Impaired Aortic Contractility to Uridine Adenosine Tetraphosphate in Angiotensin II-Induced Hypertensive Mice: Receptor Desensitization?

OBJECTIVE: We previously showed that uridine adenosine tetraphosphate (Up4A)-mediated aortic contraction is partly mediated through purinergic P2X1 receptors (P2X1R). It has been reported that the plasma level of Up4A is elevated in hypertensive patients, implying a potential role for Up4A-P2X1R signaling in hypertension. This study investigated the vasoactive effect of Up4A in aortas isolated from angiotensin (Ang) II-infused (21 days) hypertensive mice. METHODS: Blood pressure was measured by tail cuff plethysmography. Aortas were isolated for isometric tension measurements, and protein expression was analyzed by western blot. RESULTS: Mean and systolic arterial pressures were elevated by ~50% in Ang II-infused mice. Protein levels of both AT1R and P2X1R were upregulated in Ang II-infused aortas. Surprisingly, Up4A (10-9-10-5 M)-induced concentration-dependent contraction was significantly impaired in Ang II-infused mice. Studies in control mice revealed that both P2X1R (MRS2159) and AT1R (losartan) antagonists significantly attenuated Up4A-induced aortic contraction. In addition, desensitization of AT1R by prior Ang II (100 nM) exposure had no effect on Up4A-induced aortic contraction. However, subsequent serial exposure responses to Up4A-induced aortic contraction were markedly reduced, suggesting a desensitization of purinergic receptors. This desensitization was further confirmed in control mice by prior exposure of aortas to the P2X1R desensitizer α, ß-methylene ATP (10 µM). CONCLUSION: Despite upregulation of AT1R and P2X1R in hypertension, Up4A-mediated aortic contraction was impaired in Ang II-infused mice, likely through the desensitization of P2X1R but not AT1R. This implies that vascular P2X1R activity, rather than plasma Up4A level, may determine the role of Up4A in hypertension.

High salt diet exacerbates vascular contraction in the absence of adenosine A2A receptor.

High salt (4% NaCl, HS) diet modulates adenosine-induced vascular response through adenosine A(2A) receptor (A(2A)AR). Evidence suggests that A(2A)AR stimulates cyp450-epoxygenases, leading to epoxyeicosatrienoic acids (EETs) generation. The aim of this study was to understand the vascular reactivity to HS and underlying signaling mechanism in the presence or absence of A(2A)AR. Therefore, we hypothesized that HS enhances adenosine-induced relaxation through EETs in A(2A)AR⁺/⁺, but exaggerates contraction in A(2A)AR⁻/⁻. Organ bath and Western blot experiments were conducted in HS and normal salt (NS, 0.18% NaCl)-fed A(2A)AR⁺/⁺ and A(2A)AR⁻/⁻ mice aorta. HS produced concentration-dependent relaxation to non-selective adenosine analog, NECA in A(2A)AR⁺/⁺, whereas contraction was observed in A(2A)AR⁻/⁻ mice and this was attenuated by A1AR antagonist (DPCPX). CGS 21680 (selective A(2A)AR agonist) enhanced relaxation in HS-A(2A)AR⁺/⁺ versus NS-A(2A)AR⁺/⁺, which was blocked by EETs antagonist (14,15-EEZE). Compared with NS, HS significantly upregulated the expression of vasodilators A(2A)AR and cyp2c29, whereas vasoconstrictors A1AR and cyp4a in A(2A)AR⁺/⁺ were downregulated. In A(2A)AR⁻/⁻ mice, however, HS significantly downregulated the expression of cyp2c29, whereas A1AR and cyp4a were upregulated compared with A(2A)AR⁺/⁺ mice. Hence, our data suggest that in A(2A)AR⁺/⁺, HS enhances A(2A)AR-induced relaxation through increased cyp-expoxygenases-derived EETs and decreased A1AR levels, whereas in A(2A)AR⁻/⁻, HS exaggerates contraction through decreased cyp-epoxygenases and increased A1AR levels.

Modulation of A2A adenosine receptors and associated Galphas proteins by ZM 241385 treatment of porcine coronary artery.

Functional regulation and expression of the adenosine A2A receptor and associated G-protein were investigated in porcine coronary artery exposed to an A2A receptor antagonist, ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol). The arteries were incubated for 3 days in culture medium in the absence (control) and presence (treated) of 10 microM ZM 241385. Changes in isometric tension by adenosine receptor agonists were evaluated in endothelium-free tissues. ZM 241385-treatment produced a statistically significant rightward displacement of CGS-21680, NECA, and CAD concentration-response curves compared with the respective controls (P < 0.05). The EC50, expressed in nM, values in treated and control tissues were: 617.3 +/- 23 versus 24.9 +/- 1.5 for CGS-21680 (2-(p-(2-carboxyethyl)phenethylamino)-5'N-ethylcarboxamidoadenosine), 27.4 +/- 6.3 versus 3.06 +/- 0.8 for NECA (5'-N-ethylcarboxamidoadenosine), and 5786.2 +/- 160 versus 89.2 +/- 24.1 for CAD (chloroadenosine). However, the relaxing effect of forskolin remained unchanged in treated and control tissues. The concentration-response curves for NECA, CAD, and CGS-21680 were also displaced to the right when cAMP levels were measured in treated and control smooth muscle cells while no differences were observed with forskolin. Quantitative Western blot analysis demonstrated that the density of A2A receptors increased in ZM 241385-treated artery. We also showed a significant decrease in Galphas protein levels after ZM 241385 treatment compared with control. Taken together, these data indicate that prolonged blockade of A2A receptors in the coronary artery leads to desensitization of the functional effects of adenosine agonists by a mechanism that involves decreases in cAMP production. This was associated with an up-regulation of A2A receptors and a decrease in Galphas protein expression.

Role of endothelial adenosine receptor-mediated vasorelaxation in ethanol-induced hypotension in hypertensive rats.

Our previous findings showed that chronic ethanol feeding lowers blood pressure in spontaneously hypertensive rats. The present study investigated the role of the adenosine receptor-endothelial nitric oxide (NO) pathway in the hypotensive response to ethanol. Changes in blood pressure were evaluated in radiotelemetered pair-fed rats receiving liquid diet with or without ethanol (2.5% or 5%, w/v) for 12 weeks. The vasorelaxant activity of the adenosine analogue 5'-N-ethylcarboxamidoadenosine (NECA) in isolated aortic rings obtained from ethanol and control rats were evaluated. Ethanol (2.5% and 5%) lowered blood pressure in a dose-dependent manner. The hypotension started at week 1, reached its maximum at week 4 and remained so thereafter. In aortas with intact endothelium, NECA (10(-10) to 10(-4) M) produced a concentration-dependent relaxation of the phenylephrine-precontracted aortas. Compared with control rats, ethanol (2.5% and 5%) caused significant and concentration-related increases in NECA responses. This effect of ethanol was attenuated by the adenosine receptor antagonist 8-sulfophenyltheophylline and the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA). Further, endothelium denudation abolished the ethanol-evoked enhancement of NECA responses. The vasorelaxant responses to acetylcholine or sodium nitroprusside in aortic rings were not influenced by ethanol. In conclusion, the present findings suggest that chronic ethanol enhances the NO-dependent vasorelaxant responses to adenosine receptor activation and this may explain, at least partly, the mechanism of the hypotensive effect of ethanol in spontaneously hypertensive rats.