Our research program is directed towards studying the biochemistry and physiology of the nerve terminal. The neurohophysial nerve terminals are used as the model system, and secretion of vasopressin and oxytocin from isolated intact posterior pituitaries and isolated neurosecretosomes, and ionic channels on the neurosecretory vesicle membrane are studied. The secretion of peptides from intact neural lobes was found to be facilitated by increasing the stimulus frequency. Experiments using K+ channel blockers suggested that action potential widening caused by inactivation of transient K+ channels may form the molecular basis of this frequency-dependent facilitation. A neurotoxin from Dendorsis Angusticeps which blocks transient K+ channels was found to enhance hormone secretion under low frequency stimulation, suggesting that these channels may be involved in the innate facilitation. Secretion from oxytocin terminals was found to be inhibited by the opiate kappa receptor agonist, dynorphin, released from vasopressin terminals. The nerve endings (neurosecretosomes) were shown to contain kappa type opiate receptors. The preparation of neurosecretosomes has been maintained under tissue culture conditions for over a week in order to study hormone secretion and binding of ion channel blockers and receptor probes in these nerve endings. This preparation is also suitable for high resolution fluorescence and differential interference contrast microscopy and analysis of ionic channels. Patch clamp studies on intermediate lobe cells revealed the presence of three different types of calcium channels. A method was developed for classification of these three types of channels based on their inactivation kinetics. Bilayer incorporation studies using isolated neuro-secretory vesicles have led to the identification of a calcium-activated cation channel with large conductance (greater than 400 PS) and an anion channel on these membranes. These channels may be important in the mechanism of exocytotic secretion. A novel high affinity calcium binding protein was identified in nerve endings of the posterior pituitary and partially purified.
