Impaired body calcium metabolism with low bone density and compensatory colonic calcium absorption in cecectomized rats

An earlier study reported that cecal calcium absorption contributes less than 10% of total calcium absorbed by the intestine, although the cecum has the highest calcium transport rate compared with other intestinal segments. Thus, the physiological significance of the cecum pertaining to body calcium metabolism remains elusive. Herein, a 4-wk calcium balance study in cecectomized rats revealed an increase in fecal calcium loss with marked decreases in fractional calcium absorption and urinary calcium excretion only in the early days post-operation, suggesting the presence of a compensatory mechanism to minimize intestinal calcium wasting. Further investigation in cecectomized rats showed that active calcium transport was enhanced in the proximal colon but not in the small intestine, whereas passive calcium transport along the whole intestine was unaltered. Since apical exposure to calcium-sensing receptor (CaSR) agonists similarly increased proximal colonic calcium transport, activation of apical CaSR in colonic epithelial cells could have been involved in this hyperabsorption. Calcium transporter genes, i.e., TRPV6 and calbindin-D(9k), were also upregulated in proximal colonic epithelial cells. Surprisingly, elevated serum parathyroid hormone levels and hyperphosphatemia were evident in cecectomized rats despite normal plasma calcium levels, suggesting that colonic compensation alone might be insufficient to maintain normocalcemia. Thus, massive bone loss occurred in both cortical and trabecular sites, including lumbar vertebrae, femora, and tibiae. The presence of compensatory colonic calcium hyperabsorption with pervasive osteopenia in cecectomized rats therefore corroborates that the cecum is extremely crucial for body calcium homeostasis.     

Chronic metabolic acidosis upregulated claudin mRNA expression in the duodenal enterocytes of female rats

Previous investigations showed that chronic metabolic acidosis (CMA) increased the paracellular permeability of ion and neutral hydrophilic molecules in the duodenum of rats and small intestinal-like cell lines. Since proteins of the claudin family have been known to regulate the paracellular transport in several epithelia, an increase in the paracellular permeability during CMA may have resulted from changes in the pattern of claudin expression. The present study aimed to investigate the expression profile of 22 claudins in the duodenum of female Sprague-Dawley rats given 1.5% NH(4)Cl for 21 days to induce CMA. Arterial blood gas analysis revealed plasma pH values of 7.40 in normal rats and 7.31 in acidotic rats. Blood chemistry showed increases in the total plasma calcium, free-ionized calcium and magnesium, indicating a typical adaptive response of animals to CMA. RT-PCR demonstrated mRNA expressions of claudin-1 to -12, -14, -15, -17 to -20, -22 and -23 in duodenum of normal rats. Claudin-16 was not expressed in normal duodenum, but was strongly expressed in the kidney. Claudin-13 expression was seen only in the cecum, colon, liver and kidney of mice. After 21-day CMA, mRNA expressions of claudin-2, -3, -6, -8, -11, -12, -14, -19 and -22 were significantly enhanced, whereas expressions of other claudins were not changed. Confocal laser-scanning microscopy demonstrated that duodenal enterocytes of normal rats expressed claudin-3 protein on the paracellular membrane. The distribution of claudin-3 protein along the paracellular membrane was markedly increased in CMA, especially near the apical surface. Our results, therefore, provided novel evidence that 21-day CMA markedly altered claudin profile in the duodenum of rats by upregulating specific claudin expression.     

Impaired bone formation and osteopenia in heterozygous β<Superscript>IVSII-654</Superscript> knockin thalassemic mice

β-thalassemia caused by the C→T mutation at nucleotide 654 of the intron 2 (β^IVSII-654) results in aberrant splicing of β-globin RNA, leading to an almost absence of β-globin synthesis. Although trabecular and cortical bone loss was previously reported in β-thalassemic mice with deletion of β-globin gene, the microscopic changes in trabecular structure in β^IVSII-654 thalassemic mice remained elusive. Here, we investigated the macroscopic and microscopic bone changes in 12-week-old β^IVSII-654 knockin thalassemic mice by dual-energy X-ray absorptiometry (DXA) and histomorphometric analysis, respectively. DXA revealed a decrease in bone mineral density in the lumbar vertebrae and tibial metaphysis, but not in the femoral diaphysis, suggesting that β^IVSII-654 thalassemia predominantly led to osteopenia at the trabecular site, but not the cortical site. Further histomorphometric analysis of the tibial secondary spongiosa showed that trabecular bone volume was significantly decreased with the expansion of marrow cavity. Decreases in osteoblast surface, osteoid surface, mineral apposition rate, mineralizing surface, and mineralized volume were also observed. Moreover, trabecular bone resorption was markedly enhanced as indicated by increases in the osteoclast surface and eroded surface. It could be concluded that β^IVSII-654 thalassemia impaired bone formation and enhanced bone resorption, thereby leading to osteopenia especially at the trabecular sites, such as the tibial metaphysis.     

Long-term prolactin exposure differentially stimulated the transcellular and solvent drag-induced calcium transport in the duodenum of ovariectomized rats

Prolactin, having been shown to stimulate transcellular active and solvent drag-induced calcium transport in the duodenum of female rats, was postulated to improve duodenal calcium transport in estrogen-deficient rats. The aim of the present study was, therefore, to demonstrate the effects of long-term prolactin exposure produced by anterior pituitary (AP) transplantation on the duodenal calcium transport in young (9-week-old) and adult (22-week-old) ovariectomized rats. We found that ovariectomy did not alter the transcellular active duodenal calcium transport in young and adult rats fed normal calcium diet (1.0% w/w Ca) but decreased the solvent drag-induced duodenal calcium transport from 75.50 +/- 10.12 to 55.75 +/- 4.77 nmol.hr(-1).cm(-2) (P < 0.05) only in adult rats. Long-term prolactin exposure stimulated the transcellular active calcium transport in young and adult AP-grafted ovariectomized rats fed with normal calcium diet by more than 2-fold from 7.56 +/- 0.79 to 16.54 +/- 2.05 (P < 0.001) and 9.78 +/- 0.72 to 15.99 +/- 1.75 (P < 0.001) nmol.hr(-1).cm(-2), respectively. However, only the solvent drag-induced duodenal calcium transport in young rats was enhanced by prolactin from 95.51 +/- 10.64 to 163.20 +/- 18.03 nmol.hr(-1).cm(-2) (P < 0.001) whereas that in adult rats still showed a decreased flux from 75.50 +/- 10.12 to 47.77 +/- 5.42 nmol.hr(-1).cm(-2) (P < 0.05). Because oral calcium supplement has been widely used to improve calcium balance in estrogen-deficient animals, the effect of a high-calcium diet (2.0% w/w Ca) was also investigated. The results showed that stimulatory action of long-term prolactin on the transcellular active duodenal calcium transport in both young and adult rats was diminished after being fed a high-calcium diet. The same diet also abolished prolactin-enhanced solvent drag-induced duodenal calcium transport in young and further decreased that in adult AP-grafted ovariectomized rats. We concluded that the solvent drag-induced duodenal calcium transport in adult rats was decreased after ovariectomy. Long-term prolactin exposure stimulated the transcellular active duodenal calcium transport in both young and adult rats whereas enhancing the solvent drag-induced duodenal calcium transport only in young rats. Effects of prolactin were abolished by a high-calcium diet.     

Prolactin-stimulated transepithelial calcium transport in duodenum and Caco-2 monolayer are mediated by the phosphoinositide 3-kinase pathway

Prolactin (PRL) has been shown to stimulate intestinal calcium absorption but the mechanism was still unknown. This study aimed to investigate the mechanism and signaling pathway by which PRL enhanced calcium transport in the rat duodenum and Caco-2 monolayer. Both epithelia strongly expressed mRNAs and proteins of PRL receptors. Ussing chamber technique showed that the duodenal active calcium fluxes were increased by PRL in a dose-response manner with the maximal effective dose of 800 ng/ml. This response diminished after exposure to LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor. Caco-2 monolayer gave similar response to PRL with the maximal effective dose of 600 ng/ml. By nullifying the transepithelial potential difference, we showed that the voltage-dependent paracellular calcium transport did not contribute to the PRL-enhanced flux in Caco-2 monolayer. In contrast, the calcium gradient-dependent paracellular transport and calcium permeability were increased by PRL. Effects of PRL on Caco-2 monolayer were abolished by PI3K inhibitors (LY-294002 and wortmannin), but not by inhibitors of MEK (U-0126) or JAK2 (AG-490). To investigate whether the PRL-enhanced paracellular transport was linked to changes in the epithelial charge selectivity, the permeability ratio of sodium and chloride (P(Na)/P(Cl)) was determined. We found that PRL elevated the P(Na)/P(Cl) in both epithelia, and the effects were blocked by PI3K inhibitors. In conclusion, PRL directly and rapidly stimulated the active and passive calcium transport in the rat duodenum and Caco-2 monolayer via the nongenomic PI3K-signaling pathway. This PRL-enhanced paracellular calcium transport could have resulted from altered charge selectivity.     

Duodenal calcium transporter mRNA expression in stressed male rats treated with diazepam, fluoxetine, reboxetine, or venlafaxine

Chronic stress has been reported to decrease bone density and intestinal calcium absorption, but its underlying mechanism remains elusive. Since long-term exposure to glucocorticoids, major stress hormones from adrenal gland, is known to downregulate the mRNA expression of intestinal calcium transporter TRPV6, the present study aimed to demonstrate whether decreases in mRNA expressions of duodenal calcium transporter genes were observed in male rats subjected to restraint stress for 4?weeks. The results from quantitative real-time PCR showed that restraint stress significantly downregulated the mRNA expressions of apical calcium channels (TRPV6 and Ca(v)1.3), cytoplasmic calcium-binding protein (calbindin-D(9k)), and basolateral calcium pump (PMCA(1b)), but not the expression of TRPV5 or NCX1. The mRNA expressions of paracellular genes, ZO-1, occludin, and claudin-3, were not altered by restraint stress. Since several antidepressant or anxiolytic drugs effectively alleviate stress-induced depressive and anxiety symptoms, we further hypothesized that these drugs may also enhance calcium transporter gene expression in stressed rats. As expected, 4-week daily administration of 10?mg/kg fluoxetine, 10?mg/kg reboxetine, or 10?mg/kg venlafaxine differentially increased calcium transporter mRNA expression in stressed rats, whereas 2?mg/kg diazepam had no such effect. It could, therefore, be concluded that 4-week restraint stress downregulated some important calcium transporter mRNA expression in the duodenal epithelial cells of male rats, which could be prevented by oral administration of fluoxetine, reboxetine, and venlafaxine. The present findings may be applied to help alleviate the stress-induced bone loss and osteoporosis by restoring intestinal calcium absorption to provide calcium for bone formation.     

Proliferation and mRNA expression of absorptive villous cell markers and mineral transporters in prolactin‐exposed IEC‐6 intestinal crypt cells

During pregnancy and lactation, prolactin (PRL) enhances intestinal absorption of calcium and other minerals for fetal development and milk production. Although an enhanced absorptive efficiency is believed to mainly result from the upregulation of mineral transporters in the absorptive villous cells, some other possibilities, such as PRL‐enhanced crypt cell proliferation and differentiation to increase the absorptive area, have never been ruled out. Here, we investigated cell proliferation and mRNA expression of mineral absorption‐related genes in the PRL‐exposed IEC‐6 crypt cells. As expected, the cell proliferation was not altered by PRL. Inasmuch as the mRNA expressions of villous cell markers, including dipeptidylpeptidase‐4, lactase and glucose transporter‐5, were not increased, PRL was not likely to enhance crypt cell differentiation into the absorptive villous cells. In contrast to the previous findings in villous cells, PRL was found to downregulate the expression of calbindin‐D_9k, claudin‐3 and occludin in IEC‐6 crypt cells, while having no effect on transient receptor potential vanilloid family channels‐5/6, plasma membrane Ca²⁺‐ATPase (PMCA)‐1b and Na⁺/Ca²⁺ exchanger‐1 expression. In conclusion, IEC‐6 crypt cells did not respond to PRL by increasing proliferation or differentiation into villous cells. The present results thus supported the previous hypothesis that PRL enhanced mineral absorption predominantly by increasing transporter expression and activity in the absorptive villous cells. Copyright © 2012 John Wiley & Sons, Ltd.     

Endogenous prolactin modulated the calcium absorption in the jejunum of suckling rats

Prolactin has been reported to stimulate intestinal calcium absorption in young and mature, but not aging rats. The present study was performed on suckling rats to elucidate the actions of endogenous prolactin on calcium absorption in various intestinal segments. Before measuring the calcium fluxes, 9-day-old rats were administered for 7 days with 0.9% NaCl, s.c. (control), 3 mg/kg bromocriptine, i.p., twice daily to abolish secretion of endogenous prolactin, or bromocriptine plus exogenous 2.5 mg/kg prolactin, s.c. Thereafter, the 16-day-old rats were experimented upon by instilling the 45Ca-containing solution into the intestinal segments. The results showed that, under a physiological condition, the jejunum had the highest rate of calcium absorption compared with other segments (1.4 +/- 0.35 micromol.h-1.cm-1, p < 0.05). The duodenum and ileum also manifested calcium absorption, whereas the colon showed calcium secretion. Lack of endogenous prolactin decreased lumen-to-plasma and net calcium fluxes in jejunum from 2.07 +/- 0.31 to 1.19 +/- 0.12 and 1.40 +/- 0.35 to 0.88 +/- 0.18 micromol.h-1.cm-1 (p < 0.05), respectively, and exogenous prolactin restored the jejunal calcium absorption to the control value. Endogenous prolactin also had an effect on the duodenum but, in this case, exogenous prolactin did not reverse the effect of bromocriptine. However, neither ileal nor colonic calcium fluxes were influenced by prolactin. Because luminal sodium concentration has been demonstrated to affect calcium absorption in mature rats, the effect of varying luminal sodium concentrations on calcium fluxes in suckling rats was evaluated. The jejunum was used due to its highest rate of calcium absorption. After filling the jejunal segments with 124 (control), 80, 40 mmol/L Na+-containing or Na+-free solution, increases in calcium absorption were found to be inversely related to luminal sodium concentrations in both control and bromocriptine-treated rats. The plasma concentration of 45Ca under luminal sodium free condition was also higher than that of the control condition (2.26% +/- 0.07% vs. 2.01% +/- 0.09% administered dose, p < 0.05). However, 3H-mannitol, a marker of the widening of tight junction that was introduced into the lumen, had a stable level in the plasma during an increase in plasma 45Ca, suggesting that the widening of tight junction was not required for enhanced calcium absorption. In conclusion, calcium absorption in suckling rats was of the highest rate in the jejunum where endogenous prolactin modulated calcium absorption without increasing the paracellular transport of mannitol.