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W C Hymer

Publications and source records attributed to W C Hymer.

At least 37 records · Page 2Linked to original sources
PubMed1991

Prolactin-induced mitogenesis of lymphocytes from ovariectomized rats.

PRL can induce interleukin-2 (IL-2) receptor expression in splenocytes from ovariectomized (OVX) female rats. In this further study of the effects of PRL on lymphocytes in vitro we found that PRL induced IL-2 receptor expression, IL-2 production, and proliferation of splenocytes and thymocytes from OVX rats. Cells from male rats were not affected. The proliferative response, as measured by [3H]thymidine incorporation, depended on the concentration of PRL and the presence of adherent cells in the culture. After a 48-h incubation with PRL (1 microgram/ml), splenocytes from OVX rats incorporated essentially the same amount of [3H]thymidine as cells incubated with the polyclonal T-cell mitogen Concanavalin-A (ConA). As determined by autoradiography, approximately 40% of the splenocytes responded to PRL or to ConA. After incubation of splenocytes and thymocytes with PRL, bioactive IL-2 was detected in culture medium only from cells of OVX female rats, while incubation with ConA caused IL-2 production by lymphocytes from both male and OVX rats. However, ConA induced IL-2 activity sooner than PRL. Immunofluorescent-flow cytometric analysis revealed time-dependent increases in percentages of IL-2 receptor-positive splenocytes as well as increases in percentages of total T-cells and cells of the CD8 and, to a lesser extent, the CD4 subclass after PRL stimulation.

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PubMed1990

Separation of rat pituitary secretory granules by continuous flow electrophoresis.

The separation of growth hormone-containing cytoplasmic secretory granules from the rat pituitary gland by continuous flow electrophoresis is described. The results are consistent with the hypothesis that granule subpopulations can be separated due to differences in surface charge; these, in turn, may be related to the oligomeric state of the hormone.

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PubMed1990

Prolactin induction of interleukin-2 receptors on rat splenic lymphocytes.

A case for the involvement of PRL in the regulation of the immune system is strong. However, no mechanism by which PRL exerts this regulation has yet been identified. We studied the in vitro effects of PRL on splenocytes from ovariectomized (OVX) rats and discovered that PRL induced the formation of interleukin-2 (IL-2) cell surface receptors. However, PRL did not induce IL-2 secretion. This response, which was dependent on the concentration of PRL, also depended upon the estrogen status of the splenocyte donor; thus, splenocytes from OVX rats or rats in diestrus responded to PRL, whereas those from estrogen-treated OVX rats or rats in estrus did not. We propose that in vivo exposure of PRL, under certain physiological conditions, may prime a pool of splenocytes to express IL-2 cell surface receptors, allowing these cells to be responsive to variations in local concentrations of IL-2.

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PubMed1990

Growth hormone aggregates in the rat adenohypophysis.

Although it has been known for some time that GH aggregates are contained within the rat anterior pituitary gland, the role that they might play in pituitary function is unknown. The present study examines this issue using the technique of Western blotting, which permitted visualization of 11 GH variants with apparent mol wt ranging from 14-88K. Electroelution of the higher mol wt variants from gels followed by their chemical reduction with beta-mercaptoethanol increased GH immunoassayability by about 5-fold. With the blot procedure we found 1) that GH aggregates greater than 44K were associated with a 40,000 x g sedimentable fraction; 2) that GH aggregates were not present in glands from thyroidectomized rats, but were in glands from the thyroidectomized rats injected with T4; 3) that GH aggregates were uniquely associated with a heavily granulated somatotroph subpopulation isolated by density gradient centrifugation; and 4) that high mol wt GH forms were released from the dense somatotrophs in culture, since treatment of the culture medium with beta-mercaptoethanol increased GH immunoassayability by about 5-fold. Taken together, the results show that high mol wt GH aggregates are contained in secretory granules of certain somatotrophs and are also released in aggregate form from these cells in vitro.

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PubMed1990

A new tissue-slicing method for the study of function and position of somatotrophs contained within the male rat pituitary gland.

A tissue-slicing method has been developed for obtaining quantitative information relative to the function and distribution of somatotrophs in the individual pituitary of the male rat. The adenohypophysis was cut along midtransverse, coronal, and sagittal planes, dividing the gland into eight distinct slices. Basal (unstimulated) GH release was measured from sections placed in gyrotory incubation for three consecutive periods (20 min each). Response to rat hypothalamic GH-releasing hormone (rhGHRH) was studied in slices placed in perifusion and in dispersed cells placed in static incubation. Tissues and cells were analyzed by light and electron microscopy and flow cytometry. Cells retained good morphological integrity and viability (greater than 85%) after incubation (1 h) or perifusion (5 h). Somatotrophs were evenly distributed sagittally and rostrocaudally, but were unevenly distributed dorsoventrally; mean values for dorsal and ventral sections were 45.3 +/- 2.5% and 38.9 +/- 1.9%, respectively. Differential basal GH release was observed after the first gyrotory incubation period; values differed by as much as 3-fold. These differences became less apparent with subsequent incubations. Pituitary slices in perifusion showed a differential response to rhGHRH. For some sections GH release increased up to 2-fold; in other sections the response was either negligible or absent. Dissociated cells from sections also showed differential response to rhGHRH; increases ranged from 160-250% above unstimulated values. These studies demonstrate that tissue slicing is a useful method for investigating pituitary cell function in vitro. The correlation between function and position suggests that heterogeneity of somatotrophs is related in part to their location in the gland.

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PubMed1989

Prolactin variants in the rat adenohypophysis.

Prolactin (PRL) heterogeneity in the rat pituitary gland was first reported in 1978. Since then, multiple forms of the hormone in the rat pituitary gland have been reported by other investigators. Some of these variants have been ascribed to posttranslational modifications, while the origin of the others is as yet undefined. In this study, the nature, distribution and origin(s) of PRL size variants in the rat adenohypophysis were defined by Western blotting, Northern blot hybridization analysis and pulse-chase approaches. Thirteen structurally related variants, grouped into species with apparent molecular weights (Mr) 97-34,000, 31,000, 24,000 and 22-17,000, were consistently obtained on the basis of their electrophoretic mobilities in nonreducing polyacrylamide gels. Many of the Mr forms were disulphide-linked aggregates. Northern blot analysis suggested that the 24,000 monomeric PRL was encoded by the 0.9 kb mRNA species, while the 45,000 variant could be encoded by the 1.9 kb mRNA. Four PRL variants in the Mr range of 25-46,000 were synthesized concurrently by the mammotrophs in culture. Newly synthesized high Mr variants were also released into the medium and could be detected as early as 1 min after synthesis. Collectively these results suggest that structurally related PRL variants in the rat pituitary gland are due to a combination of posttranscriptional and posttranslational modification of a single gene product. In addition high Mr variants are preferentially secreted over low Mr forms, probably through an alternative secretory pathway.

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PubMed1988

Expression of the rat growth hormone gene in transfected CV-1 cells.

Expression of the cloned rat growth hormone gene was studied in transfected CV-1 cells. Cell lines expressing rat growth hormone synthesized and secreted the 22 kilodalton form and the 20 kilodalton variant. The results confirm that the 20 kilodalton variant arises from an alternatively spliced mRNA rather than expression from a variant gene in rat. The 5' end of the mRNA from expressing cell lines was located upstream of the normal initiation site in rat. Transcription initiated within a 5' flanking AT rich sequence. Results presented indicate that transcription from normally silent promoter or promoter-like sequences dictated rat growth hormone gene expression in transfected cells. Finally, no hormone was expressed by CV-1 cells transfected with a plasmid containing the rat growth hormone gene in the same transcriptional reading frame as the neomycin resistance gene of pSV2neo indicating an effect of cloning orientation on expression in CV-1 cells.

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PubMed1988

Effects of bromocriptine on prolactin cellular hypertrophy, proliferation and secretory activity in diethylstilbestrol-induced pituitary tumors.

Pituitary tumors induced by chronic diethylstilbestrol (DES) treatment in female F344 rats were treated subsequently with bromocriptine (BC). Effects of BC on separable subpopulations of lactotrophs were examined. Enzymatically dissociated cells from individual pituitaries were assessed regarding total number, relative lactotroph population, intracellular prolactin (PRL) content, PRL release in primary culture, and density alterations by separation in Ficoll-Hypaque or after sedimentation at unit gravity. In addition to the treatment and analysis of in situ tumors, the effects of BC treatment in vitro were assessed, using tumor cells which were first separated on Ficoll-Hypaque. Cell proliferation was assessed by cell cycle analysis, using DNA measurement by laser flow cytometry. BC treatment of tumors reversed the effects of DES on pituitary weight, PRL content and in vitro PRL release. Total cell recovery was not affected by BC, but cell separation showed that BC reduced the number of larger PRL-containing cells. Cell cycle analysis showed a decrease in numbers of cells in S and G2 cycle phases after BC in only one of four experiments. BC had an effect on proliferation in only the upper gradient fractions, containing the smallest cells. Culture of Ficoll-separated tumor cells revealed greater PRL release among lighter/smaller cells. BC treatment inhibited PRL release from both light and dense cells. The results establish that PRL cell hypertrophy, as well as hyperplasia, results from DES treatment. Bromocriptine treatment reverses this hypertrophy concomitant with inhibiting PRL synthesis and release. Reversal of proliferation in tumor cells is not a major effect of bromocriptine treatment.

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PubMed1988

Heterogeneity in mammotrophs prepared from diethylstilbestrol-induced prolactinomas.

Pituitary glands from ovariectomized F-344 rats bearing diethylstilbestrol-containing capsules for 70-100 days were dissociated and separated into three size classes by unit gravity sedimentation. The average percentage of large mammotrophs in fraction I was 86 +/- (SE) 2%, the percentage of intermediate-sized mammotrophs in fraction II was 86 +/- 4%, and the percentage of small mammotrophs in fraction III was 72 +/- 3%. By electron microscopy, cells in fractions I and II contained cytoplasmic secretory granules, whereas those in fraction III were either sparsely granulated or agranular. Cells retained their morphology after culture. Biological (B) and immunological (I) assays of PRL released into the medium after 24-h culture both showed that fraction I and II cells released more hormone than fraction III cells. The B/I ratios of released PRL were consistently greater than 1. Analysis of the separated mammotrophs by flow cytometry indicated major differences in light scatter profiles, both before and after culture. Since these differences have been shown to reflect secretory granule content, changes in perpendicular light scatter after culture were interpreted to indicate that small mammotrophs accumulated granules, whereas some of the large mammotrophs lost granules. Results from a cell blot assay indicated that fraction I and II cells secreted more hormone than fraction III cells. This same rank order was found when live cells were stained and analyzed for surface PRL. However, the reverse plaque assay yielded different information, viz. more plaques were found around fraction III cells than in fraction I or II cells. Finally, PRL contained in alkaline extracts of fraction I, II, and III cells was analyzed by Western blotting after electrophoresis under nondenaturing, denaturing, or denaturing and reducing conditions. Fraction III cells contained a prominent and unique PRL variant that had low mobility in native gels and an apparent mol wt in the range of 10-14K. After 24 h the culture medium from fraction III cells also contained this low mol wt variant. Our data indicate considerable heterogeneity in form and function within the mammotroph population of diethylstilbestrol-induced prolactinomas.

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PubMed1988

Cell bioprocessing in space: applications of analytical cytology.

Cell bioprocessing in space consists of the preparation, cultivation, purification and investigation of cells and their products in the microgravity environment of orbital space flight. Inertial acceleration is used as an independent variable to explore the limits of specific bioprocessing functions, such as cell growth and secretion, gravity-dependent phenomena in cell bioreactors, cell fusion, the influence of thermal convection on processes at cellular dimensions, the electrophoretic separation of cell subpopulations and subcellular particles, and two-phase partitioning of cells, bioparticles, and macromolecules. Analytical cytology techniques are under development for on-orbit application to future cell growth and separation experiments, such as those anticipated in the Space Station era.

Acceleration↗
PubMed1987

Alternative splicing model for the synthesis and secretion of the 20 kilodalton form of rat growth hormone.

The characterization of a 20 kilodalton (20 kD) variant of rat growth hormone is reported. The 20 kD variant from rat pituitary gland extracts was identified on Western immunoblots of polyacrylamide gels. It was also shown that pituitary tissue maintained in culture secretes the 20 kD form. A rat growth hormone cDNA fragment was used as a probe in S1 nuclease mapping experiments of rat pituitary poly (A) mRNA to detect the presence of two growth hormone mRNAs in the rat pituitary gland. The protected mRNAs correspond to the predicted sizes that would encode the 22 kD and 20 kD forms of growth hormone. The site of variation between the mRNAs maps to a potential alternative 3' splice site in the 5' end of exon 3 of the coding sequence. The results support the hypothesis that the 20 kD variant in rat is the product of an mRNA alternatively spliced in exon 3, as is the case for the human growth hormone.

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PubMed1987

An enzyme immunoassay for rat growth hormone: applications to the study of growth hormone variants.

A sensitive and specific competitive enzyme immunoassay (EIA) for rat growth hormone was developed using reagents from the National Institutes of Arthritis, Diabetes, Digestive Diseases and Kidney, Bethesda, Md. In this assay soluble growth hormone and growth hormone adsorbed to a solid-phase support compete for monkey anti-growth hormone antibody binding sites. The immobilized antibody-growth hormone complex is detected and quantified using goat anti-monkey immunoglobin G covalently conjugated to horse radish peroxidase. Therefore a high concentration of soluble growth hormone in the sample will result in low absorbance detection from the colored products of the enzyme reaction. 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 27 hours. A sensitivity range of 0.19 ng to 25 ng in the region of 10 to 90% binding was obtained. Near 50% binding (3 ng) the intraassay coefficient of variation (CV) was 5.54% and the interassay CV was 5.33%. The correlation coefficient (r2) between radioimmunoassay and EIA was 0.956 and followed the curve Y = 0.78X + 1.9. Selected applications were described as follows. Alkaline extracts of pituitary tissue increase 2 fold in GH content after mercaptoethanol treatment. Alkaline extracts of pituitary tissue chromatographed on HPLC molecular sieving columns showed selective enhancement of rat growth hormone content based upon molecular weight. Fractions representing a molecular weight greater than 200 kD were enhanced 6 fold. Fractions whose molecular weight range was 22 kD to 50 kD were enhanced 2 fold. This assay provides a reliable alternative to RIA and offers the major advantage of eliminating radioactive reagents and counting equipment.

Animals↗
PubMed1987

Heterotransplantation of human prostatic tissue.

Pieces of human prostatic tissue (approximately 1 mm3) were encapsulated in XM-50 Amicon hollow fibers and either implanted into the testis of an adult male rat or placed in culture. The protein synthetic capacity of such tissue pieces removed 1-42 days later was monitored by TCA precipitation, SDS-PAGE, and autoradiography. The results showed that tissue pieces retained their functional protein synthetic elements after this procedure. A newly developed solid-phase enzyme immunoassay for human prostatic antigen (PA), described in this report, was used to monitor PA levels in the serum of the recipient host or culture media. In some instances PA was detected 1-2 weeks postimplantation, a result that implies maintenance of functional secretory elements in vivo. Finally, morphology of tissue pieces 1-2 weeks postimplantation sometimes showed the presence of ductal epithelia and stromal elements in distribution patterns typical of those seen in fresh biopsy samples. We conclude that the function and structure of prostatic tissue implanted intrastesticularly compares favorably with that maintained in conventional explant culture. As such, the hollow fiber method offers promise for a new way of monitoring hormonal and/or chemotherapy testing of human prostatic tissue.

Animals↗
PubMed1987

Continuous flow electrophoretic separation of proteins and cells from mammalian tissues.

A new continuous flow electrophoretic separator for cells and macromolecules was built and tested in laboratory experiments and in the microgravity environment of space flight. Buffer flows upward in a 120-cm long flow chamber, which is 6 cm wide X 1.5 mm thick in the laboratory version and 16 cm wide X 3.0 mm thick in the microgravity version. Electrophoretic subpopulations are collected in 197 fractions spanning 16 cm at the upper end of the chamber. The electrode buffer is recirculated through front and back cooling chambers, which are also electrode chambers. Ovalbumin and rat serum albumin were used as test proteins in resolution and throughout tests; resolution of these two proteins at 25% total w/v concentration in microgravity was the same as that found at 0.2% w/v concentration in the laboratory. Band spreading caused by Poiseuille flow and conductance gaps was evaluated using polystyrene microspheres in microgravity, and these phenomena were quantitatively the same in microgravity as in the laboratory. Rat anterior pituitary cells were separated into subpopulations enriched with cells that secrete specific hormones; growth-hormone-secreting cells were found to have high electrophoretic mobility, whereas prolactin-secreting cells were found to have low electrophoretic mobility. Cultured human embryonic kidney cells were separated into several electrophoretic subfractions that produced different plasminogen activators; a medium-high-mobility subpopulation and a medium-low-mobility subpopulation each produced a different molecular form of urokinase, whereas a high- and an intermediate-mobility subpopulation produced tissue plasminogen activator. Canine pancreatic islets of Langerhans cells were separated into subpopulations, which, after reaggregation into pseudoislets, were found to be enriched with cells that secrete specific hormones; insulin-secreting beta cells were found in lowest mobility fractions, whereas glucagon-secreting alpha cells were found in the highest mobility fractions. Results of particle electrophoresis experiments were comparable in microgravity and in the laboratory, since cell densities that overloaded the carrier buffer (resulting in zone sedimentation) were avoided, and a 500-fold increase in protein throughput was achieved without compromising resolution in microgravity.

Animals↗
PubMed1987

Changes in pituitary growth hormone cells prepared from rats flown on Spacelab 3.

Anterior pituitaries from "small" (250 g) and "large" (400 g) rats flown on the 7-day Spacelab 3 mission were pooled and trypsinized into two single-cell suspensions. Compared with ground-based controls, flight cells appeared to contain more intracellular growth hormone (GH) but release less GH over a 6-day culture period. After implantation into hypophysectomized rats, both sets of flight cells released only 50% of the GH compared with the control cells. Glands from large flight rats contained 44% somatotrophs compared with 37% for controls; small animals showed no difference. There were no striking differences in somatotroph ultrastructure between cells in the four groups. Western blot analysis indicated that there were no major differences in immunoactive GH variants. High-performance liquid chromatography fractionation of culture media indicated that small flight cells released much less of a high-molecular weight variant rich in GH bioactivity. The results suggest that GH cells from rats exposed to microgravity may experience secretory dysfunction. The possibility that this occurs directly at the pituitary cell level is discussed.

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PubMed1987

Cell blotting: a new approach to quantify hormone secretion from individual rat pituitary cells.

A new technique is described for quantification of PRL secretion from individual pituitary cells incubated 1-6 h on ImmobilonTM, a high protein binding capacity transfer membrane. After incubation, the membrane is processed according to procedures commonly used in Western blotting. Individual cells and their "zone of secretion" can be seen by light microscopy. Purified hormone, incubated and processed in a similar fashion, is used to generate a PRL standard curve. An OasysTM Image Analysis System is used to measure area and intensity of released hormone. PRL release ranged from 0.1-1.5 pg/cell, depending upon time of incubation and cell donor. Heterogeneity of hormone release from single cells was obvious.

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PubMed1986

Pituitary lactotroph sedimentation profiles and in vitro secretory activity after ablation of the medial basal hypothalamus.

Pituitary cells from adult male rats subjected to chronic (6 and 10 weeks) medial hypothalamic ablation (MHA) were analyzed by unit gravity sedimentation to assess distribution of size and density of lactotrophs, and for subsequent in vitro prolactin (PRL) release in primary culture. Tinctorial staining (Herlant's tetrachrome) showed that initial preparations of cells from MHA rats were small and relatively undifferentiated. MHA cells did not sediment as far into the gradient as did cells from intact control pituitaries. Intracellular PRL content was lower in all gradient fractions of MHA cells. At 6 weeks after surgery, peak recovery of PRL was also in the upper portions of the gradient. In the 10-weeks group, however, peak PRL recovery from MHA cells was in a population that sedimented further, but more restrictedly, in comparison with control cells. At both postsurgical intervals, the majority of tinctorially or immunocytochemically identified lactotrophs from lesioned rats were lower in the gradient, indicating enlarged and denser cells. Relative numbers of lactotrophs (per pituitary) were increased 10 weeks after MHA. In vitro PRL release, over a maximum of 21 days culture, was comparable for cells from MHA rats and intact controls, according to daily per cell secretion rates and "production index" (hormone released/initial hormone content). By comparison, luteinizing hormone (LH) release was suppressed in culture compared to intact controls, and LH was recovered from gradient fractions of smaller cells. The results indicate that chronic removal of hypothalamic influence results in gradual prolactin cell hypertrophy and decreased hormone retention and in relative increase in numbers.(ABSTRACT TRUNCATED AT 250 WORDS)

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