Protein kinase c and insulin regulation of red blood cell na+/h+
exchange.
Ceolotto, Giulio, Paul Conlin, Giulio Clari, Andrea Semplicini, and
Mitzy Canessa.
Endocrine Hypertension Division, Brigham and Women's Hospital,
Department of Medicine, Harvard Medical School, Boston, MA,
Departments of Clinical Medicine and Biochemistry, University of of
Padova Medical School, Padova, Italy and Departamento de
Biolog[acute]ia, Facultad de Ciencias, Universidad de Chile,
Santiago, Chile
APStracts 3:0354C, 1996.
Insulin activation of red blood cell (RBC) Na+/H+ (NHE) and Na+/Li+
(NLiE) exchanges is mimicked by okadaic acid, thus suggesting that it
may change the state of phosphorylation of serine/threonine NHE
residues. To investigate the role of the serine/threonine protein
kinase C (PKC), in insulin regulation we evaluated the effect of
phorbol 12-myristate 13-acetate (PMA,1 [mu]M) and insulin on PKC
activity (pmoles/mg protein x min), membrane protein phosphorylation
and the activation kinetics of both exchangers. Our studies revealed
that PMA decreased cytosolic PKC activity (4.1 +/-0.6 to 2.3 +/-0.5,
n=9, p<0.001), increased membrane PKC activity (42.3 +/-5 to
132 +/-12, n=11, p<0.001), and enhanced serine phosphorylation
of bands 3, 4.1, and 4.9 membrane proteins. PMA markedly reduced the
Km for intracellular H+ (415 +/-48 to 227 +/-38 nM,n=11,
p<0.01) but had no effect on the maximal transport rate (Vmax)
of NHE and the Km for Na+ of NLiE. NHE activation and PKC activity
were affected differently by insulin (100 [mu]U/ml) and PMA. Insulin
increased the Vmax of NHE and the Km for Na+ of NLiE but had no
effect on the Km for intracellular H+ and membrane PKC activity.
These findings lead us to conclude that in the human RBC, NHE is
modulated by PKC and insulin through different biochemical
mechanisms.
Received 10 April 1996; accepted in final form 18 October 1996.
APS Manuscript Number C197-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 13 November 1996