title: Multiple cytosolic calcium buffers in posterior pituitary nerve terminals.
abstract: Cytosolic Ca(2+) buffers bind to a large fraction of Ca(2+) as it enters a cell, shaping Ca(2+) signals both spatially and temporally. In this way, cytosolic Ca(2+) buffers regulate excitation-secretion coupling and short-term plasticity of release. The posterior pituitary is composed of peptidergic nerve terminals, which release oxytocin and vasopressin in response to Ca(2+) entry. Secretion of these hormones exhibits a complex dependence on the frequency and pattern of electrical activity, and the role of cytosolic Ca(2+) buffers in controlling pituitary Ca(2+) signaling is poorly understood. Here, cytosolic Ca(2+) buffers were studied with two-photon imaging in patch-clamped nerve terminals of the rat posterior pituitary. Fluorescence of the Ca(2+) indicator fluo-8 revealed stepwise increases in free Ca(2+) after a series of brief depolarizing pulses in rapid succession. These Ca(2+) increments grew larger as free Ca(2+) rose to saturate the cytosolic buffers and reduce the availability of Ca(2+) binding sites. These titration data revealed two endogenous buffers. All nerve terminals contained a buffer with a Kd of 1.5-4.7 µM, and approximately half contained an additional higher-affinity buffer with a Kd of 340 nM. Western blots identified calretinin and calbindin D28K in the posterior pituitary, and their in vitro binding properties correspond well with our fluorometric analysis. The high-affinity buffer washed out, but at a rate much slower than expected from diffusion; washout of the low-affinity buffer could not be detected. This work has revealed the functional impact of cytosolic Ca(2+) buffers in situ in nerve terminals at a new level of detail. The saturation of these cytosolic buffers will amplify Ca(2+) signals and may contribute to use-dependent facilitation of release. A difference in the buffer compositions of oxytocin and vasopressin nerve terminals could contribute to the differences in release plasticity of these two hormones.
authors.affiliation: Biophysics PhD Program, Department of Neuroscience, and Physiology PhD Program, University of Wisconsin, Madison, WI 53705.
authors.forename: Shane M
authors.initials: SM
authors.lastname: McMahon
authors.affiliation: Biophysics PhD Program, Department of Neuroscience, and Physiology PhD Program, University of Wisconsin, Madison, WI 53705 Biophysics PhD Program, Department of Neuroscience, and Physiology PhD Program, University of Wisconsin, Madison, WI 53705.
authors.forename: Che-Wei
authors.initials: CW
authors.lastname: Chang
authors.affiliation: Biophysics PhD Program, Department of Neuroscience, and Physiology PhD Program, University of Wisconsin, Madison, WI 53705 mbjackso@wisc.edu.
authors.forename: Meyer B
authors.initials: MB
authors.lastname: Jackson
mesh_terms: D000818:Animals; D064032:Calbindin 2 / Q000378:metabolism; D064026:Calbindins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D003593:Cytoplasm / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials; D009411:Nerve Endings / Q000378:metabolism*; D010525:Peripheral Nerves / Q000378:metabolism*; D010902:Pituitary Gland / Q000294:innervation*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D064032:Calbindin 2; D064026:Calbindins; D002118:Calcium