Calcium Buffering in the Nerve Terminals of the Posterior Pituitary

Abstract

Nerve terminals possess a variety of mechanisms to regulate Ca2+ signaling, and these mechanisms in turn provide a variety of avenues for regulating exocytosis and neurosecretion. Endogenous Ca2+ buffers are important Ca2+ regulating molecules that can have an especially profound influence on the spatiotemporal dynamics of intracellular Ca2+ signals. These proteins rapidly bind free Ca2+ within several hundred nanometers of open Ca2+ channels, limiting the rise of free Ca2+ in the nerve terminal and restricting its spatial spread. Ca2+ sequestration and extrusion mechanisms subsequently act to restore resting levels of free Ca2+. By modulating Ca2+, these Ca2+ buffering, sequestration and extrusion mechanisms play an important role in regulation Ca2+ dependent exocytosis. In the present work, intracellular Ca2+ signaling in the peptidergic nerve terminals of the rat posterior pituitary was investigated using two-photon fluorescence imaging with the Ca2+-sensitive fluorescent dye fluo-8. Ca2+ imaging together with simultaneous measurement of Ca2+ current by patch clamp recording provided a direct assessment of the cytoplasmic Ca2+ buffering capacity. These experiments showed that the cytoplasmic Ca2+ buffering capacity declined as cytosolic [Ca2+] rose, and thus indicated that the Ca2+ dye and endogenous buffers saturated in the range of [Ca2+] studied. These data were interpreted using a model for the [Ca2+] dependence of buffering capacity that takes binding site saturation into account. This analysis yielded values for Kd of 1850 ± 485 nM and concentration of 243 ± 46 μM for the endogenous Ca2+ buffer (n = 16). This provides insight into some of the essential properties of the cytoplasmic Ca2+ regulatory machinery in the posterior pituitary. This endogenous buffer will shape the spatiotemporal profile Ca2+ rises induced by action potentials and regulate the amount of neuropeptide release.