. In addition, ATP/ADP apyrase and inhibitors of P2X(f) inhibited regulatory volume decrease (RVD). These data are consistent with the presence of a P2X receptor that plays a role in the regulation of cell volume and K+ channels in frog renal proximal tubule cells.     

Inhibitory effects of U73122 and U73343 on Ca2+ influx and pore formation induced by the activation of P2X7 nucleotide receptors in mouse microglial cell line

P2X7 receptors are ATP-gated ion channels and play important roles in microglial functions in the brain. Activation of P2X7 receptors by ATP or its agonist BzATP induces Ca2+ influx from extracellular space, followed by the formation of non-selective membrane pores that is permeable to larger molecules, such as fluorescent dye. To determine whether phospholipase C (PLC) is involved in the activation of P2X7 receptors in microglial cells, U73122, a specific inhibitor of PLC, and its inactive analogue U73343 were examined on ATP and BzATP-induced channel and pore formation in an immortalized C57BL/6 mouse microglial cell line (MG6-1). ATP induced both a transient and a sustained increase in the intracellular Ca2+ concentration ([Ca2+]i) in MG6-1 cells, whereas BzATP evoked only a sustained increase. U73122, but not U73343, inhibited the transient [Ca2+]i increase involving Ca2+ release from intracellular stores through PLC activation. In contrast, both U73122 and U73343 inhibited the sustained [Ca2+]i increase either prior or after the activation of P2X7 receptor channels by ATP and BzATP. In addition, these U-compounds inhibited the influx of ethidium bromide induced by ATP and BzATP, suggesting possible PLC-independent blockage of the process of P2X7-associated channel and pore formations by U-compounds in C57BL/6 mouse microglial cells.     

Purinergic receptors mediate two distinct glutamate release pathways in hippocampal astrocytes

The purinergic P2X(7) receptor (P2X(7)R) can mediate glutamate release from cultured astrocytes. Using patch clamp recordings, we investigated whether P2X(7)Rs have the same action in hippocampal astrocytes in situ. We found that 2- and 3-O-(4-benzoylbenzoyl)ATP (BzATP), a potent, although unselective P2X(7)R agonist, triggers two different glutamate-mediated responses in CA1 pyramidal neurons; they are transient inward currents, which have the kinetic and pharmacological properties of previously described slow inward currents (SICs) due to Ca(2+)-dependent glutamate release from astrocytes, and a sustained tonic current. Although SICs were unaffected by P2X(7)Rs antagonists, the tonic current was inhibited, was amplified in low extracellular Ca(2+), and was insensitive to glutamate transporter and hemichannel inhibitors. BzATP triggered in astrocytes a large depolarization that was inhibited by P2X(7)R antagonists and amplified in low Ca(2+). In low Ca(2+) BzATP also induced lucifer yellow uptake into a subpopulation of astrocytes and CA3 neurons. Our results demonstrate that purinergic receptors other than the P2X(7)R mediate glutamate release that evokes SICs, whereas activation of a receptor that has features similar to the P2X(7)R, mediates a sustained glutamate efflux that generates a tonic current in CA1 neurons. This sustained glutamate efflux, which is potentiated under non-physiological conditions, may have important pathological actions in the brain.     

ATP binding at human P2X1 receptors. Contribution of aromatic and basic amino acids revealed using mutagenesis and partial agonists

P2X receptors comprise a family of ATP-gated ion channels with the basic amino acids Lys-68, Arg-292, and Lys-309 (P2X(1) receptor numbering) contributing to agonist potency. In many ATP-binding proteins aromatic amino acids coordinate the binding of the adenine group. There are 20 conserved aromatic amino acids in the extracellular ligand binding loop of at least 6 of the 7 P2X receptors. We used alanine replacement mutagenesis to determine the effects of individual conserved aromatic residues on the properties of human P2X(1) receptors expressed in Xenopus oocytes. ATP evoked concentration-dependent (EC(50) approximately 1 microm) desensitizing currents at wild-type receptors and for the majority of mutants there was no change (10 residues) or a <6-fold decrease in ATP potency (6 mutants). Mutants F195A and W259A failed to form detectable channels at the cell surface. F185A and F291A produced 10- and 160-fold decreases in ATP potency. The partial agonists 2',3'-O-(4-benzoyl)-ATP (BzATP) and P(1),P(5)-di(adenosine 5')-pentaphosphate (Ap(5)A) were tested on a range of mutants that decreased ATP potency to determine whether this resulted predominantly from changes in agonist binding or gating of the channel. At K68A and K309A receptors BzATP and Ap(5)A had essentially no agonist activity but antagonized, or for R292A potentiated, ATP responses. At F185A receptors BzATP was an antagonist but Ap(5)A no longer showed affinity for the receptor. These results suggest that residues Lys-68, Phe-185, Phe-291, Arg-292, and Lys-309 contribute to ligand binding at P2X(1) receptors, with Phe-185 and Phe-291 coordinating the binding of the adenine ring of ATP.     

Effects of protons on macroscopic and single-channel currents mediated by the human P2X7 receptor

Human P2X7 receptors (hP2X7Rs) belong to the P2X family, which opens an intrinsic cation channel when challenged by extracellular ATP. hP2X7Rs are expressed in cells of the inflammatory and immune system. During inflammation, ATP and protons are secreted into the interstitial fluid. Therefore, we investigated the effect of protons on the activation of hP2X7Rs. hP2X7Rs were expressed in Xenopus laevis oocytes and activated by the agonists ATP or benzoyl-benzoyl-ATP (BzATP) at different pH values. The protons reduced the hP2X7R-dependent cation current amplitude and slowed the current deactivation depending on the type and concentration of the agonist used. These effects can be explained by (i) the protonation of ATP, which reduces the effective concentration of the agonist ATP⁴⁻ at the high- and low-affinity ATP activation site of the hP2XR, and (ii) direct allosteric inhibition of the hP2X7R channel opening that follows ATP⁴⁻ binding to the low-affinity activation site. Due to the hampered activation via the low-affinity activation site, a low pH (as observed in inflamed tissues) leads to a relative increase in the contribution of the high-affinity activation site for hP2X7R channel opening.     

Endothelial P2X7 receptors’ expression is reduced by schistosomiasis

Endothelial cells control vascular tone, permeability and leukocyte transmigration and are modulated by pro-inflammatory mediators. Schistosomiasis is an intravascular disease associated with inflammation, therefore altering endothelial cells’ phenotype. Purinergic P2X7 receptors (P2X7R) play an important role in inflammation; however, the impact of the disease upon endothelial P2X7R function or expression has not been explored. Using ethidium bromide uptake to investigate P2X7R function, we observed that the effects of ATP (3 mM) and the P2X7R agonist 3′-O-(4-benzoyl)-ATP (BzATP) were smaller in mesenteric endothelial cells from the Schistosoma mansoni-infected group than in the control group. In the control group, BzATP induced endothelial nitric oxide production, which was blocked by the P2X7R antagonists KN-62 and A740003. However, in the infected group, we observed a reduced effect of BzATP and no effect of both P2X7R antagonists, suggesting a downregulation of endothelial P2X7R in schistosomiasis. We observed similar results in both infected and P2X7R^−/− groups, which were also comparable to data obtained with KN-62- or A740004-treated control cells. Data from Western blot and immunocytochemistry assays confirmed the reduced expression of P2X7R in the infected group. In conclusion, our data show a downregulation of P2X7R in schistosomiasis infection, which likely limits the infection-related endothelial damage.     

The human SH-SY5Y neuroblastoma cell-line expresses a functional P2X7 purinoceptor that modulates voltage-dependent Ca2+ channel function

Fura-2 imaging of purinergic stimulation of non-differentiated neuronal human SH-SY5Y cells resulted in a rapid elevation in intracellular Ca2+ ([Ca2+]i) that was dependent on extracellular Ca2+. The rank order of agonists (200 micro m) was as follows: 2',3'-O-(4-benzoyl-benzoyl)-ATP (BzATP) > ATP4- > ATP; whereas 2-(methylthio)-ATP, ADP, UTP and alpha,beta-methylene-ATP and beta,gamma-methylene-ATP were ineffective. The response to BzATP was inhibited by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic-acid (PPADS, 1 micro m), 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-l-tyrosyl)-4-phenylpiperazine (KN-62, 100 nm) and 8-(3-benzamido-4-4-methylbenzamido)-naphthalene-1,3,5-trisulfonic-acid (suramin, 200 micro m). The presence of a P2X7 receptor was confirmed by western blot studies using anti-P2X7. EC50 for BzATP was 212 +/- 6 micro m. BzATP > 30 micro m induced an initial, transient increase in [Ca2+]i before a plateau level was reached. BzATP < 30 micro m only produced a monophasic increase to the plateau level. The transient phase was reduced by the introduction of nimodipine (3 micro m) and to a smaller degree by omega-conotoxin GVIA (1 micro m) despite an almost equal presence of L and N-type Ca2+-channels. In whole-cell voltage-clamp studies at - 90 mV, BzATP (300 micro m) produced a fast activating inward current with a similar pharmacology as observed with Fura-2 imaging. Current clamp studies showed a dose-dependent depolarization to BzATP and ATP4-. BzATP also triggered transmitter release. Thus, the human neuronal SH-SY5Y cell line expresses a functional P2X7 receptor coupled to activation of Ca2+-channels.     

P2X Receptor Chimeras Highlight Roles of the Amino Terminus to Partial Agonist Efficacy,the Carboxyl Terminus to Recovery from Desensitization,and Independent Regulation of Channel Transitions

P2X receptor subtypes can be distinguished by their sensitivity to ATP analogues and selective antagonists. We have used chimeras between human P2X1 and P2X2 receptors to address the contribution of the extracellular ligand binding loop, transmembrane segments (TM1 and TM2), and intracellular amino and carboxyl termini to the action of partial agonists (higher potency and efficacy of BzATP and Ap₅A at P2X1 receptors) and antagonists. Sensitivity to the antagonists NF449, suramin, and PPADS was conferred by the nature of the extracellular loop (e.g. nanomolar for NF449 at P2X1 and P2X2-1EXT and micromolar at P2X2 and P2X1-2EXT). In contrast, the effectiveness of partial agonists was similar to P2X1 levels for both of the loop transfers, suggesting that interactions with the rest of the receptor played an important role. Swapping TM2 had reciprocal effects on partial agonist efficacy. However, TM1 swaps increased partial agonist efficacy at both chimeras, and this was similar for swaps of both TM1 and 2. Changing the amino terminus had no effect on agonist potency but increased partial agonist efficacy at P2X2-1N and decreased it at P2X1-2N chimeras, demonstrating that potency and efficacy can be independently regulated. Chimeras and point mutations also identified residues in the carboxyl terminus that regulated recovery from channel desensitization. These results show that interactions among the intracellular, transmembrane, and extracellular portions of the receptor regulate channel properties and suggest that transitions to channel opening, the behavior of the open channel, and recovery from the desensitized state can be controlled independently.     

Apically Sorted P2X7 Receptors Mediate Purinergic-Induced Pore Formation Preferentially in Apical Domains of the Plasma Membrane

Treatment of human epithelial cervical cells CaSki attached on filters with the P2X7-receptor (P2X7-R) agonist BzATP induced acute transient influx of calcium, most likely the result of P2X7-R channel activation, followed by slower sustained calcium influx. In cultures incubated in the presence of ethidium bromide (EB), BzATP induced slow and sustained influx of the dye with a time-course similar to the late slow calcium influx, suggesting P2X7-R pore formation. The acute and late calcium effects of BzATP were greater if the agonist was added to the luminal solution, facing the apical membrane of the cells. The EB effect of BzATP initially occurred in the apical membrane, while effects in the basolateral membrane were delayed and smaller. These results suggest that in polarized epithelial cells under steady-state conditions the P2X7-R is located in the apical membrane, and activation of the receptor induces formation of P2X7-R pores preferentially in the apical membrane.     

Apically sorted P2X7 receptors mediate purinergic-induced pore formation preferentially in apical domains of the plasma membrane

Treatment of human epithelial cervical cells CaSki attached on filters with the P2X7-receptor (P2X7-R) agonist BzATP induced acute transient influx of calcium, most likely the result of P2X7-R channel activation, followed by slower sustained calcium influx. In cultures incubated in the presence of ethidium bromide (EB), BzATP induced slow and sustained influx of the dye with a time-course similar to the late slow calcium influx, suggesting P2X7-R pore formation. The acute and late calcium effects of BzATP were greater if the agonist was added to the luminal solution, facing the apical membrane of the cells. The EB effect of BzATP initially occurred in the apical membrane, while effects in the basolateral membrane were delayed and smaller. These results suggest that in polarized epithelial cells under steady-state conditions the P2X7-R is located in the apical membrane, and activation of the receptor induces formation of P2X7-R pores preferentially in the apical membrane.     

Modeling P2Y receptor-Ca response coupling in taste cells

Here we elaborated an analytical approach for the simulation of dose-response curves mediated by cellular receptors coupled to PLC and Ca²⁺ mobilization. Based on a mathematical model of purinergic Ca²⁺ signaling in taste cells, the analysis of taste cells responsiveness to nucleotides was carried out. Consistently with the expression of P2Y₂ and P2Y₄ receptors in taste cells, saturating ATP and UTP equipotently mobilized intracellular Ca²⁺. Cellular responses versus concentration of BzATP, a P2Y₂ agonist and a P2Y₄ antagonist, implicated high and low affinity BzATP receptors. Suramin modified the BzATP dose-response curve in a manner that suggested the low affinity receptor to be weakly sensitive to this P2Y antagonist. Given that solely P2Y₂ and P2Y₁₁ are BzATP receptors, their high sensitivity to suramin is poorly consistent with the suramin effects on BzATP responses. We simulated a variety of dose-response curves for different P2Y receptor sets and found that the appropriate fit of the overall pharmacological data was achievable only with dimeric receptors modeled as P2Y₂/P2Y₄ homo- and heterodimers. Our computations and analytical analysis of experimental dose-response curves raise the possibility that ATP responsiveness of mouse taste cells is mediated by P2Y₂ and P2Y₄ receptors operative mostly in the dimeric form.