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)-receptor activation decreases NaCl absorption by cortical THALs through activation of NOS and increased production of NO. We found that the alpha(2)-receptor 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), whereas 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 methyl 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 NOS 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 pmol. mm(-1). min(-1)). When we blocked the alpha(2)-receptors with rauwolscine (1 microM), 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 mediate the effects of clonidine in the THAL. We investigated the mechanism of NOS activation and found that intracellular calcium concentration did not increase in response to clonidine, whereas pretreatment with 150 nM wortmannin 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 microM LY-83583, an inhibitor of soluble guanylate cyclase, blocked clonidine-mediated inhibition of THAL J(Cl). In conclusion, alpha(2)-receptor stimulation decreases THAL J(Cl) by increasing NO release and stimulating guanylate cyclase. These data suggest that alpha(2)-receptors act as physiological regulators of THAL NO synthesis, thus inhibiting chloride transport and participating in the natriuretic and diuretic effects of clonidine in vivo.