The urothelium of the bladder has been previously considered a passive barrier. However, in recent years, a large number of reports have shown that the urothelium plays an important role in bladder mechanosensory transduction (de Groat, 2004; Birder, 2005). In response to mechanical stimuli, two critical concomitant intracellular events occur in urothelial cells: an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the release of signaling molecules such as NO, ATP, and acetylcholine (ACh). The release of ATP from urothelial cells directly activates purinergic receptors on bladder afferent terminals to increase their excitability, thereby enhancing sensory function of the bladder (Yu and de Groat, 2008). Although several studies and a few reviews have been conducted on mechanosensing in urothelial cells (Merrill et al., 2016; Janssen et al., 2017), the exact cellular mechanisms by which urothelial cells sense mechanical stimuli are not fully understood.

The activation of mechanosensitive channels (MSCs) on urothelial cells is the first step in initiating mechanotransduction. These channels detect and transduce external mechanical forces into intracellular Ca²⁺ elevation and ATP release. Most MSCs are nonselective cation channels with high Ca²⁺ permeability that contribute to the elevation of intracellular Ca²⁺. Several classes of membrane-bound MSCs have been implicated in the urothelial mechanotransduction. These channels include acid-sensing ion channels (Sadananda et al., 2009); transient receptor potential vanilloid (TRPV1 or TRPV4) channels (Birder et al., 2002; Mochizuki et al., 2009); epithelial Na⁺ channels (Du et al., 2007); the Piezo1 channel (Miyamoto et al., 2014); Piezo2 channel (Marshall et al., 2020); pannexin hemichannels (Timoteo et al., 2014; McLatchie and Fry, 2015); and connexin channels (Shimura et al., 2018).

In vivo, changes in intravesical pressure or bladder wall tension are physiologically relevant mechanical stimuli for the urothelium. To simulate these stimuli in vitro, common strategies include stretching of urothelial cells cultured on coverslips using various customized devices (Miyamoto et al., 2014; Guan et al., 2018) or exposing urothelial cells to high hydrostatic pressure (Olsen et al., 2011). For example, custom designed chambers, attached to an extension device, were used to stretch urothelial cells in one study (Miyamoto et al., 2014). In another study, urothelial cells cultured on plastic dishes were exposed to sustained hydrostatic pressure (5–20 cm H₂O; Olsen et al., 2011).

The above approaches of delivering mechanical stimuli are valuable in revealing the underlying mechanisms of urothelial mechanotransduction. However, they require technically complicated setups. In other reports in the literature, exposure of urothelial cells to low-osmolality solutions to induce cell swelling was also a frequent strategy to alter cell membrane stress (Haeberle et al., 2008; Wu et al., 2011). Delivering hypoosmotic shock to cells is simple. However, this modality might not present a physiologically relevant stimulus. Although low-osmolarity urine is occasionally present in the bladder in vivo, it may not lead to the swelling of urothelial cells. This is because of the presence of specialized lipid molecules and uroplakin proteins in the apical membrane of umbrella cells, which reduce the permeability of the cells to water.

Several studies have reported that cells can be mechanically stimulated by creating an air-liquid interface (ALI). The ALI is produced by reducing the volume of liquid covering cells, and a thin film of liquid on the cells is drawn down to such proximity that tension forces at the ALI cause cell deformation (Patrick et al., 2001; Ramsingh et al., 2011). In the study by Ramsingh et al. (2011), lung epithelial cells were exposed to ALI forces by briefly introducing an air bubble into a closed perfusion chamber or by tilting the culture dish containing the cells. Such stimuli produced reversible cell deformation, a transient increase in [Ca²⁺]_i, and ATP release. In another study, a microfluidic device was developed to create an ALI by liquid evaporation (Huang et al., 2016), to investigate ALI-induced intracellular Ca²⁺ response in a human leukemic cell line (HL-60).

In the present study, we developed another method to create an ALI in a perfusion cell chamber, by simply stopping influx, but maintaining efflux of the perfusion, until a thin layer of liquid was created on the urothelial cells. We found that the ALI can induce a remarkable increase in [Ca²⁺]_i and ATP release from urothelial cells cultured on glass coverslips, and the responses were repeatable and did not affect cell viability. We aimed to investigate the underlying cellular mechanisms of mechanical stimulus-induced increase in [Ca²⁺]_i with this easily-operated model. Our main focus was on the potential involving channels or receptors located either on the cell membrane or those associated with intracellular Ca²⁺ stores. We found that both Ca²⁺ influx via membrane-bound TRPV4 or pannexin 1 channels, and Ca²⁺ release from intracellular Ca²⁺ stores via IP3 and ryanodine receptors contribute to an ALI-induced [Ca²⁺]_i increase. Furthermore, ATP released from urothelial cells also contribute to a [Ca²⁺]_i increase via autocrine or paracrine mechanisms.

In the current study, the cellular mechanisms underlying ALI-induced increase in [Ca²⁺]_i of cultured urothelial cells were investigated using various pharmacological blockers. Our main findings were as follows: (1) Both Ca²⁺ influx and Ca²⁺ release from intracellular Ca²⁺ stores contribute to the [Ca²⁺]_i increase; (2) TRPV4 or pannexin 1 channels are the two membrane-bound MSCs involved in Ca²⁺ influx; (3) activation of both IP3 and ryanodine receptors on the ER contribute to Ca²⁺ release from intracellular Ca²⁺ stores; and (4) ATP release also contributes to the [Ca²⁺]_i increases by activating P2X and P2Y receptors via autocrine or paracrine mechanisms.

In vitro, commonly used approaches to deliver mechanical stimulation to urothelial cells include: stretching the cells with various custom designed devices (Miyamoto et al., 2014; Guan et al., 2018); exposing the cells to high hydrostatic pressure; or simply subjecting the cells to a hypoosmotic shock (Olsen et al., 2011). Using these approaches, valuable information has been obtained regarding urothelial cell mechanotransduction. For example, TRPV4 or Piezo1 channels have been revealed as the MSCs responsible for sensing mechanical stimuli (Mochizuki et al., 2009; Miyamoto et al., 2014). In addition, the activation of pannexin 1 channels has been considered an important mechanism of ATP release (Penuela et al., 2013). However, the above approaches usually require technically complicated setups.

In the current study, another form of mechanical stimulation was established, i.e., creation of an ALI on urothelial cells. The ALI evoked similar cellular responses in urothelial cells as the mechanical modalities mentioned above, i.e., [Ca²⁺]_i increase and ATP release. Moreover, the whole procedure did not cause cell lysis, which was evidenced by the remarkable [Ca²⁺]_i increase in response to ATP after inductions of ALI. Thus, an ALI can induce the [Ca²⁺]_i increase and ATP release, but does not necessarily cause damage to the plasma membrane. Although the chances that the bladder urothelium is exposed to an ALI are not common under physiological conditions, this may occur during some invasive procedures, like bladder surgeries under laparoscopy or during cystoscopy.

An ALI may also be frequently encountered in urothelial cell culture experiments. For example, when the culture medium is changed. In the current study, we did not observe to what extent cell deformation was evoked by the ALI. When the cell is exposed to an ALI, surface tension forces up to 320 Newtons/in² could be exerted on the cell membrane (Ludwig et al., 2008), and the pressure generated by surface tension is large enough to cause significant cell deformation. Thus, we established a simple procedure to deliver mechanical stimulation to cultured urothelial cells, which can be used as a valuable model to investigate the cellular mechanisms of [Ca²⁺]_i increase or ATP release.

Tension-induced Ca²⁺ entry into cells is reportedly achieved through the activation of MSCs (Olsen et al., 2011; Leybaert and Sanderson, 2012; Jafarnejad et al., 2015). In the current study, the involvement of MSC channels in the ALI-induced [Ca²⁺]_i increase could be supported by the observation of the almost complete blockade of the ALI-induced [Ca²⁺]_i increase by GdCl₃. As a common nonspecific MSC blocker, Gd³⁺ has been shown to block many MSC channels, including several types of TRP channels (White et al., 2016), pannexin 1 channels (Jiang and Penuela, 2016), as well as mechano-insensitive channels like those associated with purinergic receptors (Khakh et al., 2001).

In previous studies, TRPV4 (Mochizuki et al., 2009), epithelial Na⁺ (ENa; Du et al., 2007), Piezo1 (Miyamoto et al., 2014), Piezo 2 (Marshall et al., 2020), connexin (Shimura et al., 2018), and pannexin 1 channels (Timoteo et al., 2014; McLatchie and Fry, 2015) have been implicated in mechanotransduction of urothelial cells. In agreement with these previous reports, our observations of the significant reduction of the [Ca²⁺]_i increase by RR, as well as the highly TRPV4-selective antagonist, suggest the involvement of TRPV4 channels. The expression of TRPV4 in the urothelium further supports the contribution of TRPV4. However, our observations that the ENaC antagonist (amiloride), the Piezo channel antagonist (GsMTx4), and the connexin channel antagonist (carbenoxolone) could not block the ALI-induced [Ca²⁺]_i increase invalidates the involvement of these MSCs.

These results are in contrast with those of previous reports, which state that Piezo1, Piezo2 (Marshall et al., 2020), ENaC, or connexin channels (Shimura et al., 2018) contribute to mechanosensory transduction of the bladder urothelium (Du et al., 2007; Miyamoto et al., 2014). These inconsistent results may be attributed to the different ways in which the cells were stimulated. In the study Miyamoto et al. (2014), mouse urothelial cells were horizontally elongated by extending the cell chamber. In the current study, the apical surface of urothelial cells were exposed to the ALI, such that any curved cell surface was subjected to a net component of tension forces that acted perpendicularly to the cell membrane (Figure 1B). Thus, the inconsistency in results may imply that urothelial cells use different mechanosensing channels in response to various forms of tension forces (horizontal vs. perpendicular).

Pannexin 1 channels are characterized by their mechanosensitivity and their ability to form large pores that are permeable to ATP and other molecules (Penuela et al., 2013). Therefore, they are reportedly involved in the nonvesicular release of ATP in the bladder urothelium, in response to mechanical stimulation (McLatchie and Fry, 2015). Consistent with this theory, our observation that ¹⁰panx, the specific pannexin 1 channel antagonist, significantly attenuated both the ALI-induced [Ca²⁺]_i increase (by 39.3%) and ATP release indicates the involvement of pannexin 1 channels. Furthermore, our immunohistochemical analysis demonstrated the expression of the pannexin 1 channel in urothelial cells.

We found that the ALI-induced [Ca²⁺]_i increase was reduced by 22% in Ca²⁺-free HBSS and was reduced by 62.4% following the application of thapsigargin, a blocker that irreversibly inhibits the ER Ca²⁺-ATPase pumps. These results suggest that compared with Ca²⁺ entry, Ca²⁺ release from the ER calcium stores are the dominant source of the ALI-induced [Ca²⁺]_i increase. The contribution of Ca²⁺ release from the ER calcium stores can also be supported by the observation that ALI induced [Ca²⁺]_i was significantly reduced by application of BAPTA-AM to clamp intracellular Ca²⁺ levels (Figure 4Bc). The IP3 receptor (IP3R) and ryanodine receptor on the ER have been implicated in Ca²⁺ release from intracellular calcium stores, and play an important role in Ca²⁺-induced Ca²⁺ release (CICR; Galione and Churchill, 2002). In agreement with this idea, when the ER calcium stores were blocked with the antagonist, IP3R (Xest-C), or the ryanodine receptor, the ALI-induced [Ca²⁺]_i increase was reduced by 71.6 and 67.7%, respectively. The CICR was mediated by the activation of ryanodine receptors located on the ER, which can be blocked by high concentrations of ryanodine (100 μM; Bouchard et al., 2003). In the current study, the blocking effects of Xest-C and ryanodine may imply the involvement of the CICR mechanism in the ALI-induced [Ca²⁺]_i increase.

One report on a human urothelial cell line (T24 cells) showed that ATP released upon cell stretching contributes to a major part of Ca²⁺ signaling (Guan et al., 2018). In agreement with this finding, we also observed that ALI-evoked ATP release contributed to the ALI-induced [Ca²⁺]_i increase via paracrine or autocrine mechanisms. This conclusion can be supported by the following evidence: (1) the ALI-induced [Ca²⁺]_i increase was significantly reduced by ATP hydrolysis with apyrase and (2) the [Ca²⁺]_i increase was significantly reduced by blocking the P2X and P2Y purinergic receptors with PPADS. Thus, autocrine ATP signaling works like an amplifier to increase the concentration of cytosolic Ca²⁺. We did not examine which type of purinergic receptors is involved with these processes. However, previous studies have shown that the P2X receptor that mediates the increase in [Ca²⁺]_i is dependent on extracellular Ca²⁺. On the other hand, P2Y receptors are G-protein-coupled receptors that can induce the release of Ca²⁺ from intracellular stores via the IP3 pathway (von Kugelgen and Hoffmann, 2016). Based on the dominant role of intracellular Ca²⁺ release from the ER in the ALI-induced [Ca²⁺]_i increase, also based on our observation that the amplitude of ATP evoked [Ca²⁺]_i increase in zero Ca²⁺ bath is not significantly different from that in normal HBSS bath (data not shown), we propose that P2Y receptors may have a greater contribution to the process than P2X receptors.

Based on the current results, a working model has been proposed (Figure 7). The process is likely initiated with the activation of TRPV4 or pannexin 1 channels and Ca²⁺ influx, which in turn activates IP3 or ryanodine receptors on the ER to induce Ca²⁺ release from intracellular Ca²⁺ stores to further increase [Ca²⁺]_i. Activation of the pannexin 1 channels or [Ca²⁺]_i increase leads to ATP release through nonvesicular or vesicular mechanisms, respectively, which further enhances the increase in [Ca²⁺]_i by activating at P2X and P2Y receptors via autocrine or paracrine mechanisms.

Notably, the method used to create the ALI-induced plasma membrane deformation by causing changes in the perfusion volume may alter the equilibrium and concentration of signaling molecules close to the plasma membrane, thereby altering their autocrine/paracrine effects. Typically, this is the case with the ATP concentration; transient reduction of the perfusion fluid volume may increase the concentration of the nucleotide. Compared with other techniques, this is a disadvantage of the ALI method to induce urothelial cell deformation and is a limitation of the current study. Another limitation is the pharmacological approach employed for most of our experiments to determine the associated channels or receptors. Some of the antagonists used in the current study may not be very specific.

In summary, we revealed the cellular mechanisms underlying membrane tension force induced [Ca²⁺]_i increase in urothelial cells. Changes of any one of the associated channels or receptors (Figure 7) may affect bladder mechanotransduction and sensory function. Although the chances that the bladder urothelium exposed to an ALI is uncommon under physiological conditions, detrusor overactivity that occurs in overactive bladder conditions can increase deformation in the urothelium, which could trigger an increase in [Ca²⁺]_i and the release of ATP, independent of urine volume.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by the Ethics Committee of the Second Hospital, Cheeloo College of Medicine, Shandong University [KYLL-2016(GJ)A-0027].

XZ and SW participated in research design. JW, ZC, SW, and MZ conducted experiments. JW, XZ, SZu, ZC, and MZ performed data analysis. XZ, SZh, and JW wrote the manuscript. All authors contributed to the article and approved the submitted version.