The TRPV4 Cation Channel Mediates Stretch-evoked Ca2+ Influx and ATP Release in Primary Urothelial Cell Cultures

Masayuki Takeda,Kayoko Fujishita,Makoto Tominaga,Kunitoshi Uchida,Tsutomu Mochizuki,Schuichi Koizumi,Koji Shibasaki,Takaaki Sokabe,Keiji Naruse,Isao Araki

doi:10.1074/jbc.m109.020206

Abstract

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, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) evoked Ca(2+) influx in wild-type (WT) urothelial cells, but not in TRPV4-deficient (TRPV4KO) cells. We established a cell-stretch 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 4alpha-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.

Highlights

Transient receptor potential vanilloid 4 (TRPV4),3 a member of the TRP superfamily of cation channels, is a Ca2ϩ-permeable channel activated by a wide variety of physical and chemical stimuli [1, 2]
Primary 3-day urothelial cell cultures from both WT and TRPV4deficient mice revealed expression of ck7, which is an intermediate filament protein present in all urothelial layers and considered a urothelial marker [38] (Fig. 1A). trpv4 expression was much more abundant than other thermosensitive TRP channels in WT urothelium, whereas no trpv4 expression was detected in TRPV4-deficient cells (Fig. 1, A and B). trpa1 seemed to be present in both cell types, while the expression of trpv1 and trpm8 genes was undetectable, unlike previous reports [21, 29, 39]
Immunocytochemical analysis of primary urothelial cells obtained from both WT and TRPV4-deficient mice revealed that these cultured cells originated from urothelial cells with positive signals for cytokeratin 7 (CK7), and WT urothelial cells were stained with an anti-TRPV4 antibody as did the tissues, but TRPV4-deficient cells were negative for TRPV4 staining (Fig. 2B)

Summary

To whom correspondence may be addressed

Mechano-sensor, because the channel opens in response to hypotonicity-induced cell swelling [3,4,5] and shear stress [6]. TRPV1-deficient mice displayed a higher frequency of low amplitude non-voiding bladder contractions in comparison with wild-type (WT) mice [22], suggesting that TRPV1 is required for detection of bladder stretch, which involves stretch-evoked release of ATP and nitric oxide. The release of both mediators was reduced in the bladders of TRPV1-deficient mice. We examined the functional contribution of TRPV4 to stretch-dependent urothelial cell responses and stretch-evoked ATP release in vitro. We demonstrated that urothelial cells sense mechanical stretch stimuli via TRPV4 channels, which induces robust Ca2ϩ influx and contributes to ATP release upon extension

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION