核苷酸和P2Y2受体与伤害性感受的研究进展

P2受体为以ATP、UTP及其类似物激活的受体，分为促离子型P2X受体和促代谢型P2Y受体两大类，其各自又分为许多亚型。许多证据显示，P2Y，特别是P2Y2受体，在痛觉信息调节和痛觉过敏中起着重要作用。ATP和UTP作为P2Y2受体的天然激动剂，是伤害性感受和痛觉过敏信息传递中的重要介质。P2Y2受体参与疼痛的调节可能与辣椒素受体有关。膜片钳分析小鼠背根神经节神经元证明了是P2Y2而非P2Y1受体在ATP诱发的TRPV1介导的热痛过敏反应中起作用。原位杂交组织化学研究结果也显示大鼠腰段脊髓背根神经节中TRPV1mRNA与P2Y2mRNA共同表达。这些证据证明了促代谢型P2Y，受体介导背根神经节神经元中核苷酸对VR1的增敏效应及参与伤害性感受的调节。     

P2Y receptors activated by diadenosine polyphosphates reestablish Ca(2+) transients in achondroplasic chondrocytes

Achondroplasia is the most common type of dwarfism, characterised by a mutation in the gene that encodes the fibroblast growth factor receptor 3 (FGFR3). Achondroplasia mainly affects the chondrocytes and therefore bones do not grow properly since intracellular pathways are altered. In this sense, defective calcium signaling by mutant FGFR3 has been previously described. The purpose of this study was to investigate the presence of purinergic P2Y receptors and how the activation of these receptors can have influence on defective calcium signaling observed in achondroplasic chondrocytes. The presence of P2Y receptors was determined by immunocytochemical and western blot techniques. Calcium mobilization after stimulation with nucleotides, dinucleotides, or, FGF9 application, was measured using the ratiometric dye fura-2/AM and fluorescence imaging. Our results demonstrate the expression of P2Y(1), P2Y(2), P2Y(6) and P2Y(11) receptors in achondroplasic chondrocytes, as well as the activation of these receptors after nucleotides and dinucleotides exposure. The altered calcium signaling of achondroplasic chondrocytes was confirmed, since FGF9 treatment fails to induce calcium mobilization. However, achondroplasic chondrocytes pre-treated with Ap(4)A are able to respond with increases in intracellular calcium after FGF9 stimulation. These findings show the rescue effect of diadenosine tetraphosphate (Ap(4)A), acting by means of P2Y receptors, on defective calcium response triggered by achondroplasic FGFR3.     

P2Y purinergic potentiation of glucose-induced insulin secretion and pancreatic beta-cell metabolism

Purine nucleotides and their analogs increase insulin secretion through activation of pancreatic beta-cell P2Y receptors. The present study aimed at determining the role of glucose metabolism in the response to P2Y agonists and whether ATP-activated K+ channels (KATP channels) are involved in this response. The experiments were performed in the rat isolated pancreas, perfused with a Krebs-bicarbonate buffer supplemented with 2 g/l bovine serum albumin under dynamic glucose conditions from 5 mmol/l baseline to 11 mmol/l. ADPbetaS (0.5 micromol/l) was selected as a stable and selective P2Y agonist. This compound, ineffective on the 5 mmol/l glucose background, induced a significant threefold increase in insulin release triggered by the glucose challenge. The effect of ADPbetaS was markedly reduced (P <0.001) in the presence of an inhibitor of glucose metabolism. In addition to glucose, the ADP analog also amplified the beta-cell insulin response to 15 mmol/l methyl pyruvate (P <0.05), but it was ineffective on the insulin response to 2.5 mmol/l methyl succinate. A nonmetabolic stimulus was applied using tolbutamide (185 micromol/l). Insulin secretion induced by the KATP channel blocker was strongly reinforced by ADPbetaS (P <0.001), which prompted us to check a possible interplay of KATP channels in the effect of ADPbetaS. In the presence of diazoxide 250 micromol/l and 21 mmol/l KCl, ADPbetaS still amplified the second phase of glucose-induced insulin secretion (P <0.001). We conclude that P2Y receptor activation is able to promote insulin secretion through a mechanism, involving beta-cell metabolism and a rise in intracellular calcium; this effect does not result from a direct inhibitory effect on KATP channels.     

ATP induces c-fos expression in C6 glioma cells by activation of P(2Y) receptors

BACKGROUND: Extracellular ATP functions in the enteric nervous system as a neurotransmitter, and recent evidence suggests ATP may regulate development through effects on cellular proliferation. METHODS: The action of ATP at purinoceptors and the role of second messenger pathways in c-fos mRNA expression in C6 glioma cells were investigated using the techniques of Northern and Western blotting. RESULTS: Treatment of C6 cells with ATP caused a time- and dose-dependent increase in c-fos expression. The rank order of agonist potency was ATP = ADP > gammasATP > alphabetaATP > betagammaATP > AMP = UTP. The ATP-induced c-fos increment was inhibited by three P(2Y) receptor antagonists-suramin, reactive blue, and DIDS-by 99+/-3, 89+/-7, and 61+/-14%, respectively. The ATP-stimulated c-fos expression was attenuated by phospholipase C inhibitor (U73122), protein kinase C (PKC) down-regulation (4alpha-phorbol 12-myristate 13-acetate and chelerythrine), mitogen-activated protein (MAP) kinase inhibition (apigenin), an inhibitor of MAP kinase kinase (PD98059), down-regulation of adenylate cyclase (SQ22536), and inhibition of type II protein kinase A (8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate), but was not affected by inhibition of type I protein kinase A (8-bromoadenosine-3',5'-cyclic monophosphorothioate) and inhibitors of calmodulin kinase (KN93 and KN62). Phosphorylated MAP kinase was increased in cells exposed to ATP. This effect was suppressed by chelerythrine. CONCLUSIONS: These studies demonstrate that ATP-induced c-fos mRNA expression is under multifactorial regulation.     

大鼠嘌呤P2Y2受体在神经系统分布的实验研究

目的探讨P2Y2受体在脊髓、背根神经节和坐骨神经的表达和具体分布，研究三磷酸腺苷（adenosine triphosphate．ATP）对神经再生的作用机制提供理论基础。方法将6只SD乳鼠的脊髓、背根神经节和坐骨神经标本置于含有0．1％二乙基焦碳酸的4%多聚甲醛液中快速固定、石蜡包埋、超薄切片。实验组：根据P2Y2受体mRNA的碱基序列，制作非放射性标记的核苷酸探针，原位杂交后将切片置于含有NBT溶液（50mg／ml硝基兰四唑溶于二甲基甲酰胺）、BCIP溶液（75mg／ml溴绿一吲哚磷酸溶于二甲基酰胺）的检测缓冲液中染色，镜下观察P2Y2受体的具体分布。对照组：切片刚不含探针的杂交缓冲液覆盖、染色观察。结果实验组：脊髓灰质前角神经元胞浆中可见P2Y2受体的表达，呈弱阳性；背根神经节雪旺细胞的胞浆和胞核中均可见P2Y2受体的表达，且呈强阳性；对照组：无P2Y2受体表达。实验组与对照组坐骨神经原位杂交尢P2Y2受体的表达。结论P2Y2受体主要分布在脊髓、背根神经节，细胞外ATP可以通过P2Y2受体作用于脊髓和背根神经节细胞。     

P2Y2 receptors and GRK2 are involved in oscillatory fluid flow induced ERK1/2 responses in chondrocytes

Mechanical loading is an important factor regulating cartilage metabolism maintained by chondrocytes. However, some of its underlying mechanisms remain poorly understood. In this study, we employed a chondrogenic cell line ATDC5 to investigate roles of P2Y₂ and GRK2 in chondrocyte mechanotransduction. We first confirmed the expression of chondrocyte markers in differentiated ATDC5 cells. We then exposed both differentiated and undifferentiated ATDC5 cells to oscillatory fluid flow, and found that differentiated ATDC5 cells responded to oscillatory fluid flow by increasing COX‐2 and aggrecan expressions. More importantly, fluid flow induced ERK1/2 response in differentiated cells was increased more than 10 times compared to those in undifferentiated cells. Furthermore, we found that P2Y₂ mRNA and protein levels in differentiated ATDC5 cells were significantly higher than those in undifferentiated cells. In contrast, GRK2 protein levels in differentiated cells were significantly lower than those in undifferentiated cells. Finally, overexpressions of P2Y₂ and GRK2 in differentiated ATDC5 cells result in a 34% increase and a 21% decrease of the ERK1/2 phosphorylation, respectively, in response to oscillatory fluid flow, suggesting important roles of P2Y₂ and GRK2 in chondrocyte mechanotransduction. © 2010 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:828–833     

The luminal P2Y receptor in the isolated perfused mouse cortical collecting duct

Extracellular nucleotides regulate renal ion transport. With the use of in vitro perfusion and [Ca(2+)](i) imaging, this study investigated whether mouse and rabbit cortical collecting ducts (CCD) respond to luminal nucleotides. In mouse CCD, luminal ATP (EC(50): 10 microM) and UTP (EC(50): 9.7 microM) increased [Ca(2+)](i) with an initial peak and a plateau. To make certain that basolateral P2 receptors were not activated by luminal nucleotides via leak diffusion, luminal trypsin (1 microM), a known agonist for basolateral proteinase-activated receptors, was perfused. Mouse CCD that were responsive to luminal ATP were nonresponsive to luminal trypsin but always showed [Ca(2+)](i) elevations by basolateral trypsin (10 or 100 nM). Luminal alpha,beta- and beta,gamma-methylene ATP, 2-methyl-S-ATP, ADP, UDP, and 2',3'-O-4-benzoylbenzoyl ATP had no effect (100 microM, n = 9). Without external Ca(2+), luminal ATP still stimulated a [Ca(2+)](i) increase. Mouse CCD also responded to basolateral ATP (EC(50): 23 microM) and UTP (EC(50): 23 microM) with smaller [Ca(2+)](i) elevations. Confocal microscopy of perfused CCD showed that luminal ATP (100 microM) rapidly increased [Ca(2+)](i) in nearly all cells (n = 6) and the same cells that responded to luminal ATP responded to basolateral ATP (100 microM). In contrast, rabbit CCD did not respond to luminal ATP/UTP (n = 8) despite ATP's known effect from the basolateral side (EC(50): 34 microM). These data indicate the expression of luminal P2Y receptors (probably P2Y(2)) in principal cells of mouse CCD but not in rabbit CCD.     

Luminal P2Y2 receptor-mediated inhibition of Na+ absorption in isolated perfused mouse CCD.

Extracellular nucleotides regulate renal transport. A luminal P2Y2 receptor in mouse cortical collecting duct (CCD) principal cells has been demonstrated elsewhere. Herein the effects of adenosine triphosphate (ATP) and uridine triphosphate (UTP) on electrogenic Na+ absorption in perfused CCD of mice kept on a low-NaCl diet were investigated. Simultaneously, transepithelial voltage (V(te)), transepithelial resistance (R(te)), and fura-2 [Ca2+]i fluorescence were measured. Baseline parameters were V(te), -16.5 +/- 1.2 mV; R(te), 80.8 +/- 7.1 Omega cm2; and equivalent short-circuit current (I(sc)), -261.0 +/- 25.1 microA/cm2 (n = 45). Amiloride (10 microM) almost completely inhibited I(sc) to -3.9 +/- 3.8 microA/cm2 (n = 10). Luminal ATP (100 microM) reduced V(te) from -16.5 +/- 2.1 to -12.5 +/- 1.93 and increased R(te) from 113.1 +/- 16.2 to 123.8 +/- 16.7 Omega cm2, which resulted in a 31.7% inhibition of amiloride-sensitive I(sc) (n = 12). Similarly, luminal UTP reversibly reduced V(te) from -22.0 +/- 2.1 to -13.6 +/- 2.1 mV and increased R(te) from 48.4 +/- 5.3 to 59.2 +/- 7.1 Omega cm2, which resulted in 49.1% inhibition of Na+ absorption (n = 6). In parallel, luminal ATP and UTP elevated [Ca2+]i in CCD, increasing the fura-2 ratio by 2.7 +/- 0.7 and 4.0 +/- 1.2, respectively. Basolateral ATP and UTP (100 microM) also inhibited amiloride-sensitive I(sc) by 21.8 (n = 14) and 20.1% (n = 8), respectively. Inhibition of luminal nucleotide-induced [Ca2+]i increase by Ca2+ store depletion with cyclopiazonic acid (3 microM) did not affect nucleotide-mediated inhibition of Na+ transport (n = 7). No evidence indicated the activation of a luminal Ca2+-activated Cl- conductance, a phenomenon previously shown in M-1 CCD cells (J Physiol 524: 77-99, 2000). In essence, these data indicate that luminal ATP and UTP, most likely via P2Y2 receptors, mediate inhibition of amiloride-sensitive I(sc) in perfused mouse CCD. This inhibition appears to occurs independently of an increase of cytosolic Ca2+.     

The P2Y13 receptor regulates extracellular ATP metabolism and the osteogenic response to mechanical loading

ATP release and subsequent activation of purinergic receptors has been suggested to be one of the key transduction pathways activated by mechanical stimulation of bone. The P2Y₁₃ receptor, recently found to be expressed by osteoblasts, has been suggested to provide a negative feedback pathway for ATP release in different cell types. Therefore, we hypothesized that the P2Y₁₃ receptor may contribute to the mediation of osteogenic responses to mechanical stimulation by regulating ATP metabolism by osteoblasts. To test this hypothesis, wild‐type (WT) and P2Y₁₃ receptor knockout (P2Y₁₃R^?/?) mice were subject to non‐invasive axial mechanical loading of the left tibiae to induce an osteogenic response. Micro‐computed tomography analysis showed mechanical loading induced an osteogenic response in both strains of mice in terms of increased total bone volume and cortical bone volume, with the P2Y₁₃R^?/? mice having a significantly greater response. The extent of the increased osteogenic response was defined by dynamic histomorphometry data showing dramatically increased bone formation and mineral apposition rates in P2Y₁₃R^?/? mice compared with controls. In vitro, primary P2Y₁₃R^?/? osteoblasts had an accumulation of mechanically induced extracellular ATP and reduced levels of hydrolysis. In addition, P2Y₁₃R^?/? osteoblasts also had a reduction in their maximal alkaline phosphatase (ALP) activity, one of the main ecto‐enzymes expressed by osteoblasts, which hydrolyzes extracellular ATP. In conclusion, deletion of the P2Y₁₃ receptor leads to an enhanced osteogenic response to mechanical loading in vivo, possibly because of the reduced extracellular ATP degradation by ALP. The augmented osteogenic response to mechanical stimulation, combined with suppressed bone remodeling activities and protection from OVX‐induced bone loss after P2Y₁₃ receptor depletion as previously described, suggests a potential role for P2Y₁₃ receptor antagonist‐based therapy, possibly in combination with mechanical loading, for the treatment of osteoporosis.     

The relationship between oxygenator exhaust P(CO2) and arterial P(CO2) during hypothermic cardiopulmonary bypass

During cardiopulmonary bypass the partial pressure of carbon dioxide in oxygenator arterial blood (P(a)CO2) can be estimated from the partial pressure of gas exhausting from the oxygenator (P(E)CO2). Our hypothesis is that P(E)CO2 may be used to estimate P(a)CO2 with limits of agreement within 7 mmHg above and below the bias. (This is the reported relationship between arterial and end-tidal carbon dioxide during positive pressure ventilation in supine patients.) During hypothermic (28-32 degrees C) cardiopulmonary bypass using a Terumo Capiox SX membrane oxygenator, 80 oxygenator arterial blood samples were collected from 32 patients during cooling, stable hypothermia, and rewarming as per our usual clinical care. The P(a)CO2 of oxygenator arterial blood at actual patient blood temperature was estimated by temperature correction of the oxygenator arterial blood sample measured in the laboratory at 37 degrees C. P(E)CO2 was measured by connecting a capnograph end-to-side to the oxygenator exhaust outlet. We used an alpha-stat approach to cardiopulmonary bypass management. The mean difference between P(E)CO2 and P(a)CO2 was 0.6 mmHg, with limits of agreement (+/-2 SD) between -5 to +6 mmHg. P(E)CO2 tended to underestimate P(a)CO2 at low arterial temperatures, and overestimate at high arterial temperatures. We have demonstrated that P(E)CO2 can be used to estimate P(a)CO2 during hypothermic cardiopulmonary bypass using a Terumo Capiox SX oxygenator with a degree of accuracy similar to that associated with the use of end-tidal carbon dioxide measurement during positive pressure ventilation in anaesthetized, supine patients.     

Clopidogrel (Plavix), a P2Y12 receptor antagonist,inhibits bone cell function in vitro and decreases trabecular bone in vivo

Clopidogrel (Plavix), a selective P2Y₁₂ receptor antagonist, is widely prescribed to reduce the risk of heart attack and stroke and acts via the inhibition of platelet aggregation. Accumulating evidence now suggests that extracellular nucleotides, signaling through P2 receptors, play a significant role in bone, modulating both osteoblast and osteoclast function. In this study, we investigated the effects of clopidogrel treatment on (1) bone cell formation, differentiation, and activity in vitro; and (2) trabecular and cortical bone parameters in vivo. P2Y₁₂ receptor expression by osteoblasts and osteoclasts was confirmed using qPCR and Western blotting. Clopidogrel at 10 µM and 25 µM inhibited mineralized bone nodule formation by 50% and >85%, respectively. Clopidogrel slowed osteoblast proliferation with dose‐dependent decreases in cell number (25% to 40%) evident in differentiating osteoblasts (day 7). A single dose of 10 to 25 µM clopidogrel to mature osteoblasts also reduced cell viability. At 14 days, ≥10 µM clopidogrel decreased alkaline phosphatase (ALP) activity by ≤70% and collagen formation by 40%, while increasing adipocyte formation. In osteoclasts, ≥1 µM clopidogrel inhibited formation, viability and resorptive activity. Twenty‐week‐old mice (n = 10–12) were ovariectomized or sham treated and dosed orally with clopidogrel (1 mg/kg) or vehicle (NaCl) daily for 4 weeks. Dual‐energy X‐ray absorptiometry (DXA) analysis showed clopidogrel‐treated animals had decreases of 2% and 4% in whole‐body and femoral bone mineral density (BMD), respectively. Detailed analysis of trabecular and cortical bone using micro–computed tomography (microCT) showed decreased trabecular bone volume in the tibia (24%) and femur (18%) of clopidogrel‐treated mice. Trabecular number was reduced 20%, while trabecular separation was increased up to 15%. Trabecular thickness and cortical bone parameters were unaffected. Combined, these findings indicate that long‐term exposure of bone cells to clopidogrel in vivo could negatively impact bone health. © 2012 American Society for Bone and Mineral Research.