We studied the effect of 17f8-estradiol (E) on the proliferation and alkaline phosphatase activity of cultured UMR106 cells, a clonal osteoblastic cell line. Growth rates were reduced and alkaline phosphatase activity was increased in cells incubated for 2 days in medium containing E (10-8 M). In contrast, E had no effect on the growth rates or alkaline phosphatase of a human fibroblastic cell line, S90E. The effect of E was not observed with low cell density or at confluence. 1,25-Dihydroxyvitamin D3 antagonized the response to E. Preincubation of the cells with dexamethasone, a potent inducer of differentiation, reversed the effect of E or 1,25-dihydroxyvitamin D3. These results indicate that cellular and/or extracellular factors such as cell density, the phase of the cell cycle, the state of differentiation, and the presence or absence of other steroids influenced the response of UMR106 cells to E. Serum was removed from the culture medium to minimize the effect of the steroids, growth factors, and nutrients present in serum. A striking stimulation of alkaline phosphatase by E occurred with serum-free conditions. This stimulation was biphasic over an E concentration from 101-2 to 10-8 M, with the peak response at 10-1' M. The action of E on UMR106 cells was metabolite-specific, since the isomer 17a-estradiol produced no effect on proliferation rates or alkaline phosphatase activity. The cyclic AMP response to parathyroid hormone (residues 1-34) was not altered by E treatment of these cells. In contrast, dexamethasone exposure did increase the cyclic AMP response to parathyroid hormone. These results demonstrate a direct effect of E on an osteoblastic cell line. They also raise the possibility that similar or identical actions of E occur in cultured normal osteoblasts.Estrogens influence the skeleton, as evidenced by the loss of bone density in postmenopausal women (1, 2) and the preventative action of exogenous estrogens on the loss of bone density in ovariectomized subjects (3). Estrogen administration also produces changes related to bone and mineral metabolism, such as increased intestinal absorption of calcium (4) and alterations in the circulating calciotropic hormones (4, 5). The mechanism of estrogens' action on the skeleton is still unknown. Most investigators have postulated an indirect pathway because of the apparent absence of estrogen receptors in bone (6-9). We report the results of experiments showing an effect of 17p-estradiol (E) on the UMR106 cell line, a well-characterized osteoblastic cell model (10-12). MATERIALS AND METHODSMaterials. The culture media and additives were obtained from the Tissue Culture Facility of the Lineberger Cancer Research Center, University of North Carolina. The additives included fetal bovine serum, penicillin, streptomycin, insulin, transferrin, selenium, and trypsin, the latter for release of adherent cells. E and 17a-estradiol (17a-E) and dexamethasone were purchased from Sigma. Parathyroid hormone, as the synthetic fragment consisting of amino acid res...

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Background: 5-HT2B receptors are localized within the myenteric nervous system, but their functions on motor/sensory neurons are unclear. To explore the role of these receptors, we further characterized the 5-HT2B receptor antagonist RS-127445 and studied its effects on peristalsis and defecation. Experimental approach: Although reported as a selective 5-HT2B receptor antagonist, any interactions of RS-127445 with 5-HT4 receptors are unknown; this was examined using the recombinant receptor and Biomolecular Interaction Detection technology. Mouse isolated colon was mounted in tissue baths for isometric recording of neuronal contractions evoked by electrical field stimulation (EFS), or under an intraluminal pressure gradient to induce peristalsis; the effects of RS-127445 on EFS-induced and on peristaltic contractions were measured. Faecal output of rats in grid-bottom cages was measured over 3 h following i.p. RS-127445 and separately, validation of the effective doses was achieved by determining the free, unbound fraction of RS-127445 in blood and brain. ), dose-dependently reduced faecal output, reaching significance at 10 and 30 mg·kg -1 (n = 6-11). In blood and brain, >98% of RS-127445 was protein-bound. Conclusions and implications: High-protein binding of RS-127445 indicates that relatively high doses are required for efficacy. The results suggest that 5-HT2B receptors tonically regulate colonic motility.

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