Na+/H+ exchanger 3 inhibitor diminishes hepcidin-enhanced duodenal calcium transport in hemizygous ß-globin knockout thalassemic mice.

Recent investigation has shown that the liver-derived iron-regulating hormone, hepcidin, can potentiate intestinal calcium absorption in hemizygous ß-globin knockout thalassemic (BKO) mice. Since the upregulation of Fe2+ and H+ cotransporter, divalent metal transporter (DMT)-1, has been shown to correlate with thalassemia-induced intestinal calcium absorption impairment, the inhibition of the apical Na+/H+ exchanger (NHE)-3 that is essential for cytoplasmic pH regulation and transepithelial sodium absorption was hypothesized to negatively affect hepcidin action. Herein, the positive effect of hepcidin on the duodenal calcium transport was evaluated using Ussing chamber technique. The results showed that BKO mice had lower absorptive surface area and duodenal calcium transport than wild-type mice. Besides, paracellular transport of zinc in BKO mice was compromised. Hepcidin administration completely restored calcium transport. Since this hepcidin action was totally abolished by inhibitors of the basolateral calcium transporters, Na+/Ca2+ exchanger (NCX1) and plasma membrane Ca2+-ATPase (PMCA1b), the enhanced calcium flux potentially occurred through the transcellular pathway rather than paracellular pathway. Interestingly, the selective NHE3 inhibitor, 100 nM tenapanor, markedly inhibited hepcidin-enhanced calcium transport. Accordingly, hepcidin is one of the promising therapeutic agents for calcium malabsorption in ß-thalassemia. It mainly stimulates the transcellular calcium transport across the duodenal epithelium in an NHE3-dependent manner.

Hepcidin and 1,25(OH)2D3 effectively restore Ca2+ transport in ß-thalassemic mice: reciprocal phenomenon of Fe2+ and Ca2+ absorption.

Previously, ß-thalassemia, an inherited anemic disorder with iron overload caused by loss-of-function mutation of ß-globin gene, has been reported to induce osteopenia and impaired whole body calcium metabolism, but the pathogenesis of aberrant calcium homeostasis remains elusive. Herein, we investigated how ß-thalassemia impaired intestinal calcium absorption and whether it could be restored by administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or hepcidin, the latter of which was the liver-derived antagonist of intestinal iron absorption. The results showed that, in hemizygous ß-globin knockout (BKO) mice, the duodenal calcium transport was lower than that in wild-type littermates, and severity was especially pronounced in female mice. Both active and passive duodenal calcium fluxes in BKO mice were found to be less than those in normal mice. This impaired calcium transport could be restored by 7-day 1,25(OH)2D3 treatment. The 1,25(OH)2D3-induced calcium transport was diminished by inhibitors of calcium transporters, e.g., L-type calcium channel, NCX1, and PMCA1b, as well as vesicular transport inhibitors. Interestingly, the duodenal calcium transport exhibited an inverse correlation with transepithelial iron transport, which was markedly enhanced in thalassemic mice. Thus, 3-day subcutaneous hepcidin injection and acute direct hepcidin exposure in the Ussing chamber were capable of restoring the thalassemia-associated impairment of calcium transport; however, the positive effect of hepcidin on calcium transport was completely blocked by proteasome inhibitors MG132 and bortezomib. In conclusion, both 1,25(OH)2D3 and hepcidin could be used to alleviate the ß-thalassemia-associated impairment of calcium absorption. Therefore, our study has shed light on the development of a treatment strategy to rescue calcium dysregulation in ß-thalassemia.

CFTR induces extracellular acid sensing in Xenopus oocytes which activates endogenous Ca²âº-activated Cl⁻ conductance.

The cystic fibrosis transmembrane conductance regulator (CFTR) produces a cyclic adenosine monophosphate (cAMP)-dependent Cl⁻ conductance of distinct properties that is essential for electrolyte secretion in human epithelial tissues. However, the functional consequences of CFTR expression are multifaceted, encompassing much more than simply supplying a cellular cAMP-regulated Cl⁻ conductance. When we expressed CFTR in Xenopus oocytes, we found that extracellular acidic pH activates a Ca²âº-dependent outwardly rectifying Cl⁻ conductance that does not reflect CFTR activity. The proton-activated Cl⁻ conductance showed biophysical and pharmacological features of a Ca²âº-dependent Cl⁻ conductance, most likely mediated by Xenopus TMEM16A. In contrast to the effects of extracellular acidification, intracellular acidification did not activate an endogenous Cl⁻ conductance. Proton/CFTR-mediated activation of human TMEM16A was also detected in HEK293 cells. The gating mutant G551D-CFTR conferred proton sensitivity, while deltaF508-CFTR enabled proton activation of TMEM16A only in Xenopus oocytes, which, unlike HEK293 cells, allow deltaF508-CFTR to be trafficked to the cell membrane. Activation of TMEM16A by lysophosphatidic acid was enhanced in the presence of CFTR but was additive with activation by extracellular protons. Because expression of CFTR-E1474X did not confer proton sensitivity, we propose that CFTR translocates a proton receptor to the plasma membrane via its PDZ-binding domain.

Electrogenic Na⁺/HCO3⁻ co-transporter-1 is essential for the parathyroid hormone-stimulated intestinal HCO3⁻ secretion.

Parathyroid hormone (PTH) was recently demonstrated to enhance the HCO(3)(-) secretion through the apical anion channel, cystic fibrosis transmembrane conductance regulator (CFTR), but how the HCO(3)(-) entered the epithelial cells was not well understood, in part, due to the lack of specific inhibitors of the basolateral HCO(3)(-) transporters. Moreover, the function of the PTH-stimulated HCO(3)(-) secretion has never been investigated in vivo. Here, we designed three specific pairs of small interfering RNA sequences to simultaneously knockdown three variants of the electrogenic Na(+)/HCO(3)(-) co-transporter (NBCe)-1 in the intestinal epithelium-like Caco-2 monolayer. The results showed that NBCe1 mRNA levels were markedly reduced, and the PTH-induced transepithelial current and voltage changes were diminished after triple knockdown as determined by quantitative real-time PCR and Ussing chamber technique, respectively. An in vivo ligated intestinal loop study further showed that there was an increased fluid secretion, presumably driven by HCO(3)(-) transport, in the ileum, but not in jejunum or colon, of rats administered intravenously with 2 µg/kg body weight of rat PTH 1-34. Therefore, the present results suggested that PTH stimulated intestinal HCO(3)(-) secretion, particularly in the ileum, by inducing the basolateral HCO(3)(-) uptake via NBCe1.