Pharmacological characterization of nucleotide P2Y receptors on endothelial cells of the mouse aorta

Abstract

Nucleotides regulate various effects including vascular tone. This study was aimed to characterize P2Y receptors on endothelial cells of the aorta of C57BL6 mice. Five adjacent segments (width 2 mm) of the thoracic aorta were mounted in organ baths to measure isometric force development. Nucleotides evoked complete (adenosine 5' triphosphate (ATP), uridine 5' triphosphate (UTP), uridine 5' diphosphate (UDP); >90%) or partial (adenosine 5' diphosphate (ADP)) relaxation of phenylephrine precontracted thoracic aortic rings of C57BL6 mice. Relaxation was abolished by removal of the endothelium and was strongly suppressed (>90%) by inhibitors of nitric oxide synthesis. The rank order of potency was: UDP approximately UTP approximately ADP>adenosine 5'-[gamma-thio] triphosphate (ATPgammaS)>ATP, with respective pD2 values of 6.31, 6.24, 6.22, 5.82 and 5.40. These results are compatible with the presence of P2Y1 (ADP>ATP), P2Y2 or P2Y4 (ATP and UTP) and P2Y6 (UDP) receptors. P2Y4 receptors were not involved, since P2Y4-deficient mice displayed unaltered responses to ATP and UTP. The purinergic receptor antagonist suramin exerted surmountable antagonism for all agonists. Its apparent pKb for ATP (4.53+/-0.07) was compatible with literature, but the pKb for UTP (5.19+/-0.03) was significantly higher. This discrepancy suggests that UTP activates supplementary non-P2Y2 receptor subtype(s). Further, pyridoxal-phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS) showed surmountable (UTP, UDP), nonsurmountable (ADP) or no antagonism (ATP). Finally, 2'-deoxy-N6-methyladenosine3',5'-bisphosphate (MRS2179) inhibited ADP-evoked relaxation only. Taken together, these results point to the presence of functional P2Y1 (ADP), P2Y2 (ATP, UTP) and P2Y6 (UDP) receptors on murine aorta endothelial cells. The identity of the receptor(s) mediating the action of UTP is not fully clear and other P2Y subtypes might be involved in UTP-evoked vasodilatation.

Figure 1

Representative tracings of concentration–response curves of purinergic agonists. ATP induced relaxation in phenylephrine precontracted blood vessels with endothelium (a), but not in endothelium-denuded rings (b). Staining with an antibody against CD31 confirmed the effectiveness of the endothelial denudation. Micrographs of a vessel with endothelium (c) and an endothelial-denuded vessel (d).

Figure 2

Cumulative concentration–response curves of phenylephrine precontracted thoracic aorta segments. (a) ATP evoked complete relaxation, whereas ADP evoked partial relaxation of the thoracic aorta. (b) UTP and UDP both evoked potent and complete relaxation. (c) ATPγS was more potent than ATP, while adenosine caused a small relaxation. Mean±s.e.m.; n=5.

Figure 3

Cumulative concentration–response curves for (a) ATP, ADP and ATPγS. (b) UTP and UDP of phenylephrine precontracted thoracic aorta segments in the presence of the combination of the NOS inhibitors LNA (300 μM) and L-NAME (300 μM). Mean±s.e.m.; n=5.

Figure 4

Cumulative dose–response curves in the thoracic aorta segments of P2Y₄^−/0 and WT mice for (a) UTP and (b) ATP. Mean±s.e.m.; n=6.

Figure 5

Cumulative dose–response curves in segments of the thoracic aorta for (a) ATP and (b) ADP in the presence of MRS2179, a selective P2Y₁ receptor antagonist. Mean±s.e.m.; n=5.

Figure 6

Cumulative dose–response curves in segments of the thoracic aorta for (a) ATP, (b) ADP, (c) UTP and (d) UDP in the presence of suramin, a nonselective purinergic receptor antagonist. Curves displayed concentration-dependent rightward shifts in the presence of suramin. Mean±s.e.m.; n=5.

Figure 7

Cumulative dose–response curves in segments of the thoracic aorta for (a) ATP, (b) ADP, (c) UTP and (d) UDP in the presence of PPADS, a nonselective purinergic receptor antagonist. PPADS showed surmountable (UTP and UDP) or nonsurmountable (ADP) antagonism, while ATP-evoked vasodilatation was not affected. Mean±s.e.m.; n=5.