Background/Aims: The effect of daily injections with genistein (naturally occurring phytoestrogen) on intestinal chloride (Cl-) secretion was measured with Ussing chamber short circuit current (Isc, µA/cm2), in C57BL/6J male and female mice, using 600 mg/kg genistein/day (600G), 300 mg/kg genistein/day (300G), 150 mg/kg genistein/day (150G) or genistein-free vehicle control (0G) for 1- or 2-weeks. Methods and Results: Injecting with 600G elicited significant increases in basal Isc in females after 1-week (ñ70 µA/cm2, n=15, p < 0.05) and in males after 2-weeks (ñ80 µA/cm2, n=5, p < 0.05) compared to their 0G counterparts. Chloride-free ringer significantly reduced basal Isc by 65% in 600G males and 72% in 600G females, suggesting that Cl- was the major anion comprising the genistein-stimulated secretion. The forskolin-stimulated (10 µM) Isc was significantly inhibited by the CFTR chloride channel inhibitors, glibenclamide (500 µM) and CFTRinh-172 (100 µM) in 600G males and females, suggesting some contribution by genistein-dependent CFTR-mediated Cl- secretion. We found no associated changes in intestinal morphology, nor change in total CFTR protein with 600G. There was a 5% increase in apical/subapical ratio in 600G males compared to controls (no change in females). Conclusion: These data suggest that male and female mice both exhibit increased Cl- secretion with 600G, however, the mechanisms mediating this are gender-dependent.
The major route for cAMP‐mediated chloride (Cl) secretion across the intestinal epithelial apical membrane is presumed to be via CFTR, a chloride channel widely known to be activated in vitro by genistein. We have shown that daily injections of genistein (600mg/kg body weight, 600G) mediate significant increases in intestinal Cl secretion (Isc) in male mice after 2 weeks and female mice after one week, when compared to control (0G). This study aimed to determine whether genistein‐induced increases in intestinal Cl secretion were attributed to; (a) changes in CFTR protein expression using standard western blot techniques and, (b) changes in intracellular signaling mechanisms using basolateral application of: MDL‐12330A (an adenylate cyclase inhibitor), KT‐5720 (a PKA inhibitor), LY‐294002 (a PI3K inhibitor) and PD‐98059 (a MAPK inhibitor). We found no effect of 1μM KT‐5720, 20μM LY‐294002 or 10μM PD‐98059 on the basal or steady‐state forskolin‐stimulated Isc in 600G male or female mice. MDL‐12330A (10μM)) significantly reduced basal Isc in 600G females from 215.8±12.6 μA/cm2 (n=15) to 157.0±21.5 μA/cm2 (n=8, P<0.05) and decreased steady‐state forskolin‐stimulated Isc from 101.8±17.5 μA/cm2 (n=15), to 35.6±8.3 μA/cm2 (n=8, P<0.05), but was without effect in 600G males. Total CFTR protein expression normalized to total actin protein expression was unchanged in jejunum removed from any groups. These data suggest that the genistein‐mediated increases in intestinal Isc; (a) are not mediated via changes in intestinal CFTR protein expression in male or female mice and, (b) are mediated by adenylate cyclase in female but not male mice. RS and SS were supported by the Midwestern University Summer Fellowship Program. LA was supported by NIH (1R15DK071625‐01A2).
Uncertainty remains over the interpretation of nano-phenomena from various biological and geological environments as living organisms. A characteristic feature of nano-phenomena is their association with a variety of mineral phases. One of the most controversial issues involving nanophenomena is the existence of Nanobacterium sanguineum isolated from human and cow sera [1]. A unique trait associated with this nano-organism is the formation of hydroxyapatite (HA). The nature and formation of the HA phase(s), however, is still not well understood in this system. It is clear that HA is associated with many pathological calcifications, including kidney stones, atherosclerosis, urolithiasis, calcification in hemodialysis patients, dental plaque, and dysfunctional calcification occurring in implanted cardiovascular devices. Although our results do not confirm bacteria controlled or induced mineralization leading to formation of these mineralized nanoforms, the association of HA with serum protein suggests a biogenic origin.[2] To investigate the interaction of nanoforms with MC3T3-E1 cells and the mechanism(s) of calcification, several sets of experiments were conducted under variable cell culture conditions. Extensive variation in morphology occurs between HA in nanoforms obtained from 10% FBS/DMEM ( Fig. 1a) and HA present in MC3T3-E1 cell cultures (Figs. 1b-f). Aggregates of altered nanoforms were present within a membranous organelle (Figure 1c, d). Occasionally, vacuole-like structures containing isolated crystals were observed that are likely a result of the disintegration of nanoforms. The original structure of nanoforms was not preserved either outside or inside of the cells. The distribution and concentration of mineralized structures within the cells differed extensively depending on the composition of the growth medium. The mineralized structures in the experiments conducted with nanoforms grown in EMEM show high concentrations of HA crystals within the cells. Larger well-ordered crystals, showing evidence of newly formed HA, are observed within membrane-bound regions (Fig. 1f). It is likely that the changes in size and morphology of the HA crystals are the result of the dissolution of the fine-grained HA crystals associated with the nanoforms obtained from serum (Fig. 1a) and the neoformation of distinct HA crystals observed in MC3T3-E1 cells (Fig. 1f). The mechanism of transformation and the involvement of MC3T3-E1 cells in this reaction are not understood. It may be that within the membrane-bound phagocytic compartments observed in the MC3T3-E1 cells, different regulatory controls are exerted over crystal growth that otherwise would occur extracellularly, thus resulting in the formation of large crystals. Further study may reveal the significance of these experiments for pathological calcification and bone formation. However, the approach used in this study provides a unique opportunity to understand the mechanisms of HA formation in both normal and pathological calcification. In addition, this process ma...
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