h-89 and 8-cyclopentyl-1-3-dimethylxanthine

Adipogenesis is stimulated in 3T3-L1 fibroblast by a combination of insulin, dexamethasone, and methylisobutylxanthine (MIX). Mitotic clonal expansion (MCE) precedes differentiation of 3T3-L1 fibroblast to adipocytes. MIX increases cAMP content, which is the activator of protein kinase A (PKA). However, PKA-independent cAMP signaling has also been described. In this paper, it was found that H89, an inhibitor of PKA, was able to block MCE but not differentiation of 3T3-L1 fibroblast. Consistently, MCE did not occur in the absence of MIX in the differentiation mixture but was recovered by overexpression of a catalytic subunit of PKA. In addition, the transfection of 3T3-L1 fibroblast with a dominant-negative mutant of PKA inhibited MCE. On the other hand, differentiation of 3T3-L1 fibroblast to adipocytes did not occur when MIX was not present in the differentiation mixture and it could not be recovered by overexpression of a catalytic subunit of PKA. Differentiation was restored by addition of either dibutyryl-cAMP (db-cAMP) or 8 CPT-2 Me-cAMP. The latter activates cAMP-EPAC but not PKA signaling. These results indicate that cAMP-PKA-independent signaling, is required for 3T3-L1 fibroblasts differentiation to adipocytes and MIX signaling through cAMP-PKA is necessary for MCE, although MCE is not essential for adipogenesis.

Topics: 1-Methyl-3-isobutylxanthine; 3T3-L1 Cells; Adipogenesis; Animals; Cell Differentiation; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dexamethasone; Fibroblasts; Insulin; Isoquinolines; Mice; Mitosis; Protein Kinase Inhibitors; Signal Transduction; Sulfonamides; Theophylline

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.

Topics: Animals; Cyclic AMP; Dose-Response Relationship, Drug; Drug Synergism; Isoquinolines; Kidney Tubules, Collecting; Osmosis; Permeability; Protein Kinase Inhibitors; Protein Kinases; Rabbits; Stereoisomerism; Sulfonamides; Theophylline; Vasopressins; Water