CYTOSOLIC PROTEIN-KINASE-A MEDIATES THE GROWTH-HORMONE (GH)-RELEASINGACTION OF GH-RELEASING FACTOR IN PURIFIED RAT SOMATOTROPHS

The growth hormone (GH)-releasing action of GH-releasing factor (GRF) is known to be cAMP-dependent. However, definitive proof for the invol vement of the cAMP-dependent enzyme protein kinase A (PKA) is still la cking. In this study, we characterized the PKA system in purified rat somatotrophs and examined its role in mediating GRF-stimulated GH rele ase under static incubation conditions. PKA enzyme activity was detect ed only in the cytosolic, but not the particulate fraction of rat soma totrophs. This cytosolic PKA activity exhibited the characteristic cAM P dependence (with ED(50) Of 0.1 mu M), ability to phosphorylate kempt ide (a synthetic peptide with a PKA phosphorylation site), and suscept ibility to inhibition by the bovine heat-stable PKA inhibitor. GRF tre atment (1 pM-1 nM) stimulated the cytosolic PKA activity and GH releas e from rat somatotrophs in a dose-dependent manner. Time-course studie s also demonstrated that activation of cAMP synthesis and PKA activity preceded the GH response to GRF. Stimulation of cytosolic PKA activit y in rat somatotrophs by the adenylate cyclase activator forskolin (10 nM-1 mu M) and membrane permeant cAMP analog db.cAMP (5 mu M-0.5 mM) mimicked the GH-releasing effect of GRF. In contrast, Rp.cAMP, a cAMP antagonist for PKA regulatory subunits, blocked both the cytosolic PKA activity as well as GRF-induced GH release. Similar inhibitions were also observed when an inhibitor for PKA catalytic subunits, H89, was u sed. Somatostatin (SRlF) (1 nM), the physiological GH-release inhibito r, suppressed the GH response to GRF without affecting the basal or GR F-stimulated PKA activity. SRIF at a higher dose (10 nM) abolished the GH-releasing effect of GRF. In this case, SRIF also induced a small b ut significant inhibition of GRF-stimulated PKA activity. Taken togeth er, the present study provides direct evidence that PKA enzyme activit y is localized only in the cytosol of rat somatotrophs and constitutes an essential component of the signal transduction mechanism for GRF-s timulated GH release. This cytosolic PKA system, however, does not app ear to be a major target for the GH-release inhibiting action of SRIF.