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The internal pH of synaptic‐like microvesicles in rat pinealocytes in culture
Author(s) -
Hayashi Mitsuko,
Yamamoto Akitsugu,
Moriyama Yoshinori
Publication year - 2002
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2002.01025.x
Subject(s) - bafilomycin , synaptic vesicle , biology , vesicle , ammonium chloride , pinealocyte , microvesicles , nigericin , biochemistry , biophysics , exocytosis , neurotransmitter , organelle , microbiology and biotechnology , secretion , chemistry , membrane , endocrinology , pineal gland , apoptosis , melatonin , microrna , gene , receptor , organic chemistry , autophagy
Synaptic‐like microvesicles (SLMVs) are morphological and functional equivalents of neuronal synaptic vesicles, and are responsible for the storage and secretion of classical neurotransmitters in various endocrine cells. Vacuolar H + ‐ATPase acidifies the internal space of these organelles and provides a driving force for the uptake of neurotransmitters. Thus, the luminal pH is an important determinant of the function of SLMVs, although its value in living cells is unknown. Here, we determined the luminal pH of SLMVs in living rat pinealocytes by means of an immunoelectronmicroscopic procedure basedon the distribution of an amphipathic amine, 3‐(2,4‐dinitroanilino)‐3′‐amino‐ N ‐methyldipropylamine (DAMP). Use of double‐labeling techniques with antibodies against 2,4‐dinitrophenol for DAMP and synaptophysin for SLMVs, and of frozen ultrathin sections enabled us to determine the number of immunogold particles for DAMP per µm 2 of SLMVs. Using the density of gold particles, the luminal pH of SLMVs was calculated to be 5.11 ± 0.01. Treatment with either 1 µ m bafilomycin A1, a specific inhibitor of vacuolar H + ‐ATPase , or 50 m m ammonium chloride, a dissipater of the transmembrane pH gradient, increased the luminal pH to 6.04 ± 0.07 and 6.05 ± 0.11, respectively. Simultaneously, the lysosomal pH was found to be 5.14 ± 0.07, which increased to 5.77 ± 0.09 and 5.93 ± 0.13 with bafilomycin A1 and ammonium chloride, respectively. It is concluded that the luminal pH of SLMVs is comparable to that of lysosomes in vivo .