Na+-k+(nh4+)-2cl- cotransport in medullary thick ascending limb.
control by pka, pkc, and 20-hete.
Amlal, Hassane, Christian Legoff, Catherine Vernimmen, Michel
Paillard, and Maurice Bichara.
Physiologie et Endocrinologie Cellulaire R[acute]enale, INSERM
Unit[acute]e 356, Universit[acute]e Pierre et Marie Curie and
H[circumflex]opital Broussais, Paris, France
APStracts 3:0057C, 1996.
Cell pH was monitored in suspensions of medullary thick ascending
limbs (MTALs) of rat kidney to determine possible effects of various
transduction pathways on apical Na+-K+(NH4+)-2Cl- cotransport, the
activity of which was measured as the bumetanide-sensitive component
of cell acidification caused by abrupt exposure to 4 mM NH4Cl. 8
-bromoadenosine 3',5'-cyclic monophosphate (cAMP) stimulated
cotransport activity through activation of cAMP-dependent protein
kinase (PKA) since the cAMP effect was abolished by N-[2-(p
-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89);
stimulation by cAMP (P<0.02) was observed even when other Na+,
Cl-, and K+ carriers were blocked by ouabain, diphenylamine-2
-carboxylate, and barium, which indicates that cotransport was
directly affected by PKA. Phorbol 12,13-dibutyrate also stimulated
cotransport activity (P<0.03), which was abolished by protein
kinase C (PKC) blockade by staurosporine. By contrast, cotransport
activity was reduced (P<0.001) by arachidonic acid or 20
-hydroxyeicosatetraenoic acid (20-HETE), as well as by an ionomycin
-induced rise in cytosolic Ca2+ ([Ca2+]i). Inhibition by arachidonic
acid or ionomycin was abolished by econazole and SKF-525A that
inhibit cytochrome-P-450-dependent monooxygenase which produces 20
-HETE from arachidonic acid in the MTAL, and the ionomycin effect was
prevented when phospholipase A2 (PLA2) was blocked by 4
-bromophenacyl-bromide or oleyloxyethyl phosphorylcholine. The results
demonstrate that MTAL apical Na+-K+(NH4+)-2Cl- cotransport is
stimulated by PKA and PKC and inhibited by 20-HETE that may be
produced after a rise in [Ca2+]i through PLA2 activation.
Received 25 July 1995; accepted in final form 29 January 1996.
APS Manuscript Number C453-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 14 February 96