A B S T R A C T Since many cell types have been shown to respond to extracellular stimulation with a rapid increase in phosphatidylinositol turnover, the present studies were undertaken to determine whether carbohydrate-stimulated insulin secretion from the isolated rat pancreatic islet is accompanied by detectable alterations in the phosphatidylinositol metabolism of this tissue. We have demonstrated that rat pancreatic islets incubated with tritiated myo-inositol rapidly incorporate radioactivity into islet phosphatidylinositol. Incubation of prelabeled islets with elevated concentrations of carbohydrates which stimulate insulin scretion (D-glucose -and D-mannose) results in a decrease in the recovery of lipidbound radioactivity, whereas incubation with carbohydrates which do not stimulate insulin secretion (D-galactose and myo-inositol) has no effect upon the recovery of lipid-bound radioactivity. Within 2 min of exposure of prelabeled islets to elevated concentrations of D-glucose, a decrease in the recovery of [2-'H]myo-inositolderived radioactivity in islet phosphatidylinositol can be demonstrated. When islets prelabeled with [2-8H]myoinositol are perifused with elevated concentrations of D-glucose or D-mannose (but not D-galactose or myoinositol) a rapid and transient increase in the rate of extracellular release of water-soluble radioactivity is observed. Since a significant fraction of the radioactivity released under these conditions is in the form of myoinositol-phosphate, cyclic myo-inositol-1,2-phosphate, and glycerophosphorylmyo-inositol, it is presumably derived This work was presented in part at the Joint Annual
To determine the role of vitamin A in fetal islet development, beta- and alpha-cell mass, apoptosis, and alpha- and beta-cell replication were measured in rats using a model of marginal vitamin A deficiency. Female rats before and during pregnancy and their offspring postweaning were fed a diet containing retinol as retinyl palmitate at a low marginal (LM, 0.25 mg/kg diet) or a sufficient (SUFF, 4.0 mg/kg diet) level. Fetal islet size, replication, apoptosis, and offspring glucose tolerance were examined. Both beta-cell area and number per islet were reduced approximately 50% in fetuses from dams fed an LM vitamin A diet compared with those from dams fed the SUFF vitamin A diet. The alpha-cell area and number per fetal islet were not affected by vitamin A deficiency. Apoptosis was not increased. The percentage of newly replicated beta-cells in the LM fetal pancreas was 42% less than that of SUFF offspring, but alpha-cell replication was not affected. To determine whether this decrease in beta-cell area affected adult glucose tolerance and insulin secretion, 65-d-old offspring were subject to glucose tolerance tests. LM rats had a 55% lower plasma insulin level and a 76% higher serum glucose than SUFF rats. The same pattern could be seen in 35-d-old rats. These findings show that vitamin A deficiency decreases beta-cell mass and this reduction can be attributed to a reduced rate of fetal beta-cell replication in LM offspring. This may contribute to impaired glucose tolerance later in adult life.
Regulation of intracellular Ca2+ in breast cancer may be important in modulating cell proliferation, differentiation, apoptosis, and cytotoxicity, as well as contributing to mechanisms of action of anticancer agents. One of these agents, the steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is intimately involved in maintaining cellular Ca2+ homeostasis. The purpose of this study was to investigate Ca2+ regulatory pathways in breast cancer cells and to determine the role of 1,25(OH)2D3 in modulating these pathways. We examined pathways for Ca2+ entry from the extracellular space and Ca2+ mobilization from intracellular stores in the estrogen-receptor negative human breast cancer cell line BT-20. Fluorescence digital video imaging and Ca2+ indicator fura-2 were employed to measure the concentration of intracellular free Ca2+ ([Ca2+]i) and Ca2+ responses at the single-cell level. We found that BT-20 breast cancer cells expressed nonselective, voltage-insensitive Ca2+ channels (VICC), as indicated by their permeability to Mn2+, response to elevated extracellular Ca2+ with an increase in [Ca2+]i, blockage by La3+ and Ni2+, and response to K+ depolarization with a slight decrease in [Ca2+]i and Ca2+ influx. There was no evidence for voltage- dependent Ca2+ channels in BT-20 cells. Endoplasmic reticulum Ca2+ stores comprised a major intracellular Ca2+ pool, as was evident after application of a Ca2+ ionophore ionomycin in nominally Ca2+-free buffer to the cells with thapsigargin-depleted Ca2+ stores. Thapsigargin depletion of Ca2+ stores did not increase influx of extracellular Ca2+, implying no significant activation of the capacitative Ca2+ entry. 1,25(OH)2D3 did not induce a rapid rise in [Ca2+]i, yet Ca2+ influx through VICC was increased. Treatment with 1,25(OH)2D3 for 4-72 h significantly increased the percentage of cells with a markedly elevated basal [Ca2+]i. Ca2+ response of those cells to thapsigargin was attenuated. Taken together, our findings show that VICC and the thapsigargin-sensitive endoplasmic reticulum Ca2+ stores are the principal pathways for Ca2+ entry and Ca2+ mobilization in the breast cancer cell line used in this study. 1,25(OH)2D3 rapidly increases Ca2+ influx through VICC and after a chronic treatment, depletes endoplasmic reticulum Ca2+ stores. Targeting of Ca2+ signaling mediated by VICC and endoplasmic reticulum Ca2+ stores may represent a novel approach to the treatment and chemoprevention of breast cancer.
Calbindin-D 28k is a 28,000 M r calcium-binding protein initially identified in avian intestine and was the first known target of vitamin D action (1). Calbindin has since been reported in many other tissues including kidney and bone and in tissues that are not primary regulators of serum calcium such as brain and pancreas (2-4). This calcium-binding protein has been conserved during evolution and is regulated by a number of different hormones and factors (3, 4). Calbindin-D 28k , a predominantly cytosolic protein, is a member of a family of high affinity calcium-binding proteins that includes calmodulin, S100 protein, and parvalbumin (5). It has been suggested that the role of calbindin in kidney and intestine is to facilitate transcellular calcium diffusion (6, 7). In brain, calbindin is not vitamin D-dependent and its proposed function is to buffer calcium, resulting in protection against calcium-mediated neurotoxicity (8, 9). In 1979 the discovery in the pancreas of a high affinity receptor for the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), was the first demonstration of a nonclassical target tissue to contain vitamin D receptors (10). Further autoradiographic and immunohistochemical analyses have shown that vitamin D receptors and calbindin-D 28k are both localized in the  cell (11-13). Although these studies and others (14 -17) established a link between the pancreatic  cell and the vitamin D endocrine system and although the importance of calcium in insulin secretion is well known, there is still little information available concerning the exact mechanism whereby vitamin D may affect  cell function. It has been suggested that the role of vitamin D in calcium metabolism of the  cell may involve a genomic effect of 1,25-(OH) 2 D 3 , including the production of calbindin.Although isolated islets and perfused pancreas from vitamin D-deficient animals have previously been used to study the effects of 1,25-(OH) 2 D 3 on  cell function (14 -17), recently we reported that the  cell line R1N1046-38 contains both calbindin and receptors for 1,25-(OH) 2 D 3 and suggested that  cell lines may provide a useful in vitro system for studying the effects of the vitamin D endocrine system on  cell function (18,19). Although interesting data have been generated in numerous studies using RIN cells, the RIN cell line may not be the best model because these cells have little or no response to glucose and the insulin content of these cells is only approximately 0.1% of the insulin content found in the normal  cell.In this study, to understand the role of calbindin-D 28k in the pancreatic  cells, calbindin was transfected and overexpressed in HC and TC cells, pancreatic  cells that secrete insulin in a regulated manner and at levels more comparable with those of normal  cells. Both cell lines are derived from transgenic mice that express the SV40 T-antigen in  cells under the
Immunoreactive calcium binding protein (iCaBP) has been localized in the rat nephron using the unlabeled antibody peroxidase-antiperoxidase immunocytochemical technique. Kidneys from normal young adult, vitamin D-deficient, and 12 day old rats were prepared by either freeze-substitution or 1% glutaraldehyde-Bouin's fixation. CaBP was localized with rabbit antiserum to chicken vitamin D-dependent intestinal CaBP. iCaBP was found specifically in the distal convoluted tubules (DCT); however, not all cells of the DCT were positive. In adult nephrons, a few scattered cells apparently belonging to the collecting tubules were positive. In the neonatal nephrons, there was also localization of iCaBP to the thick limb of Henle, suggesting a difference in the regulation of intracellular calcium during maturation. Proximal tubules, renal corpuscles, macula densa, and thin limbs of Henle gave no specific localization of iCaBP. These results present for the first time the localization of iCaBP in the mammalian nephron both in the neonate and in the young adult.
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