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P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells.

ABSTRACT: 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+)](i)) 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+)](i) 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+)](i) was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATP?S, ADP?S, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, ??MeATP, and ??MeATP. 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.

SUBMITTER: Lyubchenko T

PROVIDER: S-EPMC3043637 | biostudies-other | 2011 Feb

REPOSITORIES: biostudies-other

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P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells.

Lyubchenko Taras T Woodward Heather H Veo Kristopher D KD Burns Nana N Nijmeh Hala H Liubchenko Ganna A GA Stenmark Kurt R KR Gerasimovskaya Evgenia V EV

American journal of physiology. Cell physiology 20101020 2

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+)](i)) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes ...[more]

PMID: 20962269

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Project description:Expansion of the vasa vasorum network has been observed in a variety of systemic and pulmonary vascular diseases. We recently reported that a marked expansion of the vasa vasorum network occurs in the pulmonary artery adventitia of chronically hypoxic calves. Since hypoxia has been shown to stimulate ATP release from both vascular resident as well as circulatory blood cells, these studies were undertaken to determine if extracellular ATP exerts angiogenic effects on isolated vasa vasorum endothelial cells (VVEC) and/or if it augments the effects of other angiogenic factors (VEGF and basic FGF) known to be present in the hypoxic microenvironment. We found that extracellular ATP dramatically increases DNA synthesis, migration, and rearrangement into tube-like networks on Matrigel in VVEC, but not in pulmonary artery (MPAEC) or aortic (AOEC) endothelial cells obtained from the same animals. Extracellular ATP potentiated the effects of both VEGF and bFGF to stimulate DNA synthesis in VVEC but not in MPAEC and AOEC. Analysis of purine and pyrimidine nucleotides revealed that ATP, ADP and MeSADP were the most potent in stimulating mitogenic responses in VVEC, indicating the involvement of the family of P2Y1-like purinergic receptors. Using pharmacological inhibitors, Western blot analysis, and Phosphatidylinositol-3 kinase (PI3K) in vitro kinase assays, we found that PI3K/Akt/mTOR and ERK1/2 play a critical role in mediating the extracellular ATP-induced mitogenic and migratory responses in VVEC. However, PI3K/Akt and mTOR/p70S6K do not significantly contribute to extracellular ATP-induced tube formation on Matrigel. Our studies indicate that VVEC, isolated from the sites of active angiogenesis, exhibit distinct functional responses to ATP, compared to endothelial cells derived from large pulmonary or systemic vessels. Collectively, our data support the idea that extracellular ATP participates in the expansion of the vasa vasorum that can be observed in hypoxic conditions.

Project description:Thrombin is a procoagulant inflammatory agonist that can disrupt the endothelium-lumen barrier in the lung by causing contraction of endothelial cells and promote pulmonary cell proliferation. Both contraction and proliferation require increases in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this study, we compared the effect of thrombin on Ca(2+) signaling in human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells. Thrombin increased the [Ca(2+)](cyt) in both cell types; however, the transient response was significantly higher and recovered quicker in the PASMC, suggesting different mechanisms may contribute to thrombin-mediated increases in [Ca(2+)](cyt) in these cell types. Depletion of intracellular stores with cyclopiazonic acid (CPA) in the absence of extracellular Ca(2+) induced calcium transients representative of those observed in response to thrombin in both cell types. Interestingly, CPA pretreatment significantly attenuated thrombin-induced Ca(2+) release in PASMC; this attenuation was not apparent in PAEC, indicating that a PAEC-specific mechanism was targeted by thrombin. Treatment with a combination of CPA, caffeine, and ryanodine also failed to abolish the thrombin-induced Ca(2+) transient in PAEC. Notably, thrombin-induced receptor-mediated calcium influx was still observed in PASMC after CPA pretreatment in the presence of extracellular Ca(2+). Ca(2+) oscillations were triggered by thrombin in PASMC resulting from a balance of extracellular Ca(2+) influx and Ca(2+) reuptake by the sarcoplasmic reticulum. The data show that thrombin induces increases in intracellular calcium in PASMC and PAEC with a distinct CPA-, caffeine-, and ryanodine-insensitive release existing only in PAEC. Furthermore, a dynamic balance between Ca(2+) influx, intracellular Ca(2+) release, and reuptake underlie the Ca(2+) transients evoked by thrombin in some PASMC. Understanding of such mechanisms will provide an important insight into thrombin-mediated vascular injury during hypertension.

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P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells.

Publications

P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells.

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