1-oleoyl-2-acetylglycerol and Pituitary-Neoplasms

The phorbol ester 12-O-tetradecanoyl phorbol 13-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 4 alpha-phorbol 12,13-didecanoate (4 alpha-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 TRH-sensitive cyclase, however, TPA-responsiveness was lost. We therefore suggest that the action of TPA as it relates to activation of adenylate cyclase, is probably mediated via the Gi component of the adenylate cyclase complex, while TRH stimulates the enzyme via the classical pathway involving the stimulatory GTP binding protein (Gs).

Topics: Adenylyl Cyclases; Animals; Cell Line; Colforsin; Diglycerides; Enzyme Activation; Guanylyl Imidodiphosphate; Phorbol Esters; Pituitary Neoplasms; Polymyxin B; Prolactin; Rats; Somatostatin; Tetradecanoylphorbol Acetate; Thyrotropin-Releasing Hormone

Biochemical and spectrophotometric studies of second messenger pathways transducing TRH signals in clonal pituitary (GH) cells have shown that TRH induces rapid turnover of phosphoinositides and changes in cytoplasmic Ca2+ as well as activation of protein kinase C (PKC) and secretion of PRL. Here we have used classical microelectrode and contemporary patch pipette recording techniques under current-clamp conditions to compare the effects of TRH receptor-coupled stimulation with direct activation of PKC on the excitability of GH3/B6 cells. With high resistance microelectrodes TRH induced a complex sequence of changes in membrane properties consisting of an initial 20- to 30-mV hyperpolarization associated with an increase in membrane conductance lasting less than a minute, followed by several minutes of low amplitude fluctuations and action potential activity superimposed on a modest increase in input resistance. Active phorbol ester induced a slowly developing hyperpolarization of about 5 mV and a modest increase in input resistance, followed by several minutes of low amplitude fluctuations and spontaneous action potential activity. Both the peptide- and phorbol ester-evoked changes in excitability were attenuated or completely lost during patch recordings in the whole cell mode. Dilute aqueous lysates of the clone restored various phases of the electrical response. The low amplitude fluctuations and action potential activity phase could be induced by either TRH or phorbol ester if the cells were dialyzed with intracellular electrolyte containing PKC and at least 50 nM Ca2+. These results demonstrate that the phosphoinositide/PKC circuit activated by TRH in clonal pituitary cells has electrically detectable effects on cell excitability, and these help to explain TRH's actions on electrical activity.

Topics: Action Potentials; Animals; Calcium; Cell Line; Cell Membrane; Clone Cells; Diglycerides; Electrophysiology; Enzyme Activation; Phosphatidylinositols; Pituitary Gland; Pituitary Neoplasms; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Thyrotropin-Releasing Hormone

Interactions among lithium, calcium, and phorbol esters in the regulation of adrenocorticotropin hormone (ACTH) release were examined in a tumor cell line (AtT-20) of the anterior pituitary. Lithium, which blocks the phosphatase that converts inositol phosphates (IPs) to inositol, increases the levels of IPs in these cells and stimulates ACTH release. This ion potentiates the ability of calcium, an activator of phospholipase C, to raise levels of IPs in these cells and to stimulate ACTH secretion. Pretreatment of AtT-20 cells with calcium specifically abolishes the ACTH release response to lithium or calcium, a result suggesting that these secretagogues may act through a common mechanism to induce hormone secretion. Prior exposure of AtT-20 cells to either lithium or calcium also attenuates the ACTH release induced by phorbol ester, an activator of protein kinase C. To examine the link among lithium, calcium, phosphatidylinositol (PI) turnover, and phorbol ester-evoked ACTH secretion, AtT-20 cells were treated with 1-oleoyl-2-acetoyl-sn-3-glycerol (OAG), an analogue of the diacylgylcerols that are formed by phospholipase C during PI metabolism and that also activate protein kinase C. OAG itself does not alter ACTH release or the levels of IPs in AtT-20 cells. Pretreatment of AtT-20 cells with OAG, however, selectively blocks the ACTH release response to lithium, calcium, or phorbol ester. Furthermore, such pretreatment reduced the ability of lithium to increase levels of IPs. The results suggest that one mechanism of action of lithium is to potentiate selectively an action of calcium, possibly the stimulation of phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adrenocorticotropic Hormone; Animals; Calcium; Cell Line; Colforsin; Corticotropin-Releasing Hormone; Diglycerides; Dose-Response Relationship, Drug; Drug Interactions; Glycerides; Inositol Phosphates; Lithium; Mice; Phorbol Esters; Pituitary Gland, Anterior; Pituitary Neoplasms; Potassium; Tetradecanoylphorbol Acetate; Type C Phospholipases

To elucidate possible functions of elevation of endogenous diacylglycerol induced by thyrotropin-releasing hormone in pituitary cells, we have studied the actions of two synthetic diacylglycerols, sn-1-oleoyl-2-acetylglycerol (OAG) and sn-1,2-dioctanoylglycerol (DiC8), on cytosolic free calcium concentration ([Ca2+]i) in GH4C1 cells. OAG induced an immediate increase in [Ca2+]i which gradually reached a peak that was twice the basal level after the first min; [Ca2+]i then returned to remain at basal level after 3 min. The increase in [Ca2+]i was dependent on the concentration of OAG added with two apparent potencies; half-maximal actions on [Ca2+]i were observed at 70 nM and greater than 20 microM. The increase in [Ca2+]i induced by OAG was blocked completely by chelating extracellular calcium, or by pretreatment with calcium channel blockers. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which itself induces a rise in [Ca2+]i in these cells that is similar in time course, magnitude, and drug sensitivity to that of OAG, blocked completely the actions of subsequent exposure to OAG. Analogous results were obtained using DiC8, although DiC8 induced a transient inhibition to 75% of basal levels of [Ca2+]i after the initial increase in [Ca2+]i, and DiC8 was less potent than OAG. These data indicated that diacylglycerols induce influx of extracellular calcium in these cells, possibly by activation of voltage-dependent Ca2+ channels. Furthermore, diacylglycerols and phorbol esters appear to utilize a common pathway in eliciting these actions on [Ca2+]i, possibly involving activation of a protein kinase C. These actions of diacylglycerol provide a pathway by which thyrotropin-releasing hormone may act to enhance calcium channel activity.

Topics: Animals; Calcium; Cell Line; Cytosol; Diglycerides; Glycerides; Kinetics; Pituitary Neoplasms; Rats; Tetradecanoylphorbol Acetate; Thyrotropin-Releasing Hormone

Prolactin (PRL) release in permeable GH3 pituitary cells was stimulated by the protein kinase C activators 12-O-tetradecanoylphorbol 13-acetate (TPA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG). Both agents stimulated secretion at 10 nM Ca2+, but higher [Ca2+] (greater than 0.1 microM) potentiated TPA and OAG action. Maximal potentiation occurred at 1 microM calculated free Ca2+, and a similar value was obtained when the cytoplasmic [Ca2+] was measured with the Ca2+-sensitive dye Quin 2. Release of a secretory sulfated proteoglycan was also stimulated by TPA and OAG in permeable GH3 cells, with characteristics similar to those for PRL release. Trifluoroperazine, polymyxin B, neomycin, and 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate all inhibited both TPA- and Ca2+-stimulated PRL release, but in each case the half-maximal inhibitory concentrations were approximately 2-fold higher for TPA-stimulated release compared to Ca2+-stimulated release. Thyrotropin-releasing hormone (TRH) and guanosine 5'-Q-thiotriphosphate, which stimulate polyphosphoinositide breakdown in permeable cells, were found to be only weak stimulators of PRL release, compared to TPA and exogenous diacylglycerol. However, a much stronger effect of TRH was seen if cells were briefly treated with TRH prior to permeabilization. PRL release from TRH-pretreated permeable cells resembled TPA- and OAG-stimulated secretion, with [Ca2+] greater than 0.1 microM potentiating the effect of TRH pretreatment. These studies support the hypothesis that PRL release in GH3 cells can be stimulated directly by a diacylglycerol-activated secretory mechanism whose activity is modulated by [Ca2+].

Topics: Animals; Calcium; Cell Line; Cell Membrane Permeability; Diglycerides; Drug Synergism; Glycerides; Kinetics; Phorbols; Phosphatidylinositol Phosphates; Phosphatidylinositols; Pituitary Neoplasms; Prolactin; Proteoglycans; Rats; Tetradecanoylphorbol Acetate

We have used GH3 cells permeabilized by electric field discharge to examine the effects of Ca2+ and protein kinase C activators (phorbol ester and diacylglycerol) on prolactin (PRL) release. Ca2+ was found to stimulate PRL release approximately 4 fold at 3 microM Ca2+ with a half-maximal response at approximately .5 microM estimated free Ca2+. 12-O-tetradecanoyl phorbol-13-acetate and 1-oleoyl-2-acetyl-sn-glycerol stimulated PRL release throughout a range of Ca2+ concentrations (1 nM -3 microM), but stimulation was greater at higher Ca2+ concentrations (.1 microM to 1 microM). Both agents decreased by 1.8 fold the apparent [Ca2+] at which half-maximal stimulation of secretion occurred. Quin 2 was used to measure the free [Ca2+] of intact and permeable cells; PRL secretion at a free [Ca2+] corresponding to resting cytoplasmic [Ca2+] was 10% of maximal, while secretion at the [Ca2+] corresponding to the Ca2+ spike induced by thyrotropin-releasing hormone was approximately 25% of maximal.

Topics: Aminoquinolines; Animals; Calcium; Cell Line; Cell Membrane Permeability; Cytoplasm; Diglycerides; Enzyme Activation; L-Lactate Dehydrogenase; Pituitary Gland; Pituitary Neoplasms; Prolactin; Protein Kinase C; Protein Kinases; Rats; Tetradecanoylphorbol Acetate; Thyrotropin-Releasing Hormone