Transepithelial Cl- secretion in polarized renal A6 cells is composed of two steps: (1) Cl- entry step across the basolateral membrane mediated by Na+/K+/2Cl(-) cotransporter (NKCC) and (2) Cl- releasing step across the apical membrane via cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. We estimated CFTR Cl- channel activity and transcellular Cl- secretion by measuring 5-nitro 2-(3-phenylpropylamino)benzoate (NPPB, a blocker of CFTR Cl-channel)-sensitive transepithelial conductance (Gt) and short-circuit current (Isc), respectively. Pretreatment with 1 mu M insulin for 24 h had no effects on NPPB-sensitive Gt or Isc. On the other hand, in A6 cells treated with carbobenzoxy-L-leucyl-leucyl-L-leucinal (MG132; 100 mu M for 2 h) that inhibits endocytosis of proteins at the plasma membrane into the cytosolic space, insulin pretreatment increased the NPPB-sensitive Ise with no effects on NPPB-sensitive Gt. Genistein (100 mu M) induced sustained increases in NPPB-sensitive Gt and Isc, which were diminished by brefeldin A (a blocker of protein translocation to Golgi apparatus from endoplasmic reticulum). Co-application of insulin and genistein synergically stimulated the NPPB-sensitive Ise without any effects on NPPB-sensitive Gt. These observations suggest that: (1) insertion and endocytosis of NKCC are stimulated by insulin, (2) the insulin-induced stimulation of NKCC insertion into the basolateral membrane is offset by the stimulatory action on NKCC endocytosis from the basolateral membrane, (3) genistein stimulates insertion of both CFTR Cl- channel into the apical membrane and NKCC into the basolateral membrane, and (4) insulin and genistein synergically stimulated NKCC insertion into the basolateral membrane. (c) 2005 Elsevier Inc. All rights reserved.