guanosine-triphosphate and Pituitary-Neoplasms

Topics: Animals; Binding Sites; Cholera Toxin; DNA Mutational Analysis; Enzyme Activation; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Mutation; Neoplasm Proteins; Oncogenes; Pituitary Neoplasms; Protein Conformation; Proto-Oncogene Proteins p21(ras)

Multiple physiologic estrogens (estradiol, estriol, and estrone), as well as xenoestrogenic compounds (including alkylphenols and bisphenol A), can act via nongenomic signaling initiated by liganding of the plasma membrane estrogen receptor-α (mERα). We examined heterotrimeric G protein involvement leading to extracellular-regulated kinase (ERK) activation in GH3/B6/F10 rat anterior pituitary tumor cells that express abundant mERα, and smaller amounts of mERβ and GPR30. A combination of microarrays, immunoblots, and quantitative immunoassays demonstrated the expression of members of all α, β, and γ G protein classes in these cells. Use of selective inhibitors showed that the G(αi) subtype was the primary initiator of downstream ERK signaling. Using antibodies against the GTP-bound form of G(α) protein subtypes i and s, we showed that xenoestrogens (bisphenol A, nonylphenol) activated G(αi) at 15-30s; all alkylphenols examined subsequently suppressed activation by 5min. GTP-activation of G(αi) for all estrogens was enhanced by irreversible cumulative binding to GTPγS. In contrast, G(αs) was neither activated nor deactivated by these treatments with estrogens. ERα and G(αi) co-localized outside nuclei and could be immuno-captured together. Interactions of ERα with G(αi) and caveolin I were demonstrated by epitope proximity ligation assays. An ERα/β antagonist (ICI182780) and a selective disruptor of caveolar structures (nystatin) blocked estrogen-induced ERK activation.. Xenoestrogens, like physiologic estrogens, can evoke downstream kinase signaling involving selective interactions of ERα with G(αi) and caveolin I, but with some different characteristics, which could explain their disruptive actions.

Topics: Animals; Benzhydryl Compounds; Caveolae; Caveolin 1; Cell Line, Tumor; Cell Membrane; Estrogen Receptor alpha; Estrogens; Estrogens, Non-Steroidal; Extracellular Signal-Regulated MAP Kinases; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine Triphosphate; Immunoblotting; MAP Kinase Signaling System; Microscopy, Fluorescence; Oligonucleotide Array Sequence Analysis; Phenols; Pituitary Neoplasms; Protein Binding; Rats

The divalent cation selective ionophores A23187 and ionomycin were compared for their effects on the Ca2+ contents, nucleotide contents, and protein synthetic rates of several types of cultured cells. Both ionophores reduced amino acid incorporation by approximately 85% at low concentrations (50-300 nmol/L) in cultured mammalian cells without reducing ATP or GTP contents. At these concentrations A23187 and ionomycin each promoted substantial Ca2+ efflux, whereas at higher concentrations a large influx of the cation was observed. Ca2+ influx occurred at lower ionophore concentrations and to greater extents in C6 glioma and P3X63Ag8 myeloma than in GH3 pituitary cells. The ATP and GTP contents of the cells and their ability to adhere to growth surfaces declined sharply at ionophore concentrations producing increased Ca2+ influx. Prominent reductions of nucleotide contents occurred in EGTA-containing media that were further accentuated by extracellular Ca2+. Ionomycin produced more Ca2+ influx and nucleotide decline than comparable concentrations of A23187. The inhibition of amino acid incorporation and mobilization of cell-associated Ca2+ by ionomycin were readily reversed in GH3 cells by fatty acid-free bovine serum albumin, whereas the effects of A23187 were only partially reversed. Amino acid incorporation was further suppressed by ionophore concentrations depleting nucleotide contents. Mitochondrial uncouplers potentiated Ca2+ accumulation in response to both ionophores. At cytotoxic concentrations Lubrol PX abolished protein synthesis but did not cause Ca2+ influx. Nucleotide depletion at high ionophore concentrations is proposed to result from increased plasmalemmal Ca2+-ATPase activity and dissipation of mitochondrial proton gradients and to cause intracellular Ca2+ accumulation. Increased Ca2+ contents in response to Ca2+ ionophores are proposed as an indicator of ionophore-induced cytotoxicity.

Topics: Adenosine Triphosphate; Animals; Blood Proteins; Calcimycin; Calcium; Detergents; Guanosine Triphosphate; Ionophores; Mice; Mitochondria; Nucleotides; Pituitary Neoplasms; Polidocanol; Polyethylene Glycols; Protein Biosynthesis; Sclerosing Solutions; Serum Albumin; Tumor Cells, Cultured

The expression of the pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide (PACAP/VIP) receptor subtypes was evaluated in the normal rat pituitary gland and in different rat spontaneous transplantable SMtTW tumours (SMtTW2 which expresses prolactin (PRL), SMtTW10 which expresses GH and SMtTW3 which expresses both PRL and GH) by measurement of PACAP/VIP-stimulated adenylate cyclase activity and detection of the presence of mRNA coding for the different receptor forms. In normal glands, the order of potency of the peptides suggested that adenylate cyclase activity was mediated through interaction with PACAP selective receptors (PACAP I receptors); mRNAs coding for the PACAP I receptor, but also for the PACAP II VIP2 receptor, were detected. In SMtTW2 tumours, the functional response was close to that observed in the presence of PACAP II VIP2 receptors; mRNAs coding for PACAP I and PACAP II VIP1 and PACAP II VIP2 receptors were detected. In the SMtTW10 tumours, the functional response was complex but compatible with the involvement of PACAP I and PACAP II receptors; mRNAs coding for the PACAP I and PACAP II VIP1 receptors were detected. In the SMtTW3 tumour, the profile was similar to that of the normal pituitary gland and the mRNA coding for the PACAP I receptor only was detected. Thus, while the control of normal pituitary gland adenylate cyclase activity by PACAP and VIP was mediated by PACAP-selective receptors, in spontaneous transplantable tumours a variable profile was observed and PACAP, as well as VIP1 and VIP2 receptors, may contribute to the responses.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Colforsin; DNA Primers; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Neoplasm Transplantation; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Pituitary Gland; Pituitary Neoplasms; Polymerase Chain Reaction; Rats; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Peptide, Type II; Reference Values; RNA, Messenger; Sodium Fluoride; Transcription, Genetic

1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of protein phosphatase involvement in the late stages of the secretory pathway for adrenocorticotrophin (ACTH) secretion. The effects of the type 1 and 2 phosphatase inhibitor calyculin A upon calcium-, guanine nucleotide- and phorbol 12-myristate 13-acetate (PMA)-stimulated ACTH secretion from electrically-permeabilized AtT-20 cells were studied. 2. Calyculin A (1 nM-1 microM) inhibited both calcium (10 microM)- and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) (100 microM)-evoked ACTH secretion from permeabilized cells in a concentration-dependent manner. These effects were maximal with 100 nM calyculin A. 3. ACTH secretion was stimulated from electrically-permeabilized cells when the cytosolic free calcium ion concentration, controlled by calcium-EGTA buffers, was raised over the concentration range of 100 nM to 10 microM. This calcium-stimulated ACTH secretion was inhibited by co-incubation with calyculin A (100 nM). 4. GTP-gamma-S (10 nM-100 microM) stimulated ACTH secretion from permeabilized cells at concentrations greater than 1 microM GTP-gamma-S. Co-incubation with calyculin A (100 nM) inhibited this stimulation of ACTH secretion observed at these concentrations of GTP-gamma-S. 5. PMA (100 nM) significantly stimulated ACTH secretion from permeabilized cells in the absence of either calcium and guanine nucleotides and this action was enhanced by calyculin A (100 nM). Furthermore, an inhibition of GTP-gamma-S (100 microM)-stimulated ACTH secretion observed in the presence of calyculin A (100 nM) was not observed in the presence of PMA (100 nM). 6. The results of the present study indicate that dephosphorylation by phosphatases plays an important role in stimulus-secretion coupling in AtT-20 cells and is involved in mediating the effects of GE upon the secretory apparatus in these cells. Furthermore, the point of regulation of the secretory response by PKC which underlies the ability of PKC to amplify the calcium/GE system may lie distal to both GE and these phosphatases.

Topics: Adrenocorticotropic Hormone; Animals; Calcium; Cells, Cultured; Dose-Response Relationship, Drug; Guanine Nucleotides; Guanosine Triphosphate; Marine Toxins; Mice; Oxazoles; Phosphoprotein Phosphatases; Pituitary Neoplasms; Radioimmunoassay; Tetradecanoylphorbol Acetate

We used the PCR amplification technique in an attempt to characterize further the dopamine D2L receptor expressed in the prolactin-secreting pituitary MMQ cell clone, derived from the prolactin- and ACTH-secreting Buffalo rat 7315 alpha pituitary tumour. By semiquantitative PCR amplification we were unable to detect the mRNA encoding the D2S receptor isoform, which derives from the well-known process of alternative splicing, producing two D2 receptor subtypes (D2L and D2S) in such tissues as the anterior pituitary and the corpus striatum. Although the pharmacology of the D2 receptor has been established in many studies on both native receptors and transfected receptor isoforms, because of the lack of tissues naturally expressing only one receptor isoform, MMQ cells represent the first example of cells uniquely or prevalently expressing only the D2L receptor, conceivably coupled to its native transduction mechanisms. These considerations prompted us to evaluate the pharmacology and the second messenger systems known to be modulated by dopamine. Scatchard analysis of [3H]spiperone binding resulted in a linear plot, consistent with the existence of a single class of binding sites, with a Kd of 0.055 +/- 0.002 nM and a Bmax of 27 +/- 3.5 fmol/mg protein. Competition experiments confirmed the GTP-dependence and the order of potency for agonist and antagonist ligands consistent with binding to a D2 receptor. The inhibitory effects of dopamine on adenylyl cyclase activity, inositol phosphate production and intracellular free calcium concentrations, the latter presumably via the opening of K+ channels, and prolactin secretion, as well as the reversal of the effect by the D2-selective antagonist (-)sulpiride and pretreatment with pertussis toxin, are consistent with the known biological actions of dopamine at D2 receptors. Based on our observations, the MMQ cell line can be considered a useful tool for investigating ligand-receptor interactions to develop new selective dopaminergic D2L ligands for the therapy of dopamine-related disorders such as schizophrenia, depression, Parkinson's disease and drug addiction.

Topics: Adenylyl Cyclases; Animals; Base Sequence; Calcium; Clone Cells; Cloning, Molecular; Dopamine Agonists; Dopamine Antagonists; Guanosine Triphosphate; Hemolytic Plaque Technique; Inositol Phosphates; Ion Channel Gating; Membrane Potentials; Molecular Sequence Data; Neoplasm Proteins; Pituitary Neoplasms; Polymerase Chain Reaction; Potassium Channels; Prolactin; Radioligand Assay; Rats; Rats, Inbred BUF; Receptors, Dopamine D2; Second Messenger Systems; Signal Transduction; Tumor Cells, Cultured

The 7315c cell, derived from a rat anterior pituitary tumor, expresses an angiotensin II (AII) receptor. [3H]AII binds to 7315c membranes specifically and saturably (Kd = 2.1 +/- 0.6 x 10(-6) M, Bmax = 282 +/- 33 fmol/mg of protein). GTP diminished the affinity of the membranes for [3H]AII (Kd = 4.1 +/- 0.4 x 10(-9) M, Bmax = 210 +/- 26 fmol/mg of protein). [3H]AII binding was displaced by AII (Ki = 1.3 +/- 0.6 x 10(-9) M), angiotensin III (AIII) (Ki = 0.9 +/- 0.4 x 10(-9) M), and the nonpeptide AII antagonist DuP753 (Ki = 1.4 +/- 0.6 x 10(-8) M). In contrast, a second nonpeptide AII ligand, PD123177, did not compete for [3H]AII binding sites. In intact cells, AII and AIII stimulated inositol trisphosphate (IP3) production (EC50 = 1.1 +/- 0.6 x 10(-8) M and 1.1 +/- 0.5 x 10(-8) M, respectively); this response to AII was antagonized by DuP753 (Ki = 1.7 +/- 0.3 x 10(-7) M). Pertussis toxin treatment failed to affect the ability of AII to stimulate IP3 production. In a crude membrane preparation, GTP was required for maximal AII-induced IP3 stimulation; guanosine thio-diphosphate abolished the agonist-GTP stimulation of IP3 production, in a concentration-dependent fashion. AII and AIII also inhibited adenylyl cyclase (EC50 = 2.9 +/- 1.1 x 10(-8) M and 6.0 +/- 1.0 x 10(-8) M, respectively). DuP753 antagonized the inhibition by AII of adenylyl cyclase (Ki = 2.8 +/- 0.4 x 10(-8) M). PD123177 failed to antagonize AII-induced cyclase inhibition. Pertussis toxin treatment abolished the AII and AIII inhibition of adenylyl cyclase. GTP was required for AII-induced inhibition of adenylyl cyclase. These data suggest that, in 7315c cells, a single subtype of AII receptor, identified by DuP753, is capable of regulating two different guanine nucleotide-binding protein (G protein) signalling pathways; one G protein, which is insensitive to pertussis toxin, stimulates IP3 production and the other G protein, which is sensitive to pertussis toxin, inhibits adenylyl cyclase.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Angiotensin II; Angiotensin Receptor Antagonists; Animals; Biphenyl Compounds; Cell Membrane; GTP-Binding Proteins; Guanosine Triphosphate; Imidazoles; Losartan; Pertussis Toxin; Phosphatidylinositols; Pituitary Gland, Anterior; Pituitary Neoplasms; Rats; Rats, Inbred BUF; Receptors, Angiotensin; Signal Transduction; Sulfhydryl Reagents; Tetrazoles; Tritium; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

An invasive TSH-secreting adenoma inducing mild hyperthyroidism was diagnosed in a 16-year-old male. Initial surgical treatment led to a temporary clinical and biological improvement. Recurrence of the thyrotoxicosis was treated with the somatostatin analogue, SMS 201-995 (octreotide) with normalization of the serum thyroid hormone levels with a dose of 200 micrograms per day. With immunoelectron microscopy, the tumour cells appeared poorly granulated with small secretory granules located at the periphery of the cells; only part of those were immunoreactive with an anti-TSH beta monoclonal antibody. No specific TRH binding site was found in a tumour membrane preparation. By quantitative autoradiography, somatostatin specific binding sites were as numerous in the TSH-secreting tumour as in control GH-secreting tumours. Binding kinetics and guanosine triphosphate dependency of the binding were equivalent in the TSH and GH tumours tested. Although all of the tumour cells displayed the same ultrastructural features, some were non-immunoreactive, suggesting that they could secrete an altered form of TSH. The absence of TRH receptors in the tumour cells is in accordance with previous reports on this type of tumour. We confirm the efficiency of octreotide treatment in this case of neoplastic TSH inappropriate secretion. The therapeutic effect of octreotide goes along with the presence of a high density of guanine nucleotide-dependent somatostatin binding sites in the tumour cells.

Topics: Adenoma; Adolescent; Antibodies, Monoclonal; Autoradiography; Growth Hormone; Guanosine Triphosphate; Humans; Hyperthyroidism; Male; Microscopy, Electron; Octreotide; Pituitary Neoplasms; Receptors, Neurotransmitter; Receptors, Somatostatin; Thyrotropin; Thyrotropin-Releasing Hormone

The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and thyroliberin exerted additive stimulatory effects on prolactin release and synthesis in rat adenoma GH4C1 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'-[beta,gamma-imino]triphosphate or forskolin, nor did it affect the enzyme's apparent affinity (basal, 7.2 mM; thyroliberin-enhanced, 2.2 mM) for free Mg2+. The TPA-mediated adenylate cyclase activation was entirely dependent on exogenously added guanosine triphosphate. ED50 (dose yielding half-maximal activation) was 60 microM. 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, 4 alpha-phorbol didecanoate, by the protein kinase C inhibitor polymyxin B and by pertussis toxin, while thyroliberin-sensitive adenylate cyclase remained unaffected. Experimental conditions known to translocate protein kinase C to the plasma membrane and without inducing adenylate cyclase desensitization, increased both basal and thyroliberin-stimulated enzyme activities, while absolute TPA-enhanced adenylate cyclase was maintained. Association of extracted GTP-binding inhibitory protein, Gi, from S49 cyc- murine lymphoma cells with GH4C1 cell membranes yielded a reduction of basal and hormone-stimulated adenylate cyclase activities, while net inhibition of the cyclase of somatostatin was dramatically enhanced. However, TPA restored completely basal and hormone-elicited adenylate cyclase activities in the Gi-enriched membranes. Finally, TPA completely abolished the somatostatin-induced inhibition of adenylate cyclase in both hybrid and non-hybrid membranes. These data suggest that, in GH4C1 cells, protein kinase C stimulation by phorbol esters completely inactivates the n alpha i subunit of the inhibitory GTP-binding protein, leaving the n beta subunit functionally intact. It can also be inferred that thyroliberin conveys its main effect on the adenylate cyclase through activation of the stimulatory GTP-binding protein, Gs.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Alkylation; Animals; Calcium; Colforsin; Cyclic AMP; Enzyme Activation; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Pertussis Toxin; Pituitary Neoplasms; Polymyxin B; Prolactin; Protein Kinase C; Rats; Somatostatin; Tetradecanoylphorbol Acetate; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured; Virulence Factors, Bordetella

The neuropeptide bombesin acts on a variety of target cells to stimulate the processes of secretion and cell proliferation. In this study we determined whether bombesin receptors interact with known guanine nucleotide-binding proteins in four different cell types: GH4C1 pituitary cells, HIT pancreatic islet cells, Swiss 3T3 fibroblasts, and rat brain tissue. Maximal concentrations of nonhydrolyzable GTP analogs decreased agonist binding to bombesin receptors in membranes from all four sources. In GH4C1 and HIT cell membranes GTP analogs inhibited bombesin receptor binding with IC50 values of about 0.1 microM, whereas GDP analogs were approximately 10-fold less potent. In contrast, GMP and the nonhydrolyzable ATP analog adenylyl-imidodiphosphate had no effect at 100 microM. Equilibrium binding experiments in GH4C1 and HIT cell membranes indicated a single class of binding sites with a dissociation constant (Kd) for [125I-Tyr4]bombesin of 24.4 +/- 7.0 pM and a binding capacity of 176 +/- 15 fmol/mg protein. Guanine nucleotides decreased the apparent affinity of the receptors without significantly changing receptor number. Consistent with this observation, guanine nucleotides also increased the rate of ligand dissociation. Pretreatment of GH4C1 or HIT cells with either pertussis toxin (100 ng/ml) or cholera toxin (500 ng/ml) for 18 h did not affect agonist binding to membrane bombesin receptors, its regulation by guanine nucleotides, or bombesin stimulation of hormone release. Although pertussis toxin pretreatment has been reported to block bombesin stimulation of DNA synthesis in Swiss 3T3 cells, it did not alter the binding properties of bombesin receptors in Swiss 3T3 membranes or inhibit the rapid increase in intracellular [Ca2+] produced by bombesin in these cells. In summary, our results indicate that the bombesin receptor interacts with a guanine nucleotide-binding protein which exhibits a different toxin sensitivity from those which regulate adenylate cyclase as well as those which couple some receptors to phospholipases.

Topics: Adenylate Cyclase Toxin; Algorithms; Animals; Bombesin; Brain; Cholera Toxin; Fibroblasts; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guanylyl Imidodiphosphate; Islets of Langerhans; Kinetics; Mice; Pertussis Toxin; Pituitary Neoplasms; Rats; Receptors, Bombesin; Receptors, Neurotransmitter; Receptors, Somatostatin; Thionucleotides; Tumor Cells, Cultured; Virulence Factors, Bordetella

The role of GTP on somatostatin-induced K+ current increase was examined in dissociated human pituitary tumor cells obtained from three acromegalic patients. Pituitary cells in culture were voltage-clamped by using the patch clamp technique in the whole-cell configuration. Somatostatin (100 nM) increased the membrane permeability to K+ ions and inhibited hormone secretion. A current-voltage relation of the somatostatin-induced K+ current showed an inward rectification when the concentration of extracellular K+ ions was increased. The amplitude of the somatostatin-induced K+ current decreased during recording when the patch pipette solution did not contain GTP; addition of 100 microM GTP to the patch pipette solution prevented this reduction. Intracellular application of 100 microM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] evoked an inward rectifying K+ conductance in the absence of somatostatin. After the GTP[gamma S]-induced K+ conductance reached a steady level, application of somatostatin did not further increase the K+ conductance. In pertussis toxin-treated cells GTP[gamma S] did not evoke K+ conductance. It was concluded that somatostatin-induced K+ channels were regulated by a GTP-binding protein.

Topics: Acromegaly; Adenoma; Drug Interactions; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Membrane Potentials; Pertussis Toxin; Pituitary Neoplasms; Potassium; Somatostatin; Thionucleotides; Tumor Cells, Cultured; Virulence Factors, Bordetella

The molecular mechanisms of somatostatin (SRIF) desensitization were investigated in the anterior pituitary tumor cell line AtT-20. Previous studies have shown that pretreatment of AtT-20 cells with SRIF analogs desensitizes the cells to SRIF inhibition of hormone release, cyclic AMP formation and calcium influx. This desensitization may involve a change in the properties of the SRIF receptors. Pretreatment of AtT-20 cells with Trp8-SRIF reduced the binding of the SRIF analog [125I]CGP 23996 (des-Alal, Gly2-[desamino-Cys3, Tyr11]-3, 14-dicarbasomatostatin) to AtT-20 cell membranes. The loss of [125I]CGP 23996 binding was dependent on the time of Trp8-SRIF treatment and was reversible. The ability of GTP analogs to inhibit [125I]CGP 23996 binding was reduced after Trp8-SRIF treatment, suggesting that the SRIF receptor and the inhibitory G proteins become uncoupled during desensitization. This is indicated further by the decrease in SRIF stimulation of GTPase activity and SRIF inhibition of forskolin-stimulated adenylyl cyclase activity in desensitized membranes. The reduction and recovery of SRIF inhibition of adenylyl cyclase activity after Trp8-SRIF pretreatment has a similar time course as the changes in [125I]CGP 23996 binding. GTP inhibition of forskolin-stimulated adenylyl cyclase activity is also reduced in SRIF-desensitized membranes. The loss of the GTP effect occurs rapidly and does not fully recover after Trp8-SRIF pretreatment. The levels of ADP-ribosylation of inhibitory GTP binding protein, the relative quantity of the alpha subunits of the inhibitory G proteins and their electrophoretic mobility after 2-dimensional gel electrophoretic analysis, are not altered in SRIF-desensitized membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclase Inhibitors; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Male; Pituitary Neoplasms; Receptors, Neurotransmitter; Receptors, Somatostatin; Somatostatin; Tumor Cells, Cultured

The agonist high- and low-affinity states of the dopamine (DA) receptor were investigated with apomorphine competition for [3H]spiperone binding to DA receptors in 7315a tumors grown in intact female rats, while the antagonist site was investigated with saturation of [3H]spiperone binding. Such as for the intact pituitary, the antagonist binding site density in 7315a tumors was not affected by NaCl and/or Gpp(NH)p, and its binding affinity was increased in the presence of NaCl. The DA receptor in 7315a tumors existed in high- and low-affinity agonist states, and the two apomorphine sites had similar affinities in tumoral and intact tissue. However, the proportion of the high affinity state was slightly lower in the 7315a tumor compared to intact tissue. Tumor (7315a) growth in ovariectomized rats was slower than in intact animal; chronic 17 beta-estradiol treatment inhibited growth of these tumors. Prolactin (PRL) concentration and density of DA receptors were higher in tumors grown in ovariectomized than in intact female rats, whereas both decreased after 23 days of 17 beta-estradiol treatment. Estradiol treatment decreased the affinity of the high- and the low-apomorphine binding sites, whereas their proportions remained unchanged. Thus, changes of DA receptors and 7315a tumor growth seem to be related; however, their relationship is complex.

Topics: Animals; Binding Sites; Estradiol; Female; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Pituitary Neoplasms; Prolactin; Rats; Rats, Inbred BUF; Receptors, Dopamine; Sodium Chloride; Spiperone

Somatostatin (SS) inhibits secretion from many cells, including clonal GH3 pituitary cells, by a complex mechanism that involves a pertussis toxin (PTX)-sensitive step and is not limited to its cAMP lowering effect, since secretion induced by cAMP analogs and K+ depolarization are also inhibited. SS also causes membrane hyperpolarization which may lead to decreases in intracellular Ca2+ need for secretion. Using patch clamp techniques we now demonstrate: 1) that both (SS) and acetylcholine applied through the patch pipette to the extracellular face of a patch activate a 55-picosiemens K+ channel without using a soluble second messenger; 2) that, after patch excision, the active state of the ligand-stimulated channel is dependent on GTP in the bath, is abolished by treatment of the cytoplasmic face of the patch with activated PTX and NAD+, and after inactivation by PTX, is restored in a GTP-dependent manner by addition of a nonactivated human erythrocyte PTX-sensitive G protein, and 3) that the 55-picosiemens K+ channel can also be activated in a ligand-independent manner with guanosine [gamma-thio] triphosphate (GTP gamma S) or with Mg2+/GTP gamma S-activated erythrocyte G protein. We call this protein GK. It is an alpha-beta-gamma trimer of which we have previously shown that the alpha-subunit is the substrate for PTX and that it dissociates on activation with Mg2+/GTP gamma S into alpha-GTP gamma S plus beta-gamma. A similarly activated and dissociated preparation of GS, the stimulatory regulatory component of adenylyl cyclase, having a different alpha-subunit but the same beta-gamma-dimer, was unable to cause K+ opening.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carbachol; Cell Membrane; Clone Cells; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Ion Channels; Membrane Potentials; Pituitary Gland, Anterior; Pituitary Neoplasms; Potassium; Rats; Receptors, Muscarinic; Somatostatin; Thionucleotides

Dopamine receptors in the 7315a transplantable rat anterior pituitary tumor were characterized using radioligand binding assays with [3H]spiroperidol ([3H]SPD) and assays of adenylate cyclase activity. Scatchard analysis of the binding of [3H]SPD yielded linear plots and a Kd value of 73 pM. Studies of the inhibition of the binding of [3H]SPD were performed with a series of competing ligands, including the antagonists domperidone, (+)-butaclamol and sulpiride and the agonists dopamine, bromocriptine and N-propylnorapomorphine. Inhibition curves for the antagonists gave Hill coefficients of approximately 1, consistent with the presence of only a single class of binding sites with a high affinity for [3H]SPD. In contrast, the Hill coefficient for dopamine was significantly less than 1. When assays were carried out in the presence of 300 microM GTP, the inhibition curve for dopamine was shifted to the right and the Hill coefficient increased to approximately 1. An effect of GTP on the affinity of a receptor for agonists is consistent with the existence of at least two agonist affinity states. Inhibition of the binding of [3H]SPD by the partial agonist bromocriptine was not affected when assays were carried out in the presence of GTP. The uniform low affinity of the selective serotonin antagonist ketanserin for these sites indicated that the radioligand was not labeling serotonin-2 receptors in this tissue. A good correlation was observed between the Ki values for competing ligands measured in the tumor and in homogenates of rat striatal tissue. Dopamine was shown to inhibit forskolin-stimulated adenylate cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclase Inhibitors; Animals; Apomorphine; Colforsin; Dopamine; Female; Guanosine Triphosphate; Pituitary Gland, Anterior; Pituitary Neoplasms; Rats; Rats, Inbred BUF; Receptors, Dopamine; Receptors, Dopamine D2; Spiperone

The mechanisms by which somatostatin (SRIF) inhibits CRF-induced ACTH secretion from AtT20 cells were characterized by comparing the effects of SRIF on cAMP production, adenylate cyclase activity, and activation of cAMP-dependent protein kinase isoenzymes with its effects on ACTH release. In isolated membranes, CRF (100 nM) stimulated adenylate cyclase activity 4- to 5-fold. SRIF inhibited CRF-stimulated adenylate cyclase in a concentration-dependent manner. However, maximal inhibition was 50%. SRIF did not inhibit basal adenylate cyclase or forskolin-stimulated cyclase in the absence of guanine nucleotides and had only small effects on forskolin-stimulated cyclase when assayed in the presence of guanine nucleotides. CRF (100 nM) induced small rises (2-fold) in intracellular cAMP levels which produced maximal ACTH release. SRIF inhibited basal and CRF-stimulated ACTH release in a concentration-dependent manner, and there was a good correlation between inhibition of ACTH release and inhibition of the activation of cAMP-dependent protein kinases in these cells. Thus, the effect of SRIF on CRF-induced ACTH release appeared to result from its effect on inhibition of adenylate cyclase. In the presence of 3-methylisobutylxanthine (MIX), CRF increased cAMP levels 20-fold and activated a greater proportion of cAMP-dependent protein kinase, but did not stimulate ACTH release more than CRF alone. Under these conditions, SRIF (100 nM) inhibited cAMP accumulation by 90%. ACTH release was also inhibited, but higher concentrations of SRIF were required to block ACTH release compared to cells incubated in the absence of MIX. Sufficient cAMP levels were achieved so that activation of cAMP-dependent protein kinases was only partially blocked. There was still sufficient cAMP to activate cAMP-dependent protein kinase to an extent equal to that seen with CRF without MIX. Similar effects of SRIF on cAMP accumulation and protein kinase activation were seen when cells were stimulated with forskolin. Our results demonstrate that SRIF inhibits ACTH release from AtT20 cells by inhibiting hormone-sensitive adenylate cyclase and thereby prevents the activation of cAMP-dependent protein kinases. However, under conditions where cAMP-dependent protein kinases are still sufficiently active to induce ACTH secretion, high concentrations of SRIF can inhibit ACTH release by a mechanism independent of cAMP-dependent protein kinase.

Topics: 1-Methyl-3-isobutylxanthine; Adenylyl Cyclases; Adrenocorticotropic Hormone; Animals; Cell Line; Colforsin; Corticotropin-Releasing Hormone; Cyclic AMP; Enzyme Activation; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guanylyl Imidodiphosphate; Isoenzymes; Mice; Pituitary Neoplasms; Protein Kinases; Somatostatin; Thionucleotides

Numerous hormones are known to rapidly activate polyphosphoinositide turnover in target cells by promoting phosphodiesteratic cleavage of the phospholipids; however, little is known about the enzymology of receptor-mediated phosphoinositide breakdown. In the present study, thyrotropin-releasing hormone (TRH) stimulation of polyphosphoinositide turnover has been characterized in electrically permeabilized, [3H]myoinositol-labeled GH3 cells. The permeable cells allow the influence of small molecular weight (Mr less than or equal to 1000) cofactors to be determined. We present evidence for the following: 1) TRH stimulates inositol phosphate generation in permeable cells; 2) optimal hormone-stimulated inositol phosphate generation requires Mg2+, ATP, and Ca2+; 3) Mg2+ and ATP requirements reflect polyphosphoinositide kinase reactions; 4) in the absence of MgATP, TRH stimulates the phosphodiesteratic breakdown of pre-existing polyphosphoinositides in a reaction which requires only low Ca2+ (10(-7) M); 5) hormone activation is potentiated in the presence of the stable guanine nucleotide, GTP gamma S; neither TRH-stimulated nor GTP gamma S-potentiated hydrolysis is inhibited by cholera or pertussis toxin treatment. These results demonstrate that hormone-induced phospholipid hydrolysis involves activation of a phosphoinositide phosphodiesterase; activation results in lowering the Ca2+ requirement of the phosphodiesterase such that maximal activity is observed at Ca2+ levels characteristic of a resting cell (10(-7) M). Furthermore, TRH regulation of polyphosphoinositide hydrolysis is modulated by guanine nucleotides; however, nucleotide regulation appears to involve a GTP-binding factor (Np) other than Ns or Ni.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Calcium; Cell Line; Cell Membrane Permeability; Cholera Toxin; Dose-Response Relationship, Drug; Electric Stimulation; Enzyme Activation; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hydrolysis; Inositol Phosphates; Lithium; Pertussis Toxin; Phosphoinositide Phospholipase C; Phosphoric Diester Hydrolases; Pituitary Neoplasms; Protein Kinases; Rats; Thionucleotides; Thyrotropin-Releasing Hormone; Virulence Factors, Bordetella

Thyrotropin-releasing hormone (TRH) stimulated a rapid rise in inositol trisphosphate (IP3) formation and prolactin release from 7315c tumor cells. The potencies (half-maximal) of TRH in stimulating IP3 formation and prolactin release were 100 +/- 30 and 140 +/- 30 mM, respectively. Pretreatment of the cells with pertussis toxin (for up to 24 h) had no effect on either process. Pretreatment of the cells with cholera toxin (30 nM for 24 h) also failed to affect basal or TRH-stimulated IP3 formation. TRH was also able to stimulate IP3 formation with a half-maximal potency of 118 +/- 10 nM in a lysed cell preparation of 7315c cells; the TRH-stimulated formation of IP3 was enhanced by GTP. 5'-Guanosine gamma-thiotriphosphate (GTP gamma S) and 5'-guanylyl imidodiphosphate (Gpp(NH)p), nonhydrolyzable analogs of GTP, stimulated IP3 formation in the absence of TRH with half-maximal potencies of 162 +/- 50 and 7500 +/- 4300 nM, respectively. In contrast to the lack of effect of pertussis toxin on the TRH receptor system, treatment of 7315c cells with pertussis toxin for 3 h or longer completely abolished the ability of morphine, an opiate agonist, to inhibit either adenylate cyclase activity or prolactin release. During this 3-h treatment, pertussis toxin was estimated to induce the endogenous ADP ribosylation of more than 70% of Ni, the inhibitory GTP-binding protein. GTP gamma S and Gpp(NH)p inhibited cholera toxin-stimulated adenylate cyclase activity (presumably by acting at Ni) with half-maximal potencies of 25 +/- 9 and 240 +/- 87 nM, respectively. Finally, Gpp(NH)p was also able to inhibit the [32P]ADP ribosylation of Ni with a half-maximal potency of 300 nM. These results suggest that a novel GTP-binding protein, distinct from Ni, couples the TRH receptor to the formation of IP3.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Cell Line; Female; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guanylyl Imidodiphosphate; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Pertussis Toxin; Pituitary Neoplasms; Prolactin; Rats; Receptors, Cell Surface; Receptors, Thyrotropin-Releasing Hormone; Thionucleotides; Thyrotropin-Releasing Hormone; Type C Phospholipases; Virulence Factors, Bordetella

Thyrotropin-releasing hormone (TRH), like numerous other Ca2+-mobilizing agonists, has been found to stimulate polyphosphoinositide hydrolysis in responsive cells. The present studies further clarify the mechanism of action of this peptide hormone by demonstrating direct in vitro effects of TRH on polyphosphoinositide hydrolysis in GH3 pituitary cell membranes. Membranes from [3H]myoinositol-labeled cells were found to generate inositol bis- and tris- but not monophosphate upon incubation. Inositol polyphosphate generation was stimulated 2-3-fold by nanomolar concentrations of TRH in a reaction which was potentiated by micromolar concentrations of GTP; hormone-stimulated hydrolysis observed in the absence of GTP was fully antagonized by guanosine 5'-O-(2-thiodiphosphate). Guanosine 5'-O-(3-thiotriphosphate), Ca2+, and sodium fluoride also activated phosphoinositide hydrolysis in vitro. Stimulated inositol polyphosphate generation was accompanied by stimulated 1,2-diacylglycerol formation. Evidence that both phosphatidylinositol 4,5-bisphosphate as well as phosphatidylinositol 4-phosphate served as substrates for the activated phosphoinositide phosphodiesterase is presented. Pretreatment of GH3 cells with cholera or pertussis toxin did not influence stimulated hydrolysis in membranes. It is concluded that the TRH receptor directly regulates polyphosphoinositide hydrolysis in GH3 cell plasma membranes by a GTP-dependent process. The GTP dependence does not appear to be mediated through a cholera or pertussis toxin substrate and may involve a novel GTP-binding protein (NP).

Topics: Animals; Cell Line; Cell Membrane; Cholera Toxin; Diglycerides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hydrolysis; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Phosphatidylinositol Phosphates; Phosphatidylinositols; Pituitary Gland; Pituitary Neoplasms; Rats; Thionucleotides; Thyrotropin-Releasing Hormone

The NAD analog N'-methylnicotinamide adenine dinucleotide (N'AD) is formed in intact human promyelocytic leukemia HL-60 and in rat pituitary tumor GH3 cells during treatment of the cultured cells with the nicotinamide derivative N'-methylnicotinamide (N'CH3NAm). N'AD formation is associated with the induced maturation of HL-60 cells and increased hormone production by GH3 cells during treatment with the nicotinamide derivative. N'AD is detected by HPLC analysis of cytoplasmic extracts as a peak which elutes near NAD. Four facts indicate that this compound is N'AD. First, a compound which elutes with identical time retention is produced by transglycosylation during reaction of NAD with pig brain NAD glycohydrolase in the presence of excess N'CH3NAm. Second, the putative N'AD is degraded by prolonged digestion with the NAD glycohydrolase to ADP-ribose. Third, N'AD formation is prevented by addition of nicotinamide along with N'CH3NAm to compete with binding of N'CH3NAm to the NAD glycohydrolase. Fourth, radioactive precursor labeling demonstrates that it contains adenosine, but it is not labeled by radioactive nicotinamide. The biological relevance of N'AD formation was evaluated. The appearance of N'AD precedes development of HL-60 maturation, and NAD levels increase, not decrease, as observed in other cell types, during treatment with N'CH3NAm. Therefore, we propose that N'AD, not the pyridine base itself, is the active species in inducing maturation. The results provide support of a role for NAD metabolism, probably ADP-ribosylation, in the regulation of HL-60 maturation and in hormone production by pituitary cells.

Topics: Adenosine Triphosphate; Animals; Cell Line; Guanosine Triphosphate; Humans; Leukemia, Myeloid, Acute; NAD; NAD+ Nucleosidase; Niacinamide; Nucleotidyltransferases; Pituitary Neoplasms; Poly(ADP-ribose) Polymerases; Rats; Substrate Specificity; Time Factors

Adenomatous cells obtained from a pituitary tumor induced in Fisher 344/Lis rats by the subcutaneous implantation of estrone (E1) were found to secrete large amounts of prolactin (PRL). The secretion of PRL was stimulated by thyrotropin-releasing hormone (TRH) and low concentrations of dopamine (DA), while micromolar concentrations of DA were inhibitory. High affinity binding sites for 3H-spiroperidol (3H-SPIR) were found to be present on the cells and to conform to the criteria of dopaminergic receptors. An adenylate cyclase (AC) present in the cells could be activated by a guanyl nucleotide and was inhibited by DA in the presence of guanosine 5'-triphosphate (GTP). Fractionation of the adenomatous cells by Percoll gradients identified two groups of cells capable of secreting PRL and bearing 3H-SPIR binding sites. These data indicate that this rat pituitary adenoma may be a model for human prolactinomas that might be utilized for the study of the mechanism of action of dopaminergic drugs.

Topics: Adenoma; Animals; Bromocriptine; Disease Models, Animal; Dopamine; Estrone; Female; Guanosine Triphosphate; Pituitary Neoplasms; Prolactin; Rats; Rats, Inbred F344; Receptors, Dopamine; Thyrotropin-Releasing Hormone

Cells of the 7315c tumor released immunoreactive PRL (IR-PRL). Cholera toxin enhanced this release. Morphine and other opiate agonists inhibited IR-PRL release from both untreated and cholera toxin-treated tumor cells. The opiate-induced inhibition of IR-PRL release was concentration dependent and naloxone sensitive. Cholera toxin also enhanced the adenylate cyclase activity of 7315c tumor tissue. Opiates inhibited enzyme activity in both untreated and cholera toxin-treated 7315c tissue in a concentration-dependent and naloxone-sensitive manner. FK 33824 was more potent than [D-Ala2,D-Leu5]enkephalin in inhibiting IR-PRL release and adenylate cyclase activity. In cholera toxin-treated 7315c tumor tissue, GTP was required for opiate-induced inhibition of adenylate cyclase activity. Nonhydrolyzable analogs of GTP inhibited toxin-stimulated cyclase activity in the absence of an opiate. These results suggest that the 7315c tumor possesses a mu-opiate receptor; stimulation of this receptor inhibits both IR-PRL release and adenylate cyclase activity. An inhibitory guanyl nucleotide component may link the mu-opiate receptor to adenylate cyclase.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cholera Toxin; Female; Guanosine Triphosphate; Kinetics; Morphine; Naloxone; Narcotic Antagonists; Pituitary Neoplasms; Prolactin; Rats; Rats, Inbred BUF; Receptors, Opioid; Receptors, Opioid, mu

The effects of forskolin, an adenylate cyclase activator, were investigated on adrenocorticotropin (ACTH) secretion from AtT-20/ D16 -16 mouse pituitary tumor cells. Forskolin increased adenylate cyclase activity in these cells in the absence of added guanyl nucleotide, an effect blocked by somatostatin. Cyclic AMP synthesis and ACTH secretion increased in a concentration-dependent manner, not only in the clonal cells, but in primary cultures of rat anterior pituitary as well. Somatostatin inhibited cyclic AMP synthesis and ACTH secretion in response to forskolin. When forskolin was coapplied with corticotropin releasing factor, cyclic AMP synthesis was potentiated and ACTH secretion additive. The calcium channel blocker, nifedipine, inhibited forskolin, and 8-bromocyclic AMP stimulated ACTH secretion. These data suggest that ACTH secretion may be regulated at the molecular level by changes in cyclic AMP formation, which in turn regulate a calcium gating mechanism.

Topics: Adenylyl Cyclases; Adrenocorticotropic Hormone; Animals; Calcimycin; Cell Line; Cells, Cultured; Colforsin; Cyclic AMP; Diterpenes; Guanosine Triphosphate; Kinetics; Male; Mice; Pituitary Gland, Anterior; Pituitary Neoplasms; Rats; Rats, Inbred Strains; Somatostatin

We have investigated the effects of SRIF and human pancreatic GH-releasing factor-44 (hpGRF-44) on adenylate cyclase (AC) activity of male rat anterior pituitaries (in which somatotrophs are present in large proportion) and of human GH-secreting pituitary adenomas (which are almost homogeneously constituted by somatotrophs). The adenoma's responsiveness to both agents in terms of secretion was previously demonstrated in in vitro experiments. SRIF inhibited in a dose-dependent fashion the GH release from monolayer cultures of the tumors. The inhibition ranged from 32-66% at the maximal effective concentration (10(-6) M). hpGRF-44 stimulated GH release in a dose-dependent fashion. The stimulation was 78-172% at 10(-7) M. SRIF and hpGRF-44 markedly affected AC activity in both systems. SRIF elicited a pronounced inhibition of the enzyme activity in a dose-dependent manner. The inhibition was about 40% in the rat and ranged from 16-49% in adenomas at the maximal effective concentration (10(-5) M SRIF). The inhibitory effect was GTP-dependent. hpGRF-44 markedly stimulated AC activity. The stimulation was dose dependent and GTP dependent. The stimulation was about 650% in the rat and 26-350% in adenomas at the maximal effective concentration (10(-6) M). These results suggest the presence of a dually regulated (by SRIF and hpGRF-44) AC in GH-secreting cells; an involvement of cAMP in the intracellular mechanisms transducing the signals of SRIF and hpGRF-44 in somatotrophs.

Topics: Adenylyl Cyclases; Animals; Bucladesine; Cell Line; Dose-Response Relationship, Drug; Growth Hormone; Growth Hormone-Releasing Hormone; Guanosine Triphosphate; Humans; Male; Pancreas; Peptide Fragments; Pituitary Gland, Anterior; Pituitary Neoplasms; Rats; Rats, Inbred Strains; Somatostatin

Thyrotropin-releasing hormone (TRH) stimulates prolactin production by GH4C1 rat pituitary tumor cells, which possess high-affinity membrane receptors for the peptide. TRH caused up to a 50% increase in the activity of a low-Km GTPase in membranes from GH4C1 cells. The TRH stimulatory effect was maximal at GTP concentrations of 1 microM or lower. TRH caused an increase in GTPase activity of between 0.2 and 20 pmol of GTP hydrolyzed per mg of protein per min, depending on GTP concentration, while TRH binding was 0.3 pmol/mg of protein. TRH did not stimulate GTPase activity in membranes from GH12C1, or GH-Y cells, two pituitary lines lacking TRH receptors. Stimulation of GTPase depended on occupancy of the TRH receptor; half-maximal increases in GTPase activity required 46 nM TRH and 25 nM [N3-methyl-His]TRH, but the TRH free acid was inactive. The apparent Kds of these peptides for receptors were similar when measured under the same conditions. The fact that TRH binding to receptors is regulated by guanyl nucleotides, together with the demonstration of TRH stimulation of low-Km GTPase activity, suggests that the TRH receptor is associated with a guanyl nucleotide regulatory protein in the lactotroph membrane.

Topics: Animals; Cell Line; Cell Membrane; GTP Phosphohydrolases; Guanosine Triphosphate; Kinetics; Phosphoric Monoester Hydrolases; Phosphorus Radioisotopes; Pituitary Neoplasms; Rats; Thyrotropin-Releasing Hormone

A thyroliberin (TRH)-responsive particulate bound adenylyl cyclase is present in two rat anterior pituitary tumor cell strains (GH4C1 and GH3) which synthesize and secrete prolactin. At a given Mg2+ concentration, ATP and the guanyl nucleotides GTP and guanyl 5'-yl-imidodiphosphate (GMP-P(NH)P) caused a dose-dependent increase in adenylyl cyclase activity. The maximum response to thyroliberin occurred with ATP and GTP at concentrations above 0.30 mM and 2 microM, respectively. The maximal stimulatory effect of thyroliberin on adenylyl cyclase activity was 2-fold in the presence of GTP. GMP-P(NH)P increased the basal enzyme activity 4- to 10-fold over and above that of equimolar concentrations of GTP but supported poorly the TRH-induced response. Mg2+ caused a dose-dependent increase in the basal enzyme activity and reduced TRH and fluoride-induced responses. Also, Mn2+ and Co2+ stimulated the basal adenylyl cyclase activity while Zn2+, Ca2+, and Cu2+ inhibited the enzyme, and neither cations supported the TRH response. Half-maximal stimulation of the adenylyl cyclase by TRH and half-maximum binding of [3H]TRH to membranes at 35 degrees C were 102 and 56 nM, respectively. Pretreatment with TRH decreased the apparent Vmax of the enzyme and the maximal binding of [3H]TRH. Of 6 TRH analogs tested, only one was able to displace [3H]TRH from its receptor and to increase the adenylyl cyclase activity. We suggest that adenylyl cyclase activation is an early event in the stimulus secretion coupling between TRH and prolactin-producing GH cells.

Topics: Adenylyl Cyclases; Animals; Cell Line; Dose-Response Relationship, Drug; Enzyme Activation; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Magnesium; Membranes; Pituitary Neoplasms; Prolactin; Rats; Receptors, Cell Surface; Receptors, Thyrotropin-Releasing Hormone; Thyrotropin-Releasing Hormone

Topics: Adenoma; Adenylyl Cyclases; Egtazic Acid; Gastrointestinal Hormones; Guanosine Triphosphate; Humans; In Vitro Techniques; Pituitary Neoplasms; Prolactin; Vasoactive Intestinal Peptide