This is a revised proposal for investigation of adenosine receptors in the small intestine. The project is intended to test the general hypothesis that adenosine modulates neuronal activity and transmitter release by interacting with a heterogeneous population of receptors on myenteric neurons that may be coupled to the adenylate cyclase/cAMP system. The project will use electrophysiological and biochemical methods as well as enteric synaptosomes to study the transduction of adenosinergic messages in microdissected myenteric plexus and enzymatically dissociated ganglion preparations. The project is designed to classify the receptor subtypes which modulate the synaptic activity of AH/type 2 and S/type 1 neurons. The coupling of these receptors to adenylate cyclase will be tested in cAMP-dependent modulation studies with the novel protein kinase A agonist/antagonist pair Sp-adenosine-3'-5'-cyclomonophosphothioate (cAMPs)/Rp-cAMPs, pertussis toxin, specific cAMP-dependent phosphodiesterase inhibitors, the cyclase inhibitor MDL 12,330A, pituitary adenylate cyclase-activating polypeptide and cAMP measurements in dissociated myenteric ganglia. The role of the phosphoinositide/protein kinase C system in adenosinergic responses will be studied with protein kinase C stimulators and inhibitors. The action potential duration in AH/type 2 neurons will serve as a model system to study direct effects on calcium influx. The hypothesis that only AH/type 2 neurons possess somal adenosine receptors will be tested in studies that compare the morphology, electrophysiology and responses to adenosine. Endogenous adenosine may exert a tonic inhibitory tone on activity of myenteric neurons and the receptors involved may be linked to adenylate cyclase in AH/type 2 neurons. This hypothesis will be tested with interventions intended to block the actions of endogenous adenosine. Slow synaptic excitation in AH/type 2 neurons is believed to occur through activation of the cAMP second messenger system. The proposed studies will characterize the adenosine receptors that are involved in suppression of 5-HT-mediated slow excitatory postsynaptic potentials (slow EPSPs), elicited in AH/type 2 neurons by focal stimulation of interganglionic connectives. Inhibition of endogenous 5-HT release by purinergic agonists will be studied in isolated ganglia and enteric synaptosomes. Integration of the data from these unique model systems, will clarify the signaling mechanisms for adenosine in myenteric neurons. A model of neuronal hypoxia will also provide insight into the mechanisms by which endogenous adenosine suppresses neurotransmission in pathologic states of the gut such as ischemia and inflammation.
