Uridine adenosine tetraphosphate-induced contraction is increased in renal but not pulmonary arteries from DOCA-salt hypertensive rats

Abstract

Uridine adenosine tetraphosphate (Up(4)A) was reported as a novel endothelium-derived contracting factor. Up(4)A contains both purine and pyrimidine moieties, which activate purinergic (P2)X and P2Y receptors. However, alterations in the vasoconstrictor responses to Up(4)A in hypertensive states remain unclear. The present study examined the effects of Up(4)A on contraction of isolated renal arteries (RA) and pulmonary arteries (PA) from DOCA-salt rats using isometric tension recording. RA from DOCA-salt rats exhibited increased contraction to Up(4)A versus arteries from control uninephrectomized rats in the absence and presence of N(G)-nitro-l-arginine (nitric oxide synthase inhibitor). On the other hand, the Up(4)A-induced contraction in PA was similar between the two groups. Up(4)A-induced contraction was inhibited by suramin (nonselective P2 antagonist) but not by diinosine pentaphosphate pentasodium salt hydrate (Ip(5)I; P2X(1) antagonist) in RA from both groups. Furthermore, 2-thiouridine 5'-triphosphate tetrasodium salt (2-ThioUTP; P2Y(2) agonist)-, uridine-5'-(γ-thio)-triphosphate trisodium salt (UTPγS; P2Y(2)/P2Y(4) agonist)-, and 5-iodouridine-5'-O-diphosphate trisodium salt (MRS 2693; P2Y(6) agonist)-induced contractions were all increased in RA from DOCA-salt rats. Protein expression of P2Y(2)-, P2Y(4)-, and P2Y(6) receptors in RA was similar between the two groups. In DOCA-salt RA, the enhanced Up(4)A-induced contraction was reduced by PD98059, an ERK pathway inhibitor, and Up(4)A-stimulated ERK activation was increased. These data are the first to indicate that Up(4)A-induced contraction is enhanced in RA from DOCA-salt rats. Enhanced P2Y receptor signaling and activation of the ERK pathway together represent a likely mechanism mediating the enhanced Up(4)A-induced contraction. Up(4)A might be of relevance in the pathophysiology of vascular tone regulation and renal dysfunction in arterial hypertension.

Fig. 1.

Contractile responses to uridine adenosine tetraphosphate (Up₄A) and phenylephrine (PE) are augmented in renal arteries (RA) but not pulmonary arteries (PA)from DOCA-salt hypertensive rats. Concentration-response curves for Up₄A (A, B) and PE (C, D) in RA (A, C) and PA (B, D) arterial rings obtained from DOCA-salt (DOCA) and normotensive control [uninephrectomized (Uni)] rats are shown. Data are means ± SE from 6 to 8 experiments; the SE is included only when it exceeds the dimension of the symbol used. *P < 0.05, DOCA vs. Uni.

Fig. 2.

Contractile responses to Up₄A in the presence of nitric oxide synthase (NOS) inhibitor are augmented in RA but not PA from DOCA-salt hypertensive rats. Concentration-response curves for Up₄A in isolated rings of RA (A) or PA (B) in the presence of 10⁻⁴ M N^G-nitro-l-arginine (l-NNA) are shown. C, D: contractile response to Up₄A in the presence of NOS inhibitor is suppressed by purinergic (P2)Y antagonist but not by P2X antagonist in RA. Effects of 10⁻⁴ M suramin (C) or 10⁻⁴ M diinosine pentaphosphate pentasodium salt hydrate (Ip₅I; D) on concentration-response curves for Up₄A in RA obtained from DOCA-salt and Uni rats in the presence of 10⁻⁴ M l-NNA. Data are means ± SE from 3 to 8 experiments; the SE is included only when it exceeds the dimension of the symbol used. The concentration-response curves for Up₄A depicted in A are replotted in C and D. *P < 0.05 vs. Uni l-NNA group; #P < 0.05 DOCA l-NNA vs. DOCA l-NNA suramin group; †P < 0.05, Uni l-NNA suramin vs. DOCA l-NNA suramin group.

Fig. 3.

Contractile responses to P2Y agonists in the presence of NOS inhibitor are augmented in RA from DOCA-salt hypertensive rats. Concentration-response curves for selective P2Y receptor agonist [2-thiouridine 5′-triphosphate tetrasodium salt (2-ThioUTP; P2Y₂ agonist; A), uridine-5′-(γ-thio)-triphosphate trisodium salt (UTPγS; P2Y₂/P2Y₄ agonist; B), and 5-iodouridine-5′-O-diphosphate trisodium salt (MRS 2693; P2Y₆ agonist; C)] in isolated rings of RA obtained from DOCA-salt and Uni rats in the presence of 10⁻⁴ M l-NNA are shown. Data are means ± SE from 4 or 5 experiments; the SE is included only when it exceeds the dimension of the symbol used. *P < 0.05, DOCA vs. Uni.

Fig. 4.

The receptor expressions of P2Y₂, P2Y₄, and P2Y₆ are similar between RA from DOCA-salt and Uni rats. Analysis of P2Y₂ (A), P2Y₄ (B), and P2Y₆ (C) protein expression in RA obtained from DOCA-salt and Uni rats is shown. Left: representative Western blots for P2Y₂, P2Y₄, P2Y₆, and β-actin. Proteins were subjected to 10% SDS-PAGE and then transferred to nitrocellulose membranes. They were then incubated with a primary antibody specific for P2Y₂ (42 kDa), P2Y₄ (78 kDa), P2Y₆ (100 kDa), or β-actin (42 kDa) and also with a secondary anti-rabbit or anti-mouse antibody. Right: bands were quantified as described in materials and methods. Ratios were calculated for the optical density of each receptor over that of β-actin. Data are means ± SE from 4 experiments.

Fig. 5.

Contractile response to Up₄A in the presence of NOS inhibitor is suppressed by ERK inhibition in RA. Effects of 10⁻⁵ M PD98059 on concentration-response curves for Up₄A in RA obtained from DOCA-salt and Uni rats in the presence of 10⁻⁴ M l-NNA are shown. Data are means ± SE from 5 experiments; the SE is included only when it exceeds the dimension of the symbol used. *P < 0.05 vs. Uni l-NNA group; #P < 0.05 DOCA l-NNA vs. DOCA l-NNA PD98059 group; †P < 0.05, Uni l-NNA PD98059 vs. DOCA l-NNA PD98059 group.

Fig. 6.

ERK1/2 phosphorylation is increased in Up₄A-treated RA from DOCA-salt hypertensive rats. A: representative Western blots for phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, and β-actin. B and C: bands were quantified as described in materials and methods. Ratios were calculated for the optical density of p-ERK1/2 over that of the corresponding total ERK1/2 (B) or for the optical density of total ERK1/2 over that of the corresponding β-actin (C). Data are means ± SE from 5 to 8 experiments. *P < 0.05 vs. Uni; #P < 0.05 vs. DOCA.