Polarized secretion of endothelin-1 and big ET-1 in MDCK cells is inhibited by cell Na+ flux and disrupted by NH4Cl.
We recently found that TGF-beta increases ET-1 secretion in MDCK, a renal tubular cell line. The secretion of ET-1, by a confluent monolayer of MDCK cells grown on a filter, to the basolateral side of the epithelium was two times greater than that to the apical side. However, TGF-beta-increased ET-1 and big ET-1 secretion were exclusively released to the basolateral side. We investigated the mechanism of polarized secretion of ET-1 and big ET-1 in MDCK cells. After 24 h of incubation with 80 pM TGF-beta, basolateral secretion of both ET-1 and big ET-1 increased two to threefold without a significant increase on the apical side. TGF-beta-stimulated ET-1 and big ET-1 secretion in the basolateral bath were inhibited when 10 microM amiloride (Na+ channel blocker) or 1 microM ouabain (Na+, K(+)-ATPase inhibitor) was added for 3 h. Polarized secretion of ET-1 and big ET-1 was not affected. In contrast, 10 mM NH4Cl or 0.2 mM chloroquine (both lysosomal function inhibitors) reduced TGF-beta-stimulated ET-1 secretion in the basolateral bath whereas big ET-1 secretion in the apical bath increased two times. Thus, the polarity of big ET-1 secretion was reversed. In addition, the conversion rate from big ET-1 to ET-1 was significantly decreased from 80 to 55% by inhibiting lysosomal function. Prepro-ET-1 mRNAs 3 h after these perturbations were virtually unaltered. Our data show that ET-1 and big ET-1 secretion may be regulated by cell Na+ flux and that lysosomal functions may play some roles in the conversion of big ET-1 to mature ET-1 and in polarized secretion of big ET-1. Translational or posttranslational regulation may play an additional role in ET-1 secretion as well as the mRNA level.
Uchida
S
S
Fourth Department of Internal Medicine, University Tokyo School of Medicine, Japan.
Horie
M
M
Yanagisawa
M
M
Matsushita
Y
Y
Kurokawa
K
K
Ogata
E
E
D000643:Ammonium Chloride / Q000494:pharmacology*; D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D004285:Dogs; D019332:Endothelin-1; D016232:Endothelins / Q000096:biosynthesis / Q000378:metabolism*; D007684:Kidney Tubules / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D011061:Poly A / Q000378:metabolism; D011498:Protein Precursors / Q000096:biosynthesis / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*
D019332:Endothelin-1; D016232:Endothelins; D011498:Protein Precursors; D000643:Ammonium Chloride; D011061:Poly A; D012313:RNA; D012964:Sodium