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Abstract
We investigated the effects of testosterone on FSH secretion from male rat anterior pituitary cells in culture at the single cell level. Anterior pituitary cells cultured with or without 10 ng/ml testosterone for 72 h were mono-dispersed and subjected to cell immunoblot assays for FSH. Cell blots specific for FSH were quantified by means of a microscopic image analyzer. The number of FSH-secreting cells detected as immunoreactive cell blots on the transfer membrane represented 4·1% of total pituitary cells applied on the membrane. The amount of FSH secreted by single cells varied from <20 to >8 000 fg/cell/h. The number of FSH-secreting cells was not changed by the addition of 10 ng/ml testosterone into the culture medium. Testosterone administration increased the mean FSH secretion by 64% after 3 h incubation, resulting in a shift to the right in the frequency distribution of FSH secretion from single cells. The total amount of FSH, namely the sum of FSH secreted by each FSH-secreting cell, was increased by 92% by the addition of testosterone. However, mean amounts of FSH secretion by the top ten cells of the largest secretor subgroup (>5 pg/cell/3 h) were not different between control and testosterone-treated groups.
The present study analyzed, for the first time, FSH secretion from rat anterior pituitary cells at the single cell level. The results suggest that stimulation by testosterone of FSH secretion in vitro is not due to an increase in the number of FSH-secreting cells but to an increase in FSH secretion from each cell.
Journal of Endocrinology (1996) 148, 427–433
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ABSTRACT
The regulation of thyroglobulin (Tg) and its specific mRNA by interluekin-1 (IL-1) in cultured human thyrocytes was investigated. Specific binding of 125I-labelled IL-1 on thyrocytes was confirmed by solid-phase binding assay. Thyrocytes dispersed from Graves' thyroid tissues were incubated with TSH with or without recombinant human IL-1. TSH stimulated Tg release from cultured human thyrocytes in a dose- and time-dependent manner. Both IL-1α and β inhibited TSH-induced Tg release at concentrations ranging from 0·01 to 10 U/ml. The suppressive activities of IL-1α and β were similar. They did not alter the basal level of Tg release. Unstimulated human thyrocytes did not contain any detectable Tg mRNA, but TSH-stimulated thyrocytes expressed a single species of Tg mRNA (8·5 kb). Both IL-lα and β inhibited TSH-induced Tg mRNA in a dose-responsive manner. IL-1 (10 U/ml) caused maximal suppression of TSH-induced Tg mRNA to nearly basal levels. In contrast, the γ-actin mRNA hybridization signal was not altered in control or treated cells. Furthermore, IL-1 stimulated [3H]thymidine uptake into thyrocyte DNA. These results demonstrate that IL-1 directly inhibits TSH-induced Tg gene expression and provide further support for a functional role of IL-1 as a local modulator of thyroid hormone synthesis.
Journal of Endocrinology (1989) 122, 177–183
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The Otsuka Long-Evans Tokushima fatty (OLETF) rat is a new spontaneous non-insulin-dependent diabetes mellitus (NIDDM) model rat strain developed in Tokushima, Japan. After 18 weeks of age, decreases of 45% and 40% respectively in insulin- and phorbol ester-stimulated [3H]2-deoxyglucose (DOG) uptake were observed, compared with those in Long-Evans Tokushima (LETO) rats (control). Insulin-specific binding and 95 kDa autophosphorylation of insulin receptor in OLETF rats were not different from those in LETO rats. Insulin-induced diacylglycerol (DG) production and Mono Q column-purified protein kinase C (PKC) translocation in adipocytes of OLETF rats were decreased compared with those of LETO rats. Insulin-induced PKC beta translocation from cytosol to membrane was also decreased in adipocytes of OLETF rats. Increases of the PKC beta I, beta II, epsilon and zeta isoforms in membranes of OLETF rats were markedly smaller than those of LETO rats. Analysis of mRNA levels of PKC isoforms in adipocytes of OLETF rats showed decreases of basal level and insulin-induced delayed responses of PKC beta I, beta II, epsilon and zeta mRNA in OLETF rats. On the other hand, insulin- or phorbol ester-induced phosphatidylinositol 3-kinase (PI 3-kinase) activation was decreased in adipocytes of OLETF rats compared with those of LETO rats. These results suggest that insulin resistance in OLETF rats, a spontaneous NIDDM model rat, may be associated with deterioration of insulin-induced DG-PKC signaling and subsequent decrease in PI 3-kinase activation.
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Abstract
We have recently provided evidence for the desensitization of oxytocin receptors in human myometrial cells. In the present study, we have investigated the possible mechanisms by which oxytocin (OT) might regulate OT receptor density. The steady state level of OT binding in cultured myometrial cells was 220 × 103 binding sites/cell, but this was time-dependently reduced to 27 × 103 sites/cell by exposure to OT for up to 20 h. Similarly, OT exposure decreased the binding of OT to cell membranes. In contrast, Western blotting data showed that the total amount of OT receptor protein was not affected by OT treatment for up to 48 h. Flow cytometry experiments demonstrated that OT receptors are not internalized during prolonged exposure of the cells to OT. However, RNase protection assays and Northern analysis showed that OT receptor mRNA was reduced by OT treatment to reach a new low steady state level with a time course similar to that of the disappearance of cell surface OT binding sites. Possible mechanisms involved in mRNA down-regulation include transcriptional suppression and destabilization of mRNA by RNA binding proteins.
Journal of Endocrinology (1997) 154, 7–18
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Search for other papers by E T Kimura in
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Abstract
While the multifunctional proteins of the transforming growth factor-β (TGF-β) family have a potent antiproliferative effect on thyroid follicular cell growth, increased expression of TGF-β in proliferating thyroid cells and in thyroid tumours has recently been described, suggesting a secondary counter-regulatory role of these proteins. We have studied further this apparent paradox in vitro using FRTL-5 cells, 5 continuous cell strains from feline multinodular goitres (MNG) and 29 primary cultures prepared from human MNG.
While dose dependent inhibition of FRTL-5 cell growth was confirmed, a variable fraction of these cells was naturally resistant towards TGF-β1, thus explaining the large interassay variability of growth inhibition (36 to 98% within 2 days, n=19). After 40 days of continuous exposure, FRTL-5 cells became fully refractory towards TGF-β1 inhibition, due to the selective growth of naturally resistant subclones, as demonstrated for example by microscopic observation of three-dimensionally growing collagen-embedded cell clusters. The refractoriness could still be demonstrated even after several cell passages. In addition, 2 out of 5 feline thyroid cell strains obtained from feline MNG and 18 out of 29 primary cultures from human MNG showed a high degree of refractoriness towards TGF-β.
We conclude that constitutively TGF-β resistant cells may occur in thyroid glands and that persistent TGF-β refractoriness may secondarily be acquired. Resistant cells may escape regular growth control mechanisms and hence may contribute to the notorious heterogeneity of thyroid growth and to nodular transformation.
Journal of Endocrinology (1996) 149, 485–496
