To study the role of the epithelial calcium channel transient receptor potential vanilloid type 6 (TRPV6) and the calcium-binding protein calbindin-D9k in intestinal calcium absorption, TRPV6 knockout (KO), calbindin-D9k KO, and TRPV6/calbindin-D(9k) double-KO (DKO) mice were generated. TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice have serum calcium levels similar to those of wild-type (WT) mice ( approximately 10 mg Ca2+/dl). In the TRPV6 KO and the DKO mice, however, there is a 1.8-fold increase in serum PTH levels (P < 0.05 compared with WT). Active intestinal calcium transport was measured using the everted gut sac method. Under low dietary calcium conditions there was a 4.1-, 2.9-, and 3.9-fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D9k KO mice, respectively (n = 8-22 per group; P > 0.1, WT vs. calbindin-D9k KO, and P < 0.05, WT vs. TRPV6 KO on the low-calcium diet). Duodenal calcium transport was increased 2.1-fold in the TRPV6/calbindin-D9k DKO mice fed the low-calcium diet (P < 0.05, WT vs. DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or KO mice. 1,25-Dihydroxyvitamin D3 administration to vitamin D-deficient null mutant and WT mice also resulted in a significant increase in duodenal calcium transport (1.4- to 2.0-fold, P < 0.05 compared with vitamin D-deficient mice). This study provides evidence for the first time using null mutant mice that significant active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D9k, thus challenging the dogma that TRPV6 and calbindin-D9k are essential for vitamin D-induced active intestinal calcium transport.

uptake. The increase in Ca 2ϩ uptake was due to the increase in surface expression of TRPV5. When the thiazide-sensitive Na ϩ -Cl Ϫ cotransporter NCC was coexpressed, the effect of WNK4 on TRPV5 was weakened by NCC in a dose-dependent manner. Although the WNK4 disease-causing mutants E562K, D564A, Q565E, and R1185C retained their ability to upregulate TRPV5, the blocking effect of NCC was further strengthened when wild-type WNK4 was replaced by the Q565E mutant, which causes FHH with hypercalciuria. We conclude that WNK4 positively regulates TRPV5-mediated Ca 2ϩ transport and that the inhibitory effect of NCC on this process may be involved in the pathogenesis of hypercalciuria of FHH caused by gene mutation in WNK4.epithelial calcium channel; CaT1; calcium reabsorption; WNK1; TRPV6 A NOVEL SERINE/THREONINE protein kinase family characterized by the absence of a conserved lysine residue in the kinase domain is formed by WNK [with no lysine (K)] kinases (34). Mutations in WNK1 and WNK4 genes, two members of the family of four WNK genes, which are linked to familial hyperkalemic hypertension (FHH, also known as pseudohypoaldosteronism type II or Gordon's syndrome) (32), highlight the physiological significance of these kinases. Mutations in WNK1 are deletions in the first intron, which result in overexpression of WNK1. Mutations in WNK4 are missense mutations, most of which are clustered in a small region rich in negatively charged amino acids after a coiled-coil stretch following its kinase domain, with the exception of R1185C, which is located close to the carboxy terminus. FHH patients carrying WNK1 or WNK4 mutations have common manifestations, including hyperkalemia, hypertension, mild metabolic acidosis, low renin, and normal glomerular filtration rate (1,22). Despite the aforementioned similarities in the two forms of FHH, there is a significant difference in Ca 2ϩ metabolism between FHH patients carrying WNK1 gene mutations and those carrying WNK4 gene mutations. Affected patients carrying the Q565E mutation in the WNK4 gene exhibit marked hypercalciuria compared with unaffected subjects in the same family (21,22). In contrast, in FHH patients with a WNK1 intronic deletion mutation, urinary Ca 2ϩ content is similar to that of the unaffected subjects (1).WNK4 has been shown to be a multifunctional protein regulating renal ion transport. WNK4 decreases activity of the thiazide-sensitive Na ϩ -Cl Ϫ cotransporter (NCC, also known as TSC and NCCT) (33, 36), the renal outer medullary K ϩ (ROMK) channel (16), and TRPV4, an osmolarity-sensitive Ca 2ϩ -permeable channel (7). On the other hand, WNK4 increases paracellular Cl Ϫ permeability, likely through phosphorylation of claudins (15, 35). The disease-causing Q565E mutant exhibits impaired ability to suppress NCC (36) and enhanced ability to suppress the ROMK channel (16). These findings explain the hypertension and hyperkalemia characteristic of FHH; however, the mechanism underlying hypercalciuria in patients carrying the Q565E mutation of the WNK4 gene is uncle...

Among the multiple organ disorders caused by the severe acute respiratory syndrome coronavirus (SARS-CoV), acute lung failure following atypical pneumonia is the most serious and often fatal event. We hypothesized that two of the hydrophilic structural coronoviral proteins (S and E) would regulate alveolar fluid clearance by decreasing the cell surface expression and activity of amiloride-sensitive epithelial sodium (Na(+)) channels (ENaC), the rate-limiting protein in transepithelial Na(+) vectorial transport across distal lung epithelial cells. Coexpression of either S or E protein with human alpha-, beta-, and gamma-ENaC in Xenopus oocytes led to significant decreases of both amiloride-sensitive Na(+) currents and gamma-ENaC protein levels at their plasma membranes. S and E proteins decreased the rate of ENaC exocytosis and either had no effect (S) or decreased (E) rates of endocytosis. No direct interactions among SARS-CoV E protein with either alpha- or gamma-ENaC were indentified. Instead, the downregulation of ENaC activity by SARS proteins was partially or completely restored by administration of inhibitors of PKCalpha/beta1 and PKCzeta. Consistent with the whole cell data, expression of S and E proteins decreased ENaC single-channel activity in oocytes, and these effects were partially abrogated by PKCalpha/beta1 inhibitors. Finally, transfection of human airway epithelial (H441) cells with SARS E protein decreased whole cell amiloride-sensitive currents. These findings indicate that lung edema in SARS infection may be due at least in part to activation of PKC by SARS proteins, leading to decreasing levels and activity of ENaC at the apical surfaces of lung epithelial cells.

The transient receptor potential cation channel, subfamily V, member 5 (TRPV5) gene, which encodes the Ca 2ϩ channel in the apical membrane of distal convoluted tubule and connecting tubule of the kidney, exhibits an unusually high frequency of nonsynonymous single nucleotide polymorphisms (SNPs) among African Americans. To assess the functional impacts of the nonsynonymous SNP variations in TRPV5, these variants were analyzed with radiotracer 45 (15,16,27). Mice lacking Trpv5 resulted in a 6-to 10-fold increase in urinary Ca 2ϩ excretion, and ultimately in defects in bone mineralization (16).Urinary Ca 2ϩ excretion is an important factor for kidney stone formation and bone health. For instance, urine Ca 2ϩ excretion correlates with bone loss in calcium-stone-forming patients with idiopathic hypercalciuria (3). Interestingly, African Americans exhibit lower urinary Ca 2ϩ excretion than whites (8,25,29,34,35,40,45), and the risk of kidney stones in African American is lower than that in whites (32,36,38). Furthermore, African Americans have higher bone mass (5, 41) and lower incidence of osteoporosis-related fractures than whites (4, 6, 7). The mechanism underlying the lowered urinary Ca 2ϩ in African Americans is not well understood. A lower 25-hydroxyvitamin D concentration in African Americans could result in an increased parathyroid hormone level and in turn renal Ca 2ϩ conservation. However, Braun and colleagues (8) observed significantly lower urinary Ca 2ϩ excretion in adolescent African American girls than white girls in a wide range of controlled Ca 2ϩ intake, whereas the 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and parathyroid hormone values were not significantly different between the two groups. The difference in bone mass and urinary Ca 2ϩ excretion between blacks and whites is not restricted to Americans; a similar finding was reported in South Africa (10). Thus genetic rather than social and environmental factors may play a major role in the superior renal Ca 2ϩ conservation mechanism in African descendents.Because TRPV5 is a key protein that regulates Ca 2ϩ reabsorption, genetic variations of TRPV5 may influence urinary Ca 2ϩ excretion. Interestingly, TRPV5 gene is one of the four contiguous genes, including EPHB6, TRPV6, TRPV5, and KEL, in chromosome 7q34-35 with striking evidence of a recent selective sweep in European Americans based on the analysis on the single nucleotide polymorphisms (SNPs) of over 100 genes from 24 African Americans and 23 European Americans provided by SeattleSNPs program (1, 37). Akey and colleagues (2) further showed that the TRPV6 haplotype, defined by three nonsynonymous SNPs, is nearly fixed in populations outside Africa, suggesting that these variations may confer a selective advantage, e.g., efficiency in Ca 2ϩ absorption from dairy products or resistance to a pathogen, after early humans migrated out of Africa. The TRPV6 variant with the three nonsynonymous SNPs exhibited increased Ca 2ϩ transport ability and may play a role in absorptive hypercalciuria and ki...

Increasing popularity of marine parks as tourist attractions brought with it a number of concerns. Considerable attention has been paid to investigate issues, such as the ethics of keeping marine mammals in captivity, welfare of captive marine mammals, and the educational and conservational abilities of marine parks. Little research has been conducted to explore the public's awareness and opinions of these issues. Public awareness is an important tool to understand the quality of a marine park's products and services. This study was designed to investigate the public's awareness of welfare of captive marine mammals, educational and conservational purposes of marine parks, and to examine public awareness and opinions of Dunlap and Van Liere's New Environmental Paradigm. A total of 120 respondents from St. Catharines, Canada completed either a visitor or a nonvisitor questionnaire. Results indicated that most people were aware of the issues of welfare of captive marine mammals and educational opportunities offered by marine parks, but showed less awareness of the conservational issues. However, results also indicated that respondents were well aware of, and agree with, the concerns expressed in the New Environmental Paradigm.