Non-transferrin-bound iron (NTBI) overtaken by heart cells might be a key cause leading to iron-mediated injury in heart disorders. NTBI uptake by heart cells might be mediated by divalent metal transporter 1 (DMT1). The understanding of the role ofDMT1in heart iron metabolism is fundamental for elucidating the cause resulting in excessive iron in the heart. The study was to evaluate effects of age and dietary iron on DMT1 mRNA expression and protein synthesis in rat heart. DMT1 mRNA expression was determined by RT-PCR and sequence analysis, and DMT1 protein by Western blot analysis. DMT1 mRNAs with or without iron-responsive element (IRE) both were found in rat heart. Expression of two forms ofDMT1mRNAs was the lowest at the age of post-natal day (PND) 7, and then increased with the age, reaching the highest at PND196 (non-IRE form) and PND63 (IRE form), respectively. During different ages, the levels ofDMT1(IRE)mRNAwere higher than those ofDMT1(non-IRE)mRNAand were significantly correlated with the non-heme iron contents in the heart. After fed a high iron for 6 weeks, the rats had a sixfold elevation in heart iron and 22% (non-IRE from) and 40% (IRE from) reduction in DMT1protein compared to the controls. Alow iron diet for 6-weeks caused cardiac hypertrophy and heart iron deficiency and also an increase in levels of two forms of DMT1 proteins. However, iron status had no significant effect on DMT1 (IRE) and DMT1 (non-IRE) mRNAs expression in the heart, although it can significantly influence heart transferrin receptor (TfR) mRNA expression. The results demonstrated that DMT1 mRNAs expression in the heart is age-dependent and that two forms of DMT1 mRNAs both are regulated by iron on the post-transcriptional level only.

AIM: To study the changes of cell proliferation and apoptosis in rat jejunal epithelium at different ages. METHODS: Cell proliferation and apoptosis of the jejunal mucosal and glandulous epithelia from birth to postnatal 12th month were observed using immunocytochemistry (ICC), and TUNEL method. The height of villus, the thickness of muscle layer and the number of goblet cells in jejunal mucosal and glandulous epithelia were measured by BeiHang analytic software and analyzed by STAT. RESULTS: (1) Proliferating cell nuclear antigen (PCNA) positive cells of jejunal glandulous recess were found and increased in number from birth to the postnatal 3rd month. The number of PCNA positive cells peaked in the postnatal 3rd month, and decreased from then on. (2) The number of apoptotic cells also peaked in the postnatal 3rd month, showing a similar trend to that of the PCNA positive cells. (3) The height of jejunal villus increased after birth, peaked in the postnatal 3rd month and decreased from then on. The jejunal muscle layer became thicker in the postnatal 3rd week and the postnatal 12th month. The number of goblet cells of the jejunal mucosal and glandulous epithelia had a linear correlation with age. CONCLUSION: (1) PCNA positive cells are distributed in the jejunal glandulous recess. (2) Apoptotic cell number peaks in the postnatal 3rd month, indicating that cell proliferation and apoptosis are developed with the formation of digestive metabolism as rat grows to maturity. (3) The thickness of jejunal muscle layer increases to a maximum in the postnatal 3rd week, which may be related to the change in diet from milk to solid food. (4) The number of goblet cells increases rapidly in the postnatal 3rd week, probably due to ingestion of solid food.

Ferroportin1 (FP1 or MTP1/IREG1), the product of the SLC40A1 gene, is a main iron export protein in mammals. Its mRNA contains an iron response element (IRE) in its 50 untranslated region, but the way this gene is regulated by iron is still unclear. The existence of FP1 in the brain has been recently confirmed. To better understand the role of this important transmembrane iron exporter in brain iron homeostasis, we investigated the effects of iron and nitric oxide (NO) on FP1 expression and that of a FP1 antibody on iron release in nerve growth factortreated rat PC12 cells.We found that FP1 expression was down-regulated by iron loading but stimulated by iron chelation and treatment with a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In addition, a significant decrease in iron release was found in cells treated with a FP1 antibody. Our findings imply that regulation of FP1 by iron in the cells is at the transcriptional level, rather than by an IRE/IRP-mediated pathway. Based on our results and published data, it is suggested that the transcriptional and translational (IRP/IRE pathway) mechanisms of FP1 expression might both operate in a tissue-specific manner and that FP1 might have a role in iron export from PC12 cells.

AIM: To determine the method of growing small intestinal epithelial cells in short-term primary culture and to investigate the effect of extracellular iron concentration ([Fe3+]) on calcium absorption and the relationship between the rising intracellular calcium concentration ([Ca2+]i) and cell apoptosis in human intestinal epithelial Caco-2 cells. METHODS: Primary culture was used for growing small intestinal epithelial cells. [Ca2+]i was detected by a confocal laser scanning microscope. The changes in [Ca2+]i were represented by fluorescence intensity (FI). The apoptosis was evaluated by flow cytometry. RESULTS: Isolation of epithelial cells and preservation of its three-dimensional integrity were achieved using the digestion technique of a mixture of collagenase XI and dispase I. Purification of the epithelial cells was facilitated by using a simple differential sedimentation method. The results showed that proliferation of normal gut epithelium in vitro was initially dependent upon the maintenance of structural integrity of the tissue. If 0.25% trypsin was used for digestion, the cells were severely damaged and very difficult to stick to the Petri dish for growing. The Fe3+ chelating agent desferrioxamine (100, 200 and 300μmol/L) increased the FI of Caco-2 cells from 27.50±13.18 (control, n=150) to 35.71±13.99 (n=150, P<0.01), 72.19±35.40 (n=150, P<0.01) and 211.34±29.03 (n=150, P<0.01) in a concentration-dependent manner. There was a significant decrease in the FI of Caco-2 cells treated by ferric ammonium citrate (FAC, a Fe3+ donor; 10, 50 and 100 μmol/L). The FI value of Caco-2 cells treated by FAC was 185.85±33.77 (n=150, P<0.01), 122.73±58.47 (n=150, P<0.01), and 53.29±19.82 (n=150, P<0.01), respectively, suggesting that calcium absorption was influenced by [Fe3+]. Calcium ionophore A23187 (0.1, 1.0 and 10μmol/L) increased the FI of Caco-2 cells from 40.45±13.95 (control, n=150) to 45.19±21.95 (n=150, P<0.01), 89.87±43.29 (n=150, P<0.01) and 104.64±51.07 (n=150, P<0.01) in a concentration-dependent manner. The positive apoptotic cell number of the Caco-2 cells after being treated with A23187 increased from 0.32% to 0.69%, 0.90% and 1.10%, indicating that the increase in the positive apoptotic cell number was positively correlated with [Ca2+]i. CONCLUSION: Ca2+ absorbability is increased with the decrease of extracellular iron concentration Fe3+ and hindered with the increase of Fe3+ consistence out of them. Furthermore, increase of [Ca2+]i can induce apoptosis in Caco-2 cells.

AIM: To study the distribution of the constitutive nitric oxide synthase (NOS) in the jejunum of adult rat. METHODS: The distribution of endothelial NOS (eNOS) was detected by immunohistochemistry. Immunofluorescence histochemical dual staining technique were used for studying the distribution of neuronal NOS (nNOS) and eNOS. The dual stained slides were observed under a confocal laser scanning microscope. RESULTS: Positive neuronal NOS (nNOS) and endothelial NOS (eNOS) cells were found to be distributed in lamina propria of villi, and the epithelial cell was not stained. eNOS was mainly located in submucosal vascular endothelia, while nNOS was mainly situated in myenteric plexus. Some cells in the villi had both nNOS and eNOS. More than 80% of the cells were positive for both nNOS and eNOS, the rest cells were positive either for nNOS or for eNOS. CONCLUSION: The two constitutive nitric oxide synthases are distributed differently in the jejunum of rat. nNOS distributed in myenteric plexus is a neurotransmitter in the non-adrenergic non-cholinergic (NANC) inhibitory nerves. eNOS distributed in endothelial and smooth muscle cells of blood vessels plays vasodilator role. eNOS and nNOS are coexpressed in some cells of lamina propria of villi. NO generated by those NOS is very important in the physiological and pathological process of small intestine.