P2Y(1) and P2Y(2) receptors are coupled to the NO/cGMP pathway to vasodilate the rat arterial mesenteric bed

Abstract

1. To assess the role of nucleotide receptors in endothelial-smooth muscle signalling, changes in perfusion pressure of the rat arterial mesenteric bed, the luminal output of nitric oxide (NO) and guanosine 3',5' cyclic monophosphate (cGMP) accumulation were measured after the perfusion of nucleotides. 2. The rank order of potency of ATP and analogues in causing relaxation of precontracted mesenteries was: 2-MeSADP=2-MeSATP>ADP>ATP=UDP=UTP>adenosine. The vasodilatation was coupled to a concentration-dependent rise in NO and cGMP production. MRS 2179 selectively blocked the 2-MeSATP-induced vasodilatation, the NO surge and the cGMP accumulation, but not the UTP or ATP vasorelaxation. 3. mRNA encoding for P2Y(1), P2Y(2) and P2Y(6) receptors, but not the P2Y(4) receptor, was detected in intact mesenteries by RT-PCR. After endothelium removal, only P2Y(6) mRNA was found. 4. Endothelium removal or blockade of NO synthase obliterated the nucleotides-induced dilatation, the NO rise and cGMP accumulation. Furthermore, 2-MeSATP, ATP, UTP and UDP contracted endothelium-denuded mesenteries, revealing additional muscular P2Y and P2X receptors. 5. Blockade of soluble guanylyl cyclase reduced the 2-MeSATP and UTP-induced vasodilatation and the accumulation of cGMP without interfering with NO production. 6. Blockade of phosphodiesterases with IBMX increased 15-20 fold the 2-MeSATP and UTP-induced rise in cGMP; sildenafil only doubled the cGMP accumulation. A linear correlation between the rise in NO and cGMP was found. 7. Endothelial P2Y(1) and P2Y(2) receptors coupled to the NO/cGMP cascade suggest that extracellular nucleotides are involved in endothelial-smooth muscle signalling. Additional muscular P2Y and P2X receptors highlight the physiology of nucleotides in vascular regulation.

Figure 1

The 2-MeSATP and UTP-induced vasodilatations are endothelium dependent, but only the 2-MeSATP relaxation is antagonised by MRS 2179. Representative recordings show that 30 nM 2-MeSATP or 1 μM UTP induced sustained relaxations that are endothelium dependent (A and B); after removal of the endothelium E (−), these nucleotides consistently vasoconstricted this territory. While the 2-MeSATP vasodilatation was antagonized by 100 nM MRS 2179 (C), the 1 μM UTP-induced relaxation was not blocked by this drug (D). The recordings shown in A–D are derived from separate mesenteries; in each case, the nucleotide was assessed before and after endothelium removal, or perfusion with MRS 2179. Closed dots denote mesentery pre-contraction with 10 μM noradrenaline, while the open dots indicate a precontraction with 1 μM noradrenaline in endothelium-denuded preparations. (E) shows 2-MeSATP and UTP concentration-response vasorelaxations with and without endothelium (n=3–6 per agonist) or perfusion with 100 nM MRS 2179 (n=3–8, applied a min before and during perfusion with the nucleotide). Symbols represent mean values; bars, the s.e.mean.

Figure 2

Identification of P2Y₁, P2Y₂ and P2Y₆ receptors mRNA by RT–PCR. Gels show PCR products corresponding to P2Y₁, P2Y₂ or P2Y₆ receptors based on their estimated molecular weight (MW, bp). When total tissue mRNA is extracted from mesenteries lacking the endothelial cell layer (E−), no PCR products for these receptors were observed, except for the P2Y₆ receptor subtype. Upon endothelium denudation, the PCR products for CD31, an endothelial cell marker, were not evidenced. In contrast, the smooth muscle PCR product for myosin alkali light chain (MALC) is observed with (E+) and without endothelium (E−). Identical results were attained in duplicate protocols.

Figure 3

2-MeSATP and UTP concentration-dependent vasodilatations and their corresponding productions of NO and cGMP. Concentration-dependent rise in the luminally accessible NO production (upper panels); increase in tissue cGMP production (middle panels) and vasodilatations (lower panels). Symbols represent the mean values; bars, the s.e.mean (n=3–16 per concentration of nucleotides).

Figure 4

Correlation between tissue production of cGMP and the luminally accessible NO induced by several concentrations of 2-MeSATP and UTP. Closed circles represent NO and cGMP determinations in mesenteries perfused with varying concentrations of these nucleotides in a drug-free buffer. The correlation coefficent was 0.74 (P<0.01, n=49); the slope of this curve is 0.072. To prevent cGMP degradation, we next assessed NO and cGMP determinations in mesenteries perfused with 500 μM IBMX (open circles). The correlation coefficient (r) between NO and cGMP production was 0.77 (P<0.01, n=14); the slope 2.4. Each symbol represents a different mesentery, in which both NO and cGMP were determined following a single nucleotide application.

Figure 5

MRS 2179 blocks the 2-MeSATP-induced production of NO and cGMP, without altering the same responses elicited by UTP. Rise of the luminal accessible NO released (A) and the tissue production of cGMP (B) evoked by 0.3 μM 2-MeSATP or 1 μM UTP (n=3–6) before and after application of 30 nM MRS 2179. The same mesentery was used to determine the luminal outflow of NO and the corresponding tissue content of cGMP; separate mesenteries were used to examine the effect of the nucleotides in the absence and in the presence of 30 nM MRS 2179. Columns refer to the mean values; bars, to the s.e.mean. ^**P<0.01.

Figure 6

Endothelium removal, blockade of NOS or soluble guanylyl cyclase obliterate the 2-MeSATP or UTP-induced vasodilatation and the surge in luminal NO and tissue cGMP production. Separate mesenteries were perfused with either 1 μM 2-MeSATP or 10 μM UTP. A single mesentery was used to measure luminal accessible NO and its corresponding increase in cGMP production in three separate experimental conditions: removal of the endothelial cell layer (E(−) n=4); blockade of NOS with L-nitro arginine (L-NNA n=4); inhibition of soluble guanylyl cyclase (ODQ n=4). Closed columns represent the effect of the nucleotides in drug-free buffer (controls, n=12). Columns indicate mean values; bars, to the s.e.mean. ^*P<0.05; ^**P<0.01.

Figure 7

Schematic model shows nucleotide receptors in the endothelium and the smooth muscle of the rat arterial mesenteric bed. Locally released nucleotides (ATP, ADP or UTP) act on endothelial P2Y₁ and P2Y₂ receptors promoting a concentration-dependent surge of NO triggered by the release of intracellular stored calcium activating nitric oxide synthase (NOS). The tridimensional spread of the gas diffuses to the adjacent smooth muscle where it activates soluble guanylyl cyclase (sGC) accumulating cGMP. The cyclic nucleotide ensues the relaxation of the vascular smooth muscles via cGMP-dependent kinases. The P2Y₆ receptor has also been identified both in the endothelial layer as in the smooth muscle. At present it is not clear whether the latter receptor mediates the contractions ensued by pyrimidines nucleotides. The model also depicts the presence of muscular P2X receptors. Adenosine receptors (Ado) dilate the rat arterial mesenteric bed, via a NO/cGMP independent muscular pathway.