Calcium-independent pkc isoforms may modulate parietal cell hcl
secretion.
Chew, C. S., C. J. Zhou, and J. A. Parente, Jr.
Institute of Molecular Medicine and Genetics, Medical College of
Georgia, Augusta, GA USA
APStracts 3:0203G, 1996.
Although the activation of cAMP and calcium-dependent signaling
pathways by histamine and cholinergic agonists, respectively, is a
generally recognized mechanism for increasing parietal cell HCl
secretion, the role of protein kinase C (PKC) in this process is
controversial. In this study, the acid secretory responses of gastric
glands from rabbits (measured as accumulation of the weak base, 14C
-aminopyrine (AP)) were found to be relatively resistant to a number
of PKC inhibitors including calphostin C, chelerythrine Cl,
staurosporine and the bisindolylmaleimide-like inhibitors, Ro 31
-8220, G[diaeresis]o 6976 and bisindolylmaleimide I HCl. Western
analyses of the PKC isozyme profile in highly enriched parietal cells
(98% purity) indicated that this cell type expresses abundant levels
of the novel or phorbol ester-dependent, calcium independent
isoforms, PKC and [mu], and of the atypical or calcium and phorbol
ester-independent isoforms, i, l and z. In contrast, there appeared
to be low to undetectable expression of the classical or calcium- and
phorbol ester-dependent isoforms, PKC [alpha] and b1/b2,
respectively. Relatively high concentrations of Ro 31-8220
potentiated both carbachol and histamine-stimulated AP accumulation
(IC50s 857 +/- 100 and 910 +/- 98 nM, respectively). There was a
similar dose-dependence for Ro 31-8220 inhibition of in situ
phosphorylation of a parietal cell phosphoprotein, pp66 (IC50s 750
+/- 120 nM). Unexpectedly, similar concentrations of Ro 31-8220 also
inhibited phosphorylation of the cytoskeletal, actin-membrane
crosslinking phosphoprotein, ezrin, but not other phosphoproteins.
Ezrin phosphorylation was subsequently found to be increased by
carbachol and the phorbol ester, TPA. Since carbachol and TPA
stimulate pp66 phosphorylation in a calcium-independent manner, our
results suggest that one or more novel PKC isoforms may be involved
in the negative regulation of HCl secretion. In related experiments,
PKC , but not PKC[mu], was immunolocalized by confocal microscopy to
a parietal cell compartment that bore a striking resemblance to that
containing filamentous (F)-actin. Moreover, pp66 was enriched in a
Triton X-100 insoluble parietal cell fraction, suggesting a potential
cytoskeletal localization for this unknown protein. Given their
location and sensitivities to Ro 31-8220, it is possible that pp66
and ezrin interact in a PKC-dependent manner to regulate the well
-known morphological changes that occur in concert with agonist
-dependent activation of parietal cell HCl secretion.
Received 5 June 1996; accepted in final form 6 September 1996.
APS Manuscript Number G227-6.
Article publication pending Am. J. Physiol. (Gastrointest. Liver
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 7 October 1996