The direct effect of insulin on NaCl transport in proximal tubules and thick ascending limbs of Henle's loop were examined. First, the effects of insulin on intracellular pH (pHi) in the in vitro microperfused rabbit S2 proximal straight tubules (PST) were examined using a fluorescent technique. Addition of insulin to the bath increased pHi in a dose-dependent manner at concentrations ranging from 10-11 to 10-6 mol/l, and its ED50 was 10-9 mol/l. The insulin-induced pHi increase was almost completely inhibited by 10-3 mol/l amiloride in the lumen, indicating that insulin activates luminal Na/H exchange in PST. Next, the effect of insulin on the transepithelial voltage (Vt) and lumen-to-bath Cl flux (JCl) were examined in the in vitro microperfused rabbit medullary thick ascending limbs of Henle's loop (MTAL). Insulin in the bath increased Vt in a dose-dependent manner, and its ED50 was 5×10-9 mol/l. Insulin significantly increased JCl. The insulin-mediated increase in Vt was abolished by ouabain and furosemide. Dibutyryl-cAMP (dbcAMP) increased Vt and JCl. H-8 abolished the effect of dbcAMP, while it did not inhibit the actions of insulin. Removal of extracellular Ca did not affect the effects of insulin on Vt and JCl. Chelation of intracellular Ca with BAPTA/AM inhibited the actions of insulin without affecting basal values. Calmodulin (CaM) inhibitors, trifluoperazine and W-7, inhibited the actions of insulin more than 90%. These results indicate that insulin directly increases NaCl reabsorption in the MTAL, which requires the activation of Ca-CaM system, independent of the adenylate cyclase-cAMP-PKA system. In conclusion, insulin directly stimulates NaCl reabsorption in the in vitro microperfused rabbit PST and MTAL.
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