Modulation of Regulatory and Catalytic Subunit Levels of cAMP-Dependent Protein Kinase A in Anterior Pituitary Cells in Response to Direct Activation of Protein Kinases A and C or after GnRH Stimulation

Abstract

We have previously shown that direct activation of protein kinase A (PKA) and protein kinase C (PKC) induced changes in the expression of genes coding for PKA RIIβ and Cα subunit isoforms in cultured anterior pituitary cells, suggesting the possibility of interconnected regulation at this point. To evaluate whether the cell content of PKA protein subunits could be similarly altered, the catalytic (C) and regulatory type I (RI) and type II (RII) sub-units were identified by Western blot analysis using specific immunoaffinity-purified antibodies. Activation of PKA by the permeant cyclic adenosine monophosphate (cAMP) analogue 8-Br-cAMP induced a dramatic time- and concentration-dependent decline of C sub-unit to a residual level that may represent 10-15% of that in untreated cells. The most profound decrease occurred during the first 5 h following treatment with 0.5-2 mM analogue (by 65 ± 14 and 79 ± 5%, respectively). Under identical conditions, RII was increased by about 40% at the higher concentrations, while RI increased slightly, but only at low concentrations (below 1 mM 8-Br-cAMP), and then gradually decreased. Exposure of cells to the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) also resulted in decreased levels of the PKA C subunit, however, with a different concentration-dependent profile. In particular, a decline in PKA C was most pronounced (60%) at a low concentration of TPA (10 nM) as compared with the concentrations equal to or above 20 nM (40% decrease). TPA at 10 nM also depressed notably (by 25%) the level of RII subunit, but higher concentrations were essentially ineffective, although a slight and statistically not significant elevation of the cell subunit content was observed as for RI. Simultaneous activation of both PKA and PKC pathways resulted in further depletion of PKA C and an important loss (50%) of RII, a subunit which was enhanced by the activation of either system alone. Finally, gonadotropin-releasing hormone, a neuropeptide that has the potentiality to activate both PKA and PKC signaling in gonadotropes, was able to alter PKA subunit cell content: PKA C was significantly reduced at either a subliminal (0.1 nM) or maximal (10 nM) concentration, whereas RII increased at the low concentration and decreased at the high concentration. In conclusion, these data demonstrate that the pituitary cell contents of RI, RII, and C subunits of PKA are regulated under the activation of PKA itself as well as PKC in a manner that can exhibit further alteration when both systems come simultaneously into play. Changes in the PKA subunit levels in certain cases may correlate with a variation of the mRNAs suggesting multiple control mechanisms, including an alteration of gene expression and changes in subunit degradation, synthesis, and/or turnover. These data, together with those obtained in the presence of gonadotropin-releasing hormone, provide further support for a hormonally induced interplay between PKA and PKC signaling pathways at the crucial level of PKA in the pituitary gland includ...

Pituitary Adenylate Cyclase-activating Polypeptide Stimulates Nitric-oxide Synthase Type I Expression and Potentiates the cGMP Response to Gonadotropin-releasing Hormone of Rat Pituitary Gonadotrophs

“…Anterior Pituitary Cells Culture-Anterior pituitary glands were removed from male Wistar rats (200 -220 g; Janvier, Le Genest-SaintIsle, France). The cells were enzymatically dispersed using the trypsin dissociation procedure described previously (38). Cells in 300 -350 l of Ham's F-10 medium (Biomedia, Boussens, France) supplemented with 10% fetal calf serum (Biomedia) and gentamycin (20 g/ml, Sigma) were plated in 20-mm diameter Nunc multiwell culture dishes (Polylabo, France) at a density 3 ϫ 10 6 cells for Western blot analysis.…”

Absence of pituitary adenylate cyclase-activating polypeptide-stimulated transcription of the human glycoprotein alpha-subunit gene in LbetaT2 gonadotrophs reveals disrupted cAMP-mediated gene transcription

“…Following the binding of cAMP, the catalytic and regulatory subunits dissociate and the catalytic subunit is freed to phosphorylate target proteins. Previous studies have shown that T3-1 gonadotrophs and rat anterior pituitary cells express PKA-C and PKA-RI and II (Garrel et al 1995(Garrel et al , 1997, and that the level of these subunits changes with cAMP treatment. PKA-C declines in the presence of 8-Br-cAMP whereas PKA-R increases during the same treatment paradigm (Garrel et al 1995).…”

“…Previous studies have shown that T3-1 gonadotrophs and rat anterior pituitary cells express PKA-C and PKA-RI and II (Garrel et al 1995(Garrel et al , 1997, and that the level of these subunits changes with cAMP treatment. PKA-C declines in the presence of 8-Br-cAMP whereas PKA-R increases during the same treatment paradigm (Garrel et al 1995). The recent availability of PKA-C and PKA-R subunit-specific antibodies has allowed identification of these subtypes in L T2 and T3-1 cells.…”

Acute and persistent nociceptive paw sensitisation in mice: The involvement of distinct signalling pathways

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