The intrinsic innervation of the bowel is unique and differs both structurally and functionally from any other region of the PNS. Among the major differences that set the enteric nervous system (ENS) apart form the remainder of the PNS are its ability to mediate reflexes independently of input from the CNS. Moreover, the ENS also sends axons centripetally to modulate transmission through prevertebral sympathetic ganglia. Even more strikingly, neurons of the myenteric plexus have recently been demonstrated to innervate accessory organs of digestion, including the pancreas. Although a great deal of progress has been made in identifying the many types of neuron in the ENS, critical elements of the microcircuits into which the ENS is organized have yet to be defined and virtually nothing is known about the enteric innervation of cells outside of the bowel.
The aim of the current proposal, therefore, is to define for the first time the cellular targets of projections of the ENS to another organ (the pancreas), to identify the myenteric plexus from putative sensory neurons of the submucosal plexus, and to visualize and characterize the cells that have been postulated to be """"""""command"""""""" neurons at the efferent interface between the intrinsic microcircuits of the ENS and the vagal input from the CNS. Making these experiments possible has been our recent progress in developing methods for tracing connections in microcircuits, such as those of the ENS, utilizing microinjections of the retrograde tracer, Fluoro-Gold, the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L) into individual enteric ganglia in surviving preparations and in fixed tissue, the carbocyanine dyes, DiI and/or DiO, which can be used as either anterograde or retrograde probes.
The specific aims of the proposal are to answer the following questions: [1] Which enteric neurons project out of the bowel to the pancreas: what are their targets in the pancreas and what is the relationship of submucosal and vagal efferent terminals to these neurons? [2] Upon which enteric neurons do vagal efferent axons terminate and what are the projections of these neurons? [3] What are the properties of those neurons in the submucosal plexus that project to myenteric ganglia? [4[ Upon which myenteric neurons do putative submucosal primary afferent neurons terminate and what are the projections of these myenteric neurons? Although a great deal of morbidity results form functional bowel disease these conditions are poorly understood and ineffectively treated. It is therefore important to learn more about the fundamental properties of the ENS, how it interacts with its input form the CNS, and how the ENS can affect other organs. Potential effects on other organs mediated by the EDS represents a virtually unexplored problem, because the projections of enteric neurons to organs such as the pancreas has just been recognized. Gant=R01NS27583 The copper-containing enzyme dopamine beta-hydroxylase (DBH) is an important enzyme in neurotransmitter biosynthesis catalyzing the conversion of dopamine to norepinephrine within the central and peripheral nervous systems. Abnormal levels of catecholamines are known to play an important role in hypertension and affective disorders, and a number of cases of congenital deficiency of DBH activity have been reported. The enzyme belongs to the class of copper-containing enzymes termed non-blue, in which a reduced Cu(I) intermediate acts as a site of oxygen binding and catalysis. Understanding the catalytic role of the copper centers and in particular that of Cu(I) in DBH and related Cu proteins is our major objective. Our recently developed multiple scattering method will be used to analyze EXAFS data of oxidized and reduced enzymes and their derivatives with substrates and inhibitors. This method allows the exact simulation of imidazole ligation, and conjunction with multifrequency EPR, will provide detailed information on histidine coordination and imidazole ring orientation. A novel and timely approach to the study of Cu(I) coordination chemistry will be the development and application of Fourier transform infrared and non- resonance-enhanced Raman spectroscopy to examine the interaction of the Cu(I) sites with inhibitor molecules such as CN and CO via measurement of the intraligand stretching frequencies. These studies will address important questions such as whether the Cu centers of DBH are mononuclear or dinuclear; whether the two copper atoms in the subunit are equivalent or inequivalent; and what is the role of each Cu center in catalysis. Mixed metal derivatives will be prepared to enable selective study of individual metal sites. Reaction with bifunctional inhibitors which cross-link substrate and the o2 binding site will selectively probe the copper which binds dioxygen, and cryogenic methods will be used to attempt to prepare Cu(I)-O2 complexes of the enzyme. In view of the proposed intermediacy of Cu(II)-OOH species in the catalytic mechanism, detailed studies on the reaction of H2 O2 and arylhydroperoxides with the enzyme will be undertaken. The experiments will be extended to the other non-blue copper proteins--amine oxidase, galactose oxidase, and lysyl oxidase.
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