Abstract
This chapter provides an overview of the current knowledge of neuronal and muscular features of biliary tract motility and its regulation during healthy and pathophysiological conditions. The chapter is organized into three major sections to provide a summary of current knowledge regarding the neurochemistry, neurophysiology, neurohormonal regulation, and ionic properties of gallbladder and sphincter of Oddi motility, as well as an overview of the mechanisms that are responsible for altered biliary function in pathological conditions. Postprandial gallbladder emptying is driven by vagal input to gallbladder neurons, which in turn increases the rate and strength of gallbladder smooth muscle (GBSM) action potentials. This pathway is activated by CCK stimulation of vagal afferents in the duodenum and is facilitated by CCK-enhancing vagal efferent synaptic input to gallbladder neurons via presynaptic facilitation. Gallbladder emptying between meals appears to involve the action of fibroblast growth factor on GBSM cells, which like most inhibitory neuromuscular signals in this system, involves activation of the cyclic AMP-protein kinase A pathway, and results in the opening of ATP-sensitive potassium channels. Sphincter of Oddi ganglia are much like myenteric ganglia, but they do not appear to contain intrinsic sensory neurons and therefore may not house intrinsic reflex circuitry. On the other hand, a local, duodedum-sphincter of Oddi circuity could provide a major role in decreased resistance of the sphincter of Oddi during gallbladder emptying, as duodenal myenteric neurons that project to the mucosa and express CCK receptors also project to sphincter of Oddi where they provide excitatory input to sphincter of Oddi neurons. Decreased gallbladder contractility is a fundamental feature of gallbladder pathophysiology. Cholesterol and hydrophobic bile salts, both of which are elevated in the bile of individuals with gallstone disease, decrease GBSM function by inhibiting excitatory receptor activation (cholesterol) and by activating bile salt receptors that stimulate the cyclic AMP-protein kinase A-K ATP channel pathway. It has been demonstrated in a mouse model of gallstone disease that the disruption in smooth muscle function precedes the development of inflammation.
Keywords
Biliary motility, Gallbladder neuron, Gallbladder smooth muscle, Gallbladder interstitial cell of Cajal, Sphincter of Oddi neuron, Aging, Development, Gallstone disease, Hydrophobic bile salt, Cholesterol, Gallbladder filling, Gallbladder emptying
20.1
Introduction
The prevalence of gallbladder disease makes the goal of understanding gallbladder function a clinically significant one. Gallstone disease in the United States afflicts some 6.3 million men and 14.2 million women aged 20–74 years. In other words, ~ 10%–15% of the adult US population has gallstones or have had a cholecystectomy. The direct costs associated with biliary disease (~$5.5–6.5 billion/year in the United States) are the second highest for any gastrointestinal ailment, with gastroesophageal reflux disease being the costliest, and peptic ulcer disease and colorectal cancer ranking behind gallbladder disease at 3rd and 4th. The costs of gallstone disease exceed the combined totals for chronic liver disease and cirrhosis, chronic hepatitis C, and pancreatic disease. Its frequency and costs make gallbladder disease a public health issue.
It is taken for granted that gallbladder disease is curable by cholecystectomy. Now that laparoscopic surgery is commonly available, and the recovery period is more tolerable as compared to the open laparotomy approach, an increase in inappropriate surgical interventions, including overuse of surgery occurs, and less effort is being expended toward understanding and preventing gallbladder disease. Very few laboratories in the United States, and for that matter, throughout the world, are now focusing on gallbladder motility. However, a sheer number of gallbladder surgeries that are performed (> 700,000/year in the United States alone), the risks and costs associated with these surgeries, and the side effects of gallbladder removal make the goals of developing nonsurgical approaches to prevent gallstone disease and/or restore gallbladder function very important.
A hallmark feature of gallbladder disease is decreased contractility of gallbladder smooth muscle (GBSM). Therefore, understanding GBSM physiology and the regulation of gallbladder contractility in normal and pathological conditions are very worthwhile. This chapter provides an overview of neuromuscular function in the gallbladder, along with a summary of neuromuscular control in the sphincter of Oddi, which works in concert with the gallbladder in the processes of interprandial bile storage and postprandial bile flow.
20.2
The Gallbladder
20.2.1
Properties of Gallbladder Ganglia and Neurons
The gallbladder wall consists of three layers: mucosa, muscularis, and serosa. A ganglionated plexus that lies at the interface between the muscularis and serosal layers provides the local innervation of the muscular and epithelial tissues of the organ, and in some species, ganglia are also sparsely distributed in the lamina propria of the mucosal layer ( Fig. 20.1 ). The main neuronal plexus is comprised of an array of small, irregularly shaped ganglia that are arranged in no discernible pattern. Extrinsic nerves, which include sensory fibers, vagal preganglionic fibers, and sympathetic postganglionic fibers, enter the gallbladder by following blood vessels in peri- and paravascular nerve bundles that interconnect with the ganglionated plexus. The muscularis and mucosal layers also contain nerve fibers that represent a mixture of projections from intrinsic neurons, sympathetic postganglionic nerves, and sensory fibers.
The neurophysiological properties of gallbladder neurons have been determined by intracellular recording techniques in whole mount preparations of guinea pig, opossum, and human gallbladders. Gallbladder neurons rarely exhibit spontaneous activity and they are rapidly accommodating, regardless of the amplitude or duration of the current pulse. These properties suggest that extrinsic nerves, hormones, or inflammatory mediators drive gallbladder neurons. Therefore, gallbladder neurons only release their neurotransmitters onto target tissues in response to incoming commands.
Gallbladder neurons receive fast and slow excitatory synaptic inputs. The fast excitatory postsynaptic potential (EPSP) is mediated by the release of acetylcholine from vagal preganglionic nerve endings, and often causes action potential generation. The slow EPSP is a prolonged depolarization that is not associated with neuronal firing. Therefore, the vagal preganglionic axons represent the principal driving force for generating neuronal output from gallbladder ganglia to the smooth muscle. As described below, the efficacy of ganglionic transmission can be modulated by physiological signals that act presynaptically on vagal terminals or postsynaptically on gallbladder neurons.
Immunocytochemical and histochemical studies have demonstrated that a variety of neuroactive compounds are expressed by gallbladder neurons. In every species studied to date, including human, dog, guinea pig, opossum, and Australian possum, all gallbladder neurons are immunoreactive for choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine, indicating that all gallbladder neurons are cholinergic. Other additional neuroactive compounds and associated synthetic enzymes have been detected in these neurons, including tachykinins, neuropeptide Y (NPY), nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), orphanin FQ (OFQ), cocaine and amphetamine regulated transcript peptide (CART), galanin (GAL), and somatostatin (SOM). The coexpression patterns of these compounds among gallbladder neuronal subpopulations are species specific. For a detailed overview, and appropriate references, the reader is referred to Balemba et al.
20.2.2
Properties of GBSM and Interstitial Cells of Cajal
Although the gallbladder is a tonic organ, individual GBSM cells have phasic electrical and contractile properties ( Fig. 20.2 ). Gallbladder tone appears to result from the collective action of independently paced contractions of individual bundles of GBSM cells. Within the muscularis layer, smooth muscle cells are not arranged in sheets; rather, the muscularis consists of smooth muscle bundles that are interposed and oriented in various directions like a ball of string. The smooth muscle cells within a given bundle are arranged in parallel, and they appear to be electrically coupled since they have a very low-input resistance when evaluated with intracellular electrodes in ex vivo preparations, and they exhibit rapid Ca 2 + transients, called flashes, that occur simultaneously in all of the smooth muscle cells of a given bundle.
20.2.2.1
Electrical and Ionic Properties of GBSM Cells
The electrical and ionic properties of GBSM cells have been elucidated by intracellular microelectrode recording studies of intact preparations and by patch clamp recording studies involving isolated myocytes ( Fig. 20.3 ). Rhythmic spontaneous action potentials are a feature of GBSM cells, and these events consist of a rapid spike followed by a plateau phase. GBSM action potentials are briefer and occur at a higher frequency than the slow wave potentials of gastrointestinal smooth muscle, with a plateau duration of 300–500 ms and a frequency of about 0.4 Hz. A number of ionic currents have been detected in GBSM, but two currents that provide the major influence on the contour of the action potential are the large conductance, dihydropyridine-sensitive (L-type) Ca 2 + channel, and the delayed rectifier K + (K V ) channel. While dihydropyridine-sensitive Ca 2 + channels and K V channels are clearly involved in the action potential of GBSM, the generation and pacing of the action potentials involve specialized pacemaker cells [see subsection below on interstitial cells of Cajal (ICC)].
Inhibition of ether-a-go-go-related gene (ERG) K + or nonselective cation channels also affects GBSM activity. Transcript and immunoreactivity for ERG channels are detected in GBSM, and in the presence of ERG channel blockers, the membrane potential is depolarized, the plateau phase of the action potential is prolonged, and multiple spikes are generated.
Action potential generation in GBSM is critically dependent on the resting membrane potential (about − 50 mV), which is ~ 35 mV more positive than the K + equilibrium potential, and a steady state, nonselective cation current contributes to the maintenances of the membrane potential of these cells. With L-type Ca 2 + and K V channels inhibited, we identified a novel spontaneously active cation conductance in GBSM that is mediated primarily by Na + influx. GBSM cells are hyperpolarized, and action potentials are eliminated when this current is inhibited by Na 2 + substitution, indicating that this nonselective cation conductance contributes to the regulation of GBSM excitability and contractility.
Ca 2 + -activated K + (BK) channels and ATP-sensitive K + (K ATP ) channels are also expressed by GBSM. While BK channel blockers do not appear to affect the membrane potential or action potential contour in GBSM, BK channels have been identified in patch clamp studies of GBSM, and they are responsible for spontaneous transient outward currents that are easily detectable in these cells. BK channel currents are activated by local release of Ca 2 + through ryanodine receptor channels (RyR) of the endoplasmic reticulum. While the functional relevance of these Ca 2 + “sparks” and associated transient outward currents in GBSM are not completely understood, they likely contribute to the regulation of GBSM excitability as they are inhibited by cholecystokinin (CCK).
K ATP channels appear to play a critical role in GBSM hyperpolarization and relaxation, and activation of their current mediates the action of inhibitory inputs to the GBSM. Exposure of intact GBSM to the K ATP channel activators lemakalim or pinacidil leads to a prolonged hyperpolarization that is associated with elimination of spontaneous action potentials, and the K ATP channel blocker, glibenclamide, blocks this effect. In isolated cells, the K ATP channel openers activate a glibenclamide-sensitive current. These channels appear to mediate the actions of inhibitory agonists in gallbladder muscle because the inhibitory effects of calcitonin gene-related peptide (CGRP) and histamine H 2 receptor agonists are blocked by glibenclamide. Activation of the K ATP channel involves the activation of the cyclic AMP-adenylate cyclase-protein kinase A signal transduction cascade, whereas activation of protein kinase C inhibits K ATP channel function.
20.2.2.2
Calcium Handling in GBSM
Influx of Ca 2 + into GBSM, and Ca 2 + release from intracellular stores are critical events for normal GBSM function since gallbladder excitability and contractility depend on [Ca 2 + ] i increases. Two types of rhythmic spontaneous Ca 2 + transients have been identified in Ca 2 + imaging studies of intact whole mount preparations of GBSM cells, Ca 2 + flashes, and Ca 2 + waves. Ca 2 + flashes are rapid Ca 2 + transients that are associated with the Ca 2 + influx that occurs during spontaneous action potentials in GBSM. Flashes occur simultaneously in all of the smooth muscle cells within a given bundle, but flashes in nonintersecting bundles exhibit asynchronous pacing.
Ca 2 + waves are intracellular Ca 2 + transients that propagate within a given smooth muscle cell, and these events appear to correspond to subthreshold depolarizations of GBSM cells. Like the spontaneous action potentials of GBSM, Ca 2 + flashes and waves are sensitive to L-type Ca 2 + channel and inositol triphosphate (IP 3 ) inhibitors. Excitatory agonists enhance Ca 2 + flash and wave activity in a phospholipase C-dependent manner, suggesting that they play a role in spontaneous excitability and pacemaking in GBSM. Interestingly, while Ca 2 + flashes are typically asynchronous among separate muscle bundles, they become synchronized in the presence of excitatory agonists. The spatiotemporal patterns of these events support a model in which asynchronous electrical and contractile activity of GBSM bundles throughout the muscularis layer is responsible for maintenance of net tone in the organ. Furthermore, synchronous global electrical rhythms that likely result from excitatory agonist stimulation may contribute to gallbladder emptying.
There are functional studies indicating that Ca 2 + entry through L-type Ca 2 + channels is required for GBSM to respond to excitatory neuro-hormonal inputs. However, CCK-induced contraction involves Ca 2 + release from intracellular stores, which have both inositol triphosphate (IP 3 ) receptors and RyRs, and are dependent on sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pumps for refilling and maintenance of intracellular Ca 2 + stores. As described above, Ca 2 + release from intracellular stores can also cause relaxation via release of Ca 2 + through RyR, as sparks, and activation of BK channels, resulting in membrane hyperpolarization and relaxation. These opposing actions of Ca 2 + , which is stored in a single intracellular domain, are likely to involve separate localizations of the IP 3 and RyR Ca 2 + release channels in GBSM cells. This allows for a more versatile and complex role of intracellular Ca 2 + mobilization in this cellular model. L-type Ca 2 + channels appear to be important for the maintenance of intracellular Ca 2 + stores in GBSM since emptying of these stores induces the activation of L-type Ca 2 + , as well as capacitative, channels. This could account for the inhibitory effect of Ca 2 + channel blockers on the actions of excitatory agonists in gallbladder muscle. Alterations in Ca 2 + handling (related to both Ca 2 + influx and release mechanisms) can be responsible for gallbladder stasis in inflammation.
20.2.2.3
ICC in the Gallbladder
As described above, GBSM cells exhibit spontaneous, rhythmic electrical activity and Ca 2 + transients, and these events are associated with contractions of individual muscle bundles. These events are not affected by inhibition of neuronal activity with tetrodotoxin, indicating that they are not neurally mediated.
There are strong morphological and physiological data in support of the concept that specialized cells, comparable to the ICC of the gastrointestinal tract, exist in the gallbladder, and that these cells generate spontaneous rhythmic activity in the gallbladder muscularis. The tyrosine kinase receptor protein, kit, which has been used as a marker of ICC in the gut, is detectable infusiform-shaped cells that are present in GBSM, oriented in parallel to the muscle cells. The gold standard for identification of ICC is their ultrastuctural features, and at the electron microscopic level, cells with features of ICC have been identified. These features include electron dense cytoplasm and an abundance of mitochondria and calveoli, and these cells are observed in direct contact with one another, and with smooth muscle cells.
Physiologically, Ca 2 + imaging studies provide strong evidence for the existence of ICC with a pacemaker function in the gallbladder. Like smooth muscle, these cells exhibit Ca 2 + flashes, but interestingly, the flashes persist in ICC-like cells in the presence of gap junction blockers, whereas they are diminished in smooth muscle cells. This indicates that these cells can generate spontaneous activity, but smooth muscle cells cannot, and it is consistent with our finding that in isolated GBSM cells, we do not detect spontaneous rhythmic activity even when the cells are warmed to 36°C. Collectively, the findings described above indicate that the spontaneous, rhythmic activity that is detected in GBSM, and which corresponds to smooth muscle bundle contractions, is generated by specialized ICC-like cells, and is not an intrinsic property of GBSM. Tan and colleagues have reported a decrease in kit expression in gallbladders from subjects undergoing cholecystectomy for gallstone disease, indicating that a decrease in ICC could contribute to hypomotility in this disorder, as has been reported for slow transit constipation and gastroparesis.
20.2.3
Neurohormonal Mechanisms Associated With Gallbladder Emptying
20.2.3.1
Hormonal CCK
Postprandial gallbladder contraction is triggered by the release of CCK from enteroendocrine cells in the epithelial lining of the duodenum. In 1928, Ivy and Oldberg solidly established the concept that CCK acts as a hormone to cause gallbladder emptying ; however, it is also likely that CCK acts at several sites to promote gallbladder contraction. The most direct means of gallbladder contraction is for hormonal CCK to act on receptors located on GBSM, which expresses the CCK 1 , but not the CCK 2 receptor. It is likely that the low-affinity CCK 1 receptor is present in GBSM, since the EC 50 for the CCK-induced gallbladder contraction is in the 10–50 nM range. It is unclear whether CCK receptors on GBSM are a normal physiological site of action for CCK, because postprandial concentrations of CCK in the serum are in the 10–20 pM range, which is below the threshold for a direct action of CCK on gallbladder muscle strips. Furthermore, meal-induced gallbladder contractions and contractions induced by physiological concentrations of CCK in vivo are significantly attenuated by neural blockade in several species, including human. Consistent with this, hexamethonium, which blocks the vagal preganglionic input to gallbladder neurons, inhibits CCK- and meal-induced gallbladder contractions. Therefore, a neural mechanism is likely involved in the prokinetic effects of postprandial CCK release.
While gallbladder neurons do not respond directly to the peptide, CCK has a potent presynaptic excitatory effect on nerve terminals in gallbladder ganglia. CCK increases acetylcholine release from vagal efferent nerves terminating on gallbladder neurons. The concentration-effect relationship of the presynaptic action of CCK in gallbladder ganglia indicates that CCK can act physiologically at this site.
It is likely that postprandial gallbladder contractions are also promoted by CCK, released from enteroendocrine cells, acting via a paracrine mechanism on vagal afferent nerve fibers in the lamina propria of the duodenum. Subdiaphragmatic vagal afferent fibers are sensitive to CCK, and postprandial physiological responses, such as increased gastric motility and pancreatic secretion, have been attributed to CCK-mediated increases in vagal afferent activity. Following a meal, CCK stimulates vagal afferent nerve fibers, which act in the vagal motor complex to increase the rate of firing of vagal preganglionic neurons.
20.2.3.2
Acetylcholine
As described above, all gallbladder neurons are cholinergic, and release of acetylcholine likely represents the major neuronal signal for GBSM contraction. Electric field stimulation (EFS) of nerves in gallbladder muscle strips elicits a contraction that is blocked by the muscarinic receptor antagonist, atropine. M 3 muscarinic receptors, mediate the contraction via activation of the phospholipase C-inositoltriphosphate pathway.
20.2.3.3
Tachykinins
Tachykinin-containing nerves are widely distributed in the gallbladder, including extrinsic afferent nerves coexpressing CGRP as well as gallbladder neurons and their axons, and it is likely that tachykinins can elicit action in gallbladder ganglia and in the muscularis. In gallbladder ganglia, tachykinins depolarize gallbladder neurons and increase their excitability via activation of the neurokinin-3 (NK 3 ) receptor. Antagonism of the NK 3 receptor attenuates capsaicin-induced depolarizations and slow EPSPs indicating that tachkinins are released in gallbladder ganglia and are likely to mediate long-lasting excitatory synaptic events. These results suggest that the tachykinin/CGRP-immunoreactive sensory fibers could serve as the afferent limb of a local axon reflex circuit within the wall of the gallbladder. It is possible that in response to inflammation or elevated intraluminal pressure, tachykinins and CGRP may be released within ganglia by sensory fibers and act directly on intrinsic neurons to facilitate ganglionic transmission. Physiological or pathophysiological modifications of the peptidergic content of the afferent innervation could result in changes in the modulation of efferent nerves or smooth muscle contractility.
Tachykinins also produce a direct, concentration-dependent contraction of the isolated gallbladder muscle strips that likely involves NK 2 receptors. As for acetylcholine, this contractile response involves the phospholipase C-inositol triphosphate pathway. The fact that tachykinins are coexpressed with acetylcholine in gallbladder neurons indicates that these compounds may act together to promote gallbladder emptying upon vagal stimulation. However, since most of the neurally mediated contractile response of gallbladder muscle strips is blocked by atropine, it is not clear how much of a role tachykinins play in neurally mediated gallbladder contractions, and therefore, gallbladder emptying.
20.2.4
Neurohormonal Mechanisms Associated With Gallbladder Filling
The triggers and mechanisms that regulate gallbladder filling are not as well understood as those that mediate gallbladder emptying. Until somewhat recently, some felt that gallbladder filling was a passive process involving the Law of Laplace. However, a candidate for hormonally mediated gallbladder relaxation during filling, fibroblast growth factor (FGF), has emerged. Also, several neuroactive compounds that are capable of eliciting gallbladder relaxation have been identified in gallbladder nerves. These include CGRP, norepinephrine, VIP, PACAP, and nitric oxide (NO), all of which can induce GBSM relaxation when applied to muscle strips. However, it is difficult to resolve which of these neurotransmitters could act physiologically to promote gallbladder filling, as many of their effects on the gallbladder have been observed at supraphysiological concentrations, and because they are costored with excitatory neurotransmitters. Described below are several potential mechanisms that could contribute to this process.
20.2.4.1
Fibroblast Growth Factor
Murine FGF15 and its human ortholog, FGF19, play an active role in liver bile acid biosynthesis. In the intestine, bile acids activate the nuclear farnesoid X receptor (FXR), which in turn leads to transcription and release of FGF. Recently, strong evidence has been provided for a role of FGF in gallbladder relaxation and filling. The gallbladders of FGF15 null mice and mice devoid of the FGF4 receptor are contracted and almost devoid of bile, and administration of FGF15 or its human ortholog, FGF19, in these mice leads to gallbladder filling. Furthermore, FGF relaxes precontracted GBSM strips, and administration of FGF15 or FGF19 in vivo leads to a rapid and dramatic increase in gallbladder volume. The precise mechanisms are not yet understood as FGF19-induced gallbladder filling in mice lacking the FGF4 receptor, and FGF15 is not expressed in the extrahepatic biliary tract. However, the findings to date do support the concept that FGF15/19 released from the ileum, in response to bile salts, causes gallbladder relaxation and filling.
20.2.4.2
Potential Inhibitory Actions of Gallbladder Neurons
Subsets of gallbladder neurons are VIP or PACAP-immunoreactive and also immunoreactive for NOS. VIP is released by nerves in the gallbladder in response to electrical stimulation of the vagus nerves. VIP and PACAP cause relaxation of resting or precontracted gallbladder muscle strips from several species, including human, probably through the activation of adenylyl cyclase and increased cAMP levels. A broad range of intracellular processes are affected by cAMP to decrease GBSM excitability and induce relaxation, including smooth muscle hyperpolarization, resulting from activation of K ATP and BK conductances ; reduction of [Ca 2 + ] i due to the inhibition of both Ca 2 + influx and Ca 2 + release from sarcoplasmic reticulum; and desensitization of the contractile machinery to Ca 2 +.
Lines of evidence for nitrergic relaxation of GBSM include the findings that inhibition of NOS increases gallbladder tone, increases agonist-induced contractions, and abolishes electrically induced nonadrenergic, noncholinergic relaxation. Furthermore, NOS inhibition reduces stimulation-induced neurogenic relaxations, and NO donors have an inhibitory effect on gallbladder tone, which are mediated through guanylate cyclase activation.
Carbon monoxide (CO) released from intrinsic gallbladder neurons may also play a role in gallbladder filling. It has been demonstrated that gallbladder neurons express the synthetic enzyme, hemeoxygenase 2, CO relaxes gallbladder muscle strips, and inhibition of hemeoxygenase 2 diminishes nonadrenergic-noncholinergic relaxations.
Despite these findings, it is difficult to conceive how gallbladder neurons could provide an unambiguous inhibitory signal to GBSM since all gallbladder neurons express ChAT, and are therefore likely to be cholinergic and excitatory. Activation of these neurons would lead to release of acetylcholine plus other compounds such as NO and/or VIP, thus providing the GBSM with a mixed signal.
20.2.4.3
Inhibitory Actions of Sensory Nerves in the Gallbladder
The neuroactive peptide CGRP, which is present in extrinsic sensory fibers, can decrease tension in gallbladder muscle strips and cause a hyperpolarization of GBSM cells. However, as described above for inhibitory actions of intrinsic gallbladder neurons, it is unlikely that CGRP released from gallbladder afferent fibers contributes to physiological relaxation of the gallbladder because tachykinins are costored with CGRP in these nerve fibers, and, as described above, tachykinins have an excitatory effect on GBSM ( Fig. 20.4 ).
20.2.4.4
Inhibitory Actions of Sympathetic Nerves in the Gallbladder
Norepinephrine released from sympathetic nerves could contribute to gallbladder relaxation and filling. Splanchnic nerve stimulation decreases gallbladder tone. EFS-induced relaxation of cat gallbladder muscle strips is blocked by the β-adrenoreceptor antagonist, propranolol, whereas the relaxation is unaltered by VIP receptor antagonists or by NOS inhibition. In addition to directly relaxing GBSM, sympathetic nerves can facilitate gallbladder filling by decreasing vagal tone in the organ. Sympathetic nerves innervating the gallbladder activate α 2 adrenoreceptors located on vagal terminals in gallbladder ganglia and a suppression of vagal synaptic input to gallbladder neurons. Consistent with this, stimulation of sympathetic nerves at low intensities results in a decrease in the excitatory response to vagal stimulation.
20.2.4.5
Glucagon-Like Peptides
Glucagon-like peptides (GLPs) are secreted in the distal ileum from enteroendocrine L cells in response to nutrients and bile acids. They control metabolism via actions on structurally related yet distinct G-protein-coupled receptors, GLP-1 and GLP-2. GLP-1 regulates gut motility, appetite, islet function, and glucose homeostasis, whereas GLP-2 enhances intestinal nutrient absorption. GLP-1R agonists are used to treat diabetes and obesity, and a GLP-2R agonist is approved to treat short bowel syndrome. An investigation of the actions of GLP receptor activation on gallbladder motility has demonstrated that GLP-2 receptor activation increases gallbladder volume in vivo and decreases spontaneous activity in GBSM bundles ex vivo. Like the inhibitory transmitters, which elicit their actions via an adenylate cyclase-protein kinase A-K ATP channel pathway, the GLP-2 receptor signals through this same pathway. Also, as with the FGF agonists in development, compounds that act on GLP receptors should be assessed for their effects on gallbladder motility.
20.2.5
Interprandial Gallbladder Motility
Between meals, the gallbladder undergoes periods of increased pressure in synchrony with Phase II of the migrating myoelectric complex (MMC). The purpose of this interprandial gallbladder motor response, which occurs simultaneously with increased antral and duodenal motor activity, is thought to be associated with clearing cellular and undigested debris from the intestines, and also with helping maintain the enterohepatic circulation of bile salts. Increases in gallbladder motility associated with Phase II of the MMC, as well as those induced by motilin, are attenuated by atropine or hexamethonium, indicating that this response is neurally mediated. It is not yet clear whether this involves a vagal reflex or if it is mediated by the neural circuitry that exists between the gut and the gallbladder. Acute inhibition of vagal reflex activity leads to a decrease in MMC-related gallbladder emptying ; however, MMC-related contractile activity is relatively normal after chronic vagotomy.
20.2.6
Muscle Contractility in the Developing and Aging Gallbladder
Smooth muscle development normally proceeds in a well-orchestrated manner before and after birth. Rapid growth and maturation of smooth muscle-containing organ systems are hallmarks of fetal development. The maturation of smooth muscle during fetal and postnatal development correlates with the need for increasing contractile capacity brought about by emerging demands for organ function. In the GI tract, smooth muscle cells appear as early as at 5 weeks of gestation, and smooth muscle layers thicken with increasing gestation, which continues well beyond after birth. The source(s) of Ca 2 + for smooth muscle contraction in neonatal animals is not well understood, but reports to date present support for both Ca 2 + -dependent and Ca 2 + -independent contractions. Agonists and high K + contract neonatal gallbladder strips, but the contractility was significantly lower than in adult strips despite the finding that Ca 2 + release in neonatal GBSM is higher than in adult tissue. The higher level of Ca 2 + mobilization is related to a number of factors, including relatively high intracellular Ca 2 + stores in neonatal cells, a lower efficiency of plasmalemma Ca 2 + extrusion mechanisms, and an increased release of Ca 2 + via the SERCA pump in response to phospholamban (PLB) phosphorylation. Furthermore, Ca 2 + sensitization is lower in neonatal cells, which is a likely contributor to the lower contractile activity despite the elevated Ca 2 + availability. These data emphasize that GI smooth muscle contractility early at birth is not upregulated by Ca 2 + sensitization mechanisms and mainly relies on Ca 2 + release from a fully loaded sarcoplasmic reticulum, the result of undeveloped Ca 2 + extrusion mechanisms and PLB-mediated SERCA inhibition.
The prevalence of GI motor dysfunctions related to motility is higher in older than in younger adults. However, few studies have investigated the cellular and molecular mechanisms responsible for such dysfunctions in aging and the effects of aging in GBSM. The study of Gomez Pinilla et al., shows that when gallbladder strips from aged animals were electrically stimulated, a significant decrease in EFS-induced contraction at all of the frequencies tested was recorded and there was a long-lasting off-relaxation in the strips from older animals, but not in the younger animals. The aged-related impairment in gallbladder contractility could involve changes in the intrinsic innervation of the organ, alterations in the smooth muscle contractility itself, or a combination of these mechanisms. In fact, acetylcholine-induced response was significantly reduced in aged animals suggesting an age-related impairment in the myogenic response to the neurotransmitter released from intrinsic nerves. However, when the expression of an EFS induced response to acetylcholine-induced contraction was calculated, the response was higher in strips from aged animals, indicating an increase in the release of acetylcholine or a decrease in the release of inhibitory neurotransmitters. A lack of the release of NO was shown in aged strips that also showed an increase in sensory innervation in aged strips after both glibenclamide treatment or capsaicin desensitization suggesting that, in aging, EFS stimulates CGRP-containing sensory nerves, which could contribute to the reduction of gallbladder responsiveness to electrical stimuli via physiological antagonism. The fact that capsaicin effects were mediated by the CGRP-1 receptor (blocked by human CGRP8-37) and its downstream target K ATP channels (blocked by glibenclamide) clearly suggests that hypersensitivity of inhibitory sensory fibers containing CGRP contribute to gallbladder motility disorders in aging.
Calcium sensitization is a pathway that leads to smooth muscle contraction independently of changes in [Ca 2 + ] i by means of inhibition of myosin light chain phosphatase. Aging has negative impacts on the gallbladder contractile response to acetylcholine, which could be due to the impairment of calcium sensitization pathways that evokes smooth muscle contraction by inhibition of myosin light chain (MLC) phosphatase and the consequent dephosphorylation of MLC20 in the presence of basal (Ca 2 + -dependent or Ca 2 + -independent) or increased MLC kinaseactivity. In keeping with this, the study of Gomez-Pinilla et al. describes that calcium sensitization signaling is negatively affected by aging in the guinea pig gallbladder, which reduces contractility in aged strips due to a decrease in the phosphorylation of MLC20. This impairment in calcium sensitization that contributes to the reduced gallbladder contractility that is associated with aging is driven by a reduction in the protein expression of the proteins of both ROCK and PKC pathways. These findings, together with results obtained in gallbladders from newborn guinea pigs, draw the complete picture about the state of calcium sensitization along life. In guinea pig gallbladder, calcium sensitization pathway increases from birth to adult ages and after that it suffers a decline. However, calcium handling runs in the opposite direction, with a reduction after birth and partial maintenance with aging. In the three age points, the state of calcium sensitization matches better with the contractile capacity than calcium signaling does.
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