SODIUM REGULATION OF NEUROHORMONE SECRETION

拟议研究的长期目标是了解

The long range goal of the proposed research is to understand the mechanism(s) of sodium regulation of neurohormone/neurotransmitter release under resting conditions and in response to stimulation. The importance of elevated cytosolic Na+ to facilitation of neurotransmitter secretion following tetanic stimulation is well established. Current models suggest that the facilitatory action of Na+ results from alteration of Ca2+ regulation. Recently, however, we showed that intracellular Na+ ([Na+]i) regulates the rate of resting vasopressin (AVP) secretion from isolated neurohypophysial nerve endings under Ca2+ clamp conditions. This has led to the unconventional hypothesis that Na+ itself can regulate secretion and to the remarkable possibility that Na+ may modulate Ca2+-induced (i.e. stimulated) secretion. The proposed experiments will utilize nerve endings of the hypothalamo-neurohypophysial system, which posses unique anatomical advantages allowing resolution of the molecular events of the secretory process in nerve endings, to be studied in greater detail than in any other nerve endings. The specific aims are: 1) to utilize time-resolved membrane capacitance (Cm) measurements, under whole cell patch clamp on individual nerve endings to determine the Na+ requirements for exocytosis. Intracellular [Na+] will be measured by fluorescence spectroscopy. This approach will provide a quantitative evaluation of [Na+]i on the rate and extent of exocytosis and allow determination of the [Na+]i required at an exocytotic release site. 2) Using ionic substitution protocols on populations of nerve endings and radioimmunoassay for AVP release, we will determine the extent to which Na+ modulates Ca2+-induced secretion. 3) Using the intact neural lobe preparation we will investigate the effect of physiologic patterning of action potentials on the amplitude and time course of changes in [Na+]i. We will then measure, on single nerve endings, the secretory activity induced by similar changes using Cm measurements. We will attempt to quantitate the endogenous buffer capacity using fluorescence spectroscopy and measurements of Na+ currents. 4) We will attempt to determine if Na+-induced AVP secretion utilizes unique secretory mechanisms or shares mechanisms in common with Ca2+-induced secretion. Regulation by ligand receptor interactions or by antibodies against specific granule proteins of Na+-induced secretion in a manner similar to Ca2+ induced secretion will be determined. Similarity of regulation may suggest commonality of mechanism. The proposed experiments are crucial to describe the normal physiology of nerve endings both as they relate to learning, memory and disease of the nervous system and in understanding basic cellular mechanisms.