Protein kinase C heterogeneity in GH4C1 rat pituitary cells. Characterization of a Ca2(+)-independent phorbol ester receptor (original) (raw)
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Molecular Endocrinology, 1987
pearing as a doublet of 78/80 kilodaltons. Long-term treatment (24 h) of cells with 0.6 HM TPA caused the specific loss of immunologically reactive PKC. Consistently, TPA pretreatment decreased the amount of phosphatidylserine-dependent protein kinase activity measured in vitro by 90%. In control cells, vasopressin (AVP) stimulated ACTH secretion and potentiated ACTH secretion stimulated by CRF. After a 24-h treatment with 0.6 MM TPA, secretory responses to AVP and the potentiating effect of AVP on CRF action were completely abolished. In contrast, CRF action on ACTH secretion, thought to be mediated by cAMP, was unaffected. Similarly, forskolin-and 8 bromo-cAMP-induced ACTH secretion remained unchanged after TPA pretreatment. These results indicate a crucial role for PKC in mediating the effects of AVP on ACTH secretion and on the potentiating action of AVP on CRF-induced secretion from corticotropic cells of the anterior pituitary. (Molecular Endocrinology 1: 555-560, 1987)
The Journal of biological chemistry, 1987
The demonstration that activators of the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), such as phorbol esters and diacylglycerols, can provoke luteinizing hormone (LH) release from pituitary gonadotropes, suggests a possible role for protein kinase C in stimulus-release coupling. We now report that administration of phorbol myristate acetate (PMA) to pituitary cell cultures causes a sustained reduction in Triton X-100-extracted protein kinase C activity. Further, phorbol ester- and diacylglycerol-stimulated LH release, as well as inhibition by PMA of gonadotropin-releasing hormone (GnRH)-stimulated inositol phosphate production, were reduced by pretreatment with PMA. The effects of phorbol ester pretreatment on PMA-stimulated LH release and protein kinase C activity were dose-dependent, sustained (greater than or equal to 24 h) and specific (no measurable effect with 4 alpha-phorbol didecanoate). The effect on PMA-stimulated LH release was apparently Ca2+...
European Journal of Pharmacology: Molecular Pharmacology, 1994
Ca*+-dependent protein kinase C (PKC) activity, diacylglycerol levels and PKCcy, /?,, /?,, and y expression were analyzed in the pituitary of female rats treated with estradiol alone (2 months) or in combination with quinagolide in the second month. Polymerase chain reaction (PCR) and Western blot analysis revealed the presence of PKCU, p, and /3,, isoenzymes in the rat pituitary gland but not of PKCy isoenzymes. Increases in pituitary weight and plasma prolactin levels induced by estradiol were associated with an increase in diacylglycerol pituitary content (1.55 + 0.06 versus 1.12 f 0.17 nmol diacylglycerol/mg protein in controls, P < 0.01). Cotreatment with quinagolide reversed these effects. Changes in PKC activity were accompanied by parallel changes in PKCa and p, expressions. Estradiol treatment increased the expression of these isoforms whereas cotreatment with quinagolide antagonized these effects. PKCP,, expression was not affected. In conclusion, Ca*+-dependent PKC activity and protein expression are increased in hyperplastic pituitary cells, suggesting the involvement of this class of PKCs in the rat pituitary cell proliferation and/or hormonal secretion. This is further assessed by the fact that the dopamine receptor agonist treatment decreases activity and expression of these PKCs in parallel with the decrease in hormonal secretion and cell proliferation.
Biochemical and Biophysical Research Communications, 1990
Protein kinase C (PKC) molecular species of GH4C 1 cells were analyzed after separation by hydroxyapatite column chromatography. A novel Ca2+-independent PKC, nPKC e, was identified together with two conventional Ca2+-dependent PKCs, PKC o~ and /31I by analysis of kinase and phorbol ester-binding activities, immunoblotting using isozyme-specific antibodies, and Northern blotting. These PKCs are downregulated differently when cells are stimulated by outer stimuli; phorbol esters deplete PKC/3 II and nPKC s from the ceils more rapidly than PKC c~, whereas thyrotropin-releasing hormone (TRH) at 200 nM depletes nPKC s exclusively with a time course similar to that induced by phorbol esters. However, translocation of PKC c~ and /3II to the membranes is elicited by both TRH and phorbol esters. These results suggest that TRH and phorbol ester activate PKCc~ and /3II differently but that nPKCe is stimulated similarly by both stimuli• Thus, in GH4C 1 ceils, Ca2+-independent nPKC e may play a crucial role distinct from that mediated by Ca 2+-dependent PKCct and fllI in a cellular response eficited by both TRH and phorbol esters•
Comparison of patterns of prolactin release in GH4C1 cells and primary pituitary cultures
Molecular and Cellular Endocrinology, 1985
The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA, an activator of C-kinase), the cation ionophore A23187, forskolin (an activator of adenylate cyclase) and thyrotropin-releasing hormone (TRH) on prolactin release from anterior pituitary cells in primary culture were investigated and compared to the effects of these same agents on prolactin release from GH4C, cells. In both GH,C, cells and primary pituitary cultures, 100 nM TRH increased prolactin release 3-to 5-fold within 4 min after the stimulation started. This peak response was followed by a fall to a sustained increased rate of release approximately 1.5-fold above the basal rate. The decline after the early peak was slower in primary cultures than in GH,C, cells. Addition of 20 PM A23187 to primary cultures caused a rapid 2-to 4-fold increase in release that fell to basal values within 12 min after the stimulation started. In GH4C, cells, A23187 caused a rise in prolactin release of less than 2-fold that was sustained longer than the rise seen in primary cultures. Perifusion of either type of cells with 50 nM TPA caused a rapid 2-to 2.5-fold increase in release that also was sustained for 30 min or more in both types of cells. Perifusion with combined TPA and A23187 caused a 3-to 5-fold increase in rate of release from each cell type that declined rapidly to a 2-fold sustained release in primary cultures, and that declined more slowly in GH,C, cells. Forskolin, 1 PM, had only a small effect by itself, but potentiated the effect of TPA or combined TPA and A23187 in both types of cells. Forskolin did not potentiate the effect of TRH in primary cultures. Although there are differences in responses of the 2 types of cells, the overall patterns of response were similar. The data are consistent with a model in which 2 parts of the Ca2' messenger system participate in the action of TRH: an increase in cytosolic Ca2 + and an activation of protein kinase C. GH,C, cells are a subclone of the rat pituitary cell strain GH,, which was adapted to culture from a pituitary tumor (Tashjian et al., 1968). GH&, and GH, cells have been used extensively * A recipient of Research Career Development Award HD-00272. To whom all correspondence should be addressed.
The Journal of Physiology, 1999
In many neuroendocrine cells of the anterior pituitary gland, activation of the protein kinase C (PKC) intracellular signalling pathway leads to sustained cellular excitability and neurosecretion although the cellular mechanisms and targets for PKC are poorly understood (Ozawa & Sand, 1986; Mason et al. 1988). In anterior pituitary corticotrophs PKC mediates the sustained phase of adrenocorticotrophin (ACTH) secretion stimulated by activation of the phospholipase C pathway by the hypothalamic secretagogue vasopressin (Carvallo & Aguilera, 1989; Oki et al. 1990). Vasopressin elicits a biphasic elevation of intracellular free calcium (Corcuff et al. 1993; Tse & Lee, 1998) and during the sustained phase of calcium influx stimulates PKC translocation and enhances PKC activity at the plasma membrane, an effect that is mimicked by the cell-permeant PKC-activating phorbol esters (Carvallo & Aguilera, 1989). In AtT20 mouse corticotroph cells phorbol-ester-mediated activation of PKC has been proposed to exert effects both distal and proximal to voltage-dependent calcium influx, which may result from activation of different PKC isoforms (McFerran et al. 1995) to elicit ACTH release (Phillips & Tashjian, 1982; Woods et al. 1992; Clark & Kempainen, 1994; McFerran et al. 1995). Intracellular free calcium measurements in AtT20 cells suggest that PKC-induced calcium influx results, at least in part, from inhibition of TEA-sensitive potassium conductances. This inhibition results in membrane depolarization and subsequent, indirect, enhancement of voltage-gated calcium influx (Reisine & Guild, 1987; Reisine, 1989). However, ionic conductances regulated by PKC activation in corticotrophs have not been identified. In rat GHÚC1 pituitary cells activation of PKC results in inhibition of the TEA-sensitive large conductance calciumand voltage-activated potassium (BK) channels (Shipston & Armstrong, 1996), which act as immediate negative feedback regulators of voltage-dependent calcium influx in several systems (Robitaille et al. 1993; Yazejian et al. 1997). Furthermore, BK channels are an important target for cellular regulation by two distinct, physiologically relevant, intracellular signalling pathways in AtT20 corticotrophs. Activation of the cAMP-dependent protein kinase pathway results in inhibition of BK channels leading to a robust
Protein Kinase C Mediates the Effect of Vasopressin in Pituitary Corticotrophs
Molecular Endocrinology, 1989
The role of protein kinase C (PKC) on vasopressin (VP) action was investigated by inhibition of endogenous PKC using prolonged incubation of the cells with phorbol ester, and by direct measurement of PKC activity in pituitary cells. Preincubation of the cells for 6 h with 100 ni wi TPA at 37 C resulted in a 90% decrease in total PKC activity. In the PKCdepleted cells, cAMP responses to stimulation with 100 nwi CRF for 30 min were normal, but the potentiating effects of VP and PMA on CRF-stimulated cAMP production were abolished. The stimulation of ACTH secretion by VP and PMA alone was also abolished in PKC-depleted cells. PKC activity in cytosolic and detergent-solubilized membrane fractions from enriched pituitary corticotrophs obtained by centrifugal elutriation, was directly measured by enzymatic assays and by immunoblotting techniques. Basal PKC activity was higher in the cytosol than in the membranes (8.43 ± 0.47 and 1.93 ± 0.11 pmol 32 P incorporated/10 min, respectively). After incubation of the cells with VP for 15 min or [ 3 H] phorbol-12-myristate-13-acetate (PMA) for 30 min, PKC activity in cytosol was decreased by 40% and 89%, respectively, while the activity in the membrane was increased by 138% and 405%, respectively. Such VP-and PMA-induced translocation of PKC was also observed when the enzyme content in the cytosol and the membranes was measured by immunoblotting using a specific anti-PKC antibody and [ 12S l]protein A. Autoradiographic analysis of immunoblots revealed an 80 kilodalton band characteristic of PKC, with OD higher in the cytosolic than in the membrane fractions. Consistent with the enzymatic assay, 15-min treatment with VP or PMA caused a marked decrease in OD of cytosolic PKC, accompanied by a corresponding increase in the membrane-associated enzyme. These data demonstrate that PKC is essential for the effect of VP in the pituitary corticotroph. This observation, in conjunction with the ability of VP to cause redistribution