alpha(2)-Adrenergic-mediated tubular NO production inhibits thick ascending limb chloride absorption

Stimulation of alpha (2)-adrenergic receptors inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (THAL) expresses alpha (2)-receptors. We hypothesized that selective alpha (2)-re ceptor activation decreases NaCl absorption by cortical THALs through activ ation of NOS and increased production of NO. We found that the alpha (2)-re ceptor agonist clonidine (10 nM) decreased chloride flux (J(Cl)) from 119.5 +/- 15.9 to 67.4 +/- 13.8 pmol.mm(-1.)min(-1) (43% reduction; P < 0.02), w hereas removal of clonidine from the bath increased J(Cl) by 20%. When NOS activity was inhibited by pretreatment with 5 mM N-G-nitro-L-arginine methy l ester, the inhibitory effects of clonidine on THAL J(Cl) were prevented ( 81.7 +/- 10.8 vs. 71.6 +/- 6.9 pmol.mm(-1).min(-1)). Similarly, when the NO S substrate L-arginine was deleted from the bath, addition of clonidine did not decrease THAL J(Cl) from control (106.9 +/- 11.6 vs. 132.2 +/- 21.3 pm ol.mm(-1).min(-1)). When we blocked the <alpha>(2)-receptors with rauwolsci ne (1 muM), we found that the inhibitory effect of 10 nM clonidine on THAL J(Cl) was abolished, verifying that alpha (2), rather than I-1, receptors m ediate the effects of clonidine in the THAL. We investigated the mechanism of NOS activation and found that intracellular calcium concentration did no t increase in response to clonidine, whereas pretreatment with 150 nM wortm annin abolished the clonidine-mediated inhibition of THAL J(Cl), indicating activation of phosphatidylinositol 3-kinase and the Akt pathway. We found that pretreatment of THALs with 10 muM LY-83583, an inhibitor of soluble gu anylate cyclase, blocked clonidine-mediated inhibition of THAL J(Cl). In co nclusion, alpha (2)-receptor stimulation decreases THAL J(Cl) by increasing NO release and stimulating guanylate cyclase. These data suggest that alph a (2)-receptors act as physiological regulators of THAL NO synthesis, thus inhibiting chloride transport and participating in the natriuretic and diur etic effects of clonidine in vivo.