Dilation of rat preglomerular microvessels (PGMV) by activation of adenosine A2A receptors (A2AR) is coupled to epoxyeicosatrienoic acid (EET) release. We have investigated the commonality of this signal transduction pathway, i.e., sequential inhibition of G(salpha), adenylyl cyclase, PKA, and Ca2+-activated K+ (KCa) channel activity, to the vasoactive responses to A2AR activation by a selective A2A agonist, CGS-21680, compared with those of 11,12-EET. Male Sprague-Dawley rats were anesthetized, and microdissected arcuate arteries (110-130 microm) were cannulated and pressurized to 80 mmHg. Vessels were superfused with Krebs solution containing NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin and preconstricted with phenylephrine. We assessed the effect of 3-aminobenzamide (10 microM), an inhibitor of mono-ADP-ribosyltranferases, on responses to 11,12-EET (3 nM) and CGS-21680 (10 microM) and found that both were inhibited by approximately 70% (P<0.05), whereas the response to SNP (10 microM) was unaffected. Furthermore, 11,12-EET (100 nM), like cholera toxin (100 ng/ml), stimulated ADP-ribose formation in homogenates of arcuate arteries compared with control. SQ-22536 (10 microM), an inhibitor of adenylyl cyclase activity, and myristolated PKI (14-22) amide (5 microM), an inhibitor of PKA, decreased activity of 11,12-EET and CGS-21680. Incubation of 11,12-EET (3 nM-3 microM) with PGMV resulted in an increase in cAMP levels (P<0.05). The responses to both 11,12-EET and CGS-21680 were significantly reduced by superfusion of iberiotoxin (100 nM), an inhibitor of KCa channel activity. Thus in rat PGMV activation of A2AR is coupled to EET release upstream of adenylyl cyclase activation and EETs stimulate mono-ADP-ribosyltransferase, resulting in Gsalpha protein activation.

1 Activation of rat adenosine 2A receptors (A 2A R) dilates preglomerular microvessels (PGMV), an effect mediated by epoxyeicosatrienoic acids (EETs). 2 Incubation of PGMV with a selective A 2A R agonist, 2-p-(2-carboxyethyl) phenethylamino-5 0 -Nethylcarboxamidoadenosine (CGS 21680; 100 mM), increased isolated PGMV EET levels to 7.5771.53 ng mg À1 protein from 1.0670.22 ng mg À1 protein in controls (Po0.05), without affecting hydroxyeicosatetraenoic acid (HETE) levels (10.870.69 vs 11.0270.74 ng mg À1 protein).

Epoxyeicosatrienoic acids (EETs) are vasodilator, natriuretic, and antiinflammatory lipid mediators. Both cis- and trans-EETs are stored in phospholipids and in red blood cells (RBCs) in the circulation; the maximal velocity (V(max)) of trans-EET hydrolysis by soluble epoxide hydrolase (sEH) is threefold that of cis-EETs. Because RBCs of the spontaneously hypertensive rat (SHR) exhibit increased sEH activity, a deficiency of trans-EETs in the SHR was hypothesized to increase blood pressure (BP). This prediction was fulfilled, since sEH inhibition with cis-4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid (AUCB; 2 mg·kg(-1)·day(-1) for 7 days) in the SHR reduced mean BP from 176 ± 8 to 153 ± 5 mmHg (P < 0.05), whereas BP in the control Wistar-Kyoto rat (WKY) was unaffected. Plasma levels of EETs in the SHR were lower than in the age-matched control WKY (16.4 ± 1.6 vs. 26.1 ± 1.8 ng/ml; P < 0.05). The decrease in BP in the SHR treated with AUCB was associated with an increase in plasma EETs, which was mostly accounted for by increasing trans-EET from 4.1 ± 0.2 to 7.9 ± 1.5 ng/ml (P < 0.05). Consistent with the effect of increased plasma trans-EETs and reduced BP in the SHR, the 14,15-trans-EET was more potent (ED(50) 10(-10) M; maximum dilation 59 ± 15 μm) than the cis-isomer (ED(50) 10(-9) M; maximum dilation 30 ± 11 μm) in relaxing rat preconstricted arcuate arteries. The 11,12-EET cis- and trans-isomers were equipotent dilators as were the 8,9-EET isomers. In summary, inhibition of sEH resulted in a twofold increase in plasma trans-EETs and reduced mean BP in the SHR. The greater vasodilator potency of trans- vs. cis-EETs may contribute to the antihypertensive effects of sEH inhibitors.

Abstract-Adenosine-induced renovasodilation in Dahl rats is mediated via activation of adenosine 2A receptors (A 2A Rs) and stimulation of epoxyeicosatrienoic acid (EET) synthesis. Unlike Dahl salt-resistant rats, salt-sensitive rats exhibit an inability to upregulate the A 2A R-EET pathway with salt loading; therefore, we examined the effect of in vivo inhibition of the A 2A R-EET pathway on blood pressure and the natriuretic response to salt-loading in Dahl salt-resistant rats. N-Methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 20 mg/kg per day), an epoxygenase inhibitor, or ZM241385 (ZM; 5 mg/kg per day), an A 2A R antagonist, was given daily as an IV bolus dose for 3 days before and after placing rats on high salt intake (2% saline). After 3 days of high salt, systolic blood pressure per 24 hours increased from 108Ϯ2 mm Hg to 136Ϯ5 mm Hg and 140Ϯ4 mm Hg when treated with MS-PPOH or ZM, respectively (PϽ0.001). Plasma levels of EETs and dihydroxyeicosatrienoic acids during salt loading and MS-PPOH (29.3Ϯ1.8 ng/mL) or ZM treatment (9.8Ϯ0.5 ng/mL) did not increase to the same extent as in vehicle-treated rats (59.4Ϯ1.7 ng/mL; PϽ0.001), and renal levels of EETsϩdihydroxyeicosatrienoic acids were 2-fold lower with MS-PPOH or ZM treatment. On day 3 of the high salt intake, MS-PPOH-and ZM-treated rats exhibited a positive Na ϩ balance, and plasma Na ϩ levels were significantly increased (163.3Ϯ1.2 and 158.1Ϯ4.5 mEq/L, respectively) compared with vehicle-treated rats (142.1Ϯ1 mEq/L), reflecting a diminished natriuretic capacity. These data support a role for the A 2A R-EET pathway in the adaptive natriuretic response to modulate blood pressure during salt loading.