Biomedical subjects

J W Funder

Publications and source records attributed to J W Funder.

At least 217 records · Page 12Linked to original sources

PubMed1985

Kallikrein gene expression in the rat anterior pituitary.

The report of 'kallikrein-like' activity in the rat neuro-intermediate lobe (N-IL) and its possible involvement in pro-opiomelanocortin processing led us to explore the expression of the kallikrein gene(s) in the pituitary. Using 32P-labelled rat pancreatic kallikrein cDNA, we have shown positive hybridization for rat anterior pituitary poly(A)+ RNA, of identical size on Northern blots (approximately 1.0 kb) to rat kidney poly(A)+ RNA run in parallel. Prior adrenalectomy or ovariectomy decreased the level of kallikrein mRNA seen in the anterior pituitary; total RNA from rat N-IL showed no significant hybridization. On hybridization histochemistry the anterior pituitary was strongly positive, and the neural and intermediate lobes negative. The previously reported kallikrein-like activity in the N-IL is therefore probably due to a non-kallikrein kininogenase; in the anterior pituitary, kallikrein may have a physiological role in limited precursor proteolysis, but lack kininogen activity.

Animals↗

PubMed1985

In-vivo metabolites of spironolactone and potassium canrenoate: determination of potential anti-androgenic activity by a mouse kidney cytosol receptor assay.

We have established a sensitive and specific radioreceptor assay for androgen receptor active materials in plasma, using tritiated methyltrienolone ([3H]R1881) as tracer, and spayed mouse kidney cytosol receptor as the binding species. On radioreceptor assay, plasma from mice chronically administered spironolactone contained approximately 10 times higher levels of androgen receptor active material than from mice administered potassium canrenoate. In parallel bioassays (antagonism of the effect of testosterone on seminal vesicle weight), spironolactone was greater than 4 times as potent an antiandrogen as potassium canrenoate. Administered potassium canrenoate circulates as canrenoic acid, in equilibrium with its lactonized congener canrenone. Since over 80% of administered spironolactone is irreversibly converted to canrenone/canrenoic acid, its much higher anti-androgen activity on radioreceptor assay and bioassay may point to the generation of unidentified, minor metabolites with very high affinity for androgen receptors and/or a very long plasma half-life.

Animals↗

PubMed1985

N-acetyl endorphin in rat spermatogonia and primary spermatocytes.

In previous reports modest levels of beta-endorphin have been found by radioimmunoassay in rat testis, and localized by immunofluorescence to the interstitial cells. We have confirmed these previous reports and extended them by showing that the majority of testicular endorphins are acetylated forms, N-acetyl gamma-endorphin, N-acetyl alpha-endorphin, and N-acetyl beta-endorphin1-27. In addition, N-acetylated endorphins are not found in interstitial cells, but are confined to spermatogonia and primary spermatocytes.

Aging↗

PubMed1985

Localization of arginine vasopressin-neurophysin II messenger ribonucleic acid in the hypothalamus of control and Brattleboro rats by hybridization histochemistry with a synthetic pentadecamer oligonucleotide probe.

By using 32P-labeled oligonucleotide probes for in situ DNA-RNA hybridization, we have defined the distribution of messenger RNA (MRNA) for arginine vasopressin-neurophysin II (AVP-NPII) in the rat hypothalamus. With a specific pentadecamer nucleotide probe, strong specific hybridization is seen in supraoptic, suprachiasmatic, and paraventricular nuclei in both Sprague-Dawley and Brattleboro rats. This finding, of equivalent distribution of AVP-NPII mRNA in Brattleboro and control hypothalami, confirms and extends the recent report of equivalent levels of AVP-NPII mRNA by Northern blot analysis.

Animals↗

PubMed1985

Ovarian immunoreactive beta-endorphin and estrous cycle in the rat.

Adult female Sprague-Dawley rats were killed at different stages of a 4-day estrous cycle, and ovaries and anterior pituitaries examined for content of immunoreactive beta-endorphin by RIA and for localization by indirect immunofluorescence. Two anti-beta-endorphin antisera, both recognizing different antigenic determinants of human-beta-endorphin, showed intense immunofluorescence staining of cells localized predominantly in ovarian corpora lutea. At proestrus, both large and small luteal cells stained positively but only the large luteal cells were immunofluorescence positive at other stages of the estrous cycle. In addition, less intense staining of granulosa cells was occasionally observed in secondary and antral follicles; scattered cells in the interstitium were also weakly positive. In contrast, cells of primordial and primary follicles, and those of theca tissue were consistently negative. Ovarian levels of immunoreactive beta-endorphin were found to be lowest at estrus (2.1 +/- 0.18 ng/g; n = 8, mean +/- SE) and significantly raised in stepwise manner over metestrus and diestrus to a peak (approximately 4 X estrous levels) at proestrus; in contrast, immunoreactive beta-endorphin content of anterior pituitaries remained unaltered during the same period. Sephadex G-50 gel chromatography of ovarian extracts revealed three distinct peaks of immunoreactive beta-endorphin, a minor peak in the void volume, and two major peaks of unequal size eluting at mol wt approximately 11.5K and approximately 3.5K. The major species of low molecular weight immunoreactive beta-endorphin on reverse phase HPLC was beta-endorphin1-31. We conclude from the findings that, in adult rat ovaries, luteal, granulosa, and interstitial cells are responsible for the production of immunoreactive beta-endorphin and that this production, being related to the estrous cycle, is presumably under the direct or indirect influence of gonadotropins.

Animals↗

PubMed1985

Vasopressin-neurophysin II gene expression in the ovary: studies in Sprague-Dawley, Long-Evans and Brattleboro rats.

The neurohypophysial hormones oxytocin and arginine vasopressin (AVP) have been identified on immunological criteria in the ovary. Confirmation of extraneuronal synthesis requires the demonstration in the tissue of the specific messenger RNA (mRNA) for the preprohormone. Using a synthetic pentadecamer nucleotide probe, highly specific for the 5' region of rat neurophysin II (NP II), we have demonstrated the presence of AVP-NP II mRNA in the ovary of Sprague-Dawley, Long-Evans and Brattleboro rats, with an apparent molecular weight identical to that seen for hypothalamus. These findings, together with the presence of immunoreactive AVP in the ovaries but not hypothalami of Brattleboro rats, suggest that tissue-specific differences in AVP-NP II gene expression occur at the translational as well as transcriptional level.

Animals↗

PubMed1984

Interaction of dopamine receptor ligands with subtypes of the opiate receptor.

There are currently several studies in which individual dopamine receptor ligands have been reported to bind with relatively low affinity to opiate receptors. To extend these studies, and to examine the opiate receptor subclass selectivity of such agents, we have examined the ability of six dopamine receptor ligands (prochlorperazine, chlorpromazine, haloperidol, bromocriptine, pimozide and metoclopramide) to compete with four tritiated tracers - [3H]naloxone, [3H][D-Ala2,D-Leu5]enkephalin, [3H]morphine and [3H]ethylketocyclazocine - for binding to rat brain membrane opiate receptors. The dopamine receptor ligands displaced the labelled opiates in a dose-dependent manner, with ED50 values of 3 microM to 3 mM. Pimozide was consistently the most potent (ED50 3-14 microM), and metoclopramide the least potent (ED50 35 microM to 3.5 mM). Dopamine receptor agonists and antagonists thus interact with opiate receptors with no clear subclass selectivity, and with similar hierarchies of inhibitory potency in each of the various opiate receptor systems.

Animals↗

PubMed1984

Naloxone inhibits both glucocorticoid and [D-Ala2,Met5]enkephalinamide reversal of behavioural effect of adrenalectomy.

Intact rats treated immediately after a 15-min swimming exposure with i.m. 1 or 10 mg/kg naloxone (but not with 10 mg/kg of MRZ2593, a quaternary analogue) showed significant reduction in immobility (55, 30%) compared with controls (70%) during a 5-min retest 24 h later. This effect of naloxone was seen when administered 1 h prior to retest. Adrenalectomized rats, as previously shown, are only approximately 30% immobile on retest; administration of [D-Ala2,Met5]enkephalinamide (50 micrograms i.m.) or dexamethasome (6 micrograms i.m.) elevated immobility to 66 and 69% respectively. Administration of naloxone (0.1-10 mg/kg) progressively antagonized the effect of both [D-Ala2,Met5]enkephalinamide and dexamethasone, to 36 and 41% immobility on retest at 10 mg/kg. We conclude that the incorporation of information post-stress normally involves at least two opioidergic pathways, and that the integrity of one of these pathways is an absolute requirement for such incorporation to occur, based on the naloxone studies. In contrast, either glucocorticoids or enkephalin analogues are sufficient to restore behaviour post-adrenalectomy, suggesting that corticosteroid-dependent neural mechanisms may substitute for this second opioidergic pathway.

Adrenalectomy↗

PubMed1984

Oestrogen administration induces progesterone receptors in lactating rat mammary gland.

Displaceable binding of the synthetic progestin [3H]ORG 2058 (16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20 dione) was found in ammonium sulphate treated cytosols from uterus and mammary gland from lactating rats. Specificity studies, however, showed that in mammary gland but not uterus this binding was exclusively to glucocorticoid receptors, and that specific progesterone receptors were undetectable. In contrast, after in vivo administration of oestradiol benzoate in oil (4 micrograms at -48, -24h), [3H]ORG 2058 bound predominantly to specific, high affinity progesterone receptors in lactating mammary gland. Thus though in lactation progesterone receptors are normally absent from the mammary gland, they can be induced by oestrogen administration.

Animals↗

PubMed1984

Glucocorticoid effects on newly synthesized proteins in muscle and non-muscle cells cultured from neonatal rat hearts.

To analyze direct effects of steroids on the rates of synthesis (and/or degradation) of newly synthesized proteins of the rat heart, we have used high resolution two-dimensional gel electrophoresis and autoradiography. A collective steroid domain of nineteen proteins, comprising fifteen with an increased rate of synthesis and four with a decreased rate of synthesis, was consistently seen in cultures of cardiac muscle and non-muscle cells from neonatal rats following 24 h incubation with 10(-7) dexamethasone. Similarly, incubation with 10(-7) M sex steroids, mineralocorticoids, and other glucocorticoids including the highly selective compound RU26988, established the glucocorticoid-specificity of the response. Different subsets of this glucocorticoid domain were seen for collagenase- or trypsin-dispersed primary cultures of cardiac muscle and non-muscle cells or for passaged cultures of cardiac non-muscle cells. Six polypeptides were consistently induced in all cardiac cultures, regardless of cell morphology. Two polypeptides were consistently induced only in those cultures containing cardiac non-muscle cells, whereas protein l, of identical Mr(approximately 52K) and pI (approximately 5.3) to desmin, was induced only in cultures of spontaneously contractile cardiac muscle cells. The glucocorticoid domain proteins described herein represent direct steroid effects on cardiac cells and are therefore candidate mediators of physiological glucocorticoid effects on, for example, differentiation and contractility.

Aldosterone↗

PubMed1984

A common 43 K protein induced by glucocorticoids in a variety of cells and tissues.

We have investigated the domain of adrenal steroid action in a variety of mammalian cells and tissue by high resolution two-dimensional gel electrophoresis and autoradiography. Following in vivo or in vitro treatment with steroids, minced tissue or cells were pulsed with [35S]methionine, and they newly synthesized polypeptides compared with controls by visual inspection of partial protein maps. We have found a similar protein (Mr approximately 41-44K, pI approximately 6.3-6.5) to be consistently increased by dexamethasone in 3 rat tissues and 9 human, bovine and rat cell lines. The protein was constitutively synthesized in all targets; quantitative measurements on the magnitude of response show a 2-fold induction by dexamethasone in all systems, in vivo and in vitro. Glucocorticoid-specific hormones but not sex steroids increase the rate of synthesis; deoxycorticosterone has agonist or antagonist effects on 43K synthesis in different systems studied. Co-electrophoresis experiments indicated that 43K proteins co-migrate in at least 3 rat cell lines from tissues with diverse functions. The functional significance of this common glucocorticoid-induced protein is unknown; its ubiquity and electrophoretic properties suggest a highly conserved, common indicator of glucocorticoid action in diverse target cells and tissues, rather than a tissue-specific response.

Androstanols↗

PubMed1984

Overlapping but not identical protein synthetic domains in cardiovascular cells in response to glucocorticoid hormones.

We have compared, by two-dimensional gel electrophoresis, the effects of glucocorticoids on newly synthesized proteins of cultured cardiovascular cells (rat synthetic- and contractile-state vascular smooth muscle, rat cardiac muscle and non-muscle, and bovine endothelium) and rat hepatoma (H4) cells, as a non-cardiovascular target. In each cell type, glucocorticoids consistently affected a particular set of proteins termed the glucocorticoid domain for that target tissue. Cardiovascular cells exhibited overlapping but non-identical glucocorticoid domains; these domains varied in number of proteins, proportion of induced and repressed proteins, and overlap (extent of shared protein responses) between the various cardiovascular cell types studied. On physico-chemical and co-electrophoresis data the glucocorticoid domain of cardiovascular cells also overlapped with that of hepatoma cells, but to a lesser extent. The glucocorticoid domain in cardiac muscle cells included five proteins not found in cardiac non-muscle cells. Contractile-state vascular smooth muscle cells showed some, but not all, of the proteins affected by glucocorticoids in synthetic-state cells; the only response seen in the contractile but not vascular smooth muscle cells was a protein of Mr-52K and pI-5.3, which was also induced in spontaneously contractile cardiac muscle. Further characterization of contractile and synthetic vascular smooth muscle and cardiac muscle proteins affected by glucocorticoids is necessary to ascertain their role(s) in specialized cardiovascular functions.

Animals↗

PubMed1984

Extravascular CBG-like sites in rat kidney and mineralocorticoid receptor specificity.

To determine the intrarenal distribution of extravascular corticosteroid-binding globulin- (CBG) like sites, we measured the specific binding of [3H]corticosterone to supernatants prepared from papilla-inner medulla, outer medulla, and cortex from adrenalectomized rats. Renal papilla-inner medulla of exhaustively perfused kidneys contained 8-20 times higher concentrations of such sites than cortex or outer medulla. The ontogeny and steroid specificities of such sites were identical to those of plasma CBG; their molar concentration in the inner medulla-papilla was 4 times that of plasma CBG, consistent with local accumulation of CBG. Cytosols from all three zones bound [3H]aldosterone in the presence of RU-26988, a highly specific synthetic glucocorticoid, with high affinity (Kd 4 degrees C, 0.3-0.4 X 10(-9) M) and limited capacity (papilla-inner medulla, 23-34; outer medulla, 15-16; cortex, 12-17 fmol/mg). In papilla-inner medulla the apparent specificity of such [3H]aldosterone binding sites was aldosterone greater than corticosterone greater than dexamethasone, in contrast to aldosterone = corticosterone greater than dexamethasone in outer medulla and cortex. We propose that extravascular CBG confers mineralocorticoid specificity on the papilla-inner medulla [3H]aldosterone binding sites. In addition, given the higher levels of extravascular than intravascular CBG and the recurrent vascular architecture of the renal medulla-papilla, a countercurrent exchange model is proposed for renewable sequestration of corticosterone in the region.

Aldosterone↗

PubMed1984

Ontogeny of renal mineralocorticoid receptors and urinary electrolyte responses in the rat.

We examined the ontogeny of cytosol mineralocorticoid receptors in renal inner medulla-papilla (IM-PAP) and outer medulla-cortex (OM-CX) and of a biological response to administered corticosteroids (urinary K+/Na+ ratio). Rats at early (7-9 days), intermediate (13-15 days), and late (23-30 days) stages of maturation were used. Young rats were found to be essentially insensitive to aldosterone, in contrast to rats aged 13-15 days and older. Scatchard analysis of IM-PAP and OM-CX cytosol showed equivalent high-affinity aldosterone binding sites at all ages. The affinity of these sites was constant (Kd, 4 degrees C approximately 0.5 nM) and their concentration varied only slightly over maturation. In a series of in vivo nuclear transfer studies, the hyposensitivity to aldosterone in young rats did not seem to be due to a difference in the nuclear binding of aldosterone-receptor complexes. The resistance of the immature (7-9 days) rat kidney to aldosterone thus appears to be a postreceptor phenomenon. The maturational events allowing expression of mineralocorticoid-inducible genes in the kidney remain to be determined.

Adrenalectomy↗

PubMed1984

Immunoreactive beta-endorphin in a subpopulation of mouse spleen macrophages.

Using radioimmunoassay and immunofluorescence with antibodies to beta-endorphin (beta EP) and ACTH, we have shown that a subpopulation of mouse spleen cells, expressing Mac-1, a marker of macrophage differentiation, contains immunoreactive (ir)-beta EP, ir-ACTH, and smaller amounts of presumptive higher molecular weight forms of both. Neither nonadherent spleen cells, nor adherent or nonadherent cells from peripheral blood, contained detectable levels of these peptides. These findings suggest that beta EP and ACTH may be synthesized in a subpopulation of spleen macrophages, and are consistent with the possibility that these or related peptides may modulate lymphocyte function in the specific microenvironment of the spleen.

Adrenocorticotropic Hormone↗

PubMed1984

Estrogen lowers immunoreactive beta-endorphin in the neurointermediate lobe of the rat pituitary.

The effects of ovariectomy and sex steroids on tissue levels of immunoreactive beta-endorphin (ir-beta EP) were examined. Adult female Sprague-Dawley rats were sham operated, ovariectomized (OVX), adrenalectomized (ADRX), or ADRX-OVX. Animals were given estradiol (E2) or vehicle (oil) by six daily im injections, and tissue levels of immunoreactive beta-endorphin (ir-beta EP) determined. In OVX animals, neurointermediate lobe (NIL) content of ir-beta EP was double that in controls; plasma ir-beta EP was modestly but significantly raised, but levels in the anterior pituitary (AP) remained unaltered. The effects of ovariectomy on NIL and plasma ir-beta EP were reversed by E2 in a dose-related manner. ADRX-OVX animals showed elevations of AP and NIL ir-beta EP to levels double those in SHAM, and plasma levels 5 times higher. E2 administration to ADRX-OVX rats normalized ir-beta EP in NIL, but not that in AP nor plasma. These findings suggest that the ovary may exert a specific tonic influence on NIL ir-beta EP in the rat, and that this inhibition appears to be mediated, at least in part, through ovarian estrogen.

Adrenalectomy↗

PubMed1984

A pituitary tumor producing high molecular weight adrenocorticotropin-related peptides: clinical and cell culture studies.

The tissue-specific processing of proopiomelanocortin (POMC), the precursor of ACTH, beta-endorphin, and their related peptides, is currently of considerable interest. We report a patient with a large aggressive pituitary tumor, Cushing's syndrome, and hyperpigmentation managed by transsphenoidal hypophysectomy, bilateral adrenalectomy, and sellar irradiation. Preoperatively, plasma levels of immunoreactive ACTH (ir-ACTH; 280 ng/liter) and beta-endorphin (ir-beta EP; 520 ng/liter) were moderately elevated. Chromatography of the plasma showed two peaks of ACTH immunoreactivity, with the major peak eluting in the void volume, and two major peaks of ir-beta EP, corresponding to the elution positions of beta-lipotropin and beta-endorphin standards. Plasma ir-ACTH and ir-beta EP were not suppressed by high doses of glucocorticoid or bromocriptine, a degree of autonomy more commonly found with POMC production from ectopic sources than that from pituitary tumors. Tissue removed at operation was enzymatically dispersed, and the cells were cultured in suspension, propagated, and passaged sequentially for over 20 passages. Using this cell line, we demonstrated that the biosynthesis of POMC, its pattern of processing, and the release of POMC/ir-beta EP/ir-ACTH in vitro were consistent with the in vivo evidence of autonomous secretion and abnormal processing of POMC by this pituitary tumor.

Adenoma↗

PubMed1984

Thymic sensitivity to sex hormones develops post-natally; an in vivo and an in vitro study.

In vitro thymic organ cultures were used to examine the effects of the sex hormones estradiol and dihydrotestosterone on thymocytes. In contrast with the marked loss of cortical thymocytes seen in vivo with these hormones, no effect was apparent in vitro even at concentrations up to 10(-6) M. The glucocorticoid dexamethasone caused severe depletion in vivo and in vitro. Thymic androgen and estrogen receptors were determined; in the newborn animals up to 2 wk of age, receptor levels were barely detectable. The possibility of indirect modulation of thymic function by steroids in vivo was investigated by culturing thymic lobes in media containing serum from animals treated with these hormones. Only sera from dexamethasone-injected animals caused changes in cell size, number, viability, or phenotype in the culture system. The mechanism for the previously reported effects of sex steroids on the neonatal thymus therefore remains to be elucidated.

Aging↗