Synergic action of insulin and genistein on Na+/K+/2Cl- cotransporter in renal epithelium.
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 microM 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 microM for 2 h) that inhibits endocytosis of proteins at the plasma membrane into the cytosolic space, insulin pretreatment increased the NPPB-sensitive Isc with no effects on NPPB-sensitive Gt. Genistein (100 microM) 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 Isc 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.
Ueda-Nishimura
Tomoko
T
Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.
Niisato
Naomi
N
Miyazaki
Hiroaki
H
Naito
Yuji
Y
Yoshida
Norimasa
N
Yoshikawa
Toshikazu
T
Nishino
Hoyoku
H
Marunaka
Yoshinori
Y
D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D020126:Brefeldin A / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000737:chemistry; D004357:Drug Synergism; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism*; D019833:Genistein / Q000494:pharmacology*; D006056:Golgi Apparatus / Q000378:metabolism; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D007477:Ions; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D009579:Nitrobenzoates / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D013696:Temperature; D013997:Time Factors; D014982:Xenopus laevis
D018118:Chloride Channels; D007328:Insulin; D007477:Ions; D007976:Leupeptins; D009579:Nitrobenzoates; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; C013407:benzamil; D020126:Brefeldin A; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D000584:Amiloride; D019833:Genistein; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde