NO Inhibits Nacl Absorption by Rat Thick Ascending Limb Through Activation of Cgmp-Stimulated Phosphodiesterase.

Abstract

P96 NO regulates salt balance by inhibiting NaCl reabsorption along the nephron. In the isolated, perfused rat thick ascending limb (THAL), adding arginine to the bath stimulates endogenous NO production which inhibits NaCl absorption. While we reported the NO-signaling pathway in collecting ducts, it has not been studied in THALs. We hypothesized that endogenous NO inhibits NaCl transport by increasing cGMP production which in turn activates cGMP dependent protein kinase (PKG). THALs from rats were isolated and perfused, and net chloride flux ( J Cl - ) was measured. L-arginine (0.5 mM) added to the bath decreased J Cl - from 154.4±9.9 to 101.9±14.1 a 35.2% decrease ( n =7; p < 0.05). Next we tested the effect of the soluble guanylate gyclase (sGC) inhibitor LY-83583 on L-Arg induced inhibition of J Cl - . In the presence of 10 μM LY-83583 adding L-Arg to the bath did not affect THAL J Cl - (143.7±28.1 vs. 136.7±22.2 pmol mm -1 min -1 ; n =6). 10 μM LY-83583 alone did not affect THAL J Cl - ( n =4). We then tested whether the cGMP-stimulated phosphodiesterase (PDE II) inhibitor EHNA blocked the effects of L-Arg. In the presence of 50 μM EHNA, L-Arg reduced chloride flux by only 11% (146.2±7.3 vs. 129.1±6.8 pmol mm -1 min -1 ; p < 0.06; n =5). 50 μM EHNA alone did not change THAL J Cl - ( n =4). Since cGMP activates PKG as well as PDE II, we tested whether the PKG inhibitor KT-5823 could prevent the L-Arg effect. In the presence of 2 μM KT-5823, L-Arg decreased J Cl - from 132.5±14.1 to 91.1±8.8 pmol mm -1 min -1 , an inhibition of 31% ( n =9; p < 0.05). To determine whether all of the effects of L-arg are mediated by cGMP, we tested its effect on THAL J Cl - in the presence of cGMP. In the presence of 50 μM dibutyryl-cGMP, J Cl - was 116.3±9.7 pmol mm -1 min -1 . After L-Arg was added to the bath, J Cl - was 100.7±8.3 pmol mm -1 min -1 ( n.s ; n =5). We concluded that: 1) NO reduces THAL J Cl - by activating sGC, increasing cGMP and stimulating PDE II which likely decreases cAMP; 2) activation of PKG is not necessary for NO to decrease J Cl - ; 3) the cascade activated by NO in the THAL is different from that activated in the collecting duct.