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Biomedical subjects

J W Funder

Publications and source records attributed to J W Funder.

At least 163 records · Page 9Linked to original sources
PubMed1988

Analysis of renal and hippocampal type I and type II receptors by fast protein liquid chromatography.

Type I and Type II adrenal steroid receptors from rat renal and hippocampal cytosols were studied by the technique of Fast Protein Liquid Chromatography. Type I receptors were labelled with [3H]aldosterone plus excess RU26988, and Type II receptors with [3H]dexamethasone. On a Mono Q anion exchange column the molybdate-stabilized renal and hippocampal Type I receptors both eluted as single symmetrical peaks at 0.27 M NaCl, with a recovery of approximately 90% and 60-fold purification (renal) and 10-15-fold (hippocampal). Molybdate-stabilized Type II binding sites from both hippocampal and renal cytosols co-eluted with the Type I sites. On Superose gel filtration renal Type I receptor-steroid complexes consistently eluted two fractions later than hippocampal Type I complexes, suggesting that the renal complexes are smaller; Type II receptor-steroid complexes from both cytosols co-eluted, consistently one fraction behind hippocampal Type I sites. Sequential gel filtration and anion exchange chromatography achieved a 1000-fold purification of renal Type I binding sites, with an overall recovery of 10%.

Androstanols↗
PubMed1988

Alpha-lactalbumin production by rat mammary gland: prepartum emergence of resistance to glucocorticoids and progestins.

Explants of mammary glands from pregnant or lactating rats were shown to differ markedly in their response to glucocorticoid and progestin in terms of alpha-lactalbumin production. Tissue from day 14 pregnant rats had low initial tissue levels and basal production of alpha-lactalbumin, and showed a dose-biphasic response to glucocorticoids, with significantly higher production at 3 nM than 300 nM RU26988; the progestin ORG 2058 inhibited production. In explants from lactating rats (day 1 and day 5) tissue content and basal production of alpha-lactalbumin were markedly increased compared with day 14 pregnant, but were unaltered by glucocorticoid or progestin. Animals near parturition (day 19, day 20) showed a transitional pattern, with considerable variation between animals. Basal production and tissue content correlated closely; as levels rose, the tissues became progressively less responsive to the effects of both classes of steroid. We interpret these data as evidence for a switch, in terms of glucocorticoid control of alpha-lactalbumin gene expression pre-partum; the mechanism(s) underlying this switch remain to be explored.

Androstanols↗
PubMed1988

Steroid binding to cardiac type I receptors: in vivo studies.

In vitro, Type I (mineralocorticoid) receptors show equivalently high affinity for aldosterone, corticosterone and cortisol in cytosol preparations from a wide range of tissues. This steroid non-selectivity is also found for hippocampal Type I receptors in vivo; in contrast, Type I receptors in physiological mineralcorticoid target tissues are highly aldosterone-specific in vivo. In the present study we show that Type I receptors in the heart are non-selective in vivo, like those in hippocampus, and therefore presumably act as high-affinity glucocorticoid receptors rather than mineralocorticoid receptors.

Aldosterone↗
PubMed1988

Immunoreactive ACTH, beta-endorphin, and cortisol levels in plasma following spinal manipulative therapy.

This study examines the possibility of a humorally mediated analgesic response to spinal manipulative therapy by determination of plasma levels of beta-endorphin, adrenocorticotropic hormone (ACTH), and cortisol before and after intervention. Forty male subjects (20 symptomatic, 20 asymptomatic) were allocated into four equal groups. Two treatment groups were given spinal manipulative therapy, and two groups underwent a sham procedure. Blood samples were taken via indwelling butterfly needles pre- and postintervention in all four groups, and levels of immunoreactive ACTH, immunoreactive beta-endorphin, and cortisol determined by radioimmunoassay. No differences in ACTH or beta-endorphin were found between sham and treated groups, or between pre- and postintervention in any group; cortisol levels fell over the course of the study in all groups. The findings thus appear to exclude a humoral role for beta-endorphin in mediating the analgesic response to spinal manipulative therapy; in addition, they suggest that such therapy is not a stressor that activates the hypothalamo-pituitary-adrenal axis.

Adolescent↗
PubMed1988

Effect of 3 day infusion of ACTH or CRF on the pituitary-adrenal axis.

1. The effects of 72 h subcutaneous infusion of graded doses of rat CRF and ACTH(1-24) were studied in rats of initial weight 150-170 g. Rat CRF was infused at 30, 100 or 300 ng/h, and ACTH(1-24) at 125, 250 or 500 ng/h. 2. There was a progressive though modest increase in adrenal weight for all CRF doses, and an associated reduction of thymus weight. Circulating ir-ACTH, ir-beta-endorphin and corticosterone levels, and adrenal DNA content, were not increased after 3 days. Adrenal RNA and protein content were increased at the highest CRF dose used. 3. ACTH infusion caused a progressive increase in adrenal weight and thymic involution which was marked at the higher doses; circulating corticosterone levels were not significantly altered by the lowest dose but were significantly raised by the higher doses. As expected, plasma ir-beta-endorphin was suppressed to low levels with all doses. Adrenal DNA did not alter but there were progressive increases in adrenal protein and RNA. 4. There was a marked difference in gain between the two infusion regimens in terms of all parameters measured, suggesting that potent mechanisms exist to temper the pituitary-adrenal response to markedly different levels of peripheral CRF input. The damped effect of CRF infusion compared with that of ACTH may represent desensitization of CRF receptors at the pituitary; alternatively, it may reflect binding and substantial inactivation of CRF in peripheral blood.

Adrenal Glands↗
PubMed1988

Immunoreactive dynorphin is regulated by estrogen in the rat anterior pituitary.

The pituitary and hypothalamic content of dynorphin was determined by radioimmunoassay and characterized by high-performance liquid chromatography (HPLC) in adult female Sprague-Dawley rats, intact and ovariectomized with and without estrogen treatment. Animals were given estradiol benzoate, or vehicle (oil) by six daily intramuscular injections. Anterior pituitary content of immunoreactive (ir)-dynorphin in ovariectomized rats was approximately twice that of intact animals, and consisted of a single HPLC peak co-eluting with dynorphin 32. Administration of estradiol benzoate (0.06-6 micrograms/day) caused a marked decrease of ir-dynorphin in the anterior lobe of castrate female rats, with a half-maximal effect at 0.2 microgram/day; levels were restored to those seen in intact animals with 6 micrograms estradiol benzoate per day, an effect which was not influenced by concomitant administration of progesterone (1 mg/day), or bromocriptine (100 micrograms/day). In the hypothalamus and neuro-intermediate lobe multiple peaks of immunoreactive dynorphin were seen, coeluting with dynorphin A 1-8, dynorphin A 1-17 and dynorphin 32. Neither castration nor estrogen treatment altered ir-dynorphin content in these tissues. These findings suggest that the ovary exerts a specific modulating influence on AP ir-dynorphin in the rat, and that in addition this inhibition appears to be mediated by ovarian estrogen.

Animals↗
PubMed1988

Beta endorphin and dynorphin levels in rat pituitary and hypothalamus: age studies.

The content of immunoreactive (ir)-beta-endorphin, ir-dynorphin 1-17, and ir-dynorphin 1-8 was determined in hypothalamus, anterior pituitary, and neurointermediate lobe of rats 3-24 months of age. In the anterior pituitary ir-beta-endorphin showed a progressive rise with age (0.5 +/- 0.06 ng/tissue at 3 months to 1.02 +/- 0.23 at 24 months); a similar change was seen in ir-adrenocorticotropic hormone content of the same tissues. No age-related change in neurointermediate lobe ir-endorphin content was observed. In the hypothalamus, the ir-beta-endorphin content fell progressively from 3 to 18 months, but was restored at 24 months to levels indistinguishable from those at 3 months (16.2 +/- 4.3 ng/tissue compared with 11.3 +/- 4.0 at 24 months). The ir-dynorphin content did not change progressively over the age span examined, except in the case of anterior pituitary content of ir-dynorphin 1-17 which fell progressively between 3 and 18 months (from 1.65 +/- 0.15 ng/tissue at 3 months to 0.73 +/- 0.12). Radioimmunoassay following high-performance liquid chromatography analysis of extracts of the tissues showed little variation of the immunoreactive forms with age, with two notable exceptions. In the hypothalamic extracts from 24-month-old rats the ratio of the nonacetylated ir-endorphin to the acetylated ir-endorphin was lower than in equivalent extracts from 3-month-old rats. In anterior pituitary extracts from 3-month-old rats, ir-dynorphin 1-17 appeared as two peaks (putative 6K and 4K species) of approximately equal size.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1988

Ontogeny of corticotropin-releasing factor and arginine vasopressein in the rat.

The ontogeny in the rat hypothalamus of arginine vasopressin (AVP) and corticotropin-releasing factor (CRF) was studied to determine whether these two peptides develop in parallel, given the synergistic effect of AVP with CRF on adrenocorticotropic hormone (ACTH) release. Hypothalami and extrahypothalamic cerebrum of 15-day fetal--40-day postpartum rats were extracted in acetic acid for radioimmunoassay. Both AVP- and CRF-like immunoreactivity was detected in whole brain extracts of 15-day fetuses. Hypothalamic CRF levels were low during fetal life, fluctuated around the time of birth, and then progressively rose to 63% of adult levels by day 21 postpartum. Extrahypothalamic levels of brain CRF paralleled hypothalamic levels. Hypothalamic AVP levels began to rise at fetal day 19 in a stepwise manner, with the greatest rise in levels occurring after day 14 postpartum. At day 21 postpartum hypothalamic levels were only 17% of adult. Extrahypothalamic AVP levels initially paralleled those in the hypothalamus but reached a plateau after day 4 post-partum, and did not rise again until after day 21. Hypothalamic CRF and AVP thus do not develop strictly in parallel; the adult ratios of these peptides (approximately 1:40) were never seen during development, perhaps of significance in the observed blunting of the hypothalamic-pituitary-adrenal response to stress in infant rats.

Animals↗
PubMed1988

Luteinizing hormone-beta mRNA levels are regulated primarily by gonadotropin-releasing hormone and not by negative estrogen feedback on the pituitary.

Long-term effects of gonadotropin-releasing hormone (GnRH) and/or estrogen on pituitary mRNA levels for the beta-subunit of luteinizing hormone (LH-beta) were determined in anterior pituitary glands from ovariectomized (OVX) ewes. The relative roles of these two factors were assessed by studying hypothalamopituitary disconnected (HPD) ewes with appropriate hormonal treatments. Levels of LH-beta mRNA were increased by ovariectomy and substantially reduced by HPD. Treatment of OVX-HPD ewes with pulses of GnRH (250 ng each 2 h) for 1 week restored LH-beta mRNA levels to OVX levels, whereas treatment with estrogen alone did not alter the low levels found in OVX-HPD ewes. Combined GnRH and estrogen treatment for one week produced LH-beta mRNA levels that were similar to those found in OVX-HPD ewes given GnRH alone; plasma LH pulse amplitudes were also similar in these two groups. From these data we conclude that the long-term negative feedback effect of estrogen to reduce LH secretion is due to a primary inhibition of GnRH secretion and is not a pituitary effect of estrogen. Long-term regulation of LH-beta mRNA is thus primarily regulated by GnRH.

Animals↗
PubMed1988

Innervation of the sheep adrenal cortex: an immunohistochemical study with rat corticotrophin-releasing factor antiserum.

Using indirect immunohistochemistry and an antiserum raised against rat corticotrophin-releasing factor (CRF) we have outlined an asymmetric network of cells and varicose fibers in sheep adrenal cortex. This network was not associated with the larger splanchnic nerves, but was occasionally found in small bundles or with blood vessels; in most instances fibers were found weaving independently through cortical parenchyma. A plexus of fibers was found in the zona reticularis, with a few fibers ramifying into adjacent medulla. Uni or bipolar cells were found throughout the cortex, with the greatest frequency at the corticomedullary junction; a multipolar-type cell was also found in this area. Staining of varicose structures and most cells was abolished by incubation with excess rat CRF 1-41, but not by ovine CRF or a range of other peptides. Though the immunoreactive species has not as yet been identified, it may thus share homology with sequences present in rat but not ovine CRF.

Adrenal Cortex↗
PubMed1988

Studies of the regulation of the hypothalamic-pituitary-adrenal axis in sheep with hypothalamic-pituitary disconnection. I. Effect of an audiovisual stimulus and insulin-induced hypoglycemia.

These studies were undertaken to characterize the secretion of adrenocorticotropin (ACTH), immunoreactive (ir) beta-endorphin (ir-beta-EP) and ir alpha-melanocyte-stimulating hormone (ir-alpha-MSH) from the surgically isolated ovine pituitary in response to an audiovisual stress (barking dog, 3 min) and insulin hypoglycemia. The studies were performed in 4 ovariectomized, hypothalamo-pituitary-disconnected (HPD) and 4 sham-HPD ewes bearing indwelling jugular venous catheters. Basal concentrations of the three pro-opiomelanocortin (POMC) peptides and plasma cortisol were significantly increased in the HPD animals. When the control ewes were exposed to the audiovisual stimulus, plasma ACTH, ir-beta-EP and ir-alpha-MSH levels were increased 2.5-, 10-, and 5-fold 1 min after the stress; plasma cortisol attained maximal values at 5 min. In contrast, plasma levels of the three POMC peptides were not significantly increased in the HPD animals, although a rise in plasma cortisol occurred. The administration of regular insulin (5 units/kg i.v.) to control ewes caused plasma ACTH, ir-beta-EP, and ir-alpha-MSH levels to increase 17-, 22-, and 67-fold at 50 min; plasma cortisol values were maximal at 60 min. In contrast, the elevated basal levels of POMC peptides in the HPD animals were not significantly increased by the hypoglycemia, but a significant elevation of plasma cortisol was seen. We conclude that: (1) the increase in ACTH in intact animals after an audiovisual emotional stress and hypoglycemia, and the abolition of this increase by HPD, indicates that both stimuli, each acting through distinct neuroanatomical pathways, increase the net corticotropin-releasing activity of the hypothalamus; (2) the rise in plasma cortisol in HPD animals after stress suggests that peripheral humoral factors may release additional small amounts of ACTH from the anterior pituitary, and (3) the finding of increased basal ACTH levels after HPD suggests that POMC peptide synthesis and secretion by the anterior pituitary is tonically regulated by an inhibitory factor of hypothalamic origin.

Adrenocorticotropic Hormone↗
PubMed1988

Post-translational processing of pro-opiomelanocortin in the Brattleboro (di/di) rat pituitary.

In homozygous (di/di) Brattleboro rats, pro-opiomelanocortin (POMC) synthesis and processing, and the release of its products, is by definition not under the control of hypothalamic arginine vasopressin (AVP) in contrast with normal rats or heterozygote (di/+) littermates. To explore the role of AVP on these parameters, we have compared homozygotes and heterozygotes in terms of POMC mRNA levels, pituitary content of immunoreactive (ir)-ACTH, ir-beta-endorphin (beta-EP), alpha-melanocyte-stimulating hormone (alpha-MSH) and ir-N-acetyl-EP (NacEP), plasma levels of ir-ACTH and ir-beta-EP, and RP-HPLC profiles of the various forms of ir-alpha-MSH and ir-NacEP. Homozygous rats had immeasurably low levels of hypothalamic ir-AVP, in contrast with their heterozygote littermates; hypothalamic ir-corticotropin releasing factor did not differ between strains. No difference between-strains was seen in levels of POMC mRNA; elevated levels of all pituitary peptides, except ir-ACTH, were found in di/di rats; plasma levels of both ir-ACTH and ir-beta-EP were lower in di/di rats; pituitary ir-alpha-MSH RP-HPLC profiles were similar in both strains, but those for ir-NacEP showed a striking increase in Nac alpha-EP in di/di rats. We interpret these data as evidence for decreased degradation/retarded release of POMC products from the di/di anterior pituitary, for increased processing of POMC products to shorter forms in both anterior pituitary and neuro-intermediate lobe, and thus for a role of AVP in the processing of POMC, as well as POMC synthesis and ACTH/beta-EP release.

Adrenocorticotropic Hormone↗
PubMed1988

Inhibition of growth of B16 melanoma by glucocorticoids does not result directly from receptor-mediated inhibition of tumour cells.

The ability of dexamethasone (DM) to slow the growth of B16 melanoma in C57B1 mice was confirmed. Inhibition was dose-related and was demonstrated in the use of established tumours as well as initial transplants. Although glucocorticoid receptors (GR) were present in cultured tumour cells, DM did not reduce growth of B16 in vitro, even at high concentration. This dissociation of effect on growth in vivo and in vitro suggests that DM slows B16 tumour growth in vivo by mechanisms other than GR-mediated inhibition of tumour cells.

Animals↗
PubMed1988

Melatonin and glucocorticoid hormones.

The synthetic glucocorticoid dexamethasone (2.5, 5 or 10 micrograms/day), administered in the drinking water for 7 days, significantly reduced body weight gain, thymus weight and adrenal weight in female rats. Concomitant administration of melatonin (0.2 or 1.0 mg, i.p.) at either 8.00 or 16.00 h did not alter the glucocorticoid effect on any of these parameters. In competition binding studies melatonin did not compete with 3H-dexamethasone for binding to glucocorticoid receptors in thymus cytosol from adrenalectomized rats. By these indices, therefore, melatonin does not appear to be a glucocorticoid antagonist.

Adrenalectomy↗
PubMed1987

Genital fibroblasts from normal individuals and patients with androgen insensitivity: demonstration of reduced or absent levels of a specific protein (Mr approximately 41K, pI approximately 6) in receptor negative cells.

Genital fibroblasts were obtained from normal individuals and from patients with a variety of syndromes of defective androgenization (complete androgen insensitivity, partial androgen insensitivity, microgenitalia, hypospadias, infertility). Cells were labelled with [35S]methionine, and patterns of protein synthesis compared by two-dimensional gel electrophoresis, with isoelectrofocusing electrophoresis gels or non-equilibrated pH gradient electrophoresis (NEPHGE) gels as the first dimension. A protein (mol. wt approximately 41K, pI approximately 6) was found on NEPHGE gels to be reduced or absent in fibroblasts in which androgen receptor levels were abnormal. The protein was unaltered by prior incubation with 1-100 nM dihydrotestosterone for 48 h, and was present in cells both from normal controls, and from patients with abnormal sexual differentiation showing normal androgen receptor levels. The coincidence of low or absent 41K with low or absent androgen receptors suggested the possibility that it may constitute a steroid-binding moiety of the androgen receptor. To test this possibility cytosols from normal foreskins or normal cultured fibroblasts were adsorbed with testosterone-sepharose affinity resin to remove androgen receptors. Cytosols so treated showed levels of 41K on NEPHGE indistinguishable from those in untreated cytosols, or in cytosols treated with underivatized sepharose. We therefore conclude that the 41K protein, while an accurate marker of the presence or absence of androgen receptors over a range of clinical disorders, is neither an androgen-induced protein nor an androgen-binding protein.

Androgens↗
PubMed1987

Equivalent affinity of aldosterone and corticosterone for type I receptors in kidney and hippocampus: direct binding studies.

In studies from several laboratories evidence has been adduced that renal Type I (mineralocorticoid) receptors and hippocampal "corticosterone-preferring" high affinity glucocorticoid receptors have similar high affinity for both aldosterone and corticosterone. In all these studies the evidence for renal mineralocorticoid receptors is indirect, inasmuch as the high concentrations of transcortin (CBG) in renal cytosol make studies with [3H]corticosterone as a probe difficult to interpret, given its high affinity for CBG. We here report direct binding studies, with [3H]aldosterone and [3H]corticosterone as probes, on hippocampal and renal cytosols from adrenalectomized rats, in which tracer was excluded from Type II dexamethasone binding glucocorticoid receptors with excess RU26988, and from CBG by excess cortisol 17 beta acid. In addition, we have compared the binding of [3H]aldosterone and [3H]corticosterone in renal cytosols from 10-day old rats, in which CBG levels in plasma and kidney are extremely low. Under conditions where neither tracer binds to type II sites or CBG, they label an equal number of sites (kidney 30-50 fmol/mg protein, hippocampus approximately 200 fmol/mg protein) with equal, high affinity (Kd 4 degrees C 0.3-0.5 nM). Thus direct tracer binding studies support the identity of renal Type I mineralocorticoid receptors and hippocampal Type I (high affinity, corticosterone preferring) glucocorticoid receptors.

Aldosterone↗