AJP - Gastrointestinal and Liver Physiology, Vol 254, Issue 4 543-G551, Copyright © 1988 by American Physiological Society
Ca2+ - and cAMP-induced protein phosphorylation in lacrimal gland basolateral membranes
D. A. Dartt, A. K. Mircheff, M. Donowitz and G. W. Sharp
Eye Research Institute, Boston, Massachusetts 02114.
Basolateral plasma membranes play an integral role in coupling of stimulus
to secretion of fluid and protein from the lacrimal gland. To determine if
basolateral plasma membranes contain Ca2+- or adenosine 3', 5'-cyclic
monophosphate (cAMP)-dependent protein kinases, which could phosphorylate
specific proteins important for secretion, a purified preparation of
basolateral plasma membranes was prepared from rat exorbital lacrimal
glands by differential and density gradient centrifugation. Phosphorylation
of basolateral plasma membrane proteins was studied in the presence of
[gamma-32P]ATP and was analyzed by sodium dodecyl sulfate-poly-acrylamide
gel electrophoresis. Increasing the Ca2+ concentration in the presence of
calmodulin stimulated phosphorylation of a 52,000-Mr peptide with a maximal
increase in phosphorylation obtained at 3 and 66 microM free Ca2+. The
phenothiazines trifluoperazine and promethazine inhibited phosphorylation
of this 52,000-Mr peptide; 50% inhibition was obtained at 15 and 95 microM,
respectively. Increasing the cAMP level from 0 to 10 microM stimulated
phosphorylation of another peptide of 91,000 Mr. This effect could be
reproduced by guanosine 3', 5'-cyclic monophosphate, but only at 100
microM. The cAMP concentration causing 50% of maximal phosphorylation was
0.3 microM. We conclude that lacrimal gland basolateral plasma membranes
contain Ca2+/calmodulin- and cAMP-dependent protein kinases and protein
substrates.
