P2Y14 Receptor as a Target for Neutrophilia Attenuation in Severe COVID-19 Cases: From Hematopoietic Stem Cell Recruitment and Chemotaxis to Thrombo-inflammation.

The global SARS-CoV-2 pandemic starting in 2019 has already reached more than 2.3 million deaths. Despite the scientific community's efforts to investigate the COVID-19 disease, a drug for effectively treating or curing patients yet needs to be discovered. Hematopoietic stem cells (HSC) differentiating into immune cells for defense express COVID-19 entry receptors, and COVID-19 infection hinders their differentiation. The importance of purinergic signaling in HSC differentiation and innate immunity has been recognized. The metabotropic P2Y14 receptor subtype, activated by UDP-glucose, controls HSC differentiation and mobilization. Thereon, the exacerbated activation of blood immune cells amplifies the inflammatory state observed in COVID-19 patients, specially through the continuous release of reactive oxygen species and extracellular neutrophil traps (NETs). Further, the P2Y14 subtype, robustly inhibits the infiltration of neutrophils into various epithelial tissues, including lungs and kidneys. Here we discuss findings suggesting that antagonism of the P2Y14 receptor could prevent the progression of COVID-19-induced systemic inflammation, which often leads to severe illness and death cases. Considering the modulation of neutrophil recruitment of extreme relevance for respiratory distress and lung failure prevention, we propose that P2Y14 receptor inhibition by its selective antagonist PPTN could limit neutrophil recruitment and NETosis, hence limiting excessive formation of oxygen reactive species and proteolytic activation of the kallikrein-kinin system and subsequent bradykinin storm in the alveolar septa of COVID-19 patients.

Targeting the purinergic pathway in breast cancer and its therapeutic applications.

Breast cancer (BC) is the most frequent cause of death among women, representing a global public health problem. Here, we aimed to discuss the correlation between the purinergic system and BC, recognizing therapeutic targets. For this, we analyzed the interaction of extracellular nucleotides and nucleosides with the purinergic receptors P1 and P2, as well as the influence of ectonucleotidase enzymes (CD39 and CD73) on tumor progression. A comprehensive bibliographic search was carried out. The relevant articles for this review were found in the PubMed, Scielo, Lilacs, and ScienceDirect databases. It was observed that among the P1 receptors, the A1, A2A, and A2B receptors are involved in the proliferation and invasion of BC, while the A3 receptor is related to the inhibition of tumor growth. Among the P2 receptors, the P2X7 has a dual function. When activated for a short time, it promotes metastasis, but when activated for long periods, it is related to BC cell death. P2Y2 and P2Y6 receptors are related to BC proliferation and invasiveness. Also, the high expression of CD39 and CD73 in BC is strongly related to a worse prognosis. The receptors and ectonucleotidases involved with BC become possible therapeutic targets. Several purinergic pathways have been found to be involved in BC cell survival and progression. In this review, in addition to analyzing the pathways involved, we reviewed the therapeutic interventions already studied for BC related to the purinergic system, as well as to other possible therapeutic targets.

Differential regulation of atrial contraction by P1 and P2 purinoceptors in normotensive and spontaneously hypertensive rats.

In the normotensive rat atrium, adenosine-5'-triphosphate and uridine-5'-triphosphate exert a biphasic effect consisting of an initial negative inotropic effect (NIE) followed by a subsequent positive inotropic effect (PIE). We comparatively studied these responses in normotensive Wistar rats (NWRs) and spontaneously hypertensive rats (SHRs). Compared with NWRs, the NIE responses in the atria were lower and the PIE responses were higher in SHRs. The P1 purinoceptor antagonist, D 8-cyclopentyl-1,3-dipropylxanthine, partially blocked the NIE responses of both ATP and UTP and mildly enhanced the PIE responses in both NWRs and SHRs. Furthermore, the P2 purinoceptor blockers suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium salt induced a pronounced block of the PIE responses in both atria types. The PIE responses to ATP were inhibited more efficiently by nifedipine. These responses were depressed by ryanodine and, to a lesser extent, carbonyl cyanide 3-chlorophenylhydrazone in SHR atria compared with NWR atria. The higher responses in SHR rats suggest the existence of an augmented endoplasmic reticulum Ca(2+) store and faster mitochondrial Ca(2+) cycling in SHR atria compared with NWR atria. These data support the hypothesis that a dysfunction of purinergic neurotransmission and enhanced sympathetic activity are contributing factors in the pathogenesis of hypertension.

Modulation of P2 receptors on pancreatic ß-cells by agonists and antagonists: a molecular target for type 2 diabetes treatment.

Morbidity and mortality from diabetes mellitus (DM) are serious worldwide concerns. By the year 2030, the estimated number of diabetic patients will reach a staggering 439 million worldwide. Diabetes mellitus type 2 (DM2), which involves disturbances in both insulin secretion and resistance, is the most common form of diabetes and affects approximately 5 to 7% of the world's population. When a patient with DM2 cannot regulate his or her blood glucose levels through diet, weight loss, or exercise, oral medications, such as hypoglycemic agents (i.e., sulphonylureas, biguanides, alpha glucosidase inhibitors and thiazolidinediones), are crucial. Here, we discuss some physiological aspects of P2 receptors on pancreatic ß-cells, which express a variety of P2 receptor isoforms. These receptors enhance glucose-dependent insulin release. In addition, we speculate on the potential of purinergic compounds as novel or additional treatments for Type 2 Diabetes mellitus.

Alteration of purinergic neurotransmission in isolated atria of streptozotocin-induced diabetic rats.

Cardiac dysfunctions are described in diabetes. However, the role of purinergic neurotransmission in diabetes-related cardiovascular diseases is unknown. The purpose of this study was to evaluate the purinergic neurotransmission in isolated atria from streptozotocin-induced diabetic rats. The animals were grouped as control and diabetic with 30 days (D30) and 60 days (D60) after streptozotocin-induced diabetes. The isolated left and right atria were used in functional experiments. The effects of adenosine triphosphate, uridine diphosphate, and adenosine were evaluated on atrial inotropism and chronotropism. The antagonists 8-cyclopentyl-1,3-dipropylxanthine and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate were also used, as blockers of P1 and P2 receptors, respectively. A negative inotropic effect followed by a positive inotropic effect was induced by adenosine triphosphate in isolated atria. This negative inotropic effect was decreased by 25% in left atria of D30. Additionally, the apparent affinity for adenosine was diminished in left atria of D30, suggesting changes in P1 receptor function. No changes were found in the right atria of D30 stimulated by adenosine. The left atria and right atria stimulated by uridine diphosphate showed an increased inotropic effect of 92% and 17%, respectively. No changes were observed in left and right atria of D30 stimulated by uridine diphosphate. Our data showed the involvement of purinergic neurotransmission in diabetes-related cardiovascular changes.

Modulation of arterial pressure by P2 purinoceptors in the paraventricular nucleus of the hypothalamus of awake rats.

In the present study we evaluated the role of purinergic mechanisms in the PVN on the tonic modulation of the autonomic function to the cardiovascular system as well on the cardiovascular responses to peripheral chemoreflex activation in awake rats. Guide-cannulae were bilaterally implanted in the direction of the PVN of male Wistar rats. Femoral artery and vein were catheterized one day before the experiments. Chemoreflex was activated with KCN (80 µg/0.05 ml, i.v.) before and after microinjections of P2 receptors antagonist into the PVN. Microinjection of PPADS, a non selective P2X antagonist, into the PVN (n=6) produced a significant increase in the baseline MAP (99±2 vs 112±3 mmHg) and HR (332±8 vs 375±8 bpm) but had no effect on the pressor and bradycardic responses to chemoreflex activation. Intravenous injection of vasopressin receptors antagonist after microinjection of PPADS into the PVN produced no effect on the increased baseline MAP. Simultaneous microinjection of PPADS and KYN into the PVN (n=6) had no effect in the baseline MAP, HR or in the pressor and bradycardic responses to chemoreflex activation. We conclude that P2 purinoceptors in the PVN are involved in the modulation of baseline autonomic function to the cardiovascular system but not in the cardiovascular responses to chemoreflex activation in awake rats.

Pharmacological properties of a pore induced by raising intracellular Ca2+.

Recent studies on the P2X(7) receptor in 2BH4 cells and peritoneal macrophages have demonstrated that the raise in intracellular Ca(2+) concentration induces a pore opening similar to P2X(7) receptor pore. Herein, we have investigated whether the pore activated by the elevation of intracellular Ca(2+) concentration is associated to P2X(7) receptor. Using patch clamp in cell attached, whole cell configuration, and dye uptake, we measured the pore opening in cell types that express the P2X(7) receptor (2BH4 cells and peritoneal macrophages) and in cells that do not express this receptor (HEK-293 and IT45-RI cells). In 2BH4 cells, the stimulation with ionomycin (5-10 microM) increased intracellular free Ca(2+) concentration and induced pore formation with conductance of 421 +/- 14 pS, half-time (t(1/2)) for ethidium bromide uptake of 118 +/- 17 s, and t(1/2) for Lucifer yellow of 122 +/- 11 s. P2X(7) receptor antagonists did not block these effects. Stimulation of HEK-293 and IT45-RI cells resulted in pore formation with properties similar to those found for 2BH4 cells. Connexin hemichannel inhibitors (carbenoxolone and heptanol) also did not inhibit the pore-induced effect following the increase in intracellular Ca(2+) concentration. However, 5-(N,N-hexamethylene)-amiloride, a P2X(7) receptor pore blocker, inhibited the induced pore. Moreover, intracellular signaling modulators, such as calmodulin, phospholipase C, mitogen-activated protein kinase, and cytoskeleton components were important for the pore formation. Additionally, we confirmed the results obtained for electrophysiology by using the flow cytometry, and we discarded the possibility of cellular death induced by raising intracellular Ca(2+) at the doses used by using lactate dehydrogenase release assay. In conclusion, increased concentration in intracellular Ca(+2) induces a novel membrane pore pharmacologically different from the P2X(7) associated pore and hemigap-junction pore.

A comparative study of ectonucleotidase and P2 receptor mRNA profiles in C6 cell line cultures and C6 ex vivo glioma model.

Glioblastoma multiforme is the most common type of primary brain tumour and has the worst clinical outcome. Nucleotides represent an important class of extracellular molecules involved in cell proliferation, differentiation and apoptosis. Alterations in purinergic signalling have been implicated in pathological processes, such as cancer, and glioma cell lines are widely employed as a model to study the biology of brain tumours. Increasing evidence, however, suggests that glioma cell lines may not present all the phenotypic and genetic characteristics of the primary tumours. We have compared the biological characteristics of C6 rat glioma cells in culture and the same cells after their implantation in the rat brain and growth in culture (denominated as the C6 ex vivo culture model). Parameters evaluated included cell morphology, differentiation, angiogenic markers, purinergic receptors and ecto-nucleotidase mRNA profile/enzymatic activity. Analysis of the C6 glioma cell line and C6 ex vivo glioma cultures revealed distinct cell morphologies, although cell differentiation and angiogenic marker expressions were similar. Both glioma models co-expressed multiple P2X and P2Y receptor subtypes with some differences. In addition, the C6 glioma cell line and C6 ex vivo glioma cultures exhibited similar extracellular ATP metabolism and cell proliferation behaviour when exposed to cytotoxic ATP concentrations. Thus, the disruption of purinergic signalling is a feature shown not only by glioma cell lineages, but also by primary glioma cultures. Our results therefore suggest the participation of the purinergic system in glioma malignancy.

Pharmacological properties of purinergic receptors and their effects on proliferation and induction of neuronal differentiation of P19 embryonal carcinoma cells.

We have used P19 embryonal carcinoma cells as in vitro model for early neurogenesis to study ionotropic P2X and metabotropic P2Y receptor-induced Ca(2+) transients and their participation in induction of proliferation and differentiation. In embryonic P19 cells, P2Y(1), P2Y(2) and P2X(4) receptors or P2X-heteromultimers with similar P2X(4) pharmacology were responsible for ATP and ATP analogue-induced Ca(2+) transients. In neuronal-differentiated cells, P2Y(2,) P2Y(6), P2X(2) and possibly P2X(2)/P2X(6) heteromeric receptors were the major mediators of the elevations in intracellular free calcium concentration [Ca(2+)](i). We have collected evidence for the involvement of metabotropic purinergic receptors in proliferation induction of undifferentiated and neural progenitor cells by using a BrdU-incorporation assay. ATP-, UTP-, ADP-, 2-MeS-ATP- and ADP-betaS-induced proliferation in P19 cells was mediated by P2Y(1) and P2Y(2) receptors as judged from pharmacological profiles of receptor responses. ATP-provoked acceleration of neuronal differentiation, determined by analysis of nestin and neuron-specific enolase gene and protein expression, also resulted from P2Y(1) and P2Y(2) receptor activation. Proliferation- and differentiation-induction involved the activation of inositol-trisphosphate sensitive intracellular Ca(2+) stores.

Na+ modulates anion permeation and block of P2X7 receptors from mouse parotid glands.

We previously reported that mouse parotid acinar cells display anion conductance (I(ATPCl)) when stimulated by external ATP in Na+-free extracellular solutions. It has been suggested that the P2X7 receptor channel (P2X7R) might underlie I(ATPCl). In this work we show that I (ATPCl) can be activated by ATP, ADP, AMP-PNP, ATPgammaS and CTP. This is consistent with the nucleotide sensitivity of P2X7R. Accordingly, acinar cells isolated from P2X7R( -/- ) mice lacked I(ATPCl). Experiments with P2X7R heterologously expressed resulted in ATP-activated currents (I(ATP-P2X7)) partially carried by anions. In Na(+)-free solutions, I (ATP-P2X7) had an apparent anion permeability sequence of SCN(-) > I(-) congruent with NO3(-) > Br(-) > Cl(-) > acetate, comparable to that reported for I(ATPCl) under the same conditions. However, in the presence of physiologically relevant concentrations of external Na+, the Cl(-) permeability of I(ATP-P2X7) was negligible, although permeation of Br(-) or SCN(-) was clearly resolved. Relative anion permeabilities were not modified by addition of 1 mM: carbenoxolone, a blocker of Pannexin-1. Moreover, cibacron blue 3GA, which blocks the Na(+) current activated by ATP in acinar cells but not I(ATPCl), blocked I(ATP-P2X7) in a dose-dependent manner when Na+ was present but failed to do so in tetraethylammonium containing solutions. Thus, our data indicate that P2X7R is fundamental for I(ATPCl) generation in acinar cells and that external Na+ modulates ion permeability and conductivity, as well as drug affinity, in P2X7R.

Differential role of extracellular histidines in copper, zinc, magnesium and proton modulation of the P2X7 purinergic receptor.

The P2X7 receptor is a non-selective cationic channel activated by extracellular ATP, belonging to the P2X receptor family. To assess the role of extracellular histidines on the allosteric modulation of the rat P2X7 receptor by divalent metals (copper, zinc and magnesium) and protons, these amino acid residues were singly substituted for corresponding alanines. Wild-type and mutated receptors were injected to Xenopus laevis oocytes; metal-related effects were evaluated by the two-electrode voltage-clamp technique. Copper inhibited the ATP-gated currents with a median inhibitory concentration of 4.4 +/- 1.0 micromol/L. The inhibition was non-competitive and time-dependent; copper was 60-fold more potent than zinc. The mutant H267A, resulted in a copper resistant receptor; mutants H201A and H130A were less sensitive to copper inhibition (p < 0.05). The rest of the mutants examined, H62A, H85A, and H219A, conserved the copper-induced inhibition. Only mutants H267A and H219A were less sensitive to the modulator action of zinc. Moreover, the magnesium-induced inhibition was abolished exclusively on the H130A and H201A mutants, suggesting that this metal may act at a novel cationic modulator site. Media acidification inhibited the ATP-gated current 87 +/- 3%; out of the six mutants examined, only H130A was significantly less sensitive to the change in pH, suggesting that His-130 could be involved as a pH sensor. In conclusion, while His-267 is critically involved in the copper or zinc allosteric modulation, the magnesium inhibitory effects is related to His-130 and His-201, His-130 is involved in proton sensing, highlighting the role of defined extracellular histidines in rat P2X7 receptor allosteric modulation.

Activation of P2Y1 receptor triggers two calcium signaling pathways in bone marrow erythroblasts.

In this study, we describe the presence of P2 receptor subtypes and Ca2+ signaling in erythroblasts. ATP and ADP produced a biphasic increase of intracellular Ca2+ concentration ([Ca2+]i), with an initial transient phase followed by a sustained phase. Reverse transcription polymerase chain reaction (RT-PCR) showed the expression of P2Y1, P2Y2 and P2Y12. The selective P2Y1 receptor antagonist 2'-deoxy-N6-methyl-adenosine-3',5'-diphosphate (MRS2179) and the G(i) protein inhibitor pertussis toxin blocked Ca2+ increase. The initial transient [Ca2+]i increase phase was sensitive to the 1,4,5-inositol trisphosphate (IP3) receptor blocker 2-aminoethoxy-diphenylborate (2-APB), while the sustained phase was sensitive to the protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF109203X) and calcium calmodulin kinase II (CaMKII) inhibitor 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62). In addition, the PKC activator phorbol-12,13-dibutyrate (PDBu) produced increase of [Ca2+]i. Flow cytometry analysis showed the expression of Ca2+-dependent PKC alpha, betaI, gamma and phospho-CaMKII. These results suggest that the activation of the P2Y1 receptor triggers two different [Ca2+]i increase pathways, one IP3-dependent and the other kinase-dependent.

Extracellular histidine residues identify common structural determinants in the copper/zinc P2X2 receptor modulation.

To assess the mechanism of P2X2 receptor modulation by transition metals, the cDNA for the wild-type receptor was injected to Xenopus laevis oocytes and examined 48-72 h later by the two-electrode voltage-clamp technique. Copper was the most potent of the trace metals examined; at 10 microm it evoked a 25-fold potentiation of the 10 microm ATP-gated currents. Zinc, nickel or mercury required 10-fold larger concentrations to cause comparable potentiations, while palladium, cobalt or cadmium averaged only 12- and 3-fold potentiations, respectively. Platinum was inactive. The non-additive effect of copper and zinc at 10-100 microm suggests a common site of action; these metals also shifted to the left the ATP concentration-response curves. To define residues necessary for trace metal modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X2 receptor. The H120A and H213A mutants were resistant to the modulator action of copper, zinc and other metals with the exception of mercury. Mutant H192A showed a reduction but not an abrogation of the copper or zinc potentiation. H245A showed less affinity for copper while this mutant flattened the zinc-induced potentiation. Mutant H319A reduced the copper but not the zinc-induced potentiation. In contrast, mutants H125A, H146A, H152A and H174A conserved the wild-type receptor sensitivity to trace metal modulation. We propose that His120, His192, His213 and His245 form part of a common allosteric metal-binding site of the P2X2 receptor, which for the specific coordination of copper, but not zinc, additionally involves His319.

Heavy metals modulate the activity of the purinergic P2X4 receptor.

To further characterize the nature of the regulatory metal-binding sites of the rat P2X(4) receptor, several transition heavy metals were tested to examine their ability to mimic the facilitator action of zinc or the inhibitory action of copper. cDNA coding for the rat P2X(4) receptor was injected into Xenopus laevis oocytes; the two-electrode voltage-clamp technique was used to measure and quantify the ATP-evoked currents in the absence or presence of the metals. Cadmium facilitated the ATP-gated currents in a reversible and voltage-independent manner; maximal potentiation occurred within less than 1 min. Cadmium displaced leftward, in a concentration-dependent manner, the ATP concentration-response curve. In contrast, mercury reduced the ATP-gated currents in a reversible, time, and concentration manner. Maximal inhibition occurred after about 5 min of metal application. Cobalt also augmented the ATP-evoked currents, but its action was long lasting and did not reverse even after 45 min of metal washout. Other metals such as lead, nickel, manganese, silver, or gallium did not significantly alter the ATP-gated currents. The co-application of cadmium plus zinc or mercury plus copper caused additive effects. Mutation of H140 by alanine (H140A) augmented both the cadmium-induced facilitation and the mercury-induced inhibition. In contrast, the H241A mutant showed characteristics indistinguishable from the wild type. The H286A mutant showed a normal cadmium-induced potentiation, but an increased mercury inhibition. Out of the metals examined, only cadmium mimicked closely the action of zinc, evidencing commonalities. While mercury mimicked the action of copper, both metals apparently interact at distinct metal-binding sites. The present findings allow us to infer that heavy metals modulate the P2X(4) receptor by acting in at least three separate metal-binding sites.

Are second messengers crucial for opening the pore associated with P2X7 receptor?

Stimulation of the P2X7 receptor by ATP induces cell membrane depolarization, increase in intracellular Ca2+ concentration, and, in most cases, permeabilization of the cell membrane to molecules up to 900 Da. After the activation of P2X7, at least two phenomena occur: the opening of low-conductance (8 pS) cationic channels and pore formation. At least two conflicting hypotheses have been postulated to reconcile these findings: 1) the P2X7 pore is formed as a result of gradual permeability increase (dilation) of cationic channels, and 2) the P2X7 pore represents a distinct channel, possibly activated by a second messenger and not directly by extracellular nucleotides. In this study, we investigated whether second messengers are necessary to open the pore associated with the P2X7 receptor in cells that expressed the pore activity by using the patch-clamp technique in whole cell and cell-attached configurations in conjunction with fluorescent imaging. In peritoneal macrophages and 2BH4 cells, we detected permeabilization and single-channel currents in the cell-attached configuration when ATP was applied outside the membrane patch in a condition in which oxidized ATP and Lucifer yellow were maintained within the pipette. Our data support Ca2+ as a second messenger associated with pore formation because the permeabilization depended on the presence of intracellular Ca2+ and was blocked by BAPTA-AM. In addition, MAPK inhibitors (SB-203580 and PD-98059) blocked the permeabilization and single-channel currents in these cells. Together our data indicate that the P2X7 pore depends on second messengers such as Ca2+ and MAP kinases.

P2X7 receptors stimulate AKT phosphorylation in astrocytes.

1. Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X(7) subtype, are coupled to the phosphoinositide 3-kinase (PI3K)/Akt pathway in astrocytes. 2. P2Y and P2X receptor agonists ATP, uridine 5'-triphosphate (UTP) and 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP) stimulated Akt phosphorylation in primary cultures of rat cortical astrocytes. BzATP induced Akt phosphorylation in a concentration- and time-dependent manner, similar to the effect of BzATP on Akt phosphorylation in 1321N1 astrocytoma cells stably transfected with the rat P2X(7) receptor. Activation was maximal at 5 - 10 min and was sustained for 60 min; the EC(50) for BzATP was approximately 50 microM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. 3. The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X(7) receptor antagonist Brilliant Blue G and the P2X receptor antagonist iso-pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid, but was unaffected by trinitrophenyl-ATP, oxidized ATP, suramin and reactive blue 2. 4. Results with specific inhibitors of signal transduction pathways suggest that extracellular and intracellular calcium, PI3K and a Src family kinase are involved in the BzATP-induced Akt phosphorylation pathway. 5. In conclusion, our data indicate that stimulation of astrocytic P2X(7) receptors, as well as other P2 receptors, leads to Akt activation. Thus, signaling by nucleotide receptors in astrocytes may be important in several cellular downstream effects related to the Akt pathway, such as cell cycle and apoptosis regulation, protein synthesis, differentiation and glucose metabolism.

Characterisation of P2Y(1)-like receptor in cultured rat pineal glands.

The rat pineal gland possesses P2 receptors which potentiate the effect of noradrenaline-induced N'-acetyl-5-hydroxytryptamine (N'-acetyl-5-HT) production. In the current study, this receptor was characterised according to agonist selectivity and signal transduction mechanisms. 2-MethylthioATP (2MeSATP), 2-chloroATP (2-ClATP), adenosine 5'-O-2-thiodiphosphate, (ADPbetaS), ATP and ADP, but not UTP, potentiated noradrenaline-induced N'-acetyl-5-HT production in a concentration-dependent manner. 2MeSATP neither induced the production of adenosine 3':5'-cyclic monophosphate (cyclic AMP), nor inhibited its formation when the glands were stimulated by forskolin. The phospholipase C inhibitor 1-[6-[[(17beta)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), but not the inactive analogue, 1-[6-[[(17beta)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione (U73343), blocked the 2MeSATP effect. The P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-dissulphonic acid (PPADS), which inhibits phospholipase C-coupled P2Y(1) receptors, blocked the 2MeSATP effect. In conclusion, our data strongly suggest that the P2-like receptor that is present in rat pinealocytes and which is responsible for the potentiation of noradrenaline-induced N'-acetyl-5-HT production is a P2Y(1)-like receptor, coupled to a G protein which stimulates phospholipase C.

P2Z purinoceptor-associated pores induced by extracellular ATP in macrophages and J774 cells.

Millimolar concentrations of extracellular ATP (ATPo) can induce the permeabilization of plasma membranes of macrophages and other bone marrow-derived cells to low-molecular-weight solutes, a phenomenon that is the hallmark of P2Z purinoceptors. However, patch-clamp and whole cell electrophysiological experiments have so far failed to demonstrate the existence of any ATPo-induced P2Z-associated pores underlying this permeabilization phenomenon. Here, we describe ATPo-induced pores of 409 +/- 33 pS recorded using cell-attached patch-clamp experiments performed in macrophages and J774 cells. These pores are voltage dependent and display several properties of the P2Z-associated permeabilization phenomenon: they are permeable to both large cations and anions, such as tris(hydroxymethyl)aminomethane, N-methyl-D-glucamine, and glutamate; their opening is favored at temperatures higher than 30 degrees C; they are blocked by oxidized ATP and Mg2+; and they can be triggered by 3'-O-(4-benzoylbenzoyl)-ATP but not by UTP or ADP. We conclude that the pores described in this report are associated with the P2Z permeabilization phenomenon.

Antinociceptive effect of purine nucleotides.

The antinociceptive effect of purine nucleotides administered systematically (sc) was determined using the formalin and writhing tests in adult male albino mice. The mechanisms underlying nucleotide-induced antinociception were investigated by preinjecting the animals (sc) with specific antagonists for opioid (naloxone, 1 mg/kg), purinergic P1 (caffeine, 5, 10, of 30 mg/kg); theophylline, 10 mg/kg) or purinergic P2 receptors (suramin, 100 mg/kg; Coomassie blue, 30-300 mg/kg; quinidine, 10 mg/kg). Adenosine, adenosine monophosphate (AMP), diphosphate (ADP) and triphosphate (ATP) caused a reduction in the number of writhes and in the time of licking the formalin-injected paw. Naloxone had no effect on adenosine- or adenine nucleotide-induced antinociception. Caffeine (30 mg/kg) and theophylline (10 mg/kg) reversed the antinociceptive action of adenosine and adenine nucleotide derivatives in both tests. P2 antagonists did not reverse adenine nucleotide-induced antinociception. These results suggest that antinociceptive effect of adenine nucleotides is mediated by adenosine.