Dept. of Pediatrics, University of Colorado Denver, Research 2, Box B131, Aurora, CO 80045, USA.
Am J Physiol Cell Physiol. 2011 Feb;300(2):C266-75. doi: 10.1152/ajpcell.00237.2010. Epub 2010 Oct 20.
Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca(2+) concentration (Ca(2+)) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca(2+) signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of Ca(2+) via Ca(2+) influx through plasma membrane channels as well as Ca(2+) mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca(2+) responses in both cytosolic and nuclear compartments. An increase in Ca(2+) was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPβS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αβMeATP, and βγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca(2+) responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion.
细胞外 ATP 和 ADP 已被证明对肺动脉 adventitial vasa vasorum 内皮细胞 (VVEC) 具有强烈的血管生成作用。然而，细胞外核苷酸介导的血管生成的分子信号机制仍未完全阐明。由于细胞内 Ca(2+)浓度 (Ca(2+)) 的升高是细胞增殖所必需的，并且发生在对细胞外核苷酸的反应中，因此本研究旨在阐明参与 VVEC 中嘌呤能受体亚型的 Ca(2+)信号转导和细胞外核苷酸介导的有丝分裂反应。我们的数据表明，用细胞外 ATP 刺激 VVEC 会导致通过质膜通道的 Ca(2+)内流以及从细胞内储存中动员 Ca(2+)，从而升高 Ca(2+)。此外，细胞外 ATP 诱导细胞质和核区室中的同时 Ca(2+)反应。观察到对各种嘌呤能受体激动剂（包括 ATP、ADP、ATPγS、ADPβS、UTP、UDP、2-甲硫基-ATP (MeSATP)、2-甲硫基-ADP (MeSADP) 和 BzATP）的反应增加了 Ca(2+)，但对腺苷、AMP、二腺苷四磷酸、αβMeATP 和 βγMeATP 没有反应。使用 RT-PCR，我们在 VVEC 中鉴定了 P2Y1、P2Y2、P2Y4、P2Y13、P2Y14、P2X2、P2X5、P2X7、A1、A2b 和 A3 嘌呤能受体的 mRNA。用 P2Y1 选择性拮抗剂 MRS2179 和 P2Y13 选择性拮抗剂 MRS2211 预孵育 VVEC，以及用百日咳毒素孵育，可在不同程度上减弱激动剂诱导的细胞内 Ca(2+)反应和 ERK1/2、Akt 和 S6 核糖体蛋白的激活，表明 P2Y1 和 P2Y13 受体在 VVEC 生长反应中起主要作用。考虑到嘌呤能信号在调节血管生成和血管稳态中的广泛生理意义，我们的发现表明 P2Y1 和 P2Y13 受体可能是涉及 vasa vasorum 扩张的病理性血管重塑的新型和特异性治疗靶点。
