Peptide secretion probably occurs from most neurons. It modulates CNS function as well as serving neuroendocrine regulation. Knowledge of peptide secretory mechanisms is limited to a very few experimentally tractable systems. This work proposes rigorous experiments on a uniquely suitable invertebrate preparation to understand how secretion of neuropeptides is related to physiological stimuli, the electrical responses and changes of cytoplasmic [Ca++] and [Na+] that these produce in the nerve terminals, and the possible modulation of these by hormones. The X-organ - sinus gland (XOSG) of the crab (Cardisoma carnifex) will be used. Analogous to the vertebrate hypothalamic-neurohypophyseal system in its role and physiology, it is purely peptidergic with 3 major hormonal activities that can be immunocytologically localized and quantified in perfusates by bioassay or ELISA. The XOSG is easily isolated; terminals and preterminal swellings form a neurohemal organ (the SG), and many are large enough for intracellular recording. Terminals can be dissociated for patch-clamping. Somata dissociated from the XO grow in defined culture. For each of the 3 hormonal activities, crustacean hyperglycemic hormone, red-pigment concentrating hormone and molt-inhibiting hormone, the following questions are to be addressed: Is the rate of basal secretion governed by [Ca++]i or [Na+]i, and what controls these? Is the response to stimulation accounted for by entry of Ca++, or is release from internal stores involved? Does [Na+]i influence evoked secretion? Does release occur only at contacts with blood spaces, or at other sites as well? Is secretion modulated by hormones, and at what sites and by what mechanisms? What limits the amount of releasable hormone, and is there peripheral restocking? In isolated XOSGs, for the 3 hormonal activities, basal secretion and secretion evoked by raised [K+]o and axonal stimulation will be followed by analysis of perfusate aliquots. The effects of ionic composition, pharmacological agents and temperature that alter these will then also be tested for effects on electrical responses. """"""""Whole terminal"""""""" patch-clamp will characterize ionic currents of isolated terminals which will then be identified immunocytologically. Fura-2 and SBFI will be used to optically assess [Ca]i and [Na]i in terminals and cultured neurons. Capacitance measurements and computer-aided, video-enhanced microscopy will be used to track secretion from terminals and growth cones. Immunocytologically identified terminals will be characterized at the EM level together with changes resulting from secretory activity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS015453-10
Application #
3396269
Study Section
Neurology C Study Section (NEUC)
Project Start
1979-07-01
Project End
1996-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
10
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Hawaii
Department
Type
Organized Research Units
DUNS #
121911077
City
Honolulu
State
HI
Country
United States
Zip Code
96822
Duan, S; Cooke, I M (2000) Glutamate and GABA activate different receptors and Cl(-) conductances in crab peptide-secretory neurons. J Neurophysiol 83:31-7
Duan, S; Cooke, I M (1999) Selective inhibition of transient K+ current by La3+ in crab peptide-secretory neurons. J Neurophysiol 81:1848-55
Meyers, D E; Cooke, I M (1997) Comparison of Ca2+ currents of peptidergic neurons developing differing morphology with time in culture. J Exp Biol 200:723-33
Richmond, J E; Penner, R; Keller, R et al. (1996) Characterization of the Ca2+ current in isolated terminals of crustacean peptidergic neurons. J Exp Biol 199:2053-9
Richmond, J E; Codignola, A; Cooke, I M et al. (1996) Calcium- and barium-dependent exocytosis from the rat insulinoma cell line RINm5F assayed using membrane capacitance measurements and serotonin release. Pflugers Arch 432:258-69
Sher, E; Codignola, A; Rogers, M et al. (1996) Noradrenaline inhibition of Ca2+ channels and secretion in single patch-clamped insulinoma cells. FEBS Lett 385:176-80
Richmond, J E; Sher, E; Keller, R et al. (1995) Regulation of calcium currents and secretion by magnesium in crustacean peptidergic neurons. Invert Neurosci 1:215-21
Richmond, J E; Sher, E; Cooke, I M (1995) Characterization of the Ca2+ current in freshly dissociated crustacean peptidergic neuronal somata. J Neurophysiol 73:2357-68
Keller, R; Grau, S; Cooke, I M (1995) Quantitation of peptide hormone in single cultured secretory neurons of the crab, Cardisoma carnifex. Cell Tissue Res 281:525-32
Graf, R A; Cooke, I M (1994) Outgrowth morphology and intracellular calcium of crustacean neurons displaying distinct morphologies in primary culture. J Neurobiol 25:1558-69

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