Transient receptor potential channels have recently been implicated in physiological functions in a urogenital system. In this study, we investigated the role of transient receptor potential vanilloid 4 (TRPV4) channels in a stretch sensing mechanism in mouse primary urothelial cell cultures. The selective TRPV4 agonist, 4␣-phorbol 12,13-didecanoate (4␣-PDD) evoked Ca 2؉ influx in wild-type (WT) urothelial cells, but not in TRPV4-deficient (TRPV4KO) cells. We established a cellstretch system to investigate stretch-evoked changes in intracellular Ca 2؉ concentration and ATP release. Stretch stimulation evoked intracellular Ca 2؉ increases in a stretch speed-and distance-dependent manner in WT and TRPV4KO cells. In TRPV4KO urothelial cells, however, the intracellular Ca 2؉ increase in response to stretch stimulation was significantly attenuated compared with that in WT cells. Stretch-evoked Ca 2؉ increases in WT urothelium were partially reduced in the presence of ruthenium red, a broad TRP channel blocker, whereas that in TRPV4KO cells did not show such reduction. Potent ATP release occurred following stretch stimulation or 4␣-PDD administration in WT urothelial cells, which was dramatically suppressed in TRPV4KO cells. Stretch-dependent ATP release was almost completely eliminated in the presence of ruthenium red or in the absence of extracellular Ca 2؉ . These results suggest that TRPV4 senses distension of the bladder urothelium, which is converted to an ATP signal in the micturition reflex pathway during urine storage. Transient receptor potential vanilloid 4 (TRPV4),3 a member of the TRP superfamily of cation channels, is a Ca 2ϩ -permeable channel activated by a wide variety of physical and chemical stimuli (1, 2). TRPV4 was originally viewed as an osmo-or mechano-sensor, because the channel opens in response to hypotonicity-induced cell swelling (3-5) and shear stress (6). Alternatively, TRPV4 can be activated by diverse chemical stimuli such as synthetic phorbol ester 4␣-phorbol 12,13-didecanoate (4␣-PDD) (7), a botanical agent (bisandrographolide A), anandamide metabolites such as arachidonic acid and epoxyeicosatrienoic acids, as well as moderate warmth (Ͼ27°C) (8 -10). TRPV4 is widely expressed throughout the body, including renal epithelium, auditory hair cells, skin keratinocytes, hippocampus neurons, endothelial cells, and urinary bladder epithelium, thereby contributing to numerous physiological processes such as osmoregulation (11, 12), hearing (13), thermal and mechanical hyperalgesia (14, 15), neural activity in the brain (16), skin barrier recovery (17), and cell volume regulation (18). Therefore, the TRPV4 channel is now considered a multimodal transducer in various tissues and cells.Non-neuronal cells within the urinary bladder wall (notably the transitional epithelial cells (urothelial cells)) function as a barrier against ions, solutes, and infection and also participate in the detection of physical and chemical stimuli (19 -21). The urothelium expresses various sensory receptors and channe...

Proliferation, differentiation, and tube formation by endothelial progenitor cells in response to shear stress. Endothelial progenitor cells (EPCs), circulating in peripheral blood, migrate toward target tissue, differentiate, and contribute to the formation of new vessels. In this study, we report that shear stress generated by blood flow or tissue fluid flow can accelerate the proliferation, differentiation, and capillary-like tube formation of EPCs. When EPCs cultured from human peripheral blood were subjected to laminar shear stress, the cells elongated and oriented their long axes in the direction of flow. The cell density of the EPCs exposed to shear stress was higher, and a larger percentage of these cells were in the G 2-M phase of the cell cycle, compared with EPCs cultured under static conditions. Shear stress markedly increased the EPC expression of two vascular endothelial growth factor receptors, kinase insert domain-containing receptor and fms-like tyrosine kinase-1, and an intercellular adhesion molecule, vascular endothelial-cadherin, at both the protein and mRNA levels. Assays for tube formation in the collagen gels showed that the shear-stressed EPCs formed tubelike structures and developed an extensive tubular network significantly faster than the static controls. These findings suggest that EPCs are sensitive to shear stress and that their vasculogenic activities may be modulated by shear stress. blood vessels; angiogenesis; neovascularization; mechanical stress; vascular endothelial growth factor THE FORMATION OF NEW BLOOD vessels in postnatal life has generally been considered to be mediated by the sprouting of endothelial cells (ECs) from preexisting vessels, a process referred to as angiogenesis. However, recent studies have indicated that a vasculogenesis process involving the in situ differentiation of endothelial precursor cells and their subsequent organization into new vessels is also responsible for postnatal neovascularization (10, 22, 27). The existence of bone marrow-derived endothelial progenitor cells (EPCs) circulating in the peripheral blood has been demonstrated in adult humans (6, 26). EPCs have the capacity to circulate, proliferate, and differentiate into mature ECs in response to a variety of growth factors, including VEGF, and other cytokines (7, 16, 18). Transplantation studies have revealed that EPCs can be incorporated into sites of active neovascularization in ischemic hindlimbs and myocardium and contribute to both tumor growth and the formation of new blood vessels (5,20,40). However, the role of EPCs in supporting postnatal vasculogenesis is under intensive investigation, and the factors regulating the migration, proliferation, differentiation, and vessel formation of EPCs are not yet known.During the process of EPC incorporation into tissues and neovascularization, the cells are exposed to fluid shear stress, a mechanical force generated by blood flow or interstitial fluid flow (42). Ample evidence has shown that shear stress modulates mature EC function and gene...