Gary M. Mawe, PhD - US grants

A great deal of morbidity is caused by diseases of the gallbladder; nevertheless, the control of the motile and absorptive functions of the organ are incompletely understood. Although the musculature of the gallbladder responds to hormones, such as cholecystokinin (CCK), there is also a prominent ganglionated neural plexus in the gallbladder wall. The functions of this intrinsic nervous system, and its interaction with hormones re unknown. In order to gain a complete understanding of the physiology of the gallbladder, it is important to understand how the nervous system of the gallbladder is organized, to what extent it receives input form external sources (such as the CNS, sympathetic, and sensory ganglia), and whether or no the actions of hormones on the gallbladder are mediated in whole or in part through neurons. In the current proposal, therefore, the structure of the ganglionated plexus of the gallbladder is analogous to that of the gut (the enteric nervous system (ENS) has a unique structure and chemistry, which reflect an ability to mediate reflex activity independently of control by the CNS) or whether gallbladder ganglia instead are parasympathetic relay ganglia. These studies will include: (1). analysis of the organization of gallbladder ganglia (presence or absence of collagen, basal laminae, method of axonal ensheathment by supporting cells); (2). determination of whether the supporting cells f gallbladder ganglia are glial or Schwann cells; (3). determination of whether or not specialized capillary endothelia provide a blood-tissue barrier for gallbladder ganglia; (4). characterization of the unusual tyrosine hydroxylase-containing neurons of the gallbladder ganglia. The extrinsic sources of neural input to the gallbladder will be investigated through the use of retrograde tracers. This study will test the novel hypothesis that the gallbladder projects to and receives input from the ENS. The membrane properties and types of synaptic input to the intrinsic neurons of the gallbladder will be studied with intracellular microelectrodes. An aim of this investigation will be to compare the physiological properties of gallbladder neurons to neurons of the ENS on the one hand, and to extraenteric autonomic ganglia on the other. Finally, the effects of CCK and putative neurotransmitters on gallbladder neurons will be analyzed electro-physiologically to attempt to determine the role that the neurons play in the action of these compounds on the gallbladder. Previous studies of the effects of drugs and hormones on neurons of the gallbladder and on gallbladder motility have involved application of the compounds while recording intraluminal pressure or contraction of gallbladder muscle strips; however, the contractile response to given substance is likely to be the net result of a complicated series of actions on a variety of the neurons that comprise the intrinsic circuits of the gallbladder's nervous system. It is therefore essential to study the effects of drugs, transmitter candidates, or hormones on individual neurons, as will be done in the current proposal.

[unreadable] DESCRIPTION (provided by applicant): A hallmark of biliary tract disease is a decrease in the tone of gallbladder smooth muscle (GBSM), and a decrease in the responsiveness of GBSM to excitatory agonists. Despite the importance of GBSM excitation, contraction (EC) coupling, little is known about this process. The objectives of this proposal are to establish the mechanisms for GBSM excitation and how this translates to muscle contraction. The first Specific Aim is to determine the cellular mechanisms that are responsible for electrical rhythmicity in GBSM. Our preliminary data indicate that the gallbladder rhythmicity departs radically from the mechanisms that dominate control in other parts: of the GI tract. The novel possibility that the action potential of GBSM is terminated by ERG K+ channel activation will be explored. The ERG channel is a target of a number of clinically relevant drugs including the prokinetic agent, cisapride. We will be particularly interested in determining whether the GBSM ERG channel differs pharmacologically from the cardiac muscle channel, with the hopes of ultimately guiding studies on the development of GBSM specific ERG blockers. The second Specific Aim will determine the mechanisms by which excitatory agonists such as acetylcholine and CCK stimulate GBSM, with a focus on non-selective cation channels, specifically: TRP channels. The role of K+ channel inhibition in the excitatory: effects of agonists will also be determined. The third Specific Aim will determine how excitatory agonists alter the pattern of calcium signaling in GBSM. We will test the novel concept that excitatory agonists shift calcium signaling from sparks - a smooth muscle relaxation mechanism through activation of K+ channels - to calcium waves, a signal that contributes to contraction. We propose an integrated approach using state-of-the-art techniques to determine GBSM function from single molecules to the intact tissue. Together with our previous studies on gallbladder nerves, we expect to provide a comprehensive view of gallbladder function, which should illuminate new pathways to handle biliary tract disease.

Between 10 and 20 percent of the US population suffers from diseases of the extrahepatic biliary system. This system, which includes the gallbladder, the biliary ducts, and the sphincter of Oddi (SO), must work as a unit to store and concentrate bile between meals, and deliver bile to the lumen of the duodenum following meals. Little is known about the role of nerves in the control of motility in this system, especially with regard to the SO. The SO is a smooth muscle sphincter that surrounds the opening of the common bile duct as it passes through the wall of the duodenum. This sphincter has several functions, including regulation of the flow of bile and pancreatic juices into the gut, and preventing the regurgitation of intestinal contents into the bile or pancreatic ducts. The function of the SO is influenced by extrinsic and intrinsic nerves, as well as by hormones. Within the wall of the sphincter, there exists a plexus of, nerves that comprises clusters of nerve cells, or ganglia. This ganglionated plexus, which is the target of extrinsic neural inputs, is likely to be a primary regulator of the contractile state of the SO. The properties of this neural plexus, and the mechanisms by which it controls the SO, are largely unknown. However, related studies indicate that hormones that relax the SO actually act, at least in part, on neurons in this plexus, rather than directly on smooth muscle. This indicates that the ganglionated plexus of the gallbladder may be a site where external inputs are received and integrated, and where the final signals that control the motility of the SO are generated. The studies proposed in this grant are designed to elucidate the morphological, neurochemical, and physiological features of the intrinsic neural elements of the SO. Studies of the ultrastructure of SO ganglia and the structure of individual neurons within these ganglia will be done. Also, the neurotransmitters that act on the neurons of the SO, and those that are generated by SO neurons, will be determined immunohistochemically. Retrograde tracing studies will be done to determine the sources of extrinsic neural input to the ganglionated plexus of the SO, and to determine whether the circuitry exists for synaptic interaction between neurons in the SO and those in the wall of the gallbladder. Intracellular recording techniques will be used to determine the electrical properties of SO neurons and the types of synaptic input that they receive. These techniques will also be employed to determine how hormonal and sympathetic inputs effect the neurons of the SO, and therefore, how they might influence the output of SO ganglia. In these studies, CCK and norepinephrine will be applied to SO neurons, and both direct and presynaptic actions of the compounds will be assessed. The studies that are outlined in this proposal represent the most in-depth study ever, of the structure and neurochemistry of the ganglionic plexus of any gastrointestinal sphincter. These are also the first studies undertaken, using intracellular recording techniques, to assess the electrical and synaptic properties of the neurons of a smooth muscle sphincter of the bowel.

DESCRIPTION (provided by applicant): Intestinal inflammation leads to changes in a variety of functions, including motility, secretion and sensitivity. Neural circuits of the bowel regulate ll of these functions, and it is likely that changes in these reflex circuits contribute to the symptos suffered by afflicted individuals. In the past 8 years, we have evaluated inflammation-induced changes along the circuitry of the colon in a step-wise fashion, and we have identified fundamental changes at several sites, including: (1) increased serotonin availability in the mucosal layer; (2) intrinsic sensory neuron hyperexcitability; (3) facilitation of synaptic signals between neurons; and (4) attenuated inhibitory purinergic neuromuscular transmission. Furthermore, we have elucidated the mechanisms that underlie many of these changes, determined what changes persist following recovery from inflammation, and linked changed in neural function to altered motility patterns. In this grant application, we are proposing to build upon our findings and those of others to examine novel mechanisms by which gut functions and bone density can be affected by inflammation, and explore innovative approaches to prevent or reverse these changes and to minimize mucosal damage during the inflammatory response. The first aim of this grant application is designed to test the hypothesis that the inflammation- induced decrease in purinergic neuromuscular transmission involves a decrease in purine synthesis and release as the result of oxidative stress damage to mitochondria in the muscular is of the inflamed colon. Experiments proposed in the second aim is based on our recent discovery that 5-HT4 receptors (5HT4Rs) are highly expressed in the colonic epithelium, and that activation of these receptors induces 5-HT, mucus, and Cl- secretion, and promotes propulsive motility. We will test the hypothesis that activation of 5-HT4Rs on cells in the epithelial lining has a protective effect, attenuating the severity of colitis and protecting colonc motor function. Specific aim 3 is based on the knowledge that various forms of intestinal inflammation are associated with decreased bone density, and the recent discovery that circulating gut-derived serotonin has a negative impact on bone formation. We will test the hypothesis that the inflammation-induced increase in mucosal serotonin availability contributes to the decrease in bone density, and that these effects can be reduced by modulating mucosal serotonin signaling or by inhibiting the 5-HT1B receptor, which is expressed by pro-osteoblasts. This proposal involves an integrated approach, using state-of-the-art techniques, to investigate novel concepts related to the neuromuscular and serotonin signaling in the gut, and to examine a potential damaging relationship between mucosal serotonin and bone integrity. The findings of these studies will greatly improve our understanding of purinergic neuromuscular transmission and mucosal serotonin signaling in the gut.

Project Summary/Abstract Individuals with multiple sclerosis (MS) frequently suffer from autonomic dysfunction in addition to the neuromuscular ailments that represent their hallmark symptoms, and one of the most common and debilitating of their autonomic problems is constipation. Our theory is that constipation develops as a form of collateral damage from the central nervous system (CNS) inflammatory response that occurs in MS. During this inflammatory response, the patient's immune system generates antibodies that target proteins in the cellular debris of dying neurons and glial cells in affected areas, and these autoantibodies could have actions at distant sites. The enteric nervous system (ENS) is unique in that it contains intrinsic reflex circuitry that regulates motility, secretion and vascular tone in the gut, independently of CNS innervation. The ENS contains glial cells that share transcriptome features of oligodendrocytes and astrocytes, including proteolipid protein and myelin basic protein. These are two known antigenic targets in MS that could be incidentally targeted where they are also expressed in the ENS. This grant proposal is designed to test the hypothesis that autoantibodies generated in MS patients can target proteins on neuronal and glial membranes in the ENS, resulting in altered enteric neural reflex activity leading to constipation. In support of our hypothesis and the feasibility of our proposed studies, we have demonstrated that GI function is altered in mice with experimental autoimmune encephalomyelitis (EAE), the predominant mouse model of MS, in ways that are consistent with the development of constipation. Furthermore, we have found that blood samples from MS patients and EAE mice contain antibodies that bind to neurons and glial cells in the GI tract. In our proposed studies, we will examine the features of GI dysfunction in EAE mice, and confirm that the changes in gut motility involve circulating autoantibodies. We will determine the ENS cell types that MS autoantibodies bind to, and what effects these antibodies have on GI function in naïve mice. We will also directly examine the physiological activities of glial cells and neurons in the GI tract, and we will elucidate how these activities are altered in EAE mice, and in response to treatment with autoantibodies from MS patients. In these studies, we will also use colons from mice in which a calcium indicator protein is expressed by enteric glial cells or select populations of enteric neurons, and we will use intracellular recording to evaluate the strength of excitatory and inhibitory neuromuscular transmission. Finally, we will test potential therapeutic approaches for their effectiveness in alleviating constipation in the MS mouse model. Understanding the mechanisms responsible for constipation in MS is a clinically relevant goal since it is a critical step toward developing an effective therapeutic solution for the GI ailments of affected MS patients and others with autoimmune-mediated dysmotility.

Project Summary/Abstract Functional and inflammatory disorders of the gastrointestinal tract represent a significant burden to patients and to society. These disorders are difficult to treat, and recurrence of symptoms is common. As a result, there are ongoing efforts to develop more effective treatment options. The gut microbiome is now recognized as a dynamic entity that can influence a wide variety of physiological processes, ranging from the integrity of the intestinal epithelial barrier to brain neurochemistry and behavior. The clear success of fecal microbiome transplants in the treatment of C. difficile infections has highlighted the potential of utilizing the microbiome as a means of improving health. The studies that are included in this grant application are designed to exploit specific biochemical properties of certain bacterial strains to test novel treatment strategies for functional bowel disorders. We will test the hypothesis that tryptophan-synthesizing bacteria increase active tryptophan metabolites that enhance colonic motility through the activation of 5-HT4 receptors. Tryptophan is an essential amino acid that is typically acquired from the diet, but can also be produced by enteric bacteria. In addition to being incorporated into proteins, it serves as a precursor molecule for serotonin (5-HT), or it can be enzymatically converted to form kynurenine and its derivatives. In the epithelium of the intestine, enterochromaffin cells convert tryptophan to 5-HT. When 5-HT is released from these cells it triggers enteric reflexes that increase propulsive motility and epithelial secretion. Changes in 5-HT signaling have been identified in functional and inflammatory conditions of the gut, and drugs targeting 5-HT receptors have been developed for the treatment of constipation and diarrhea. In Specific Aim 1 of this proposal, we will test the hypothesis that treatment of mice with a bacterial strain that is known to produce tryptophan, and which has been used safely in probiotic formulations, results in increased mucosal 5- HT levels and enhanced intestinal motility. Specific Aim 2 of this proposal is aimed at determining the mechanisms by which tryptophan producing bacteria promote motility. The 5-HT receptor that is most directly linked to propulsive motility is the 5-HT4 receptor. We will test whether this receptor is involved and determine what tissues express the receptor that mediate this effect. We will also examine what changes in motility patterns and neuronal activity are associated with enhanced propulsive motility, and we will test whether increasing the tryptophan metabolite, tryptamine, can enhance the motility augmenting effect of tryptophan-producing bacteria. The results of these studies could provide a predictive translational strategy for the use of probiotic bacteria, with known biochemical features, for the treatment of constipation.