Unlike the GI tract, the GB muscle layer only consists of a single layer of SMCs. GB muscle fibers are separated by different amounts of connective tissue and orientated in different directions (35). GSMCs are shuttle-shaped, with abundant thin (actin and calponin) and thick filaments (myosin) in the cell body. Typical binding of actin and myosin results in cross-bridges, which form the basic unit of smooth muscle movement (36). α-Smooth muscle actin (α-SMA) is frequently used as a specific marker for smooth muscle (37). Another characteristic structure of GSMCs is the plasma membrane-sarcoplasmic reticulum (SR) junction, which are invaginations of the plasma membrane containing signaling molecules and ion channels (38).

Research on GB ICCs began in the 21st century. In 2006, Sun et al (39) first confirmed the existence of ICCs in CD1 mouse GB by c-Kit antibody labeling in combination with methylene-blue staining. Later, ICCs were also identified in human extrahepatic bile ducts, where they are more densely aggregated than in the GB (40,41). Light microscopy indicated that ICCs are typically elongated with oval-shaped cell bodies and 1-3 long processes extending from their poles, or exhibit a triangular cell body with several slender lateral branches (42). The fusiform ICCs form a multiple connecting network that is oriented parallel to adjacent muscle fibers in the GB muscularis layer. TEM scanning revealed that ICCs possess large nuclei, a well-developed smooth endoplasmic reticulum, abundant free perinuclear mitochondria, distinctive caveolae, free ribosomes and intermediate filaments without thick filaments, which are adjacent to SMCs and nerve endings (43). Recently, two identified genes, Ano1 and Na⁺-K⁺-Cl⁻ cotransporter (NKCC1), were found to be highly expressed in GB, representing a new and highly selective molecular marker for studying the distribution and fate of ICCs (44,45).

In 2007, Hinescu et al (41) first described TCs in human GB in detail. In the human adult GB, TCs are mostly placed near small vessels in the subepithelial region of the lamina propria and between smooth muscle bundles in the muscularis (46). TEM is considered the most accurate method for identifying TCs (15,26,47). In TEM images, TCs exhibit a variable tiny body with several dichotomously branched, extremely long and thin telopodes. The shape of the cytoplasm varies, including fusiform, pyriform and triangular shapes depending on the number of telopodes, which have a moniliform profile characterized by the alternation of thin tracts with dilations. Hematoxylin and eosin staining revealed long and extremely thin prolongations undetectable by light microscopy. The thin segments are called podomers, while the dilated regions are called podoms. Podoms hold functional units consisting of numerous mitochondria, endoplasmic reticulum and caveolae. CD34 and PDGFRα are considered reliable markers of TCs in the GI tract (48,49). In addition, TCs selectively express the small conductance Ca²⁺-activated K⁺ channel SK3 in the gut, which exhibits significant changes in functionality in the context of GI disease (50). TCs always form networks and provide mechanical support in the GI wall. However, the distribution of TCs in GB across various species remains controversial and further study is required to elucidate it.

Intracellular voltage recordings from intact guinea pig GSMCs revealed that characteristic action potentials (APs) have four distinct components: A resting membrane potential of -40 to -50 mV, a rapidly depolarizing (rarely exceeds 0 mV) and transient repolarizing spike, followed by a slowly sustained declining plateau phase, and finally complete repolarization (52).

GSMCs exhibit rhythmic spontaneous APs (0.3 to 0.4 Hz) started by Ca²⁺ entry, mainly through voltage-dependent Ca²⁺ channels (VDCCs) (52). The AP spike results from activation of L-type VDCCs in the absence of a T-type Ca²⁺ current in guinea pig GSMCs (53). The open state of L-type Ca²⁺ channels is regulated by neurotransmitters and drugs (54,55). For instance, L-type Ca²⁺ channel blockers such as nifedipine may abolish spontaneous AP and inhibit GB contraction. L-type channels are critical for proper GSMC function, providing the major source of contractile Ca²⁺. Depolarization of I_cat, a spontaneously active Na⁺-mediated nonselective cation channel, was indicated to maintain the resting membrane potential and increase contractility of GBSM, thus stabilizing GB tone (56).

The repolarization of APs is determined by voltage-gated K⁺ (K_v) channels and ether-a-go-go-related gene (ERG) K⁺ channels (57,58). Potassium reflux via K_v channels is responsible for the repolarization of APs and regulates the contraction of GBSM. These channels demonstrate relatively low sensitivity to aminopyridines but are inhibited by quinine (59). ERG, which encodes a delayed rectifier K⁺ channel in GB, contributes to repolarization of both the rapid spike and plateau phase (60). ERG channel blockers prolong repolarization of the plateau phase, increasing basal contractility of GSMCs and their response to receptor activation (57).

Other potassium channels identified in GSMCs include ATP-sensitive K⁺ (K_ATP) channels and large-conductance Ca²⁺-activated K (BK_Ca) channels. Activation of the K_ATP channel causes prolonged hyperpolarization, reducing the frequency of GBSM APs and associated spontaneous GBSM contractions (61). The K_ATP channel appears to have a major role in receptor-mediated relaxation of GBSM, as it is responsible for the inhibitory effects of calcitonin gene-related peptide (CGRP) and agonists of H2 receptors for histamine (62,63). In GSMCs, localized Ca²⁺ release events from ryanodine-sensitive receptors (RyR), also called Ca²⁺ sparks, antagonize GSMC excitability by activating BK_Ca channels in the nearby plasma membrane (see below) (64). Spontaneous transient activation of BK_Ca currents causes transient membrane hyperpolarization of GSMCs that was, in part, inhibited by cholecystokinin (CCK). Additional cellular mechanisms underlying bile acid-induced GBSM relaxation in vivo and in vitro potentially include activation of BK_Ca channels to generate outward currents, thus counteracting contraction (65).

GSMCs also express the SK3 channel. SK3 likely physically associates with ORAI calcium release-activated calcium modulator 1 (Orai1), a plasma membrane protein, to form a signaling complex. Ca²⁺ influx through Orai1 activates SK3 to induce membrane hyperpolarization in GBSM (66). This hyperpolarizing effect of the Orai1-SK3 complex may serve to prevent excessive contraction in response to contractile agonists.

GBSM excitation-contraction (E-C) coupling is dependent on an increase in the intracellular concentration of Ca²⁺ [Ca²⁺]_i, which is caused by an influx of extracellular Ca²⁺ through VDCCs and/or receptor-operated Ca²⁺ channels, as well as the release of Ca²⁺ from the SR (67). The influx of extracellular Ca²⁺ required for E-C coupling may enter cells through VDCCs, capacitative calcium entry (CCE) or nonselective cation channels (NSCCs).

The predominant class of VDCC in GSMCs is the L-type Ca²⁺ channel. As previously described, global cytosolic [Ca²⁺]_i is largely dictated by the open state probability of plasmalemmal L-type Ca²⁺ channels, while calcium entry through VDCCs is determined by the cell membrane potential (68). The depletion of intracellular calcium stores activates CCE, a Ca²⁺ entry mechanism at the plasma membrane (69). Thapsigargin, a sarcoplasmic Ca²⁺-ATPase inhibitor, is able to prevent the accumulation of Ca²⁺ by the SR. Activation of extracellular Ca²⁺-dependent responses and Ca²⁺ influx by thapsigargin is regarded as evidence in favor of the involvement of CCE (70). Contractile responses to Ca²⁺ re-addition following depletion of SR Ca²⁺ stores with thapsigargin strongly supports CCE as a source of activating Ca²⁺ for GBSM contraction (71). In addition, actin reorganization is proven to participate in the implementation of CCE, supporting a conformational coupling model for this process in naive SMCs (72). NSCCs in GSMCs demonstrate high selectivity for Ca²⁺ over monovalent cations, leading to activation of VDCCs mediating extracellular Ca²⁺ entry and contraction (73). Transient receptor potential (TRP) channels are a large family of NSCCs widely expressed in GSMCs (74). TRPP2 protein belongs to the TRP superfamily and is encoded by the polycystin 2 gene (75). In guinea pig GB muscle strips, knockdown of TRPP2 significantly reduced carbachol-evoked Ca²⁺ release (27). Accumulating evidence demonstrates that TRPP2 not only mediates intracellular Ca²⁺ release, but also regulates extracellular Ca²⁺ influx to enhance [Ca²⁺]_i (76-78). Furthermore, TRP protein family C (TRPC) is a candidate channel involved in CCE (28). The expression of TRPC protein depends on cytosolic Ca²⁺ levels through activation of Ca²⁺/calmodulin-dependent kinases and cAMP-response element binding protein.

Calcium influx and release from the SR, also known as intracellular stores, are crucial for GSMC contractility, which primarily depends on increases in [Ca²⁺]_i (79). Intracellular calcium release from the SR involves the participation of two ligand-gated channel/receptor complexes [inositol 1,4,5-trisphosphate receptors (IP₃R) and RyR] and is regulated by sarcoplasmic/endoplasmic reticulum calcium ATPase (80,81). Calcium release via IP₃R is activated by IP₃, which is generated in response to numerous G-protein-coupled receptors (GPCRs) and tyrosine kinase-linked receptor activators, including neurotransmitters, hormones and drugs. RyR mediates the rapid release of calcium from intracellular stores into the cytosol, which is essential for numerous cellular functions, including E-C coupling in muscle. Three types of rhythmic spontaneous Ca²⁺ transients were determined by laser confocal imaging of intracellular Ca²⁺ in GBSM whole-mount preparations (31,64,79). Ca²⁺ flashes reflect calcium entry associated with spontaneous APs and simultaneously occur in all GSMCs in the given bundle, although they are asynchronous among nonintersecting bundles. Ca²⁺ waves are rhythmic Ca²⁺ transients propagating within GSMCs that are asynchronous between individual muscle cells in the given bundle; apparently, these waves correspond to subthreshold depolarization of GSMCs. Both flashes and waves triggered by Ca²⁺ release from the SR occur through IP₃ receptors, which is amplified by calcium-induced calcium release (CICR) and VDCCs (82). Superimposed Ca²⁺ waves induce Ca²⁺ flashes, while the summation of spontaneous transient depolarizations results in APs. In the guinea pig GB, rapid Ca²⁺ transients occur simultaneously in all the GSMCs of a given bundle, but without synchronization between muscle bundles (38). Of note, synchronous Ca²⁺ flashes occur among smooth muscle bundles in the presence of CCK or muscarinic agonists. These findings indicate that the net tone in the GB originates from asynchronous, multifocal contractions of bundles throughout the tissue wall, while synchronous electrical rhythms occurring in all muscle bundles may contribute to GB emptying. Therefore, flashes and waves are critical in maintaining the basal tone and neurohormonal-induced stimulation of GB motility and emptying. Ca²⁺ release from intracellular stores not only induces contraction, it also induces relaxation. Ca²⁺ sparks are another type of focal, nonpropagating calcium transients caused by the coordinated opening of a cluster of RyR. In GB, Ca²⁺ sparks do not lead to any elevation in global [Ca²⁺]_i. Instead, transient localized [Ca²⁺]_i elevations through opening of BK_Ca channels cause SMC hyperpolarization and relaxation (64).

GSMC contraction is also regulated by Ca²⁺-independent mechanisms via protein kinase C (PKC)/CPI-17 or RhoA/Rho-kinase (ROCK)-mediated pathways. The regulation of MLC phosphorylation by MLCK causes SMC contraction, whereas inhibition of MLCP may enhance the extent of MLC phosphorylation and SMC contraction and increase Ca²⁺ sensitivity, a phenomenon known as Ca²⁺ sensitization (83). In the classical PKC/CPI-17 pathway, G proteins cause activation of phospholipase C (PLC), diacylglycerol output and activation of PKC. PKC phosphorylates CPI-17, an inhibitor of MLCP activity, resulting in GBSM contraction (84,85). ROCK also regulates GSMC contraction by regulating the Ca²⁺ sensitization mechanism. Contractions induced by carbachol and CCK are mediated by GPCR muscarinic M₃ receptors and CCK₁ receptors in guinea-pig GBSM (86,87). The selective ROCK inhibitor Y-27632 significantly inhibited GBSM contractions evoked by carbachol and CCK in vitro (30). In human GB, Y-27632 markedly reduced 5-hydroxytryptamine, neurokinin A and KCl-induced contractions (88). The results of these studies indicate that a RhoA/ROCK-mediated pathway has a role in the regulation of GSMCs.

ICCs have an important role in producing and propagating rhythmic electrical activity and GB motility. Isolated ICCs display spontaneous electrical rhythmicity similar to the electrical activity of intact muscles. In fact, electrical coordination between regions of SMCs must occur through the integrity of ICC networks due to the lack of ion channels to regenerate or actively propagate SWs (43,94). In GBSMs, SWs may also be recorded from SMCs due to electrical coupling with ICCs. The function of SWs is to change the membrane potential from a state of low open probability for VDCCs to depolarization, which means APs, when there is an increased probability of associated ionic channel opening (9). A Ca²⁺ imaging study by Lavoie et al (43) indicated that the intensity of fluo-4 fluorescence in ICCs was higher than that of the surrounding GSMCs, while rhythmic Ca²⁺ flashes were synchronized in any given GBSM bundle and associated with ICCs. More importantly, gap junction blockers may eliminate or markedly disrupt spontaneous rhythmic Ca²⁺ flashes in GBSM, but persist in ICCs, whereas the selective Kit tyrosine kinase inhibitor imatinib mesylate disrupted or abolished APs and Ca²⁺ flashes in both cell types, as well as associated GBSM contractions. These results demonstrate that the spontaneous rhythmic activity detected in GBSM, which corresponds to smooth muscle bundle contractions, is generated by specialized ICCs and not an intrinsic property of GSMCs. Taken together, ICCs conduct pacemaker SWs into neighboring GSMCs, causing membrane depolarization, opening of the VDCC, intracellular Ca²⁺ release and activation of the contractile apparatus of GB. To date, no specific 'pacing region' has been identified in the GB.

ICCs serve as pacemaker cells and express a specialized apparatus that includes Ca²⁺-activated Cl⁻ channels (CaCCs), T-type voltage-dependent Ca²⁺ channels, NSCCs, NKCC1, inward rectifier K⁺ channels and Na⁺/Ca²⁺ exchanger (NCXs). SWs recorded from ICCs have fast upstroke depolarizations with large amplitudes and a sustained plateau potential.

SWs in ICCs are mediated by activation of Ano1 channels and NSCCs. ICC depolarization depends upon activation of CaCCs encoded by the ANO1 gene, such that loss or block of Ano1 abolishes the electrical activity of SWs in intact smooth muscles (95). Periodic activation of Ano1 channel clusters generates spontaneous transient inward currents (STICs) and subsequently initiates coordinated activation of CaCCs that summates to cause the depolarization responses known as SWs (96). The calcium entry from RyR and IP3R of ICCs during CICR appears to be the signal coupled to activation of CaCC, as these channels are sensitive to [Ca²⁺]_i (97). Of note, research on cultured ICCs indicated that NSCCs, not CaCCs, generated the inward current responsible for SWs (95,96,98,99). This may be explained by rapid loss of Ano1 expression in cell culture and alteration of the autorhythmicity retained by ICCs compared with the pacemaker activity of cells in situ. Unitary potentials, which are small irregular noisy fluctuations in membrane potential, may be the primary pacemaker activity that underlies SWs. These electric events were insensitive to concentrations of niflumic acid (the inhibitor of CaCC) that blocked SWs (99). The Ca²⁺-inhibited NSCC-activated STICs observed from isolated ICCs may be responsible for unitary potentials (95). Accordingly, NSCC may contribute to the pacemaker current and generation of electrical SWs in GI smooth muscles. T-type Ca²⁺ channels coordinate Ca²⁺ release from stores in ICCs, thus controlling the openings of Ano1 channels responsible for SW currents (100). The mechanism of SW propagation in tissues has been explored by using muscle strips and partitioned recording chambers. Reduced extracellular Ca²⁺ or antagonists of T-type Ca²⁺ channels inhibit SW upstroke depolarization velocity and propagation (101). These results suggest that SWs propagate through the ICC network by a voltage-dependent mechanism that relies on activation of T-type Ca²⁺ channels (38). ICCs have been demonstrated to express genes encoding inward rectifying K⁺ channels, and this inwardly rectifying conductance contributes to the regulation of resting potentials and excitability of SMCs (102).

The plateau component of SWs was dependent on the Cl⁻ current through CaCCs, while the activation of Ano1 channels results in efflux of Cl⁻ during SWs (103). Thereby, a mechanism must exist for the recovery of Cl⁻ loss. IHC confirmed that NKCC1 is expressed at high levels in ICCs (104). Inhibition of NKCC1 with bumetanide and gene knockout of NKCC1 both diminished the plateau component of SWs without directly affecting Ano1 or T-type Ca²⁺ channels (45,105). In isolated GB ICCs, inhibitors of mitochondrial NKCC1 also abolished spontaneous rhythmic activity, suggesting that NKCC1 may have an important role in maintaining the Cl⁻ gradient supporting the driving force for the inward current mediated by Ano1 (106). Furthermore, NKCC1 may elongate the plateau phase by activation of reverse-mode NCX. NCX, an ion transport protein, extrudes Ca²⁺ in parallel with the plasma membrane ATP-driven Ca²⁺ pump (107). NCX has dynamic features in the SW cycle, in which Ca²⁺ exit helps to maintain the basal [Ca²⁺]_i between SWs and deactivate Ano1 channels at the end of the plateau; furthermore, Ca²⁺ entry sustains the activation of Ano1 channels during the plateau phase of SWs (108). The longevity of the plateau phase is related to the duration of time that NCX remains in Ca²⁺ entry mode. However, the underlying molecular mechanisms of SWs in GB ICCs remain to be further elucidated.

GB neurons are relatively unexcitable, driven instead by vagal inputs and modulated by hormones, peptides released from sensory fibers, and inflammatory mediators (118).

CCK, an important gut hormone secreted by enteroendocrine I-cells of the upper small intestine, mainly exerts its physiological functions in GB through the activation of GPCRs identified as CCK₁ receptors. CCK₁ receptors have been identified in both GSMCs and ICCs of human and guinea pig GB and are responsible for the stimulation of contraction (131,132). Previous electrophysiological studies of the GB demonstrated that CCK has presynaptic facilitatory effects within neural ganglia to increase acetylcholine (ACh) release from vagal terminals onto GB neurons, and also stimulates vagal afferent nerve fibers in the duodenum, thus increasing stimulation of vagal preganglionic neurons (133). Furthermore, CCK induces a decrease in resistance of the sphincter of Oddi, a determinant of GB emptying (134). In brief, CCK coordinates the pressure gradient in the biliary system by promoting GB emptying and relaxing the sphincter of Oddi, ultimately facilitating bile evacuation during the feeding period.

Co-expression of TKs with ACh in GB neurons indicates that these factors may act together to promote GB emptying following afferent nerve stimulation (130). M₃ receptors are the major muscarinic receptor in GB and M₄ receptors appear to enhance carbamylcholine-induced contractility of GBSM (135). Release of ACh from neurons results in the contraction of GBSM via activation of M₃ receptors on GSMCs. Activation of M₃ receptors leads to phosphatidylinositol hydrolysis by the G protein-coupled PLC pathway and inhibits cAMP accumulation (136). In human GB, M₃ muscarinic receptors are mainly regulated by voltage-gated Ca²⁺ channels and ROCK (137). The TKs contract the guinea pig GB in vivo and in vitro by acting on NK2 receptors (138). TKs-induced muscle contraction involves activation of PKC, for which stimulation of inositol phospholipid hydrolysis was associated with the state of NK2 receptors (139).

Bradykinins and their receptors (B₁ and B₂) are potent mediators of inflammation, smooth muscle contraction and nociception. In human and guinea pig GB, bradykinin has been demonstrated to evoke a robust contraction via B₂ receptor activation (140,141). Bradykinin-induced contraction of GBSM in vitro relies on the synthesis of prostanoids, whose activation evokes inflammatory responses either by direct stimulation of effector cells or through the release of other mediators, including prostanoid, NO and peptide neurotransmitters. By contrast, B₁ receptors are rarely expressed in normal GB and their upregulation most probably depends on the inflammatory state of the tissue. Activation of B₁ receptors has been related to the maintenance of chronic pain and inflammation (142). Thus, the kinins system has a major role in evoking contraction in normal and, in particular, inflamed GB by stimulating both B₁ and B₂ receptors.

The physiological source of ATP in GB remains elusive and it is possible that ATP functions as a neurotransmitter (143). ATP is known to act on two different classes of P2 receptors, P2X ion channels and G-protein-coupled P2Y receptors (144). The dominant role of G protein-coupled P2Y₄ receptors in ATP-induced contraction has been confirmed in guinea pigs. ATP likely stimulates P2Y₄ receptors within GSMCs and, in turn, prostanoid production via cyclooxygenase-1, leading to increased excitability of GBSM (145). In the guinea pig, high levels of P2X₂ and P2X₃ expression are found in sensory fibers of the paravascular plexus. Double labelling IHC revealed that P2X₂ and P2X₃-immunoreactive neurons were also immunoreactive for VIP, CGRP and nNOS (146).

Neurotransmitters that have an inhibitory effect on GBSM include calcitonin, CGRP, VIP, PACAP and NO. Humoral factors that relax the GB include pancreatic polypeptide (PP), SST and fibroblast growth factor (FGF)15 in mice or FGF19 in humans.

CGRP may induce concentration-dependent relaxation of GB in vivo, but has no effect on resting GB pressure (147). CGRP did not affect the release of CCK and the excitatory effect of CGRP was completely abolished by pretreatment with atropine. This implies that the site where CGRP activates contractile activity is on intramural cholinergic neurons rather than GSMCs. This relaxation is primarily due to the opening of K_ATP channels, as well as the cAMP pathway (62,148). The increased levels of NO observed when CGRP was present suggest NO is also involved in the CGRP-induced relaxation response (149). NO has been proposed to serve as a neurotransmitter in non-adrenergic non-cholinergic nerves. Synthesized by nNOS, NO stimulates soluble guanylate cyclase enzyme in GSMCs, leading to the formation of 3′,5′-cyclic-guanosine monophosphate (cGMP), which mediates GB relaxation (150). Endogenous carbon monoxide (CO) produced in the GB may act as a mediator in relaxation reactions by increasing cGMP levels (151). Of note, despite persistent nNOS expression in heme oxygenase 2-knockout mice, their responses to stimulation are nearly abolished, whereas exogenous CO restored normal responses, indicating that NO does not function in the absence of CO generation (152).

VIP and PACAP are members of a VIP-secretin-glucagon superfamily of structurally related peptide hormones that exert their physiological actions through three GPCRs: PAC₁, VPAC₁ and VPAC₂ (153). VIP is thought to work as a neurotransmitter of vagus nerve terminals, which relaxes GBSM, decreases GB pressure and inhibits CCK-induced contractions (127,154,155). PACAP was able to produce both contraction and relaxation of CCK-induced GB preparations according to the resting GB tone (156). The dual effects of PACAP are likely mediated through a different type of receptor. Specifically, PACAP induces GB contraction through binding of PAC₁ receptors in unstimulated strips, while the relaxant effect of PACAP in CCK-contracted muscle strips appears to be directly mediated by GSMCs through VPAC₂ receptors (157).

Other gut hormones, such as the NPY family, SST and neurotensin (NT), also enhance GB relaxation (158-160). However, it remains elusive whether these hormones regulate GB tone through direct effects on ICCs, GSMCs and TCs, as there is no direct evidence that their respective specific receptors are expressed in GB. The NPY family contains biological active peptides of the gut-brain axis, including NPY, peptide YY (PYY) and PP (161). In guinea pigs, sympathetic postganglionic nerves are immunoreactive for NPY (125). These nerves likely represent the principal source of inhibitory neural input to the GB, leading to a decline of GB tone (162). PYY and PP are almost exclusively expressed in the GI tract. PYY is a GI peptide secreted from endocrine L cells localized in the distal small intestine, colon and rectum (163). PYY was able to abolish the cephalic phase of postprandial GB emptying and probably acts via vagal-dependent rather than CCK-dependent pathways (164). PP is postprandially secreted from the pancreas, in which it is synthesized by endocrine F cells of the pancreatic islets. Similar to PP, PYY infusion results in increased volume and filling of the GB (165). Circulating PP binds to Y4 receptors in the dorsal vagal complex and affects the hepatic vagal afferent, leading to the inhibition of GB contraction and pancreatic exocrine secretion (166). SST, a peptide with potent inhibitory actions on GB contraction, enhances GB relaxation and reduces plasma excitatory gut hormone (ACh and CCK) secretion during the late postprandial phase (167). SST at a pathological concentration was able to inhibit the GB motor response to intrinsic excitatory innervation in vitro (168). NT, a peptide consisting of 13 amino acids, may either stimulate or inhibit GB motility, depending on the dose and species (169). NT induced a dose-dependent contraction of isolated GB of guinea pigs, and these contractile effects resulted from the excitement of cholinergic neurons in the myenteric plexus of GB (170). However, intravenous infusion of NT caused relaxation of the GB in humans (160). Of note, this contractile response was not observed in vitro (171).

Recently, bile acids (BAs) have been recognized as signaling molecules capable of regulating GB filling through two different mechanisms: The BAs-Takeda GPCR 5 (TGR5) pathway and the FGF15/19-farnesoid X receptor (FXR) pathway. TGR5 expression was identified in both enteroendocrine L cells and GSMCs (172,173). First, separate BAs were able to directly bind TGR5 in GSMCs, promoting GB filling. In addition, BAs in the intestinal lumen stimulated TGR5 on enteroendocrine L cells, which released glucagon-like peptide 2 (GLP-2) that subsequently activated GLP-2 receptors on GSMCs, ultimately mediating relaxation (174). BAs also activate the FXR expressed by enterocytes, thereby mediating the synthesis and release of FGF15/19 into the blood and subsequent stimulation of FGF receptors on GSMCs, inducing GB relaxation (175). Of note, activation of FXR of enteroendocrine L cells may inhibit GLP-2 release, and this effect may antagonize BA-induced relaxation of GB under certain circumstances.

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 Ca²⁺ 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 Ca²⁺ sensitization observed in AC by desensitizing Ca²⁺ pools and impairing the functional status of plasma membrane Ca²⁺ 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 PGE₂ and the severity of inflammation was associated with the concentration of PGE₂ (197). Symptoms of AC are significantly reduced during the first 24 h by the cyclooxygenase inhibitor indomethacin (198). Furthermore, PGE₂ 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 H₂O₂ formation (200,201). Exogenous H₂O₂ 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 H₁ and H₂ receptors. Activation of H₁ receptors depolarizes GSMCs, whereas activation of H₂ 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 B₁ receptors by GSMCs may contribute to the typical symptoms that underline biliary colic during the cholecystitis state (142).