Interstitial cells (ICs) in the bladder have attracted much research attention in recent years (Andersson and Mccloskey, 2014; Koh et al., 2018). Based on their location, two populations of ICs in the bladder have been characterized: LP-ICs, which are between the urothelium and detrusor and ICs in the detrusor (detrusor-ICs) (Andersson and Mccloskey, 2014; Gevaert et al., 2014). LP-ICs can be sub-grouped further into ICs in the upper lamina propria (ULP-ICs, which are immediately beneath the urothelium) or sub-urothelial ICs and ICs in the deep lamina propria (DLP-ICs, which lie between the ULP and detrusor) (Gevaert et al., 2014).

Several molecular markers are used to characterize bladder ICs: the broad mesenchymal marker vimentin (Vim) (Davidson and Mccloskey, 2005; Gevaert et al., 2014), the myogenic differentiation marker alpha-smooth muscle actin (Monaghan et al., 2012; Neuhaus et al., 2018) (α-SMA), platelet-derived growth factor receptor alpha (PDGFRα) (Monaghan et al., 2012; Gevaert et al., 2014) and the protooncogene c-Kit (Mccloskey and Gurney, 2002). It should be noted that c-Kit, the assumed specific marker for interstitial cells of Cajal (ICC), recently has been found to be expressed only on mast cells in urinary bladder (Gevaert et al., 2017). Upper lamina propria in the human bladder are characterized as Vim + /αSMA + /PDGFRα + /c-kit-. In contrast, DLP-ICs are Vim + /αSMA-/PDGFRα + /c-kit-. Detrusor-ICs have a similar phenotype to that of DLP-ICs (Monaghan et al., 2012; Gevaert et al., 2014; Steiner et al., 2018).

Considerable progress has been made regarding the cellular markers, calcium signaling, ion channels, and receptor expression of bladder ICs (Andersson and Mccloskey, 2014; Koh et al., 2018), but their exact physiologic functions in the bladder are not known. Based on morphology, spatial distribution, and limited functional data, ULP-ICs and DLP-ICs are thought to have important roles in afferent signaling processing in the bladder mucosa (Fry et al., 2007; Gevaert et al., 2011; Andersson and Mccloskey, 2014), whereas detrusor-ICs have been proposed to modulate detrusor spontaneous contractions or excitability (Koh et al., 2018).

In addition to their sensitivity to adenosine triphosphate (ATP), low pH, and acetylcholine (ACh) (Fry et al., 2007; Johnston et al., 2008), ULP-ICs have been shown to have mechanical sensitivity, and that mechanical stimuli such as stretch, shear stress, or hypotonicity, can evoke an increase in the intracellular Ca²⁺ concentration [(Ca²⁺)_i] (Neuhaus et al., 2020). That study also suggested that ULP-ICs are the active elements in afferent signaling processing in the bladder. Active participation of ULP-ICs is also supported by a study that reported increased ATP release from cultured ICs from pig bladders by hypotonic stimulation (Cheng et al., 2011). In line with those findings, ULP-ICs network has been proposed to function as a stretch receptor for perception of physical and chemical stimuli (Wiseman et al., 2003; Vannucchi and Traini, 2018). Although purinergic (P2X3 or P2Y6) (Sui et al., 2006) or muscarinic receptors have been found in bladder ICs, it is not clear if other receptors that can perceive mechanical or chemical stimuli are present on LP-ICs.

Transient receptor potential (TRP) channels are important sensors for cells in response to mechanical and chemical stimuli or temperature change. TRPA1, TRPV1, TRPV2, TRPV4, and TRPM8 expressed in bladder sensory afferents or urothelial cells have pivotal roles in the sensory function of the bladder (Vanneste et al., 2021). However, few studies have investigated the expression and function of TRP channels in bladder ICs, particularly in ULP-ICs. Recently, the bladder ICs of humans, guinea pigs, and pigs have been shown express TRPA1 (Steiner et al., 2018). TRPA1 expression has also been found in Vim + ICs of the ureter (Weinhold et al., 2018) and prostate gland (Gratzke et al., 2010) of humans. However, whether TRPA1 is functionally active in LP-ICs is not known.

The recently recognized mechanically sensitive channels Piezo1/Piezo2 have been shown to be expressed in the human urothelium as well as in neurons of dorsal-root ganglia innervating the bladder, and have been implicated in mechanical sensory transduction in the bladder (Miyamoto et al., 2014; Marshall et al., 2020). We examined if these sensory channels (TRPA1, TRPV1, TRPV2, TRPV4, TRPM8, and Piezo1/Piezo2) are expressed in LP-ICs of the human bladder. Real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence staining, and Ca²⁺ imaging were utilized. Vim and α-SMA were used as molecular markers for ICs.

We measured expression of TRPA1, TRPV1, TRPV2, TRPV4, TRPM8, and Piezo1/Piezo2 channels in human-bladder LP-ICs at mRNA, protein, and functional levels. To the author’s knowledge, this is the first study demonstrating the functional expression of TRPA1, TRPV2, TRPV4, and Piezo1 channels in human-bladder LP-ICs. Most importantly, activation of these channels resulted in ATP release from LP-ICs. Our observation suggests that LP-ICs could sense mechanical and chemical stimuli via these sensory channels, and then impact the activity of surrounding urothelial cells or nerve endings in a paracrine fashion. Our results provide further evidence for the active role of LP-ICs in the processing of sensory signals in the bladder mucosa.

In addition to ICs or stromal cells, bladder ICs have been called ICC, ICC-like, myofibroblast-like, fibroblast-like cells (Koh et al., 2018; Vannucchi and Traini, 2018), and telocytes (Vannucchi and Traini, 2018). This heterogeneity in terminology leads to considerable confusion between research teams working in this area. Nevertheless, ICs termed differently have a common property: positive staining with the broad mesenchymal marker Vim. Thus, Vim + cells were identified as ICs and the common term ICs was adopted in our study.

Two populations of ICs from the human bladder have been identified: α-SMA + /Vim + /PDGFRα + /TRPA1 + in the ULP, and α-SMA-/Vim + /PDGFRα + /TRPA1 + in the DLP and detrusor muscle (Monaghan et al., 2012; Gevaert et al., 2014; Steiner et al., 2018). Thus, α-SMA is the key marker to differentiate ULP-ICs from DLP-ICs. In agreement with this concept, αSMA + ICs were located mainly in the ULP of the human bladder wall (Figure 1B). For our cultured LP-ICs, >90% were α-SMA+, which suggests that cultured ICs were mainly from the ULP. The predominant population in cultured ICs was ULP-ICs, which could have been because DLP-ICs account for a minority of the LP-IC population in the human bladder (Figure 1B). α-SMA- cultured ICs (∼10% of the total) might be from the DLP.

For TRPV1 and TRPM8 expression, there is a disparity between the immunofluorescence staining (Figure 3) and the functional data (Figure 4). The lack of responses to capsaicin and menthol in LP-ICs (Figure 4) is in contrasts to the expression of TRPM8 and TRPV1 demonstrated by immunocytochemistry or RT-qPCR (Figure 2). Lamina propria may mimic urothelial cells that TRPV1 or TRPM8 expressions were demonstrated at mRNA level, but no functional expressions were found (Xu et al., 2009; Everaerts et al., 2010; Shabir et al., 2013). The reasons for the absence of capsaicin and menthol response in LP-ICs is not clear for us, probably because the mRNA expression level of TRPV1and TRPM8 are relatively low.

TRPA1-immunoactivity has been demonstrated in the bladder ICs of humans, guinea pigs, and pigs (Steiner et al., 2018). We demonstrated TRPA1 expression in human LP-ICs at mRNA and protein levels (Figures 2, 3). Furthermore, TRPA1 functional expression was found in 65.5% of LP-ICs in our study (Figure 4). Initially, TRPA1 was characterized as a noxious cold receptor. Subsequently, TRPA1 was identified as an important chemical sensor to painful or potentially harmful stimuli (Andersson, 2019). Thus, TRPA1 channels in LP-ICs may have an important role as sensors of toxic and irritant substances produced in bladder wall or pass from urine into the bladder wall if the urothelial barrier is disrupted during bladder infection or interstitial cystitis. In support of this idea, we found that H₂O₂, the product of oxidative stress (ROS), can induce an increase in [Ca²⁺]_i in LP-ICs, and this effect could be inhibited by pretreatment of TRPA1 antagonist (HC030031,30 μM; Figures 6C,D).

TRPV2, TRPV4, and Piezo1 are mechanical or stretch sensors (Andersson, 2019; Jiang et al., 2021). In our study, functional expression of these channels was found in most human LP-ICs. Most importantly, activation of these channels by their agonists promoted ATP release in LP-ICs. Studies have shown that ULP-ICs express the Cx43 protein and form gap junctions, thus behaving as a functional syncytium to propagate chemical or electrical signals (Fry and Vahabi, 2016). This ULP-IC functional network has also been proposed to act as a stretch receptor for the perception of local or bladder-wall distension (Wiseman et al., 2003; Vannucchi and Traini, 2018). In support of this notion, sub-urothelial ICs have been shown to have mechanical sensitivity, and that mechanical stimuli (e.g., stretch, shear stress, hypotonicity) induce an increase in [Ca²⁺]_i (Neuhaus et al., 2020). Our study further showed that stretch evoked intracellular Ca²⁺ increase could be inhibited by the antagonist of TRPV2, TRPV4, and Piezo1, respectively (Figure 6). This finding provides direct evidence that TRPV2, TRPV4, and Piezo1 may be the stretch sensors for ULP-ICs perceiving local or bladder filling-induced wall stretching.

Another important finding of our study is that activation of TRPA1, TRPV2, TRPV4, and Piezo1 channels by their agonists could elicit ATP release from LP-ICs. Bladder filling-induced ATP release from urothelial cells activating P2X3 receptors of sensory afferents has been considered the key underlying mechanism for generation of a bladder-filling sensation (Yu and de Groat, 2008). Given the close contact of ULP-ICs with sub-urothelial sensory nerves (Wiseman et al., 2003), we propose that ATP released from ULP-ICs may also have an important role in sensory afferent activation during bladder-filling. ULP-ICs are located immediately underneath urothelial cells, and urothelial cells and ICs are responsive to ATP (Supplementary Figure 2B). Thus, bidirectional communications between the urothelium and ULP-ICs may occur via ATP, and the two elements may form a stage for amplification of sensory signals and detection of bladder-filling (Fry et al., 2007).

In addition to the paracrine fashion (via ATP release) discussed above, the modulating effects of ULP-ICs on afferent activity in the bladder may also result from the mechanically contracting and stimulating impacts on sensory afferents. In the present study and previous studies (Monaghan et al., 2012; Gevaert et al., 2014; Steiner et al., 2018), ULP-ICs were found to contain the contracting element α-SMA. ULP-ICs will contract if [Ca²⁺]_i is increased in response to chemical or mechanical stimuli and, finally, alter gain of the sensory pathway.

Under identical recording conditions to that of LP-ICs, only TRPV4 was functionally expressed in our cultured human urothelial cells. This finding is consistent with other studies showing TRPV4 (but not TRPV1) being functionally expressed on human (Shabir et al., 2013), mouse (Everaerts et al., 2010) and guinea pig (Xu et al., 2009) urothelial cells. No functional expression of TRPV1 in human urothelial cells may suggest that capsaicin effects observed on human bladder may result from its action (activation or desensitization) on TRPV1 channels in primary sensory afferents (Fowler et al., 1992). However, expression of TRPV2 and Piezo1 channels in urothelial cells may have a species difference because functional expression of TRPV2 and Piezo1 are found in urothelial cells in mice (Everaerts et al., 2010) and rats (data not shown). Only TRPV4 is functionally expressed in human urothelial cells, which is in stark contrast to LP-ICs, in which TRPA1, TRPV2, TRPV4 and Piezo1 channels are functionally expressed. Given the important role of these channels in the sensing of chemical and mechanical stimuli, we propose that the role of the network of ULP-ICs may be even more important than that of urothelial cells in the perception of chemical or mechanical signals in the bladder mucosa.

In summary, we found that human suburothelial ICs functionally express TRPA1, TRPV2, TRPV4 and Piezo1 channels, and release ATP when these channels are activated. Our study suggests that LP-ICs can perceive stretch or chemical stimuli in the bladder LP via activation of TRPA1, TRPV2, TRPV4, and Piezo1 channels. Bidirectional communications may be present between LP-ICs and surrounding urothelial or sensory afferents in a paracrine manner. Given the recognized role of the urothelium in sensory function of the bladder, LP-ICs may work together with urothelial cells for perception and transduction of mechanical or chemical signals in human-bladder mucosa.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved [KYLL-2016(GJ)A-0027] by the Ethics Committee of the Second Hospital, Cheeloo College of Medicine of Shandong University (Jinan, China). All patients provided written informed consent for their tissue to be used in our experiments.

XZ and GC participated in the research design. MZ, ZC, LL, SZ, WS, and ND conducted the experiments. JW, JL, WW, SZ, WS, and NG performed the data analysis. XZ, GC, and MZ wrote the first manuscript, and all authors revised it critically to meet the standard for publication. All authors contributed to the article and approved the submitted version and are accountable for all aspects of this work.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.