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Biomedical subjects

W R Robertson

Publications and source records attributed to W R Robertson.

At least 109 records · Page 6Linked to original sources
PubMed1986

Effects of physiological concentrations of parathyroid hormone on acid phosphatase activity in cultured rat bone cells.

The mechanism by which parathyroid hormone (PTH) induces osteoclastic bone resorption is still incompletely understood. Recent evidence suggests that the hormone exerts its effects indirectly, via the osteoblasts. Bone cells isolated from fetal rat calvaria by enzymatic digestion were used. Two heterogeneous cell populations were isolated by equilibrium density centrifugation on Percoll gradients and maintained by differential culture conditions. These two populations, which are morphologically distinguishable from one another by light and electron microscopy, have been characterized previously both biochemically and with regard to their hormonal (PTH and calcitonin) responses. We have called them type C cells (containing cells with some of the properties of osteoclasts) and type B cells (containing osteoblast-like cells, as well as fibroblasts, chondrocytes and other stromal cells). In the present study, we have further characterized the functional relationship between the two cell populations, with particular regard to the hormonal responses of type C cultures. Acid phosphatase, measured cytochemically in individual cells, was used as a marker for C cell responses. C cells had significantly higher levels of acid phosphatase activity than either B cells or spleen macrophages. Calcitonin (0-10 pg/ml) decreased C cell acid phosphatase activity but was without effect on B cells or spleen macrophages. Co-culture of C cells with B cells produced increased enzyme activity only in the former; this effect could be mimicked if fibroblasts replaced B cells and cell contact was essential for this response. PTH (0-10 pg/ml) raised enzyme activity further in C cells only when they were cultured with B cells. When C cells were cultured so that they shared medium, but were not in contact, with B cells, PTH (2 pg/ml) still increased enzyme activity in the former. Fibroblasts were ineffective in this system. Spleen macrophages were also unresponsive to PTH when substituted for C cells. Calcitonin (10 pg/ml) blocked the effects of PTH on C cells. These results indicate that macrophages are probably not a significant proportion of the C cell population, and that PTH may produce increased acid phosphatase activity in C cells via a humoral factor produced by cells present in B cell cultures.

Acid Phosphatase↗
PubMed1985

On the site of action of the anti-adrenal steroidogenic effect of cyproterone acetate.

Cyproterone acetate (CA) inhibited 1-24 ACTH (50 ng/l)-stimulated cortisol production by dispersed guinea-pig adrenal cells in a dose-related manner. Inhibition occurred over the range 10(-6) to 5 X 10(-5) moles/l. The concentration of drug which induced 50% inhibition was 4.6 X 10(-6) moles/l. The sites of action of this anti-steroidogenic effect have been established. Dispersed adrenal cells were challenged with the cortisol precursor steroids (all at 10(-5) moles/l) pregnenolone (Pe), 17 alpha hydroxypregnenolone (17-OH Pe), 17 alpha-hydroxyprogesterone (17-Po), and 11 deoxycortisol or 1-24 ACTH (100 ng/1) in the absence and presence of increasing concentrations of CA (10(-5) to 10(-4) moles/l). In the absence of drug, the steroid precursors or ACTH provoked cortisol secretion greater than 10-fold that secreted by cells incubated in their absence. ACTH-stimulated cortisol secretion was inhibited greater than 68% at concentrations of CA greater than 10(-5) moles/l. CA (10(-5) moles/l) had no significant effect on steroid-stimulated cortisol production when the delta 4, 3 ketosteroids 17-OH Po and 11-deoxycortisol were used, but depressed secretion by greater than 61% (P less than 0.001) when the delta 5 3 beta hydroxysteroids (Pe, 17-OH Pe) were employed. Increasing CA concentrations to 10(-4) moles/l had little effect on cortisol secretion provoked by 11-deoxycortisol, but significantly (P less than 0.05) depressed cortisol secretion stimulated by 17-OH Po. These results suggest that the major site of action of CA is delta 5, 3 beta hydroxysteroid dehydrogenase (3 beta-HSD) with a secondary effect on 21-hydroxylase activity. To confirm these findings cortisol secretion provoked by 17, 21, dihydroxypregnenolone (17, 21 diOH Pe) and 21 deoxycortisol (21-DOC) was measured in the absence and presence of increasing concentrations of CA. At the lowest concentration of CA (5 X 10(-6) moles/l), cortisol secretion provoked by 17, 21 diOH Pe was inhibited by 28% (P less than 0.01) whereas secretion provoked by 21-DOC was not significantly affected. At the highest concentration of CA (10(-4) moles/l), the relative inhibition was 80% for 17, 21 diOH Pe and 38% for 21-DOC. We conclude that cyproterone acetate inhibits adrenal steroidogenesis at both 3 beta-HSD and 21-hydroxylase, the degree of inhibition being more pronounced at 3 beta-HSD.

3-Hydroxysteroid Dehydrogenases↗
PubMed1985

A simple in vitro approach to the estimation of the biopotency of drugs affecting adrenal steroidogenesis.

Dispersed guinea-pig adrenal cells can be maximally stimulated to secrete cortisol by adrenocorticotrophin (ACTH greater than 50 ng/l). Further, this stimulation appears to be specific to ACTH alone, with other naturally occurring chemicals (e.g. steroids, protein hormones) at supra-physiological concentrations being without effect on cortisol production. The effect of drugs of differing structure and therapeutic function (aminogluthethimide, metyrapone, trilostane, 17-ketotrilostane, danazol, epostane, megestrol acetate, stanozolol and etomidate) on ACTH-stimulated (50 ng/l) cortisol production has been tested in this system. All the drugs depressed steroid output in a similar dose-related fashion. The concentration of drug which inhibited cortisol output by 50% was (mumol/l, mean +/- SEM): etomidate 0.097 +/- 0.002: epostane 0.44 +/- 0.02: 17-ketotrilostane 0.55 +/- 0.04: trilostane 1.3 +/- 0.1: metyrapone 3.5 +/- 0.6: megestrol acetate, 11 +/- 2: danazol 22 +/- 2: aminogluthethimide 41 +/- 5: stanozolol 50 +/- 4. Thus, etomidate, an anaesthetic, is more potent than the established anti-steroidogenic drugs metyrapone, aminogluthethimide and trilostane. Further, direct anti-steroidogenic effects have been demonstrated for megestrol acetate and stanozolol for the first time. We conclude that this technique offers a promising new approach to the assessment of biological potency of drugs affecting endocrine tissues.

Adrenal Glands↗
PubMed1985

Some characteristics of human adrenal microsomal 21-hydroxylase activity.

The following general characteristics of 21-hydroxylase activity were determined using pooled microsomes obtained from three glands. Enzyme activity exhibited a broad pH dependence, being optimal between pH 7.4-pH 7.8, and was maximal with NADPH in the range 2 to 4.75 X 10(-4)mol/l. No microsomal 21-hydroxylase activity was detected in the absence of NADPH or substrate and when heat denatured microsomes were employed. Enzyme activity was depressed by greater than 75% in the presence of 100% oxygen or nitrogen. In a second set of experiments, microsomal fractions were prepared individually from 7 glands. In the presence of 17 alpha-hydroxy progesterone (2.0 X 10(-7) and 2.0 X 10(-6)mol/l) product formation was linear with time for up to 90 s when the microsomal protein concentration was 5, 10 and 20 micrograms/ml. Between 5 and 30% of the substrate was converted during the first 60 s. In 5/7 of the glands the addition of the autologous cytosol (20 micrograms protein/ml) was without effect, and enzyme activity (using a 60 s reaction and either 2.0 X 10(-7) or 2 X 10(-6)mol/l 17 alpha-hydroxy progesterone was directly proportional to the microsomal protein concentration (range 0-20 micrograms/ml). With the other 2 adrenals 21-hydroxylation was not proportional to the same range of microsomal protein concentrations, although it became so upon the addition of cytosol, which significantly augmented activity. There was considerable variation in enzyme activity between glands from different individuals (Vmax ranging from 2.6 to 16.6 X 10(-9) mol/min/mg protein) and in the apparent Km's (from 0.22 to 1.1 X 10(-6)mol/l). In the two preparations sensitive to cytosol, the Vmax increased 2-fold, and the Km was 3 times lower. Cytosol was without effect upon the kinetic characteristics of the other 5 microsomal preparations. Ascorbic acid (1 X 10(-3) mol/l) depressed enzyme activity by 25-43% whereas oxidised and reduced glutathione (1 X 10(-3) mol/l) showed a slight and variable effect upon 21-hydroxylation.

17-alpha-Hydroxyprogesterone↗
PubMed1985

Effect of propofol, thiopentone and etomidate on adrenal steroidogenesis in vitro.

The i.v. anaesthetic agents propofol, thiopentone and etomidate inhibited ACTH-stimulated production of cortisol by guineapig dispersed adrenal cells in a dose-related manner. For two of the drugs, propofol and thiopentone, inhibition occurred over a similar concentration range: 2 X 10(-5) - 5 X 10(-4) mol litre-1. With etomidate, inhibition occurred over a much lower concentration range (5 X 10(-8) - 5 X 10(-6) mol litre-1). The concentrations of anaesthetic which induced 50% inhibition of cortisol secretion were propofol 1.7 X 10(-4), thiopentone 1.6 X 10(-4), and etomidate 1.0 X 10(-7) mol litre-1.

Adrenal Glands↗
PubMed1985

On the stability in vitro of bioactive human adrenocorticotrophin in blood and plasma.

The disappearance in vitro of ACTH from whole human blood and plasma held at 22 degrees C has been monitored using a dispersed adrenal cell bioassay and an unextracted radioimmunoassay. In three normal subjects after a metyrapone test and two patients with Addison's disease, endogenous bioactive ACTH levels were unchanged for at least 1 h in blood and 2 h in plasma. Moreover greater than 50% of the bioactive plasma ACTH was still present in the plasma samples from the patients with Addison's disease after 24 h incubation at ambient temperatures. Human pituitary ACTH (1-39), spiked into plasma from dexamethasone suppressed subjects to give a concentration of 250 ng/l, was stable by bioassay for at least 2 h. No loss of biological activity was observed on subjecting plasma from a patient with Addison's disease or spiked plasma to two cycles of thawing at 37 degrees C and freezing at -70 degrees C or thawing at 20 degrees C and freezing at -20 degrees C. Some loss of bioactivity (20%) occurred on subjecting the patient's, but not ACTH-spiked plasma to four cycles of thawing at 20 degrees C/freezing at -20 degrees C. We conclude that bioactive ACTH (endogenous or exogenous) may be more stable in vitro in human blood and plasma than has been previously thought. If our studies can be confirmed in a larger series then it may be that conditions for handling blood specimens for ACTH assays could be reappraised.

Addison Disease↗
PubMed1985

Measurement of low concentrations of bovine thyrotrophin by iodide uptake and organification in porcine thyrocytes.

Thyrocytes isolated from porcine thyroids by mechanical and enzymatic dispersion and cultured in Eagle's minimal essential medium, supplemented with 5% (v/v) fetal calf serum, glutamine and cortisol, formed a continuous monolayer within 48 h. This monolayer was without cytochemical peroxidase and diaphorase (NADPH reoxidation) activity. In the presence of bovine thyrotrophin (bTSH; 50 mu./l) the cells developed a follicular-like architecture which was maximal at 4 days before reverting back to a uniform monolayer at 6 days. There were no detectable changes in the total DNA content over this period. The follicular structures had marked diaphorase and peroxidase activity, the latter being apically distributed. Concomitant with follicle formation bTSH induced uptake and organification of iodide presented to the cells during the last 6 h of culture. The extent of this process depended on the dose of bTSH and the duration of stimulation. The most sensitive effects for both iodide uptake and organification occurred with 1 mu. bTSH/l and were maximal with 100 mu./l. Uptake and organification were increased 20 +/- 8-fold and 9.6 +/- 2-fold (n = 10) respectively over the control with 100 mu./l and the doses of bTSH at which a half maximal response was seen (ED50) were 15 +/- 2 and 7 +/- 1 (S.D) mu./l (n = 10) respectively. On changing the culture medium to a serum-free system using HB101 culture medium the stimulation time for the most sensitive bTSH effect was reduced to 2.5 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

On the biopotency and site of action of drugs affecting endocrine tissues with special reference to the anti-steroidogenic effect of anaesthetic agents.

Dispersed guinea-pig adrenal cells or mouse Leydig cells were stimulated with a saturating dose of adrenocorticotrophin (ACTH, 50 ng/1) or luteinizing hormone (LH, 5IU/1), respectively. The incubations were performed in the presence of increasing concentrations (10(-9) - 5 X 10(-4)mol/l) of the anaesthetic agents propofol, thiopentone and etomidate. At the end of this stimulation period, cortisol (from the adrenal preparation) or testosterone (from the Leydig cell culture) were assayed by radioimmunoassay. Propofol, thiopentone and etomidate all inhibited ACTH-stimulated cortisol secretion in a dose-related fashion. Similar inhibition of LH-stimulated testosterone output was found with propofol and thiopentone whereas etomidate was without effect at any concentration employed, an observation in accordance with its known site of action, 11 beta-hydroxylase, an enzyme which is not involved in the biosynthesis of testosterone. The concentration (mumol/l) of anaesthetics which gave 50% inhibition (ED50) of ACTH-stimulated cortisol secretion was 0.1 +/- 0.002 (n = 7), 160 +/- 18 (n = 3) and 170 +/- 18 (n = 3) (mean +/- s.e.m.) for etomidate, thiopentone and propofol, respectively. The corresponding values for the LH stimulated testosterone output from the Leydig cell preparations were 186 (thiopentone) and 180 (propofol) mumol/l. In a separate series of experiments adrenal cells were stimulated with (a) the cortisol precursor steroids (all at 10(-5)mol/l) pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone and 11-deoxycortisol, (b) dibutyryl cAMP (10(-3)mol/l) or (c) ACTH (100 ng/l) in the presence and absence of either etomidate (5 X 10(-5)mol/l), propofol (2.5 X 10(-4)mol/l) or thiopentone (5 X 10(-4)mol/l). All the stimulators increased cortisol production by > 7-fold over that seen in their absence. Propofol depressed ACTH and dibutyryl cAMP induced cortisol output by > 60% (P < 0.05) but was without effect when the steroid precursors were used, suggestive of an inhibition between the sequence involving ACTH binding -> pregnenolone production. In contrast, etomidate and thiopentone reduced cortisol secretion by > 40% (P < 0.05) regardless of the stimulator used, indicating that at least one site of action was at the level of the final enzymic step of cortisol synthesis, i.e. 11beta-hydroxylase.

Adrenocorticotropic Hormone↗
PubMed1984

Computerized continuous monitoring of cytochemical enzyme reaction product formation by the Vickers M85A microdensitometer.

An experimental set-up for the continuous monitoring of cytochemical enzyme reaction rates is described. The design consists of a 32K Commodore PET microcomputer, upgraded to Basic 4 and containing a graphics facility. The microcomputer is interfaced with a Vickers M85A microdensitometer, which has been modified and equipped with a Zeiss X63 water immersion objective. The software allows for the automatic transfer of density readings from the M85A to the microcomputer, and for the continuous monitoring of the enzyme reaction as its proceeds at short time intervals (i.e. down to 3.3 s). The relationship between the absorbance change of the enzyme final reaction product and time is printed both numerically and graphically. Initial velocity rates are calculated by regression analysis, and statistical evaluation of grouped data is possible.

20-Hydroxysteroid Dehydrogenases↗
PubMed1984

The cytochemical assay of NAD+ kinase activity in the follicular cells of guinea-pig thyroid gland.

A quantitative cytochemical assay for NAD+ kinase-like activity in the guinea-pig thyroid gland is described. The NADP+ produced by the activity of the kinase was used to drive the NADP+-dependent enzyme glucose-6-phosphate dehydrogenase which is endogenous to the tissue. The activity of glucose-6-phosphate dehydrogenase is greatly in excess of that of the kinase and was unaffected by the constituents of the kinase incubation medium (ATP, Mg2+ and NAD+) either alone or in combination. Kinase activity was dependent both on ATP and Mg2+, with maximal activity seen when the Mg-ATP ratio was between 1:1 and 4:1. Free ATP inhibited the activity of the enzyme. Enzyme activity was exhibited over a broad pH range (7-9) with a peak at pH 8.2. The sulphydryl-blocking agents, p-chloromercuribenzoate, iodoacetate and iodoacetamide (at 1 mM), completely abolished kinase activity but were without effect on glucose-6-phosphate dehydrogenase activity. N-ethylmaleimide and citrate (both at 1 mM) had no effect on either kinase or glucose-6-phosphate dehydrogenase activities.

Adenosine Triphosphate↗
PubMed1984

The bioavailability and metabolism of trilostane in normal subjects, a comparative study using high pressure liquid chromatographic and quantitative cytochemical assays.

High pressure liquid chromatographic (HPLC) analysis of plasma taken over 8 h from ten normal male subjects medicated with 120 mg of trilostane revealed that the drug is rapidly metabolised into at least one metabolite, 17-keto trilostane. Both compounds were detected in the blood stream at concentrations greater than 2 X 10(-7) M within an hour and were cleared from the blood by 6-8 h. Approximately 3 times the concentration of metabolite was detected compared to the parent compound in most samples analysed. There were large subject to subject variations in the handling of drug. Standard curves of pure 17-keto trilostane and trilostane were parallel as assessed by cytochemical bioassay. This assay is based upon the inhibition of 3 beta-hydroxysteroid dehydrogenase activity in unfixed tissue sections of the dioestrous rat ovary. The relative potency of the metabolite compared to trilostane was 1.71 (95% confidence 1.5-2.0) over the dose range 0.15-1.5 microM. Thus, the metabolite may be the major active agent when trilostane is administered for clinical purposes. In a further 4 volunteers, who also received 120 mg trilostane and were sampled over an 8 h period, plasma was analysed independently by HPLC and cytochemical assays. In the majority of cases the bioactivity recorded (relative to a trilostane standard curve) was substantially higher than the molar sum of circulating trilostane and 17-keto-trilostane (as assessed by HPLC). However, if the relative potency of 17-keto-trilostane is taken into consideration, correlation between the two assays was excellent (r = 0.947, n = 18, P less than 0.001). This also suggests that no further active metabolites were present in the plasma samples. The drug profiles seen in the second study were essentially the same as described for the first 10 volunteers. The combination of a bioassay, which detects trilostane-like bioactivity, and HPLC, which reveals the type of metabolism, should aid our understanding of the clinical value of this potentially important drug.

Adult↗
PubMed1984

Cortisol production by dispersed guinea-pig adrenal cells; a specific, sensitive and reproducible response to ACTH....and its fragments.

Naturally occurring steroids and peptide hormones, tested at supraphysiological concentrations, were without effect on basal and human (h) 1-39 ACTH (NIBSC code 74/555, 25 ng/l (5.5 X 10(-12) mol/l] stimulated cortisol production. Further, low concentrations of angiotensin II, N-pro-opiocortin (N terminal fragment 1-76) and gamma-MSH all of which have been reported to synergise with ACTH with regard to cortisol production, were without significant effect alone or in combination with ACTH over the range 2.2 X 10(-13) to 5.5 X 10(-12) mol/l. The activity of h 1-39 was compared with that of the ACTH related peptides 1-24, 1-18, 1-17, 1-16, 1-13-NH2 (alpha MSH), 1-10 and 4-10. The dose responses were parallel and the same maximal cortisol output was observed with all the peptides except the 1-10 fragment. Half maximal stimulation occurred at 3.1 X 10(-12) (1-24), 4.4 X 10(-12) (h 1-39), 1.5 X 10(-11) (1-39), 3.3 X 10(-10) (1-18), 5 X 10(-9) (1-13-NH2), 8 X 10(-9) (1-17), 2 X 10(-7) (1-16) and 1 X 10(-5) (4-10) mol/l respectively. Interference by the above ACTH-derived peptides in cortisol secretion by the cells in response to 5.5 X 10(-12) mol/l h 1-39 ACTH was minimal over the range 5.2 X 10(-12)-2.2 X 10(-6) mol/l. The sensitivity of the adrenal cells to h 1-39 ACTH was such that 2 ng/l (4.4 X 10(-13) mol/l) provoked cortisol secretion over the control (P less than 0.05, n = 17). The coefficient of variation within assay for each dose on the full standard curve (2.2 X 10(-13)-1.1 X 10(-10) mol/l) was less than 10% (n = 6). Half maximal stimulation was given by 14.5 ng/l (3.2 X 10(-12) mol/l). Between control and 1.1 X 10(-10) mol/l ACTH there was a 32 +/- 8 (mean +/- SD, n = 9) fold change in cortisol production.

Adrenal Glands↗
PubMed1984

A cytochemical section bioassay for plasma trilostane: an orally active inhibitor of 3 beta-hydroxysteroid dehydrogenase activity.

Trilostane is a competitive inhibitor of 3 beta-hydroxysteroid dehydrogenase activity in vitro and an orally active inhibitor of steroidogenesis in vivo. A cytochemical section bioassay for this drug (4 alpha, 5-epoxy-17 beta-hydroxy-3-oxo-5 alpha-androstane-2 alpha-carbonitrile) in human plasma has been developed. The assay is based upon the inhibition of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity in the corpus luteum of unfixed tissue sections (6 micron) of the mature dioestrous rat ovary. Trilostane is extracted from plasma by a simple chloroform procedure. The standard curve (0.15-2.5 mumol/l pure trilostane) exhibits a four-fold change in 3 beta-HSD activity and is constructed in the presence of extract from control plasma pools. The detection limit of the plasma assay is 2 mumol/l trilostane after allowing for dilution effects and recovery losses. This level gives a significantly different response from the zero quality control (P less than 0.05) and has an inter-assay coefficient of variation of less than 15%. No false positives have been recorded to date and the majority of samples from subjects receiving therapeutic doses of the drug have levels of greater than 2 mumol/l. Intra- and inter-assay coefficients of variation using both plasma quality controls and unknown samples are 4% (n = 15) and 13.9% (n = 27) respectively. Recovery is 80 +/- 9% (n = 13) and 79 +/- 10% (n = 10) for plasma quality controls containing 12 and 4.8 mumol/l trilostane respectively. Dilutions (up to 1:5) of plasma extracts are parallel to the standard curve and the assay throughput is approximately 16 samples/week technician.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1984

Preliminary experiences with a bioassay for adrenocorticotrophin (ACTH) in unextracted human plasma using dispersed guinea-pig adrenal cells.

A simple, accurate and precise dispersed cell bioassay suitable for the assay of ACTH levels greater than 50 ng/1 in unextracted human plasma is described. Isolated adrenal cells were prepared by tryptic digestion of the guinea-pig adrenal gland and cortisol production by these cells was controlled specifically by ACTH. The cortisol response to 2 ng/1 of human pituitary ACTH (1-39) was always significantly (P less than 0.05, n = 17) different from the control. ACTH concentrations between 1 and 20 ng/1 gave a 14 +/- 3 fold (n = 17) increment in cortisol response. Dilutions of test plasmas were parallel with standard ACTH (1-39) when the plasma concentrations were maintained throughout at 4% (1 in 25 dilution) by the addition of dexamethasone-suppressed plasma ('ACTH free'). Using a 25 fold plasma dilution, the limit of detection of ACTH in unextracted plasma was 50 ng/1. Recovery of ACTH (1-39) spiked into dexamethasone suppressed plasma at concentrations of 250 and 100 ng/1 was 97 +/- 10% (n = 17) and 114 +/- 15% (n = 17) respectively. The within assay coefficient of variation (CV) of both quality control and patients' samples never exceeded 9% while between assay variation was less than or equal to 13%. The sample throughput was 30 plasma samples/week/technician.

Addison Disease↗
PubMed1984

On the site of action of the anti-adrenal steroidogenic effect of etomidate and megestrol acetate.

The sites of action of the anti-steroidogenic action of etomidate and megestrol acetate have been established with a novel in vitro approach based upon the inhibition of cortisol (Co) secretion by dispersed guinea-pig adrenal cells. The cells were challenged with the Co precursor steroids (all at 10(-5) mol/l) pregnenolone (Pe), 17-hydroxy-pregnenolone (17-Pe), progesterone (Po), 17-hydroxyprogesterone (17-Po), 21-deoxycortisol (21-DOC) and 11-deoxycortisol (11-DOC), or 1-24 adrenocorticotrophin (ACTH, 100 ng/l) in the presence or absence of either etomidate, megestrol acetate, metyrapone or trilostane (all at 5 X 10(-5) mol/l). In the absence of drug, the steroid precursors or ACTH provoked a cortisol secretion of greater than 14 times that secreted by cells incubated in their absence. ACTH-stimulated Co secretion was inhibited by greater than 85% by all the drugs employed. In the presence of trilostane and megestrol acetate, Co secretion provoked by the delta 4 3-keto steroids (Po, 17-Po, 21-DOC and 11-DOC) was similar to the controls. However, with the delta 5, 3 beta-hydroxy steroids, 17-Pe and Pe, Co secretion was inhibited by greater than 57% in the presence of these drugs. In contrast, etomidate and metyrapone inhibited Co secretion by greater than 60% when 11-deoxycortisol was employed, indicative of a block at 11 beta-hydroxylase, the final step in the cortisol biosynthetic pathway. Similar results were seen with Pe, 17-Pe, Po and 17-Po, all of which are converted to cortisol via a biosynthetic route which includes catalysis by 11 beta-hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1983

Basal steroid production by the zona reticularis of the guinea-pig adrenal cortex.

The zonae glomerulosa and fasciculata (ZG/ZF) have been separated by microdissection from the zona reticularis (ZR) of the guinea-pig adrenal cortex. Using non-proliferative organ culture and specific radioimmunoassay the basal secretion of 12 steroids from each zone have been studied. The ZG/ZF secreted 9 times more cortisol (in pmol/mg dry weight 5 h culture) than the ZR. In contrast the androgens (androstenedione, 5-androstenediol, dehydroepiandrosterone, dehydroepiandrosterone-sulphate and testosterone) were preferentially produced (by a factor of 3) by the ZR which also secreted more of all the other steroids studied, with the exception of progesterone and 17-hydroxyprogesterone. The mean molar ratios of total androgen output to cortisol secretion were 1:1.7 and 1:45 for the ZR and ZF respectively. Using computerised image analysis the relative volumes of the ZG/ZF and ZR in the cortex were calculated to be 65 and 35% respectively. The medulla occupies 4.6% of the total gland. From the zonal composition of the cortex, the relationship between wet and dry weight for each zone and the basal secretion rate of each steroid/mg dry weight/5 h culture, we calculated that the ZR could be responsible for 7 and 66% of the cortisol and total androgen secretion respectively from the guinea-pig adrenal gland. It is concluded that the ZF and ZR are functionally as well as morphologically distinct.

Adrenal Cortex↗
PubMed1983

Microheterogeneity of luteinizing hormone in pituitary glands from women of pre- and postmenopausal age.

Luteinizing hormone from the pituitaries of women of premenopausal (Pre-M) and postmenopausal (Post-M) age has been studied to further understand the variations in the quality of plasma LH in women of different ages. The median and range for bioactive LH was 101: 85-155 IU/mg protein and 83: 52-201 IU/mg protein for the Pre- and Post-M groups respectively. The ratios of LH bioactivity/immunoreactivity (B:I) were 1.04 +/- 0.13 (Pre-M) and 0.97 +/- 0.02 (Post-M). Broad range isoelectrophoretic profiles of the pituitary extracts revealed a heterogeneous population of coincident bioactive and immunoreactive LH peaks. Recoveries from the columns were similar in both groups and B:I values of the LH material throughout the columns were between 0.5 and 2.0 in over 90% of the fractions. Between 31.8 and 36.6% of the LH from both groups appeared between pH 7 and pH 8. The most striking difference between the two groups was that greater than 34% of the bioactive LH from the Post-M pituitaries, but less than 6% of the LH from the other group had migrated as a major discrete peak to an acidic region of pH less than 6. We conclude that although there is no difference in total LH activity in the pituitaries from the two groups, substantially more 'acidic' LH material is stored in the postmenopausal pituitaries. These observations are consistent with the recent finding of greater circulating levels of acidic LH in plasma from post-menopausal women.

Adult↗