Slow excitatory synaptic transmission is a mechanism neural communication in the enteric nervous system and is likely to participate in 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 and other autonomic neuropathies. Despite the clear significance, very little is known about the 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 myenteric slow synaptic potentials. The specific hypothesis to be tested is 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. The specific goals of these studies are to develop primary cultures of myenteric neurons from guinea pig ileum. Cultured neurons will be characterized using conventional electrophysiological and immunohistochemical 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 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. In order to determine if changes in intracellular calcium (Ca2+) disposition underlie alterations in K+ channel activity, intracellular Ca2+ levels will be studied using fura-2, a Ca2+-sensitive fluorescent dye. Simultaneous measurements of membrane current and Ca+ concentration will be accomplished using a photomultiplier tube and appropriate data acquisition/analysis hardware and software. Cell-attached and cell-free patch recordings will be used to study single K+ channels. These studies will allow direct measurement of voltage, Ca+ 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. These studies will begin to identify the specific molecular mechanisms by which enteric neurons regulate their own activity.
