A high-salt diet increases renal endothelin (ET) production and thick ascending limb (THAL) endothelial nitric oxide synthase (eNOS) expression. ET stimulates THAL eNOS expression via ET(B) receptors. The tonicity of the renal medulla is highly variable, and hyperosmolality stimulates ET-1 synthesis by endothelial cells. We hypothesized that a high-salt diet raises medullary osmolality, increases ET release by the THAL, and thus enhances eNOS expression. Seven days of high salt (1% NaCl in drinking water) increased eNOS expression in THALs by 125 +/- 31%. High salt increased outer medullary osmolality from 362 +/- 13 to 423 +/- 6 mosmol/kg H(2)O (P < 0.05). Bosentan, a dual-ET receptor antagonist, blocked the increase in THAL eNOS expression caused by high salt (2.66 +/- 0.44 absorbance units with bosentan vs. 5.15 +/- 0.67 for vehicle; P < 0.05). Conscious systolic blood pressure did not differ between the two groups. In primary cultures of medullary THALs, raising osmolality from 300 to 350 and 400 mosmol/kg H(2)O using NaCl increased eNOS expression by 39 +/- 11% (P < 0.05) and 71 +/- 16%, respectively (P < 0.05). In primary cultures of THALs, raising osmolality from 300 to 400 mosmol/kg H(2)O for 1 h increased ET-1 release from 62 +/- 7 to 113 +/- 2 pg/mg protein (P < 0.05). BQ-788, an ET(B) receptor antagonist (1 muM), blocked the stimulatory effect of 400 mosmol/kg H(2)O on eNOS expression (70 +/- 13% vs. -5 +/- 10%; paired difference, 74 +/- 15%; P < 0.05). BQ-788 alone had no significant effect. We concluded that high salt stimulates THAL eNOS expression by increasing outer medullary osmolality, ET-1 release by the THAL and ET(B) receptor activation. This may be an important regulatory mechanism of THAL NaCl absorption when dietary salt intake is increased.