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).

20-HETE, a potent vasoconstrictor, is generated by cytochrome P-450 omega-hydroxylases and is the principal eicosanoid produced by preglomerular microvessels. It is released from preglomerular microvessels by ANG II and is subject to metabolism by cyclooxygenase (COX). Because low-salt (LS) intake stimulates the renin-angiotensin system and induces renal cortical COX-2 expression, we examined 20-HETE release from renal arteries (interlobar and arcuate and interlobular arteries) obtained from 6- to 7-wk-old male Sprague-Dawley rats fed either normal salt (0.4% NaCl) or LS (0.05% NaCl) diets for 10 days. With normal salt intake, the levels of 20-HETE recovered were similar in arcuate and interlobular arteries and interlobar arteries: 30.1 +/- 8.5 vs. 24.6 +/- 5.3 ng. mg protein(-1). 30 min(-1), respectively. An LS diet increased 20-HETE levels in the incubate of either arcuate and interlobular or interlobar renal arteries only when COX was inhibited. Addition of indomethacin (10 microM) to the incubate of arteries obtained from rats fed an LS diet resulted in a two- to threefold increase in 20-HETE release from arcuate and interlobular arteries, from 39.1 +/- 13.2 to 101.8 +/- 42.6 ng. mg protein(-1). 30 min(-1) (P < 0.03), and interlobar arteries, from 31.7 +/- 15.1 to 61.9 +/- 29.4 ng. mg protein(-1). 30 min(-1) (P < 0.05) compared with release of 20-HETE when COX was not inhibited. An LS diet enhanced vascular expression of cytochrome P-4504A and COX-2 in arcuate and interlobular arteries; COX-1 was unaffected. Metabolism of 20-HETE by COX is proposed to represent an important regulatory mechanism in setting preglomerular microvascular tone.

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