melitten and Pituitary-Neoplasms

ATP-sensitive K+ (KATP) channels have been characterized in pituitary GH3 cells with the aid of the patch-clamp technique. In the cell-attached configuration, the presence of diazoxide (100 microM) revealed the presence of glibenclamide-sensitive KATP channel exhibiting a unitary conductance of 74 pS. Metabolic inhibition induced by 2,4-dinitrophenol (1 mM) or sodium cyanide (300 microM) increased KATP channel activity, while nicorandil (100 microM) had no effect on it. In the inside-out configuration, Mg-ATP applied intracellularly suppressed the activity of KATP channels in a concentration-dependent manner with an IC50 value of 30 microM. The activation of phospholipase A2 caused by mellitin (1 microM) was found to enhance KATP channel activity and further application of aristolochic acid (30 microM) reduced the mellitin-induced increase in channel activity. The challenging of cells with 4,4'-dithiodipyridine (100 microM) also induced KATP channel activity. Diazoxide, mellitin and 4,4'-dithiodipyridine activated the KATP channels that exhibited similar channel-opening kinetics. In addition, under current-clamp conditions, the application of diazoxide (100 microM) hyperpolarized the membrane potential and reduced the firing rate of spontaneous action potentials. The present study clearly indicates that KATP channels similar to those seen in pancreatic beta cells are functionally expressed in GH3 cells. In addition to the presence of Ca(2+)-activated K+ channels, KATP channels found in these cells could thus play an important role in controlling hormonal release by regulating the membrane potential.

Topics: 2,4-Dinitrophenol; Action Potentials; Adenoma; Adenosine Triphosphate; Animals; Aristolochic Acids; Calcium Chloride; Diazoxide; Disulfides; Enzyme Activation; Ion Transport; Magnesium; Melitten; Nicorandil; Phenanthrenes; Phospholipases A; Phospholipases A2; Pituitary Gland, Anterior; Pituitary Neoplasms; Potassium; Potassium Channels; Pyridines; Rats; Sodium Cyanide; Tumor Cells, Cultured

The selective immunosuppressants cyclosporin A (CsA) and tacrolimus (FK506) are used in the prevention of allogenic transplant rejection and in the therapy of chronic autoimmune inflammatory pathologies. Chronic treatment with CsA leads to secondary functional and trophic alterations of multiple organs and cell systems among which endocrine ones, through insofar uncharacterized mechanisms. With the recent use of FK506 there have been reports of an improved therapeutic efficacy and a reduction of side-effects, as compared to CsA. An intriguing hypothesis is that toxic damage could be due to a systemic CsA activation of arachidonic acid (AA) metabolism, through pathways as yet only partially characterized. The side-effects of both drugs have been poorly studied on cells from tissues other than blood or kidney. We have thus proceeded to study their action on AA release in corticotropic AtT-20/D16-16 cells. The results obtained are as follows: 1) during incubation times > or =12 h, basal AA release is increased by CsA, but not FK506; the acute effect (10 min) of melittin, a PLA2 activator, is significantly potentiated starting from a 30 min pretreatment with CsA but not FK506; manoalide, a PLA2 inhibitor, antagonizes the melittin potentiation of AA release by CsA whereas the inhibition of the melittin stimulus by glucocorticoids is antagonized both by CsA and FK506. 2) during longer (>2 d) incubation times, cell growth is inhibited by CsA but not FK506. These results indicate a role for CsA, not apparent for FK506, in the activation of PLA2 and in the inhibition of cell growth. They also suggest that CsA does not have a direct (i.e. not mediated by the immune system) therapeutic effect in inflammatory processes.

Topics: Animals; Arachidonic Acid; Cell Division; Cell Size; Cell Survival; Cyclosporine; Dexamethasone; Digitonin; Dose-Response Relationship, Drug; Enzyme Activation; Immunosuppressive Agents; Interleukin-1; Melitten; Mice; Phospholipases A; Phospholipases A2; Pituitary Neoplasms; Tacrolimus; Terpenes; Time Factors; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vitamin E

The dihydropyridine calcium channel activator, BAY-K-8644, stimulates cGMP formation in ACTH-secreting mouse AtT-20 clonal corticotrophs. The recent report that calmodulin antagonists could inhibit dihydropyridine binding in several tissues suggested that these agents might also affect the cyclic nucleotide response to BAY-K-8644. In fact, TMB-8, trifluoperazine, and melittin, described as in vitro antagonists of calmodulin-dependent enzyme activities, all inhibited BAY-K-8644 induced cGMP synthesis in a concentration-dependent manner. The antagonists had no effect on cGMP formation stimulated by sodium nitroprusside or sodium azide. The calcium channel antagonist, nifedipine, did not stimulate cGMP formation nor did it alter the effect of BAY-K-8644 on accumulation of the nucleotide; one explanation thus is that the cyclase involved in cGMP formation is coupled to a low affinity binding site for BAY-K-8644, which is less accessible to other dihydropyridines. The relation of cyclic GMP formation to the function of the calcium channel in AtT-20 cells remains unknown.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Calcium Channel Blockers; Calmodulin; Cells, Cultured; Cyclic GMP; Gallic Acid; Humans; Ion Channels; Melitten; Pituitary Neoplasms; Trifluoperazine

TRH stimulates the secretion of PRL by clonal GH3 pituitary cells. The studies of the accompanying paper have shown that the secretory response during the first 30-60 min is biphasic (phase I, 0-3 min; phase II, 5-60 min) and that the phase I response may be mediated through mechanisms involving Ca+2 translocation. In previous studies, it has been shown that TRH treatment rapidly induces the breakdown of inositol phospholipids with accompanying diacylglycerol accumulation. In this paper, we present evidence for a possible role for diacylglycerol as a second messenger which mediates the phase II response to TRH. A role for lipid-dependent mechanisms in regulating PRL secretion in GH3 cells was supported by the finding that phospholipase C, phorbol esters, melittin, and exogenous diacylglycerols were effective secretagogues in GH3 cells. Secretion promoted by these agents was found to be persistent, as was the phase II response to TRH. For three of the agents examined (TRH, phorbol esters, and phospholipase C), stimulated PRL release was found to be nonadditive, suggesting the presence of some common element in the pathways by which these agents exert their effects. This lipid-linked pathway of activation was distinguished from a cAMP-mediated pathway.

Topics: Animals; Bacillus cereus; Calcium; Cell Line; Clostridium perfringens; Diglycerides; Glycerides; Melitten; Pituitary Neoplasms; Prolactin; Protein Kinase C; Protein Kinases; Rats; Tetradecanoylphorbol Acetate; Thyrotropin-Releasing Hormone; Time Factors; Type C Phospholipases