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-β 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-β-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-β, basolateral secretion of both ET-1 and big ET-1 increased two to threefold without a significant increase on the apical side. TGF-β-stimulated ET-1 and big ET-1 secretion in the basolateral bath were inhibited when 10 μM amiloride (Na⁺channel blocker) or 1 μM 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-β -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 post-translational regulation may play an additional role in ET-1 secretion as well as the mRNA level.