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Biomedical subjects

J W Funder

Publications and source records attributed to J W Funder.

At least 181 records · Page 10Linked to original sources

Rapid and specific lowering of pituitary FSH beta mRNA levels by inhibin.

Hypothalamo-pituitary disconnected sheep were given gonadotropin releasing hormone pulses every 2 h for 1 week, and the effects of inhibin on mRNA for FSH beta, LH beta, alpha-subunit and prolactin examined. Levels of FSH beta mRNA were reduced to approximately 20% 6 h after administration, and to approximately 10% by 30 h; no change was seen in LH beta, alpha-subunit or prolactin mRNA. These data show that inhibin has a very rapid and specific effect on FSH beta mRNA levels directly at the level of the pituitary gland.

Animals↗

High-affinity aldosterone binding in rat liver--a re-evaluation.

The use of sodium molybdate as a stabilizing agent, and a RU26988 to exclude [3H]aldosterone from Type II glucocorticoid receptors, has enabled us to characterize high affinity Type I aldosterone binding sites in rat liver cytosol. In liver cytosols from male rats aldosterone bound with an affinity (Kd-22 degrees C) of 0.6 nmol/l (range 0.3-0.8 nmol/l), and Nmax 1.7 fm/mg protein (s.e.m. = 0.4); specificity of binding was similar to that for Type I sites in classical aldosterone target tissues (aldosterone greater than or equal to corticosterone greater than dexamethasone). Hepatic Type I receptor levels were relatively constant in both male and female rats aged 30-120 days, with levels significantly higher in females. Parallel studies on hepatoma H4 cells showed levels of Type I sites similar to those in normal liver, suggesting a general distribution of such sites throughout liver parenchyma, rather than a concentration in a specific cell type. The function of such Type I sites, and whether or not they are aldosterone-selective in vivo and can thus act as mineralocorticoid receptors, remains to be determined.

Androstanols↗

Isolation of opiate receptor ligands in coffee.

1. We have reported previously that instant coffee contains ligands for opiate receptors with characteristics similar to those of opiate antagonists. 2. A concentrate of receptor-active ligands from instant coffee was prepared by serial treatments involving Amberlite XAD-2, flash chromatography and gel permeation chromatography. 3. Examination of the final concentrate by GC-MS showed the presence of a number of isomeric (iso)feruloylquinic acid lactones. 4. It is suggested that the synthesis and biological testing of each quinide isomer will establish which is responsible for the opiate receptor activity of instant coffee.

Animals↗

Posttranslational processing of corticotropin-releasing factor in the ovine tuberoinfundibular system and pituitary.

The present studies were undertaken to characterize the immunoreactive-corticotropin-releasing factor (ir-CRF) in two areas of the ovine tuberoinfundibular system, hypophysial portal blood, and pituitary. With an antiserum raised against synthetic ovine (o)CRF(1-41) and 125I-Tyro-oCRF(1-41) as the tracer, concentrations of ir-CRF (pg/mg wet weight, n = 5) were: paraventricular hypothalamus (PVN), 11.7 +/- 2.5; median eminence (ME), 2276 +/- 296; anterior pituitary (AP), less than 0.5; posterior pituitary (PP), 10.0 +/- 2.2. Analysis of the ir-CRF in these areas on G-75 Sephadex chromatography revealed two main peaks--a 'major' peak which coeluted with synthetic oCRF(1-41) and a 'minor' peak which eluted eight fractions later. These two immunoreactive species of CRF were also found in hypophysial portal blood. When ME extract was analyzed by reverse-phase high performance liquid chromatography (HPLC), the 'minor' peak of ir-CRF eluted before that of CRF(1-41). Since CRF contains Arg35-Lys36 within its sequence, we tested the hypothesis that the 'minor' peak of ir-CRF represented a fragment, or fragments, of the molecule derived by proteolytic cleavage at this site. Tyro-oCRF(34-41) was digested with trypsin and the reaction products were identified by amino acid analysis. Two of these products were CRF(36-41) and CRF(37-41), and both migrated in the 'minor' peak area on G-75 Sephadex chromatography and HPLC. In the CRF(1-41) RIA, serial dilution of both fragments yielded nonparallel displacement curves. However, with 125I-Tyro-oCRF(34-41) as the radiolabeled ligand and Tyro-oCRF(34-41) as the standard, serial dilutions of CRF(1-41), CRF(36-41), and CRF(37-41) generated parallel displacement curves, and the molar cross-reactivities were 90%, 45% and 10% respectively. When the ir-CRF in HPLC fractions of ovine ME was measured in the Tyro-oCRF(34-41) RIA, the molar abundance of the hexapeptide and pentapeptide could be obtained. Calculations based on the premise that the 'minor' peak was solely composed of either the hexapeptide or pentapeptide indicated that CRF(36-41) could account for up to 37% of the total ir-CRF, or that CRF(37-41) could account for up to 73% of the total immunoreactivity. On more discriminating HPLC systems, immunoreactive (ir-) oCRF in the sheep median eminence (ME) could be resolved into five different molecular forms. On three distinct chromatographic systems, four of these immunoreactive species shared retention characteristics identical with synthetic oCRF(37-41), oCRF(36-41), oCRF(16-41) and oCRF(1-41), with the fifth immunoreactive peak as yet unidentified.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Adrenocortical steroids and the brain.

In summary, a wide variety of effects of adrenal steroids on the brain have been reported and have been recently and exhaustively reviewed. From the viewpoint of endocrine physiology, however, what is often forgotten is the extraordinary difference in signal level between the two unique products of the adrenal cortex, the mineralocorticoid and glucocorticoid hormones. Levels of cortisol or corticosterone are 2-3 orders of magnitude higher than those of aldosterone, a difference that is tempered by perhaps one order of magnitude by the much higher binding of glucocorticoids to plasma protein. The signal-detecting mechanisms for the lower-intensity signal, i.e. the mineralocorticoid receptor, must therefore have powerful specificity-conferring mechanisms to enable it to recognize, bind, and respond to aldosterone. In vitro studies from a number of laboratories have shown that Type I receptors, in both classic mineralocorticoid target tissues (kidney, parotid, gut) and nontarget tissues (pituitary, hippocampus), cannot distinguish between aldosterone and corticosterone. This finding highlights the problem of aldosterone-selectivity in the kidney (Na+ transport) or the brain (Na+ appetite). In vivo studies, in contrast, show that corticosterone is very poorly taken up and/or retained in kidney, colon, parotid, and pituitary (but not in hippocampus) in mature and 10-day-old (minimal transcortin) rats, whereas aldosterone is well taken up and/or retained by all tissues, evidence for tissue-specific aldosterone selectivity in vivo. Two nonexclusive (i.e. possibly additive) models for such aldosterone selectivity are proposed, one "prebinding" and the other "postbinding". Both models accommodate the experimental findings of the nonselectivity of cytosol preparations in vitro and the stringent specificity seen in in vivo receptor and effector studies. In any real sense, the action of adrenal steroids on the brain is still largely an area of unconnected phenomenology, despite the efforts of a number of talented individuals and groups over the past two decades. Without descriptions of phenomena, even of the most basic ablation and replacement type, we have no chance of making physiological statements. It is equally important, in the attempt to make a coherent physiology, to erect a scaffolding of hypothesis that can be tested against the existing experimental findings and that can serve to suggest further studies in a logical sequence. These hypotheses themselves, and the models used to reify them, may be validated, altered, or rejected by the studies over the next few years.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Cortex Hormones↗

Mineralocorticoid specificity of renal type I receptors: in vivo binding studies.

We have injected rats with [3H]aldosterone or [3H]corticosterone, plus 100-fold excess of the highly specific glucocorticoid RU 28362, with or without excess unlabeled aldosterone or corticosterone and compared type I receptor occupancy in kidney and hippocampus. Thirty minutes after subcutaneous injection [3H]aldosterone was well retained in renal papilla-inner medulla, renal cortex-outer medulla, and hippocampus; in contrast, [3H]corticosterone was well retained only in hippocampus. Competition studies for [3H]aldosterone binding sites showed corticosterone to be a poor competitor in the kidney compared with hippocampus. Time-course studies, with rats killed 10-180 min after tracer administration, showed very low uptake/retention of [3H]corticosterone by kidney; in hippocampus [3H]corticosterone retention was similar to that of [3H]aldosterone in kidney, and retention of [3H]aldosterone by hippocampus was much more prolonged than of either tracer in any other tissue. Studies in 10-day-old rats, with very low levels of corticosteroid binding globulin (CBG), showed a high degree of aldosterone selectivity in both zones of the kidney, whereas [3H]aldosterone and [3H]corticosterone were equivalently bound in hippocampus. We interpret these data as evidence for a mechanism unrelated to extravascular CBG conferring mineralocorticoid specificity on renal type I receptors and propose two models derived from our findings consistent with such differential selectivity.

Aldosterone↗

Type I receptors in parotid, colon, and pituitary are aldosterone selective in vivo.

Previous in vivo studies have demonstrated that type I receptors in the rat kidney are aldosterone selective, whereas those in the hippocampus do not appear to discriminate between aldosterone and corticosterone. We have injected mature rats with [3H]aldosterone or [3H]corticosterone plus 100-fold excess of RU 28362, with or without unlabeled aldosterone or corticosterone, and compared type I receptor occupancy in two classic mineralocorticoid target tissues (parotid and colon) and in the pituitary. Mature rats were killed 10-180 min after tracer administration; [3H]aldosterone was well taken up and retained in all tissues, whereas [3H]corticosterone was significantly retained only in the pituitary 10 min after tracer administration. To assess a possible role for corticosterone-binding globulin (CBG) in conferring aldosterone specificity on type I receptors, 10-day-old rats (with very low levels of CBG) were similarly injected. In the colon and parotid, [3H]aldosterone binding was at least an order of magnitude higher than that of corticosterone; in the pituitary aldosterone binding was approximately three times that of corticosterone. We interpret these data as evidence that in the parotid and colon type I receptors are aldosterone selective by a non-CBG-requiring mechanism, whereas in the pituitary there appear to be both aldosterone-selective and nonselective type I sites.

Aldosterone↗

Glucocorticoids, adrenal medullary opioids, and the retention of a behavioral response after stress.

In the Porsolt model swimming test, naive rats become progressively more immobile over a 15-min initial test (0-5 min, approximately 30% immobile; 5-10 min, approximately 50%, 10-15 min, approximately 70%). Twenty-four hours later, rats have retained the response, being immobile for about 70% of a 5-min retest period. We previously reported that in this test adrenalectomized rats are indistinguishable from intact rats over the 15-min test period, but are immobile for only approximately 30% of a 5-min retest period 24 h later. The finding that this behavioral effect of adrenalectomy can be reversed by glucocorticoids or kappa-selective opioids led us to hypothesize that retention of the immobile behavior can be effected by glucocorticoids from the adrenal cortex or kappa-selective opioids from the adrenal medulla. To test this hypothesis, we have given intact rats the antiglucocorticoid RU38486 and the anticholinergics atropine and mecamylamine to block adrenal medullary discharge, either alone or simultaneously. Administered alone, neither class of antagonist affected the response; administered together, they profoundly reduced immobility to levels seen in adrenalectomized rats. Additional evidence for medullary kappa-selective opioids mediating the behavioral effect was that the administration to hypophysectomized rats of atropine plus mecamylamine or the kappa-selective antagonist MR2266 reduced immobility to the levels seen in adrenalectomized animals. We conclude that secretion from either the adrenal cortex or medulla is sufficient for the incorporation of this learned response after stress, and that in intact animals input from both zones may contribute to the terms of this behavioral response.

Adrenal Medulla↗

Concomitant dopaminergic and glucocorticoid control of pituitary proopiomelanocortin messenger ribonucleic acid and beta-endorphin levels.

Synthesis and secretion of POMC-derived peptides appear to be differentially regulated in the anterior pituitary (AP) and neurointermediate lobe (NIL). In the AP, glucocorticoids inhibit, and CRF and arginine vasopressin stimulate, synthesis of POMC and release of immunoreactive (ir)-beta-endorphin (beta EP); in the NIL, synthesis and release of POMC and its derivatives are under tonic inhibitory dopaminergic control. There is, however, evidence for some overlap of these control mechanisms under certain circumstances. In the present study we have used specific RIA and Northern blot analysis to examine the effects of chronic treatment with dopaminergic agents and dexamethasone (DM) (both alone and in combination) on AP and NIL content of ir-beta EP and POMC messenger RNA (mRNA), and/or hypothalamic ir-arginine vasopressin and ir-CRF content. In the NIL, the dopamine agonist bromocriptine reduced and the antagonist haloperidol raised both POMC mRNA and ir-beta EP content. Long term DM treatment did not alter NIL ir-beta EP content in the intact rat, but increased levels of POMC mRNA. DM abolished the haloperidol-induced increase in NIL ir-beta EP content but further increased the haloperidol-induced rise in POMC mRNA. DM treatment lowered both ir-beta EP and POMC mRNA in the AP as well as lowering levels of hypothalamic ir-CRF. In DM-treated rats, haloperidol partially restored AP ir-beta EP and POMC mRNA to control untreated levels. These findings further support the proposition that both dopaminergic agents and glucocorticoids can modulate POMC mRNA levels and/or tissue content of ir-beta EP in both the NIL and AP of the rat. The effects of DM on the NIL, both alone or with haloperidol, suggest that glucocorticoids may have both direct and indirect effects on POMC gene expression in this tissue.

Animals↗

Isolation, amino acid sequence and action of guinea-pig ACTH on aldosterone production by glomerulosa cells.

Though minor sequence differences between-species have been reported for adrenocorticotrophin, ACTH(1-39), the steroidogenic moiety ACTH(1-24) has appeared invariant in mammals. We here report the isolation, purification and amino acid sequencing of guinea-pig (GP) ACTH in which Pro24 is replaced by Ala24, and the demonstration that GP-ACTH stimulates aldosterone production to maximal levels well above those seen with human ACTH(1-39) or Synacthen, ACTH(1-24) amide, the synthetic ACTH fragment widely used for diagnostic and therapeutic purposes.

Adrenal Cortex↗

alpha-N-acetylated beta-endorphin is differentially processed in the normal and hypothalamo-pituitary-disconnected (HPD) ewe.

We have used specific radioimmunoassay and high-performance liquid chromatography (HPLC) to determine content and molecular forms of pituitary alpha-N-acetylated endorphin (NacEP) in the intact and hypothalamo-pituitary-disconnected (HPD) sheep. No significant differences were seen in anterior pituitary (AP) immunoreactive (ir-) NacEP levels (control = 4.34 +/- 0.45 ng/mg; HPD = 4.38 +/- 0.83 ng/mg; n = 4) or in HPLC profiles following pituitary disconnection. In contrast, levels of ir-NacEP in the HPD intermediate lobe (IL) (263 +/- 16.5 ng/mg; n = 4) were double those in the control (110 +/- 14 ng/mg; n = 4), accompanied by a marked change in the molecular profile. In the control IL, the major species were Nac alpha EP (30%), Nac gamma EP (30%) and Nac beta EP1-27 (35%); in HPD IL, Nac beta EP1-27 (65%) is the dominant molecular form, with Nac alpha EP (11%) and Nac gamma EP (9%) relatively minor components.

Animals↗

Mechanisms of biphasic action of glucocorticoids on alpha-lactalbumin production by rat mammary gland explants.

The highly selective Type II glucocorticoid ligand RU28362 showed a clear biphasic effect on alpha-lactalbumin (alpha-LA) production in rat mammary gland explants, with a peak at 1 nM and a return to basal levels at 30-300 nM; dexamethasone showed a similar profile. Corticosterone, which has a higher affinity for Type I than Type II receptors, produced a variable response. In six out of eleven studies this was biphasic, with a maximum at 300 nM; in five no increase above baseline was seen. Classical Type I receptor ligands--aldosterone and deoxycorticosterone--showed responses parallel to their Type II agonist activity. We interpret these data as follows occupancy of Type I receptors does not increase alpha-LA production the response to selective Type II receptor ligands is truly biphasic and one explanation of this pattern may be the existence of both "turn-on" and "turn-off" acceptor sites in the nucleus.

Aldosterone↗

Photoaffinity labelling of androgen receptors from human foreskin and cultured genital fibroblasts with tritiated methyltrienolone [3H]R1881.

Androgen receptors were partially purified by affinity chromatography of cytosols prepared from either normal foreskins or normal cultured fibroblasts. Partially purified receptors were covalently labelled with [3H]R1881 (tritiated methyltrienolone) by ultraviolet photoactivation. Gel electrophoresis of cytosols from both sources showed two peaks of specific binding, Mr approximately 40 K and approximately 85 K, under denaturing conditions. Photoaffinity labelling of the human androgen receptor may thus provide a useful tool in further studies on disorders of androgenization.

Affinity Labels↗

Cytosol and nuclear levels of thymic progesterone receptors in pregnant, pseudopregnant and steroid-treated rats.

Cytosol and nuclear levels of progesterone receptors in rat thymus and uterus were compared in mature Sprague-Dawley rats under a variety of physiological (non-oestrus, oestrus, pregnancy) and experimental circumstances (pseudopregnancy, oestrogen administration, oestrogen plus progesterone administration). Cytosols and nuclear extracts were charcoal treated to remove endogenous steroids, and incubated overnight at 4 degrees C with the synthetic progestin [3H]R5020 +/- greater than 100-fold R5020, in the presence of greater than 200-fold RU26988 (a highly specific synthetic glucocorticoid) to exclude tracer from glucocorticoid receptors. In the various states examined, levels of progesterone receptors in the uterus were an order of magnitude higher (350-1300 fmol/mg protein) than in the thymus (40-140 fmol/mg protein). In contrast, considerable parallelism between uterus and thymus was seen in terms of the effects of the various manipulations upon tissue levels of progesterone receptors, and of their distribution between cytosol and nuclear compartments.

Animals↗

Cortisol 17 beta acid, transcortin, and the heterogeneity of rat brain glucocorticoid receptors.

Binding of tracer or competing steroids to transcortin can compromise specificity studies on receptors for adrenal steroids. Recently Alexis et al. have used cortisol 17 beta acid at high concentrations to prevent steroid binding to any transcortin possibly contaminating rat brain cytosol preparations. On the basis of limited specificity studies of [3H]dexamethasone and [3H]corticosterone binding under such conditions, it was claimed that binding sites for the two steroids are indistinguishable, and it is thus unnecessary to invoke distinct binding sites for each glucocorticoid. We have extended these competition studies in the presence of cortisol 17 beta acid, and shown that in rat hippocampus Type I, corticosterone-preferring glucocorticoid receptors can be clearly distinguished both from transcortin and from Type II, dexamethasone-binding glucocorticoid receptors.

Androstanols↗

Expression of the renal kallikrein gene in mineralocorticoid-treated and genetically hypertensive rats.

On the basis of both clinical observations and experimental studies it has been proposed that renal kallikrein is a mineralocorticoid regulated protein. In other studies, changes in renal kallikrein activity have been implicated in the genesis of, and/or response to, hypertension. Using a cloned complementary DNA (cDNA) to rat pancreatic kallikrein (pcXP39) for hybridization histochemistry, and both Northern and dot blot analysis, we studied expression of the kallikrein gene in steroid-treated control animals, and in three strains of genetically hypertensive rats. No differences in renal kallikrein messenger RNA (mRNA) levels were found between adrenalectomized rats and those treated for 5-14 days with 9 alpha-fludrocortisone, corticosterone or dexamethasone, or between hypertensive rats and their appropriate controls. Since mRNA levels appear essentially invariant under such circumstances, the change in renal kallikrein activity/immunoreactivity after chronic mineralocorticoid elevation, or in hypertensive rats, presumably reflects modulation at the post-transcriptional level.

Animals↗