MRS 2365

Cyclic tensile force stimulates BMP9 synthesis and in vitro mineralization by human periodontal ligament cells.[Pubmed:30206934]

J Cell Physiol. 2019 Apr;234(4):4528-4539.

Periodontal ligament (PDL) cells are mechanosensitive and have the potential to differentiate into osteoblast-like cells under the influence of cyclic tensile force (CTF). CTF modulates the expression of regulatory proteins including bone morphogenetic proteins (BMPs), which are essential for the homeostasis of the periodontium. Among the BMPs, BMP9 is one of the most potent osteogenic BMPs. It is yet unknown whether CTF affects the expression of BMP9 and mineralization. Here, we demonstrated that continuously applied CTF for only the first 6 hr stimulated the synthesis of BMP9 and induced mineral deposition within 14 days by human PDL cells. Stimulation of BMP9 expression depended on ATP and P2Y 1 receptors. Apyrase, an ecto-ATPase, inhibited CTF-mediated ATP-induced BMP9 expression. The addition of ATP increased the expression of BMP9. Loss of function experiments using suramin (a broad-spectrum P2Y antagonist), MRS2179 (a specific P2Y 1 receptor antagonist), MRS 2365 (a specific P2Y 1 agonist), U-73122 (a phospholipase C [PLC] inhibitor), and thapsigargin (enhancer of intracytosolic calcium) revealed the participation of P2Y 1 in regulating the expression of BMP9. This was mediated by an increased level of intracellular Ca (2+) through the PLC pathway. A neutralizing anti-BMP9 antibody decreased mineral deposition, which was stimulated by CTF for almost 45% indicating a role of BMP9 in an in vitro mineralization. Collectively, our findings suggest an essential modulatory role of CTF in the homeostasis and regeneration of the periodontium.

Activation of retinal glial (Muller) cells by extracellular ATP induces pronounced increases in extracellular H+ flux.[Pubmed:29466379]

PLoS One. 2018 Feb 21;13(2):e0190893.

Small alterations in extracellular acidity are potentially important modulators of neuronal signaling within the vertebrate retina. Here we report a novel extracellular acidification mechanism mediated by glial cells in the retina. Using self-referencing H+-selective microelectrodes to measure extracellular H+ fluxes, we show that activation of retinal Muller (glial) cells of the tiger salamander by micromolar concentrations of extracellular ATP induces a pronounced extracellular H+ flux independent of bicarbonate transport. ADP, UTP and the non-hydrolyzable analog ATPgammas at micromolar concentrations were also potent stimulators of extracellular H+ fluxes, but adenosine was not. The extracellular H+ fluxes induced by ATP were mimicked by the P2Y1 agonist MRS 2365 and were significantly reduced by the P2 receptor blockers suramin and PPADS, suggesting activation of P2Y receptors. Bath-applied ATP induced an intracellular rise in calcium in Muller cells; both the calcium rise and the extracellular H+ fluxes were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin and when the PLC-IP3 signaling pathway was disrupted with 2-APB and U73122. The anion transport inhibitor DIDS also markedly reduced the ATP-induced increase in H+ flux while SITS had no effect. ATP-induced H+ fluxes were also observed from Muller cells isolated from human, rat, monkey, skate and lamprey retinae, suggesting a highly evolutionarily conserved mechanism of potential general importance. Extracellular ATP also induced significant increases in extracellular H+ flux at the level of both the outer and inner plexiform layers in retinal slices of tiger salamander which was significantly reduced by suramin and PPADS. We suggest that the novel H+ flux mediated by ATP-activation of Muller cells and of other glia as well may be a key mechanism modulating neuronal signaling in the vertebrate retina and throughout the brain.

Platelet-derived growth factor receptor-alpha-positive cells and not smooth muscle cells mediate purinergic hyperpolarization in murine colonic muscles.[Pubmed:25055825]

Am J Physiol Cell Physiol. 2014 Sep 15;307(6):C561-70.

Enteric inhibitory neurotransmission is an important feature of the neural regulation of gastrointestinal motility. Purinergic neurotransmission, via P2Y1 receptors, mediates one phase of inhibitory neural control. For decades, ATP has been assumed to be the purinergic neurotransmitter and smooth muscle cells (SMCs) have been considered the primary targets for inhibitory neurotransmission. Recent experiments have cast doubt on both of these assumptions and suggested that another cell type, platelet-derived growth factor receptor-alpha-positive (PDGFRalpha(+)) cells, is the target for purinergic neurotransmission. We compared responses of PDGFRalpha(+) cells and SMCs to several purine compounds to determine if these cells responded in a manner consistent with enteric inhibitory neurotransmission. ATP hyperpolarized PDGFRalpha(+) cells but depolarized SMCs. Only part of the ATP response in PDGFRalpha(+) cells was blocked by MRS 2500, a P2Y1 antagonist. ADP, MRS 2365, beta-NAD, and adenosine 5-diphosphate-ribose, P2Y1 agonists, hyperpolarized PDGFRalpha(+) cells, and these responses were blocked by MRS 2500. Adenosine 5-diphosphate-ribose was more potent in eliciting hyperpolarization responses than beta-NAD. P2Y1 agonists failed to elicit responses in SMCs. Small hyperpolarization responses were elicited in SMCs by a small-conductance Ca(2+)-activated K(+) channel agonist, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine, consistent with the low expression and current density of small-conductance Ca(2+)-activated K(+) channels in these cells. Large-amplitude hyperpolarization responses, elicited in PDGFRalpha(+) cells, but not SMCs, by P2Y1 agonists are consistent with the generation of inhibitory junction potentials in intact muscles in response to purinergic neurotransmission. The responses of PDGFRalpha(+) cells and SMCs to purines suggest that SMCs are unlikely targets for purinergic neurotransmission in colonic muscles.

Pharmacological characterization of the P2 receptors profile in the podocytes of the freshly isolated rat glomeruli.[Pubmed:24048730]

Am J Physiol Cell Physiol. 2013 Nov 15;305(10):C1050-9.

Calcium flux in the podocytes is critical for normal and pathophysiological regulation of these types of cells, and excessive calcium signaling results in podocytes damage and improper glomeruli function. Purinergic activation of P2 receptors is a powerful and rapid signaling process; however, the exact physiological identity of P2 receptors subtypes in podocytes remains essentially unknown. The goal of this study was to determine the P2 receptor profile in podocytes of the intact Sprague-Dawley rat glomeruli using available pharmacological tools. Glomeruli were isolated by differential sieving and loaded with Fluo-4/Fura Red cell permeable calcium indicators, and the purinergic response in the podocytes was analyzed with ratiometric confocal fluorescence measurements. Various P2 receptors activators were tested and compared with the effect of ATP, specifically, UDP, MRS 2365, bzATP, alphabeta-methylene, 2-meSADP, MRS 4062, and MRS 2768, were analyzed. Antagonists (MRS 2500, 5-BDBD, A438079, and NF 449) were tested when 10 muM ATP was applied as the EC50 for ATP activation of the calcium influx in the podocytes was determined to be 10.7 +/- 1.5 muM. Several agonists including MRS 2365 and 2-meSADP caused calcium flux. Importantly, only the P2Y1-specific antagonist MRS 2500 (1 nM) precluded the effects of ATP concentrations of the physiological range. Immunohistochemical analysis confirmed that P2Y1 receptors are highly expressed in the podocytes. We conclude that P2Y1 receptor signaling is the predominant P2Y purinergic pathway in the glomeruli podocytes and P2Y1 might be involved in the pathogenesis of glomerular injury and could be a target for treatment of kidney diseases.

Astrocytic P2Y(1) receptor is involved in the regulation of cytokine/chemokine transcription and cerebral damage in a rat model of cerebral ischemia.[Pubmed:21487414]

J Cereb Blood Flow Metab. 2011 Sep;31(9):1930-41.

After brain ischemia, significant amounts of adenosine 5'-triphosphate are released or leaked from damaged cells, thus activating purinergic receptors in the central nervous system. A number of P2X/P2Y receptors have been implicated in ischemic conditions, but to date the P2Y(1) receptor (P2Y(1)R) has not been implicated in cerebral ischemia. In this study, we found that the astrocytic P2Y(1)R, via phosphorylated-RelA (p-RelA), has a negative effect during cerebral ischemia/reperfusion. Intracerebroventricular administration of the P2Y(1)R agonist, MRS 2365, led to an increase in cerebral infarct volume 72 hours after transient middle cerebral artery occlusion (tMCAO). Administration of the P2Y(1)R antagonist, MRS 2179, significantly decreased infarct volume and led to recovered motor coordination. The effects of MRS 2179 occurred within 24 hours of tMCAO, and also markedly reduced the expression of p-RelA and interleukin-6, tumor necrosis factor-alpha, monocyte chemotactic protein-1/chemokine (C-C motif) ligand 2 (CCL2), and interferon-inducible protein-10/chemokine (C-X-C motif) ligand 10 (CXCL10) mRNA. P2Y(1)R and p-RelA were colocalized in glial fibrillary acidic protein-positive astrocytes, and an increase in infarct volume after MRS 2365 treatment was inhibited by the nuclear factor (NF)-kappaB inhibitor ammonium pyrrolidine dithiocarbamate. These results provide evidence that the P2Y(1)R expressed in cortical astrocytes may help regulate the cytokine/chemokine response after tMCAO/reperfusion through a p-RelA-mediated NF-kappaB pathway.

P2Y1 receptor activation elicits its partition out of membrane rafts and its rapid internalization from human blood vessels: implications for receptor signaling.[Pubmed:18799799]

Mol Pharmacol. 2008 Dec;74(6):1666-77.

The nucleotide P2Y(1) receptor (P2Y(1)R) is expressed in both the endothelial and vascular smooth muscle cells; however, its plasma membrane microregionalization and internalization in human tissues remain unknown. We report on the role of membrane rafts in P2Y(1)R signaling by using sodium carbonate or OptiPrep sucrose density gradients, Western blot analysis, reduction of tissue cholesterol content, and vasomotor assays of endothelium-denuded human chorionic arteries. In tissue extracts prepared either in sodium carbonate or OptiPrep, approximately 20 to 30% of the total P2Y(1)R mass consistently partitioned into raft fractions and correlated with vasomotor activity. Vessel treatment with methyl beta-cyclodextrin reduced the raft partitioning of the P2Y(1)R and obliterated the P2Y(1)R-mediated contractions but not the vasomotor responses elicited by either serotonin or KCl. Perfusion of chorionic artery segments with 100 nM 2-methylthio ADP or 10 nM [[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl] 2,3dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS 2365), a selective P2Y(1)R agonist, not only displaced within 4 min the P2Y(1)R localization out of membrane rafts but also induced its subsequent internalization. 2'-Deoxy-N(6)-methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS 2179), a specific P2Y(1)R antagonist, did not cause a similar displacement but blocked the agonist-induced exit from rafts. Neither adenosine nor uridine triphosphate displaced the P2Y(1)R from the membrane raft, further evidencing the pharmacodynamics of the receptor-ligand interaction. Vascular reactivity assays showed fading of the ligand-induced vasoconstrictions, a finding that correlated with the P2Y(1)R exit from raft domains and internalization. These results demonstrate in intact human vascular smooth muscle the association of the P2Y(1)R to membrane rafts, highlighting the role of this microdomain in P2Y(1)R signaling.

Stimulation of the P2Y1 receptor up-regulates nucleoside-triphosphate diphosphohydrolase-1 in human retinal pigment epithelial cells.[Pubmed:17626796]

J Pharmacol Exp Ther. 2007 Oct;323(1):157-64.

Stimulation of receptors for either ATP or adenosine leads to physiologic changes in retinal pigment epithelial (RPE) cells that may influence their relationship with the adjacent photoreceptors. The ectoenzyme nucleoside-triphosphate diphosphohydrolase-1 (NTPDase1) catalyzes the dual dephosphorylation of ATP and ADP to AMP. Although NTPDase1 can consequently control the balance between ATP and adenosine, it is unclear how its expression and activity are regulated. Classic negative feedback theory predicts an increase in enzyme activity in response to enhanced exposure to substrate. This study asked whether exposure to ATP increases NTPDase1 activity in RPE cells. Although levels of NTPDase1 mRNA and protein in cultured human ARPE-19 cells were generally low under control conditions, exposure to slowly hydrolyzable ATPgammaS led to a time-dependent increase in NTPDase1 mRNA that was accompanied by a rise in levels of the functional 78-kDa protein. Neither NTPDase2 nor NTPDase3 mRNA message was elevated by ATPgammaS. The ATPase activity of cells increased in parallel, indicating the up-regulation of NTPDase1 was functionally relevant. The up-regulation of NTPDase1 protein was partially blocked by P2Y1 receptor inhibitors MRS2179 (N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate) and MRS2500 [2-iodo-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate] and increased by P2Y1 receptor agonist MRS2365 [(N)-methanocarba-2MeSADP]. In conclusion, prolonged exposure to extracellular ATPgammaS increased NTPDase1 message and protein levels and increased ecto-ATPase activity. This up-regulation reflects a feedback circuit, mediated at least in part by the P2Y1 receptor, to regulate levels of extracellular purines in subretinal space. NTPDase1 levels may thus serve as an index for increased extracellular ATP levels under certain pathologic conditions, although other mechanisms could also contribute.

Induction of novel agonist selectivity for the ADP-activated P2Y1 receptor versus the ADP-activated P2Y12 and P2Y13 receptors by conformational constraint of an ADP analog.[Pubmed:15345752]

J Pharmacol Exp Ther. 2004 Dec;311(3):1038-43.

ADP is the cognate agonist of the P2Y1, P2Y12, and P2Y13 receptors. With the goal of identifying a high potency agonist that selectively activates the P2Y1 receptor, we examined the pharmacological selectivity of the conformationally constrained non-nucleotide analog (N)-methanocarba-2MeSADP [(1'S,2'R, 3'S,4'R,5'S)-4-[(6-amino-2-methylthio-9H-purin-9-yl)-1-diphosphoryloxymethyl]bicy clo[3.1.0]hexane-2,3-diol] among the three ADP-activated receptors. Each P2Y receptor was expressed transiently in COS-7 cells, and inositol lipid hydrolysis was quantified as a measure of receptor activity. In the case of the Gi-linked P2Y12 and P2Y13 receptors, a chimeric G protein, Galphaq/i, was coexpressed to confer a capacity of these Gi-linked receptors to activate phospholipase C. 2MeSADP (2-methylthio-ADP) was a potent agonist at all three receptors exhibiting EC50 values in the sub to low nanomolar range. In contrast, whereas (N)-methanocarba-2MeSADP was an extremely potent (EC50=1.2 +/- 0.2 nM) agonist at the P2Y1 receptor, this non-nucleotide analog exhibited no agonist activity at the P2Y12 receptor and very low activity at the P2Y13 receptor. (N)-Methanocarba-2MeSADP also failed to block the action of 2MeSADP at the P2Y12 and P2Y13 receptors, indicating that the (N)-methanocarba analog is not an antagonist at these receptors. The P2Y1 receptor selectivity of (N)-methanocarba-2MeSADP was confirmed in human platelets where it induced the shape change promoted by P2Y1 receptor activation without inducing the sustained platelet aggregation that requires simultaneous activation of the P2Y12 receptor. These results provide the first demonstration of a high-affinity agonist that discriminates among the three ADP-activated P2Y receptors, and therefore, introduce a potentially important new pharmacological tool for delineation of the relative biological action of these three signaling proteins.

Adenine nucleotide analogues locked in a Northern methanocarba conformation: enhanced stability and potency as P2Y(1) receptor agonists.[Pubmed:11985476]

J Med Chem. 2002 May 9;45(10):2090-100.

Preference for the Northern (N) ring conformation of the ribose moiety of nucleotide 5'-triphosphate agonists at P2Y(1), P2Y(2), P2Y(4), and P2Y(11) receptors, but not P2Y(6) receptors, was established using a ring-constrained methanocarba (a 3.1.0-bicyclohexane) ring as a ribose substitute (Kim et al. J. Med. Chem. 2002, 45, 208-218.). We have now combined the ring-constrained (N)-methanocarba modification of adenine nucleotides with other functionalities known to enhance potency at P2 receptors. The potency of the newly synthesized analogues was determined in the stimulation of phospholipase C through activation of turkey erythrocyte P2Y(1) or human P2Y(1) and P2Y(2) receptors stably expressed in astrocytoma cells. An (N)-methanocarba-2-methylthio-ADP analogue displayed an EC(50) at the hP2Y(1) receptor of 0.40 nM and was 55-fold more potent than the corresponding triphosphate and 16-fold more potent than the riboside 5'-diphosphate. 2-Cl-(N)-methanocarba-ATP and its N(6)-Me analogue were also highly selective, full agonists at P2Y(1) receptors. The (N)-methanocarba-2-methylthio and 2-chloromonophosphate analogues were full agonists exhibiting micromolar potency at P2Y(1) receptors, while the corresponding ribosides were inactive. Although beta,gamma-methylene-ATP was inactive at P2Y receptors, beta,gamma-methylene-(N)-methanocarba-ATP was a potent hP2Y(1) receptor agonist with an EC(50) of 160 nM and was selective versus hP2Y(2) and hP2Y(4) receptors. The rates of hydrolysis of Northern (N) and Southern (S) methanocarba analogues of AMP by rat 5'-ectonucleotidase were negligible. The rates of hydrolysis of the corresponding triphosphates by recombinant rat NTPDase1 and 2 were studied. Both isomers were hydrolyzed by NTPDase 1 at about half the rate of ATP hydrolysis. The (N) isomer was hardly hydrolyzed by NTPDase 2, while the (S) isomer was hydrolyzed at one-third of the rate of ATP hydrolysis. This suggests that new, more stable and selective nucleotide agonists may be designed on the basis of the (N)-conformation, which greatly enhanced potency at P2Y(1) receptors.