TYROSINE KINASE, PHOSPHATIDYLINOSITOL 3-KINASE, AND PROTEIN-KINASE-C REGULATE INSULIN-STIMULATED NACL ABSORPTION IN THE THICK ASCENDING LIMB

We have previously shown a direct stimulatory effect of insulin on NaC l absorption in the medullary thick ascending limb of Henle's loop (mT AL). To further investigate the signal transduction involved, we deter mined whether tyrosine kinase, phosphatidylinositol 3-kinase (PI3-kina se), and/or protein kinase C (PKC) regulate insulin-stimulated NaCl ab sorption in the mTAL by in vitro microperfusion methods. In control ex periments, insulin increased transepithelial voltage (V-te) and net lu men-to-bath Cl- flux (J(Cl)). Genistein and methyl 2,5-dihydroxycinnam ate, two specific tyrosine kinase inhibitors, abolished the effects of insulin. Wortmannin, a specific PI3-kinase inhibitor. inhibited the a ction of insulin. The effects of insulin also were inhibited by stauro sporin and calphostin C, which are dissimilar inhibitors of PKC. These results indicate that insulin stimulates NaCl absorption in the mTAL through tyrosine kinase, PI3-kinase, and PKC-mediated mechanisms. More over, because we have reported previously that insulin causes no detec table change in cytosolic free Ca2+ in the mTAL cells, the present res ults also suggest that insulin-induced PKC activation is not related t o inositol 1,4,5-triphosphate (IP3) production.