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Properties of P2X and P2Y receptors are dependent on artery diameter in the rat mesenteric bed.

ABSTRACT: P2 receptor mediated contractile responses have been characterized in different diameter arteries from the rat mesenteric arterial vasculature (first, second to third and fifth to sixth order for large, medium and small arteries) using wire myograph and diamtrak video imaging. alpha,ss-methylene ATP (alpha,beta-meATP) evoked transient concentration-dependent contractions in mesenteric arteries with EC(50) values of 0.4, 2.5 and 107 microM for small, medium and large arteries respectively. Suramin (10 - 100 microM) produced substantial parallel rightward shifts of the concentration-response curve to alpha,beta-meATP in small and medium-sized arteries with pA(2) of 5.1. Responses in large vessels were unaffected by suramin. Immunohistochemical analysis of arterial sections revealed no substantial differences in expression patterns of P2X receptors between different sizes of artery. P2X(1) receptors were expressed at high levels, P2X(4) and P2X(5) receptors were also detected on smooth muscle. The P2X receptor response is dominated by P2X(1) receptor in small and medium arteries but the nature of the receptor mediating the suramin insensitive alpha,beta-meATP mediated response in large arteries is unclear. The P2Y receptor agonist UTP was significantly more potent in small than in medium or large arteries (EC(50) values: 15.0 microM small, 88.5 microM diamtrak medium 1.6 mM myography medium and 1.4 mM large). Responses in both small and medium-sized vessels were reduced by suramin (30 - 100 microM). The sensitivity to UTP and suramin indicates the presence of P2Y(2) receptors. This study shows that P2 receptors do not have a homogenous phenotype throughout the mesenteric vascular bed and that the properties depend on artery size.

SUBMITTER: Gitterman DP

PROVIDER: S-EPMC1572518 | biostudies-other | 2000 Dec

REPOSITORIES: biostudies-other

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Project description:RationaleThe role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission.Experimental approachLSCM-Fluo-4/(Ca(2+))-imaging of postsynaptic Ca(2+) transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs).ResultsSynaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPβS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,β-MeATP) agonist evokes fast, slow, biphasic Ca(2+) transients or Ca(2+) oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 >> P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7, P2Y1,2,12-14R.ConclusionsPurines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are potential novel therapeutic targets for neurogastroenterology.

Project description:(1) Vasorelaxation and hyperpolarization of endothelial cells by adenosine 5'-[beta-thio]diphosphate (ADPbetaS) and adenosine 5'-[gamma-thio]triphosphate (ATPgammaS) were studied in rat-isolated mesenteric artery. Effects from stimulation of P2X receptors were avoided by desensitization with alpha,beta-methylene adenosine triphosphate. (2) ADPbetaS caused concentration- and endothelium-dependent relaxations of methoxamine-precontracted small (third generation) and main mesenteric artery. These were inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME) or a combination of apamin plus charybdotoxin (inhibitors of Ca(2+)-activated K(+) channels); L-NAME, apamin and charybdotoxin applied together abolished the response. (3) ATPgammaS induced limited relaxation (35% of methoxamine-induced tone at 10 micro M) of small mesenteric artery, which was sensitive to L-NAME or endothelium denudation. However, it almost completely relaxed the main mesenteric artery over an extended concentration range (>6 orders of magnitude) in an endothelium-dependent manner. This relaxation was inhibited by either L-NAME or a combination of apamin with charybdotoxin, and abolished by a combination of all the three inhibitors. (4) The P2Y(1) receptor antagonist MRS 2179 (2'-deoxy-N(6)-methyladenosine 3',5'-bisphosphate; 0.3-3 micro M) caused parallel rightward shifts of the concentration/relaxation curve to ADPbetaS (pA(2)=7.1). However, MRS 2179 did not inhibit, but potentiated, relaxant responses to ATPgammaS. MRS 2179 did not affect the contractile responses ATPgammaS in small mesenteric artery; ATPgammaS did not contract the main mesenteric artery. (5) ADPbetaS hyperpolarized the endothelium of the main mesenteric artery in a concentration-dependent manner. This was unaffected by L-NAME but antagonized by MRS 2179. ATPgammaS also hyperpolarized the mesenteric artery endothelium in a concentration-dependent manner but, when ATPgammaS was applied at 10 micro M, its effect was potentiated by MRS 2179 (3 micro M). (6) It is concluded that both relaxation and hyperpolarization to ADPbetaS are mediated by P2Y(1) receptors and that the endothelial hyperpolarization is related to the L-NAME-resistant relaxation. Relaxation to the P2Y(2) agonist ATPgammaS shows regional variation along the mesenteric vasculature. The mechanisms for potentiation of relaxation and hyperpolarization by ATPgammaS are unknown, but may indicate interactions between P2Y receptor subtypes.

Project description:Gastric cancer (GC) is the one of the most prevalent cancers and one of the leading causes of cancer-induced deaths. Previously, we found that the expression of purinergic P2Y2 receptor (P2Y2R) is increased in GC samples as compared to adjacent healthy mucosa taken from GC-diagnosed patients. In this work, we studied in detail purinergic signaling in the gastric adenocarcinoma-derived cell lines: AGS, MKN-45, and MKN-74, and compared them to a nontumoral epithelial cell line: GES-1. In GC-derived cells, we detected the expression of several purinergic receptors, and found important differences as compared to GES-1 cells. Functional studies revealed a strong contribution of P2Y2Rs in intracellular calcium increases, elicited by adenosine-triphosphate (ATP), uridine-triphosphate (UTP), and the P2Y2R agonist MRS2768. Responses were preserved in the absence of extracellular calcium and inhibited by P2Y2R antagonists. In GES-1 cells, ATP and UTP induced similar responses and the combination of P2X and P2Y receptor antagonists was able to block them. Proliferation studies showed that ATP regulates AGS and MKN-74 cells in a biphasic manner, increasing cell proliferation at 10-100 μM, but inhibiting at 300 μM ATP. On the other hand, 1-300 μM UTP, a P2Y2R agonist, increased concentration-dependent cell proliferation. The effects of UTP and ATP were prevented by both wide-range and specific purinergic antagonists. In contrast, in GES-1 cells ATP only decreased cell proliferation in a concentration-dependent manner, and UTP had no effect. Notably, the isolated application of purinergic antagonists was sufficient to change the basal proliferation of AGS cells, indicating that nucleotides released by the cells can act as paracrine/autocrine signals. Finally, in tumor-derived biopsies, we found an increase of P2Y2R and a decrease in P2X4R expression; however, we found high variability between seven different biopsies and their respective adjacent healthy gastric mucosa. Even so, we found a correlation between the expression levels of P2Y2R and P2X4R and survival rates of GC patients. Taken together, these results demonstrate the involvement of different purinergic receptors and signaling in GC, and the pattern of expression changes in tumoral cells, and this change likely directs ATP and nucleotide signaling from antiproliferative effects in healthy tissues to proliferative effects in cancer.

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Properties of P2X and P2Y receptors are dependent on artery diameter in the rat mesenteric bed.

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