Signal transduction pathways involved in kinin B2receptor-mediated vasodilation in the rat isolated perfused kidney

The signal transduction pathways involved in kinin B2 receptor-related vasodilation were investigated in rat isolated perfused kidneys. During prostaglandin F2α or KCl-induced constriction, the vasodilator response to a selective B2 receptor agonist, Tyr(Me)8bradykinin (Tyr(Me)8BK), was assessed. Tyr(Me)8BK produced a concentration- and endothelium-dependent relaxation that was decreased by about 30 – 40% after inhibition of nitric oxide (NO) synthase by NG-nitro-L-arginine (L-NOARG) or of cyclo-oxygenase by indomethacin; a greater decrease (about 40 – 50%) was observed after concomitant inhibition of the two pathways. High extracellular K+ diminished Tyr(Me)8BK-induced relaxation by about 75% suggesting a major contribution of endothelium-derived hyperpolarization. The residual response was almost completely suppressed by NO synthase and cyclo-oxygenase inhibition. The K+ channel inhibitors, tetrabutylammonium (non-specific) and charybdotoxin (specific for Ca2+-activated K+ channel), suppressed Tyr(Me)8BK-induced relaxation resistant to L-NOARG and indomethacin. Inhibition of cytochrome P450 (clotrimazole or 7-ethoxyresorufin) decreased the NO/prostanoids-independent relaxation to Tyr(Me)8BK by more than 60%, while inhibition of the cannabinoid CB1 receptor (SR 141716A) had only a moderate effect. Acetylcholine induced a concentration-dependent relaxation with characteristics nearly similar to the response to Tyr(Me)8BK. In contrast, the relaxation elicited by sodium nitroprusside was potentiated in the absence of NO (L-NOARG or removal of endothelium) but remained unchanged otherwise. These results indicate that the activation of kinin B2 receptors in the rat isolated kidney elicits an endothelium-dependent vasorelaxation, mainly dependent on the activation of charybdotoxin-sensitive Ca2+-activated K+ channels. In addition, cytochrome P450 derivatives appear to be involved. Keywords: Tyr(Me)8bradykinin, kinin B2 receptor, acetylcholine, endothelium, rat isolated kidney, nitric oxide, prostanoids, K channels, cytochrome P450, cannabinoid CB1 receptor Introduction After the description by Furchgott & Zawadzki (1980) of an absolute requirement of endothelium for the vasodilation elicited by acetylcholine (ACh) in rabbit aorta, endothelial cells were shown to release a variety of mediators which can act to alter the mechanical and electrical properties of the vascular smooth muscle cells (Furchgott & Vanhoutte, 1989). These factors include prostacyclin, endothelin, endothelium-derived relaxing factor and endothelium-derived hyperpolarizing factor (EDHF). Although endothelium-derived relaxing factor was rapidly recognized as nitric oxide (NO) or a compound releasing NO (Palmer et al., 1987; Ignarro et al., 1987), the identity of EDHF still remains controversial (Quilley et al., 1997; Mombouli & Vanhoutte, 1997). Endothelium-dependent vasodilation also occurs with bradykinin (BK), mainly by the activation of bradykinin B2 receptors. BK has been shown to increase the release of prostacyclin and NO from bovine aortic endothelial cells in culture (Busse et al., 1994; Blatter et al., 1995) and to produce hyperpolarization in coronary arteries with intact endothelium (Vanhoutte, 1993; Fisslthaler et al., 1999). The involvement of three major pathways, i.e. prostacyclin, NO and EDHF, in BK-induced relaxation was further documented in various blood vessels by the use of specific inhibitors of cyclo-oxygenase (COX), NO synthase (NOS) and K+ channels. These studies also revealed that, according to the vessel, each pathway did not contribute to the same degree to the vasorelaxation. In particular, NO-independent mechanisms appear to play a more important role in small resistance vessels (Garland et al., 1995). In the rat renal vasculature, we previously reported that the main response to BK existed of a relaxation via bradykinin B2 receptors (Bagate et al., 1999). BK-induced vasodilation was mimicked by Tyr(Me)8BK, a selective B2 receptor agonist (Regoli & Barabe, 1980), and was inhibited by icatibant, a specific B2 receptor antagonist. The question of kinin signalling pathways in renal vessels was previously addressed using BK (Fulton et al., 1992; Rapacon et al., 1996; Mieyal et al., 1998). The aim of the present study was to investigate these pathways in the isolated perfused rat kidney, with Tyr(Me)8BK. The kinin B2 receptor-mediated vasodilation was analysed before and after exclusion of transduction pathways by selective inhibitors. After having controlled requirement of intact endothelium, we evaluated the effects of NOS and/or COX inhibition. The NO/prostanoids-independent vasodilation was considered to be linked to so called ‘EDHF' although measurement of vascular smooth muscle cell membrane potential was not possible in our experimental preparation. However, suppression of the response with high extracellular K+ and by inhibitors of K+ channels supported the contribution of a hyperpolarizing factor. We finally investigated the effects of cytochrome P450 inhibition and cannabinoid CB1 receptor antagonism because epoxyeicosatrienoic acids (EETs) and endogenous cannabinoids are possible candidates for EDHF (Mombouli & Vanhoutte, 1997).