P2Y<Subscript>2</Subscript> and P2Y<Subscript>6</Subscript> receptor activation elicits intracellular calcium responses in human adipose-derived mesenchymal stromal cells

Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ, these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal stromal cells (AD-MSCs). The purpose of this study was to undertake a comprehensive analysis of purinergic receptor expression. Mesenchymal stromal cells were isolated from human subcutaneous adipose tissue and confirmed by flow cytometry. The expression profile of purinergic receptors was determined by quantitative real-time PCR and immunocytochemistry. The molecular basis for adenine and uracil nucleotide-evoked intracellular calcium responses was determined using Fura-2 measurements. All the known subtypes of P2X and P2Y receptors, excluding P2X2, P2X3 and P2Y₁₂ receptors, were detected at the mRNA and protein level. ATP, ADP and UTP elicited concentration-dependent calcium responses in mesenchymal cells (N?=?7–9 donors), with a potency ranking ADP (EC₅₀ 1.3 ± 1.0 μM)?>?ATP (EC₅₀ 2.2 ± 1.1 μM)?=?UTP (3.2 ± 2.8 μM). Cells were unresponsive to UDP (<?30 μM) and UDP-glucose (<?30 μM). ATP responses were attenuated by selective P2Y₂ receptor antagonism (AR-C118925XX; IC₅₀ 1.1 ± 0.8 μM, 73.0?±?8.5% max inhibition; N?=?7 donors), and UTP responses were abolished. ADP responses were attenuated by the selective P2Y₆ receptor antagonist, MRS2587 (IC₅₀ 437 ± 133nM, 81.0?±?8.4% max inhibition; N?=?6 donors). These data demonstrate that adenine and uracil nucleotides elicit intracellular calcium responses in human AD-MSCs with a predominant role for P2Y₂ and P2Y₆ receptor activation. This study furthers understanding about how human adipose-derived mesenchymal stromal cells can respond to external signalling cues.     

Localisation of P2Y<Subscript>1</Subscript> and P2Y<Subscript>4</Subscript> receptors in dorsal root,nodose and trigeminal ganglia of the rat

The presence and distribution of P2Y (nucleotide) receptor subtypes in rat sensory neurons has been investigated. RT-PCR showed that P2Y₁, P2Y₂, P2Y₄ and P2Y₆ receptor mRNA is expressed in sensory ganglia [dorsal root ganglion (DRG), nodose ganglion (NG) and trigeminal ganglion (TG)]. The regional and cellular distribution of P2Y₁ and P2Y₄ receptor proteins in these ganglia was investigated using immunohistochemistry. P2Y₁ polyclonal antibodies stained over 80% of the sensory neurons, particularly the small-diameter (neurofilament-negative) neurons. The P2Y₄ receptor antibody stained more medium- and large- (neurofilament-positive) diameter neurons than small-diameter neurons. P2Y₁ and P2Y₄ receptor immunoreactivity (P2Y₁-IR and P2Y₄-IR) was often coexpressed with P2X₃ receptor immunoreactivity (P2X₃-IR) in subpopulations of neurons. Double immunohistochemistry showed that 73–84% of P2X₃ receptor-positive neurons also stained for the P2Y₁ receptor in DRG, TG and NG while only 25–35% also stained for the P2Y₄ receptor. Subpopulations of P2Y₁-IR neurons were coexpressed with NF200, CGRP and IB₄; most P2Y₄-IR neurons were coexpressed with NF200, while only a few neurons were coexpressed with CGRP (10–20%) or with IB₄ (1–2%). The results suggest that P2Y as well as P2X receptor subtypes contribute to purinergic signalling in sensory ganglia.     

Lung injury during LPS-induced inflammation occurs independently of the receptor P2Y<Subscript>1</Subscript>

Disruption of the lung endothelial and epithelial barriers during acute inflammation leads to excessive neutrophil migration. It is likely that activated platelets promote pulmonary recruitment of neutrophils during inflammation, and previous studies have found that anti-platelet therapy and depletion of circulating platelets have lung-protective effects in different models of inflammation. Because ADP signaling is important for platelet activation, I investigated the role of the ADP-receptor P2Y₁, a G protein-coupled receptor expressed on the surface of circulating platelets, during lipopolysaccharide (LPS)-induced inflammation and lung injury in P2Y₁-null and wild-type mice. Systemic inflammation was induced by a single intraperitoneal dose of LPS (3 mg/kg), and the mice were analyzed 24 h posttreatment. The data show that the LPS-induced inflammation levels were comparable in the P2Y₁-null and wild-type mice. Specifically, splenomegaly, counts of circulating platelets and white blood cells (lymphocytes and neutrophils), and assessments of lung injury (tissue architecture and cell infiltration) were similar in the P2Y₁-null and wild-type mice. Based on my results, I conclude that lung injury during LPS-induced inflammation in mice is independent of P2Y₁ signaling. I propose that if a blockade of purinergic signaling in platelets is a potential lung-protective strategy in the treatment of acute inflammation, then it is more likely to be a result of the disruption of the signaling pathway mediated by P2Y₁₂, another G protein-coupled receptor that mediates platelet responses to ADP.     

Involvement of P2Y<Subscript>12</Subscript> receptor of stellate ganglion in diabetic cardiovascular autonomic neuropathy

Diabetes as a chronic epidemic disease with obvious symptom of hyperglycemia is seriously affecting human health globally due to the diverse diabetic complications. Diabetic cardiovascular autonomic neuropathy (DCAN) is a common complication of both type 1 and type 2 diabetes and incurs high morbidity and mortality. However, the underlying mechanism for DCAN is unclear. It is well known that purinergic signaling is involved in the regulation of cardiovascular function. In this study, we examined whether the P2Y₁₂ receptor could mediate DCAN-induced sympathetic reflexes. Our results revealed that the abnormal changes of blood pressure, heart rate, heart rate variability, and sympathetic nerve discharge were improved in diabetic rats treated with P2Y₁₂ short hairpin RNA (shRNA). Meanwhile, the expression of P2Y₁₂ receptor, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and connexin 43 (Cx43) in stellate ganglia (SG) was decreased in P2Y₁₂ shRNA-treated diabetic rats. In addition, knocking down the P2Y₁₂ receptor also inhibited the activation of p38 MARK in the SG of diabetic rats. Taken together, these findings demonstrated that P2Y₁₂ receptor in the SG may participate in developing diabetic autonomic neuropathy, suggesting that the P2Y₁₂ receptor could be a potential therapeutic target for the treatment of DCAN.     

Synthesis,characterization, and in vitro evaluation of the selective P2Y<Subscript>2</Subscript> receptor antagonist AR-C118925

The G_q protein-coupled, ATP- and UTP-activated P2Y₂ receptor is a potential drug target for a range of different disorders, including tumor metastasis, inflammation, atherosclerosis, kidney disorders, and osteoporosis, but pharmacological studies are impeded by the limited availability of suitable antagonists. One of the most potent and selective antagonists is the thiouracil derivative AR-C118925. However, this compound was until recently not commercially available and little is known about its properties. We therefore developed an improved procedure for the synthesis of AR-C118925 and two derivatives to allow up-scaling and assessed their potency in calcium mobilization assays on the human and rat P2Y₂ receptors recombinantly expressed in 1321N1 astrocytoma cells. The compound was further evaluated for inhibition of P2Y₂ receptor-induced β-arrestin translocation. AR-C118925 behaved as a competitive antagonist with pA ₂ values of 37.2 nM (calcium assay) and 51.3 nM (β-arrestin assay). Selectivity was assessed vs. related receptors including P2X, P2Y, and adenosine receptor subtypes, as well as ectonucleotidases. AR-C118925 showed at least 50-fold selectivity against the other investigated targets, except for the P2X1 and P2X3 receptors which were blocked by AR-C118925 at concentrations of about 1 μM. AR-C118925 is soluble in buffer at pH 7.4 (124 μM) and was found to be metabolically highly stable in human and mouse liver microsomes. In Caco2 cell experiments, the compound displayed moderate permeability indicating that it may show limited peroral bioavailability. AR-C118925 appears to be a useful pharmacological tool for in vitro and in vivo studies.     

P2Y<Subscript>1</Subscript>receptor switches to neurons from glia in juvenile versus neonatal rat cerebellar cortex

Background  

In the CNS, several P2 receptors for extracellular nucleotides are identified on neurons and glial cells to participate to neuron-neuron, glia-glia and glia-neuron communication.     

Distribution of P2Y<Subscript>2</Subscript> receptors in the guinea pig enteric nervous system and its coexistence with P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> receptors,neuropeptide Y,nitric oxide synthase and calretinin

The distribution of P2Y₂ receptor-immunoreactive (ir) neurons and fibers and coexistence of P2Y₂ with P2X₂ and P2X₃ receptors, neuropeptide Y (NPY), calretinin (CR), calbindin (CB) and nitric oxide synthase (NOS) was investigated with immunostaining methods. The results showed that P2Y₂-ir neurons and fibers were distributed widely in myenteric and submucous plexuses of the guinea pig stomach corpus, jejunum, ileum and colon. The typical morphology of P2Y₂-ir neurons was a long process with strong positive staining on the same side of the cell body. The P2Y₂-ir neurons could be Dogiel type 1. About 40–60% P2X₃-ir neurons were immunoreactive for P2Y₂ in the myenteric plexus and all the P2X₃-ir neurons expressed the P2Y₂ receptor in the submucosal plexus; almost all the NPY-ir neurons and the majority of CR-ir neurons were also immunoreactive for P2Y₂, especially in the myenteric plexus of the small intestine; no P2Y₂-ir neurons were immunoreactive for P2X₂ receptors, CB and NOS. It is shown for the first time that S type/Dogiel type 1 neurons with fast P2X and slow P2Y receptor-mediated depolarizations could be those neurons expressing both P2Y₂-ir and P2X₃-ir and that they are widely distributed in myenteric and submucosal plexuses of guinea pig gut.     

The P2Y<Subscript>1</Subscript> receptor is involved in the maintenance of glucose homeostasis and in insulin secretion in mice

Pancreatic cells express several P2 receptors including P2Y₁ and the modulation of insulin secretion by extracellular nucleotides has suggested that these receptors may contribute to the regulation of glucose homeostasis. To determine whether the P2Y₁ receptor is involved in this process, we performed studies in P2Y₁–/– mice. In baseline conditions, P2Y₁–/– mice exhibited a 15% increase in glycemia and a 40% increase in insulinemia, associated with a 10% increase in body weight, pointing to a role of the P2Y₁ receptor in the control of glucose metabolism. Dynamic experiments further showed that P2Y₁–/– mice exhibited a tendency to glucose intolerance. These features were associated with a decrease in the plasma levels of free fatty acid and triglycerides. When fed a lipids and sucrose enriched diet for 15 weeks, the two genotypes no longer displayed any significant differences. To determine whether the P2Y₁ receptor was directly involved in the control of insulin secretion, experiments were carried out in isolated Langerhans islets. In the presence of high concentrations of glucose, insulin secretion was significantly greater in islets from P2Y₁–/– mice. Altogether, these results show that the P2Y₁ receptor plays a physiological role in the maintenance of glucose homeostasis at least in part by regulating insulin secretion.     

P2Y<Subscript>1</Subscript> receptor modulation of endogenous ion channel function in <Emphasis Type="Italic">Xenopus</Emphasis> oocytes: Involvement of transmembrane domains

Agonist activation of the hP2Y₁ receptor expressed in Xenopus oocytes stimulated an endogenous voltage-gated ion channel, previously identified as the transient inward (T_in) channel. When human P2Y₁ (hP2Y₁) and skate P2Y (sP2Y) receptors were expressed in Xenopus oocytes, time-to-peak values (a measure of the response to membrane hyperpolarization) of the T_in channel were significantly reduced compared to oocytes expressing the hB₁-bradykinin receptor or the rat M₁-muscarinic (rM₁) receptor. Differences in activation were also observed in the T_in currents elicited by various P2Y receptor subtypes. The time-to-peak values of the T_in channel in oocytes expressing the hP2Y₄, hP2Y₁₁, or hB₁-bradykinin receptors were similar, whereas the channel had significantly shorter time-to-peak values in oocytes expressing either the hP2Y₁ or sP2Y receptor. Amino acid substitutions at His-132, located in the third transmembrane domain (TM3) of the hP2Y₁ receptor, delayed the onset of channel opening, but not the kinetics of the activation process. In addition, Zn²⁺ sensitivity was also dependent on the subtype of P2Y receptor expressed. Replacement of His-132 in the hP2Y₁ receptor with either Ala or Phe increased Zn²⁺ sensitivity of the T_in current. In contrast, truncation of the C-terminal region of the hP2Y₁ receptor had no affect on activation or Zn²⁺ sensitivity of the T_in channel. These results suggested that TM3 in the hP2Y₁ receptor was involved in modulating ion channel function and blocker pharmacology of the T_in channel.     

Effect of fibrinogen on platelet reactivity measured by the VerifyNow P2Y<Subscript>12</Subscript> assay

The VerifyNow assay is based upon the ability of activated platelets to cross-link beads coated with fibrinogen. However, fibrinogen is an abundant protein of blood, and therefore it may affect test results by competing with fibrinogen of beads for binding to platelets. To test this assumption, we assessed the influence of artificial alteration of fibrinogen level in blood samples obtained from donors (n = 9) and patients on clopidogrel therapy (n = 8) on the results of the VerifyNow P2Y₁₂ assay. Fibrinogen level was altered by adding to blood samples 1/10 volume of fibrinogen solution (10.56 g/liter) or corresponding buffer. Relative to baseline, addition of buffer significantly increased platelet reactivity, whereas addition of fibrinogen decreased it. Analysis of the relationship between change in platelet reactivity values (dBase and dPRU) and change in fibrinogen concentration (dFg) revealed strong negative correlations: dBase =–63.3 × dFg–27.1 (r =–0.924, p < 0.0005) and dPRU =–54.4 × dFg–21.8 (r =–0.764, p < 0.0005). Thus, the results of our experiments suggest that: (i) blood fibrinogen strongly influences results of the VerifyNow P2Y₁₂ assay, and (ii) correcting for fibrinogen effect may be needed to improve the accuracy of the test in the measuring of antiplatelet effect of clopidogrel therapy.     

GPR80/99, proposed to be the P2Y<Subscript>15</Subscript> receptor activated by adenosine and AMP,is not a P2Y receptor

The orphan receptor GPR80 (also called GPR99) was recently reported to be the P2Y₁₅ receptor activated by AMP and adenosine and coupled to increases in cyclic AMP accumulation and intracellular Ca²⁺ mobilization (Inbe et al. J Biol Chem 2004; 279: 19790–9). However, the cell line (HEK293) used to carry out those studies endogenously expresses A_2A and A_2B adenosine receptors as well as multiple P2Y receptors, which complicates the analysis of a potential P2Y receptor. To determine unambiguously whether GPR80 is a P2Y receptor subtype, HA-tagged GPR80 was either stably expressed in CHO cells or transiently expressed in COS-7 and HEK293 cells, and cell surface expression was verified by radioimmunoassay (RIA). COS-7 cells overexpressing GPR80 showed a consistent twofold increase in basal inositol phosphate accumulation. However, neither adenosine nor AMP was capable of promoting accumulation of either cyclic AMP or inositol phosphates in any of the three GPR80-expressing cells. A recent paper (He et al. Nature 2004; 429: 188–93) reported that GPR80 is a Gq-coupled receptor activated by the citric acid cycle intermediate, -ketoglutarate. Consistent with this report, -ketoglutarate promoted inositol phosphate accumulation in CHO and HEK293 cells expressing GPR80, and pretreatment of GPR80-expressing COS-7 cells with glutamate dehydrogenase, which converts -ketoglutarate to glutamate, decreased basal levels of inositol phosphates. Taken together, these data demonstrate that GPR80 is not activated by adenosine, AMP or other nucleotides, but instead is activated by -ketoglutarate. Therefore, GPR80 is not a new member of the P2Y receptor family.     

Evidence for the possible involvement of the P2Y<Subscript>6</Subscript> receptor in Ca<Superscript>2+</Superscript> mobilization and insulin secretion in mouse pancreatic islets

Subtypes of purinergic receptors involved in modulation of cytoplasmic calcium ion concentration ([Ca²⁺]_i) and insulin release in mouse pancreatic β-cells were examined in two systems, pancreatic islets in primary culture and beta-TC6 insulinoma cells. Both systems exhibited some physiological responses such as acetylcholine-stimulated [Ca²⁺]_i rise via cytoplasmic Ca²⁺ mobilization. Addition of ATP, ADP, and 2-MeSADP (each 100 μM) transiently increased [Ca²⁺]_i in single islets cultured in the presence of 5.5 mM (normal) glucose. The potent P2Y₁ receptor agonist 2-MeSADP reduced insulin secretion significantly in islets cultured in the presence of high glucose (16.7 mM), whereas a slight stimulation occurred at 5.5 mM glucose. The selective P2Y₆ receptor agonist UDP (200 μM) transiently increased [Ca²⁺]_i and reduced insulin secretion at high glucose, whereas the P2Y_2/4 receptor agonist UTP and adenosine receptor agonist NECA were inactive. [Ca²⁺]_i transients induced by 2-MeSADP and UDP were antagonized by suramin (100 μM), U73122 (2 μM, PLC inhibitor), and 2-APB (10 or 30 μM, IP₃ receptor antagonist), but neither by staurosporine (1 μM, PKC inhibitor) nor depletion of extracellular Ca²⁺. The effect of 2-MeSADP on [Ca²⁺]_i was also significantly inhibited by MRS2500, a P2Y₁ receptor antagonist. These results suggested that P2Y₁ and P2Y₆ receptor subtypes are involved in Ca²⁺ mobilization from intracellular stores and insulin release in mouse islets. In beta-TC6 cells, ATP, ADP, 2-MeSADP, and UDP transiently elevated [Ca²⁺]_i and slightly decreased insulin secretion at normal glucose, while UTP and NECA were inactive. RT-PCR analysis detected mRNAs of P2Y₁ and P2Y₆, but not P2Y₂ and P2Y₄ receptors.     

Prostaglandin E<Subscript>2</Subscript> (PGE<Subscript>2</Subscript>) suppresses natural killer cell function primarily through the PGE<Subscript>2</Subscript> receptor EP4

The COX-2 product prostaglandin E₂ (PGE₂) contributes to the high metastatic capacity of breast tumors. Our published data indicate that inhibiting either PGE₂ production or PGE₂-mediated signaling through the PGE₂ receptor EP4 reduces metastasis by a mechanism that requires natural killer (NK) cells. It is known that NK cell function is compromised by PGE₂, but very little is known about the mechanism by which PGE₂ affects NK effector activity. We now report the direct effects of PGE₂ on the NK cell. Endogenous murine splenic NK cells express all four PGE₂ receptors (EP1-4). We examined the role of EP receptors in three NK cell functions: migration, cytotoxicity, and cytokine release. Like PGE₂, the EP4 agonist PGE₁-OH blocked NK cell migration to FBS and to four chemokines (ITAC, MIP-1α, SDF-1α, and CCL21). The EP2 agonist, Butaprost, inhibited migration to specific chemokines but not in response to FBS. In contrast to the inhibitory actions of PGE₂, the EP1/EP3 agonist Sulprostone increased migration. Unlike the opposing effects of EP4 vs. EP1/EP3 on migration, agonists of each EP receptor were uniformly inhibiting to NK-mediated cytotoxicity. The EP4 agonist, PGE₁-OH, inhibited IFNγ production from NK cells. Agonists for EP1, EP2, and EP3 were not as effective at inhibiting IFNγ. Agonists of EP1, EP2, and EP4 all inhibited TNFα; EP4 agonists were the most potent. Thus, the EP4 receptor consistently contributed to loss of function. These results, taken together, support a mechanism whereby inhibiting PGE₂ production or preventing signaling through the EP4 receptor may prevent suppression of NK functions that are critical to the control of breast cancer metastasis.     

Hypoxic expression of NLRP3 and VEGF in cultured retinal pigment epithelial cells: contribution of P2Y<Subscript>2</Subscript> receptor signaling

Retinal hypoxia is a major condition of the chronic inflammatory disease age-related macular degeneration. Extracellular ATP is a danger signal which is known to activate the NLRP3 inflammasome in various cell systems. We investigated in cultured human retinal pigment epithelial (RPE) cells whether hypoxia alters the expression of inflammasome-associated genes and whether purinergic receptor signaling contributes to the hypoxic expression of key inflammatory (NLRP3) and angiogenic factor (VEGF) genes. Hypoxia and chemical hypoxia were induced by a 0.2%-O₂ atmosphere and addition of CoCl₂, respectively. Gene expression was determined with real-time RT-PCR. Cytosolic NLRP3 and (pro-) IL-1β levels, and the extracellular VEGF level, were evaluated with Western blot and ELISA analyses. Cell culture in 0.2% O₂ induced expression of NLRP3 and pro-IL-1β genes but not of the pro-IL-18 gene. Hypoxia also increased the cytosolic levels of NLRP3 and (pro-) IL-1β proteins. Inflammasome activation by lysosomal destabilization decreased the cell viability under hypoxic, but not control conditions. In addition to activation of IL-1 receptors, purinergic receptor signaling mediated by a pannexin-dependent release of ATP and a release of adenosine, and activation of P2Y₂ and adenosine A₁ receptors, was required for the full hypoxic expression of the NLRP3 gene. P2Y₂ (but not A₁) receptor signaling also contributed to the hypoxic expression and secretion of VEGF. The data indicate that hypoxia induces priming and activation of the NLRP3 inflammasome in cultured RPE cells. The hypoxic NLRP3 and VEGF gene expression and the secretion of VEGF are in part mediated by P2Y₂ receptor signaling.     

The P2Y<Subscript>1</Subscript> receptor-mediated leukocyte adhesion to endothelial cells is inhibited by melatonin

Extracellular ATP (released by endothelial and immune cells) and its metabolite ADP are important pro-inflammatory mediators via the activation of purinergic P2 receptors (P2Y and P2X), which represent potential new targets for anti-inflammatory therapy. Endothelial P2Y₁ receptor (P2Y₁R) induces endothelial cell activation triggering leukocyte adhesion. A number of data have implicated melatonin as a modulator of immunity, inflammation, and endothelial cell function, but to date no studies have investigated whether melatonin modulates endothelial P2YR signaling. Here, we evaluated the putative effect of melatonin on P2Y₁R-mediated leukocyte adhesion to endothelial cells and TNF-α production, using mesenteric endothelial cells and fresh peripheral blood mononuclear cells isolated from rats. Endothelial cells were treated with the P2Y₁R agonist 2MeSATP, alone or in combination with melatonin, and then exposed to mononuclear cells. 2MeSATP increased leukocyte adhesion to endothelial cells and TNF-α production in vitro, and melatonin inhibited both effects without altering P2Y₁R protein expression. In addition, assays with the Ca²⁺ chelator BAPTA-AM indicate that the effect of melatonin on 2MeSATP-stimulated leukocyte adhesion depends on intracellular Ca²⁺ modulation. P2Y₁R is considered a potential target to control chronic inflammation. Therefore, our data unveiled a new endothelial cell modulator of purinergic P2Y₁ receptor signaling.     

P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> purinoceptors in the rat enteric nervous system

Adenosine 5-triphosphate receptors are known to be involved in fast excitatory postsynaptic currents in myenteric neurons of the digestive tract. In the present study, the distribution of P2X₂ and P2X₃ receptor mRNA was examined by in situ hybridisation while P2X₂ and P2X₃ receptor protein was localised by immunohistochemical methods. In addition, P2X₂ and P2X₃ receptors were colocalised with calbindin and calretinin in the myenteric and submucosal plexus. P2X₂- and P2X₃-immunoreactive neurons were found in the myenteric and submucosal plexuses throughout the entire length of the rat digestive tract from the stomach to the colon. Approximately 60%, 70% and 50% of the ganglion cells in the myenteric plexus of the gastric corpus, ileum and distal colon, and 56% and 45% in the submucosal plexus of the ileum and distal colon, respectively, showed positive immunoreactivity to the P2X₂ receptor. Approximately 10%, 2% and 15% of the ganglion cells in the myenteric plexus of the gastric corpus, ileum and distal colon, and 62% and 40% in the submucosal plexus of the ileum and distal colon, respectively, showed positive immunoreactivity to the P2X₃ receptor. Double-labelling studies showed that about 10–25% of the neurons with P2X₂ immunoreactivity in myenteric plexus and 30–50% in the submucosal plexus were found to express calbindin or calretinin. About 80% of the neurons with P2X₃ receptor immunoreactivity in the myenteric plexus and about 40% in the submucosal plexus expressed calretinin. Approximately 30–75% of the neurons with P2X₃ receptor immunoreactivity in the submucosal plexus expressed calbindin, while none of them were found to express calbindin in the myenteric plexus.     

Cerebellar astrocytes co-express several ADP receptors. Presence of functional P2Y<Subscript>13</Subscript>-like receptors

Astrocytes exhibit a form of excitability based on variations of intracellular Ca²⁺ concentration in response to various stimuli, including ADP, ATP, UTP and dinucleotides. Here, we investigate the presence of the recently cloned ADP-sensitive receptors, P2Y₁₂ and P2Y₁₃ subtypes, which are negatively coupled to adenylate cyclase, in cerebellar astrocytes. We checked the effect of specific agonists, 2-methylthioadenosine diphosphate (2MeSADP) and ADP, on adenylate cyclase stimulation induced by isoproterenol. Both agonists significantly reduced the cAMP accumulation induced by isoproterenol. The inhibitory effect was concentration-dependent with IC₅₀ values of 46 ± 13 and 23 ± 14 nM for 2MeSADP and ADP, respectively. The experiments were carried out in the presence of MRS-2179, a specific antagonist of P2Y₁ receptor, to avoid any contribution of this receptor. Using fura-2 microfluorimetry we also proved that astrocytes responded to 2MeSADP stimulations with calcium responses in the absence and also in the presence of MRS-2179. Both effects, inhibition of adenylate cyclase and intracellular calcium mobilization, were not modified by 2MeSAMP, an antagonist of P2Y₁₂ receptor, suggesting that were mediated by P2Y₁₃-like receptors.¹These authors equally contributed to this work.     

The Role of P2Y₁ and P2Y₂ Purinoceptors in Determining Mechanosensitivity in Connective Tissue Cells

This article has no abstract.