Identification of Contractile P2Y₁, P2Y₆, and P2Y₁₂Receptors in Rat Intrapulmonary Artery Using Selective Ligands

Abstract

ATP and UDP constrict rat intrapulmonary arteries, but which receptors mediate these actions is unclear. Here, we used selective agonists and antagonists, along with measurements of P2Y receptor expression, to characterize the receptor subtypes involved. Isometric tension was recorded from endotheliumdenuded rat intrapulmonary artery rings (i.d. 200 -500 m) mounted on a wire myograph. Expression of P2Y receptor subtype expression was determined by using reverse transcriptionpolymerase chain reaction with receptor-specific oligonucleotide primers. The selective P2Y 1 agonist (N)-methanocarba-2-methylthioadenosine-5Ј-O-diphosphate (MRS2365) induced small, concentration-dependent contractions that were inhibited by the P2Y 1 antagonist N 6 -methyl-2Ј-deoxyadenosine-3Ј,5Ј-bisphosphate (MRS2179). Contractions evoked by ATP were unaffected by MRS2179, but inhibited by approximately one-third by the P2Y 12 antagonist N 6 -(2-methylthiomethyl)-2-(3,3,3-trifluoropropylthio)dichloro-methylene ATP (AR-C69931MX). Combined blockade of P2X1 and P2Y 12 receptors virtually abolished the response to ATP. ADP also evoked contractions that were abolished by AR-C69931MX. The selective P2Y 6 receptor agonist 3-(2-oxo-2-phenylethyl)-UDP (PSB 0474) evoked concentrationdependent contractions and was approximately three times more potent than UDP, but the P2Y 14 agonist UDP-glucose had no effect. Contractions evoked by UDP were inhibited by the P2Y 6 receptor antagonist N,NЈ-1,4-butanediylbis-NЈ-(3-isothiocyanatophenyl)thiourea (MRS2578), but not the cysteinyl leukotriene 1 (CysL 1 ) antagonist 3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)((3-dimethylamino-3-oxopropyl)thio)methyl)thiopropanoic acid (MK571). Higher concentrations of MRS2578 inhibited contractions to KCl, so they were not studied further. mRNA for P2Y 1 , P2Y 6 , and P2Y 12 receptors was identified. Our working model is that P2Y 12 and P2X1 receptors are present in rat intrapulmonary arteries and together mediate ATP-induced vasoconstriction. Contractile P2Y 6 , but not P2Y 14 or CysLT 1 , receptors are also present and are a major site through which UDP evokes constriction.

Extracellular Adenosine Diphosphate Ribose Mobilizes Intracellular Ca2+ via Purinergic-Dependent Ca2+ Pathways in Rat Pulmonary Artery Smooth Muscle Cells

“…The expression of multiple P2X and P2Y receptors in PASMCs have been reported previously [55][56][57], and is substantiated by the complete analysis of P2X and P2Y mRNA expression in the present study. Except the low levels of P2X6, P2Y6 and P2Y12 mRNA, transcripts of all P2X and P2Y subtypes were clearly expressed in PA smooth muscle.…”

“…Except the low levels of P2X6, P2Y6 and P2Y12 mRNA, transcripts of all P2X and P2Y subtypes were clearly expressed in PA smooth muscle. This is congruent with immunohistological detection of P2X1, P2X4 and P2X7 proteins [56,73,74], and pharmacological identification of P2X1, P2Y1, P2Y6, and P2Y12-dependent contraction in pulmonary arteries [57,75]. The large repertoire of P2X and P2Y receptors in PASMCs suggest that ADPR and ATP may utilize different combinations of purinergic receptors to mediate their specific physiological functions in pulmonary vasculature.…”

“…Many P2 receptor subtypes are expressed in a variety of cells, including PASMCs [55][56][57]. To explore the contribution of P2 receptors in the signaling for extracellular ADPR, PASMCs were treated with the non-selective P2 receptor antagonist suramin, the P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS) and the specific P2Y1 receptor antagonist MRS2179 for 20 minutes before application of ADPR.…”

Purinergic Signaling and Blood Vessels in Health and Disease

“…In endothelium-denuded rat pulmonary arteries, P2Y 2 receptors and a UDP-sensitive receptor (presumably P2Y 6 ) were identified on myocytes (Hartley et al, 1998). In a recent article (Mitchell et al, 2012), P2Y 1 , P2Y 6 , and P2Y 12 receptors were reported to mediate contraction of rat intrapulmonary arteries. An ATP-induced increase in [Ca 2+ ] i in rat pulmonary artery myocytes was mediated by P2X and P2U (P2Y 2 and/or P2Y 4 ) receptors (Guibert et al, 1996;Hartley and Kozlowski, 1997).…”

UDP-induced relaxation is enhanced in aorta from female obese Otsuka Long–Evans Tokushima Fatty rats

“…Based on molecular structure and signaling pathways, so far, the P2 purinergic receptors are subdivided into ionotropic receptors P2X (P2X [1][2][3][4][5][6][7] ) and metabotropic Gprotein-coupled receptors P2Y (P2Y 1,2,4,6,[11][12][13][14] [1,2]. Among purinoceptors, P2Y 6 receptor, a receptor for UDP, plays a pivotal role in various responses, including inflammatory and immune responses and cardiovascular physiology [1][2][3][4][5][6].…”