guanylyl-imidodiphosphate and Pituitary-Neoplasms

Topics: 1-Methyl-3-isobutylxanthine; Animals; Calcium; Cells, Cultured; Chlortetracycline; Cyclic AMP; Guanylyl Imidodiphosphate; Ion Channels; Pituitary Gland; Pituitary Neoplasms; Potassium; Prolactin; Protein Kinases; Sodium Fluoride; Thyrotropin; Thyrotropin-Releasing Hormone; Verapamil

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

The thyroliberin receptor in GH3 pituitary tumour cells is known to couple to phospholipase C via a guanine-nucleotide-binding protein (G protein). Thyroliberin is postulated also to activate adenylyl cyclase, via the stimulatory G protein (Gs). In order to study this coupling, we constructed an antisense RNA expression vector that contained part of the Gs alpha-subunit cDNA clone (Gs alpha) in an inverted orientation relative to the mouse metallothionein promoter. The cDNA fragment included part of the coding region and all of the 3' non-translated region. Transient expression of Gs alpha antisense RNA in GH3 cells resulted in the specific decrease of Gs alpha mRNA levels, followed by decreased Gs alpha protein levels. Thyroliberin-elicited adenylyl cyclase activation in membrane preparations showed a reduction of up to 85%, whereas phospholipase C stimulation remained unaffected. Activation of adenylyl cyclase by vasoactive intestinal peptide was reduced by 30-40%. Investigation of the effects of thyroliberin and vasoactive intestinal peptide on adenylyl cyclase in GH3 cell membranes pretreated with antisera against Gs alpha and Gi-1 alpha/Gi-2 alpha support the results obtained by the use of the antisense technique. We conclude that thyroliberin has a bifunctional effect on GH3 cells, in activating adenylyl cyclase via Gs or a Gs-like protein in addition to the coupling to phospholipase C.

Topics: Adenylyl Cyclases; Animals; Blotting, Western; Enzyme Activation; Gene Expression; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Immune Sera; Pituitary Neoplasms; Plasmids; Rats; Receptors, Neurotransmitter; Receptors, Thyrotropin-Releasing Hormone; RNA, Antisense; RNA, Messenger; Thyrotropin-Releasing Hormone; Transfection; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

We have investigated the modulation of different G protein alpha- and beta-subunit levels in prolactin (PRL) and growth hormone producing rat pituitary adenoma cells (GH3 cells) in culture after prolonged exposure (6-48 h) to the steroid hormones 17 beta-oestradiol and dexamethasone. Gi-3 alpha- and G beta-subunits were the only G protein subunits which increased in response to 10(-6) M oestradiol (to approximately 150 and 200% of controls, respectively), while the other alpha-subunits investigated (Gs alpha, Gi-2 alpha and G(o) alpha) remained relatively unchanged. Thyroliberin (TRH)--and guanosine 5'-[beta gamma-imido]trisphosphate (Gpp(NH)p)-elicited adenylyl cyclase (AC) activities were reduced during 6-12 h of oestradiol treatment (by 60 and 20%, respectively), while the inhibitory effect of somatostatin (SRIF) increased by approximately 100%. Dexamethasone (10(-6) M) increased levels of the stimulatory G protein Gs alpha (to approximately 340%) and decreased levels of Gi-3 alpha (to 25%). After 48 h, the AC response to TRH was reduced by approximately 70%, whereas the effect of the other modulators remained close to controls. We conclude that G protein subunits in GH3 cells are subject to specific regulation by steroid hormones and that this may be important in the tuning of the responsiveness of PRL secretion to hormones in the in vivo situation.

Topics: Adenylyl Cyclases; Animals; Dexamethasone; Estradiol; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Pituitary Neoplasms; Prolactin; Rats; Second Messenger Systems; Somatostatin; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

We have synthesized two photoreactive derivatives of somatostatin, namely [125I-Tyr11,azidonitrobenzoyl (ANB)-Lys4]somatostatin and [125I-Tyr11,ANB-Lys9]somatostatin, and used them to characterize somatostatin receptors biochemically in several cell types. Saturation binding experiments carried out in the dark demonstrated that [125I-Tyr11,ANB-Lys4]somatostatin bound with high affinity (KD = 126 +/- 39 pM) to a single class of binding sites in GH4C1 pituitary cell membranes. The affinity of this analog was similar to that of the unsubstituted peptide [125I-Tyr11]somatostatin (207 +/- 3 pM). In contrast, specific binding was not observed with [125I-Tyr11,ANB-Lys9]somatostatin. The binding of both [125I-Tyr11,ANB-Lys4]somatostatin and [125I-Tyr11]somatostatin was potently inhibited by somatostatin (EC50 = 300 pM) whereas at 100 nM unrelated peptides had no effect. Furthermore, both pertussis toxin treatment and guanyl-5'yl imidophosphate (Gpp(NH)p) markedly reduced [125I-Tyr11,ANB-Lys4]somatostatin binding. Thus, [125I-Tyr11,ANB-Lys4]somatostatin binds to G-protein coupled somatostatin receptors with high affinity. To characterize these receptors biochemically, GH4C1 cell membranes were irradiated with ultraviolet light following the binding incubation, and the labeled proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A major band of 85 kDa was specifically labeled with [125I-Tyr11,ANB-Lys4]somatostatin but not with [125I-Tyr11,ANB-Lys9]somatostatin or [125I-Tyr11]somatostatin. The binding affinity of the 85-kDa protein for [125I-Tyr11,ANB-Lys4]somatostatin was very high (Kd = 34 pM). Labeling of this protein was inhibited competitively by somatostatin (EC50 = 140 +/- 80 pM) but not by unrelated peptides. Furthermore, this band was not labeled in pertussis toxin-treated membranes or in untreated membranes incubated with Gpp(NH)p. Finally, [125I-Tyr11,ANB-Lys4]somatostatin specifically labeled bands of 82, 75, and 72 kDa in membranes prepared from mouse pituitary AtT-20 cells, rat pancreatic acinar AR4-2J cells, and HIT hamster islet cells, respectively. Thus, [125I-Tyr11,ANB-Lys4]somatostatin represents the first photolabile somatostatin analog able to bind to receptors with high affinity. Our studies demonstrate that this novel peptide covalently labels specific somatostatin receptors in a variety of target cell types.

Topics: Affinity Labels; Animals; Binding, Competitive; Cell Line; Cell Membrane; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Kinetics; Pancreas; Pertussis Toxin; Pituitary Neoplasms; Receptors, Neurotransmitter; Receptors, Somatostatin; Somatostatin; Virulence Factors, Bordetella

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

Phorbol esters alter cyclic AMP levels in a number of tissues, including the anterior pituitary. We report that membrane preparations from GH3 cells exposed to phorbol esters exhibit decreased vasoactive intestinal peptide (VIP)-stimulated and enhanced forskolin-stimulated adenylate cyclase activity. The responsiveness of adenylate cyclase activity to NaF, guanylyl-imidodiphosphate, and Mn2+ was also reduced by phorbol ester treatment. The ability of somatostatin to inhibit forskolin-stimulated adenylate cyclase activity was reduced while phorbol ester exposure had no apparent effect on somatostatin inhibition of VIP-stimulated adenylate cyclase activity. We suggest that protein kinase C alters at least two distinct components of the adenylate cyclase system. One modification disrupts hormone receptor-Gs interaction (lowering VIP efficacy) and the second perturbation augments the activity of the adenylate cyclase catalytic subunit.

Topics: Adenylyl Cyclases; Animals; Colforsin; Cyclic AMP; Enzyme Activation; Guanylyl Imidodiphosphate; Manganese; Pituitary Neoplasms; Protein Kinase C; Rats; Sodium Fluoride; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

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

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

We have investigated the structure of D2 receptors present in two prolactin-secreting, dopamine-resistant, transplantable rat pituitary tumors, 7315a and MtTW15. These receptors specifically bind with high affinity the dopamine antagonist [3H]spiroperidol when membrane bound or solubilized by [3-(3-cholamidopropyl)-dimethyl-ammonio]-1-propane sulfonate 10 mM and are pharmacologically characterized as D2 type. Target-size analysis by radiation inactivation indicated a molecular mass of approximately 100,000 and 200,000 daltons for receptors present respectively in 7315a and MtTW15 tumors either membrane bound or solubilized. The minimal size of the D2 binding site was evaluated at 94,000 daltons by photoaffinity labeling with [125I]azido-N-(p-aminophenethyl)-spiperone followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A guanine nucleotide had no effect on the displacing potency of the agonist N-propylnorapomorphine evaluated with membrane-bound or solubilized receptors obtained from either tumor. These results suggest the absence or inactivation of a guanine nucleotide binding protein in the receptorial complex of these tumors. Thus, our data indicate that a structural anomaly is present in the D2 receptorial complex of these prolactin-secreting rat pituitary tumors, which may be responsible for their resistance to the inhibitory effects of dopamine.

Topics: Affinity Labels; Animals; Apomorphine; Cell Membrane; Dopamine; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Female; Guanylyl Imidodiphosphate; Molecular Weight; Neoplasm Transplantation; Photochemistry; Pituitary Neoplasms; Prolactin; Rats; Rats, Inbred BUF; Rats, Inbred WF; Receptors, Dopamine; Receptors, Dopamine D2; Solubility; Spiperone

The effects of chronic MtTW15 pituitary adenoma implantation on beta-adrenergic responsiveness, cardiac beta-adrenoreceptors, and muscarinic receptors were studied in the rat. Five weeks after s.c. administration of tissue fragments of the MtTW15 adenoma, there was a 51 and 20% increase in the heart weight and body weight, respectively, and a 49-fold increase in the serum prolactin level as compared to the controls. At this time there was also an attenuation in the adenoma-bearing group of the ability of isoproterenol to produce a dipsogenic response and to increase the heart rate. In contrast, isoproterenol stimulated cardiac ornithine decarboxylase (ODC) activity 4.2-fold in both the control and adenoma-bearing groups. There was no change between the two groups in the cardiac ventricular beta-adrenoreceptor or muscarinic receptor concentration as measured by specific (-)-[125I]iodocyanopindolol (CYP) and (-)-[3H]quinuclidinyl benzilate (QNB) respectively. In addition, the concentrations of isoproterenol and carbachol required to inhibit by 50% (IC50) [125I]CYP and [3H]QNB binding, respectively, in the absence of 5'-guanylylimidodiphosphate (Gpp(NH)p) were not different between the two groups. In the presence of Gpp(NH)p, the isoproterenol IC50 value was not different between the two groups, whereas the carbachol IC50 value was increased slightly in the adenoma-bearing group. The data indicate that chronic MtTW15 adenoma implantation attenuated beta-mediated dipsogenic and heart rate responses but had little or no effect on cardiac ODC activity or cardiac autonomic receptor concentrations and agonist binding properties.

Topics: Adenoma; Animals; Cardiomegaly; Drinking; Guanylyl Imidodiphosphate; Heart Rate; Hypothyroidism; Isoproterenol; Male; Myocardium; Neoplasm Transplantation; Ornithine Decarboxylase; Pituitary Neoplasms; Rats; Rats, Inbred Strains; Receptors, Adrenergic, beta; Receptors, Muscarinic

We have investigated dopamine (DA) receptor agonist high- and low-affinity states in female rat estradiol-induced prolactin (PRL)-secreting pituitary tumors and intact pituitary tissue. Estradiol treatment increased the anterior pituitary weight 9-fold and plasma prolactin levels 74-fold and these measures are correlated (R = 0.745, n = 73, p less than 0.001). Competition for [3H]-spiperone binding to the DA receptor by apomorphine was compared in normal and adenomatous pituitary tissue. The inhibition constants (Ki) and the proportions of the two apomorphine sites are unchanged in tumors compared to intact pituitary tissue. Guanosine 5'-[beta-gamma-imino]triphosphate (Gpp(NH)p) causes complete conversion of the high into low affinity dopaminergic agonist site in normal pituitary and in tumors. These results suggest that rats with primary estradiol-induced pituitary tumors have normal and functional DA receptors.

Topics: Animals; Estradiol; Guanine Nucleotides; Guanylyl Imidodiphosphate; Organ Size; Pituitary Gland, Anterior; Pituitary Neoplasms; Prolactin; Rats; Receptors, Dopamine

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

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

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

Somatostatin inhibits basal and chlorpromazine stimulated adenylyl cyclase activity in homogenates of GH1 rat pituitary tumor cells. The Dtryp8-Dcys14 analogue is more potent than tyrosyl somatostatin as an inhibitor of both basal and chlorpromazine-stimulated adenylyl cyclase. Somatostatin had no effect on sodium fluoride or quanylyl-imidodiphosphate-stimulated cyclase in GH1 cell homogenates or on basal, epinephrine or prostaglandin E1 stimulated cyclase activity in sonicated BHK fibroblasts. These results indicate a specific effect of somatostatin to inhibit pituitary adenylyl cyclase activity.

Topics: Adenylyl Cyclase Inhibitors; Animals; Calcium; Cells, Cultured; Chlorpromazine; Epinephrine; Fibroblasts; Fluorides; Guanylyl Imidodiphosphate; Magnesium; Pituitary Neoplasms; Prostaglandins E; Rats; Somatostatin

Topics: Adenylyl Cyclases; Cell Line; Chlorpromazine; Enzyme Activation; Fluorides; Guanylyl Imidodiphosphate; Kinetics; Pituitary Neoplasms; Prolactin; Structure-Activity Relationship