scholarly journals CMP activates reversal of phosphatidylinositol synthase and base exchange by distinct mechanisms in rat pituitary GH3 cells

1990 ◽  
Vol 272 (3) ◽  
pp. 813-816 ◽  
Author(s):  
A B Cubitt ◽  
M C Gershengorn
Keyword(s):  
Phospholipase D ◽  
Gh3 Cells ◽  
Reverse Direction ◽  
Isotopic Equilibrium ◽  
Base Exchange ◽  
Permeabilized Cells ◽  
Rat Pituitary ◽  
Rapid Accumulation ◽  
Nucleoside Monophosphates ◽  
Exchange Activity

CMP is known to activate phosphatidylinositol (PtdIns)/inositol (Ins) base exchange and has been reported to activate reversal of PtdIns synthase also. Because it is possible that PtdIns synthase acting in the reverse direction, followed by re-incorporation of ambient Ins, could be responsible for base-exchange activity, we characterized these processes in rat pituitary GH3 cells. In permeabilized GH3 cells prelabelled with [3H]Ins and incubated in buffer with LiCl but without added Ins, CMP stimulated rapid accumulation of [3H]Ins and decreases in [3H]PtdIns; the Km for CMP was 1.7 mM. CDP and CTP were less effective, whereas 2′-CMP, 3′-CMP, other nucleoside monophosphates and cytidine did not influence this process. In permeabilized cells prelabelled to isotopic equilibrium with [3H]Ins and [32P]Pi, CMP stimulated decreases in both the 32P and 3H labelling of PtdIns, but did not increase that of [32P]phosphatidic acid. These findings demonstrate that in the absence of added Ins the effect of CMP is not via activation of base exchange nor via a phospholipase D, but by reversal of PtdIns synthase. In permeabilized cells prelabelled with [3H]Ins and [32P]Pi, unlabelled Ins inhibited loss of 32P labelling of PtdIns caused by CMP while markedly stimulating loss of 3H labelling of PtdIns and release of [3H]Ins. These data demonstrate that Ins inhibits reversal of PtdIns synthase, but stimulates base exchange. We conclude that in GH3 cells reversal of PtdIns synthase and PtdIns/Ins base exchange are both stimulated by CMP, but are distinct processes.

Phosphatidohydrolase and base-exchange activity of commercial phospholipase D

1974 ◽  
Vol 164 (2) ◽  
pp. 420-428 ◽  
Author(s):  
Masaki Saito ◽  
Elizabeth Bourque ◽  
Julian Kanfer
Keyword(s):  
Phospholipase D ◽  
Base Exchange ◽  
Exchange Activity

scholarly journals 5′-CMP stimulates phospholipase A-mediated hydrolysis of phosphatidylinositol in permeabilized pituitary GH3 cells

1991 ◽  
Vol 278 (3) ◽  
pp. 831-834 ◽  
Author(s):  
A B Cubitt ◽  
C N Thaw ◽  
M C Gershengorn
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A ◽  
Gh3 Cells ◽  
Base Exchange ◽  
Rat Pituitary ◽  
Lag Period ◽  
Hydrolysis Of

We showed previously that 5′-CMP activates PtdIns-Ins base exchange and reversal PtdIns synthase in permeabilized rat pituitary GH3 cells. Here we report another effect of 5′-CMP on PtdIns metabolism in these cells. In permeabilized GH3 cells prelabelled with [3H]Ins and incubated in buffer with LiCl and a free Ca2+ concentration of 0.1 microM but without added Ins, 5′-CMP stimulated formation of glycerophospho[3H]inositol (GroP[3H]Ins) after a lag period of at least 5 min. This effect was concentration-dependent; the apparent Km was 0.30 +/- 0.02 mM. CDP and CTP stimulated GroPIns formation less effectively than did 5′-CMP, but cytidine, 2′-CMP, 3′-CMP, 5′-AMP and 5′-GMP had no effect. 5′-CMP stimulated formation of lysoPtdIns also. In permeabilized GH3 cells prelabelled with [3H]arachidonic acid, 5′-CMP stimulated release of [3H]arachidonic acid without a measurable lag period. These data show that 5′-CMP stimulates a phospholipase A activity in permeabilized GH3 cells that hydrolyses PtdIns.

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 Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway

2004 ◽  
pp. 233-240 ◽  
Author(s):  
AM Nanzer ◽  
S Khalaf ◽  
AM Mozid ◽  
RC Fowkes ◽  
MV Patel ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Kinase ◽  
Cell Line ◽  
Kinase Inhibitor ◽  
Thymidine Incorporation ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Gh3 Cells ◽  
Rat Pituitary ◽  
Mitogen Activated Protein

OBJECTIVES: Ghrelin is a brain-gut peptide with GH-releasing and appetite-inducing activities and a widespread tissue distribution. Ghrelin is the endogenous ligand of the GH secretagogue receptor type 1a (GHS-R1a), and both ghrelin and the GHS-R1a are expressed in the pituitary. There are conflicting data regarding the effects of ghrelin on cell proliferation. A positive effect on proliferation and activation of the mitogen-activated protein kinase (MAPK) pathway has been found in hepatoma, adipose, cardiomyocyte and prostate cell lines. However, ghrelin has also been shown to have anti-proliferative effects on breast, lung and thyroid cell lines. We therefore examined the effect of ghrelin on the rat pituitary cell line GH3. METHODS: RT-PCR was used for the detection of GHS-R1a and pre-proghrelin mRNA expression in GH3 cells. The effect of ghrelin on cell proliferation was studied using [(3)H]thymidine incorporation; cell counting and the activation of the MAPK pathway were studied using immunoblotting and inhibitors of the extracellular signal-regulated kinase 1 and 2 (ERK 1/2), protein kinase C (PKC) and tyrosine phosphatase pathways. RESULTS: GHS-R1a and ghrelin mRNA expression were detected in GH3 cells. Ghrelin, at 10(-10) to 10(-6) M concentrations, significantly increased [(3)H]thymidine incorporation (at 10(-9) M, 183+/-13% (means+/-s.e.m.) compared with untreated controls), while 12-phorbol 13-myristate acetate (PMA) at 10(-7) M (used as a positive control) caused a 212+/-14% increase. A reproducible stimulatory effect of desoctanoyl ghrelin was also observed on [(3)H]thymidine incorporation (135+/-5%; P<0.01 at 10(-9) M compared with control), as well as on the cell count (control 6.8 x 10(4)+/-8.7 x 10(3) cells/ml vs desoctanoyl ghrelin (10(-9) M) 1.04 x 10(5)+/-7.5 x 10(3) cells/ml; P<0.01). Ghrelin caused a significant increase in phosphorylated ERK 1/2 in immunoblotting, while desoctanoyl ghrelin showed a smaller but also significant stimulatory effect. The positive effect of ghrelin and desoctanoyl ghrelin on [(3)H]thymidine incorporation was abolished by the MAPK kinase inhibitor U0126, the PKC inhibitor GF109203X and the tyrosine kinase inhibitor tyrphostin 23, suggesting that the ghrelin-induced cell proliferation of GH3 cells is mediated both via a PKC-MAPK-dependent pathway and via a tyrosine kinase-dependent pathway. This could also be clearly demonstrated by Western blot analysis, where a transient increase in ERK 1/2 phosphorylation by ghrelin was attenuated by all three inhibitors. CONCLUSION: We have shown a novel role for ghrelin in stimulating the proliferation of a somatotroph pituitary tumour cell line, suggesting that ERK activation is involved in mediating the effects of ghrelin on cell proliferation. Desoctanoyl ghrelin showed a similar effect. As ghrelin has been shown to be expressed in both normal and adenomatous pituitary tissue, locally produced ghrelin may play a role in pituitary tumorigenesis via an autocrine/paracrine pathway.

A background sodium conductance is necessary for spontaneous depolarizations in rat pituitary cell line GH3

1994 ◽  
Vol 266 (3) ◽  
pp. C709-C719 ◽  
Author(s):  
S. M. Simasko
Keyword(s):  
Cell Line ◽  
Calcium Current ◽  
Membrane Conductance ◽  
Potassium Currents ◽  
Pituitary Cell ◽  
Calcium Currents ◽  
Gh3 Cells ◽  
Sodium Conductance ◽  
Rat Pituitary ◽  
Voltage Dependent

The role of Na+ in the expression of membrane potential activity in the clonal rat pituitary cell line GH3 was investigated using the perforated patch variation of patch-clamp electrophysiological techniques. It was found that replacing bath Na+ with choline, tris(hydroxymethyl)aminomethane (Tris), or N-methyl-D-glucamine (NMG) caused the cells to hyperpolarize 20-30 mV. Tetrodotoxin had no effect. The effects of the Na+ substitutes could not be explained by effects on potassium or calcium currents. Although all three Na+ substitutes suppressed voltage-dependent calcium current by 10-20%, block of voltage-dependent calcium current by nifedipine or Co2+ did not result in hyperpolarization of the cells. There was no effect of the Na+ substitutes on voltage-dependent potassium currents. In contrast, all three Na+ substitutes influenced calcium-activated potassium currents [IK(Ca)], but only at depolarized potentials. Choline consistently suppressed IK(Ca), whereas Tris and NMG either had no effect or slightly increased IK(Ca). These effects on IK(Ca) also cannot explain the hyperpolarization induced by removing bath Na+. Choline always hyperpolarized cells yet suppressed IK(Ca). Furthermore, removing bath Na+ caused an increase in cell input resistance, an observation consistent with the loss of a membrane conductance as the basis of the hyperpolarization. Direct measurement of background currents revealed a 12-pA inward current at -84 mV that was lost upon removing bath Na+. These results suggest that this background sodium conductance provides the depolarizing drive for GH3 cells to reach the threshold for firing calcium-dependent action potentials.

scholarly journals Detection of two chromatin proteins which bind specifically to the 5'-flanking region of the rat prolactin gene.

1985 ◽  
Vol 5 (11) ◽  
pp. 2967-2974 ◽  
Author(s):  
B A White ◽  
G M Preston ◽  
T C Lufkin ◽  
C Bancroft
Keyword(s):  
Transcription Initiation ◽  
Genomic Clone ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Sequence Specificity ◽  
Strong Binding ◽  
Prolactin Gene ◽  
Rat Pituitary ◽  
Chromatin Proteins ◽  
Flanking Region

We employed a protein gel blotting procedure to search for nuclear proteins from rat pituitary cells that bind preferentially to the 5'-flanking region of the rat prolactin gene. By gel blots of chromatin proteins from GH3 rat pituitary tumor cells with a 32P-labeled prolactin genomic clone, we detected two major binding proteins with molecular weights of approximately 44,000 and 48,000, designated NP44 and NP48, respectively. Both NP44 and NP48 are minor chromatin proteins which are extracted at low salt concentrations (0.4 M NaCl) and exhibit a range of slightly acidic isoelectric variants. NP44 and NP48 were detected at similar levels in chromatin extracts of GH3 cells, the prolactin-negative GC cell variant of the GH3 cells, and normal rat pituitary tissue. Considerably lower levels of these two proteins were found in chromatin extracts from rat liver and rat C6 glial cells. NP44 and NP48 exhibit DNA sequence specificity, as evidenced by their strong binding to the upstream flanking region of the prolactin gene, but only very weak binding to plasmid DNA, rat prolactin or growth hormone cDNAs, or upstream flanking regions of two other rat genes. By analyzing subclones of a rat prolactin genomic clone, we established that NP44 and NP48 bind to at least two sites, which are located between 0.4 and 2.0 kilobases (region I) and between 2.0 and 4.8 kilobases (region II) upstream of the transcription initiation site. These findings are discussed in the context of a possible functional association between the strong binding of NP44 and NP48 to the prolactin 5'-flanking region and pituitary-specific expression of the prolactin gene.

scholarly journals Activities of bone morphogenetic proteins in prolactin regulation by somatostatin analogs in rat pituitary GH3 cells

2011 ◽  
Vol 332 (1-2) ◽  
pp. 163-169 ◽  
Author(s):  
Naoko Tsukamoto ◽  
Fumio Otsuka ◽  
Tomoko Miyoshi ◽  
Kenichi Inagaki ◽  
Eri Nakamura ◽  
...  
Keyword(s):  
Bone Morphogenetic Proteins ◽  
Somatostatin Analogs ◽  
Gh3 Cells ◽  
Rat Pituitary
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