Slow excitatory synaptic transmission is a mechanism of neural communication in the enteric nervous system and is likely to participate in the regulation of gut motility. Defects in postsynaptic intracellular mechanisms of slow excitatory synaptic transmission might contribute to the pathophysiology of poorly understood disorders of gut motility such as the irritable bowel syndrome, idiopathic pseudo-obstruction and disturbances in gut motility associated with diabetic autonomic neuropathies. Despite the significance, little is known about athe cellular mechanisms underlying slow synaptic excitation in myenteric neurons.The overall objective of this proposal is to characterize the receptors, the ionic basis and the intracellular transduction mechanisms producing slow synaptic potentials. The specific hypotheses to be tested are that receptors for the enteric neurotransmitters 5-hydroxytryptamine, acetylcholine and substance P are coupled via a G-protein to adenylate cyclase and phospholipase C. Activation of these enzymes results in activation of protein kinase A (PKA)) and protein kinase C (PKC). PKA and PKC phosphorylate two potassium (K+) channels whose function is to regulate neuronal resting membrane potential. The K+ channels are a voltage-independent background K+ channel and a calcium-activated K+ channel. Phosphorylation decreases channel open time leading to membrane depolarization and increased neuronal excitability. An additional mechanism for slow synaptic excitation present in 30% of myenteric neurons is by activating a chloride conductance possibly via an inositol trisphosphate- (IP3) mediated increase in intracellular Ca2+. The specific goals of these studies are to develop primary cultures of myenteric neurons from guinea pig ileum. Cultured neurons will be initially studied using conventional electrophysiological methods to establish that these neurons are similar to neurons studied in intact preparations of myenteric plexus in vitro. The initial studies will establish that primary cultures are an appropriate model for studies of slow synaptic signal transduction in myenteric nerves. Patch clamp methods will be used to study mechanisms of signal transduction. Nerve-mediated and agonist-induced responses will be studied using whole-cell voltage clamp to record membrane currents in single neurons. Drugs which inhibit or activate PKA- and PKC-dependent pathways will be applied in the extracellular medium or directly into neurons via the patch electrode. Cell-attached and cell-free patch recordings will be used to study single K+ channels. These studies will allow direct measurement of voltage, Ca2+ and phosphorylation-induced changes in K+ channel behavior. The effects of Ca2+ on channel kinetics will be studied using inside-out patches of membrane containing K+ channels. The Ca2+ concentration at the intracellular face of the membrane and the membrane potential can be controlled. The effects of channel phosphorylation on channel activity will be studied directly using inside-out patches of membrane and purified rat brain PKC and bovine brain PKA. Activation of the chloride conductance will be studied using whole cell methods and ion substitution and chloride channel blockers to characterize the chloride conductance. Drugs which activate or which activate or block IP3 sensitive Ca2+ release will be used to determine the signal transduction mechanism. These studies will begin to identify the specific molecular mechanisms by which enteric neurons regulate their own activity.
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