Evidence for a role of phospholipase A2 in the mechanism of LHRH priming in rat anterior pituitary tissue

1994 ◽  
Vol 141 (1) ◽  
pp. 15-31 ◽  
Author(s):  
F J Thomson ◽  
M S Johnson ◽  
R Mitchell ◽  
B Wolbers
Keyword(s):  
Protein Synthesis ◽  
Anterior Pituitary ◽  
Priming Effect ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Pituitary Tissue ◽  
Gonadotrophin Secretion ◽  
Lh Release ◽  
Functional Relevance

Abstract The phospholipase A2 (PLA2) inhibitors, quinacrine, p-bromophenacyl bromide, ONO-RS-082, aristolochic acid and chloracysine blocked the priming effect of LHRH, but not acute LHRH-induced gonadotrophin release measured in anterior pituitary pieces in pro-oestrous rats in vitro. These results suggest that the intracellular mechanisms underlying LHRH priming are distinct from those which mediate LH release in the present circumstances in that they involve PLA2. Furthermore, neither LHRH-induced LH release from preprimed tissue nor Ca2+-induced LH release were attenuated by quinacrine, indicating that this inhibitor does not interfere with the general Ca2+-dependent secretory apparatus of the gonadotroph and that the critical period for its action is in the induction of priming. LHRH induced the release of [3H]arachidonic acid ([3H]AA) from [3H]AA-prelabelled anterior pituitary tissue from pro-oestrous rats; a response which was sensitive to inhibitors of PLA2, of protein kinase C (PKC) and of protein synthesis. Activation of PKC also resulted in [3H]AA release which was inhibited with exactly the same pharmacological profile as the response to LHRH. Both gonadotrophin secretion and [3H]AA release responses to LHRH and to phorbol ester varied in parallel during the oestrous cycle and in ovariectomized/oestradiol-17β-replaced animals, as did their sensitivity to quinacrine and the protein synthesis inhibitor cycloheximide. These results indicate that LHRH priming is dependent on a hormonally regulated cascade involving a distinct form of PKC acting through a protein synthesis-dependent step to release AA by means of PLA2 activity. The priming effect was mimicked (at least in part) by conditioning preincubation with AA, confirming the functional relevance of this signalling cascade. Results using standard inhibitors of lipoxygenase/epoxygenase pathways were equivocal as to whether these pathways were critically involved, whilst cyclo-oxygenase inhibitors were completely without effect. The steps downstream from AA (and its possible metabolites) by which stimulus–secretion coupling is up-regulated in priming remain to be clarified. Journal of Endocrinology (1994) 141, 15–31

PRIMING EFFECT OF LUTEINIZING HORMONE RELEASING FACTOR IN VITRO: ROLE OF PROTEIN SYNTHESIS, CONTRACTILE ELEMENTS, Ca2+ AND CYCLIC AMP

1979 ◽  
Vol 81 (3) ◽  
pp. 223-234 ◽  
Author(s):  
A. J.-M. C. PICKERING ◽  
G. FINK
Keyword(s):  
Protein Synthesis ◽  
Luteinizing Hormone ◽  
Rna Polymerase Ii ◽  
Priming Effect ◽  
First Response ◽  
Gonadotrophin Secretion ◽  
Intracellular Ca ◽  
Cyclic Phosphate

The mechanism of the priming effect of luteinizing hormone releasing factor (LH-RF) upon gonadotrophin secretion was studied using short-term incubation of hemipituitary glands from pro-oestrous rats. The dependence of the priming, but not the LH releasing action of LH-RF on protein synthesis in pituitary tissue was confirmed. Cytochalasin B failed to affect the first response to LH-RF, but abolished the priming effect, suggesting that the integrity of cellular microfilaments was essential. Colchicine and vinblastine did not modify the response to LH-RF. Neither inhibitors of DNA nor the inhibitor of RNA polymerase II, α-amanitin, significantly affected the priming action of LH-RF. Normal extracellular concentrations of Ca2+ were necessary for gonadotrophin release, but the priming effect was not significantly affected by low extracellular Ca2+ and could not be elicited by raising intracellular Ca2+ concentrations. Adenosine 3′:5′-cyclic phosphate did not appear to act as a second messenger for either the gonadotrophin releasing or the priming action of LH-RF.

Oxytocin stimulates LH production by the anterior pituitary gland of the rat

1992 ◽  
Vol 132 (2) ◽  
pp. 277-283 ◽  
Author(s):  
G. Robinson ◽  
J. J. Evans ◽  
K. J. Catt
Keyword(s):  
Female Rats ◽  
Release Mechanism ◽  
Protein Synthesis Inhibitor ◽  
Pituitary Cells ◽  
Synthesis Inhibitor ◽  
Mechanical Dispersion ◽  
Lh Release

ABSTRACT Gonadotrophin-releasing activity of oxytocin has previously been demonstrated in vitro and in vivo. This study investigated whether oxytocin is also able to induce LH accumulation in pituitary cells. Following trypsin digestion and mechanical dispersion, pituitary cells from female rats were incubated with oxytocin (100 nmol/l) for 24 h. LH release stimulated by oxytocin increased (P < 0·001) progressively during the incubation indicating a different secretory pattern from the more rapid but less sustained secretion stimulated by gonadotrophin-releasing hormone. Oxytocin also enhanced (P < 0·01) total LH accumulation in the incubation system (released plus cell contents) which was apparent after 7–11 h of stimulation. The release of LH stimulated by oxytocin was reduced by the protein synthesis inhibitor cycloheximide (10 μmol/l). However, cycloheximide did not completely block oxytocin-stimulated LH release; there remained some LH release above that seen in non-stimulated controls (P < 0·01) revealing the presence of a cycloheximide-resistant component in the release mechanism. Furthermore, accumulation of total LH in 24 h incubations was suppressed (P < 0·01) by cycloheximide. The advancement in LH release which oxytocin has been shown to induce in vivo in pro-oestrous rats was accompanied by an early reduction of pituitary LH stores. However, the fall normally observed in LH content during the surge was markedly attenuated by the oxytocin treatment. Thus, loss of pituitary LH stores was less in oxytocin-treated rats than in saline-treated controls, even though net LH release into plasma was increased. Therefore, oxytocin stimulated the replenishment of LH stores. Although the mechanism(s) remains to be defined and the relationships between in-vitro and in-vivo results are as yet uncharacterized, the present study demonstrates that oxytocin treatment stimulates LH production in both dispersed cells and intact pituitaries in situ. Journal of Endocrinology (1992) 132, 277–283

The effect of gonadotrophin surge-inhibiting factor on the self-priming action of gonadotrophin-releasing hormone in female rats in vitro

1992 ◽  
Vol 134 (3) ◽  
pp. 427-436 ◽  
Author(s):  
D. W. Koppenaal ◽  
A. M. I. Tijssen ◽  
J. de Koning
Keyword(s):  
Protein Synthesis ◽  
Priming Effect ◽  
The Self ◽  
Pituitary Cells ◽  
Inhibiting Factor ◽  
Lh Release ◽  
Gonadotrophin Releasing Hormone ◽  
Functional Antagonism

ABSTRACT The present study was designed to explore further the functional antagonism between gonadotrophin-releasing hormone (GnRH) and the ovarian factor, gonadotrophin surge-inhibiting factor (GnSIF). In all experiments, pituitary tissue was exposed to various amounts of GnSIF, after which the self-priming action of GnRH was studied. GnSIF was increased in vivo by FSH treatment and increased in vitro by adding various amounts of follicular fluid (FF) to cultured pituitary cells. Treatment with 3 or 10 IU FSH suppressed the initial LH response and delayed the maximally primed LH response to GnRH. Treatment with FSH was only effective in intact rats on days 1 and 2 of dioestrus. There was no difference in the rate of maximal LH release irrespective of treatment with either FSH or saline. Since FSH treatment was ineffective in long-term ovariectomized rats, it was concluded that the initial suppressive effect of FSH on LH release was mediated by GnSIF. Cycloheximide prevented the self-priming action of GnRH by inhibiting GnRH-induced protein synthesis. The initial protein synthesis-independent GnRH-stimulated LH release, which was already suppressed by FSH treatment, remained suppressed in the presence of cycloheximide. Pretreatment with GnRH in vivo increased the protein synthesis-independent GnRH-induced LH release during subsequent incubation of the glands. This increase did not occur after FSH treatment. Pituitary cells, cultured for 20 h in medium only, failed to elicit the self-priming effect of GnRH. Preincubation with FF maintained the self-priming effect. This was independent of the concomitant presence of various amounts of oestradiol. Preincubation with bovine FF suppressed the initial GnRH-stimulated LH release dose-dependently. Porcine FF, human FF and testicular extract suppressed the release of LH in a similar way. It was concluded that GnSIF suppresses the initial LH response to continuous GnRH stimulation. Increased levels of GnSIF caused by FSH treatment also delayed the primed LH release. The mechanism of functional antagonism between GnSIF and GnRH could give rise to the occurrence of the phenomenon of GnRH self-priming. Journal of Endocrinology (1992) 134, 427–436

146 EFFECT OF PROTEIN SYNTHESIS INHIBITOR ON BOAR SPERM CAPACITATION AND FERTILIZATION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 186
Author(s):  
Y. Okudaira ◽  
H. Funahashi
Keyword(s):  
Protein Synthesis ◽  
Acrosome Reaction ◽  
Specific Inhibitor ◽  
Protein Synthesis Inhibitor ◽  
Sperm Viability ◽  
Sperm Capacitation ◽  
Synthesis Inhibitor ◽  
Mitochondrial Ribosome ◽  
Cytoplasmic Ribosome

In human, bovine, mouse, and rat sperm, translation of RNA to proteins in the mitochondrial ribosome during capacitation has been reported to be important for fertilization. The objective of this study was to examine effect of protein synthesis inhibitor (ribosome inhibitor) on boar sperm capacitation and IVF. Sperm from an ejaculated sperm-rich fraction of Berkshire boars were washed by centrifugation (1500 rpm for 35 min) in a Percoll gradient (45/90%) and then incubated in modified Medium-199 containing 0.4% BSA and 5 mM caffeine sodium benzoate, supplemented with or without a mitochondrial ribosome-specific (55S ribosome) inhibitor, chloramphenicol (CP; 0.3 mM), or a cytoplasmic ribosome-specific (80S ribosome) inhibitor, cyclohexide (CH; 3.6 mM), in an atmosphere of 5% CO2 in air at 39°C for 45 or 90 min. At 45 and 90 min after culture, sperm viability, motility, and chlortetracyclin-stained patterns (to assess the sperm functional status, capacitation, and acrosome reaction) were examined. Porcine oocytes were matured in vitro for 44 h in porcine oocyte medium supplemented with eCG, hCG, and dibutyryl cyclic adenosine monophosphate for the first 20 h. Matured oocytes after the removal of cumulus cells were co-cultured with sperm (final conc.: 2.5 × 105 cells mL–1) in the absence or presence of CP or CH for 8 h. Sperm penetrability was also determined. Statistical analyses of data from 4 replicated trials were performed by ANOVA. After 45 and 90 min of culture, neither CP nor CH affected sperm viability and motility (P > 0.05). The addition of CP after 45 and 90 min of culture significantly (P < 0.05) decreased capacitated and acrosome-reacted sperm rates, as detected by chlortetracyclin fluorescence assay (capacitated: control 9.6 v. CP 5.6%, control 17.8 v. CP 10.2%; acrosome reacted: control 4.6 v. CP 2.2%, control 9.2 v. CP 4.8%, respectively; P < 0.05). In the presence of CH, IVF rate and number of sperm per penetrated egg were decreased (control 80.8 v. CH 46.8%, 2.2 v. 1.4, respectively; P < 0.05). In the presence of CH, however, the percentage of metaphase II oocytes after co-culture with sperm for 8 h was lower than other 2 groups (control 87.6 v. CP 85.5 v. CH 74.0%; P < 0.05), and the percentage of A/T-II oocytes was higher than in the other 2 groups (control 1.1 v. CP 0 v. CH 9.4%; P < 0.05). From these results, we conclude that mitochondrial ribosome-specific inhibitor, chloramphenicol, affects capacitation and acrosome reaction but not penetration, whereas cytoplasmic ribosome-specific inhibitor, cyclohexide, decreases the number of oocytes that reach metaphase II stage and are penetrated.

scholarly journals Long-Lasting Effects of GABA Infusion Into the Cerebral Cortex of the Rat

2000 ◽  
Vol 7 (1-2) ◽  
pp. 1-8 ◽  
Author(s):  
Teresa Montiel ◽  
Daniel Almeida ◽  
Iván Arango ◽  
Eduardo Calixto ◽  
César Casasola ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Long Term Potentiation ◽  
Information Storage ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Receptor Stimulation ◽  
Paroxysmal Activity ◽  
The Brain

In electrophysiological terms, experimental models of durable information storage in the brain include long-term potentiation (LTP), long-term depression, and kindling. Protein synthesis correlates with these enduring processes. We propose a fourth example of long-lasting information storage in the brain, which we call the GABA-withdrawal syndrome (GWS). In rats, withdrawal of a chronic intracortical infusion of GABA, a ubiquitous inhibitory neurotransmitter, induced epileptogenesis at the infusion site. This overt GWS lasted for days. Anisomycin, a protein synthesis inhibitor, prevented the appearance of GWSin vivo. Hippocampal and neocortical slices showed a similar post-GABA hyperexcitabilityin vitroand an enhanced susceptibility to LTP induction. One to four months after the epileptic behavior disappeared, systemic administration of a subconvulsant dose of pentylenetetrazol produced the reappearance of paroxysmal activity. The long-lasting effects of tonicGABAAreceptor stimulation may be involved in long-term information storage processes at the cortical level, whereas the cessation ofGABAAreceptor stimulation may be involved in chronic pathological conditions, such as epilepsy. Furthermore, we propose that GWS may represent a common key factor in the addiction to GABAergic agents (for example, barbiturates, benzodiazepines, and ethanol). GWS represents a novel form of neurono-glial plasticity. The mechanisms of this phenomenon remain to be understood.

Role of corticosterone in TNF and IL-6 production in isolated perfused rat liver

1995 ◽  
Vol 268 (3) ◽  
pp. R699-R706 ◽  
Author(s):  
J. Liao ◽  
J. A. Keiser ◽  
W. E. Scales ◽  
S. L. Kunkel ◽  
M. J. Kluger
Keyword(s):  
Protein Synthesis ◽  
Rat Liver ◽  
Intravenous Infusion ◽  
Isolated Perfused Rat Liver ◽  
Protein Synthesis Inhibitor ◽  
Perfused Rat Liver ◽  
Synthesis Inhibitor ◽  
In Situ Data ◽  
Data Support

Using an isolated perfused rat liver (IPRL) preparation, we assessed whether corticosterone may contribute to the rise in tumor necrosis factor (TNF) and interleukin-6 (IL-6) in rats after injection with lipopolysaccharide (LPS) or exposure to psychological stress. Intravenous infusion of LPS into the IPRL led to dose-dependent increases in TNF and IL-6 concentrations in the effluent. Anisomycin, a protein synthesis inhibitor, completely blocked the rise in TNF and IL-6 concentration in the IPRL effluent, supporting the hypothesis that the synthesis (or release) of these cytokines was dependent on protein synthesis. Intravenous infusion of corticosterone at nonstressed (35 ng/ml) and stressed levels (350 ng/ml) increased TNF and/or IL-6 release. However, when LPS was combined with corticosterone, the lower dose of corticosterone facilitated the release of cytokines, whereas the higher dose of corticosterone suppressed the release of cytokines. We then showed that isolated Kupffer cells were capable of significant TNF and IL-6 production and that corticosterone decreased LPS-induced cytokine production in these cells. Our data support the hypothesis that the liver is an important source of circulating cytokines in response to LPS. In addition, although in vitro data generally support the hypothesis that corticosterone suppresses the production of cytokines, our in situ data support the hypothesis that physiological levels of corticosterone cause an increase in TNF and IL-6.

scholarly journals TePhe, a tellurium-containing phenylalanine mimic, allows monitoring of protein synthesis in vivo with mass cytometry

2019 ◽  
Vol 116 (17) ◽  
pp. 8155-8160 ◽  
Author(s):  
Jay Bassan ◽  
Lisa M. Willis ◽  
Ravi N. Vellanki ◽  
Alan Nguyen ◽  
Landon J. Edgar ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Direct Detection ◽  
Aromatic Amino Acids ◽  
Protein Synthesis Inhibitor ◽  
Mass Cytometry ◽  
Synthesis Inhibitor ◽  
Molecular Specificity ◽  
Noncanonical Amino Acid

Protein synthesis is central to maintaining cellular homeostasis and its study is critical to understanding the function and dysfunction of eukaryotic systems. Here we report L-2-tellurienylalanine (TePhe) as a noncanonical amino acid for direct measurement of protein synthesis. TePhe is synthetically accessible, nontoxic, stable under biological conditions, and the tellurium atom allows its direct detection with mass cytometry, without postexperiment labeling. TePhe labeling is competitive with phenylalanine but not other large and aromatic amino acids, demonstrating its molecular specificity as a phenylalanine mimic; labeling is also abrogated in vitro and in vivo by the protein synthesis inhibitor cycloheximide, validating TePhe as a translation reporter. In vivo, imaging mass cytometry with TePhe visualizes translation dynamics in the mouse gut, brain, and tumor. The strong performance of TePhe as a probe for protein synthesis, coupled with the operational simplicity of its use, suggests TePhe could become a broadly applied molecule for measuring translation in vitro and in vivo.

Oestradiol-17β modulates the actions of pharmacologically distinct forms of protein kinase C in rat anterior pituitary cells

1993 ◽  
Vol 136 (1) ◽  
pp. 105-117 ◽  
Author(s):  
F. J. Thomson ◽  
M. S. Johnson ◽  
D. J. MacEwan ◽  
R. Mitchell
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Anterior Pituitary ◽  
Ovariectomized Rats ◽  
Pituitary Tissue ◽  
Kinase C ◽  
Lh Release ◽  
Facilitatory Action

ABSTRACT Phorbol ester-induced release of LH and GH from rat anterior pituitary tissue in vitro is differentially inhibited by some, but not other, inhibitors of protein kinase C (PKC), suggesting that pharmacologically distinct species of PKC may have different functional roles in these cells. Since stimulus-induced anterior pituitary hormone release can be enhanced by oestradiol-17β (OE2) pretreatment, we investigated the effect of OE2 treatment of long-term (4 weeks) ovariectomized rats on the amount, activity and cellular actions of pharmacologically distinct PKC species in rat anterior pituitary tissue. Here we report that OE2 treatment enhanced phorbol 12,13-dibutyrate (PDBu)-induced LH but not GH release measured in vitro. This effect of OE2 on LH release may involve synthesis of additional PKCs that are not targeted by the synthetic diacylglycerol, 1,2-dioctanoyl-snglycerol (DOG). Measurements of anterior pituitary PKC activity and [3H] phorbol ester-binding studies suggested that the facilitatory action of OE2 on LH release may occur, at least in part, by altering the quantity and activity of PKC(s). Our results also demonstrate that the OE2-induced PKC(s) which facilitate LH release may be of the type that are not dependent upon raised intracellular Ca2+ for their activation and display distinct pharmacological properties (being readily activated by PDBu, but not by DOG, and are staurosporine-sensitive but H7-insensitive). This facilitatory action of OE2 on PKC-induced LH release does not appear to involve OE2-induced changes in the affinity of existing PKC(s) for PDBu, or changes in the amount of releasable LH in the pituitary prior to the stimulus. Journal of Endocrinology (1993) 136, 105–117

scholarly journals Role of lysosomal cathepsin activities in cell hypertrophy induced by NH4Cl in cultured renal proximal tubule cells.

1996 ◽  
Vol 7 (1) ◽  
pp. 73-80
Author(s):  
H Ling ◽  
S Vamvakas ◽  
M Gekle ◽  
L Schaefer ◽  
M Teschner ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cell Protein ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Renal Hypertrophy ◽  
Cytosolic Ph ◽  
Cell Hypertrophy ◽  
Renal Proximal Tubule Cells

An increase of renal ammoniagenesis has been implicated in renal hypertrophy associated with various clinical disorders such as metabolic acidosis, diabetic nephropathy, and renal insufficiency. In vivo and in vitro studies have shown that ammonia promotes hypertrophy in tubular epithelial cells. To elucidate its role on protein turnover, the effects of NH4Cl on the activities of cathepsins B, H, and L+B, as well as on protein synthesis and degradation in LLC-PK1 cells, were investigated. The results show that NH4Cl (20 mM) induced cell hypertrophy, as defined by an increase in both cell protein content and cell volume (+25.5 +/- 1.3 and +10.4 +/- 0.1% after 48 h). This hypertrophy was associated with the suppression of the activities of cathepsins B and L+B (-57.0 +/- 0.9 and -54.5 +/- 1.5% after 48 h) and a reduction of protein degradation rate (-59.7 +/- 4.1% after 48 h), but without enhanced protein synthesis. The findings were further supported with an additional experiment, showing that the protein synthesis inhibitor cycloheximide (10 microM) did not blunt NH4Cl-induced cell hypertrophy. Moreover, NH4Cl (20 mM) resulted in a persistent elevation of the lysosomal pH, whereas the rise in the cytosolic pH was only transient. This alkalinization in lysosomes may be causatively involved in the impairment of the activities of cathepsins B and L+B. In conclusion, the suppression of the activities of cathepsins B and L+B and the subsequent reduction of protein breakdown due to intralysosomal alkalinization contribute to NH4Cl-induced hypertrophy in LLC-PK1 cells.

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