Coupling switch of P2Y-IP<Subscript>3</Subscript> receptors mediates differential Ca<Superscript>2+</Superscript> signaling in human embryonic stem cells and derived cardiovascular progenitor cells

Purinergic signaling mediated by P2 receptors (P2Rs) plays important roles in embryonic and stem cell development. However, how it mediates Ca²⁺ signals in human embryonic stem cells (hESCs) and derived cardiovascular progenitor cells (CVPCs) remains unclear. Here, we aimed to determine the role of P2Rs in mediating Ca²⁺ mobilizations of these cells. hESCs were induced to differentiate into CVPCs by our recently established methods. Gene expression of P2Rs and inositol 1,4,5-trisphosphate receptors (IP₃Rs) was analyzed by quantitative/RT-PCR. IP₃R3 knockdown (KD) or IP₃R2 knockout (KO) hESCs were established by shRNA- or TALEN-mediated gene manipulations, respectively. Confocal imaging revealed that Ca²⁺ responses in CVPCs to ATP and UTP were more sensitive and stronger than those in hESCs. Consistently, the gene expression levels of most P2YRs except P2Y₁ were increased in CVPCs. Suramin or PPADS blocked ATP-induced Ca²⁺ transients in hESCs but only partially inhibited those in CVPCs. Moreover, the P2Y₁ receptor-specific antagonist MRS2279 abolished most ATP-induced Ca²⁺ signals in hESCs but not in CVPCs. P2Y₁ receptor-specific agonist MRS2365 induced Ca²⁺ transients only in hESCs but not in CVPCs. Furthermore, IP₃R2KO but not IP₃R3KD decreased the proportion of hESCs responding to MRS2365. In contrast, both IP₃R2 and IP₃R3 contributed to UTP-induced Ca²⁺ responses while ATP-induced Ca²⁺ responses were more dependent on IP₃R2 in the CVPCs. In conclusion, a predominant role of P2Y₁ receptors in hESCs and a transition of P2Y-IP₃R coupling in derived CVPCs are responsible for the differential Ca²⁺ mobilization between these cells.     

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.     

NCI-H295R,a Human Adrenal Cortex-Derived Cell Line,Expresses Purinergic Receptors Linked to Ca2+-Mobilization/Influx and Cortisol Secretion

Purinergic receptor expression and involvement in steroidogenesis were examined in NCI-H295R (H295R), a human adrenal cortex cell line which expresses all the key enzymes necessary for steroidogenesis. mRNA/protein for multiple P1 (A_2A and A_2B), P2X (P2X₅ and P2X₇), and P2Y (P2Y₁, P2Y₂, P2Y₆, P2Y₁₂, P2Y₁₃, and P2Y₁₄) purinergic receptors were detected in H295R. 2MeS-ATP (10–1000 µM), a P2Y₁ agonist, induced glucocorticoid (GC) secretion in a dose-dependent manner, while other extracellular purine/pyrimidine agonists (1–1000 µM) had no distinct effect on GC secretion. Extracellular purines, even non-steroidogenic ones, induced Ca²⁺-mobilization in the cells, independently of the extracellular Ca²⁺ concentration. Increases in intracellular Ca²⁺ concentration induced by extracellular purine agonists were transient, except when induced by ATP or 2MeS-ATP. Angiotensin II (AngII: 100 nM) and dibutyryl-cyclic AMP (db-cAMP: 500 µM) induced both GC secretion and Ca²⁺-mobilization in the presence of extracellular Ca²⁺ (1.2 mM). GC secretion by AngII was reduced by nifedipine (10–100 µM); whereas the Ca²⁺ channel blocker did not inhibit GC secretion by 2MeS-ATP. Thapsigargin followed by extracellular Ca²⁺ exposure induced Ca²⁺-influx in H295R, and the cells expressed mRNA/protein of the component molecules for store-operated calcium entry (SOCE): transient receptor C (TRPC) channels, calcium release-activated calcium channel protein 1 (Orai-1), and the stromal interaction molecule 1 (STIM1). In P2Y₁-knockdown, 2MeS-ATP-induced GC secretion was significantly inhibited. These results suggest that H295R expresses a functional P2Y₁ purinergic receptor for intracellular Ca²⁺-mobilization, and that P2Y₁ is linked to SOCE-activation, leading to Ca²⁺-influx which might be necessary for glucocorticoid secretion.     

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.     

CB<Subscript>1</Subscript> Receptors Mediated Inhibition of ATP-Induced [Ca<Superscript>2+</Superscript>]i Increase in Cultured Rat Spinal Dorsal Horn Neurons

Spinal cannabinoid receptor 1 (CB₁R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB₁R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB₁ receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca²⁺ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca²⁺]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca²⁺]i increase in cultured DH neurons. ATP-evoked [Ca²⁺]i increase in DH neurons was blocked by chelating extracellular Ca²⁺ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca²⁺]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca²⁺]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca²⁺ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca²⁺ response was mimicked by ACEA (CB₁R agonist), but was not influenced by AM1241 (CB₂R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca²⁺ mobilization was blocked by AM251 (CB₁ receptor antagonist), but was not influenced by AM630 (CB₂ receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB₁R rather than CB₂R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB₁R on P2X-channel-induced Ca²⁺ mobilization.     

Endothelial Cell E- and P-Selectin and Vascular Cell Adhesion Molecule-1 Function as Signaling Receptors

Previous studies have shown that polymorphonuclear leukocyte (PMN) adherence to endothelial cells (EC) induces transient increases in EC cytosolic free calcium concentration ([Ca²⁺]_i) that are required for PMN transit across the EC barrier (Huang, A.J., J.E. Manning, T.M. Bandak, M.C. Ratau, K.R. Hanser, and S.C. Silverstein. 1993. J. Cell Biol. 120:1371–1380). To determine whether stimulation of [Ca²⁺]_i changes in EC by leukocytes was induced by the same molecules that mediate leukocyte adherence to EC, [Ca²⁺]_i was measured in Fura2-loaded human EC monolayers. Expression of adhesion molecules by EC was induced by a pretreatment of the cells with histamine or with Escherichia coli lipopolysaccharide (LPS), and [Ca²⁺]_i was measured in single EC after the addition of mAbs directed against the EC adhesion proteins P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or platelet/endothelial cell adhesion molecule-1 (PECAM-1). Both anti–P- and anti–E-selectin mAb, as well as anti–VCAM-1 mAb, induced transient increases in EC [Ca²⁺]_i that were comparable to those induced by 200 μM histamine. In contrast, no effect was obtained by mAbs directed against the endothelial ICAM-1 or PECAM-1. PMN adherence directly stimulated increases in [Ca²⁺]_i in histamine- or LPS-treated EC. mAbs directed against leukocyte CD18 or PECAM-1, the leukocyte counter-receptors for endothelial ICAM-1 and PECAM-1, respectively, did not inhibit PMN-induced EC activation. In contrast, mAb directed against sialyl Lewis x (sLe^x), a PMN ligand for endothelial P- and E-selectin, completely inhibited EC stimulation by adherent PMN. Changes in EC [Ca²⁺]_i were also observed after adherence of peripheral blood monocytes to EC treated with LPS for 5 or 24 h. In these experiments, the combined addition of mAbs to sLe^x and VLA-4, the leukocyte counter-receptor for endothelial VCAM-1, inhibited [Ca²⁺]_i changes in the 5 h–treated EC, whereas the anti–VLA-4 mAb alone was sufficient to inhibit [Ca²⁺]_i changes in the 24 h-treated EC. Again, no inhibitory effect was observed with an anti-CD18 or anti–PECAM-1 mAb. Of note, the conditions that induced changes in EC [Ca²⁺]_i, i.e., mAbs directed against endothelial selectins or VCAM-1, and PMN or monocyte adhesion to EC via selectins or VCAM-1, but not via ICAM-1 or PECAM-1, also induced a rearrangement of EC cytoskeletal microfilaments from a circumferential ring to stress fibers. We conclude that, in addition to their role as adhesion receptors, endothelial selectins and VCAM-1 mediate endothelial stimulation by adhering leukocytes.     

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.     

Modeling P2Y receptor-Ca response coupling in taste cells

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

Protein Interacting C-Kinase 1 Modulates Surface Expression of P2Y<Subscript>6</Subscript> Purinoreceptor,Actin Polymerization and Phagocytosis in Microglia

Microglia clean up dead cells and debris through phagocytosis in the central nervous system. UDP-activated P2Y₆ receptors (P2Y₆Rs) induce the formation of phagocytic cup-like structure and P2Y₆R expression is increased during the phagocytosis. However, it remains unclear how surface expression of P2Y₆R is increased. PICK1 (protein interacting with C-kinase-1) interacts with various neurotransmitter receptors, transporters, and enzymes. We here report that PICK1 might interact with P2Y₆R. Surface P2Y₆R was reduced in microglia from PICK1-knockout mice and PICK1-knockdown BV2 cells, which was also confirmed by electrophysiological recordings, showing that P2Y₆R-mediated current was increased by PICK1 overexpression but was reduced by PICK1-knockdown in BV2 microglia. Finally, PICK1 was sufficient to affect cytoskeletal aggregation and phagocytosis both in primary microglia and BV2 cells. These results indicate that PICK1 is an important regulator of P2Y₆R expression and microglial phagocytosis.     

Purinergic receptor-mediated Ca<Superscript>2+</Superscript> signaling in the olfactory bulb and the neurogenic area of the lateral ventricles

Like in other vertebrates, the anterior part of the telencephalon of amphibians mainly consists of the olfactory bulb (OB), but different from higher vertebrates, the lateral telencephalic ventricles of larval Xenopus laevis expand deep into the anterior telencephalon. The neurogenic periventricular zone (PVZ) of the lateral ventricles generates new OB neurons throughout the animal’s lifetime. We investigated the ultrastructural organization of the PVZ and found that within a time period of 24 h, 42.54 ± 6.65% of all PVZ cells were actively proliferating. Functional purinergic receptors are widespread in the central nervous system and their activation has been associated with many critical physiological processes, including the regulation of cell proliferation. In the present study we identified and characterized the purinergic system of the OB and the PVZ. ATP and 2MeSATP induced strong [Ca²⁺]_i increases in cells of both regions, which could be attenuated by purinergic antagonists. However, a more thorough pharmacological investigation revealed clear differences between the two brain regions. Cells of the OB almost exclusively express ionotropic P2X purinergic receptor subtypes, whereas PVZ cells express both ionotropic P2X and metabotropic P1 and P2Y receptor subtypes. The P2X receptors expressed in the OB are evidently not involved in the immediate processing of olfactory information.     

Differential expression of P2Y receptors in the rat cochlea during development

Purinergic signaling has broad physiological significance to the hearing organ, involving signal transduction via ionotropic P2X receptors and metabotropic G-protein-coupled P2Y and P1 (adenosine), alongside conversion of nucleotides and nucleosides by ecto-nucleotidases and ecto-nucleoside diphosphokinase. In addition, ATP release is modulated by acoustic overstimulation or stress and involves feedback regulation. Many of these principal elements of the purinergic signaling complex have been well characterized in the cochlea, while the characterization of P2Y receptor expression is emerging. The present study used immunohistochemistry to evaluate the expression of five P2Y receptors, P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₂, during development of the rat cochlea. Commencing in the late embryonic period, the P2Y receptors studied were found in the cells lining the cochlear partition, associated with establishment of the electrochemical environment which provides the driving force for sound transduction. In addition, early postnatal P2Y₂ and P2Y₄ protein expression in the greater epithelial ridge, part of the developing hearing organ, supports the view that initiation and regulation of spontaneous activity in the hair cells prior to hearing onset is mediated by purinergic signaling. Sub-cellular compartmentalization of P2Y receptor expression in sensory hair cells, and diversity of receptor expression in the spiral ganglion neurons and their satellite cells, indicates roles for P2Y receptor-mediated Ca²⁺-signaling in sound transduction and auditory neuron excitability. Overall, the dynamics of P2Y receptor expression during development of the cochlea complement the other elements of the purinergic signaling complex and reinforce the significance of extracellular nucleotide and nucleoside signaling to hearing.     

2-Arachidonoylglycerol modulates human endothelial cell/leukocyte interactions by controlling selectin expression through CB1 and CB2 receptors

Accumulated evidence points to a key role for endocannabinoids in cell migration, and here we sought to characterize the role of these substances in early events that modulate communication between endothelial cells and leukocytes. We found that 2-arachidonoylglycerol (2-AG) was able to initiate and complete the leukocyte adhesion cascade, by modulating the expression of selectins. A short exposure of primary human umbilical vein endothelial cells (HUVECs) to 2-AG was sufficient to prime them towards an activated state: within 1 h of treatment, endothelial cells showed time-dependent plasma membrane expression of P- and E-selectins, which both trigger the initial steps (i.e., capture and rolling) of leukocyte adhesion. The effect of 2-AG was mediated by CB₁ and CB₂ receptors and was long lasting, because endothelial cells incubated with 2-AG for 1 h released the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α) for up to 24 h. Consistently, TNF-α-containing medium was able to promote leukocyte recruitment: human Jurkat T cells grown in conditioned medium derived from 2-AG-treated HUVECs showed enhanced L-selectin and P-selectin glycoprotein ligand-1 (PSGL1) expression, as well as increased efficiency of adhesion and trans-migration. In conclusion, our in vitro data indicate that 2-AG, by acting on endothelial cells, might indirectly promote leukocyte recruitment, thus representing a potential therapeutic target for treatment of diseases where impaired endothelium/leukocyte interactions take place.     

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.     

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.     

Cell density‐dependent changes in intracellular Ca2+ mobilization via the P2Y2 receptor in rat bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are an interesting subject of research because they have characteristics of mesenchymal stem cells. We investigated intracellular Ca²⁺ signaling in rat BMSCs. Agonists for purinergic receptors increased intracellular Ca²⁺ levels ([Ca²⁺]_i). The order of potency followed ATP = UTP > ADP = UDP. ATP‐induced rise in [Ca²⁺]_i was suppressed by U73122 and suramin, but not by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), suggesting the functional expression of G protein‐coupled P2Y₂ receptors. RT‐PCR and immunohistochemical studies also showed the expression of P2Y₂ receptors. [Ca²⁺]_i response to UTP changed with cell density. The UTP‐induced rise in [Ca²⁺]_i was greatest at high density. V_max (maximum Ca²⁺ response) and EC₅₀ (agonist concentration that evokes 50% of V_max) suggest that the amount and property of P2Y₂ receptors were changed by cell density. Note that UTP induced Ca²⁺ oscillation at only medium cell density. Pharmacological studies indicated that UTP‐induced Ca²⁺ oscillation required Ca²⁺ influx by store‐operated Ca²⁺ entry. Carbenoxolone, a gap junction blocker, enhanced Ca²⁺ oscillation. Immunohistochemical and quantitative real‐time PCR studies revealed that proliferating cell nuclear antigen (PCNA)‐positive cells declined but the mRNA expression level of the P2Y₂ receptor increased as cell density increased. Co‐application of fetal calf serum with UTP induced Ca²⁺ oscillation at high cell density. These results suggest that the different patterns observed for [Ca²⁺]_i mobilization with respect to cell density may be associated with cell cycle progression. J. Cell. Physiol. 219: 372–381, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Copper inhibits P2Y2-dependent Ca signaling through the effects on thapsigargin-sensitive Ca stores in HTC hepatoma cells

Purinergic P2Y₂ G-protein coupled receptors play a key role in the regulation of hepatic Ca²⁺ signaling by extracellular ATP. The concentration of copper in serum is about 20 μM. Since copper accumulates in the liver in certain disease states, the purpose of these studies was to assess the effects of copper on P2Y₂ receptors in a model liver cell line. Exposure to a P2Y₂ agonist UTP increased [Ca²⁺]_i by stimulating Ca²⁺ release from thapsigargin-sensitive Ca²⁺ stores. Pretreatment of HTC cells for several minutes with copper did not affect cell viability, but potently inhibited increases in [Ca²⁺]_i evoked by UTP and thapsigargin. During this pretreatment, copper was not transported into the cytosol, and inhibited P2Y₂ receptors in a concentration-dependent manner with the IC₅₀ of about 15 μM. These results suggest that copper inhibits P2Y₂ receptors through the effects on thapsigargin-sensitive Ca²⁺ stores by acting from an extracellular side. Further experiments indicated that these effect of copper may lead to inhibition of regulatory volume decrease (RVD) evoked by hypotonic solution. Thus, copper may contribute to defective regulation of purinergic signaling and liver cell volume in diseases associated with the increased serum copper concentration.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.     

POM-1 inhibits P2 receptors and exhibits anti-inflammatory effects in macrophages

Extracellular nucleotides can modulate the immunological response by activating purinergic receptors (P2Rs) on the cell surface of macrophages, dendritic, and other immune cells. In particular, the activation of P2X7R can induce release of cytokines and cell death as well as the uptake of large molecules through the cell membrane by a mechanism still poorly understood. Polyoxotungstate-1 (POM-1) has been proposed as a potent inhibitor of ecto-nucleotidases, enzymes that hydrolyze extracellular nucleotides, regulating the activity of P2Rs. However, the potential impact of POM-1 on P2Rs has not been evaluated. Here, we used fluorescent dye uptake, cytoplasmic free Ca²⁺ concentration measurement, patch-clamp recordings, scanning electron microscopy, and quantification of inflammatory mediators to investigate the effects of POM-1 on P2Rs of murine macrophages. We observed that POM-1 blocks the P2YR-dependent cytoplasmic Ca²⁺ increase and has partial effects on the cytoplasmic Ca²⁺, increasing dependence on P2XRs. POM-1 can inhibit the events related with ATP-dependent inflammasome activation, anionic dye uptake, and also the opening of large conductance channels, which are associated with P2X7R-dependent pannexin-1 activation. On the other hand, this compound has no effects on cationic fluorescent dye uptake, apoptosis, and bleb formation, also dependent on P2X7R. Moreover, POM-1 can be considered an anti-inflammatory compound, because it prevents TNF-α and nitric oxide release from LPS-treated macrophages.