BIOASSAY OF INHIBIN-LIKE ACTIVITY USING PITUITARY CELLS IN VITRO

1979 ◽  
Vol 80 (1) ◽  
pp. 91-102 ◽  
Author(s):  
F. H. DE JONG ◽  
S. D. SMITH ◽  
H. J. VAN DER MOLEN
Keyword(s):  
Cell Culture ◽  
Luteinizing Hormone ◽  
Male Rats ◽  
Pituitary Cells ◽  
Lh Rh ◽  
Dose Dependent ◽  
Higher Doses ◽  
Ovarian Follicular Fluid ◽  
Sensitive Method

The secretion of follicle-stimulating hormone (FSH) by the pituitary gland appears to be partially regulated by a protein hormone, inhibin, which is produced in the Sertoli cells of the testis and is also present in ovarian follicular fluid (FF). The aim of the present study was to develop a sensitive method for the detection and estimation of inhibin-like activity, using dispersed pituitary cells in culture. Pituitary cells from adult male rats were dispersed and precultured for 3 days. After renewal of the medium (2 ml), samples to be tested for inhibin-like activity were added and culture was continued for a further 3 days. A dose-dependent suppression of the concentration of FSH in the medium (λ = 0·17–0·22) was observed after addition of FF (0·05–1 μl) or Sertoli cell culture medium (SCCM, 0·05–1 ml). Luteinizing hormone (LH) concentrations were not affected with these doses of FF, but SCCM and higher doses of FF caused a significant increase in the concentration of LH in the medium. During an additional 6 h of culture in the presence of luteinizing hormone releasing hormone (LH-RH), FF and SCCM suppressed the release of both FSH (λ = 0·07–0·11) and LH in a dose-dependent way. Cellular content of FSH, but not of LH, was decreased after these treatments. These results could not be explained by damage to the pituitary cells, by degradation of FSH or LH-RH, or by effects of steroids. It is concluded that this pituitary cell culture system can be used as a sensitive method for the estimation of inhibin-like activity in FF and SCCM.

Stimulatory effect of thyrotrophin-releasing hormone (TRH) on the release of luteinizing hormone (LH) from rat anterior pituitary cells in vitro

1983 ◽  
Vol 61 (2) ◽  
pp. 186-189 ◽  
Author(s):  
Noboru Fujihara ◽  
Masataka Shiino
Keyword(s):  
Luteinizing Hormone ◽  
Anterior Pituitary ◽  
Pituitary Cells ◽  
Thyrotrophin Releasing Hormone ◽  
Releasing Hormone ◽  
Anterior Pituitary Cells ◽  
Lh Release ◽  
Lh Rh

The effect of thyrotrophin-releasing hormone (TRH, 10−7 M) on luteinizing hormone (LH) release from rat anterior pituitary cells was examined using organ and primary cell culture. The addition of TRH to the culture medium resulted in a slightly enhanced release of LH from the cultured pituitary tissues. However, the amount of LH release stimulated by TRH was not greater than that produced by luteinizing hormone – releasing hormone (LH–RH, 10−7 M). Actinomycin D (2 × 10−5 M) and cycloheximide (10−4 M) had an inhibitory effect on the action of TRH on LH release. The inability of TRH to elicit gonadotrophin release from the anterior pituitary glands in vivo may partly be due to physiological inhibition of its action by other hypothalamic factor(s).

STIMULATION OF PROSTAGLANDIN ACCUMULATION IN THE RAT ANTERIOR PITUITARY GLAND BY LUTEINIZING HORMONE RELEASING HORMONE IN VITRO

1977 ◽  
Vol 75 (2) ◽  
pp. 277-283 ◽  
Author(s):  
N. BARDEN ◽  
A. BETTERIDGE
Keyword(s):  
Luteinizing Hormone ◽  
Cyclic Amp ◽  
Anterior Pituitary ◽  
Pituitary Cells ◽  
Luteinizing Hormone Releasing Hormone ◽  
Releasing Hormone ◽  
Lh Release ◽  
Lh Rh ◽  
Stimulation Of

The addition of luteinizing hormone releasing hormone (LH-RH) to cultures of monolayers of rat anterior pituitary cells was shown to increase both the concentrations of prostaglandins E1 and E2 (PGE) in the cells and the release of LH over similar ranges of concentrations of LH-RH (10−6 to 10−10 mol/l). The peak concentration of PGE was observed after 2·5 h. The stimulation of the level of PGE in the cells by LH-RH was completely inhibited by two inhibitors of prostaglandin synthetase, which only partially inhibited the stimulation of LH release. Therefore the increased concentration of PGE was not obligatory for the effect of LH-RH on LH release. It was also shown that monobutyryl cyclic AMP stimulated the intracellular concentration of PGE and it is suggested that the stimulation of PGE levels may be mediated by increased levels of cyclic AMP in the cells after the addition of LH-RH.

Energetic dependence of NPY-induced LH secretion in a teleost fish (Dicentrarchus labrax)

1999 ◽  
Vol 277 (6) ◽  
pp. R1627-R1634 ◽  
Author(s):  
José Miguel Cerdá-Reverter ◽  
Lisa Ann Sorbera ◽  
Manuel Carrillo ◽  
Silvia Zanuy
Keyword(s):  
Glucose Metabolism ◽  
Luteinizing Hormone ◽  
Dicentrarchus Labrax ◽  
Sea Bass ◽  
Pituitary Cells ◽  
Lh Secretion ◽  
Dose Dependent Increase ◽  
Dose Dependent

The purpose of this work was to examine the role of energetic status in neuropeptide Y (NPY)-induced luteinizing hormone (LH) secretion and glucose metabolism in fish. Fasted juvenile sea bass ( Dicentrarchus labrax) were injected intraperitoneally with pig (p) NPY or pNPY + glucose, whereas fed animals were injected with pNPY alone and plasma glucose, insulin, and LH levels were examined. pNPY alone or in combination with glucose was found to induce a dose-dependent increase in LH secretion in fasted animals. Similar LH responses to pNPY were observed in vitro in dispersed pituitary cells isolated from fed and fasted animals incubated in L-15 and restricted media. Injection of pNPY + glucose in fasted animals resulted in depletion of glucose. Insulin plasma levels decreased in fasted animals coinjected with pNPY + glucose but remained stable when NPY was administrated alone to fed and fasted animals. Results suggest that 1) NPY-induced LH secretion in fish is dependent on energetic status and 2) NPY is capable of modifying glucose metabolism.

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.

Evolution of Design and Achievement of Inhibitors of the Luteinizing Hormone-Releasing Hormone as Inhibitors of Ovulation

1982 ◽  
Vol 37 (2) ◽  
pp. 246-259 ◽  
Author(s):  
Karl Folkers ◽  
John Humphries ◽  
Cyril Y. Bowers
Keyword(s):  
Luteinizing Hormone ◽  
Adult Male ◽  
Subcutaneous Tissue ◽  
Female Rats ◽  
Male Rats ◽  
Luteinizing Hormone Releasing Hormone ◽  
Releasing Hormone ◽  
Lh Rh

Abstract Detailed structure-activity studies on inhibitors of the luteinizing hormone releasing hormone (LH-RH) have been described. The most potent ovulation inhibitors have substitutions in positions 1, 2, 3, and 6. Currently four basic structural requirements for potent antiovulatory activity are: a D-aromatic amino acid, such as D-Trp or D-Phe, in position 6; a D-Phe residue in position 2; substitutions in positions 1 and 3. For inhibitors based on substitutions in positions 2, 3, and 6, the substitution of a Pro, N-Me-Leu or D-Trp residue in position 3 is equally acceptable, and gives analogues which inhibit ovulation at 750 ^g/rat. For inhibitors based on substitutions in positions 1, 2, 3, and 6, D-Trp appears necessary in position 3 in order for ovulation to be inhibited at 200 μ/rat. Many analogues based on the [residue1, D-Phe2, D-Trp3, D-Trp6]-LH-RH sequence are known which inhibit ovulation at 200 μ/rat. These include those analogues having D- <Glu, Ac-Pro, N-Ac-Hyp and N-Ac-Thr in position 1. The choice between L- or Dresidues in this position is structure dependent (Ac-L-Pro > Ac-D-Pro, D- <Glu >L- <Glu, etc.). In addition, a "protected" N-terminal residue having some polar character appears to be important. Substitution of the dipeptide residue, <Glu-Pro-, into position 1 has produced a new category of potent ovulation inhibitors based on linear peptides longer than decapeptides. Continued studies on other analogues in this later class could provide more potent inhibitors by (1) utilizing new binding sites on or in the vicinity of the LH-RH receptor(s); (2) altering transportation properties; (3) producing "pro-drugs". The substitution of N-Me-Leu into position 7 was not advantageous, presumably because of the presence of bulky D-aromatic amino acids in position 6. Nonapeptide ethylamide analogues also had very low antiovulatory potencies. The analogue [chlorambucil1, Leu2, Leu3, D-Ala6]-LH-RH acted as an agonist, but also inhibited in a modified assay in vitro. Comparative assays measuring the inhibition of LH-RH, and inhibition of ovulation have emphasized other factors of importance to inhibitor design. Although all ovulation inhibitors active at 750 or 200 /μg/rat strongly inhibited in vivo, at a ratio of analogue to LH-RH of 166:1, other analogues of comparable in vitro potency have displayed a range of antiovulatory activities. Similar discrepancies have been observed in the results of in vivo LH-RH inhibition assays. The most potent ovulation inhibitors always inhibited LH-RH at 333:1 in adult male chimpanzees, and at 100:1 in adult male rats. The dissociation of the results of the LH-RH and antiovulatory assays have been rationalized in two cases. The Cpc-analogues were active in inhibiting LH-RH in rats and in chimpanzees when given i.V., but were inactive in rats when given s.c. which is the mode of administration in the antiovulatory assay. The results for inhibition of LH-RH in vivo paralleled the results for inhibition of ovulation, and raised a question as to differences in absorption of peptides though the lipid layers of subcutaneous tissue. The reduced in vivo activities of the L-Trp3 analogues in both the LH-RH and antiovulatory assays suggest an increase in enzymatic inactivation for these compounds. [D-Phe2, Pro3, D-Phe6]-LH-RH can inhibit endogenous LH-RH in the Rhesus monkey and inhibit ovulation. Infusion of [D-Phe2, Pro3, D-Trp6]-LH-RH at 375 ^ug/day for 4 days from a s.c. implanted minipump completely inhibited ovulation in cycling female rats and decreased serum LH levels in castrated rats. In contrast with LH-RH or des-Gly10- [D-Ala6]-LH-RH ethylamide the Pro3 analogue did not block uterine implantation sites of mated rats, indicating a difference in the mechanism of contraception for LH-RH agonists and inhibitors

EFFECT OF TREATMENT WITH AN AGONIST OF LUTEINIZING HORMONE RELEASING HORMONE ON EARLY MATURATIONAL CHANGES IN PITUITARY AND TESTICULAR FUNCTION IN THE RAT

1979 ◽  
Vol 80 (2) ◽  
pp. 249-257 ◽  
Author(s):  
R. M. SHARPE ◽  
H. M. FRASER ◽  
J. SANDOW
Keyword(s):  
Luteinizing Hormone ◽  
Serum Concentration ◽  
Male Rats ◽  
Testicular Function ◽  
Luteinizing Hormone Releasing Hormone ◽  
Releasing Hormone ◽  
Testicular Weight ◽  
Effect Of Treatment ◽  
Lh Rh

Male rats aged 30 days were injected once daily for between 1 and 7 days with 50 ng (d-serine t-butyl6, des-glycine-NH210) luteinizing hormone releasing hormone ethylamide (LH-RH agonist), and pituitary and testicular function were assessed. Treatment for 7 days significantly (P < 0·02) inhibited maturational increases in the pituitary content and serum concentration of gonadotrophins, testicular luteinizing hormone (LH)-receptor concentration and the testicular capacity to secrete testosterone; the pituitary content and serum concentration of prolactin, the hypothalamic content of LH-RH and testicular weight were unaffected. In rats treated with LH-RH agonist, the initial (2 to 3 days) reduction in testicular LH-receptors and the capacity to secrete testosterone probably resulted from acutely raised levels of LH in the blood, whilst later effects may have resulted from the apparently chronic reduction in serum gonadotrophin levels. The latter may reflect a decrease in pituitary responsiveness to repeated stimulation with LH-RH agonist. Despite the extensive loss of testicular LH-receptors and diminished responsiveness, the concentration of HCG which significantly (P < 0·05) increased testosterone secretion by the testis in vitro was the same (2 pmol/l) as that for testes from control rats.

Effects of in vivo gonadal hormone environment on in vitro hGRF-40-stimulated GH release

1985 ◽  
Vol 249 (3) ◽  
pp. E276-E280 ◽  
Author(s):  
W. S. Evans ◽  
R. J. Krieg ◽  
E. R. Limber ◽  
D. L. Kaiser ◽  
M. O. Thorner
Keyword(s):  
Female Rats ◽  
Male Rats ◽  
Gonadal Hormone ◽  
Base Line ◽  
Pituitary Cells ◽  
Intact Male ◽  
Castrate Male ◽  
Basal Release

The effects of gender and the gonadal hormone environment on basal and stimulated growth hormone (GH) release by dispersed and continuously perifused rat anterior pituitary cells were examined. Cells from intact male and diestrus day 2 female rats and from castrate male rats either untreated or treated with testosterone (T) or 17 beta-estradiol (E2) were used. Basal GH release (ng/min per 10(7) cells; mean +/- SE) by cells from diestrus day 2 female rats was less than by cells from castrate rats treated with T (4.3 +/- 0.6 vs. 11.4 +/- 2.7, respectively; P less than 0.025). No other differences in basal release were detected. Concentration-response relationships were documented between human GH-releasing factor 40 (hGRF-40; 0.03-100 nM given as 2.5-min pulses every 27.5 min) and GH release. Mean (+/- SE) overall GH release (ng/min per 10(7) cells) above base line was greater by cells from intact male rats (496 +/- 92) than by cells from castrate (203 +/- 37.3; P less than 0.0001), castrate and T-treated (348 +/- 52.8; P = 0.008), or castrate and E2-treated (58.1 +/- 6.8; P less than 0.001) male rats or by diestrus day 2 rats (68.6 +/- 9.5; P = 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Human vascular smooth muscle responses mediated by α2 mechanisms in vivo and in vitro

1985 ◽  
Vol 68 (s10) ◽  
pp. 147s-150s ◽  
Author(s):  
S. Thom ◽  
J. Calvete ◽  
R. Hayes ◽  
G. Martin ◽  
P. Sever
Keyword(s):  
Smooth Muscle ◽  
Physiological Role ◽  
Smooth Muscle Contraction ◽  
Α1 Receptors ◽  
Dose Dependent ◽  
Higher Doses ◽  
Α2 Receptors ◽  
Autoregulatory Mechanism

1. The effects of compounds with α2-agonist and α2-antagonist properties on human forearm blood flow and on isolated human arterial segments have been studied. 2. The findings from these studies in vivo and in vitro did not provide evidence in support of the hypothesis that postsynaptic α2-receptors mediate smooth muscle contraction in the tissues under investigation. 3. The constriction of the forearm vascular bed in response to low intra-arterial doses of idazoxan (RX 781094), an α2-antagonist, provides evidence for a physiological role for a presynaptic α2 autoregulatory mechanism. 4. The variability of the forearm vascular responses to higher doses of idazoxan highlights the pitfalls that may have misled previous authors in their interpretation of the results of similar studies. A U-shaped dose-response curve to compounds with mixed α2-and α1-antagonist properties may be constructed, which emphasizes the importance of the dose-dependent selectivity of these antagonists at α2- and α1-receptors. 5. The effect of idazoxan on the responses of arterial segments in vitro to exogenous catecholamines was dependent on the integrity of the endothelium, and provides evidence that α2-receptors may mediate release of the endothelium-derived relaxing factor.

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