Phorbol esters induce two distinct changes in GH3 pituitary cell adenylate cyclase activity (original) (raw)

Phorbol esters increase adenylate cyclase activity and stability in pituitary membranes

Biochemical and Biophysical Research Communications, 1988

In this report, we demonstrate that calcium and phorbol esters enhance CAMP production in GH& cell homogenates. The mechanism for this is a reduction in the rate of decay of adenylate cyclase activity over the course of the assay. Purified protein kinase C can reconstitute calciumand phorbol ester-dependent adenylate cyclase. Phorbol ester-activated protein kinase C increases both the initial rate of CAMP synthesis and reduces the time-dependent decay of adenylate cyclase activity in membrane preparations. The rate of CAMP production is fit to an equation derived from a model which assumes that adenylate cyclase initially exists in a high activity state which decays exponentially into a low activity state. We suggest that protein kinase C can both prevent the decay of the high activity state and convert the low activity state into the high activity state. 0 1988 Academic Press, Inc.

Regulation of GH3pituitary tumour-cell adenylate cyclase activity by activators of protein kinase C

Biochemical Journal, 1989

The influence of protein kinase C (PKC) activation on cyclic AMP production in GH3 cells has been studied. The stimulation of cyclic AMP accumulation induced by forskolin and cholera toxin was potentiated by 4 beta-phorbol 12,13-dibutyrate (PDBu). Moreover, PDBu, which causes attenuation of the maximal response to vasoactive intestinal polypeptide (VIP), also induced a small right shift in the dose-response curve for VIP-induced cyclic AMP accumulation. PDBu-stimulated cyclic AMP accumulation was unaffected by pretreatment of cells with pertussis toxin or the inhibitory muscarinic agonist, oxotremorine. PDBu stimulation of adenylate cyclase activity required the presence of a cytosolic factor which appeared to translocate to the plasma membrane in response to the phorbol ester. The diacylglycerol-generating agents thyroliberin, bombesin and bacterial phospholipase C each stimulated cyclic AMP accumulation, but, unlike PDBu, did not attenuate the stimulation induced by VIP. These res...

Phorbol esters and thyroliberin have distinct actions regarding stimulation of prolactin secretion and activation of adenylate cyclase in rat pituitary tumour cells (GH4C1 cells)

Biochemical Pharmacology, 1988

The phorbol ester 12-0-tetradecanoyl phorbol13-acetate (TPA) enhances the effects of TRH on phase II of prolactin secretion as well as on hormone synthesis at both low and high TPA receptor occupancy. Furthermore TPA, but not the biologically inactive substance 4cu-phorbol12,13-didecanoate (4(~-PDD), stimulates the particulate bound adenylate cyclase with a time course paralleling that of TRH activation. However, the combined additions of TRH and TPA activate this cyclase in an additive manner while the Gpp(NH)p-and the forskolin-sensitive enzyme are unaffected by TPA addition. Polymyxin B, which inhibits protein kinase C, abolishes activation of adenylate cyclase by TPA without interfering with the stimulatory action of TRH. Also, when phosphatase activity is preferentially inhibited by pretreatment of the cells with sodium vanadate, the TRH-sensitive cyclase is unaltered, while TPA activation is obliterated. Maximal stimulation of adenylate cyclase by cholera toxin pretreatment, obliterated the actions of TRH and TPA. Cells pretreated with pertussis toxin retained their TRHsensitive cyclase, however, TPA-responsiveness was lost.

Continuous Activation of Pituitary Adenylate Cyclase-Activating Polypeptide Receptors Elicits Antipodal Effects on Cyclic AMP and Inositol Phospholipid Signaling Pathways in CATH.a Cells: Role of Protein Synthesis and Protein Kinases

Journal of Neurochemistry, 2002

Continuous exposure of cells to agonists develops a process that determines the extent to which the cells eventually respond to further stimuli. Here we used CATH.a cells (a catecholaminergic neuron-like cell line), which express pituitary adenylate cyclase-activating polypeptide (PACAP) receptors linked to both adenylyl cyclase and phospholipase C-ß pathways, to investigate the influence of prolonged hormonal treatment on dual signaling and gene transcription. Prolonged incubation of cells with PACAP failed to down-regulate the density and affinity of membrane binding sites and caused opposite changes in messenger systems: PACAP-stimulated cyclic AMP accumulation was attenuated in a time-and dose-dependent fashion (t 112 = 6.7 h and IC50 = 0.1 nM), whereas phosphoinositide turnover was overstimulated. Both effects were insensitive to pertussis toxin, whereas the drop in cyclic AMP concentration was also unchanged in the presence of 3-isobutyl-1 -methylxanthine, indicating that neither G-like proteins nor cyclic nucleotide phosphodiesterases play a critical role in these processes. Blockade of protein synthesis with cycloheximide, as well as inhibition by H89 of cyclic AMP-dependent protein kinase (but not by bisindolylmaleimide of protein kinase C) antagonized the influences exerted by PACAP on adenylyl cyclase activity and inositol phosphate formation. Transcription of the chimeric GAL4-CREB construct, transiently transfected into CATH.a cells, was stimulated by PACAP, and this effect was potentiated as a result of chronic PACAP treatment. The results of the present investigation provide new insight into the possible differential regulation and cross-talks of transduction signals of receptors linked to multiplex signaling. They demonstrate that prolonged exposure of CATH.a cells to PACAP results in the desensitization of the cyclic AMP pathway and superinduction of the inositol phosphate signal, through protein neosynthesis and cyclic AMP-dependent protein kinase activation. At the same time, they show that desensitization of cyclic AMP signaling not only fails to hamper, but actually amplifies PACAP-stimulated CREB-regulated transcription. Key Words: CATH.a cells-Pituitary adenylate cyclase-activating polypeptide-Cyclic AMP-Phosphoinositide turnover-Desensitization -CREB expression.

Phorbol Ester-Induced Down-Regulation of Protein Kinase C Abolishes Vasopressin-Mediated Responses in Rat Anterior Pituitary Cells

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)

Novel action of pituitary adenylate cyclase-activating polypeptide

Cellular Signalling, 2000

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal peptide/secretin family. Using microphysiometry, we have found that PACAP acutely (1 min) increased the extracellular acidification rate (ECAR) in GH4C1 cells approximately 40% above basal in a concentration-dependent manner. ECAR, maximally induced by PACAP, can be increased further by thyrotropin-releasing hormone (TRH), indicating that the signalling pathways for these two neuropeptides are not identical. In studies on the mechanism of PACAP-enhanced ECAR, we found that maximum stimulation of the cAMP/ PKA pathway by treatment with FSK, or the PKC pathway with PMA, did not inhibit the ECAR response to PACAP. The PKC inhibitor calphostin C and the MAP kinase inhibitor PD98059 had no effect on the ECAR response to PACAP. Furthermore, PACAP induced little or no change in cytosolic Ca 2ϩ ([Ca 2ϩ ] i), while TRH induced a large increase in [Ca 2ϩ ] i. However, the tyrosine kinase inhibitor genistein completely blocked PACAP-induced ECAR, suggesting involvement of tyrosine kinase(s). We conclude that PACAP causes an increase in ECAR in GH4C1 rat pituitary cells, which is not dependent on the PKA, PKC, MAP kinase or Ca 2ϩ signalling pathways, but does require tyrosine kinase activity.

Protein kinase C stimulates adenylate cyclase activity in prolactin-secreting rat adenoma (GH4C1) pituicytes by inactivating the inhibitory GTP-binding protein Gi

European Journal of Biochemistry, 1989

The phorbol ester 12-0-tetradecanoyl-phorbol 13-acetate (TPA) and thyroliberin exerted additive stimulatory effects on prolactin release and synthesis in rat adenoma GH4CI pituicytes in culture. Both TPA and thyroliberin activated the adenylate cyclase in broken cell membranes. When combined, the secretagogues displayed additive effects. TPA did not alter the time course (time lag) of adenylate cyclase activation by hormones, guanosine 5'-[/j,y-iminoltriphosphate or forskolin, nor did it affect the enzyme's apparent affinity (basal, 7.2 mM; thyroliberinenhanced, 2.2 mM) for free Mg2+. The TPA-mediated adenylate cyclase activation was entirely dependent on exogenously added guanosine triphosphate. ED5,, (dose yielding half-maximal activation) was 60 pM. Access to free Ca2+ was necessary to express TPA activation of the enzyme, however, the presence of calmodulin was not mandatory. TPA-stimulated adenylate cyclase activity was abolished by the biologically inactive phorbol ester, 4a-phorbol didecanoate, by the protein kinase C inhibitor polymyxin B and by pertussis toxin, while thyroliberinsensitive adenylate cyclase remained unaffected.