Multiple cytosolic calcium buffers in posterior pituitary nerve terminals

Cytosolic Ca²⁺ buffers bind to a large fraction of Ca²⁺ as it enters a cell, shaping Ca²⁺ signals both spatially and temporally. In this way, cytosolic Ca²⁺ 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 Ca²⁺ entry. Secretion of these hormones exhibits a complex dependence on the frequency and pattern of electrical activity, and the role of cytosolic Ca²⁺ buffers in controlling pituitary Ca²⁺ signaling is poorly understood. Here, cytosolic Ca²⁺ buffers were studied with two-photon imaging in patch-clamped nerve terminals of the rat posterior pituitary. Fluorescence of the Ca²⁺ indicator fluo-8 revealed stepwise increases in free Ca²⁺ after a series of brief depolarizing pulses in rapid succession. These Ca²⁺ increments grew larger as free Ca²⁺ rose to saturate the cytosolic buffers and reduce the availability of Ca²⁺ binding sites. These titration data revealed two endogenous buffers. All nerve terminals contained a buffer with a K_d of 1.5–4.7 µM, and approximately half contained an additional higher-affinity buffer with a K_d 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 Ca²⁺ buffers in situ in nerve terminals at a new level of detail. The saturation of these cytosolic buffers will amplify Ca²⁺ 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.