Patch clamp techniques will be used to investigate the regulation of secretion from nerve terminals of the posterior pituitary (neurohypophysis). These nerve terminals secrete two neuropeptides, vasopressin, which is involved in cardiovascular function, and oxycytocin, which is involved in reproductive function. In thin slices, single nerve terminals are accessible to patch clamp techniques, making it possible to study cellular and molecular mechanisms of secretion. Opioid and dopamine receptors can be activated to enhance or inhibit neuropeptide release from the posterior pituitary. Experiments proposed will investigate the consequences of activation of these receptors in pituitary nerve terminals, and elucidate the underlying mechanisms of transduction. The ion channels coupled to these receptors will be identified and characterized. The mechanism of coupling between receptors and effectors will be investigated by clarifying the roles of G-proteins, protein kinases, and protein phosphatases. Hypotheses will be tested concerning the influence of action potential shape on secretion and the relationship between Ca2+ entry, changes in intracellular Ca2+ concentration, and hormone secretion. These experiments will provide a better understanding of how neurotransmitters regulate neuropeptide secretion from neurosecretory neurons. The results should also be relevant to broader issues of regulation of synaptic transmission, and how drugs act on presynaptic opioid and dopamine receptors. Experiments will also explore the mechanism by which Ca2+ triggers exocytosis, and how Ca2+-sensing proteins participate in this process. Quantitative kinetic techniques will be used to measure rates of secretion under defined levels of intracellular Ca2+. Reagents will be introduced that are specific for identified proteins believed to play critical roles in Ca2+-triggered secretion. Using reagents for CAPS and synaptotagmin, two proteins with demonstrated roles in neurosecretion, experiments will provide insight into how these proteins couple excitation and secretion in nerve endings. These issues are relevant not only to the physiological regulation of blood pressure and volume, lactation, and parturition, which are controlled by the two neurohypophysial hormones, but are also relevant to a broad class of systems in which synaptic plasticity occurs, and in which mechanisms of synaptic plasticity are believed to be presynaptic.
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