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.

1,25-Dihydroxyvitamin D3-induced upregulation of the thyrotropin-releasing hormone receptor in clonal rat pituitary GH3 cells

1995 ◽  
Vol 147 (3) ◽  
pp. 397-404 ◽  
Author(s):  
L M Atley ◽  
N Lefroy ◽  
J D Wark
Keyword(s):  
Vitamin D ◽  
Fold Increase ◽  
Thyrotropin Releasing Hormone ◽  
Gh3 Cells ◽  
Pituitary Hormone ◽  
Pituitary Cells ◽  
Hormone Production ◽  
Releasing Hormone ◽  
Rat Pituitary ◽  
Trh Receptor

Abstract 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is active in primary dispersed and clonal pituitary cells where it stimulates pituitary hormone production and agonist-induced hormone release. We have studied the effect of 1,25-(OH)2D3 on thyrotropin-releasing hormone (TRH) binding in clonal rat pituitary tumour (GH3) cells. Compared with vehicle-treated cells, 1,25-(OH)2D3 (10 nmol/l) increased specific [3H]MeTRH binding by 26% at 8 h, 38% at 16 h, 35% at 24 h and reached a maximum at 48 h (90%). In dose–response experiments, specific [3H]MeTRH binding increased with 1,25-(OH)2D3 concentration and reached a maximum at 10 nmol/l. Half-maximal binding occurred at 0·5 nmol 1,25-(OH)2D3/l. The vitamin D metabolite, 25-OH D3, increased [3H]MeTRH binding but was 1000-fold less potent than 1,25-(OH)2D3. In equilibrium binding assays, treatment with 10 nmol 1,25-(OH)2D3/l for 48 h increased the maximum binding from 67·4 ± 8·8 fmol/mg protein in vehicle-treated cells to 96·7 ± 12·4 fmol/mg protein in treated cells. There was no difference in apparent Kd (1·08 ± 0·10 nmol/l for 1,25-(OH)2D3-treated and 0·97 ± 0·11 nmol/l for vehicle-treated cells). Molecular investigations revealed that 10 nmol 1,25-(OH)2D3/l for 24 h caused an 8-fold increase in TRH receptor-specific mRNA. Actinomycin D (2 μg/ml, 6 h) abrogated the 1,25-(OH)2D3-induced increase in [3H]MeTRH binding. Cortisol also increased [3H]MeTRH binding but showed no additivity or synergism with 1,25-(OH)2D3. TRH-stimulated prolactin release was not enhanced by 1,25-(OH)2D3. We conclude that the active vitamin D metabolite, 1,25-(OH)2D3, caused a time- and dose-dependent increase in [3H]MeTRH binding. The effect was vitamin D metabolite-specific and resulted from an upregulation of the TRH receptor. Further studies are needed to determine the functional significance of this novel finding. Journal of Endocrinology (1995) 147, 397–404

scholarly journals Formation of inositol phosphate isomers in GH3 pituitary tumour cells stimulated with thyrotropin-releasing hormone. Acute effects of lithium ions

1987 ◽  
Vol 248 (2) ◽  
pp. 463-470 ◽  
Author(s):  
P J Hughes ◽  
A H Drummond
Keyword(s):  
Inositol Phosphate ◽  
Cell Types ◽  
Pituitary Tumour ◽  
Thyrotropin Releasing Hormone ◽  
Gh3 Cells ◽  
Acute Effects ◽  
Releasing Hormone ◽  
Inositol Phosphate Metabolism ◽  
Trh Stimulation ◽  
Gh3 Cell

With a h.p.l.c. system, the inositol mono-, bis- and tris-phosphate isomers found in [3H]inositol-labelled GH3 cells were resolved and identified. These cells possess at least ten distinct [3H]inositol-containing substances when acid-soluble extracts are analysed by anion-exchange h.p.l.c. These substances were identified by their co-elution with known inositol phosphate standards and, to a limited extent, by examining their chemical structure. Two major inositol monophosphate (InsP) isomers were identified, namely Ins1P and Ins4P, both of which accumulate after stimulation with the hypothalamic releasing factor (TRH) (thyrotropin-releasing hormone). Three inositol bisphosphate (InsP2) isomers were resolved, of which two were positively identified, i.e. Ins(1,4)P2 and Ins(3,4)P2. TRH treatment increases both of these isomers, with Ins(1,4)P2 being produced at a faster rate than Ins(3,4)P2. The third InsP2 isomer has yet to be fully identified, although it is co-eluted with an Ins(4,5)P2 standard. This third InsP2 is also increased after TRH stimulation. In common with other cell types, the GH3 cell contains two inositol trisphosphate (InsP3) isomers: Ins(1,4,5)P3, which accumulates rapidly, and Ins(1,3,4)P3, which is formed more slowly. The latter substance appears simultaneously with its precursor, inositol 1,3,4,5-tetrakisphosphate. We also examined the effects of acute Li+ treatment on the rates of accumulation of these isomers, and demonstrated that Li+ augments TRH-mediated accumulation of Ins1P, Ins4P, Ins(1,4)P2, the presumed Ins(4,5)P2 and Ins(1,3,4)P3. These results suggest that the effects of Li+ on inositol phosphate metabolism are more complex than was originally envisaged, and support work carried out by less sophisticated chromatographic analysis.

scholarly journals Separation of multiple isomers of inositol phosphates formed in GH3 cells

1987 ◽  
Vol 242 (2) ◽  
pp. 361-366 ◽  
Author(s):  
N M Dean ◽  
J D Moyer
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Inositol Phosphates ◽  
Thyrotropin Releasing Hormone ◽  
Gh3 Cells ◽  
Releasing Hormone ◽  
Inositol 1 ◽  
Trh Stimulation ◽  
Inositol 1,4,5 Trisphosphate

A high-performance-liquid-chromatography (h.p.l.c.) separation was developed, which resolves isomers of inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) in a single run. In GH3 cells labelled with [3H]inositol, treated with Li+ and thyrotropin-releasing hormone (TRH), radiolabelled components identified as inositol 1-phosphate (I1P), inositol 2-phosphate (I2P), inositol 4-phosphate (I4P), inositol 1,4-bisphosphate [I(1,4)P2], inositol 1,3,4-trisphosphate [I(1,3,4)P3] and inositol 1,4,5-trisphosphate [I(1,4,5)P3] are present, as are multiple unidentified IP2 peaks. After TRH stimulation, both I1P and I4P increase, the increase in I4P preceding that of I1P; I(1,4)P2 and an unknown IP2 increase; and both I(1,3,4)P3 and I(1,4,5)P3 increase, the increase in I(1,4,5)P3 being rapid and transient, whereas the increase in I(1,3,4)P3 is slower and more sustained. The most rapidly appearing inositol phosphates produced after TRH stimulation are I(1,4)P2 and I(1,4,5)P3.

scholarly journals Synthesis and characterization of a high-affinity photoactivatable analogue of thyrotropin-releasing hormone

1992 ◽  
Vol 281 (1) ◽  
pp. 179-184
Author(s):  
K D Brady ◽  
A H Tashjian
Keyword(s):  
Thyrotropin Releasing Hormone ◽  
British Library ◽  
Releasing Hormone ◽  
High Affinity ◽  
Sds Page ◽  
Rat Pituitary ◽  
Single Polypeptide ◽  
Trh Analogues ◽  
Trh Receptor

An analogue of thyrotropin-releasing hormone (TRH, pGlu-His-ProNH2), i.e. pGlu-His-ProNH-(CH2)6-(4-azidosalicylamide) (TRH-ASA), has been synthesized and, in a radioiodinated form (TRH-IASA), characterized and used as a photoaffinity reagent to label the TRH receptor on rat pituitary GH4C1 cells. TRH-IASA bound to GH4C1 cells with high affinity (Kd = 8 nM), comparable with that of TRH binding. The binding of TRH-IASA was competitive with binding of TRH, two TRH analogues and a TRH receptor antagonist, chlordiazepoxide. TRH-IASA did not bind to or label GH12C1 cells, which lack functional TRH receptors. Labelling of GH4C1 cells with TRH-IASA followed by SDS/PAGE and autoradiography of membrane proteins demonstrated labelling of a single polypeptide which ran as a diffuse band between 71 and 91 kDa, centred at 76 kDa. No change in this labelling pattern was observed as a function of the length of time (between 5 min and 2 h) that GH4C1 cells were incubated with 3 nM-TRH-IASA. Using either a very short (5 s) photolysis interval or low TRH-IASA concentrations, only the 76 kDa band was labelled. Minor bands appeared only after extended photolysis and use of high TRH-IASA concentrations. We conclude that the TRH receptor from rat pituitary GH4C1 cells is a single peptide with an apparent molecular mass of 76 kDa. Details of the chemical synthesis of TRH-ASA are given in Supplementary Publication SUP 50167 (5 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1992) 281, 5.

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