AJP - Cell Physiology, Vol 263, Issue 1 C147-C153, Copyright © 1992 by American Physiological Society
cAMP-dependent protein kinase mediates hydrosmotic effect of vasopressin in collecting duct
H. M. Snyder, T. D. Noland and M. D. Breyer
Department of Veterans Affairs Medical Center, Nashville, Tennessee.
The role of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein
kinase A (PKA) in mediating the hydrosmotic effect of vasopressin in in
vitro microperfused rabbit cortical collecting ducts (CCDs) was examined.
We measured PKA substrate phosphorylation and water permeability [hydraulic
conductivity (Lp) = 10(-7) cm.atm-1.s-1], stimulated by substituted cAMP
analogues selective for a unique cAMP binding site (site A or B) on PKA
regulatory subunit (R). Synergy between site A- and site B-selective
analogues suggests involvement of PKA, because both sites must be occupied
for R to dissociate from the catalytic subunit (C), allowing
phosphorylation to proceed. As single agents, the site B-selective
analogues 8-(4-chlorophenylthio)-cAMP (8-CPT) and 8-thiomethyl-cAMP
(8-SCH3) were at least two orders of magnitude more potent than the site
A-selective analogues N6-monobutyryl-cAMP (N6-mono) or N6-benzoyl-cAMP
(N6-benz). Combinations of subthreshold concentrations of two site A
analogues (N6-mono+N6-benz) or two site B-selective analogues (8-CPT +
8-SCH3) failed to significantly increase protein phosphorylation or water
permeability. In contrast, combination of a site A plus site B analogue
synergistically stimulated both protein phosphorylation and Lp. Rp-cAMPS,
an inhibitor of cAMP binding to PKA, reduced both vasopressin (41%
inhibition)- and cAMP (56% inhibition)-stimulated water permeability. H-89
(50 microM), an inhibitor of PKA kinase activity, also blocked
cAMP-stimulated water permeability (90% inhibition). These findings suggest
that vasopressin-induced water permeability in the rabbit CCD is mediated
by PKA.
