maitotoxin and Pituitary-Neoplasms

Maitotoxin (MTX) is a water-soluble polyether, isolated from the marine dinoflagellate Gambierdiscus toxicus, that stimulates hormone release and Ca2+ influx. We have investigated the action by which MTX induces Ca2+ influx and stimulates prolactin (PRL) release from GH4C1 rat pituitary cells. PRL release elicited by MTX is abolished in a concentration-dependent manner by nimodipine, a dihydropyridine (DHP) antagonist of type L voltage-dependent calcium channels (L-VDCC), indicating that MTX-enhanced PRL release occurs via activation of type L-VDCC. As an initial approach to determine whether MTX interacts directly with VDCC, we examined whether MTX affects the binding of [3H]PN 200-110, a DHP class antagonist, in intact GH4C1 cells. MTX increased the Bmax of [3H]PN 200-110 binding to intact GH4C1 cells from 4.6 +/- 0.03 to 12.5 +/- 2.2 fmol/10(6) cells, without changing the Kd. This indicates that MTX does not bind to the DHP site, but rather suggests that MTX may have an allosteric interaction with the DHP binding site. The effect of MTX on DHP binding was largely (65%) calcium-dependent. We next examined whether MTX alters the membrane potential of GH4C1 cells using the potential sensitive fluorescent dye bisoxonol. Addition of 100 ng/ml MTX to GH4C1 cells caused a membrane depolarization within 2.5 min which reached a plateau at 5 min. The MTX-induced depolarization was not prevented by substitution of impermeant choline ions for Na+. It was similarly unaffected by K+ channel blockers or by depleting the K+ chemical concentration gradient with gramicidin, a monovalent cation pore-forming agent. By contrast, low extracellular Ca2+ totally abolished the depolarization response, and nimodipine at 100 nM substantially reduced the MTX-induced membrane depolarization. These results indicate that the predominant effect of MTX on depolarization is Ca2+ influx through L-VDCC. Taken together, our results indicate that MTX-enhanced PRL release occurs exclusively via activation of type L-VDCC in GH4C1 cells. We suggest that MTX induces an initial slow calcium conductance, possibly via an allosteric interaction with a component of the VDCC complex, which, in turn, initiates a positive feedback mechanism involving calcium-dependent membrane depolarization and voltage-dependent activation of calcium channels.

Topics: Animals; Calcium; Calcium Channels; Cell Membrane; Choline; Dose-Response Relationship, Drug; Isradipine; Kinetics; Marine Toxins; Membrane Potentials; Nimodipine; Oxocins; Pituitary Neoplasms; Potassium; Rats; Sodium; Tumor Cells, Cultured

MMQ cells, a prolactin-secreting cell line possessing dopamine receptors, were exposed to the calcium channel activator maitotoxin and dopamine or adrenoceptor agonists or antagonists. Dopamine (500 nM) or the dopamine agonists lisuride (50 nM), terguride (50 nM), and N-0437 (50 nM) decreased maitotoxin-stimulated prolactin release from perifused MMQ cells. In this system, sulpiride (100 nM), a dopamine D2 antagonist, reversed the prolactin inhibition produced by lisuride (20 nM). In static incubations of MMQ cells, lisuride (10-500 nM) inhibited maitotoxin-stimulated prolactin release in a concentration-dependent manner; this inhibition was attenuated in a concentration-related manner by sulpiride (100-500 nM). In addition, sulpiride reversed dopamine (50-500 nM), lisuride (10-500 nM), and terguride (50-500 nM) inhibition of forskolin-stimulated cAMP generation. The alpha 2-adrenergic agonist clonidine inhibited maitotoxin-stimulated prolactin release from perifused MMQ cells; this inhibition was abolished by idazoxan, an alpha 2-adrenergic antagonist. In contrast, serotonin or the serotonin antagonist methysergide had no effect on prolactin release from MMQ cells. These data indicate that activation of dopamine D2 receptors and alpha 2-adrenoceptors by classically defined pharmacological agents inhibits prolactin release and cellular cAMP levels in MMQ cells. Therefore, MMQ cells may provide a valuable model for the development of pharmacological agents and assist in the identification of the mechanisms involved in the dopaminergic inhibition of prolactin release.

Topics: Adrenergic Agonists; Cyclic AMP; Depression, Chemical; Dopamine; Humans; Lisuride; Marine Toxins; Oxocins; Pituitary Neoplasms; Prolactin; Receptors, Dopamine; Tumor Cells, Cultured

In the present study we used 235-1 cells, a prolactin secreting clone derived from a pituitary tumor. In these cells maitotoxin, a calcium channels activator, likely acting on voltage sensitive calcium channels, increases intracellular free calcium measured by Quin 2 technique. Maitotoxin stimulation of calcium flux was inhibited both by nicardipine and verapamil in a dose dependent manner. Pertussis toxin pretreatment does not modify maitotoxin activation of calcium channels, while completely abolishes nicardipine inhibition of maitotoxin induced voltage sensitive calcium channels activation, without affecting verapamil effect. These results suggest a possible involvement of a pertussis toxin sensitive G protein in dihydropyridine inhibition of voltage sensitive calcium channels.

Topics: Animals; Calcium; Dihydropyridines; Ion Channels; Marine Toxins; Nicardipine; Oxocins; Pertussis Toxin; Pituitary Neoplasms; Prolactin; Tumor Cells, Cultured; Verapamil; Virulence Factors, Bordetella

Although dopamine inhibits PRL release from the normal anterior pituitary lactotroph, a conclusive demonstration of the mechanisms involved in this response has been impeded by the presence of other cell types in the anterior pituitary. To circumvent this problem, we have isolated a clonal cell line, designated MMQ, from the 7315a rat pituitary tumor. The MMQ cell is an exemplary model for our use because it only secretes PRL. Our studies show that dopamine inhibits secretagogue-induced PRL release from these cells. In addition, dopamine decreases the intracellular cAMP concentration in MMQ cells that have been exposed to forskolin, cholera toxin, or vasoactive intestinal polypeptide, each a stimulator of cAMP generation. This inhibition is, in turn, reversed by the dopamine antagonist haloperidol and by pertussis toxin, an inactivator of the GTP-binding coupling protein. Dopamine also decreases the uptake and fractional efflux of 45Ca2+ by MMQ cells that have been exposed to the calcium channel activator maitotoxin. It seems, therefore, that dopamine decreases PRL release from MMQ cells at least in part by decreasing intracellular cAMP levels and calcium uptake. In additional experiments, we have found that MMQ cells are responsive to somatostatin, estrogen, progesterone, and acetylcholine, but not to TRH, angiotensin II, neurotensin, or bombesin. Furthermore, these cells possess a functional protein kinase-C system, as evidenced by the increase in PRL release and decrease in stimulated intracellular cAMP levels that occur in response to treatment with phorbol diesters. We suggest that the MMQ cell line will prove a useful model system for study of the biochemical effects of dopamine and other factors that modify PRL release.

Topics: Calcium; Cholera Toxin; Colforsin; Cyclic AMP; Dopamine; Enzyme Activation; Estradiol; Haloperidol; Immunohistochemistry; Marine Toxins; Oxocins; Pertussis Toxin; Pituitary Neoplasms; Prolactin; Protein Kinase C; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

Cells of the 7315a prolactin-secreting tumour express biochemically normal cell-surface receptors for dopamine. However, dopamine inhibits prolactin release from these cells only when the basal rate of prolactin release is augmented by increasing the intracellular and/or extracellular calcium concentration of the tumour cells. This suggests that dopaminergic modulation of calcium ion flux could have a central physiological role in these neoplastic cells. In 7315a cells we examined the ability of dopamine to regulate 45Ca2+ influx and fractional 45Ca2+ efflux under conditions of enhanced calcium flux using the calcium channel activator, maitotoxin. It was observed that unidirectional calcium influx stimulated by maitotoxin was significantly inhibited by dopamine. Maitotoxin stimulated fractional efflux and prolactin release from the tumour cells and dopamine simultaneously inhibited both processes by a haloperidol-reversible mechanism. Therefore, in 7315a cells dopamine receptor activation is coupled to inhibition of calcium flux as at least one component in the regulation of prolactin release. These cells may provide further opportunity to study intracellular signalling mechanisms that are modulated by dopamine receptor activity.

Topics: Animals; Calcium; Cells, Cultured; Dopamine; Marine Toxins; Oxocins; Pituitary Neoplasms; Prolactin; Rats

Arachidonate and its metabolites may play an important role in the release of prolactin. In the present study, the effect of maitotoxin, a calcium channel activator, was measured on the release of arachidonate and its metabolites from the prolactin-secreting 7315a tumor. Maitotoxin increased the release of prolactin, arachidonate, prostaglandins E2 and F2 alpha (PGE2, PGF2 alpha) and leukotriene C4 (LTC4) from 7315a cells prelabeled with [3H]arachidonate. The magnitude of the increase of prolactin and arachidonate release was decreased in low-calcium medium. The release of arachidonate from cellular phospholipids is necessary for the effect of maitotoxin on prolactin release because quinacrine, an inhibitor of arachidonate hydrolysis from phospholipids, blocked the maitotoxin-induced release of prolactin. The ability of maitotoxin to induce prolactin release appears to require metabolic transformation of arachidonate to its metabolites because BW755c, an inhibitor of the conversion of arachidonate, blocked the maitotoxin-induced prolactin release. In particular, LTC4 may be an important component of the prolactin release process because nordihydroguaiaretic acid and nafazatrom, which block the production of leukotrienes and other lipoxygenase-generated products, decreased LTC4 and prolactin release without affecting arachidonate, PGE2 or PGF2 alpha production. In contrast, indomethacin, a prostaglandin synthesis inhibitor, decreased PGE2 and PGF2 alpha production without affecting LTC4 or prolactin release. These data indicate that release of LTC4 and prolactin are closely linked events in 7315a tumor cells.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Cell Line; Indomethacin; Ion Channels; Marine Toxins; Oxocins; Pituitary Neoplasms; Prolactin; Prostaglandins; Pyrazoles; Pyrazolones; Rats; SRS-A

Reserpine exerts direct effects on several tissues, including inhibition of hormone release from rat anterior pituitary cells. To test the hypothesis that reserpine may be acting as a calcium channel antagonist, normal or GH3 rat anterior pituitary cells were preincubated in reserpine or the conventional calcium channel blocker, D-600, followed by exposure to 45Ca2+ together with stimulants of calcium uptake: maitotoxin, a potent calcium channel activator; A23187, a calcium ionophore; or 50 mMK+. After incubation, the cells were harvested by vacuum filtration and cell-associated radioactivity determined. In normal cells, reserpine blocked both basal and K+-stimulated calcium uptake. Reserpine selectively blocked maitotoxin but not A23187-induced calcium uptake. In GH3 cells 9 microM reserpine and 30 microM D-600 were equally effective in blocking maitotoxin-stimulated calcium uptake. Reserpine appears to block voltage-dependent calcium channels in pituitary cells in a concentration-dependent manner but not calcium uptake caused nonspecifically by A23187.

Topics: Animals; Calcimycin; Calcium; Calcium Channel Blockers; Cell Line; Female; Gallopamil; Ion Channels; Marine Toxins; Oxocins; Pituitary Gland, Anterior; Pituitary Neoplasms; Rats; Rats, Inbred Strains; Reserpine

Maitotoxin has been reported to activate calcium channels and stimulate calcium-dependent functions in several tissues, but a thorough investigation of 45Ca2+ fluxes is lacking. To characterize the influence of maitotoxin on 45Ca2+ flux in greater detail, we incubated dispersed GH3 pituitary tumor cells in 45Ca2+ with maitotoxin and other agents affecting calcium channels. Within 10 sec of exposure, maitotoxin induced a net calcium influx in cells at isotopic equilibrium. Calcium uptake was concentration dependent between 0.4 and 40 ng/ml maitotoxin and was inhibited by antagonists of voltage-dependent calcium channels but not by inhibitors of sodium channels. PRL and GH release from perifused GH3 cells was stimulated within 1 min by maitotoxin. We conclude that maitotoxin causes a rapid, concentration-dependent influx of calcium through presumed voltage-dependent endogenous calcium channels, culminating in enhanced hormone release. This potent toxin may provide a more precise understanding of the role of calcium in the stimulus-secretion coupling process.

Topics: Animals; Calcium; Cell Line; Gallopamil; Growth Hormone; Ion Channels; Manganese; Marine Toxins; Nifedipine; Oxocins; Pituitary Gland; Pituitary Neoplasms; Potassium; Prolactin; Rats; Time Factors

The rat pituitary tumor 7315a secretes PRL and ACTH. Although dopamine has no effect on unstimulated PRL release from this tumor, dopamine decreases the adenylate cyclase activity in tumor cell homogenates in a manner similar to that in normal pituitary tissue. However, it was observed that under basal conditions, 7315a tumor cells have an abnormal calcium metabolism because 1) basal PRL release from tumor cells is not modified by the calcium channel blocker D-600 and is only moderately decreased by low calcium, treatments that markedly decrease normal pituitary PRL release; 2) D-600 had no effect on basal 7315a tumor calcium uptake, but blocked the increase in calcium uptake due to the calcium channel activator maitotoxin; 3) increasing the medium Ca+2 concentration above 5 mM increases 7315a PRL release, whereas this treatment decreases PRL release from normal pituitary cells. Maitotoxin and the calcium ionophore A23187 increased 7315a tumor cell PRL release in a manner similar to that in normal pituitary cells. Because dopamine blocks PRL release induced by maitotoxin, A23187, or elevated medium calcium concentration in 7315a tumor cells, the refractoriness of basal 7315a tumor cell PRL release to dopamine may be due to the abnormal calcium balance of the tumor cells under basal conditions.

Topics: Adenylyl Cyclases; Animals; Calcimycin; Calcium; Cells, Cultured; Dopamine; Female; Gallopamil; Marine Toxins; Neoplasm Transplantation; Oxocins; Pituitary Gland, Anterior; Pituitary Neoplasms; Prolactin; Rats; Rats, Inbred Strains