Epithelial Calcium Channel Research Articles

Vitamin D resistance

Vitamin D is a secosteroid of nutritional origin but can also be generated in the skin by ultraviolet light. After two hydroxylations 1,25-(OH)2 vitamin D avidly binds and activates the vitamin D receptor (VDR), a nuclear transcription factor, hereby regulating a large number of genes. The generation of VDR deficient mice has expanded the knowledge on vitamin D from a calcium-regulating hormone to a humoral factor with extensive actions. The effects of the vitamin D system on calcium and bone homeostasis are largely mediated by promoting active intestinal calcium transport via the induction of the epithelial calcium channel TRPV6. Although VDR is redundant in bone, it may regulate the differentiation and function of several bone cells. In skin, VDR expression in keratinocytes is essential in a ligand-independent manner for the maintenance of the normal hair cycle. Therefore, VDR but not vitamin D deficiency results in alopecia. Moreover, 1,25-(OH)2 vitamin D impairs the proliferation not only of keratinocytes but also of many cell types by regulating the expression of cell cycle genes, leading to a G1 cell cycle arrest. In addition, VDR inactivation in mice results in high renin hypertension, cardiac hypertrophy and thrombogenesis. Finally, a dual effect of vitamin D was observed in the immune system where it stimulates the innate immune system while tapering down excessive activation of the acquired immune system. Taken together, the vitamin D endocrine system not only regulates calcium homeostasis but affects several systems mainly by altering gene expression but also by ligand-independent actions.

Hormonal and environmental regulation of epithelial calcium channel in gill of rainbow trout (Oncorhynchus mykiss)

We indirectly tested the idea that the epithelial Ca2+ channel (ECaC) of the trout gill is regulated in an appropriate manner to adjust rates of Ca2+ uptake. This was accomplished by assessing the levels of gill ECaC mRNA and protein in fish exposed to treatments known to increase or decrease Ca2+ uptake capacity. Exposure of trout to soft water ([Ca2+]=20-30 nmol/l) for 5 days (a treatment known to increase Ca2+ uptake capacity) caused a significant increase in ECaC mRNA levels and an increase in ECaC protein expression. The inducement of hypercalcemia by infusing fish with CaCl2 (a treatment known to reduce Ca2+ uptake) was associated with a significant decrease in ECaC mRNA levels, yet protein levels were unaltered. ECaC mRNA and protein expression were increased in fish treated with the hypercalcemic hormone cortisol. Finally, exposure of trout to 48 h of hypercapnia (approximately 7.5 mmHg, a treatment known to increase Ca2+ uptake capacity) elicited an approximately 100-fold increase in the levels of ECaC mRNA and a significant increase in protein expression. Immunocytochemical analysis of the gills from hypercapnic fish suggested a marked increase in the apical expression of ECaC on pavement cells and a subpopulation of mitochondria-rich cells. The results of this study provide evidence that Ca2+ uptake rates are, in part, regulated by the numbers of apical membrane Ca2+ channels that, in turn, modulate the inward flux of Ca2+ into gill epithelial cells.

Characterization of a branchial epithelial calcium channel (ECaC) in freshwater rainbow trout (Oncorhynchus mykiss)

The entry of calcium (Ca2+) through an apical membrane epithelial calcium channel (ECaC) is thought to a key step in piscine branchial Ca2+ uptake. In mammals, ECaC is a member of the transient receptor potential (TRP) gene family of which two sub-families have been identified, TRPV5 and TPRV6. In the present study we have identified a single rainbow trout (Oncorhynchus mykiss) ECaC (rtECaC) that is similar to the mammalian TRPV5 and TRPV6. Phylogenetic analysis of the protein sequence suggests that an ancestral form of the mammalian genes diverged from those in the lower vertebrates prior to the gene duplication event that gave rise to TRPV5 and TRPV6. The putative model for Ca2+ uptake in fish proposes that the mitochondria-rich cell (also termed ionocyte or chloride cell) is the predominant or exclusive site of transcellular Ca2+ movements owing to preferential localisation of ECaC to the apical membrane of these cells. However, the results of real-time PCR performed on enriched gill cell populations as well as immunocytochemistry and in situ hybridisation analysis of enriched cells, cell cultures and whole gill sections strongly suggest that ECaC is not exclusive to mitochondria-rich cells but that it is also found in pavement cells. Not only was ECaC protein localized to areas of the gill normally having few mitochondria-rich cells, but there was also no consistent co-localization of ECaC- and Na+/K+-ATPase-positive (a marker of mitochondria rich cells) cells. Taken together, the results of the present study suggest that although ECaC (mRNA and protein) does exist in trout gill, its cellular distribution is more extensive than previously thought, thus suggesting that Ca2+ uptake may not be restricted to mitochondria-rich cells as was proposed in previous models.

Milk Alkali Syndrome and the Dynamics of Calcium Homeostasis

Intensive treatment with calcium-containing antacids and milk first was used in the early 20th century for the treatment of peptic ulcer disease and sometimes was associated with toxicity, eventually known as the milk alkali syndrome. Despite the introduction of H2 blockers and proton pump inhibitors for the treatment of peptic ulcer disease, the milk alkali syndrome continues to occur but is seen more frequently in older women who are receiving treatment for osteoporosis. The milk alkali syndrome provides a unique opportunity to discuss calcium homeostasis in a setting in which the primary calcium regulatory hormones, parathyroid hormone (PTH) and calcitriol, are not overtly abnormal. A thorough understanding of the pathophysiology of the milk alkali syndrome, including its generation and maintenance, requires knowledge of intestinal calcium absorption, bone influx and efflux of calcium, and renal calcium excretion and also how these processes change with age. In this review, the pathophysiology of the milk alkali syndrome is discussed in light of recent advances in our understanding of calcium homeostasis, particularly the role of the calcium-sensing receptor (CaSR) and epithelial calcium channels that are present in various tissues such as the parathyroid gland, kidney, and intestine. The contributions of alkalosis, per se , to the generation and maintenance of hypercalcemia are discussed in detail. Almost 100 years ago, Sippy (1) developed a calcium-laden milk and antacid regimen for the treatment of peptic ulcer disease. His rationale was to neutralize the hyperacidity that was deemed responsible for peptic ulcer disease. The Sippy regimen was used for the treatment of peptic ulcer disease, a disorder that was most common in middle-aged men, until the 1970s, when nonantacid treatment first was introduced (2–4). The original recommendation by Sippy consisted of the hourly administration of milk and cream together with what became known as …

1,25–Dihydroxyvitamin D and 25–hydroxyvitamin D—mediated regulation of TRPV6 (a putative epithelial calcium channel) mRNA expression in Caco–2 cells

TRPV6 is a member of the vanilloid subfamily of transient receptor potential (TRP) proteins and likely functions as an epithelial calcium channel in calcium-transporting organs, such as the intestine, kidney, and placenta. TRPV6 mRNA expression is strongly regulated by 1,25-dihydroxyvitamin D (1,25VD), the active hormonal form of vitamin D, in intestine and in Caco-2 cells, a human colon cancer cell line. The aim of the present study was to characterise the mode of regulation of the 1,25VD-mediated TRPV6 mRNA expression and to test the effect of the precursor of 1,25VD namely 25 hydroxyvitamin D (25 VD) on TRPV6 mRNA expression in Caco-2 cells. Caco-2 cells were treated in a 2 x 2 format with 1,25VD and the transcriptional inhibitor actinomycin D (AD, 4 microg/ml), and also with translational inhibitor cycloheximide (CHX, 10 microg/ml) after 14 days in culture and TRPV6 mRNA levels were determined using reverse transcription-real time PCR.TRPV6 mRNA half life studies were performed by inhibiting transcription followed by sampling at various time points for TRPV6 mRNA. Varying concentrations of 25 VD were used to test their effect on TRPV6 mRNA in the presence of 5% FBS and also in the absence of serum (but containing insulin-transferrin-selenium mixture) for 24 h. Treatment with 10(-7) M 1,25VD for 8 h resulted in a 60-fold increase in TRPV6 mRNA and this increase could be completely blocked with AD. Treatment with CHX to inhibit de novo protein synthesis did not prevent the initiation of 1,25VD-induced TRPV6 expression, although it did reduce the extent of TRPV6 mRNA accumulation. We found that TRPV6 mRNA half-life was 8 h in Caco-2 cells and was not altered by 1,25VD treatment. Finally, we observed that treatment with 10(-6) M of the pro-hormone 25 VD for 24 h resulted in a significant increase in TRPV6 expression in Caco-2 cells, which is consistent with the presence of 1alpha-hydroxylase (CYP27B1) expression in Caco-2 cells and a possible autocrine vitamin D signaling pathway in colon cells. 1,25 dihydroxyvitamin D regulates TRPV6 expression by a process that requires new mRNA and protein synthesis and the point of regulation lies likely at the transcriptional level especially since vitamin D did not increase the half life of TRPV6 mRNA. In addition, the prohormone form of 1,25 dihydroxyvitamin D, i. e. the 25 hydroxyvitamin D, induced TRPV6 mRNA expression in Caco-2 cells.

Calcium transport in human salivary glands: a proposed model of calcium secretion into saliva

Salivary calcium plays a vital role in bio-mineralization of dental enamel and exposed dentin. In order to elucidate the yet unknown cellular and molecular mechanisms of calcium secretion in human salivary glands the presence of various relevant plasma membrane transport systems for calcium were investigated. Using an RT-PCR approach, expression of the epithelial calcium channel (CaT-Like), the calcium binding protein (calbindin-2), the endoplasmic reticulum pumps (SERCA-2 and -3), and the plasma membrane calcium ATPases (PMCA-1, -2, and -4), were found in parotid and submandibular glands. Immunohistochemistry revealed that CaT-Like is located in the basolateral plasma membrane of acinar cells; while calbindin-2, SERCA-2 and SERCA-3 were found inside the acinar cells; and PMCA-2 was found in the apical membrane and in the secretory canaliculi between the cells. Based on these findings, we propose the following model of calcium secretion in human salivary glands: (1) calcium enters the acinar cell at the basolateral side via calcium channel CaT-Like (calcium influx); (2) intracellular calcium is taken up into the endoplasmic reticulum by SERCA-2 and possibly SERCA3 or bound to calbindin-2 (intracellular calcium pool); and (3) calcium is secreted by PMCAs at the apical plasma membrane (calcium efflux).

Large-Scale in Silico and Microarray-Based Identification of Direct 1,25-Dihydroxyvitamin D3 Target Genes

1alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3] regulates calcium homeostasis and controls cellular differentiation and proliferation. The vitamin D receptor (VDR) is a ligand-regulated transcription factor that recognizes cognate vitamin D response elements (VDREs) formed by direct or everted repeats of PuG(G/T)TCA motifs separated by 3 or 6 bp (DR3 or ER6). Here, we have identified direct 1,25(OH)2D3 target genes by combining 35,000+ gene microarrays and genome-wide screens for consensus DR3 and ER6 elements, and DR3 elements containing single nucleotide substitutions. We find that the effect of a nucleotide substitution on VDR binding in vitro does not predict VDRE function in vivo, because substitutions that disrupted binding in vitro were found in several functional elements. Hu133A microarray analyses, performed with RNA from human SCC25 cells treated with 1,25(OH)2D3 and protein synthesis inhibitor cycloheximide, identified more than 900 regulated genes. VDREs lying within -10 to +5 kb of 5'-ends were assigned to 65% of these genes, and VDR binding was confirmed to several elements in vivo. A screen of the mouse genome identified more than 3000 conserved VDREs, and 158 human genes containing conserved elements were 1,25(OH2)D3-regulated on Hu133A microarrays. These experiments also revealed 16 VDREs in 11 of 12 genes induced more than 10-fold in our previous microarray study, five elements in the human gene encoding the epithelial calcium channel TRPV6, as well as novel 1,25(OH2)D3 target genes implicated in regulation of cell cycle progression. The combined approaches used here thus provide numerous insights into the direct target genes underlying the broad physiological actions of 1,25(OH)2D3.

Diabetes in rat pregnancy alters renal calcium and magnesium reabsorption and bone formation in adult offspring

We tested the hypothesis that diabetes in pregnancy can result in the in-utero reprogramming of renal calcium and magnesium handling and of bone formation in the offspring, which persists into adulthood. Male offspring of streptozotocin-treated diabetic rats (OD rats) and of control non-diabetic animals (OC rats) were investigated as neonates and at 8, 12 and 16 weeks of age. Compared with OC rats, urinary calcium and magnesium output was significantly reduced in OD rats at every age studied; Na+ and K+ outputs were unaffected. The renal expression of proteins involved in the tubular reabsorption of calcium (calcium ATPase, calbindin-D28k and epithelial calcium channel) was increased in OD animals compared with that in OC animals. Additionally, we observed that adult OD rats had lower trabecular and higher cortical femoral bone volumes, explained by deposition of bone on the endosteal surface. These data show that diabetes in pregnancy has profound effects on male offspring in terms of renal tubular calcium and magnesium reabsorption and the normal pattern of bone formation. These effects persist into adulthood. Such long-lasting effects of diabetes on kidney and the skeleton were not suspected and could have important implications for the health of children born to diabetic women.

Characterization of a Madin-Darby canine kidney cell line stably expressing TRPV5

To provide a cell model for studying specifically the regulation of Ca2+ entry by the epithelial calcium channel transient receptor potential-vanilloid-5 (TRPV5), green fluorescent protein (GFP)-tagged TRPV5 was expressed stably in Madin-Darby canine kidney type I (MDCK) cells. The localization of GFP-TRPV5 in this cell line showed an intracellular granular distribution. Ca2+ uptake in GFP-TRPV5-MDCK cells cultured on plastic supports was threefold higher than in non-transfected cells. Moreover, apical Ca2+ uptake in GFP-TRPV5-MDCK cells cultured on permeable supports was eightfold higher than basolateral Ca2+ uptake, indicating that GFP-TRPV5 is expressed predominantly in the apical membrane. Patch-clamp analysis showed the presence of typical electrophysiological features of GFP-TRPV5, such as inwardly rectifying currents, inhibition by divalent cations and Ca2+-dependent inactivation. Moreover, the TRPV5 inhibitor ruthenium red completely inhibited Ca2+ uptake in GFP-TRPV5-MDCK cells, whereas Ca2+ uptake in non-transfected cells was not inhibited. The characterized GFP-TRPV5-MDCK cell line was used to assess the regulation of TRPV5. The protein kinase C activator phorbol 12-myristate 13-acetate and the cAMP-elevating compounds forskolin/3-isobutyl-1-methylxanthine, 8-Br-cAMP and PGE2 stimulated TRPV5 activity in GFP-TRPV5-MDCK cells by 121+/-7, 79+/-5, 55+/-4 and 61+/-7%, respectively. These compounds did not affect Ca2+ uptake in non-transfected cells. In conclusion, the GFP-TRPV5-MDCK cell line provides a model to specifically study the regulation of TRPV5 activity.

The epithelial calcium channels TRPV5 and TRPV6: regulation and implications for disease

The epithelial Ca(2+) channels TRPV5 and TRPV6 represent a new family of Ca(2+) channels that belongs to the superfamily of transient receptor potential channels. TRPV5 and TRPV6 constitute the apical Ca(2+) entry mechanism in active Ca(2+) transport in kidney and intestine. The central role of TRPV5 and TRPV6 in active Ca(2+) (re)absorption makes it a prime target for regulation to maintain Ca(2+) balance. This review covers the hormonal regulation, interaction with accessory proteins and (patho)physiological implications of these epithelial Ca(2+) channels.

Effect of age, vitamin D, and calcium on the regulation of rat intestinal epithelial calcium channels

Transepithelial transport of calcium involves uptake at the apical membrane, movement across the cell, and extrusion at the basolateral membrane. Active vitamin D metabolites regulate the latter two processes by induction of calbindin D and the plasma membrane ATPase (calcium pump), respectively. The expression of calbindin D and the calcium pump declines with age in parallel with transepithelial calcium transport. The apical uptake of calcium is thought to be mediated by the recently cloned calcium channels—CaT1 (or ECaC2, TRPV6) and CaT2 (or ECaC1, TRPV5). The purpose of these studies was to determine whether there were age-related changes in intestinal calcium channel regulation and to identify the dietary factors responsible for their regulation. Young (2 months) and adult (12 months) rats were fed either a high calcium or low calcium diet for 4 weeks. The low calcium diet significantly increased duodenal CaT1 and CaT2 mRNA levels in both age groups, but the levels in the adult were less than half that of the young. The changes in calcium channel expression with age and diet were significantly correlated with duodenal calcium transport and with calbindin D levels. To elucidate the relative roles of serum 1,25(OH) 2D 3 and calcium in the regulation of calcium channel expression, young rats were fed diets containing varying amounts of calcium and vitamin D. Dietary vitamin D or exogenous 1,25(OH) 2D 3 more than doubled CaT1 mRNA levels, and this regulation was independent of dietary or serum calcium. These findings suggest that the apical calcium channels, along with calbindin and the calcium pump, may play a role in intestinal calcium transport and its modulation by age, dietary calcium, and 1,25(OH) 2D 3.

Requirement of PDZ Domains for the Stimulation of the Epithelial Ca2+ Channel TRPV5 by the NHE Regulating Factor NHERF2 and the Serum and Glucocorticoid Inducible Kinase SGK1

Renal calcium reabsorption involves the epithelial calcium channel ECaC1 (TRPV5) which is tightly regulated by 1,25(OH)2D3. As shown recently, TRPV5 is activated by the serum and glucocorticoid inducible kinase SGK1, a kinase transcriptionally upregulated by 1,25(OH)2D3. This stimulatory effect is due to enhanced TRPV5 abundance in the plasma membrane and requires the presence of the scaffold protein NHERF2 (sodium hydrogen exchanger regulating factor 2). The present study aims to define the molecular requirements for the interaction of TRPV5 with SGK1 and NHERF2. Pull-down experiments and overlay assays revealed that the TRPV5 C-tail interacts in a Ca²⁺-independent manner with NHERF2. Deletion of the second but not of the first PDZ domain in NHERF2 abrogates the stimulating effect of SGK1/NHERF2 on TRPV5 protein abundance in the plasma membrane as quantified by chemiluminescence and electrophysiology. Thus, the second PDZ domain in NHERF2 is required for stabilization at or TRPV5 targeting to the plasma membrane. The experiments demonstrate the significance of SGK1 and NHERF2 as TRPV5 modulators which are likely to participate in the regulation of calcium homeostasis by 1,25(OH)2D3.

Functional characterisation and genomic analysis of an epithelial calcium channel (ECaC) from pufferfish, Fugu rubripes

An orthologue to the mammalian epithelial calcium channels, ECaC1 (TRPV5) and ECaC2 (TRPV6), was cloned from gill of pufferfish ( Fugu rubripes) and characterised, demonstrating that this gene predates the evolution of land-living vertebrates. The F. rubripes ECaC ( FrECaC) protein displays all structural features typical for mammalian ECaCs including three ankyrin repeats, six transmembrane domains, and a putative pore region between TM V and TM VI. Functional expression of FrECaC in Madin–Darby canine kidney (MDCK) cells confirmed that the channel mediates Ca 2+ influx. FrECaC was also permeable to Zn 2+ and, to a small extent, to the Fe 2+ ion. Thus, in addition to a role in Ca 2+ uptake FrECaC might serve as a pathway for zinc and iron acquisition. FrECaC mRNA was highly abundant in the gill, but sparsely present in the intestine. Calcium absorption via FrECaC in pufferfish may be subject to the regulation of 1,25(OH) 2D 3, estrogen and progesterone as consensus cis regulatory elements for the respective steroid hormone receptors were found in the upstream regulatory region of the FrECaC gene. FrECaC gene organisation is very conserved when compared with mammalian ECaCs. Only one ECaC gene seems to exist in the F. rubripes genome, and the corresponding protein clusters together with ECaC2 from mammals upon phylogenetic analysis. Thus, the two mammalian ECaC genes may originate from a single ancestral ECaC2 gene in vertebrates appearing early in evolution.

Regulation of gene expression of epithelial calcium channels in intestine and kidney of mice by 1 α,25-dihydroxyvitamin D 3

In wild-type (VDR +/+) mice, ECaC2 expression was confirmed in the intestine and kidney, while ECaC1 expression was exclusively confined to the kidney. Both mRNAs expression of ECaC1 and ECaC2 in the kidney and ECaC2 mRNA expression in the intestine increased time- and dose-dependently in response to 1α,25(OH) 2D 3 injection in VDR +/+ mice, but not in VDR −/− mice. The mRNA levels of ECaC2 in the intestine of VDR −/− mice were remarkably reduced when compared to VDR +/+ mice, while no significant differences were observed in both mRNA levels of ECaC1 and ECaC2 in the kidney between VDR +/+ mice and VDR −/− mice. In the primary renal tubular cells (PRTC) isolated from VDR +/+ mice, both ECaCs mRNA expression increased in response to 1α,25(OH) 2D 3 treatment, but not in the PRTC of VDR −/− mice. PTH increased both ECaCs mRNA expression in the PRTC of VDR +/+ mice. These results suggest that 1α,25(OH) 2D 3 directly modulates the gene expression of ECaC1 and ECaC2 together with PTH in the kidney of mice. 1α,25(OH) 2D 3 also modulates the gene expression of ECaC2 in the intestine of mice, however, further studies are needed to elucidate the direct action of 1α,25(OH) 2D 3 on the expression of ECaC2 in the intestine.

Intestinal calcium transporter genes are upregulated by estrogens and the reproductive cycle through vitamin D receptor-independent mechanisms.

1alpha,25(OH)2-vitamin D strongly regulates the expression of the epithelial calcium channel CaT1. CaT1 expression is reduced in ERKOalpha mice and induced by estrogen treatment, pregnancy, or lactation in VDR WT and KO mice. Estrogens and vitamin D are thus independent potent regulators of the expression of this calcium influx mechanism, which is involved in active intestinal calcium absorption. Active duodenal calcium absorption consists of three major steps: calcium influx into, transfer through, and extrusion out of the enterocyte. These steps are carried out by the calcium transport protein 1 (CaT1), calbindin-D9K, and the plasma membrane calcium ATPase (PMCA1b), respectively. We investigated whether estrogens or hormonal changes during the female reproductive cycle influence the expression of these genes, and if so, whether these effects are vitamin D-vitamin D receptor (VDR) dependent. We evaluated duodenal expression patterns in estrogen receptor (ER)alpha and -beta knockout (KO) mice, as well as in ovariectomized, estrogen-treated, pregnant, and lactating VDR wild-type (WT) and VDR KO mice. Expression of calcium transporter genes was not altered in ERKObeta mice. CaT1 mRNA expression was reduced by 55% in ERKOalpha mice, while the two other calcium transporter genes were not affected. Ovariectomy caused no change in duodenal expression pattern of VDR WT and KO mice, whereas treatment with a pharmacologic dose of estrogens induced CaT1 mRNA expression in VDR WT (4-fold) and KO (8-fold) mice. Pregnancy enhanced CaTI expression equally in VDR WT and KO mice (12-fold). Calbindin-D9K and PMCA1b expression increased to a lesser extent and solely in pregnant VDR WT animals. In lactating VDR WT and KO mice, CaT1 mRNA expression increased 13 times, which was associated with a smaller increase in calbindin-D9K protein content and PMCA1b mRNA expression. Estrogens or hormonal changes during pregnancy or lactation have distinct, vitamin D-independent effects at the genomic level on active duodenal calcium absorption mechanisms, mainly through a major upregulation of the calcium influx channel CaT1. The estrogen effects seem to be mediated solely by ERalpha.

Calcium transporter 1 and epithelial calcium channel messenger ribonucleic acid are differentially regulated by 1,25 dihydroxyvitamin D3 in the intestine and kidney of mice.

We examined the expression of calcium transporter 1 (CaT1) and epithelial calcium channel (ECaC) mRNA in the duodenum and kidney of mice. Intestinal CaT1 mRNA level increased 30-fold at weaning, coincident with the induction of calbindin-D(9k) expression. In contrast, renal CaT1 and ECaC mRNA expression was equal until weaning when ECaC mRNA is induced and CaT1 mRNA levels fall 70%. Long- and short-term adaptation to changes in dietary calcium (Ca) level and 1,25 dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] injection strongly regulated duodenal calbindin D(9k) and CaT1 mRNA. Following a single dose of 1,25(OH)(2)D(3), induction of CaT1 mRNA occurred rapidly (within 3 h, peak at 6 h of 9.6 +/- 0.8-fold) and preceded the induction of intestinal Ca absorption (significantly increased at 6 h, peak at 9 h). Neither renal CaT1 nor ECaC mRNA were strongly regulated by dietary calcium level or 1,25(OH)(2)D(3) injection. Our data indicate that CaT1 and ECaC mRNA levels are differentially regulated by 1,25(OH)(2)D(3) in kidney and intestine and that there may be a specialized role for CaT1 in kidney in fetal and neonatal development. The rapid induction of intestinal CaT1 mRNA expression by 1,25(OH)(2)D(3), and the marked induction at weaning, suggest that CaT1 is critical for 1,25(OH)(2)D(3)-mediated intestinal Ca absorption.

The epithelial calcium channels, TRPV5 & TRPV6: from identification towards regulation

The epithelial calcium channels, TRPV5 and TRPV6, have been extensively studied in epithelial tissues controlling the Ca 2+ homeostasis and exhibit a range of distinctive properties that distinguish them from other TRP channels. This review focuses on the tissue distribution, the functional properties, the architecture and the regulation of the expression and activity of the TRPV5 and TRPV6 channel.

Epithelial calcium channels: from identification to function and regulation.

The epithelial calcium channels TRPV5 and TRPV6 have been studied extensively in the epithelial tissues controlling Ca(2+) homeostasis and exhibit a range of distinctive properties that distinguish them from other transient receptor potential (TRP) channels. These two novel members of the superfamily of TRP channels were cloned from vitamin D-responsive epithelia: kidney, small intestine and placenta, and identified subsequently in tissues like pancreas, bone and prostate. This review addresses the unique properties of these highly Ca(2+)-selective channels and highlights their implications for the process of transepithelial Ca(2+) transport.

Vitamin D Receptor (VDR) Knockout Mice Reveal VDR-Independent Regulation of Intestinal Calcium Absorption and ECaC2 and Calbindin D9k mRNA

To study the role of calbindin D(9k) (CaBP) and epithelial calcium channel ECaC2 in intestinal calcium (Ca) absorption, vitamin D receptor knockout (KO) and wild-type (WT) mice were fed either 0.5% Ca or a 2.0% Ca rescue diet starting at 21 d of age. Ca absorption and parameters involved in this process were measured at 60 or 90 d of age. Compared with WT, KO mice fed the 0.5% Ca diet had higher plasma parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], and lower plasma Ca and insulin-like growth factor-I (IGF-I). Duodenal Ca absorption (% Ca absorbed) in KO mice was reduced 71% relative to WT mice and was associated with 55% lower CaBP mRNA, 47% lower CaBP protein and 95% lower ECaC2 mRNA levels. Compared with WT mice, the percentage of Ca absorbed in KO mice fed the 0.5% Ca diet was inappropriately low for the level of duodenal CaBP. The 2% Ca rescue diet normalized plasma Ca, prevented osteomalacia, increased growth and plasma IGF-I levels, but did not normalize plasma PTH or 1,25(OH)(2)D(3) in KO mice. In addition, the relationship between CaBP protein and the percentage of Ca absorbed was normalized, whereas ECaC2 mRNA fell to near zero. Our data demonstrate that higher CaBP levels do not ensure high rates of duodenal Ca absorption and that transcellular Ca absorption can occur even when ECaC2 gene expression is very low. In addition, our data suggest that the 2% Ca diet promotes a vitamin D receptor-independent anabolic effect on bone formation and calcium absorption, leading to improved calcium balance even in the presence of high PTH levels.

New insights into the mechanisms of vitamin D action.

The biologically active metabolite of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is a secosteroid whose genomic mechanism of action is similar to that of other steroid hormones and is mediated by stereospecific interaction of 1,25(OH)(2)D(3) with the vitamin D receptor (VDR) which heterodimerizes with the retinoid X receptor (RXR). After interaction with the vitamin D response element (VDRE) in the promoter of target genes, transcription proceeds through the interaction of VDR with coactivators and with the transcription machinery. The identification of the steps involved in this process has been a major focus of recent research in the field. However, the functional significance of target proteins as well as the functional significance of proteins involved in the transport and metabolism of vitamin D is also of major importance. Within the past few years much new information has been obtained from studies using knockout and transgenic mice. New insight has been obtained using this technology related to the physiological significance of the vitamin D binding protein (DBP), used to transport vitamin D metabolites, as well as the physiological significance of target proteins including 25-hydroxyvitamin D(3) 24-hydroxylase (24(OH)ase), 25-hydroxyvitamin D(3)-1 alpha-hydroxylase (1 alpha-(OH)ase), VDR, and osteopontin. The crystal structure of the DBP and the ligand binding domain of the VDR have recently been reported, explaining, in part, the unique properties of these proteins. In addition novel 1,25(OH)(2)D(3) target genes have been identified including the epithelial calcium channel, present in the proximal intestine and in the distal nephron. Thus in recent years a number of exciting discoveries have been made that have enhanced our understanding of mechanisms involved in the pleiotropic actions of 1,25(OH)(2)D(3).