Nucleoside triphosphate diphosphohydrolases 1, 2, 3 and 8 (NTPDases 1, 2, 3 and 8) are the dominant ectonucleotidases and thereby expected to play important roles in nucleotide signaling. Distinct biochemical characteristics of individual NTPDases should allow them to regulate P2 receptor activation differentially. Therefore, the biochemical and kinetic properties of these enzymes were compared. NTPDases 1, 2, 3 and 8 efficiently hydrolyzed ATP and UTP with K (m) values in the micromolar range, indicating that they should terminate the effects exerted by these nucleotide agonists at P2X(1-7) and P2Y(2,4,11) receptors. Since NTPDase1 does not allow accumulation of ADP, it should terminate the activation of P2Y(1,12,13) receptors far more efficiently than the other NTPDases. In contrast, NTPDases 2, 3 and 8 are expected to promote the activation of ADP specific receptors, because in the presence of ATP they produce a sustained (NTPDase2) or transient (NTPDases 3 and 8) accumulation of ADP. Interestingly, all plasma membrane NTPDases dephosphorylate UTP with a significant accumulation of UDP, favoring P2Y(6) receptor activation. NTPDases differ in divalent cation and pH dependence, although all are active in the pH range of 7.0-8.5. Various NTPDases may also distinctly affect formation of extracellular adenosine and therefore adenosine receptor-mediated responses, since they generate different amounts of the substrate (AMP) and inhibitor (ADP) of ecto-5'-nucleotidase, the rate limiting enzyme in the production of adenosine. Taken together, these data indicate that plasma membrane NTPDases hydrolyze nucleotides in a distinctive manner and may therefore differentially regulate P2 and adenosine receptor signaling.

Previously, we demonstrated that nucleotides released upon mechanical injury to corneal epithelium activate purinergic (P2) receptors resulting in mobilization of a Ca(2+) wave. However, the tissue is extensively innervated and communication between epithelium and neurons is critical and not well understood. Therefore, we developed a co-culture of primary trigeminal neurons and human corneal limbal epithelial cells. We demonstrated that trigeminal neurons expressed a repertoire of P2Yand P2X receptor transcripts and responded to P2 agonists in a concentration-dependent manner. Mechanical injuries to epithelia in the co-cultures elicited a Ca(2+) wave that mobilized to neurons and was attenuated by Apyrase, an ectonucleotidase. To elucidate the role of factors released from each cell type, epithelial and neuronal cells were cultured, injured, and the wound media from one cell type was collected and added to the other cell type. Epithelial wound media generated a rapid Ca(2+) mobilization in neuronal cells that was abrogated in the presence of Apyrase, while neuronal wound media elicited a complex response in epithelial cells. The rapid Ca(2+) mobilization was detected, which was abrogated with Apyrase, but it was followed by Ca(2+) waves that occurred in cell clusters. When neuronal wound media was preincubated with a cocktail of N-methyl-D-aspartate (NMDA) receptor inhibitors, the secondary response in epithelia was diminished. Glutamate was detected in the neuronal wound media and epithelial expression of NMDA receptor subunit transcripts was demonstrated. Our results indicate that corneal epithelia and neurons communicate via purinergic and NMDA receptors that mediate the wound response in a highly orchestrated manner.

Purinergic signaling regulates important physiological processes and the homeostatic response to stress in the cochlea via extracellular nucleosides (adenosine) and nucleotides (ATP, UTP). Using a previously established organotypic culture model, the current study investigated the effect of purinergic P1 (adenosine) and P2 (ATP) receptor activation on the survival of the sensory hair cell population in the cochlea exposed to the ototoxic aminoglycoside neomycin. Organ of Corti explants were obtained from C57BL/6 mice at postnatal day 3 (P3) and maintained in normal culture medium (with or without purine receptor agonists or analogs) for 19.5 h prior to neomycin exposure (1 mM, 3 h) followed by a further incubation for 19.5 h in culture medium. The cochlear explants were then fixed in 4% paraformaldehyde (PFA) and sensory hair cells labeled with Alexa 488-phalloidin. Neomycin induced a substantial loss of the sensory hair cells, mostly in the middle segment of the cochlea. This neomycin-induced ototoxicity was unaffected by the addition of P2 receptor agonists (ATP and UTP) in the culture medium, whilst the addition of their slowly-hydrolyzable analogs (ATPγS, UTPγS) aggravated neomycin-induced sensory hair cell loss. In contrast, the activation of P1 receptors by adenosine or adenosine amine congener (ADAC) conferred partial protection from neomycin ototoxicity. This study demonstrates a pro-survival effect of P1 receptor stimulation, whilst prolonged activation of P2 receptors has an opposite effect. Based on these findings, we postulate that P1 and P2 receptors orchestrate differential responses to cochlear injury and that the balance of these receptors is important for maintaining cochlear homeostasis following ototoxic injury.

The role of ATP and its stable analogue ATPgammaS [adenosine-5'-o-(3-thio)triphosphate] was studied in rat hippocampal neurotransmission under normoxic conditions and during oxygen and glucose deprivation (OGD). Field excitatory postsynaptic potentials (fEPSPs) from the dendritic layer or population spikes (PSs) from the soma were extracellularly recorded in the CA1 area of the rat hippocampus. Exogenous application of ATP or ATPgammaS reduced fEPSP and PS amplitudes. In both cases the inhibitory effect was blocked by the selective A(1) adenosine receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and was potentiated by different ecto-ATPase inhibitors: ARL 67156 (6-N,N-diethyl-D: -beta,gamma-dibromomethylene), BGO 136 (1-hydroxynaphthalene-3,6-disulfonate) and PV4 [hexapotassium dihydrogen monotitanoundecatungstocobaltate(II) tridecahydrate, K(6)H(2)[TiW(11)CoO(40)].13H(2)O]. ATPgammaS-mediated inhibition was reduced by the P2 antagonist suramin [8-(3-benzamido-4-methylbenzamido)naphthalene-1,3,5-trisulfonate] at the somatic level and by other P2 blockers, PPADS (pyridoxalphosphate-6-azophenyl-2',4'-disulfonate) and MRS 2179 (2'-deoxy-N (6)-methyladenosine 3',5'-bisphosphate), at the dendritic level. After removal of both P2 agonists, a persistent increase in evoked synaptic responses was recorded both at the dendritic and somatic levels. This effect was prevented in the presence of different P2 antagonists. A 7-min OGD induced tissue anoxic depolarization and was invariably followed by irreversible loss of fEPSP. PPADS, suramin, MRS2179 or BBG (brilliant blue G) significantly prevented the irreversible failure of neurotransmission induced by 7-min OGD. Furthermore, in the presence of these P2 antagonists, the development of anoxic depolarization was blocked or significantly delayed. Our results indicate that P2 receptors modulate CA1 synaptic transmission under normoxic conditions by eliciting both inhibitory and excitatory effects. In the same brain region, P2 receptor stimulation plays a deleterious role during a severe OGD insult.

In the present work, we used a unique cortical/striatal/subventricular zone organotypic model in order to analyze the role of resident microglia in oxygen/glucose deprivation and to check the presence and modulation of several P2 receptors in the cortex. Immunofluorescence with the microglial marker OX42 and pharmacological experiments with indomethacin indicate that activation and recruitment of microglia after the insult is linked to cellular loss, mainly in the cortex. The confocal analysis with OX42 shows that, among the P2 receptors tested, P2X4, and P2X7 are expressed on microglia, while P2X1 and P2Y(1-2-12), although present in the slices, did not co-localize, whereas P2X6 is not detected. The upregulation of P2X4 and P2X7 on microglia and the toxic effect that different P2 agonists exert on cortical slices during oxygen/glucose deprivation indicate that a purinergic mechanism is related to the microglia activity; the protective effect of the P2 antagonist TNP-ATP is also described. In order to better understand the relationship between P2 receptors and OGD-activated microglia, we induced oxygen/glucose deprivation in co-cultures of organotypic slices and N9 microglia cell line. The presence of the N9 (which expresses P2X4 and P2X7 protein) in the cultures increases the damage in the cortex by 40% and the use of P2 antagonist PPADS reduced the cell damage due to the N9 activation. Our results show that microglia recruitment after a metabolic impairment is associated with cellular loss and that P2X4 and P2X7, are involved in microglia activity. The neuroprotective action exerted by TNP-ATP and PPADS and the possible use of purinergic antagonist in the pharmacological treatment of oxygen/glucose deprivation is also addressed.

COVID-19 disease is associated with progressive accumulation of SARS-CoV-2-specific mRNA, which is recognized by innate immune receptors, such as TLR3. This in turn leads to dysregulated production of multiple cytokines, including IL-6, IFN-γ, CXCL1, and TNF-α. Excessive production of these cytokines leads to acute lung injury (ALI), which consequently compromises alveolar exchange of O2 and CO2. It is therefore of considerable interest to develop novel therapies that reduce pulmonary inflammation and stem production of pro-inflammatory cytokines, potentially for COVID-19 patients that are at high risk of developing severe disease. Purinergic signaling has a central role in fine-tuning the innate immune system, with P2 (nucleotide) receptor antagonists and adenosine receptor agonists having anti-inflammatory effects. Accordingly, we focused here on the potential role of purinergic receptors in driving neutrophilic inflammation and cytokine production in a mouse model of pulmonary inflammation. To mimic the effects of SARS-CoV-2-specific RNA accumulation in mice, we administered progressively increasing daily doses of a viral mimetic, polyinosinic:polycytidylic acid [poly(I:C)] into the airways of mice over the course of 1 week. Some mice also received increasing daily doses of ovalbumin to mimic virus-encoded protein accumulation. Animals receiving both poly(I:C) and ovalbumin displayed particularly high cytokine levels and neutrophilia, suggestive of both innate and antigen-specific, adaptive immune responses. The extent of these responses was diminished by genetic deletion (P2Y14R, P2X7R) or pharmacologic modulation (P2Y14R antagonists, A3AR agonists) of purinergic receptors. These results suggest that pharmacologic modulation of select purinergic receptors might be therapeutically useful in treating COVID-19 and other pulmonary infections.

Large inter-individual variation in platelet response to endogenous agonists and pharmacological agents, including resistance to antiplatelet therapy, prompts a search for novel platelet inhibitors and development new antithrombotic strategies. The present in vitro study evaluates the beneficial effects of three adenosine receptor (AR) agonists (regadenoson, LUF 5835 and NECA), different in terms of their selectivity for platelet adenosine receptors, when used alone and in combination with P2Y12 inhibitors, such as cangrelor or prasugrel metabolite. The anti-platelet effects of AR agonists were evaluated in healthy subjects (in the whole group and after stratification of individuals into high- and low-responders to P2Y12 inhibitors), using whole blood techniques, under flow (thrombus formation) and static conditions (study of platelet activation and aggregation). Compared to P2Y12 antagonists, AR agonists were much less or not effective under static conditions, but demonstrated similar antiplatelet activity in flow. In most cases, AR agonists significantly enhanced the anti-platelet effect of P2Y12 antagonists, despite possessing different selectivity profiles and antiplatelet activities. Importantly, their inhibitory effects in combination with P2Y12 antagonists were similar in high- and low-responders to P2Y12 inhibitors. In conclusion, a combination of anti-platelet agents acting via the P1 and P2 purinergic receptors represents a promising alternative to existing antithrombotic therapy.

Extracellular ATP-induced Ca2+ signalling is critical in regulating diverse physiological and disease processes. Emerging evidence suggests high concentrations of extracellular ATP in tumour tissues. In this study, we examined the P2 receptor for ATP-induced Ca2+ signalling in human hepatocellular carcinoma (HCC) cells. Fura-2-based measurements of the intracellular Ca2+ concentration ([Ca2+]i) showed that extracellular ATP induced an increase in the [Ca2+]i in human HCC Huh-7 and HepG2 cells. NF546, a P2Y11 receptor agonist was equally effective in inducing an increase in the [Ca2+]i. In contrast, agonists for the P2X receptors (αβmeATP and BzATP), P2Y1 receptor (MRS2365) or P2Y2 receptor (MRS2768) were ineffective. In addition, ATP/NF546-induced increases in the [Ca2+]i were strongly inhibited by treatment with NF340, a P2Y11 receptor antagonist. Immunofluorescent confocal imaging and western blotting analysis consistently demonstrated the P2Y11 receptor expression in Huh-7 and HepG2 cells. Transfection with P2Y11-specific siRNA attenuated the P2Y11 receptor protein expression level and also reduced NF546-induced increase in the [Ca2+]i. Importantly, immunohistochemistry revealed that the P2Y11 receptor was expressed at very high level in human HCC tissues and, by contrast, it was barely detected in normal liver tissues. Trans-well cell migration assay demonstrated that ATP and NF546 induced concentration-dependent stimulation of Huh-7 cell migration. Treatment with NF340 prevented ATP-induced stimulation of cell migration. Taken together, our results show carcinoma-specific expression of the P2Y11 receptor and its critical role in mediating ATP-inducing Ca2+ signalling and regulating cell migration in human HCC cells.

ATP receptor-mediated Ca2+ concentration changes were recorded from neocortical neurones in brain slices from 2 week-old rats. To measure the cytoplasmic concentration of Ca2+ ([Ca2+]i) slices were incubated with fura-2/AM, and the microfluorimetry system was focused on an individual cell. During transients the intracellular level of [Ca2+]i in the majority of neocortical neurones (98 of 102) varied in the concentration range of ATP 5-2000 microM between 41. 3+/-5 and 163+/-7 nM. The rank order of efficacy for purinoreceptor agonists in concentration 100 microM was: ATPgammaS>ATP>ADP>>AMP approximately Adenosine approximately alpha,beta-methylene ATP>UTP. 10 microM PPADS, a P2-purinoreceptor antagonist, reduced the ATP-induced [Ca2+]i response by 26%+/-4%. After elimination of calcium from extracellular solution the first ATP-induced [Ca2+]i transient decreased to 65+/-8%, suggesting the participation of metabotropic P2y triggered Ca-release in the generation of the transient. Elevation of cytosolic Ca2+ by activation of plasmalemmal Ca2+ channels failed to potentiate such release indicating the absence of effective reloading of the corresponding stores. No Ca2+-induced Ca2+-release has been observed in the investigated neurons.

Intracellular Ca2+ ([Ca2+]i) signals induced by metabotropic glutamate receptor (mGluR) agonists and by purinergic agonists in cultured hippocampal astrocytes were investigated using [Ca2+]-sensitive fluorophores. The mGluR agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) and (R,S)-3,5-dihydroxyphenylglycine (DHPG) induced [Ca2+]i responses in 76 and 93% of the cells, respectively. The broad-spectrum mGluR antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) and the mGluR1 antagonists (S)-4-carboxy-3-hydroxyphenylglycine (4C3HPG) and (S)-4-carboxyphenylglycine (4CPG) suppressed the agonist-evoked [Ca2+]i response in about 25% of the cells completely and in about 60% partially, depending on the agonist concentration employed. Together with immunohistochemical receptor localisations these results suggest the presence of at least two subpopulations of class I mGluRs recruited from the truncated splice variants of mGluR1 (mGluR 1b, 1c, 1d) and/or hitherto unknown glial-specific class I mGluRs. Of the hippocampal astrocytes 88, 92 or 83% of the cells responded with a [Ca2+]i elevation (mostly oscillations) to application of ATP, ADP, or 2-methylthio-ATP (2-MeS-ATP), respectively, whereas only 14 and 5% responded to AMP and adenosine, respectively, indicating the predominance of P2 receptors. The ATP-induced [Ca2+]i signal was suppressed by suramin. Release of Ca2+ from intracellular stores was involved in the response to ATP because the cells also exhibited [Ca2+]i elevations in Ca2+-free medium. Cells did not respond to 10 microM UTP. We conclude that the P2Y subtype represents the main [Ca2+]i-linked purinoceptor in hippocampal astrocytes. Sequential application of ATP and DHPG in Ca-free medium showed that metabotropic glutamate and purinergic receptors initiate release of Ca2+ from subsets of cyclopiazonic acid-sensitive Ca2+ stores which are partly independent.

Epithelial to mesenchymal transition (EMT) occurs during embryogenesis or under pathological conditions such as hypoxia, injury, chronic inflammation, or tissue fibrosis. In renal tubular epithelial cells (MDCK), TGF-β1 induces EMT by reducing or increasing epithelial or mesenchymal marker expression, respectively. In this study, we confirmed that the cAMP analogues, 8-CPT-cAMP or N6-Ph-cAMP, inhibited the TGF-β1-driven overexpression of the mesenchymal markers ZEB-1, Slug, Fibronectin, and α-SMA. Furthermore, we showed that A1, A2A, P2Y1, P2Y11, and P2X7 purine receptor agonists modulated the TGF-β1-induced EMT through the involvement of PKA and/or MAPK/ERK signaling. The stimulation of A2A receptor reduced the overexpression of the EMT-related markers, mainly through the cAMP-dependent PKA pathway, as confirmed by cell pre-treatment with Myr-PKI. Both A1 and P2Y1 receptor stimulation exacerbated the TGF-β1-driven effects, which were reduced by cell pre-treatment with the MAPK inhibitor PD98059, according to the increased ERK1/2 phosphorylation upon receptor activation. The effects induced by P2Y11 receptor activation were oppositely modulated by PKA or MAPK inhibition, in line with the dual nature of the Gs- and Gq-coupled receptor. Differently, P2X7 receptor induced, per se, similar and not additive effects compared to TGF-β1, after prolonged cell exposure to BzATP. These results suggest a putative role of purine receptors as target for anti-fibrotic agents.

Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane--subdivided into P2Y and P2X subfamilies--whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection.

Pulmonary vascular tone is modulated by nucleotides, but which P2 receptors mediate these actions is largely unclear. The aim of this study, therefore, was to use subtype-selective antagonists to determine the roles of individual P2Y receptor subtypes in nucleotide-evoked pulmonary vasodilation and vasoconstriction. Isometric tension was recorded from rat intrapulmonary artery rings (i.d. 200-500 µm) mounted on a wire myograph. Nucleotides evoked concentration- and endothelium-dependent vasodilation of precontracted tissues, but the concentration-response curves were shallow and did not reach a plateau. The selective P2Y2 antagonist, AR-C118925XX, inhibited uridine 5'-triphosphate (UTP)- but not adenosine 5'-triphosphate (ATP)-evoked relaxation, whereas the P2Y6 receptor antagonist, MRS2578, had no effect on UTP but inhibited relaxation elicited by uridine 5'-diphosphate (UDP). ATP-evoked relaxations were unaffected by the P2Y1 receptor antagonist, MRS2179, which substantially inhibited responses to adenosine 5'-diphosphate (ADP), and by the P2Y12/13 receptor antagonist, cangrelor, which potentiated responses to ADP. Both agonists were unaffected by CGS1593, an adenosine receptor antagonist. Finally, AR-C118925XX had no effect on vasoconstriction elicited by UTP or ATP at resting tone, although P2Y2 receptor mRNA was extracted from endothelium-denuded tissues using reverse transcription polymerase chain reaction with specific oligonucleotide primers. In conclusion, UTP elicits pulmonary vasodilation via P2Y2 receptors, whereas UDP acts at P2Y6 and ADP at P2Y1 receptors, respectively. How ATP induces vasodilation is unclear, but it does not involve P2Y1, P2Y2, P2Y12, P2Y13, or adenosine receptors. UTP- and ATP-evoked vasoconstriction was not mediated by P2Y2 receptors. Thus, this study advances our understanding of how nucleotides modulate pulmonary vascular tone.

Endothelial cells release ATP in response to fluid shear stress, which activates purinergic (P2) receptor-mediated signaling molecules including endothelial nitric oxide (eNOS), a regulator of vascular tone. While P2 receptor-mediated signaling in the vasculature is well studied, the role of P2Y2 receptors in shear stress-associated endothelial cell alignment, cytoskeletal alterations, and wound repair remains ill defined. To address these aspects, human umbilical vein endothelial cell (HUVEC) monolayers were cultured on gelatin-coated dishes and subjected to a shear stress of 1 Pa. HUVECs exposed to either P2Y2 receptor antagonists or siRNA showed impaired fluid shear stress-induced cell alignment, and actin stress fiber formation as early as 6 h. Similarly, when compared to cells expressing the P2Y2 Arg-Gly-Asp (RGD) wild-type receptors, HUVECs transiently expressing the P2Y2 Arg-Gly-Glu (RGE) mutant receptors showed reduced cell alignment and actin stress fiber formation in response to shear stress as well as to P2Y2 receptor agonists in static cultures. Additionally, we observed reduced shear stress-induced phosphorylation of focal adhesion kinase (Y397), and cofilin-1 (S3) with receptor knockdown as well as in cells expressing the P2Y2 RGE mutant receptors. Consistent with the role of P2Y2 receptors in vasodilation, receptor knockdown and overexpression of P2Y2 RGE mutant receptors reduced shear stress-induced phosphorylation of AKT (S473), and eNOS (S1177). Furthermore, in a scratched wound assay, shear stress-induced cell migration was reduced by both pharmacological inhibition and receptor knockdown. Together, our results suggest a novel role for P2Y2 receptor in shear stress-induced cytoskeletal alterations in HUVECs.

Adenosine triphosphate (ATP) is coreleased with catecholamines from adrenal medullary chromaffin cells in response to sympathetic nervous system stimulation and may regulate these cells in an autocrine or paracrine manner. Increases in extracellular signal-regulated kinase (ERK) 1/2 phosphorylation were observed in response to ATP stimulation of bovine chromaffin cells. The signaling pathway involved in ATP-mediated ERK1/2 phosphorylation was investigated via Western blot analysis. ATP and uridine 5'-triphosphate (UTP) increased ERK1/2 phosphorylation potently, peaking between 5 and 15 min. The mitogen-activated protein kinase (MAPK/ERK)-activating kinase (MEK) inhibitor PD98059 blocked this response. UTP, which is selective for G-protein-coupled P2Y receptors, was the most potent agonist among several nucleotides tested. Adenosine 5'-O-(3-thio) triphosphate (ATPgammaS) and ATP were also potent agonists, characteristic of the P2Y(2) or P2Y(4) receptor subtypes, whereas agonists selective for P2X receptors or other P2Y receptor subtypes were weakly effective. The receptor involved was further characterized by the nonspecific P2 antagonists suramin and reactive blue 2, which each partially inhibited ATP-mediated ERK1/2 phosphorylation. Inhibitors of protein kinase C (PKC), protein kinase A (PKA), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), and phosphoinositide-3 kinase (PI3K) had no effect on ATP-mediated ERK1/2 phosphorylation. The Src inhibitor PP2, epidermal growth factor receptor (EGFR) inhibitor AG1478, and metalloproteinase inhibitor GM6001 decreased ATP-mediated ERK1/2 phosphorylation. These results suggest nucleotide-mediated ERK1/2 phosphorylation is mediated by a P2Y(2) or P2Y(4) receptor, which stimulates metalloproteinase-dependent transactivation of the EGFR.

1. The effects of adenosine receptor agonists upon both electrically-evoked and phenylephrine-induced contractile responses were investigated in the bisected vas deferens and the cauda epididymis of the guinea-pig. Electrical field-stimulation (10 s trains of pulses at 9 Hz, 0.1 ms duration, supramaximal voltage) elicited biphasic and monophasic contractile responses from preparations of bisected vas deferens and cauda epididymis, respectively; these responses were abolished by tetrodotoxin (300 nM). 2. In the prostatic half of the vas deferens the A1 selective adenosine receptor agonists, N6-cyclopentyladenosine (CPA) and (2S)-N6-[2-endo-norbornyl]adenosine ((S)-ENBA) and the non-selective A1/A2 adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) inhibited electrically-evoked contractions (pIC50+/-s.e.mean values 6.15+/-0.24, 5.99+/-0.26 and 5.51+/-0.24, respectively). The responses to CPA were blocked by the A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, DPCPX (100 nM). 3. In the epididymal half of the vas deferens NECA potentiated (at < or = 100 nM) and inhibited (at > or = 1 microM) electrically-evoked contractions. In the presence of the non-selective alpha-adrenoceptor antagonist phentolamine (3 microM), the alpha1-adrenoceptor antagonist, prazosin (100 nM), or at a reduced train length (3 s) NECA inhibited electrically-evoked contractions (pIC50 values 6.05+/-0.25, 5.97+/-0.29 and 5.71 +/-0.27, respectively). CPA (at 10 microM) also inhibited electrically-evoked contractions in this half of the vas deferens. In the presence of prazosin (100 nM), CPA also inhibited electrically-evoked contractions (pIC50 6.14+/-0.67); this effect was antagonized by DPCPX (30 nM, apparent pK(B) 8.26+/-0.88). In the presence of the P2 purinoceptor antagonist, suramin (300 microM), CPA (up to 1 microM) potentiated electrically-evoked contractions. 4. NECA, CPA and APNEA potentiated electrically-evoked contractions in preparations of cauda epididymis (pEC50 values 7.49+/-0.62, 7.65+/-0.74 and 5.84+/-0.86, respectively), the response to CPA was competitively antagonized by DPCPX (100 nM) with an apparent pK(B) value of 7.64+/-0.64. 5. The alpha1-adrenoceptor agonist phenylephrine elicited concentration-dependent contractile responses from preparations of bisected vas deferens and cauda epididymis. NECA (1 microM) potentiated responses to phenylephrine (< or = 1 microM) in the epididymal, but not in the prostatic half of the vas deferens. In preparations of epididymis NECA (1 microM) shifted phenylephrine concentration response curves to the left (4.6 fold). In the presence of a fixed concentration of phenylephrine (1 microM), NECA elicited concentration-dependent contractions of preparations of the epididymal half of the vas deferens and of the epididymis (pEC50 values 7.57+/-0.54 and 8.08+/-0.18, respectively). NECA did not potentiate responses to ATP in either the epididymal half of the vas deferens or the epididymis. 6. These studies are consistent with the action of stable adenosine analogues at prejunctional A1 and postjunctional A1-like adenosine receptors. The prejunctional A1 adenosine receptors only inhibit the electrically-evoked contractions of purinergic origin (an effect predominant in the prostatic half of the vas deferens). At the epididymis, where electrically-evoked contractions are entirely adrenergic, the predominant adenosine receptor agonist effect is a potentiation of alpha1-adrenoceptor-, but not of ATP-induced contractility.

Microglial cells are the primary immune effector cells in the brain. Extracellular ATP, e.g., released after brain injury, may initiate microglial activation via stimulation of purinergic receptors. In the rat nucleus accumbens (NAc), the involvement of P2X and P2Y receptors in the generation of microglial reaction in vivo was investigated. A stab wound in the NAc increased immunoreactivity (IR) for P2X(1,2,4,7) and P2Y(1,2,4,6,12) receptors on microglial cells when visualized with confocal laser scanning microscopy. A prominent immunolabeling of P2X(7) receptors with antibodies directed against the ecto- or endodomain was found on Griffonia simplicifolia isolectin-B4-positive cells. Additionally, the P2X(7) receptor was colocalized with active caspase 3 but not with the anti-apoptotic marker pAkt. Four days after local application of the agonists alpha,betameATP, ADPbetaS, 2MeSATP, and BzATP, an increase in OX 42- and G. simplicifolia isolectin-IR was observed around the stab wound, quantified both densitometrically and by counting the number of ramified and activated microglial cells, whereas UTPgammaS appeared to be ineffective. The P2 receptor antagonists PPADS and BBG decreased the injury-induced increase of these IRs when given alone and in addition inhibited the agonist effects. Further, the intra-accumbally applied P2X(7) receptor agonist BzATP induced an increase in the number of caspase-3-positive cells. These results indicate that ATP, acting via different P2X and P2Y receptors, is a signaling molecule in microglial cell activation after injury in vivo. The up-regulation of P2X(7)-IR after injury suggests that this receptor is involved in apoptotic rather than proliferative effects.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by motor and cognitive deficits, the result of dopamine (DA)-depletion within the basal ganglia. Currently, DA replacement therapy in the form of Sinemet (L-DOPA plus Carbidopa) provides symptomatic motor benefits and remains the "gold standard" for treatment. Several pharmacological approaches can enhance DA neurotransmission including the administration of DA receptor agonists, the inhibition of DA metabolism, and enhancing pre-synaptic DA release. DA neurotransmission is regulated by several receptor subtypes including signaling through the purinergic system. P2 × 4 receptors (P2 × 4Rs) are a class of cation-permeable ligand-gated ion channels activated by the synaptic release of extracellular adenosine 5'-triphosphate (ATP). P2 × 4Rs are expressed throughout the central nervous system including the dopaminergic circuitry of the substantia nigra, basal ganglia, and related reward networks. Previous studies have demonstrated that P2 × 4Rs can modulate several DA-dependent characteristics including motor, cognitive, and reward behaviors. Ivermectin (IVM) and moxidectin (MOX) are two macrocyclic lactones that can potentiate P2 × 4Rs. In this study, we sought to investigate the role of P2 × 4Rs in mediating DA neurotransmission by exploring their impact on DA-dependent behavior, specifically rotation frequency in the unilateral 6-hydroxydopamine-lesioned mouse model of DA-depletion. While we did not observe any differences in the degree of lesioning based on immunostaining for tyrosine hydroxylase between sexes, male mice displayed a greater number of rotations with L-DOPA compared to female mice. In contrast, we observed that IVM plus L-DOPA increased the number of rotations (per 10 min) in female, but not male mice. These findings highlight the potential role of pharmacologically targeting the purinergic receptor system in modulating DA neurotransmission as well as the importance of sex differences impacting outcome measures.

1. The effect of externally applied ATP on cytosolic free Ca2+ concentration ([Ca2+]i) was tested in single isolated rat neurohypophysial nerve terminals by fura-2 imaging. The release of vasopressin (AVP) and oxytocin (OT) upon ATP stimulation was also studied from a population of terminals using specific radioimmunoassays. 2. ATP evoked a sustained [Ca2+]i increase, which was dose dependent in the 1-100 microM range (EC50 = 4.8 microM). This effect was observed in only approximately 40 % of the terminals. 3. Interestingly, ATP, in the same range (EC50 = 8.6 microM), evoked AVP, but no significant OT, release from these terminals. 4. Both the [Ca2+]i increase and AVP release induced by ATP were highly and reversibly inhibited by suramin, suggesting the involvement of a P2 purinergic receptor in the ATP-induced responses. Pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), another P2 purinergic receptor antagonist, strongly reduced the ATP-induced [Ca2+]i response. 5. To further characterize the receptor, different agonists were tested, with the following efficacy: ATP = 2-methylthio-ATP > ATP-gamma-S > alpha, beta-methylene-ATP > ADP. The compounds adenosine, AMP, beta, gamma-methylene-ATP and UTP were ineffective. 6. The ATP-dependent [Ca2+]i increase was dependent on extracellular Ca2+ concentration ([Ca2+]o). Fluorescence-quenching experiments with Mn2+ showed that externally applied ATP triggered a Mn2+ influx. The ATP-induced [Ca2+]i increase and AVP release were independent of and additive to a K+-induced response, in addition to being insensitive to Cd2+. The ATP-induced [Ca2+]i increase was strongly reduced in the presence of Gd3+. These results suggest that the observed [Ca2+]i increases were elicited by Ca2+ entry through a P2X channel receptor rather than via a voltage-dependent Ca2+ channel. 7. We propose that ATP, co-released with neuropeptides, could act as a paracrine-autocrine messenger, stimulating, via Ca2+ entry through a P2X2 receptor, the secretion of AVP, in particular, from neurohypophysial nerve terminals.