Smooth muscle cells, interstitial cells and neurons in the gallbladder (GB): Functional syncytium of electrical rhythmicity and GB motility (Review).

The gallbladder (GB), an accessory organ of the gastrointestinal (GI) tract, stores and concentrates most hepatic bile between meals and regulates the outflow of bile into the duodenum postprandial. The human liver normally produces at least 1,000 ml of hepatic bile per day ( 1 ). Up to 80% of hepatic bile partitions into the GB, depending on the synergy state of the GB and sphincter of Oddi ( 2 , 3 ). The GB undergoes structural and functional changes, as well as GB dysmotility, in numerous pathological conditions, including gallstone disease, GB polyps and acute acalculous cholecystitis ( 4 - 6 ). Given that GB dysmotility is so prevalent in GB disease, a comprehensive understanding of the neurons and smooth muscles responsible for GB contractile activity is critical. GI motility patterns, including those of the GB, result from coordinated contractions of the muscular layers of the alimentary canal. Several studies found that interstitial cells of Cajal (ICCs) and platelet-derived growth factor receptor α-positive (PDGFRα + ) cells form electrical coupling complexes with smooth muscle cells (SMCs) in the GI tract. Sanders et al ( 7 ) initially proposed this structure as an SMC-ICC-PDGFRα + cell (SIP) syncytium. In this functional structure, ICCs act as periodic spontaneous pacemakers to generate a slow wave (SW), which conducts SMCs to drive phasic contractions ( 8 , 9 ). Correspondingly, PDGFRα + cell excitation causes hyperpolarization of SMCs, leading to muscle relaxation ( 10 , 11 ). Unlike skeletal muscle, there is no classical neuromuscular junction between nerve terminals of the enteric nervous system (ENS) and GI smooth muscle ( 12 ). Enteric nerve endings expand to form numerous varicosities containing neurotransmitters ( 13 , 14 ). Subsequently released neurotransmitters diffuse to the adjacent SIP syncytium to regulate GI motility. Although the integrity of the morphological structure and function of SIP syncytium are important for GI physiological function, the functions of SIP syncytium are mainly derived from evaluations of specific SIP cell types. Previously, telocytes (TCs) were considered interstitial Cajal-like cells (ICLCs) due to the similar morphology under the light microscope and immunohistochemical (IHC) features with ICCs, which were found >100 years ago and considered to be pacemakers for GI motility. Subsequently, it was demonstrated that TCs are not ICLCs, as TCs presented a distinctly different ultrastructure from ICLCs in transmission electron microscopy (TEM) images. To avoid further confusion and to give a precise identity to these cells, in 2010, Popescu and Faussone-Pellegrini ( 15 ) coined the term TCs for cells previously referred to as ICLCs. Differences in the TCs' immune phenotypes have been found to be significant in different tissues; by contrast, the ultrastructural differences of TCs are the least evident. Hence, the term TCs was proposed based on the cells' unique TEM features rather than selective immune markers. Subsequently, Vannucchi et al ( 16 ) clearly indicated that TCs express PDGFRα in the human GI tract. Based on these IHC data, TCs are frequently referred to as PDGFRα + cells and this definition is commonly used in scientific reports. Of note, as TCs express different IHC markers in different organs and even in different tissues from the same organ, it remains controversial whether TCs and PDGFRα + cells are the same cell type ( 17 - 20 ). However, in the gut, all cells identified as TCs were double-positive for CD34 and PDGFRα and shared identical ultrastructural features ( 16 , 21 ); therefore, these TCs and PDGFRα + cells are the same cell type, at least in GI tract. Further research substantiated the existence of TCs in the biliary system, including GB, extrahepatic bile duct, cystic duct, common bile duct and sphincter of Oddi ( 22 ). Current electrophysiological studies of the GI tract are mostly focused on the stomach and intestine. The concept of SIP syncytium was also first demonstrated and proposed in the GI tract ( 7 ). Although the histological anatomy and physiological functions of the GB and the stomach or intestine are not identical, they belong to the same myogenic organs of the digestive tract and their physiological functions are both dependent on the movement of their smooth muscles. More importantly, both the expression and distribution of ICCs and TCs have also been demonstrated in myogenic organs such as the GB, ureter and uterus ( 23 - 26 ). Current studies on GB electrophysiology are mainly on SMCs and ICCs ( 22 , 27 - 33 ). The mechanisms of SMCs in the motor function of the GB have been most thoroughly studied. It is currently believed that ICCs in GB have a regulatory role in the motor function of the GB, but the exact mechanism of regulation remains to be clarified. The study of TCs in the GB is even more limited to histology. However, the regulation of GB motor function is important for benign GB diseases (e.g., cholelithiasis, cholecystitis, GB polyps, GB adenomyosis). In the most recent study by our group, the presence of a unique structure containing ICCs, TCs, SMCs and neurons in the GB has been proved by multiplexed IHC ( Fig. 1 ; for methods see supplementary data ). These results indicated that the four cells were in spatial proximity to each other in mouse GB. Furthermore, c-Kit and anoctamin 1 (Ano1) were used to label ICCs, CD34 and PDGFRα to label TCs, Myh11 and Acta2 to label SMCs to analyse the single-cell RNA-sequencing of normal mice (for methods see supplementary data ) ( 34 ). The results also proved that there were three double-positive cell types (ICCs, TCs and SMCs) for their respective specific molecular markers and they formed their own cell clusters ( Fig. 2 ). All of these results demonstrated that these four types of cells are present and constitute the SMC-TC-ICC-neuron (STIN) syncytium structure in the mouse GB. Based on these findings, the functional complex was proposed as an STIN syncytium ( Fig. 3 ). The present review described various aspects of the morphology, regulation and function of STIN cells in GB and discussed pathological changes of the STIN syncytium in GB disease.

Cholelithiasis is a highly prevalent digestive system disorder with high socioeconomic costs worldwide ( 176 ). In China, the incidence of cholelithiasis is nearly 8-10% and has been gradually increasing in recent years ( 177 ). Depending on individual composition and location, gallstones contain >90% cholesterol and the remaining material is black or brown pigment stones ( 4 ). The loss of ICCs results in GB dysmotility in patients with cholesterol or pigment stones, as well as animal models of gallstone disease ( 33 , 178 ). Hypercholesterolemia is an independent risk factor for cholelithiasis, as it may increase biliary cholesterol concentrations, consequently leading to bile crystallization and, ultimately, gallstone formation ( 179 , 180 ). More importantly, cholesterol accumulation strongly damaged the density and ultrastructure of GB ICCs by inhibiting the stem cell factor (SCF)/c-Kit pathway, and disrupted membrane receptor functions of STIN cells, particularly CCK1 receptors ( 181 - 183 ). Due to impaired CCK-induced emptying, the resulting GB stasis provides a microenvironment for excess cholesterol to remain in the lumen; in turn, the elevated cholesterol content further impairs GB emptying ( 184 ). During the chronic pathogenesis of cholelithiasis, cholesterol induces an oxidative stress response with characteristic concentration dependence, resulting in inhibited proliferation and continuous apoptosis of GB ICCs ( 185 , 186 ). In vitro studies suggested that cholesterol decreases Ca 2+ channel function and the fluidity of caveolar regions, causing sequestration of excitatory receptors to support reduced binding of agonists in affected GBSM ( 187 , 188 ). High cholesterol diets also significantly inhibit ROCK expression in GMSCs, leading to the promotion of gallstone formation ( 189 ). Therefore, enhancement of ROCK expression in GSMCs may be a novel strategy for the prevention and treatment of cholelithiasis. Hydrophobic bile salts decrease GB contractility, an effect directly related to the hydrophobicity of bile salt ( 190 , 191 ). Hydrophobic bile salts hyperpolarize GSMCs by binding to the GPCR GPBAR1 (also known as TGR5) and activating cAMP-mediated opening of K ATP channels, eventually disrupting GBSM function ( 172 ). The reduction in the number of ICCs may be a consequence of the toxicity of hydrophobic bile salts, while other bile components (such as glycocholic and taurocholic acids) may exert protective effects on ICCs ( 192 ). However, whether BAs are able to directly injure ICCs requires further study. Patients with gallstones display abnormalities of the GB neural network. Specifically, IHC of GB with gallstones featured a significant decrease of neurons and enteric glial cells compared with that of GB without gallstones, while calretinin-positive neurons were not different between the two groups of patients ( 193 ). Calretinin has been identified as a marker of Dogiel type II gut neurons, which appear to behave as mechanosensors. Thus, these findings support the hypothesis that GB wall mechanics remain intact in patients with or without gallstones, whereas GB motility is impaired. Gallstones are responsible for 90-95% of cases of acute cholecystitis (AC), while ~5-10% of patients exhibit acute acalculous cholecystitis ( 5 , 194 ). The pathogenesis of AC is complex and multifactorial, but GB dysmotility is the most critical pathogenic factor, as it may cause GB ischemia, cholestasis and secondary bacterial infection. Inflammation induces alterations of Ca 2+ sensitization observed in AC by desensitizing Ca 2+ pools and impairing the functional status of plasma membrane Ca 2+ channels ( 195 ). Inflammation also reduces the expression of contractile proteins, such as F-actin in GSMCs, which may be responsible for the observed reduction in sensitivity of E-C coupling ( 195 ). Inhibition of MLCP mediated by the RhoA/ROCK pathway may also be responsible for the impairment of the contractile response ( 84 ). Hydrophobic bile salts may enhance inflammatory processes, as they may diffuse through the mucosa and affect the generation of reactive oxygen species (ROS) by GBSM, either by direct action on GSMCs or increasing numbers of inflammatory cells in the GB wall ( 196 ). Like other inflammatory processes, AC involves the release of inflammatory factors, including prostaglandins (PGs), ROS, histamine and endothelin (ET). Early studies of AC patients demonstrated that both the mucosa and muscularis of GB produce high levels of PGE 2 and the severity of inflammation was associated with the concentration of PGE 2 ( 197 ). Symptoms of AC are significantly reduced during the first 24 h by the cyclooxygenase inhibitor indomethacin ( 198 ). Furthermore, PGE 2 has been indicated to hyperpolarize GB neurons, thereby inhibiting neurogenic contractions of GB ( 199 ). Normally, ROS produced during oxidative metabolism is cleared by antioxidant mechanisms, yet oxygen-derived free radical production may exceed the capability of scavengers, resulting in ROS accumulation and pathogenic effects during inflammation. Furthermore, during inflammation, excessive production of NO through inducible NOS with concurrent ROS production increases H 2 O 2 formation ( 200 , 201 ). Exogenous H 2 O 2 causes GBSM contraction and impairs GB responses to agonists of membrane-dependent receptors, thus inducing GBSM impairment ( 201 , 202 ). Histamine is released from mast cells, which are abundant in the GB wall. In GSMCs, histamine performs diametrically opposed functions through H 1 and H 2 receptors. Activation of H 1 receptors depolarizes GSMCs, whereas activation of H 2 receptors causes hyperpolarization via K ATP channels ( 63 , 203 ). However, the net effect of histamine in GB is normally contraction ( 204 ). Although the role of histamine in AC is not fully understood, it is possible that AC is associated with increased mast cell infiltration and degranulation. ETs are bioactive peptides produced by GB epithelial cells, which have a crucial role in the early inflammatory process of AC. GB tissue ET levels are elevated, which is accompanied by an increase in GB tone ( 205 ). This pathological change precedes any histological evidence of GB inflammation. ET likely exerts an autocrine/paracrine role in the human GB via ET-a and ET-b receptors of GBSM ( 206 ). Pretreatment of the GB with an ET antagonist abrogated the development of AC. In addition, decreased GB motility in AC results from the effects of neutrophils on the development and function of the ICCs network via depression of SCF/c-Kit expression ( 207 ). Upon coculture with neutrophils in vitro , the intracellular calcium transient of ICCs was less sensitive to contraction agonists and inhibitors ( 208 ). A study of human GB strips from AC suggested that overexpression of B 1 receptors by GSMCs may contribute to the typical symptoms that underline biliary colic during the cholecystitis state ( 142 ).

In summary, regulation of the membrane potential is complex, as GSMCs are electrically coupled to ICCs and TCs. Activation of conductance in any STIN cell affects the excitability of the syncytium. Individual STIN cells express intrinsic electrophysiological mechanisms and a variety of receptors for neurotransmitters, hormones, paracrine substances and inflammatory mediators. Similar to other GI SMCs, GSMCs rely on the formation of cross-bridges between actin and myosin for the development of force to empty the GB. The onset of GSMC depolarization requires SWs generated and propagated by GB ICCs. TCs (also known as PDGFRα + cells) exert an inhibitory effect on the excitability of SMCs through SK3 channels in the GI tract. However, the specific role of TCs in GB has yet to be studied and is a potential topic for future electrophysiological studies of GB. Therefore, the integrated output of the STIN syncytium sets the transient excitability of GSMCs. The primary risk factor for benign GB disease is GB dysmotility. Loss and dysfunction of STIN cells have been observed in patients and animal models with cholelithiasis and cholecystitis, suggesting that impairment of the STIN syncytium may be a critical pathogenic factor in benign GB disease. However, to date, there remains a lack of breakthroughs in the study of STIN syncytium. Thus, further research to better understand the pharmacology and physiology of the STIN syncytium is required.