Effect of Ca2+ deprivation on release of luteinizing hormone induced by luteinizing hormone releasing hormone from female rat pituitary glands in vitro

1982 ◽  
Vol 94 (1) ◽  
pp. 11-20 ◽  
Author(s):  
J. de Koning ◽  
A. M. I. Tijssen ◽  
J. A. M. J. van Dieten ◽  
G. P. van Rees
Keyword(s):  
Protein Synthesis ◽  
Initial Phase ◽  
Phase Response ◽  
Ovariectomized Rats ◽  
Second Phase ◽  
Pituitary Glands ◽  
Lh Release ◽  
Lh Rh ◽  
Intact Rats

Continuous exposure of hemi-pituitary glands from intact female rats to LH releasing hormone (LH-RH) in vitro displayed three phases in the pattern of LH release: during the first hour release of LH was low (first phase response), then it increased to a higher level during the second hour and remained constant during the next 2 h (second phase response), after which there was a refractoriness of LH release (third phase response). The initial phase response of pituitary glands from intact rats was blocked by EGTA (a Ca2+ chelator) but there was a small but significant increase in the rate of LH release during the second phase response. This increase could be prevented by inhibition of protein synthesis by cycloheximide. Cycloheximide and EGTA did not affect basal release of LH by glands from intact rats, neither did EGTA affect the high basal release of LH by glands from ovariectomized rats. However, the LH-RH-induced release of LH from pituitary glands of ovariectomized rats, which did not show the initial phase of low LH release, was completely suppressed by EGTA throughout a 4-h incubation period. The pattern of LH release stimulated by the combination of N6-monobutyryl cyclic AMP and theophylline (mbcAMP/theophylline) showed an initial phase of low LH release lasting 4 h after which it increased. The magnitude of the effect was small compared with the action of LH-RH. As it did with LH-RH, EGTA completely blocked the initial response, but allowed a small increase in the rate of LH release thereafter; this increase could also be blocked by inhibition of protein synthesis. Addition of EGTA to media during pretreatment of pituitary glands from intact rats with either LH-RH or mbcAMP/theophylline did not impair the facilitatory effect of these secretagogues on the responsiveness of the glands to subsequent exposure to LH-RH and cycloheximide and normal Ca2+ levels. The restoration of Ca2+ levels after withdrawal neither affected basal nor LH-RH-induced release of LH. Exclusion of Ca2+ from the media during a 6-h incubation of pituitary glands from intact rats with LH-RH prevented the glands from becoming refractory to subsequent stimulation by LH-RH, which occurs when normal Ca2+ concentrations are present. The results suggested that extracellular Ca2+ is obligatory for LH release and the induction of refractoriness by LH-RH. In contrast, that part of the action of LH-RH which is cyclic AMP-mediated and protein synthesis-dependent is not affected by withdrawal of extracellular Ca2+.

STUDIES ON A PROTEIN SYNTHESIS DEPENDENT STEP IN LH RELEASE BY LH-RH

1979 ◽  
Vol 92 (4) ◽  
pp. 648-657 ◽  
Author(s):  
J. de Koning ◽  
J. A. M. J. van Dieten ◽  
A. M. I. Tijssen ◽  
G. P. van Rees
Keyword(s):  
Protein Synthesis ◽  
Female Rats ◽  
Protein Factor ◽  
Active Concentration ◽  
Long Time ◽  
Pituitary Glands ◽  
Lh Release ◽  
Lh Rh ◽  
Inhibitor Of Protein Synthesis

ABSTRACT The hypothesis that LH-RH induces LH release partly through a protein synthesis dependent step (protein factor) was further investigated using two different experimental designs. First, during incubation of pituitary glands of intact dioestrous female rats with a maximally active concentration of LH-RH, the inhibitor of protein synthesis cycloheximide was added at various times after the beginning of the incubation. The results show that it takes a relatively long time, i.e. more than 1 h of exposure to LH-RH before the amount of the protein factor has increased sufficiently to allow a maximal LH secretion. Secondly, LH-RH was injected iv after which the protein factor was assayed by incubating the pituitary glands with a maximally active concentration of LH-RH in the presence of cycloheximide and measuring LH release in vitro. It was found that 1 h after the injection sufficient protein factor was present to permit an elevated response to LH-RH. This response could be suppressed by injecting cycloheximide prior to LH-RH. When the interval between injection of LH-RH and beginning of the incubation was increased to 2 h, LH release in vitro decreased again. However, ovariectomy immediately before LH-RH injection resulted in maintenance of the elevated response to LH-RH in vitro, indicating a role of the ovaries in this phenomenon.

The involvement of ovarian factors in maintaining the pituitary glands of female rats in a state of low LH responsiveness to LHRH

1987 ◽  
Vol 112 (2) ◽  
pp. 265-273 ◽  
Author(s):  
J. de Koning ◽  
A. M. I. Tijssen ◽  
G. P. van Rees
Keyword(s):  
Pituitary Gland ◽  
Phase Response ◽  
The Self ◽  
Female Rats ◽  
Ovariectomized Rats ◽  
Lag Phase ◽  
Second Phase ◽  
Rat Pituitary ◽  
Pituitary Glands

ABSTRACT The effects of discontinuation and restoration of ovarian influences on the pituitary LH response to LHRH in vitro were investigated. When female rat pituitary glands taken on day 2 of dioestrus were incubated with LHRH the release of LH was low during the first hour (lag phase response) and afterwards a progressive, protein synthesis-dependent increase took place (second phase response), this being the self-priming action of LHRH. Short-term discontinuation (less than 1 day) of ovarian influences on the rat pituitary gland in vivo (ovariectomy) or in vitro (incubation in medium only) resulted in an increased LHRH-induced LH response during the lag phase. The biphasic LH response or the self-priming action of LHRH disappeared completely after long-term discontinuation of ovarian influences on the pituitary gland, LH release being at its maximum from the start of the incubation. The biphasic response was reinstated when ovaries were implanted under the kidney capsules of ovariectomized rats. Auto-implantation of an ovary into the spleen immediately after bilateral ovariectomy did not, however, prevent the disappearance of the LHRH self-priming action. Ovarian activity responsible for the presence of the low LH response during the lag phase was thus effectively removed by the liver, but inhibin-like activity suppressing serum FSH levels remained present. Silicone elastomer implants (s.c.) containing oestradiol-17β, implanted for 4 weeks, did not reverse the loss of the biphasic LH response to LHRH. It is concluded that liver-labile factors released by the ovaries keep the pituitary gland in a state of low responsiveness to LHRH. By giving a sufficiently high LHRH stimulus this inhibitory effect is neutralized and transition to a highly responsive state can be achieved. The ovarian factor(s) is not identical to inhibin or oestradiol-17β. J. Endocr. (1987) 112, 265–273

LH-RH-dependent synthesis of protein necessary for LH release from rat pituitary glands in vitro

1976 ◽  
Vol 5 (1-2) ◽  
pp. 151-160 ◽  
Author(s):  
J. De Koning ◽  
J.A.M.J. Van Dieten ◽  
G.P. Van Rees
Keyword(s):  
Rat Pituitary ◽  
Pituitary Glands ◽  
Lh Release ◽  
Lh Rh

Release and synthesis of FSH by pituitary glands of female rats in vitro

1982 ◽  
Vol 100 (4) ◽  
pp. 499-503 ◽  
Author(s):  
A. A. J. Jenner ◽  
J. de Koning ◽  
G. P. van Rees
Keyword(s):  
Protein Synthesis ◽  
Slow Rate ◽  
Female Rats ◽  
Intact Female ◽  
Ovx Rats ◽  
Basal Release ◽  
Pituitary Glands ◽  
Independent Synthesis ◽  
Intact Rats

Abstract. Anterior hemi-pituitary glands from intact female and ovariectomized (OVX) rats were incubated with or without a maximally effective dose of LRH. During an 8 h incubation, LRH-stimulated release of FSH by pituitary glands from intact rats was biphasic: an initial slow rate of release and, from 2 to 8 h, an enhanced rate of release. Basal release was low up to 4 h, after which a marked increase of the rate of release was measured: from 6 to 8 h there was no difference between the rates of basal and LRH-stimulated release. Basal and LRH-stimulated release of FSH by pituitary glands from OVX rats were high and approximately constant during an 8 h incubation. Both basal and LRH-stimulated release by glands from intact as well as OVX rats were protein synthesis dependent. During the incubations an LRH-independent synthesis of FSH was measured. The results suggest that this synthesis is involved, either directly or indirectly, in increasing the rate of basal release of FSH after 4 h. A comparison of release and synthesis of FSH with those of LH reveals characteristic differences.

Release of LH in vitro from anterior pituitary glands of ovariectomized rats by LRH or elevated potassium concentration after pre-treatment with oestradiol benzoate in vivo

1982 ◽  
Vol 99 (2) ◽  
pp. 206-210 ◽  
Author(s):  
A. M. I. Tijssen ◽  
J. de Koning ◽  
G. P. van Rees
Keyword(s):  
Ovariectomized Rats ◽  
Bicarbonate Buffer ◽  
High K ◽  
Oestradiol Benzoate ◽  
Negative Effect ◽  
Pituitary Glands ◽  
Lh Release ◽  
Pre Treatment ◽  
Positive Effect

Abstract. Pituitary glands from ovariectomized rats which had been pre-treated with oestradiol benzoate (OeB) or solvent oil were incubated in Krebs-Ringer bicarbonate buffer with glucose containing either LRH (1000 ng/ml) or a high K+ concentration (50 mM). OeB (7 μg sc) or oil was injected at 2.5 or 6.5 h before the beginning of the incubation experiment or during the three preceding days (three daily injections). Depending upon the period during which the pituitary glands had been exposed to OeB LH release induced by LRH was inhibited (negative effect of OeB) or augmented (positive effect). When the glands were incubated in medium containing high K+, only the negative effect of OeB pre-treatment was seen. It is concluded that that part of LRH-induced LH release which is mimicked by high K+ is involved in the negative effect of OeB, but not in its positive effect.

DEPENDENCE ON PROTEIN SYNTHESIS OF THE N6-MONOBUTYRYL CYCLIC AMP PLUS THEOPHYLLINE MEDIATED RELEASE OF LUTEINIZING HORMONE INDUCED BY LUTEINIZING HORMONE RELEASING HORMONE FROM RAT PITUITARY GLANDS IN VITRO

1981 ◽  
Vol 88 (3) ◽  
pp. 329-338 ◽  
Author(s):  
J. DE KONING ◽  
J. A. M. J. VAN DIETEN ◽  
A. M. I. TIJSSEN ◽  
G. P. VAN REES
Keyword(s):  
Protein Synthesis ◽  
Luteinizing Hormone ◽  
Cyclic Amp ◽  
Female Rats ◽  
Releasing Hormone ◽  
Pituitary Tissue ◽  
Pituitary Glands ◽  
Lh Release ◽  
Lh Rh ◽  
Lh Releasing Hormone

The involvement of cyclic AMP in the action of LH releasing hormone (LH-RH) on LH secretion was studied by incubating pituitary glands from adult female rats on day 2 of dioestrus with 1 mm-N6-monobutyryl cyclic AMP (mbcAMP) and 10 mm-theophylline for periods of up to 10 h. This treatment induced a pattern of LH release similar to that observed in the presence of a low concentration of LH-RH (0·1 ng LH-RH/ml), i.e. an initial 4 h period during which the release of LH was minimal was followed subsequently by an increased rate of release. In this system inhibition of protein synthesis by cycloheximide (25 μg/ml) did not impair the initial response of the pituitary tissue but the increase in the rate of LH release during the second phase of the response was blocked. Preincubation with mbcAMP and theophylline increased the responsiveness of the pituitary tissue to LH-RH. This action could be prevented by including cycloheximide during the preincubation period, whereas addition of this drug during the incubation with LH-RH no longer impaired the increased responsiveness. The size of the sensitizing action of mbcAMP and theophylline mediated through the induction of protein synthesis was comparable with that of a high concentration of LH-RH. From the absence of a significant change in total LH during the preincubation period, it was concluded that the increased responsiveness was not the result of newly synthesized LH. The present results suggest a role or roles for cyclic AMP in the secretion of LH induced by LH-RH. Besides an effect on the formation of a factor related to the synthesis of protein, other than LH which has a permissive role in the acute release of LH, cyclic AMP might also be concerned in the secretion process through a pathway which does not involve synthesis of protein.

DISCREPANCY BETWEEN RADIOIMMUNOASSAY AND RADIOLIGAND-RECEPTOR ASSAY OF LUTEINIZING HORMONE RELEASED IN VITRO BY PITUITARY TISSUE FROM MALE RATS OF DIFFERENT AGES

1975 ◽  
Vol 65 (2) ◽  
pp. 265-273 ◽  
Author(s):  
R. M. SHARPE ◽  
M. SHAHMANESH ◽  
M. G. ELLWOOD ◽  
M. HARTOG ◽  
P. S. BROWN
Keyword(s):  
Luteinizing Hormone ◽  
Close Agreement ◽  
Male Rats ◽  
Male Rat ◽  
Receptor Assay ◽  
Pituitary Glands ◽  
Lh Release ◽  
Radioligand Receptor Assay ◽  
Lh Rh

SUMMARY Anterior pituitary glands from male rats aged 21, 40, 60 or 95 days were incubated in medium containing 0, 2 or 20 ng luteinizing hormone-releasing hormone (LH-RH)/ml. Incubates were assayed for LH by radioimmunoassay (RIA), by the radioligand-receptor assay (RLA) using testicular homogenates as the source of receptor and, in some instances, by the ovarian ascorbic acid depletion assay (OAAD). Irrespective of the dose of added LH-RH, glands from rats aged 40 and 60 days always showed a higher release of LH, as determined by RLA, than glands from animals aged 21 or 95 days. Measurement by RIA showed a similar pattern to RLA in the basal release of LH, but in the presence of LH-RH showed little difference in LH release by glands from rats aged 40, 60 or 95 days. The LH release caused by the higher concentration of LH-RH was always greater when measured by RLA than by RIA. Assay of comparable incubates by OAAD showed close agreement with RLA estimates in four incubations (mean index of discrimination 1·07; range 0·86–1·18) and consistent disagreement with RIA estimates (1·64; range 1·38–1·99). In contrast to the results with incubates, homogenates of pituitary glands from male rats of various ages showed close agreement of estimates by RLA, RIA and OAAD. These results suggest that RIA underestimates the LH-RH-stimulated release of LH in vitro from the male rat pituitary during some stages of sexual maturation.

Effect of pre-treatment of long-term ovariectomized rats with anti-LRH on the response of the pituitary gland in vitro to the analogue of LRH D-ser-(tBu)6-des-gly10-ethylamide-LRH (Buserelin®)

1983 ◽  
Vol 104 (3) ◽  
pp. 272-278 ◽  
Author(s):  
G. P. van Rees ◽  
J. A. M. J. van Dieten ◽  
J. de Koning ◽  
A. F. P. M. de Goey
Keyword(s):  
Pituitary Gland ◽  
Ovariectomized Rats ◽  
Rabbit Serum ◽  
Normal Rabbit Serum ◽  
Basal Release ◽  
Pituitary Glands ◽  
Lh Release ◽  
Pre Treatment

Abstract. Ovariectomized rats were injected iv with an antiserum against LRH or normal rabbit serum. AntiLRH caused a decrease of plasma LH and FSH. After 24 or 48 h, the rats were decapitated and the pituitary glands incubated in the presence of an analogue of LRH which reacts minimally with anti-LRH (Buserelin). Pretreatment with anti-LRH caused an increased response of pituitary LH release to Buserelin. Similar results were obtained with regard to FSH. In this case, however, basal release of FSH was lowered by pre-treatment with antiLRH. Pituitary LH and FSH contents were not affected by anti-LRH, but synthesis of LH and FSH in vitro was smaller than in control glands obtained from rats pretreated with normal rabbit serum.

Time-course of release and synthesis of luteinizing hormone, follicle-stimulating hormone and prolactin from rat pituitary glands in vitro

1983 ◽  
Vol 96 (2) ◽  
pp. 165-170 ◽  
Author(s):  
Marta E. Apfelbaum
Keyword(s):  
Time Course ◽  
Ovariectomized Rats ◽  
Second Phase ◽  
Hormone Synthesis ◽  
Continuous Release ◽  
Rat Pituitary ◽  
Pituitary Glands ◽  
Secondary Phenomenon ◽  
Two Phases

To study the time-course of release and synthesis of LH, FSH and prolactin, anterior pituitary glands from ovariectomized rats were incubated for different time-intervals between 0 and 4 h. Comparable patterns of release and synthesis of LH and FSH as a function of the incubation time were observed. It was possible to distinguish two phases in the profiles of secretion of both gonadotrophins. During the first phase, from 0–2 h, release was associated with a corresponding decrease of the hormone concentrations within the gland but no overall changes in total LH and FSH production. During the second phase, between 2 and 4 h, a progressive reaccumulation of both gonadotrophins occurred in spite of the continuous release of hormones into the medium, reflecting formation of new immunoassayable material. These results suggest that the increased synthesis ensues as a secondary phenomenon arising from the release of the hormones from the tissue. The time-course of release and synthesis of prolactin showed different dynamics during the course of incubation. High levels of prolactin were released and synthesized when the adenohypophysis was incubated in vitro. Considerably larger amounts of this hormone were found in the medium than in the tissue from the first hour of incubation. After a lag of about 40 min synthesis of prolactin was increased in parallel with its release. This led to the assumption that both prolactin synthesizing and releasing processes occurred simultaneously from the early stages of the incubation. Comparatively, prolactin-secreting cells had a very fast and LH-secreting cells a low rate of turnover; FSH-secreting cells were intermediate between the two. These results indicate that (1) the increase in release of LH, FSH and prolactin into the medium precedes that of hormone synthesis and (2) the initial depletion of the pituitary gland as a result of hormone release could act as a stimulus for synthesis, leading to the reestablishment of hormonal storage levels.

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