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.

scholarly journals Phospholipase A activity in the skin. Modulators of arachidonic acid release from phosphatidylcholine

1979 ◽  
Vol 184 (2) ◽  
pp. 283-290 ◽  
Author(s):  
V A Ziboh ◽  
J T Lord
Keyword(s):  
Arachidonic Acid ◽  
Prostaglandin E2 ◽  
Specific Activity ◽  
Feedback Regulation ◽  
Phospholipase A ◽  
Particulate Fraction ◽  
Prostaglandin F2 Alpha ◽  
Low Concentrations ◽  
Freeze Dried ◽  
Hydrolysis Of

The distribution of the hydrolysis of 1-acyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine and the simultaneous biosynthesis of prostaglandins by subcellular fractions from human and rat skin membrane preparations were determined. The phospholipase A2 activity was distributed among the subcellular particulate preparations with the highest specific activity in the 105000g particulate fraction. The activity was optimal at pH 7.5 in the presence of 1.0 mM-CaCl2 and was inhibited by EDTA. The hydrolysis of phosphatidylcholine by the skin 105000g particulate fraction was inhibited by cortisol and triamcinolone acetonide and it was stimulated by histamine, bradykinin, retinoic acid and cholera enterotoxin (freeze-dried Vibrio cholerae). Furthermore hydrolysis of phosphatidylcholine by the skin phospholipase A was also enhanced by low concentrations of prostaglandin E2 and prostaglandin F2 alpha. These last results suggest that the amplication of the hydrolysis of phosphatidylcholine by prostaglandin E2 and prostaglandin F2 alpha, with the consequent release of arachidonic acid (the substrate of prostaglandin synthesis) is likely a positive-feedback regulation of the arachidonic acid-prostaglandin cascade.

Mobilization of arachidonic acid in thrombin-stimulated human platelets

1990 ◽  
Vol 68 (2) ◽  
pp. 520-527 ◽  
Author(s):  
V. G. Mahadevappa ◽  
Frank Sicilia
Keyword(s):  
Arachidonic Acid ◽  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Selective Inhibition ◽  
Human Platelets ◽  
Phospholipase A ◽  
Serine Esterase ◽  
Membrane Phospholipids ◽  
Esterase Inhibitors ◽  
Hydrolysis Of

In the present work we investigated the effect of serine esterase inhibitors such as 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC) and phenylmethylsulfonyl fluoride (PMSF), as well as the effect of mepacrine on thrombin-induced mobilization of arachidonic acid (AA) in human platelets. The inhibitor NCDC (0.6 mM) completely abolished the thrombin-induced activation of phospholipase C, phospholipase A2, and transacylase enzymes, whereas the pretreatment of platelets with PMSF (2 mM) resulted in a highly selective inhibition of phospholipase A2 and transacylase activities, with no marked effect on thrombin-induced activation of phospholipase C. The thrombin-induced release of [3H]AA from phosphatidylcholine and phosphatidylinositol was reduced by 90 and 56%, respectively, in the presence of PMSF. This inhibitor also caused a parallel inhibition in the accumulation of [3H]AA (85%) with little effect on thrombin-induced formation of [3H]phosphatidic acid (5%), whereas mepacrine (0.4 mM) caused a selective inhibition of phospholipase A2 and transacylase activities with concomitant stimulation of [3H]phosphatidic acid formation in intact human platelets. These results demonstrate that NCDC and PMSF (serine esterase inhibitors) do not affect agonist-induced activation of phospholipases that mobilize arachidonic acid through a common site. Our results further demonstrate that the inhibition of [3H]AA release observed in the presence of NCDC, PMSF, and mepacrine is primarily due to their direct effects on enzyme activities, rather than due to their indirect effects through formation of complexes between inhibitors and membrane phospholipids. Based upon these results, we also conclude that the combined hydrolysis of phosphatidylcholine and phosphatidylinositol by phospholipase A2 serves as a major source for eicosanoid biosynthesis in thrombin-stimulated human platelets.Key words: deacylation, phospholipids, thrombin, platelets, phospholipase A2.

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 Inositol phospholipid arachidonic acid metabolism in GH3 pituitary cells

1986 ◽  
Vol 236 (1) ◽  
pp. 235-242 ◽  
Author(s):  
D T Dudley ◽  
A A Spector
Keyword(s):  
Arachidonic Acid ◽  
Molecular Species ◽  
Arachidonic Acid Metabolism ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Trh Stimulation ◽  
Inositol Phospholipids ◽  
Rat Pituitary ◽  
Arachidonic Acids ◽  
Phosphatidylinositol 4

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.

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.

scholarly journals Depletion of arachidonic acid from GH3 cells. Effects on inositol phospholipid turnover and cellular activation

1987 ◽  
Vol 246 (3) ◽  
pp. 669-679 ◽  
Author(s):  
D T Dudley ◽  
D E Macfarlane ◽  
A A Spector
Keyword(s):  
Arachidonic Acid ◽  
Cell Activation ◽  
Gh3 Cells ◽  
Arachidonic Acid Content ◽  
Cellular Activation ◽  
Inositol Phospholipids ◽  
Rat Pituitary ◽  
Long Time ◽  
Transient Events

We have adapted rat pituitary GH3 cells to grow in delipidated culture medium. In response, esterfied linoleic acid and arachidonic acid become essentially undetectable, whereas eicosa-5,8,11-trienoic acid accumulates and oleic acid increases markedly. These changes occur in all phospholipid classes, but are particularly pronounced in inositol phospholipids, where the usual stearate/arachidonate profile is replaced with oleate/eicosatrienoate (n − 9) and stearate/eicosatrienoate (n − 9). Incubation of arachidonate-depleted cells with 10 microM-arachidonic acid for only 24 h results in extensive remodelling of phospholipid fatty acids, such that close-to-normal compositions and arachidonic acid content are achieved for the inositol phospholipids. In comparison studies with arachidonic acid-depleted or -repleted cells, it was found that the arachidonate content does not affect thyrotropin-releasing-hormone (TRH)-stimulated responses measured at long time points, including [32P]Pi labelling of phosphatidylinositol and phosphatidic acid, stimulation of protein phosphorylation, and basal or TRH-stimulated prolactin release. However, transient events such as stimulated breakdown of inositol phospholipids and an initial rise in diacylglycerol are enhanced by the presence of arachidonate. These results show that arachidonic acid itself is not required for operation of the phosphatidylinositol cycle and is not an obligatory intermediate in TRH-mediated GH3 cell activation. It is possible that any structural or functional role of arachidonic acid in these processes is largely met by replacement with eicosatrienoate (n − 9). However, since arachidonate in inositol phospholipids facilitates their hydrolysis upon stimulation by TRH, arachidonic acid apparently may have a specific role in the recognition of these lipids by phospholipase C.

Arachidonic acid mobilizes calcium and stimulates prolactin secretion from GH3 cells

1984 ◽  
Vol 246 (5) ◽  
pp. E458-E462 ◽  
Author(s):  
R. N. Kolesnick ◽  
I. Musacchio ◽  
C. Thaw ◽  
M. C. Gershengorn
Keyword(s):  
Arachidonic Acid ◽  
Saturated Fatty Acids ◽  
Arachidonic Acid Metabolism ◽  
Prolactin Secretion ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Alpha Linolenic Acid ◽  
Rat Pituitary ◽  
Experimental Approaches ◽  
Rat Pituitary Cells

Because arachidonic acid and/or its metabolites may be intracellular effectors of calcium-mediated secretion, we studied whether arachidonic acid added exogenously mobilizes calcium and stimulates prolactin secretion from GH3 cells, cloned rat pituitary cells. Arachidonic acid caused efflux of 45Ca from preloaded cells and stimulated prolactin secretion. The concentration dependencies of these effects were similar; stimulation was attained with 3 microM arachidonic acid. To determine indirectly whether these effects may be caused by arachidonic acid itself, not via conversion to metabolites, two experimental approaches were used. First, inhibitors of arachidonic acid metabolism, eicosatetraynoic acid and indomethacin, did not inhibit arachidonic acid-induced prolactin secretion. And second, alpha-linolenic acid, which cannot be converted to arachidonic acid, and linoleic acid, but not saturated fatty acids of equal chain length, stimulated 45Ca efflux and prolactin secretion. These data demonstrate that arachidonic acid added exogenously causes Ca2+ mobilization and prolactin secretion from GH3 cells and suggest that arachidonic acid itself, not via metabolism, may be a cellular regulator of prolactin secretion.

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