On the involvement of cyclic AMP and extracellular Ca2+ in the regulation of hormone release from rat pituitary tumour (GH3) cells in culture

1987 ◽  
Vol 7 (2) ◽  
pp. 93-105 ◽  
Author(s):  
Kjersti Sletholt ◽  
Egil Haug ◽  
Kaare M. Gautvik
Keyword(s):  
Cyclic Amp ◽  
Pituitary Tumour ◽  
Gh3 Cells ◽  
Ionophore A23187 ◽  
Channel Blockers ◽  
Hormone Release ◽  
Calmodulin Antagonist ◽  
Rat Pituitary ◽  
Combined Action ◽  
Dose Dependent

Thyroliberin (TRH), dibutyryl cyclic AMP (db-cAMP), and 3-isobutyl-l-methylxanthine (MIX) had a stimulatory effect on prolactin (PRL) and growth hormone (GH) release from GH 3 cells. Half-maximal and maximal effects were observed for TRH at 2.5 nM and 10 nM; for db-cAMP at 0.6 mM and 5 mM, respectively. MIX (0.1 mM-1 mM) induced a dose-dependent accumulation of cellular cyclic AMP, while the hormone release was already maximally stimulated at 0.1 mM MIX. The maximal effects on hormone release of TRH and db-cAMP, but not of TRH and MIX, were additive. The Ca2+ channel blockers Co2+ (5 mM) and verapamil (100 μM) and the Ca2+ chelator EGTA (4 mM) abolished the stimulatory effect of TRH (1 μM) on hormone release. Co2+ and verapamil, but not EGTA, inhibited the stimulatory effect of db-cAMP (5 mM) on hormone release. The inhibitory effects of Co2+ and verapamil on GH release were counteracted by the combination of TRH and db-cAMP. For PRL release Co2+, but not verapamil, was able to inhibit the combined action of TRH and db-cAMP. Co2+, verapamil, and EGTA eliminated the stimulatory effect of MIX (1 mM) on PRL release while only Co2+ and EGTA affected the GH release. Hormone release in the presence of MIX plus verapamil or EGTA, but not Co2+, was increased by TRH. The calmodulin antagonist trifluoperazine (TFP) at 30 μM inhibited basal hormone release and hormone release stimulated by TRH (1 μM), db-cAMP (5 mM), and MIX (1 mM). The Ca2+ ionophore A23187 (5 μM) had a stimulatory effect on basal hormone release which was abolished by 30 μM TFP.

Role of Ca2+ and Na+ on luteinizing hormone release from the calf pituitary

1988 ◽  
Vol 255 (4) ◽  
pp. E469-E474
Author(s):  
J. P. Kile ◽  
M. S. Amoss
Keyword(s):  
Luteinizing Hormone ◽  
Ionophore A23187 ◽  
Channel Blockers ◽  
Hormone Release ◽  
Primary Cells ◽  
Rat Pituitary ◽  
Voltage Dependent ◽  
Lh Release ◽  
Ca2 Channels

It has been proposed that gonadotropin-releasing hormone (GnRH) stimulates Ca2+ entry by activation of voltage-independent, receptor-mediated Ca2+ channels in the rat gonadotroph. Little work has been done on the role of calcium in GnRH-induced luteinizing hormone (LH) release in species other than the rat. Therefore, this study was done to compare the effects of agents that alter Ca2+ or Na+ entry on LH release from calf anterior pituitary primary cells in culture. GnRH (100 ng/ml), Ca2+ ionophore A23187 (2.5 microM), and the depolarizing agent ouabain (0.1-10 microM) all produced significant increases (P less than 0.05) in LH release; these effects were significantly reduced when the cells were preincubated with the organic Ca2+ channel blockers nifedipine (1-10 microM) and verapamil (1-10 microM) and with Co2+ (0.01-1 mM). The effect of ouabain was inhibited by tetrodotoxin (TTX; 1-10 nM) as well as by nifedipine at 0.1-10 microM. In contrast to its effect on rat pituitary LH release, TTX significantly inhibited GnRH-stimulated LH release at 1-100 nM. These results suggest that GnRH-induced LH release may employ Ca2+ as a second messenger in bovine gonadotrophs and support recent speculation that GnRH-induced Ca2+ mobilization may in part be voltage dependent.

scholarly journals Stereospecific mobilization of intracellular Ca2+ by inositol 1,4,5-triphosphate. Comparison with inositol 1,4,5-trisphosphorothioate and inositol 1,3,4-trisphosphate

1988 ◽  
Vol 253 (3) ◽  
pp. 901-905 ◽  
Author(s):  
J Strupish ◽  
A M Cooke ◽  
B V L Potter ◽  
R Gigg ◽  
S R Nahorski
Keyword(s):  
Pituitary Tumour ◽  
Gh3 Cells ◽  
Maximal Response ◽  
The Novel ◽  
3T3 Cells ◽  
High Concentration ◽  
Rat Pituitary ◽  
Active Isomer ◽  
Swiss 3T3 ◽  
First Time

The stereo specificity of myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to mobilize Ca2+ from an intracellular store has been examined in permeabilized rat pituitary-tumour GH3 and Swiss 3T3 cells. A comparison of D-Ins(1,4,5)P3 with the synthetic enantiomer L-Ins(1,4,5)P3 and the racemate DL-Ins(1,4,5)P3 clearly demonstrates the marked stereospecificity of the response. Whereas D-Ins(1,4,5)P3 released 30-50% of non-mitochondrially-bound Ca2+ with a EC50 (concentration producing 50% of maximal response) of 200 nM, the L isomer was both substantially less potent and efficacious. A high concentration of the L isomer (10 microM) did not significantly shift the dose-response curve for the D isomer in Swiss 3T3 cells, suggesting that the less active isomer is probably a very weak agonist. Other studies revealed, in contrast with previous work, that the other naturally occurring isomer, D-Ins(1,3,4)P3, was essentially inactive in releasing Ca+, whereas a novel 5-phosphatase-resistant analogue, DL-myo-inositol 1,4,5-trisphosphorothioate, was a relatively potent full agonist in GH3 cells. These data reveal, for the first time, the stereoselectivity of the intracellular receptor associated with Ca2+ release. They also provide evidence for the activity of the novel phosphorothioate analogue of Ins(1,4,5)P3, but suggest that D-Ins(1,3,4)P3 is not involved in cellular Ca2+ mobilization.

Effects of trifluoperazine on prolactin release and cyclic AMP formation and degradation in GH4C1 pituitary cells

1987 ◽  
Vol 116 (1) ◽  
pp. 27-35
Author(s):  
Kjersti Sletholt ◽  
Jan Gordeladze ◽  
Egil Haug ◽  
Kaare M. Gautvik
Keyword(s):  
Inhibitory Effect ◽  
Pituitary Cells ◽  
Hormone Release ◽  
Prolactin Release ◽  
Camp Phosphodiesterase ◽  
Calmodulin Antagonist ◽  
Adenyl Cyclase Activity ◽  
Biphasic Effect ◽  
Basal Release ◽  
Dose Dependent

Abstract. In GH4C1 cells, the calmodulin antagonist trifluoperazine (TFP) showed a dose-dependent, biphasic effect on the basal release of PRL. An inhibition of PRL release was observed with 15–50 μmol/l TFP, whereas a concentration of 100 μmol/l and above had a stimulatory effect. The increase in basal hormone release evoked by TRH (1 μmol/l) and high extracellular concentration of K+ (50 mmol/l) was eliminated by 30 μmol/l TFP. The stimulatory effect of 100 μmol/l TFP on basal hormone release was not affected by addition of TRH (1 μmol/l) or K+ (50 mmol/l). The Ca2+ antagonists Co2+ (5 mmol/l) and verapamil (100 μmol/l), and the Ca2+ chelator EgTA (4 mmol/l) abolished the stimulatory effect of TRH (1 μmol/l) and of K+ (50 mmol/l on PRL release, whereas only Co2+ inhibited the stimulation caused by 100 μmol/l TFP. TFP (75 μmol/l) caused a transient increase in the concentration of cellular cAMP. Incubation of intact GH4C1 cells with TFP (75 μmol/l), had an inhibitory effect on both the low and the high affinity form of cAMP phosphodiesterase. Basal as well as TRH-stimulated adenyl cyclase activity were inhibited by TFP, and this effect was counteracted by addition of calmodulin.

scholarly journals Vasoactive-intestinal-polypeptide-stimulated adenosine 3′,5′-cyclic monophosphate accumulation in GH3 pituitary tumour cells. Reversal of desensitization by forskolin

1984 ◽  
Vol 221 (3) ◽  
pp. 789-796 ◽  
Author(s):  
S Guild ◽  
A H Drummond
Keyword(s):  
Cyclic Amp ◽  
Vasoactive Intestinal Polypeptide ◽  
Regulatory Protein ◽  
Phosphodiesterase Inhibitor ◽  
Pituitary Tumour ◽  
Tumour Cells ◽  
Gh3 Cells ◽  
Low Concentrations ◽  
Cyclic Amp Accumulation ◽  
Intestinal Polypeptide

The interaction between forskolin and vasoactive intestinal polypeptide (VIP) in the regulation of cyclic AMP production in GH3 pituitary tumour cells was investigated. Both forskolin (10nM-10 microns) and VIP (10pM-10nM) increased the cyclic AMP content of GH3 cells. Forskolin (50-100nM) was additive with VIP in stimulating cyclic AMP accumulation when low concentrations (less than 1 nM) of the peptide were used, but exhibited a synergistic interaction with higher VIP concentrations (10-100 nM). These effects on cyclic AMP accumulation were reflected in a leftward shift in the concentration-response curve for VIP-stimulated prolactin release from GH3 cells, a process known to be regulated by intracellular cyclic AMP concentrations. The synergy observed did not appear to be related to changes in cyclic nucleotide phosphodiesterase activity, since it was even more marked in the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor. Studies of the time-course of VIP-induced changes in GH3-cell cyclic AMP content revealed that, with high concentrations of VIP, production ceased within 2 min of addition. This attenuation of cyclic AMP synthesis was still observed in the presence of isobutylmethylxanthine, but was markedly inhibited by low concentrations of forskolin (50-100nM). The results suggest that VIP-induced cyclic AMP production rapidly becomes desensitized. This process, which is prevented by forskolin, may be related to changes in the ability of the guanine nucleotide regulatory protein to couple receptor occupancy to activation of adenylate cyclase.

scholarly journals Metabolism of inositol bis-, tris-, tetrakis- and pentakis-phosphates in GH3 cells

1988 ◽  
Vol 250 (2) ◽  
pp. 493-500 ◽  
Author(s):  
N M Dean ◽  
J D Moyer
Keyword(s):  
Inositol Phosphates ◽  
Pituitary Tumour ◽  
Tumour Cells ◽  
Gh3 Cells ◽  
Rat Pituitary ◽  
Potential Sources ◽  
A Minor ◽  
Gh3 Cell

Previous studies demonstrated a multiplicity of isomers of inositol phosphates in GH3 rat pituitary tumour cells. In order to determine their origin, we have investigated the metabolism of radiolabelled inositol phosphates (IPn) in GH3 cell homogenates by using h.p.l.c. I(1,4,5)P3 is either phosphorylated to I(1,3,4,5)P4 (in the presence of ATP) or dephosphorylated to I(1,4)P2 (in the absence of ATP). I(1,4)P2 is dephosphorylated to I(4)P (greater than 95%). I(1,3,4,5)P4 hydrolysis yields two products. By using dual-labelled [32P, 3H]I(1,3,4,5)P4 with 32P in either the 3 or the 4/5 position, we have identified the probable configuration of these isomers. The predominant (greater than 97%) IP3 formed is I(1,3,4)P3, with a minor I(1,4,5)P3 peak. Subsequent I(1,3,4)P3 hydrolysis yields two IP2 isomers [the major (approximately equal to 85%) is I(3,4)P2; the minor (approximately equal to 15%) is I(1,3)P2] and two IP isomers (the major (approximately equal to 90%) is I(3)P [L-I(1)P], the minor I(4)P). IP5 is very slowly dephosphorylated to and IP4 of undetermined isomeric configuration. We have also examined GH3 cell lipids for the presence of phosphoinositides either more highly phosphorylated than PIP2 (as potential sources of the IP4/IP5 and IP6 in these cells) or phosphorylated in positions other than 1, 4 and 5, and have been unable to find evidence of either. These data suggest two main routes of metabolism for I(1,4,5)P3 in GH3 cells: either (1) phosphorylation to I(1,3,4,5)P4, and the subsequent consecutive dephosphorylation to I(1,3,4)P3, I(3,4)P2 and finally L-I(1)P [D-I(3)P]; or (2) dephosphorylation to I(1,4)P2 and, subsequently, I(4)P.

scholarly journals Thyrotropin-releasing hormone receptor occupancy determines the fraction of the responsive pool of inositol lipids hydrolysed in rat pituitary tumour cells

1990 ◽  
Vol 271 (2) ◽  
pp. 331-336 ◽  
Author(s):  
A B Cubitt ◽  
E Geras-Raaka ◽  
M C Gershengorn
Keyword(s):  
Receptor Occupancy ◽  
Pituitary Tumour ◽  
Thyrotropin Releasing Hormone ◽  
Gh3 Cells ◽  
Releasing Hormone ◽  
Receptor Complexes ◽  
Trh Stimulation ◽  
Rat Pituitary ◽  
Hydrolysis Of ◽  
Trh Receptor

We report that there are distinct thyrotropin-releasing hormone (TRH)-responsive and -unresponsive pools of inositol (Ins) lipids in rat pituitary tumour (GH3) cells, and present evidence that the size of the responsive pool is determined by the number of activated TRH-receptor complexes. By use of an experimental protocol in which cycling of [3H]Ins is inhibited and resynthesis occurs with unlabelled Ins only, we were able to measure specifically the effects of TRH on the hydrolysis of the Ins lipids present before stimulation. A maximally effective dose of TRH (1 microM) caused a time-dependent decrease in 3H-labelled Ins lipids that attained a steady-state value of 42 +/- 1% of the initial level between 1.5 and 2 h. After 2 h, even though there was no further decrease in 3H-labelled Ins lipids, and no increase in [3H]Ins or [3H]Ins phosphates, turnover of Ins lipids, as assessed as incorporation of [32P]Pi into PtdIns, continued at a rate similar to that in cells incubated without LiCl or unlabelled Ins. These data indicate that Ins lipid turnover was not desensitized during prolonged TRH stimulation. Depletion of lipid 3H radioactivity by TRH occurred at higher TRH doses on addition of the competitive antagonist chlordiazepoxide. Addition of 1 microM-TRH after 3 h of stimulation by a sub-maximal (0.3 nM) TRH dose caused a further decrease in 3H radioactivity to the minimum level (40% of initial value). We propose that the TRH-responsive pool of Ins lipids in GH3 cells is composed of the complement of Ins lipids that are within functional proximity of activated TRH-receptor complexes.

scholarly journals Stimulation of prolactin synthesis and of adenosine 3':5'-cyclic phosphate formation by prostaglandins and thyroliberin in cultured rat pituitary cells

1976 ◽  
Vol 156 (1) ◽  
pp. 111-117 ◽  
Author(s):  
K M Gautvik ◽  
M Kriz
Keyword(s):  
Cyclic Amp ◽  
Prostaglandin E2 ◽  
Combined Treatment ◽  
Pituitary Tumour ◽  
Cell System ◽  
Pituitary Cells ◽  
Maximal Increase ◽  
Rat Pituitary ◽  
Prolactin Synthesis ◽  
Stimulation Of

1. The effects of prostaglandins E2 and F2alpha on prolactin synthesis were examined in a clonal strain of rat pituitary tumour cells, and compared with those of thyroliberin. 2. The prostaglandins and thyroliberin gave a dose-related and time-dependent stimulation of prolactin synthesis. The maximal effects (about twofold increases) were observed after 54h of treatment with 25nM-prostaglandin E2 and 2.5nM-prostaglandin F2alpha. A similar stimulation of prolactin synthesis was observed after 250nM-thyroliberin. The combined treatment with prostaglandins and thyroliberin did not increase prolactin synthesis over and above that obtained with each compound alone. 3. After removal of prostaglandins E2 and F2alpha there was a complete reversal of prolactin synthesis to pre-stimulation values 18h later (t1/2less than or equal to 9h). The rapid reversible effect of prostaglandins was in contrast with that of thyroliberin, where prolactin synthesis returned to control values with a t1/2 of about 42 h. 4. Prostaglandin E2 (5mum) and thyroliberin (5mum) increased cellular concentrations of cyclic AMP eight- and four-fold respectively. Maximal effects were observed after 2-5min of incubation. The increases in cyclic AMP were biphasic; normal values were obtained 60 min after the start of incubation with prostaglandin E2 or thyroliberin. 5. The dose/response curve showed that prostaglandin E2 caused maximal increase of cyclic AMP at 50nM. Concentrations of prostagland in E2 that caused half-maximal stimulation of cyclic AMP accumulation and of prolactin synthesis were 4 and 5nM respectively. 6. Combined treatment with prostaglandin E2 and thyroliberin in concentrations that separately caused maximal cyclic AMP increases did not result in a further increase in this cyclic nucleotide. 7. These results are consistent with a role of cyclic AMP in mediating the effects or prostaglandins and thyroliberin on prolactin synthesis. However, if cyclic AMP is involved as a common intracellular mediator of prolactin synthesis, it cannot alone explain all the effects of prostaglandins and thyroliberin in this cell system.

Effects of metabolic inhibitors on hormone release, cyclic AMP levels, and oxygen consumption in rat pituitary cells in culture

1987 ◽  
Vol 115 (1) ◽  
pp. 96-104
Author(s):  
Kjersti Sletholt ◽  
Claes Magnusson ◽  
Egil Haug ◽  
Kaare M. Gautvik
Keyword(s):  
Oxygen Consumption ◽  
Hormone Secretion ◽  
Inhibitory Effect ◽  
Metabolic Inhibitors ◽  
Pituitary Cells ◽  
Antimycin A ◽  
Hormone Release ◽  
Calmodulin Antagonist ◽  
Rat Pituitary ◽  
Rat Pituitary Cells

Abstract. The metabolic inhibitors antimycin A (2 μmol/l), dinitrophenol (0.5 mmol/l), and iodoacetate (6 mmol/l) were tested for their effects on hormone release, cAMP levels, and oxygen consumption in clonal strains of rat pituitary cells (GH3 cells). Basal release of growth hormone (GH) and prolactin (PRL) was reduced by all three inhibitors, and thyrotropin-releasing hormone (TRH) (l μmol/l) and K+ (50 mmol/l) stimulated hormone release were blocked. Trifluoperazine, a calmodulin antagonist, inhibited basal GH and PRL release at concentrations up to 30 μmol/l and stimulated above 50 μmol/l. The stimulatory effect of 80 μmol/l trifluoperazine on basal hormone release was eliminated by antimycin A, dinitrophenol, and iodoacetate, whereas the inhibitory effect of antimycin A, dinitrophenol and iodoacetate on basal hormone was not affected by 30 μmol/l trifluoperazine. None of the inhibitors had any effect on the level of cellular cAMP (i.e. intracellular plus extracellular). Oxygen consumption of GH3 cells was blocked by antimycin A, reduced by 25% by iodoacetate and increased by about 100% by dinitrophenol. In contrast, hormone secretion stimulated by TRH and K+ was not accompanied by any measurable alteration in oxygen consumption. Trifluoperazine (⩾ 80 μmol/l) reduced the basal oxygen consumption and blocked the stimulatory effect of dinitrophenol on oxygen consumption. In conclusion, inhibition of the energy generation of GH and PRL-producing cells severely affects the action of secretagogues, although stimulated hormone secretion may not be accompanied by any measurable increase in oygen consumption. The cellular energy supporting hormone secretion is mostly generated via oxidative phosphorylation.

scholarly journals Pertussis toxin blocks the inhibitory effect of muscarinic cholinergic agonists on cyclic AMP accumulation and prolactin secretion in GH3 anterior-pituitary tumour cells

1984 ◽  
Vol 223 (1) ◽  
pp. 145-149 ◽  
Author(s):  
B L Brown ◽  
R J H Wojcikiewicz ◽  
P R M Dobson ◽  
A Robinson ◽  
L I Irons
Keyword(s):  
Cyclic Amp ◽  
Pertussis Toxin ◽  
Anterior Pituitary ◽  
Pituitary Tumour ◽  
Inhibitory Effect ◽  
Prolactin Secretion ◽  
Gh3 Cells ◽  
Muscarinic Agonists ◽  
Cyclic Amp Accumulation ◽  
Muscarinic Cholinergic

The inhibition of prolactin secretion and cyclic AMP accumulation in GH3 cells by muscarinic agonists was blocked by preincubation of the cells with pertussis toxin (islet-activating protein). There was a lag of approx. 80 min in the onset of the effect on secretion. These results suggest that muscarinic agonists decrease prolactin secretion by inhibiting adenylate cyclase activity.

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