Masashi Imai

The sensation of pressure, mechanosensation, in vertebrates remains poorly understood on the molecular level. The ion channel TRPV4 is in the TRP family and is a candidate for a mechanosensitive calcium-permeable channel. It is located in dorsal root ganglia. In the present study, we show that disrupting the Trpv4 gene in mice markedly reduced the sensitivity of the tail to pressure and acidic nociception. The threshold to noxious stimuli and the conduction velocity of myelinated nerve responding to stimuli were also impaired. Activation of unmyelinated nerve was undetected. However, the mouse still retained olfaction, taste sensation, and heat avoidance. The TRPV4 channel expressed in vitro in Chinese hamster ovary cells was opened by low pH, citrate, and inflation but not by heat or capsaicin. These data identify the TRPV4 channel as essential for the normal detection of pressure and as a receptor of the high-threshold mechanosensory complex.

The Ca2+-permeable cation channel TRPV4, which is part of the Trp family located in the circumventricular organs, is activated by cell swelling. To investigate the role of TRPV4 in osmotic sensation, we disrupted the TRPV4 gene in mice and examined the effect on osmotic metabolism. Disruption of the mouse TRPV4 gene did not influence either water intake behavior or serum osmolality. Short-term salt ingestion, however, seemed to impair the transient free water clearance. The level of serum arginine vasopressin (AVP) of TRPV4-/- mice was not significantly changed under normal conditions but was significantly increased under stimulated conditions. Incubation of brain slices with graded hyperosmolality suggested an exaggerated response of AVP secretion in TRPV4-/- mice. Thus TRPV4 in the brain may transmit a negative signal to AVP secretion similar to an inhibitory pass through the baroregulatory system. Thus, in the regulation of serum osmolality, TRPV4 is a swell-activated channel that appears to play a role in reversion toward hyposmolality.

Endothelin (ET) is a potent and long-acting vasoconstrictor peptide consisting of 21 amino acids and recently isolated from a medium of cultured porcine endothelial cells. To determine the possible sites of ET action, we have conducted autoradiography and receptor binding assays with 125I-labeled ET in rat tissues. The displaceable binding sites of the ligand were widely distributed, not only in the arteries and heart but also in various other organs, e.g., brain, kidney, lung, adrenal gland, and intestine. The systemically injected ET did not cross the blood-brain barrier, whereas the ligand, applied in vitro, was mainly located in the hypothalamic and thalamic areas, lateral ventricular region, subfornical organ, globus pallidus, and caudate putamen. Both membrane preparations from the brain stem including diencephalon and from the heart ventricle had similar, specific, and high-affinity binding sites for 125I-ET. We suggest that ET is involved in the regulation of a large variety of organ functions and may also act as a neuropeptide.

Claudin-2 is highly expressed in tight junctions of mouse renal proximal tubules, which possess a leaky epithelium whose unique permeability properties underlie their high rate of NaCl reabsorption. To investigate the role of claudin-2 in paracellular NaCl transport in this nephron segment, we generated knockout mice lacking claudin-2 (Cldn2(-/-)). The Cldn2(-/-) mice displayed normal appearance, activity, growth, and behavior. Light microscopy revealed no gross histological abnormalities in the Cldn2(-/-) kidney. Ultrathin section and freeze-fracture replica electron microscopy revealed that, similar to those of wild types, the proximal tubules of Cldn2(-/-) mice were characterized by poorly developed tight junctions with one or two continuous tight junction strands. In contrast, studies in isolated, perfused S2 segments of proximal tubules showed that net transepithelial reabsorption of Na(+), Cl(-), and water was significantly decreased in Cldn2(-/-) mice and that there was an increase in paracellular shunt resistance without affecting the apical or basolateral membrane resistances. Moreover, deletion of claudin-2 caused a loss of cation (Na(+)) selectivity and therefore relative anion (Cl(-)) selectivity in the proximal tubule paracellular pathway. With free access to water and food, fractional Na(+) and Cl(-) excretions in Cldn2(-/-) mice were similar to those in wild types, but both were greater in Cldn2(-/-) mice after i.v. administration of 2% NaCl. We conclude that claudin-2 constitutes leaky and cation (Na(+))-selective paracellular channels within tight junctions of mouse proximal tubules.

The recently cloned epithelial Ca2+ channel (ECaC), which is expressed primarily in 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-responsive Ca2+ -transporting epithelia, is postulated to constitute the rate-limiting step in active Ca2+ reabsorption. In the present study, the effect of 1,25(OH)(2)D(3) was investigated on ECaC mRNA and protein levels in kidneys of rats that were raised on a vitamin D-depleting diet. This diet decreased the serum 1,25(OH)(2)D(3) concentration significantly, which was accompanied by a marked drop in serum Ca2+ level. Both 1,25(OH)(2)D(3) and Ca2+ levels were normalized within 48 h after 1,25(OH)(2)D(3) administration. In 1,25(OH)(2)D(3)-deficient rats, ECaC mRNA and protein levels of the kidney cortex were significantly decreased compared with the repleted animals, suggesting that 1,25(OH)(2)D(3) exerts its stimulatory effect on Ca2+ reabsorption via increased ECaC expression. In agreement with this observation, the elucidated human ECaC promoter contains several consensus vitamin D-responsive elements. ECaC was restricted to the apical membrane of the distal part of the distal convoluted and the connecting tubule. This conclusion was based on only minor overlap with the localization of the thiazide-sensitive NaCl co-transporter and complete co-localization with the 1,25(OH)(2)D(3)-dependent Ca2+ binding protein, calbindin-D(28K). In conclusion, ECaC, present in the distal part of the nephron, is an important target for 1,25(OH)(2)D(3)-mediated Ca2+ reabsorption.