NITRIC-OXIDE GENERATION BY RENAL PROXIMAL TUBULES - ROLE OF NITRIC-OXIDE IN THE CYTOTOXICITY OF LIPID-A

Lipid A, the biologically active component of lipopolysaccharide, stim ulated nitric oxide (NO) production by isolated rat proximal tubules ( as measured by NO2- release) in a time-dependent manner. At a concentr ation of 50 mu g/ml, lipid A stimulated NO2- generation and guanosine 3',5'-cyclic phosphate (cGMP) production within 5 min. Both of these e ffects were blocked by N-G-methyl-L-arginine (L-NMMA), an inhibitor of NO synthase or by 8-(N, N'-diethylamino)-octyl-3,4,5-trimethoxybenzoa te (TMB-8), an inhibitor of intracellular Ca++ release. Because an inc rease in NO production may be cytotoxic, we examined the cytotoxic pot ential of lipid A. At 90 min, lipid A (50 mu g/ml) produced significan t lactate dehydrogenase release (42 +/- 5%) compared to control (25 +/ - 5%; P < .05). Both L-NMMA (1 mM) and TMB-8 (100 mu M) completely pro tected against lipid A-induced cytotoxicity. TMB-8 but not L-NMMA inhi bited the rise intracellular Ca++ concentration ([Ca++](i)) in isolate d proximal tubules elicited by lipid A. L-NMMA but not TMB-8 inhibited proximal tubule soluble NO synthase activity. Thus, in the proximal t ubule, lipid A stimulates a rise in [Ca++](i) that in turn activates c onstitutive NO synthase. Furthermore, these events lead ultimately to NO-dependent cytotoxicity. Therefore, these findings suggest the poten tial for lipopolysaccharide to have a direct impact on proximal tubule physiology and renal function in vivo and support the potential thera peutic benefits of NO synthase inhibitors in the treatment of endotoxe mia.