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M R Walters

Publications and source records attributed to M R Walters.

At least 73 records · Page 4Linked to original sources
PubMed1983

Possible significance of new target tissues for 1,25-dihydroxyvitamin D3.

A sensitive sucrose gradient procedure provided evidence for specific 3.6S 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] receptor-like binding components with low affinity for 25(OH)D3 in several reproductive target tissues in the rat, including testis, uterus and probably epididymis. Thus, the presence of 1,25(OH)2D3 receptors in non-vitamin D endocrine tissues is not restricted to the hormone secreting glands. Estrogen-stimulated uterine growth paralleled increased levels of the putative 1,25(OH)2D3 receptors, providing in vivo evidence for the concept of a relationship between these phenomena. However, a similar correlation was not observed in the growing testis (prepubertal vs mature rats). Whether this result stems from receptor localization in a cell type prevalent in the adult or a true dissociation between testis growth and receptor levels is unclear. Finally, significant levels (10.3 +/- 1.0% vs intestinal mucosa) of the putative 1,25(OH)2D3 receptors were found in the heart, an organ in which calcium mediates many specialized functions. Taken collectively, these observations on new target tissues of quite different overall function suggest that 1,25(OH)2D3 and its receptors may play a role in intracellular calcium homeostasis and possibly in regulating specialized intracellular functions of calcium.

Aging↗
PubMed1982

1,25-Dihydroxyvitamin D receptors in an established bone cell line. Correlation with biochemical responses.

A stable cell line derived from mouse bone (cell line MMB-1) has been used for studies of the cellular receptor for 1,25-dihydroxyvitamin D3 in osteoblasts. Previous studies have demonstrated that collagen synthesis in the MMB-1 cell line is specifically inhibited by 1,25-dihydroxyvitamin D3 as well as by other bone-regulating hormones. Incubation of cell homogenates with [3H]1,25-dihydroxyvitamin D3 indicated the presence of a specific receptor which was located primarily in the chromatin fraction. Optimum conditions for the receptor assay required the inclusion of 500 kallikrein-inactivating units of Trasylol/ml and 10 mM NaMoO4. Under these conditions the receptors were stable for 2 h at 23 degrees C and for 24 h at 4 degrees C. Cellular content of receptors was dependent upon the state of confluency of the cells: fully confluent cells contained minimal concentrations of receptors. In cultures of 70-80% confluency, the 1,25-dihydroxyvitamin D3 receptors demonstrated linear Scatchard plots with Kd = 0.4 nM. Peak receptor activity was found at 3.7 S in linear sucrose gradient fractions of cell homogenates. The synthesis of collagen by MMB-1 cells was inhibited by 1,25-dihydroxyvitamin D3 in direct proportion to the concentration of cellular receptors at varying levels of culture confluence. The data indicate that MMB-1 cells contain cytoplasmic/nuclear receptors for 1,25-dihydroxyvitamin D3 which are similar to the receptors found in other target tissues for this hormone and suggest that these receptors are mediators of the effects of 1,25-dihydroxyvitamin D3 on collagen synthesis.

Animals↗
PubMed1982

Effect of vitamin D status on the equilibrium between occupied and unoccupied 1,25-dihydroxyvitamin D intestinal receptors in the chick.

The dynamic equilibrium between in vivo occupied and unoccupied 1,25-dihydroxyvitamin D(3)[1,25(OH)(2)D(3)] receptors of the chick intestinal mucosa was investigated by the exchange assay previously reported [(1980). J. Biol. Chem.255: 9534-9537]. These parameters and their correlation to biological response, i.e., the levels of intestinal vitamin D-dependent calcium binding protein (CaBP), were assessed under different physiological conditions. After a single 1,25(OH)(2)D(3) injection (3.25 nmol), occupied receptor levels increased sharply to a maximum between 1 and 2 h, followed by a rapid decline. A single dose of 1alpha-hydroxy-vitamin D(3) [1alpha(OH)D(3)], an analog that requires 25-hydroxylation for biological activity, resulted in a protracted, albeit lower, response with maximal receptor occupancy at 6 h and half maximal levels 24 h after injection. The intestinal receptor occupancy patterns mirrored the serum 1,25(OH)(2)D(3) levels after either 1,25(OH)(2)D(3) or 1alpha(OH)D(3) treatment. Additionally, time-course (half-life) of blood disappearance of 1,25(OH)(2)D(3) and occupied receptor levels were similar (1.9 and 2.3 h, respectively), suggesting that the amount of occupied 1,25(OH)(2)D(3) receptor is determined by a simple equilibrium between serum 1,25(OH)(2)D(3) and unoccupied receptors. A dose-response study after intramuscular 1,25(OH)(2)D(3) injection yielded a hyperbolic curve with an apparent plateau at 70% receptor occupancy, corresponding to 5 nmol 1,25(OH)(2)D(3) injected. Half-maximal occupancy was reached after a dose of 1 nmol 1,25(OH)(2)D(3), corresponding to 1.5 ng 1,25(OH)(2)D(3)/ml serum. From this value the apparent K(d) in vivo is 3.7 nM, which is similar to that determined in vitro. A 10-fold increase in the 1alpha(OH)D(3) dose resulted in less than a doubling of the levels of serum 1,25(OH)(2)D(3), occupied 1,25(OH)(2)D(3) receptors, or CaBP. Under all experimental conditions, there was a positive correlation between occupied receptor and CaBP levels; however, the slope of the lines depended on the times chosen for the assays due in part to the lag period for CaBP induction and its accumulation within the cell. Conversely, the correlation between serum 1,25-(OH)(2)D(3) levels and occupied receptor levels yielded a single regression line independent of the observation time. Short and long-term treatment with different vitamin D metabolites, estrogen, progesterone, or cortisol did not affect the levels of total intestinal 1,25(OH)(2)D(3) receptor. Under normal physiological conditions, only 10-15% of the total 1,25(OH)(2)D(3) receptor population was occupied by ligand. These studies provide a basis for further investigations of physiological and biochemical parameters of the vitamin D endocrine system and their clinical applications.

Animals↗
PubMed1981

Sugar and amino acid transport from the rat uterine lumen: effects of estrogen and progesterone.

Sugar and amino acid transport and diffusion from the uterine lumen were evaluated in either castrated rats 4 or 24 h after iv injection of 17 beta-estradiol (0.1--10 microgram) or in estrogen-primed castrated rats 12 or 24 h after iv injection of progesterone (25--250 microgram). Uptake phenomena were evaluated by selectively exposing the uterine luminal surface to a Ringer's solution containing [U-14C]D-mannitol and [3H]3-O-methyl-D-glucose or [3H]alpha-aminoisobutyric acid for a 30-sec incubation. Diffusion (D-mannitol uptake) decreased significantly 4 h (but not 24 h) after estrogen and 12 and 24 h after progesterone treatment. [3H]3-O-methyl-D-glucose transport increased significantly both 4 h (120--380%) and 24 h (50--200%) after estrogen. Although [3H]3-O-methyl-D-glucose transport was not significantly changed from that in the vehicle-injected control 12 or 24 h after progesterone, comparison of the transport at 12 h (reduced) to that at 24 h (increased) revealed a significant difference in the responses to progesterone treatment at these two times. [3H]alpha-aminoisobutyric acid transport increased significantly 4 and 24 h after only a pharmacological dose of 17 beta-estradiol and was unchanged 24 h after progesterone administration. In summary, uterine luminal diffusion and transport phenomena are hormone sensitive, with estrogen exerting a more pronounced effect on transport than does progesterone. Thus, through modulation of luminal transport mechanisms, these hormones may regulate substrate levels and, hence, their availability to the developing conceptus during different reproductive states in the rat.

Aminoisobutyric Acids↗
PubMed1981

Glucocorticoid receptors: documentation in the rat uterus.

The question of the presence of specific glucocorticoid receptors in the rat uterus was reassessed. A high speed supernatant obtained from uteri of ovariectomized or intact 45-day old rats was incubated 18 h at 4 c in the presence of [3H]triamcinolone acetonide ([3H]TA) with or without excess dexamethasone, followed by a 10 min exposure to dextran-coated charcoal. A specific uterine glucocorticoid receptor was detected with a Kd=5.1 nM and the number of binding sites equal to 1.1 pmol/uterus. [3H]dexamethasone was observed to underestimate the number of specific receptor sites, probably due to the instability of the dexamethasone-receptor complex during treatment with charcoal. In addition, unlabeled TA was unsatisfactory for determining nonspecific binding because of the cross-reactivity with the progesterone receptor also present in uterine cytosol. The uterine glucocorticoid receptor exhibited the appropriate steroid specificity. Sucrose density gradient analysis of uterine cytosol revealed peaks of dexamethasone-competable [3H]TA binding at 7.2S and 5.75 in low salt and at 4S in 0.4M KCL. Similar components were observed in cytosol and nuclear fractions, respectively, 20 min after in vivo injection of 25 mu Cl [3H]TA. The physical characteristics of the specific glucocorticoid receptor were consistent with those determined for other receptor proteins. The documentation of the presence of glucocorticoid receptors in the rat uterus is important physiologically because of numerous reports of glucocorticoid effects on this reproductive tissue. If receptors could not be demonstrated, extensive studies of the mechanism of these effects would be necessary for a full understanding of uterine endocrinology.

Adrenalectomy↗
PubMed1978

Stoichiometric translocation of the rat uterine progesterone receptor.

In several previous studies nuclear accumulation of the progesterone receptor was significantly lower than the quantity depleted from the cytosol one h after progesterone injection. The results presented herein indicate that this apparent lack of stoichiometry is due to loss of detectable receptor from the nucleus during the nuclear washes and after the assay incubation. This decrease in measured receptor results both from solubilization of the receptor-progesterone complex into the supernatant and from dissociation of [3H] ligand from the receptor. Conversely, no significant quantities of receptor were detected in the mitochondrial/microsomal fraction, preincubation nuclear washes, second ethanol extraction of the nuclear pellet, and soluene digest of the extracted pellet. Thin layer chromatography of the radioactive ligand bound to the nuclear receptor after in vitro exchange confirmed that virtually all the specific binding was due to [3H] progesterone, in spite of a 30% conversion to other metabolites in the incubation fluid.

Animals↗