A Rapid, Sensitive and Reliable Assay for Inhibin Bioactivity

A rapid 2-day quantitative assay for inhibin bioactivity based on FSH secretion from pituitary cells of immature female rats is described. The bioassay exhibited steeper slopes, improved precision and greater (fourfold) sensitivity compared with a previously established pituitary FSH cell content assay. Whole pituitary glands were used for the preparation of pituitary cells and the method for cell dispersion required a single enzymatic treatment with trypsin. Cells (180000 viable cells per well) were dispensed into culture media containing inhibin and incubated for 48 h. Media were removed and assayed for FSH by radioimmunoassay. Using a ram rete testis fluid preparation as standard the inhibin dose-response curves of 25 consecutive experiments showed indices of precision of - O� 08(mean)[range - 0�04 to - 0�17] and Finney's G values of 0�017[0�003-0�06]. The mean ED40 was 0�17 units of in hi bin activity per well with interassay variation of 16�2% at this point of the dose-response curve. The assay had a practical capacity of 400 wells, permitting the measurement of dose-response curves of at least 40 unknowns with three dose points and triplicate wells per dose. The assay is specific for inhibin-containing preparations from several animal species. Overall, the assay is simple, precise, and sensitive, indicative of its applicability to the measurement of inhibin samples with low inhibin bioactivity and to the screening of large numbers of fractions during inhibin purification.

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Biphasic modulation of pituitary sensitivity to GnRH by oestrogens: the effects of A- and D-ring substitution on LH release in cultured pituitary cells

Acta Endocrinologica ◽

10.1530/acta.0.1070317 ◽

1984 ◽

Vol 107 (3) ◽

pp. 317-327 ◽

Cited By ~ 7

Author(s):

G. Emons ◽

R. Knuppen ◽

P. Ball ◽

K.J. Catt

Keyword(s):

Dose Response ◽

High Sensitivity ◽

Female Rats ◽

Pituitary Cells ◽

Response Curves ◽

Methyl Group ◽

Lh Release ◽

Dose Response Curves ◽

Sensitizing Effect ◽

Almost All

Abstract. The sensitizing effect of oestrogens on GnRH-stimulated LH release was evaluated in pituitary cells from adult female rats, cultured for 2 days in the presence of 10−13 to 10−6 m concentrations of oestradiol and selected A- and D-ring modified oestrogens. With almost all steroids tested, bell-shaped dose-response curves with comparable LH-maxima but different EDmax values were obtained for the LH response to a submaximal GnRH stimulus (5 × 10−10 m). Maximal LH response to 5 × 10−10 m GnRH were found at the following oestrogen concentrations: oestradiol and 4-hydroxyoestradiol = 10−11 m; 2-methyloestradiol = 10−9 m; 2-hydroxyoestradiol = 10−8 m; with 4-methyloestradiol no significant maximum was observed. When cells were pretreated with 10−13, 10−11 and 10−9 m oestradiol, or 4-hydroxyoestradiol, or 2-hydroxyoestradiol, and exposed to increasing concentrations of GnRH (10−11 to 10−7 m), an almost 10-fold decrease in the ED50 for GnRH was observed after pretreatment with 10−11 m oestradiol and 4-hydroxyoestradiol. With 2-hydroxyoestradiol at this concentration, the sensitizing effect was much less pronounced. Increasing the steroid concentration to 10−9 m slightly decreased the effect of oestradiol and 4-hydroxyoestradiol, whereas it increased the effect of 2-hydroxyoestradiol. Thus, at the target cell 4-hydroxyoestradiol has the same potency as oestradiol, while 2-hydroxyoestradiol is significantly less active. The sensitizing effect of oestradiol is only slightly decreased by the presence of a methyl group in position 2, but is markedly reduced by a methyl group in position 4. Our results also demonstrate the high sensitivity of the pituitary to oestrogen-induced enhancement of GnRH-stimulated gonadotrophin release, as well as the decrease of the positive effect at high oestrogen concentrations. The bell-shaped dose-response curves for oestrogen action should be taken into account when evaluating the effects of oestrogens and their derivatives upon gonadotrophin release from the pituitary gland.

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Regulation of gonadotrophin secretion by inhibin, testosterone and gonadotrophin-releasing hormone in pituitary cell cultures of male monkeys

Journal of Endocrinology ◽

10.1677/joe.0.1590103 ◽

1998 ◽

Vol 159 (1) ◽

pp. 103-110 ◽

Cited By ~ 9

Author(s):

U Fingscheidt ◽

GF Weinbauer ◽

HL Fehm ◽

E Nieschlag

Keyword(s):

Dose Response ◽

Pituitary Cells ◽

Basal Secretion ◽

Response Curves ◽

Gonadotrophin Secretion ◽

Lh Release ◽

Gonadotrophin Releasing Hormone ◽

Dose Response Curves ◽

Male Rhesus

The effects of bovine inhibin, testosterone and GnRH on gonadotrophin secretion by primate pituitary cells were characterized in vitro using pituitaries from six male rhesus monkeys and one male cynomolgus monkey. The effect of inhibin on basal secretion of FSH and LH was investigated. Dose-response curves in monkeys and rats were compared. GnRH dose-response curves in the presence and absence of testosterone were also examined in monkeys. In monkey pituitary cells, testosterone at a concentration of 10(-7) M had no effect on LH or FSH secretion. Inhibin suppressed FSH secretion to 50.8% of that of controls with no effect on LH. In rats, FSH secretion was suppressed to 45.0% of that of controls with a median effective dose (ED50, 95% range) of 1.298 (1.064-1.584) U/ml, compared with 1.024 (0.7204-1.455) U/ml in monkeys. In monkey pituitary cells, LH release was stimulated 9.9-fold and FSH 3.3-fold by GnRH. Testosterone had no effect on basal or GnRH-stimulated gonadotrophin release. These results support the view that the pituitary is not the target organ for the negative feedback action of testosterone in the male. In vitro, inhibin is the major regulator of FSH secretion at the pituitary level.

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Differential effects of cyclic nucleotide analogues and GnRH on LH synthesis and release

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1981.241.1.e6 ◽

1981 ◽

Vol 241 (1) ◽

pp. E6-E13

Author(s):

T. C. Liu ◽

G. L. Jackson

Keyword(s):

Dose Response ◽

Cyclic Nucleotides ◽

Ovariectomized Rats ◽

Detectable Effect ◽

Response Curves ◽

Cyclic Monophosphate ◽

Pituitary Glands ◽

Lh Release ◽

Dose Response Curves ◽

Medium Dose

We compared the effects of gonadotropin-releasing hormone (GnRH) and cyclic nucleotides on biosynthesis and release of luteinizing hormone (LH) by rat anterior pituitary glands (APs). APs from ovariectomized rats were incubated in the presence of test compounds, [3H]glucosamine, and [14C]alanine. GnRH significantly (P less than 0.01) increased both incorporation of [3H]glucosamine into LH (glycosylation) and release of immunoreactive LH (IR-LH) and [3H]LH. None of the cyclic nucleotides alone at 5 mM significantly stimulated release of IR-LH. Adenosine 3',5'-cyclic monophosphate (cAMP), N6,O2'-dibutyryl cAMP (DBcAMP), and 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP) stimulated (P less than 0.01) glycosylation, whereas 8-Br-AMP, 8-bromoguanosine 5'-monophosphate (8-Br-GMP), cGMP, and 8-Br-cGMP had no detectable effect. Release of [3H]LH was increased by GnRH, 8-Br-cAMP, and DBcAMP, but not by other nucleotides. None of the cyclic nucleotides elevated [14C]LH, whereas GnRH elevated it (P less than 0.01) in the medium. Dose-response curves for glycosylation in response to 8-Br-cAMP and GnRH were parallel. In contrast, dose-response curves for release of both [3H]LH and IR-LH were nonparallel. It appears that cAMP can mimic the action of GnRH on LH glycosylation, but may have little direct effect on LH release.

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Relationship among sensitivity to adrenaline, plasma corticosterone level; and estrous cycle in rats

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y95-076 ◽

1995 ◽

Vol 73 (5) ◽

pp. 602-607 ◽

Cited By ~ 10

Author(s):

M. L. V. Rodrigues ◽

F. K. Marcondes ◽

R. C. Spadari-Bratfisch

Keyword(s):

Estrous Cycle ◽

Dose Response ◽

Corticosterone Level ◽

Plasma Corticosterone ◽

Female Rats ◽

Extraneuronal Uptake ◽

Response Curves ◽

Experimental Conditions ◽

Dose Response Curves ◽

Right Atria

The dose–response curves to the chronotropic effect of adrenaline obtained in right atria isolated from female rats indicated an order of increasing sensitivity to adrenaline, at the pD2 level, according to the estrous cycle, as follows: estrus ≤ metestrus ≤ diestrus ≤ proestrus. Inhibition of neuronal and extraneuronal uptake shifted the dose–response curves to adrenaline to the left only in right atria isolated from rats during estrus or metestrus. Moreover, under these experimental conditions, right atria were subsensitive to adrenaline during proestrus, in contrast to metestrus. Plasma corticosterone levels were lower during estrus and higher at proestrus. There was a positive correlation between right atria sensitivity to adrenaline and plasma corticosterone levels and estrous cycle phases. Our results also suggest that in the rat right atria during proestrus, as opposed to the other phases of the estrous cycle, there was an endogenous inhibition of extraneuronal uptake together with some alteration at the adrenoceptor level and (or) at intracellular mechanisms beyond receptors.Key words: adrenergic response, female, adrenaline, chronotropism, right atria.

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Severe uremia depresses the ability of perifused rat pituitary cells to secrete growth hormone in response to growth hormone releasing hormone.

Journal of the American Society of Nephrology ◽

10.1681/asn.v85742 ◽

1997 ◽

Vol 8 (5) ◽

pp. 742-748

Author(s):

J Rodríguez ◽

F Santos ◽

M J García de Boto ◽

E García ◽

V Martínez ◽

...

Keyword(s):

Growth Hormone ◽

Dose Response ◽

Secretory Response ◽

Pituitary Cells ◽

Maximal Effect ◽

Response Curves ◽

Secretory Rate ◽

Releasing Hormone ◽

Gh Secretion ◽

Dose Response Curves

To examine whether growth hormone (GH) secretion is impaired by chronic renal failure (CRF) and to gain some insight into the influence of uremia itself and associated malnutrition, the GH secretory response of dispersed anterior pituitary cells perifused with GH-releasing hormone (GHRH) was investigated in 5/6 nephrectomized (UREM, N = 15) and three groups (N = 15 each) of normal renal function, sham-operated rats under three different nutritional conditions: fed "ad libitum" (SAL), pair-fed with a diet similar to the UREM group (SPF), and pair-fed with a diet similar to the UREM group in terms of protein ingestion but calorically supplemented up to intake of SAL group (SPF+). Ten days after nephrectomy, UREM rats had severe CRF, as shown by much higher (P < 0.0001) serum urea nitrogen concentrations (X +/- mean +/- SE) than sham groups (59 +/- 6 versus 8 +/- 0, 9 +/- 0, and 5 +/- 0 mmol/L, respectively), and they were growth retarded, as shown by lower gains (P < 0.0001) in weight (13.5 +/- 2.5 versus 62 +/- 2.1, 20.5 +/- 1.9, and 50.4 +/- 1.0 g) and length (2.9 +/- 0.2 versus 5.8 +/- 0.1, 3.6 +/- 0.1, and 5.6 +/- 0.1 cm). Perifusion studies showed similar basal GH secretory rate (ng/min/10(7) cells) in the four groups. A fixed sequence of progressively increasing GHRH doses resulted in a lower overall mean GH secretion in UREM rats (15.8 +/- 1.6 ng/min/10(7) cells), as compared with SAL (50.8 +/- 9.0 ng/min/10(7) cells, P < 0.01), SPF (33.0 +/- 3.3 ng/min/10(7) cells, P < 0.05), and SPF+ (49.4 +/- 5.1 ng/min/10(7) cells, P < 0.01) groups. Analysis of dose-response curves showed that the maximal secretory response was produced by the same concentration of GHRH (10 nM) in the four groups and was lower (P < 0.01) in UREM than SAL and SPF+ rats (34.9 +/- 5.0 versus 115.7 +/- 28.4 and 98.9 +/- 9.8 ng/min/10(7) cells). The concentration of GHRH that caused the half of maximal effect was identical, close to 1 nM, in the four groups of animals. This study provides direct evidence that the ability of pituitary cells to secrete GH in response to GHRH is depressed in severe CRF. The lower secretory capacity of pituitary gland is only partly dependent on caloric malnutrition associated with CRF. Data of dose-response curves suggest that decreased GH secretion may be related to a lesser number of pituitary receptors for GHRH.

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ÜBER DIE DURCH UNSPEZIFISCHE SUBSTANZEN BEDINGTE ÄNDERUNG DER QUANTITATIVEN UND QUALITATIVEN WIRKSAMKEIT VON GONADOTROPINEN BEI DER BIOLOGISCHEN AKTIVITÄTSBESTIMMUNG

Acta Endocrinologica ◽

10.1530/acta.0.0380181 ◽

1961 ◽

Vol 38 (2) ◽

pp. 181-199 ◽

Cited By ~ 1

Author(s):

K. Walter ◽

S. Wysocki

Keyword(s):

Dose Response ◽

Chorionic Gonadotrophin ◽

Female Rats ◽

Response Curves ◽

Immature Female ◽

Ovarian Weight ◽

Immature Rats ◽

Reference Preparation ◽

Slope Difference ◽

Dose Response Curves

ABSTRACT The activity of chorionic gonadotrophin (HCG) injected in solutions containing different nonspecific substances (polyvinylpyrrolidone, gelatine, tragacanth = »depot-media« is compared with the activity of HCG in distilled water. Intact immature female rats are used for the bioassays, which differ only in injection schedule or the response measured (uterine resp. ovarian weight). Depending on the injection schedule used and the response measured the results of the various bioassays in intact immature rats are different, when one and the same depot-medium is used. Significant indices of discrimination as well as significant slope differences of dose-response curves are observed. The addition of a biologically inactive kaolin extract prepared according to the »crude kaolin-acetone method« to a gonadotrophic extract from the urine of postmenopausal women produced a significant decrease in activity in one type of assay, and a significant slope difference of dose-response curves in another one. In view of these results the difficulties arising for the demonstration of qualitative differences in gonadotrophic preparations and for expressing bioassay-results in terms of the International Reference Preparation are discussed.

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Dose-response Curves in the Fibrin Plate Assay Fibrinolytic Activity of Proteases

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647705 ◽

1974 ◽

Vol 32 (02/03) ◽

pp. 356-365 ◽

Cited By ~ 12

Author(s):

F Haverkate ◽

D. W Traas

Keyword(s):

Dose Response ◽

Fibrinolytic Activity ◽

Enzyme Concentration ◽

Response Curves ◽

Agar Gel ◽

Porcine Tissue ◽

Plate Assay ◽

Fibrin Plate ◽

Different Types ◽

Dose Response Curves

SummaryIn the fibrin plate assay different types of relationships between the dose of applied proteolytic enzyme and the response have been previously reported. This study was undertaken to determine whether a generally valid relationship might exist.Trypsin, chymotrypsin, papain, the plasminogen activator urokinase and all of the microbial proteases investigated, including brinase gave a linear relationship between the logarithm of the enzyme concentration and the diameter of the circular lysed zone. A similar linearity of dose-response curves has frequently been found by investigators who used enzyme plate assays with substrates different from fibrin incorporated in an agar gel. Consequently, it seems that this linearity of dose-response curves is generally valid for the fibrin plate assay as well as for other enzyme plate bioassays.Both human plasmin and porcine tissue activator of plasminogen showed deviations from linearity of semi-logarithmic dose-response curves in the fibrin plate assay.

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BIOASSAY OF THYROID HORMONE USING HYPOTHYROID TADPOLES

Acta Endocrinologica ◽

10.1530/acta.0.0410143 ◽

1962 ◽

Vol 41 (1) ◽

pp. 143-153 ◽

Cited By ~ 2

Author(s):

U. Henriques

Keyword(s):

Thyroid Hormone ◽

Dose Response ◽

Developmental Rate ◽

Response Curves ◽

Sodium Salts ◽

Dose Response Curves

ABSTRACT A bioassay of thyroid hormone has been developed using Xenopus larvae made hypothyroid by the administration of thiourea. Only tadpoles of uniform developmental rate were used. Thiourea was given just before the metamorphotic climax in concentrations that produced neoteni in an early metamorphotic stage. During maintained thiourea neotoni, 1-thyroxine and 1-triiodothyronine were added as sodium salts to the water for three days and at the end of one week the stage of metamorphosis produced was determined. In this way identical dose-response curves were obtained for the two compounds. No qualitative differences between their effects were noted except that triiodothyronine seemed more toxic than thyroxine in equivalent doses. Triiodothyronine was found to be 7–12 times as active as thyroxine.

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