Effects of lithium and phorbol on the dynamics of LH release from dispersed sheep pituitary cells

1986 ◽  
Vol 111 (1) ◽  
pp. 167-173 ◽  
Author(s):  
L. Starling ◽  
R. P. McIntosh ◽  
E. A. Mclntosh
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Inositol Phosphates ◽  
Pituitary Cells ◽  
Kinase C ◽  
Dependent Protein ◽  
Lh Release ◽  
Gonadotrophin Releasing Hormone ◽  
Lh Cells ◽  
Activate Protein Kinase

ABSTRACT The possible involvement of polyphosphoinositides in the stimulation of LH release was investigated. Dispersed sheep pituitary cells were incubated in test-tubes, or perifusedns in columns, with gonadotrophin-releasing hormone (GnRH) and Li+, or with a phorbol ester, and the amounts and patterns of LH release over time compared. Treatment with Li+ (10 mmol/l), which is known to increase levels of inositol phosphates in gonadotrophs, was shown to have effects only on the responses of desensitized cells, significantly decreasing the rate at which the cells desensitize (P<0·005) and decreasing the response to supramaximal levels of GnRH stimulus (P<0·01). It is suggested that these effects could be due to increased levels of inositol monophosphate, inositol bisphosphate inositol 1,3,4-trisphosphate. Responses to single or repeated pulses of GnRH at 18-, 30- and 60-min intervals were not significantly altered. Phorbol 12-myristate 13-acetate (PMA), an activator of the calcium and phospholipid-dependent protein kinase (protein kinase C), was specifically active in releasing LH with a half-maximal stimulating dose of approximately 3 nmol/l. Phorbol 12,13-diacetate, which is structurally similar to PMA but does not activate protein kinase C, did not release LH, except at high levels in freshly dispersed cells. The timing of PMA-stimulated LH release was similar to that for GnRH-stimulated release, and PMA was able to release greater amounts of LH than could GnRH. This suggests that activation of protein kinase C is likely to be important in the GnRH-stimulated release of LH from gonadotrophs. It also shows that the desensitization to GnRH stimulation observed after 10 min is unlikely to be caused by lack of releasable LH. Cells desensitized to maximally stimulating levels of GnRH still responded strongly to PMA stimulation, indicating that the desensitization to GnRH stimulation involves a step in the transduction mechanism before activation of protein kinase C. J. Endocr. (1986) 111, 167–173

Arachidonic acid-induced LH release is ATP-independent and insensitive to N-ethyl maleimide

1992 ◽  
Vol 132 (1) ◽  
pp. 77-82 ◽  
Author(s):  
P. V. Kaye ◽  
P. A. van der Merwe ◽  
R. P. Millar ◽  
J. S. Davidson
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Pituitary Cells ◽  
Cell Permeabilization ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Kinase C ◽  
Lh Release ◽  
Stimulation Of

ABSTRACT The mechanism of arachidonic acid (AA)-induced LH release was characterized using sheep pituitary cells in primary culture permeabilized with Staphylococcal α-toxin. In intact cells, exogenous AA evoked release of LH in a manner which was partially dependent on extracellular Ca2+. At similar concentrations, AA also caused cell permeabilization as monitored by efflux of [3H]2-deoxyglucose metabolites. In α-toxin-permeabilized cells where cytosolic Ca2+ was clamped at resting levels, AA retained its ability to cause LH release. Unlike the stimulation of exocytosis produced by Ca2+, phorbol ester or cyclic AMP, AA-evoked release was independent of ATP and was not inhibited by pretreatment with N-ethyl maleimide. These findings indicated that exogenous AA does not cause LH release by Ca2+ influx or mobilization or by activating protein kinase C. The results suggest that LH release induced by exogenous AA is probably due to its detergent-like properties, and does not represent true exocytosis. Journal of Endocrinology (1992) 132, 77–82

Studies on the subcellular mechanisms mediating the negative estradiol effect on GnRH-induced LH-release by rat pituitary cells in culture

1989 ◽  
Vol 121 (3) ◽  
pp. 350-360 ◽  
Author(s):  
Günter Emons ◽  
Ernst U. Frevert ◽  
Olaf Ortmann ◽  
Ulrich Fingscheidt ◽  
Rita Sturm ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Actinomycin D ◽  
Female Rats ◽  
Coupling Mechanism ◽  
Pituitary Cells ◽  
Kinase C ◽  
Negative Effect ◽  
Lh Release

Abstract. Cultured pituitary cells from adult female rats were treated for 4 or 24 h in the absence or presence of E2 (10−9 mol/1) with increasing concentrations of the transcription inhibitor actinomycin-D or the translation inhibitor puromycin. During the last 4 h of incubation, LH release was stimulated with 5 × 10−10 mol/1 GnRH. The positive E2 effect observed after 24 h treatment with the steroid was clearly abolished by actinomycin-D at concentrations ≥10−10 mol/1 and by puromycin at concentrations ≥ 10−5 mol/1. These findings indicate that the positive E2 effect on GnRH-induced LH release is fully dependent on intact mRNA and protein synthesis. The negative E2 effect observed after 4 h treatment with the steroid was not affected by actinomycin-D and abolished by puromycin only at concentrations ≥ 10−4 mol/1. Similar results were obtained, when cells had been treated for 4 h with actinomycin-D or puromycin before the 4 h E2 treatment started. Thus, the negative E2 effect seems to be independent of mRNA synthesis and dependent on protein synthesis to a lesser extent than the positive E2 effect. In an attempt to identify positively the subcellular mechanism via which E2 exerts its negative effect, several steps in the GnRH stimulus secretion coupling mechanism were checked whether or not they are modulated by E2. The negative effects of E2 (10−9 mol/1) on LH release induced by GnRH (10−10, 10−9 mol/1) and by the activators of voltage dependent Ca2+ channels K+ (64 mmol/1) or veratridine (3.3 × 10−5 mol/1) were comparable to those of the calcium antagonist verapamil (10−6 mol/1). These findings supported the speculation that E2 might act on the Ca2+ channels. The LH release induced by the Ca2+ ionophores A 23 187 (10 −4 mol/1) or ionomycin (6.6 × 10−5 mol/1), however, was also significantly reduced by 10−9 mol/1 E2, indicating that the steroid modulated a mechanism secondary to the increase of intracellular Ca2+. Also GnRH (10−9, 10−8 mol/1) induced accumulation of [3H]inositol phosphates was not influenced by E2 (10−9 mol/1) treatment, though the steroid exerted a significant negative effect on the LH release by these cells, indicating that phosphatidylinositol-4,5-biphosphate breakdown is not the point of attack for the estrogen. The LH release induced by the activation of protein kinase C with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 10−7 mol/1) or with the synthetic diacylglycerol 1,2-dioleyl-sn-glycerol (DOG, 5 × 10−5 mol/1, 10−4 mol/1) was significantly reduced by E2 treatment. These findings give rise to the speculation that E2 might exert its negative effect on GnRH-induced LH release in cultured pituitary cells at the level of protein kinase C or a mechanism secondary to the activation of this enzyme.

scholarly journals Ethanol-induced phospholipase C activation is inhibited by phorbol esters in isolated hepatocytes

1988 ◽  
Vol 251 (3) ◽  
pp. 865-871 ◽  
Author(s):  
J B Hoek ◽  
R Rubin ◽  
A P Thomas
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phorbol Esters ◽  
Inositol Phosphates ◽  
Isolated Hepatocytes ◽  
Intact Cells ◽  
Protein Kinase C Inhibitors ◽  
Kinase C ◽  
Activate Protein Kinase

Ethanol causes a transient activation of the phosphoinositide-specific phospholipase C in intact hepatocytes and mimics the action of receptor-mediated agonists [Hoek, Thomas, Rubin & Rubin (1987) J. Biol. Chem. 262, 682-691]. Preincubation of the hepatocytes with phorbol esters which activate protein kinase C prevented this effect of ethanol: phorbol ester treatment inhibited the ethanol-induced phosphorylase activation, the increase in intracellular free Ca2+ concentrations measured in quin 2-loaded hepatocytes, and the changes in concentrations of inositol phosphates, phosphoinositides and phosphatidic acid. Several lines of evidence indicate that these effects were mediated by protein kinase C. Phorbol esters acted in a concentration range where they activate protein kinase C; phorbol esters that do not activate protein kinase C were not effective in inhibiting the effects of ethanol. The permeant diacylglycerol oleoyl-acetylglycerol also inhibited the effects of ethanol, but other diacylglycerols were not effective in the intact cells. The inhibition of ethanol-induced Ca2+ mobilization by phorbol esters was prevented by preincubating the cells with the protein kinase C inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H7) and sphingosine. H7 also enhanced the Ca2+ mobilization induced by ethanol in cells that were not pretreated with phorbol esters, indicating that the transient nature of the ethanol-induced Ca2+ mobilization may be due to an activation of protein kinase C caused by the accumulation of diacylglycerol. These data support a model whereby ethanol activates the phosphoinositide-specific phospholipase C, possibly by affecting receptor-G-protein-phospholipase C interactions in the membrane.

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