AJP - Endocrinology and Metabolism, Vol 254, Issue 4 E449-E453, Copyright © 1988 by American Physiological Society
An assessment of phosphodiesterase activity in situ after treatment of hepatocytes with hormones
T. W. Gettys, P. F. Blackmore and J. D. Corbin
Howard Hughes Medical Institute, Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
The role of phosphodiesterase activation in controlling adenosine
3',5'-cyclic monophosphate (cAMP) levels within hepatocytes was
investigated by preloading hepatocytes with the hydrolyzable cAMP analogue
8-para-chlorophenylthio-cAMP (8-pCl phi S-cAMP) and measuring disappearance
of the analogue after treating the cells with various hormones. Incubation
of hepatocytes with 15 nM 8-pCl phi S-cAMP increased the intracellular
concentration of the analogue at 0.5 and 2 min, but by 5 min the
concentration plateaued and remained constant or declined slightly at 7 and
10 min. Treatment of hepatocytes with 5 nM glucagon led to a rapid 50%
decline in intracellular concentration of the analogue. However, 6 nM
insulin produced no detectable change in analogue concentration, and a
combination of 5 nM glucagon and 6 nM insulin produced no greater lowering
of 8-pCl phi S-cAMP than did glucagon alone. Treatment of hepatocytes with
the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (50 microM)
blocked approximately 30% of the glucagon-mediated decrease in 8-pCl phi
S-cAMP concentration, and in separate cell incubations, it blocked 50% of
the cAMP lowering produced by 125 nM 8-pCl phi S-cAMP. Treatment of
analogue-preloaded hepatocytes with effective concentrations of
phenylephrine, vasopressin, or angiotensin resulted in no change in
intracellular analogue or cAMP concentrations.(ABSTRACT TRUNCATED AT 250
WORDS)
