The urothelium is a sensory structure that contributes to mechanosensation in the urinary bladder. Here, we provide evidence for a critical role for the Piezo1 channel, a newly identified mechanosensory molecule, in the mouse bladder urothelium. We performed a systematic analysis of the molecular and functional expression of Piezo1 channels in the urothelium. Immunofluorescence examination demonstrated abundant expression of Piezo1 in the mouse and human urothelium. Urothelial cells isolated from mice exhibited a Piezo1-dependent increase in cytosolic Ca(2+) concentrations in response to mechanical stretch stimuli, leading to potent ATP release; this response was suppressed in Piezo1-knockdown cells. In addition, Piezo1 and TRPV4 distinguished different intensities of mechanical stimulus. Moreover, GsMTx4, an inhibitor of stretch-activated channels, attenuated the Ca(2+) influx into urothelial cells and decreased ATP release from them upon stretch stimulation. These results suggest that Piezo1 senses extension of the bladder urothelium, leading to production of an ATP signal. Thus, inhibition of Piezo1 might provide a promising means of treating bladder dysfunction.

We report the phenotype of mice with targeted disruption of the Trpv6 (Trpv6 KO) epithelial calcium channel. The mice exhibit disordered Ca2+ homeostasis, including defective intestinal Ca2+ absorption, increased urinary Ca2+ excretion, decreased BMD, deficient weight gain, and reduced fertility. Although our Trpv6 KO affects the closely adjacent EphB6 gene, the phenotype reported here is not related to EphB6 dysfunction. Introduction: The mechanisms underlying intestinal Ca2+ absorption are crucial for overall Ca2+ homeostasis, because diet is the only source of all new Ca2+ in the body. Trpv6 encodes a Ca2+-permeable cation channel responsible for vitamin D–dependent intestinal Ca2+ absorption. Trpv6 is expressed in the intestine and also in the skin, placenta, kidney, and exocrine organs. Materials and Methods: To determine the in vivo function of TRPV6, we generated mice with targeted disruption of the Trpv6 (Trpv6 KO) gene. Results: Trpv6 KO mice are viable but exhibit disordered Ca2+ homeostasis, including a 60% decrease in intestinal Ca2+ absorption, deficient weight gain, decreased BMD, and reduced fertility. When kept on a regular (1% Ca2+) diet, Trpv6 KO mice have deficient intestinal Ca2+ absorption, despite elevated levels of serum PTH (3.8-fold) and 1,25-dihydroxyvitamin D (2.4-fold). They also have decreased urinary osmolality and increased Ca2+ excretion. Their serum Ca2+ is normal, but when challenged with a low (0.25%) Ca2+ diet, Trpv6 KO mice fail to further increase serum PTH and vitamin D, ultimately developing hypocalcemia. Trpv6 KO mice have normal urinary deoxypyridinoline excretion, although exhibiting a 9.3% reduction in femoral mineral density at 2 months of age, which is not restored by treatment for 1 month with a high (2%) Ca2+ “rescue” diet. In addition to their deranged Ca2+ homeostasis, the skin of Trpv6 KO mice has fewer and thinner layers of stratum corneum, decreased total Ca2+ content, and loss of the normal Ca2+ gradient. Twenty percent of all Trpv6 KO animals develop alopecia and dermatitis. Conclusions: Trpv6 KO mice exhibit an array of abnormalities in multiple tissues/organs. At least some of these are caused by tissue-specific mechanisms. In addition, the kidneys and bones of Trpv6 KO mice do not respond to their elevated levels of PTH and 1,25-dihydroxyvitamin D. These data indicate that the TRPV6 channel plays an important role in Ca2+ homeostasis and in other tissues not directly involved in this process.

Amino acid homeostasis depends on specific amino acid transport systems, many of which have been characterized at the molecular level. However, the classical System IMINO, defined as the Na⁺-dependent proline transport activity that escapes inhibition by alanine, had not been identified at the molecular level. We report here the functional characteristics and tissue distribution of Sodium/Imino-acid Transporter 1 (SIT1), which exhibits the properties of classical System IMINO. SIT1, the product of the slc6a20 gene, is a member of the SLC6 Na⁺- and Cl^–-dependent neurotransmitter transporter family whose function has remained unknown. When expressed in Xenopus oocytes, rat SIT1 mediated the uptake of imino acids such as proline (K_0.5 ∼ 0.2 mm) and pipecolate, as well as N-methylated amino acids (e.g. MeAIB, sarcosine). SIT1-mediated proline transport was pH-independent and insensitive to inhibition by alanine or lysine. Proline transport was Na⁺-dependent, Cl^–-stimulated, and voltage-dependent. Li⁺, but not H⁺, could substitute for Na⁺. Human SIT1 also functioned as a Na⁺-dependent proline transporter. Rat SIT1 mRNA was expressed in epithelial cells of duodenum, jejunum, ileum, stomach, cecum, colon, and kidney proximal tubule S 3 segments. SIT1 mRNA was also expressed in the choroid plexus, microglia, and meninges of the brain and in the ovary. Previous reports have documented the marked urinary hyperexcretion of proline in newborn rodents and man. We found that SIT1 was dramatically up-regulated in the kidneys of 3-day-old mice, accounting for the maturation of proline reabsorption in the mouse. The human slc6a20 gene coding SIT1 is an appropriate target for investigation of hereditary forms of iminoaciduria in man.

Maternal–fetal calcium (Ca2+) transport is crucial for fetal Ca2+ homeostasis and bone mineralization. In this study, the physiological significance of the transient receptor potential, vanilloid 6 (TRPV6) Ca2+ channel in maternal–fetal Ca2+ transport was investigated using Trpv6 knockout mice. The Ca2+ concentration in fetal blood and amniotic fluid was significantly lower in Trpv6 knockout fetuses than in wildtypes. The transport activity of radioactive Ca2+ (45Ca) from mother to fetuses was 40% lower in Trpv6 knockout fetuses than in wildtypes. The ash weight was also lower in Trpv6 knockout fetuses compared with wildtype fetuses. TRPV6 mRNA and protein were mainly localized in intraplacental yolk sac and the visceral layer of extraplacental yolk sac, which are thought to be the places for maternal–fetal Ca2+ transport in mice. These expression sites were co-localized with calbindin D9K in the yolk sac. In wildtype mice, placental TRPV6 mRNA increased 14-fold during the last 4 days of gestation, which coincides with fetal bone mineralization. These results provide the first in vivo evidence that TRPV6 is involved in maternal–fetal Ca2+ transport. We propose that TRPV6 functions as a Ca2+ entry pathway, which is critical for fetal Ca2+ homeostasis.