PubMed Health⌕ Search

Biomedical subjects

W C Hymer

Publications and source records attributed to W C Hymer.

At least 55 records · Page 3Linked to original sources
PubMed1986

Characterization of a substance in rat serum which modulates prolactin release in vitro.

A prolactin (PRL) inhibitory substance(s) present in rat serum has previously been shown to inhibit the release of PRL from rat pituitary cells in vitro (Hymer and Signorella, 1981). In this study, addition of rat serum to rat pituitary cells cultured in serum-free medium inhibited the release of PRL in dose-dependent manner. Neither the form of the released PRL molecule, nor its biological or immunological activity, was altered by rat serum treatment. Serum prepared from clotted blood previously incubated at 37 degrees C always contained more PRL inhibitory activity than serum from blood incubated at 4 degrees C; whole blood serum, but not plasma-derived serum, contained inhibitory activity. The inhibitor therefore comes from a formed element in the blood. Results of several experiments indicated that buffy coat cells, adherent blood cells and peritoneal cells released the inhibitor, but platelets and RBCs did not; it is suggested that a macrophage/monocyte is probably the cellular source of the PRL inhibitor. Gel permeation high-performance liquid chromatography (HPLC) of rat serum indicated that the inhibitory activity was associated with high molecular weight material, i.e. 115,000-215,000. The PRL inhibitory substance was trypsin sensitive, not extractable with butanol/diisopropyl ether, precipitated at 40% saturated ammonium sulfate, associated with material having an isoelectric point of 4.96 +/- 0.26, and not adsorbed with protein-A. These characteristics suggest the proteinaceous nature of the inhibitory substance. The inhibitor did not suppress PRL release until 9 h after addition to cultured cells, but decreased PRL synthesis was evident within 4 h of treatment. The inability of the dopamine antagonist, haloperidol, to block the inhibitory effect of rat serum indicated that the material does not exert its effect through the dopamine receptor.

Animals↗
PubMed1986

Age-related alterations in the number and function of pituitary lactotropic cells from intact and ovariectomized rats.

We have sought to determine whether the reported age-related increase in in vivo and in vitro PRL secretion in intact and/or postpubertally ovariectomized female rats results from increased number and/or function of lactotropic cells Immunocytochemical staining with antirat-PRL serum, goat antirabbit serum, and peroxidase-antiperoxidase using H2O2 and 4-chloro-1-naphthol or 3'3'-diaminobenzidine hydrochloride as substrates was used to compare the percentages of lactotropes in primary suspensions of heterogeneous adenohypophyseal cells from groups of intact and 3-day ovariectomized mature (6 to 7 month) Wistar rats at estrus (E) or diestrus1-2 (D) with those from the corresponding old (22 to 24 month) rats in constant estrus (CE) or constant diestrus (CD). For both intact and ovariectomized old vs. mature rats, there were small (30-35%) but significant (P less than 0.005) increases in lactotrope number. Lactotrope number did not differ (P greater than 0.05) between E vs. D rats, or CE vs. CD rats, either before or after ovariectomy; moreover, ovariectomy per se did not alter (P greater than 0.05) lactotrope number in any of the groups. Electron microscopic examination of lactotropic cells derived from ovariectomized CE rats revealed ultrastructural changes compatible with estrogenic hyperstimulation; such alterations were not observed in cells from the corresponding E rats. Basal and 17 beta-estradiol (10(-10) M)-stimulated in vitro PRL secretion was measured for 4 days in primary cultures from each of the above groups of rats. After correction for lactotrope number, PRL secretion was greater (P less than 0.01) from cells of intact and ovariectomized CD vs. D rats, whereas 17 beta-estradiol-stimulated (P less than 0.001) but not basal (P greater than 0.05) PRL secretion was greater from the corresponding CE vs. E rats. There was no effect (P greater than 0.05) of reproductive status or of short term ovariectomy on in vitro PRL secretion. These data suggest that aging in the female rat is associated with alterations in both the number and function of pituitary lactotropic cells.

Aging↗
PubMed1986

Flow cytometric analysis and sorting of live male rat anterior pituitary cell types by forward angle and perpendicular light scatter.

Light scatter patterns produced by living cells in the flow cytometer are known to provide useful information with regard to their size and internal structure. The purpose of this study was to determine if light scatter signals produced by live male rat anterior pituitary cells could be used as markers to aid in the identification and separation of different hormone-containing cell types. The typical light scatter pattern (4 X 10(5) cells/sample X 15 min) had three ridges in the forward angle light scatter (FALS) perpendicular light scatter (PLS) bivariate cell distribution. FALS signals could be correlated with the size of different cell types and PLS signals with their content of cytoplasmic secretory granules. Agranular cells dominated the low PLS ridge while moderately granulated PRL cells and heavily granulated GH cells dominated the medium and high PLS ridges, respectively. These light scatter patterns were reproducible both within and between different cell suspensions. Inclusion of dopamine in the pituitary gland dissociation medium, a treatment known to increase intracellular PRL contents of mammotrophs, increased the intensity of the PLS signals of a large population of cells, presumably PRL cells. Pituitary cells prepared from different aged male rats also showed changes in light scatter. Cell sorting on the basis of FALS-PLS signals established the relationship between cell type and light scatter pattern.

Age Factors↗
PubMed1986

Flow cytometric analysis and sorting of live female rat anterior pituitary cell types by forward angle and perpendicular light scatter: effect of 17 beta-estradiol.

Administration of physiological concentrations of 17 beta-estradiol (E2) to ovariectomized rats resulted in changes in the forward angle light scatter (FALS) and perpendicular light scatter (PLS) signals of anterior pituitary lobe cells. One day after steroid treatment some cells showed increases in PLS signals to an intermediate ridge position lying between the two ridges seen for cells prepared from oil-treated ovariectomized rats. Over the first 5 days of E2 treatment, the major effect was a progressive increase in the FALS signal of cells in this intermediate ridge position. Increases in PLS signals continued over the 12-day treatment period. Since the results of a cell sorting experiment showed that PRL cells predominated in the intermediate scatter ridge, it was concluded that E2 increases both size (FALS) and secretory granule content (PLS) of mammotrophs. Cells in the low and high scatter regions were unaffected by E2. Those in the low scatter region were shown, by both electron microscopy and light microscopic immunochemistry, to be enriched in agranular, unstained endothelial-follicular cells. The high-scatter region contained predominantly GH cells. Administration of E2 to pituitary cells cultured in serum-free or serum-containing media for 1-12 days had no effect on the FALS-PLS bivariate distribution. However, the light scatter ridges produced from cells in culture were compressed into a single ridge of narrow PLS and broad FALS signal intensity with increasing time in culture.

Adrenocorticotropic Hormone↗
PubMed1985

Flow cytometric immunofluorescence of rat anterior pituitary cells.

We have developed a flow cytometric immunofluorescence technique for the quantification of growth hormone (GH), prolactin (PRL), and luteinizing hormone (LH) producing cells. The procedure requires about 24 hours and can objectively count 50,000 cells in about 3 minutes. It is based on indirect-immunofluorescence (fluorescein) of intracellular hormone using an EPICS V cell sorter. The fluorescein distributions are gated on DNA content (propidium iodide) to eliminate counting cell clumps. Cells from the same suspensions were stained immunocytochemically and counted microscopically (1,000-2,000 cells/sample). Immunofluorescence and immunocytochemistry correlated to within a few percent for GH and PRL cells. Cell suspensions from adult males and females with or without castration and a diethylstilbestrol (DES)-induced primary pituitary tumor were used to test the method. A major finding of this study was the objective identification of two populations of PRL producing cells, i.e., lightly and intensely stained cells. On the other hand, the fluorescence distribution of PRL cells from DES-induced pituitary tumors did not fall into two distinct populations but, rather, represented a broad continuum. This method should prove useful in studying the dynamics of pituitary cell populations under various physiological and pharmacological conditions.

Animals↗
PubMed1984

Alteration by prolactin of surface charge and membrane fluidity of rat 13762 mammary ascites tumor cells.

Intraperitoneal injection of ovine prolactin (100 micrograms/d) in Fischer 344 rats bearing transplantable 13762 mammary ascites tumor (MAT) cells modifies the surface charge density and membrane fluidity of the tumor cells. In each of five experiments the mean electrophoretic mobility (epm) of MAT cells taken from prolactin-treated rats was significantly lower than that of cells from nonhormone-treated controls. Prolactin concentrations were increased in vivo by (a) direct intraperitoneal injection of ovine prolactin; (b) subcutaneous implantation of diethylstilbestrol-containing silastic capsules to produce pituitary prolactin secreting tumors; or (c) a single subcutaneous injection of polyestradiol phosphate, a long-acting estrogen. In an effort to establish that the prolactin effect was a direct one, two in vivo protocols were used: (a) MAT cells were coincubated with anterior pituitary halves obtained from nontumor-bearing littermates; or (b) rat or ovine prolactin was added to serum-free culture media containing MAT cells. In both protocols, the epm of the prolactin-treated cells was significantly lower. The isoelectric focusing pH of whole cells was increased by prolactin treatment from 4.93 to 5.12, consistent with a reduction in the number of surface carboxyl groups. The fluidity of membranes of treated cells was drastically increased, as measured by spin-label probe rotation rates. These combined results imply that the hormone exerts its effect by stimulating events in the cell that lead to a reduction of the average density of carboxylic acid residues on the tumor cell surface.

Animals↗
PubMed1984

An enzyme-linked immunosorbent assay for rat prolactin.

A sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) for rat prolactin was developed using reagents from the National Institute of Arthritis, Diabetes, Digestive Diseases and Kidney. In this assay soluble prolactin and prolactin adsorbed to a solid-phase support compete for rabbit anti-prolactin antibody binding sites. Therefore, a high concentration of soluble prolactin in the sample will result in a low concentration of antibody immobilized to the adsorbed prolactin. The immobilized antibody-prolactin complex is detected and quantified using goat anti-rabbit immunoglobulin G covalently conjugated to the enzyme horseradish peroxidase. Assay parameters were optimized by investigating the concentration of reagents and the reaction kinetics in each of the assay steps. The assay can be performed in 24 h. A sensitivity range of 0.06 to 6 ng in the region of 90 to 10% binding was obtained. Near 50% binding (0.6 ng), the intraassay coefficient of variation (CV) was 4.2% and the interassay CV was 7.6%. The correlation between radioimmunoassay and the ELISA was 0.868. Selected applications of the assay are described. The assay should prove a useful alternative to the radioimmunoassay in those instances where steps involving the use of 125I become limiting, for example, iodination facility and gamma counter availability or prolonged reagent storage.

Animals↗
PubMed1984

Electrophoretic separation of kidney and pituitary cells on STS-8.

A Continuous Flow Electrophoresis System (CFES) was used on Space Shuttle flight STS-8 to separate specific secretory cells from suspensions of cultured primary human embryonic kidney cells and rat pituitary cells. The objectives were to isolate the subfractions of kidney cells that produce the largest amounts of urokinase (plasminogen activator), and to isolate the subfractions of rat pituitary cells that secrete growth hormone, prolactin, and other hormones. Kidney cells were separated into more than 32 fractions in each of two electrophoretic runs. Electrophoretic mobility distributions in flight experiments were spread more than the ground controls. Multiple assay methods confirmed that all cultured kidney cell fractions produced some urokinase, and five to six fractions produced significantly more urokinase than the other fractions. Several fractions also produced tissue plasminogen activator. The pituitary cells were separated into 48 fractions in each of the two electrophoretic runs, and the amounts of growth hormone (GH) and prolactin (PRL) released into the medium for each cell fraction were determined. Cell fractions were grouped into eight mobility classes and immunocytochemically assayed for the presence of GH, PRL, ACTH, LH, TSH, and FSH. The patterns of hormone distribution indicate that the specialized cells producing GH and PRL are isolatable due to the differences in electrophoretic mobilities.

Animals↗
PubMed1984

Effects of estrogen-induced hyperprolactinemia on endocrine and sexual functions in adult male rats.

Chronic estrogen treatment can lead to development of prolactin (PRL) secreting pituitary tumors. We have tested the ability of diethylstilbestrol (DES) to produce persistent hyperprolactinemia (hyperPRL) in adult male rats and examined the effects of this treatment on hypothalamic-pituitary-testicular function, adenohypophyseal structure, copulatory behavior and fertility. Silastic capsules containing approximately 5 mg DES were subcutaneously implanted into adult male CDF (F-344)/CrlBR rats and removed 15 or 20 weeks later. Extreme hyperPRL, as well as suppression of plasma LH and FSH levels, persisted after DES capsules were removed. In contrast, plasma testosterone levels increased rapidly after removal of DES capsules and reached normal levels within 4-6 weeks. Copulatory behavior was assessed on two occasions between 7 and 14 weeks after removal of the DES capsules and was found to be suppressed in DES-treated rats, as evidenced by significant increases in latencies to mount, to intromit and to ejaculate. Moreover, when the animals were placed with normal females, the interval until conception was significantly greater in DES-treated than in control males. In spite of these differences in copulatory behavior, 10 of 11 DES-treated males were fertile. At autopsy, 44 weeks after capsule implantation (i.e. 24 or 29 weeks after capsule removal), DES-treated rats had marked enlargement of the anterior pituitary, increased weights of the lateral prostate and the adrenals, increased levels of testicular hCG-binding sites, reduced concentration of dopamine and norepinephrine in the median eminence and increased concentration of LHRH in the preoptic area.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗
PubMed1984

Effect of pituitary hollow fiber units and thyroid supplementation on growth in the little mouse.

Hollow fiber units containing allogeneic pituitary cells were implanted intracranially into heterozygous (lit/+) and homozygous, mutant (lit/lit) C57BL/6J "little" weanling mice. Over the 48 days of the experiment, heterozygous mice with pituitary cell implants had a lower percentage weight gain than control mice. Homozygous, mutant mice with cell implants, however, made significant weight gains over mutant controls. Long bone lengths were lower, and organ and carcass weights were higher, in heterozygous mice receiving pituitary cell implants than in control mice, but corresponding measurements in mutant mice with and without implants were not significantly different. Supplementation of the diet with thyroid powder increased the percentage weight gain during the latter half of the 48-day period in both genotypes with and without implanted cells. Thyroid-supplemented mutant mice with pituitary cell implants had significantly higher organ and carcass weights than other mutant groups. The little mouse may serve as a model for pituitary studies and for the treatment of isolated growth hormone deficiency type 1 in man.

Animals↗
PubMed1983

A study of cell electrophoresis as a means of purifying growth hormone secreting cells.

Growth hormone secreting cells of the rat anterior pituitary are heavily laden with granules of growth hormone and can be partially purified on the basis of their resulting high density. Two methods of preparative cell electrophoresis were investigated as methods of enhancing the purification of growth hormone producing cells: density gradient electrophoresis and continuous flow electrophoresis. Both methods provided a two- to four-fold enrichment in growth hormone production per cell relative to that achieved by previous methods. Measurements of electrophoretic mobilities by two analytical methods, microscopic electrophoresis and laser-tracking electrophoresis, revealed very little distinction between unpurified anterior pituitary cell suspensions and somatotroph-enriched cell suspensions. Predictions calculated on the basis of analytical electrophoretic data are consistent with the hypothesis that sedimentation plays a significant role in both types of preparative electrophoresis and the electrophoretic mobility of the growth hormone secreting subpopulation of cells remains unknown.

Animals↗
PubMed1983

Characterization of estrogen-induced adenohypophyseal tumors in the Fischer 344 rat.

Pituitary tumors were induced in F344 female rats by chronic treatment with diethylstilbestrol (DES, 8-10 mg) implanted subcutaneously in silastic capsules. Over a range of 1-150 days of DES treatment, pairs of control and DES-treated rats were sacrificed, and their pituitaries dissociated enzymatically into single-cell preparations. The cell populations were examined regarding total cell recovery correlated with gland weight, intracellular prolactin (PRL) content and subsequent release in primary culture, immunocytochemical PRL staining, density and/or size alterations via separation on Ficoll-Hypaque and by unit gravity sedimentation, and cell cycle analysis, after acriflavine DNA staining, by laser flow cytometry. Total cell yields from DES-treated pituitaries increased from 1.3 times control yields at 8 days of treatment to 58.9 times control values by day 150. Intracellular PRL content ranged from 1.9 to 9.4 times control levels, and PRL release in vitro was significantly and consistently higher than controls, after at least 8 days of DES exposure. Beyond 8 days of DES exposure, the immunochemically PRL-positive proportion of cells increased to over 50% of the total population. Increased density and/or size and PRL content were indicated for the majority of the PRL cell population in both types of separation protocols. All these effects of DES were more pronounced among previously ovariectomized animals. The data extend the findings of other investigators, further establishing the DES-induced tumor as a model for study of PRL cellular control mechanisms.

Animals↗
PubMed1981

Feedback effects of mammary gland tumors on the host pituitary prolactin cell.

Prolactin (PRL) cells in pituitary glands of F-344 female rats bearing transplantable mammary gland adenocarcinomas (13762, 3230) were smaller, contained less intracellular hormone, and released less hormone in culture relative to non tumor-bearing littermates. In some instances removal of the tumor by surgery or chemotherapy (PAM) resulted in restoration of function in vitro. On the other hand, PRL cells from pituitaries of breast cancer patients were often hypertrophied. Since these patients had mastectomies prior to hypophysectomy, the data are consistent with the hypothesis that the tumor exerts negative feedback at the pituitary or hypothalamic level. In support of this hypothesis we have discovered a potent PIF in rat serum. In addition, an experimental protocol involving a) encapsulation of pituitary cells in Amicon hollow fibers, b) their implantation in ectopic sites and c) their subsequent in vitro culture yields data which suggest that the brain of the tumor bearing rat suppresses PRL release. It is suggested that decreased PRL cell function in animals bearing certain mammary tumors ultimately favors metastatic activity of the tumor cell.

Animals↗
PubMed1981

Rat serum inhibits prolactin release from rat pituitary mammotrophs in culture.

Rat pituitary cells maintained in culture for 3 days in alpha-MEM supplemented with 17% horse serum produce and release 4-8X the quantity of prolactin (PRL) originally contained in the cells at the time of plating. Addition of small (200 microliter) quantities of rat serum repeatedly suppressed PRL release by approximately 50%. Inhibition was related to dose of rat serum. Other studies showed (a) that the cells could recover from serum-induced inhibition, (b) that the effect was independent of the age of the pituitary and/or serum donor, (c) that GH release was unaffected by rat serum addition, (d) that the effect could not be attributed to proteases in the serum preparations and (e) that addition of T3 to the culture medium prevented the inhibitory response. The inhibitory material(s) in rat serum is stable on freezing; is non-dialyzable; is heat-labile; is stable over 3 days incubation at 37 degrees C; and is associated with molecules between 85 000 and 146 000 dalton. Inhibitory activity was generated upon incubating whole clotted blood at 37 degrees C for 23 h. It is suggested that the inhibitory material might (a) be released from platelets during clotting or (b) be generated from an inactive precursor molecule cleaved by proteases released during the clotting process.

Aging↗
PubMed1981

Pituitary hollow fiber units in vivo and in vitro.

A new technique is described for the study of mammalian pituitary cells in vivo and in vitro. The method involves encapsulation of freshly trypsinized rat, sheep or human pituitary cells in XM-50 Amicon hollow fibers followed by their intracranial implantation into hypophysectomized rats. These pituitary fiber units promoted recipient growth for 3 weeks before weight gains plateaued. Body composition analyses showed that significant quantities of protein, fat and ash accounted for the weight gain. Morphological study of the capsule contents 7-39 days postimplantation revealed the presence of intact somatotrophs and corticotrophs. The hollow fibers may provide an immunologically privileged site by virtue of the fact that the 50,000 dalton pores making up the lumen surface permit pituitary hormones to diffuse from the capsule, but theoretically do not permit immunoglobulin molecules to penetrate the capsule. Growth of hypox rats receiving capsules containing allogeneic rat pituitary cells, sheep cells or pieces of human postmortem pituitary support this concept. Furthermore, rats implanted with human PRL adenoma cells had detectable quantities of circulating hPRL 100 days postimplantation. It is suggested that the pituitary hollow fiber units function when they come in contact with a ventricular surface of a hypox animal. With these units, it will be possible to study function of the same group of pituitary cells in vitro and in vivo.

Acrylic Resins↗