Cell Deformation at the Air-Liquid Interface Evokes Intracellular Ca2+ Increase and ATP Release in Cultured Rat Urothelial Cells

Abstract

Urothelial cells have been implicated in bladder mechanosensory transduction, and thus, initiation of the micturition reflex. Cell deformation caused by tension forces at an air-liquid interface (ALI) can induce an increase in intracellular Ca2+ concentration ([Ca2+]i) and ATP release in some epithelial cells. In this study, we aimed to examine the cellular mechanisms underlying ALI-induced [Ca2+]i increase in cultured urothelial cells. The ALI was created by stopping the influx of the perfusion but maintaining efflux. The [Ca2+]i increase was measured using the Ca2+ imaging method. The ALI evoked a reversible [Ca2+]i increase and ATP release in urothelial cells, which was almost abolished by GdCl3. The specific antagonist of the transient receptor potential vanilloid (TRPV4) channel (HC0674) and the antagonist of the pannexin 1 channel (10panx) both diminished the [Ca2+]i increase. The blocker of Ca2+-ATPase pumps on the endoplasmic reticulum (thapsigargin), the IP3 receptor antagonist (Xest-C), and the ryanodine receptor antagonist (ryanodine) all attenuated the [Ca2+]i increase. Degrading extracellular ATP with apyrase or blocking ATP receptors (P2X or P2Y) with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) significantly attenuated the [Ca2+]i increase. Our results suggest that both Ca2+ influx via TRPV4 or pannexin 1 and Ca2+ release from intracellular Ca2+ stores via IP3 or ryanodine receptors contribute to the mechanical responses of urothelial cells. The release of ATP further enhances the [Ca2+]i increase by activating P2X and P2Y receptors via autocrine or paracrine mechanisms.

Polycystin-2 Is Required for Chondrocyte Mechanotransduction and Traffics to the Primary Cilium in Response to Mechanical Stimulation

“…TRPV4 inhibition with GSK205 inhibits chondrocyte matrix production in response to compressive mechanical stimulation, while the agonist GSK1016790A (GSK101) promotes matrix production in the absence of stimulation [51]. Moreover, TRPV4-deficient mice have an increased risk of obesity-related OA [52]. TRPV4 activation can trigger ATP release in multiple cell types, including chondrocytes [53][54][55].…”

Functional Expression of Transient Receptor Potential and Piezo1 Channels in Cultured Interstitial Cells of Human-Bladder Lamina Propria

“…Culture of urothelial cells was undertaken as described in our previous study ( Wen et al, 2021 ). Briefly, the mucosa (1.5 cm × 1.5 cm) dissected from the bladder wall was placed in Minimum Essential Medium containing dispase and HEPES (2.5 mg/mL) overnight at 4°C.…”

“…Fura 2-AM was dissolved in Hank’s balanced salt solution containing (in mM): 138 NaCl, 5 KCl, 0.3 KH 2 PO 4 , 4 NaHCO 3 , 2 CaCl 2 , 1 MgCl 2 , 10 HEPES, and 5.6 glucose, pH 7.4. Ca 2+ imaging was undertaken as described in our previous study ( Wen et al, 2021 ). Briefly, coverslips were placed in a recording chamber.…”

“…Samples of perfusate were collected 2 min before and immediately after agonist stimulation during calcium imaging study. The ATP concentration was measured using luciferin–luciferase bioluminescence, as described previously ( Wen et al, 2021 ). Briefly, a mixture of 100 μL of luciferin–luciferase was added to 100 μL of sample according to manufacturer instructions using the CellTiterGlo™ Luminescent Cell Viability Assay kit (Promega, Fitchburg, WI, United States).…”

“…For Ca 2+ imaging and immunocytochemistry experiments, cells were plated onto poly-L-lysine (Sigma-Aldrich)-coated glass coverslips (8 mm in diameter) and grown to 80% confluence and 50% confluence, respectively. Culture of urothelial cells was undertaken as described in our previous study (Wen et al, 2021). Briefly, the mucosa (1.5 cm × 1.5 cm) dissected from the bladder wall was placed in Minimum Essential Medium containing dispase and HEPES (2.5 mg/mL) overnight at 4 • C. Urothelial cells were scraped and placed in trypsin (0.25% wt/vol) for 5 min at 37 • C, and dissociated by trituration.…”