5'-CMP stimulates phospholipase A-mediated hydrolysis of phosphatidylinositol in permeabilized pituitary GH3 cells.
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ABSTRACT:
SUBMITTER: Cubitt AB
PROVIDER: S-EPMC1151421 | biostudies-other | 1991 Sep
REPOSITORIES: biostudies-other
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Project description:[3H]Inositol-labelled GH3 rat anterior pituitary tumour cells were permeabilized with digitonin and were incubated at 37 degrees C in the presence of ATP and Mg2+. [3H]Polyphosphoinositide breakdown and [3H]inositol phosphate production were stimulated by hydrolysis-resistant GTP analogues and by Ca2+. Of the nucleotides tested, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) was the most effective stimulus. Activation by GTP gamma S appeared to be mediated by a guanine nucleotide-binding (G) protein as GTP gamma S-stimulated [3H]inositol phosphate production was inhibited by other nucleotides with a potency order of GTP = GDP = guanosine 5'-[beta-thio]diphosphate greater than ITP greater than GMP greater than UTP = CTP = adenosine 5'-[gamma-thio]triphosphate. The stimulatory effects of 10 microM-GTP gamma S on [3H]inositol phosphate levels were reversed by spermine and spermidine with IC50 values of approx. 0.25 and 2 mM respectively. Putrescine was inhibitory only at higher concentrations. Similarly, GTP gamma S-induced decreases in [3H]polyphosphoinositide levels were reversed by 2.5 mM-spermine. The inhibitory effects of spermine were not overcome by supramaximal concentrations of GTP gamma S. In contrast, [3H]inositol phosphate production stimulated by addition of 0.3-0.6 mM-Ca2+ to incubation media was only partially inhibited by spermine (5 mM), and spermine was not inhibitory when added Ca2+ was increased to 1 mM. These data show that polyamines, particularly spermine, inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis with a marked selectivity towards the stimulatory effects of GTP gamma S.
| S-EPMC1135342 | biostudies-other
Project description: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.
| S-EPMC1149780 | biostudies-other
Project description:Metformin is an anti-hyperglycemic agent used to treat diabetes, and recent evidence suggests it has antitumor efficacy. Because growth hormone-secreting pituitary adenoma (GH-PA) patients have a high incidence of diabetes frequently treated with metformin, we assessed the antitumor effect of metformin on GH-PA. We found that metformin effectively inhibited proliferation and induced apoptosis in the GH-PA cell line GH3. We detected a decrease in mitochondrial membrane potential (MMP), an increase in expression of pro-apoptotic proteins, and a decrease in expression of an anti-apoptotic protein in metformin-treated GH3 cells, which suggests involvement of the mitochondrial-mediated apoptosis pathway. Inhibition of AMPK, which is activated by metformin, failed to reverse the antiproliferative effect. ATF3 was upregulated by metformin, and its knockdown significantly reduced metformin-induced apoptosis. In addition, GH secretion was inhibited by metformin through suppression of STAT3 activity independently of AMPK. Metformin also significantly suppressed cellular proliferation and GH secretion in primary human GH-PA cells. Metformin also significantly inhibited GH3 cell proliferation and GH secretion in vivo. ATF3 upregulation and p-STAT3 downregulation were confirmed in xenografts. These findings suggest metformin is a potentially promising therapeutic agent for the treatment of GH-PA, particularly in patients with diabetes.
| S-EPMC5514928 | biostudies-literature
Project description:Endothelin-1 (ET-1) is a novel vasoconstricting and cardiotonic peptide that is synthesized by the vascular endothelium. Bovine aortic endothelial cells which secrete ET in vitro lack membrane receptor sites for the peptide. Endothelial cells from rat brain microvessels that do not secrete ET in vitro express large amounts of high-affinity receptors for 125I-labelled ET-1 (Kd 0.8 nM). The ET receptor is recognized by sarafotoxin S6b and the different ET peptides with the following order of potency: ET-1 (Kd 0.5 nM) approximately equal to ET-2 (Kd 0.7 nM) greater than sarafotoxin S6b (Kd 27 nM) greater than ET-3 (Kd 450 nM). This structure-activity relationship is different from those found in vascular smooth muscle cells, renal cells and cardiac cells. ET-1 stimulates DNA synthesis in brain capillary endothelial cells. It is more potent than basic fibroblast growth factor. The action of ET on endothelial cells from microvessels involves phosphatidylinositol hydrolysis and intracellular Ca2+ mobilization. These observations suggest that brain endothelial cells might be an important target for ET.
| S-EPMC1131147 | biostudies-other
Project description:Generation of a phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] gradient within the plasma membrane is important for cell polarization and chemotaxis in many eukaryotic cells. The gradient is produced by the combined activity of phosphatidylinositol 3-kinase (PI3K) to increase PI(3,4,5)P(3) on the membrane nearest the polarizing signal and PI(3,4,5)P(3) dephosphorylation by phosphatase and tensin homolog deleted on chromosome ten (PTEN) elsewhere. Common to both of these enzymes is the lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], which is not only the substrate of PI3K and product of PTEN but also important for membrane binding of PTEN. Consequently, regulation of phospholipase C (PLC) activity, which hydrolyzes PI(4,5)P(2), could have important consequences for PI(3,4,5)P(3) localization. We investigate the role of PLC in PI(3,4,5)P(3)-mediated chemotaxis in Dictyostelium. plc-null cells are resistant to the PI3K inhibitor LY294002 and produce little PI(3,4,5)P(3) after cAMP stimulation, as monitored by the PI(3,4,5)P(3)-specific pleckstrin homology (PH)-domain of CRAC (PH(CRAC)GFP). In contrast, PLC overexpression elevates PI(3,4,5)P(3) and impairs chemotaxis in a similar way to loss of pten. PI3K localization at the leading edge of plc-null cells is unaltered, but dissociation of PTEN from the membrane is strongly reduced in both gradient and uniform stimulation with cAMP. These results indicate that local activation of PLC can control PTEN localization and suggest a novel mechanism to regulate the internal PI(3,4,5)P(3) gradient.
| S-EPMC2096598 | biostudies-literature
Project description:Previous studies showed that phorbol esters and thyrotropin-releasing hormone (TRH) stimulated phosphatidylcholine synthesis via protein kinase C in GH3 pituitary cells [Kolesnick (1987) J. Biol. Chem. 262, 14525-14530]. In contrast, 1,2-diacylglycerol-stimulated phosphatidylcholine synthesis appeared independent of protein kinase C. The present studies compare phosphatidylcholine synthesis stimulated by these agents with inhibition via the cyclic AMP system. The potent phorbol ester phorbol 12-myristate 13-acetate (PMA, 10 nM) increased [32P]Pi incorporation into phosphatidylcholine at 30 min to 159 +/- 6% of control. The adenylate cyclase activator cholera toxin (CT; 10 nM) and the cyclic AMP analogue dibutyryl cyclic AMP (1 mM) abolished this effect. CT similarly abolished TRH-induced phosphatidylcholine, but not phosphatidylinositol, synthesis. This is the first report of inhibiton of receptor-mediated phosphatidylcholine synthesis by the cyclic AMP system. The 1,2-diacylglycerol 1,2-dioctanoylglycerol (diC8) also stimulated concentration-dependent phosphatidylcholine synthesis. DiC8 (3 micrograms/ml) induced an effect quantitatively similar to that of maximal concentrations of PMA and TRH, whereas a maximal diC8 concentration (30 micrograms/ml) stimulated an effect 3-4-fold greater than these other agents. CT decreased the effect of diC8 (3 micrograms/ml) by 80%. Higher diC8 concentrations overcame the CT inhibition. Similar results were obtained with dibutyryl cyclic AMP. Additional differences were found between low and high concentrations of diC8. Low concentrations of diC8 failed to induce additive phosphatidylcholine synthesis with maximal concentrations of PMA, whereas high concentrations were additive. Hence, low concentrations of 1,2-diacylglycerols appear to be regulated similarly to phorbol esters, and higher concentrations appear to act via a pathway unavailable to phorbol esters.
| S-EPMC1131237 | biostudies-other
Project description:Thyrotropin-releasing hormone (TRH; thyroliberin) stimulated rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by a phosphodiesterase (phospholipase C) in GH3 cells, a prolactin-secreting rat pituitary tumour cell line. TRH caused a rapid decrease in the level of PtdIns(4,5)P2 to 60% of control and stimulated a marked transient increase in inositol 1,4,5-trisphosphate, the unique product of phosphodiesteratic hydrolysis of PtdIns(4,5)P2, to a peak of 410% of control at 15 s. TRH also caused decreases in phosphatidylinositol 4-monophosphate (PtdIns4P) and phosphatidylinositol (PtdIns) to 65% and 93% of control at 15 s respectively. Inositol 1,4-bisphosphate was increased to a peak of 450% at 30 s; inositol 1-monophosphate and inositol were not elevated until 30 s and 1 min respectively after TRH addition. To study whether PtdIns(4,5)P2 hydrolysis may be caused by an elevation in cytosolic Ca2+ concentration, the changes induced by TRH in the levels of inositol sugars were compared with the effects of membrane depolarization by high extracellular [K+]. The elevation in cytosolic [Ca2+] induced by K+ depolarization did not change the level of inositol 1,4,5-trisphosphate. These data suggest that phosphodiesteratic hydrolysis of PtdIns(4,5)P2 may be the initial event in TRH stimulation of inositol lipid metabolism in GH3 cells and that PtdIns(4,5)P2 hydrolysis is not stimulated by an elevation in cytosolic Ca2+ concentration. The decreases in PtdIns4P and PtdIns may be due to enhanced conversion of PtdIns into PtdIns4P into PtdIns(4,5)P2 or to their direct hydrolysis by phosphomonoesterases and/or phosphodiesterases. These results are consistent with the hypothesis that TRH-stimulated PtdIns(4,5)P2 breakdown causes Ca2+ mobilization leading to prolactin secretion.
| S-EPMC1152503 | biostudies-other
Project description:The phospholipase D (PLD)-mediated synthesis of phosphatidylethanol (PtdEtOH) and the hydrolysis of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) were examined in drug-sensitive and multidrug-resistant lines of MCF-7 human breast carcinoma cells. In drug-sensitive (MCF-7/WT) cells, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) failed to enhance either the synthesis of PtdEtOH or the hydrolysis of either phospholipid. In the drug-resistant (MCF-7/MDR) cells, 100 nM PMA greatly enhanced both the synthesis of PtdEtOH (approximately 21-fold) and the hydrolysis of PtdEtn (approximately 29-fold), but had no effect on the hydrolysis of PtdCho. The PLD activators sphingosine and H2O2 were found to elicit only a slight (1.28-1.4-fold) stimulatory effect on PtdCho hydrolysis in both the MCF-7/WT and MCF-7/MDR cell types, and had only a small effect on PtdEtn hydrolysis in the MCF-7/WT cells as well. However, these agents significantly (approximately 2.6-3.5-fold) stimulated PtdEtn hydrolysis in the MCF-7/MDR cells. These data indicate that MCF-7/MDR cells contain a PtdEtn-specific PLD activity which can be selectively stimulated by PMA, sphingosine and H2O2.
| S-EPMC1137280 | biostudies-other
Project description:Inositol phospholipids in cultured GH3 cells, a prolactin secreting, thyrotropin-releasing hormone (TRH) sensitive rat pituitary cell line, exhibit a preferential selectivity for incorporating arachidonic acid. Fatty acid composition data show that all inositol phospholipids are enriched in stearic and arachidonic acids to a much greater degree than other cellular phospholipids. Incubation of GH3 cells with radioactive stearate, oleate, arachidonate, eicosapentaenoate or docosahexaenoate also showed that much more stearate and arachidonate were incorporated into inositol phospholipids. In short term incubations with tracer amounts of radioactive arachidonate, incorporation was initially into phosphatidylinositol (PtdIns), with phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] being labelled at later times. During longer incubations, all of the inositol phospholipids reach equilibrium at about 10 h, and the resulting specific activities of the three fractions were similar. These findings suggest that arachidonate is incorporated initially into PtdIns and that PtdIns is then phosphorylated. There was no release of either arachidonate or eicosanoid products when GH3 cells were incubated with TRH. However, TRH stimulation of 32P-labelled GH3 cells resulted in rapid breakdown of PtdIns(4,5)P2 and PtdIns4P, with concomitant increases in [32P]phosphatidic acid and [32P]PtdIns. When the [32P]PtdIns was further analysed by argentation chromatography to separate PtdIns molecular species, it was found that tetraenoic (stearate/arachidonate) species accounted for 80% of the stimulated labelling. The selectivity for arachidonate incorporation into inositol phospholipids coupled with turnover of the arachidonate-containing molecular species suggests that inositol phospholipids containing arachidonic acid or the diacylglycerol resulting therefrom may play a vital cellular role in GH3 cells. This role may involve the operation of the PtdIns cycle itself rather than a stimulated release of arachidonate for eicosanoid formation.
| S-EPMC1146811 | biostudies-other
Project description:Sprouty (Spry) proteins are negative regulators of receptor tyrosine kinase signaling; however, their exact mechanism of action remains incompletely understood. We identified phosphatidylinositol-specific phospholipase C (PLC)-? as a partner of the Spry1 and Spry2 proteins. Spry-PLC? interaction was dependent on the Src homology 2 domain of PLC? and a conserved N-terminal tyrosine residue in Spry1 and Spry2. Overexpression of Spry1 and Spry2 was associated with decreased PLC? phosphorylation and decreased PLC? activity as measured by production of inositol (1,4,5)-triphosphate (IP(3)) and diacylglycerol, whereas cells deficient for Spry1 or Spry1, -2, and -4 showed increased production of IP(3) at baseline and further increased in response to growth factor signals. Overexpression of Spry 1 or Spry2 or small-interfering RNA-mediated knockdown of PLC?1 or PLC?2 abrogated the activity of a calcium-dependent reporter gene, suggesting that Spry inhibited calcium-mediated signaling downstream of PLC?. Furthermore, Spry overexpression in T-cells, which are highly dependent on PLC? activity and calcium signaling, blocked T-cell receptor-mediated calcium release. Accordingly, cultured T-cells from Spry1 gene knockout mice showed increased proliferation in response to T-cell receptor stimulation. These data highlight an important action of Spry, which may allow these proteins to influence signaling through multiple receptors.
| S-EPMC2947483 | biostudies-literature