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P. N. JOHN and A. W. ROGERS

SUMMARY

The distribution of radioactivity was studied autoradiographically in the uterus of the ovariectomized rat from 1 to 7 h after the s.c. injection of [3H]progesterone. Luminal and glandular epithelia were less radioactive than stroma or muscle. Grain densities over nuclei were the same as those over cytoplasm in the epithelial tissues and the muscle. Pretreatment with non-radioactive progesterone did not alter the pattern of distribution of radioactivity though in one experiment grain densities were significantly decreased in the pretreated animal; this decrease involved nucleus and cytoplasm in epithelial cells and in the muscle.

The interpretation of grain densities after the administration of [3H]progesterone is complicated by the presence of labelled metabolites. Further experiments were therefore carried out with [3H]megestrol acetate, a progestin which is not significantly metabolized in the uterus or plasma during the first 3 h after injection s.c. At 30 min, 1 and 2 h after [3H]megestrol acetate administration, the stroma and muscle were twice as radioactive as the epithelial tissues of the uterus. Nuclear and cytoplasmic grain densities were the same, both in the luminal epithelium and the stroma. Pretreatment with non-radioactive progesterone decreased the observed grain densities by 20–30% in all tissues including the extracellular spaces of the stroma. Though stromal cells were more radioactive than the surrounding extracellular spaces 1 h after [3H]megestrol acetate, the luminal epithelium was significantly less radioactive.

These results are consistent with the hypothesis that binding sites for progesterone are at relatively low concentrations in the uterus of the ovariectomized rat, that they are widely distributed in the uterus, and that after binding to these sites progestins are distributed between nucleus and cytoplasm in approximately equal proportions.

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K. BROWN-GRANT, P. N. JOHN, and A. W. ROGERS

SUMMARY

Concentration of radioiodide in the cells of the luminal epithelium of the ovariectomized rat after a single injection of progesterone was abolished by 300 μg cycloheximide/100 g body weight given with or 4 h after the progesterone. Actinomycin D at 32 or 80 μ/100 g body weight given with the progesterone did not inhibit the development of the iodide concentrating mechanism. Oestradiol given 16 h before or 2 h after progesterone inhibited the iodide response in a dose-dependent manner. The incorporation of [3H]uridine into histologically fixed material was significantly increased in all tissues of the uterus 1 h after progesterone. The incorporation of [3H]lysine into histologically fixed material was increased significantly in luminal epithelium at 3 h and in all uterine tissues at 7 and 24 h. These findings are consistent with the view that gene transcription may be stimulated by progesterone, resulting in the synthesis of proteins in the different tissues of the uterus. In the luminal epithelium however, progesterone may also stimulate protein synthesis and the development of the iodide response in the absence of gene transcription.

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JG Mabley, JM Cunningham, N John, MA Di Matteo, and IC Green

The aim of this study was to examine if the growth factor, transforming growth factor beta 1 (TGF beta 1), could prevent induction of nitric oxide synthase and cytokine-mediated inhibitory effects in the insulin-containing, clonal beta cell line RINm5F. Treatment of RINm5F cells for 24 h with interleukin-1 beta (IL-1 beta) (100 pM) induced expression of nitric oxide synthase and inhibited glyceraldehyde-stimulated insulin secretion. Combinations of IL-1 beta (100 pM), tumour necrosis factor-alpha (100 pM) and interferon-gamma (100 pM) reduced RINm5F cell viability (determined by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium (MTT) reduction assay) and de novo protein synthesis, as measured by incorporation of radiolabelled amino acids into perchloric acid-precipitable protein. Pretreatment of RINm5F cells with TGF beta 1 (10 pM) for 18 or 24 h, prior to the addition of either IL-1 beta or combined cytokines, prevented cytokine-induced inhibition of insulin secretion, protein synthesis and the loss of cell viability. TGF beta 1 pretreatment inhibited cytokine-induced expression and activity of nitric oxide synthase in RINm5F cells as determined by Western blotting and by cytosolic conversion of radiolabelled arginine into labelled citrulline and nitric oxide. Chemically generated superoxide also induced expression of nitric oxide synthase possibly due to direct activation of the nuclear transcription factor NF kappa B, an effect prevented by both an antioxidant and TGF beta 1 pretreatment. In conclusion, the mechanism of action of TGF beta 1 in blocking cytokine inhibitory effects was by preventing induction of nitric oxide synthase.

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John N Stabley, Rhonda D Prisby, Bradley J Behnke, and Michael D Delp

Bone health and cardiovascular function are compromised in individuals with type 2 diabetes mellitus (T2DM). The purpose of this study was to determine whether skeletal vascular control mechanisms are altered during the progression of T2DM in Zucker diabetic fatty (ZDF) rats. Responses of the principal nutrient artery (PNA) of the femur from obese ZDF rats with prediabetes, short-term diabetes, and long-term diabetes to endothelium-dependent (acetylcholine) and -independent (sodium nitroprusside) vasodilation and potassium chloride, norepinephrine (NE), and a myogenic vasoconstrictor were determined in vitro. Few changes in the PNA vasomotor responses occurred for the prediabetic and short-term diabetic conditions. Endothelium-dependent and -independent vasodilation were reduced, and NE and myogenic vasoconstriction were increased in obese ZDF rats with long-term diabetes relative to lean age-matched controls. Differences in endothelium-dependent vasodilation of the femoral PNA between ZDF rats and controls were abolished by the nitric oxide synthase inhibitor N G-nitro-l-arginine methyl ester. The passive pressure–diameter response of the femoral PNA was also lower across a range of intraluminal pressures with long-term T2DM. Regional bone and marrow perfusion and vascular conductance, measured in vivo using radiolabeled microspheres, were lower in obese ZDF rats with long-term diabetes. These findings indicate that the profound impairment of the bone circulation may contribute to the osteopenia found to occur in long bones during chronic T2DM.

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Melyssa R Bratton, James W Antoon, Bich N Duong, Daniel E Frigo, Syreeta Tilghman, Bridgette M Collins-Burow, Steven Elliott, Yan Tang, Lilia I Melnik, Ling Lai, Jawed Alam, Barbara S Beckman, Steven M Hill, Brian G Rowan, John A McLachlan, and Matthew E Burow

The estrogen receptor α (ERα) is a transcription factor that mediates the biological effects of 17β-estradiol (E2). ERα transcriptional activity is also regulated by cytoplasmic signaling cascades. Here, several Gα protein subunits were tested for their ability to regulate ERα activity. Reporter assays revealed that overexpression of a constitutively active Gαo protein subunit potentiated ERα activity in the absence and presence of E2. Transient transfection of the human breast cancer cell line MCF-7 showed that Gαo augments the transcription of several ERα-regulated genes. Western blots of HEK293T cells transfected with ER±Gαo revealed that Gαo stimulated phosphorylation of ERK 1/2 and subsequently increased the phosphorylation of ERα on serine 118. In summary, our results show that Gαo, through activation of the MAPK pathway, plays a role in the regulation of ERα activity.