The internal pH of synaptic-like microvesicles in rat pinealocytes in culture

Mitsuko Hayashi, Akitsugu Yamamoto, Yoshinori Moriyama

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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 based on 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 μm2 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 μM bafilomycin A1, a specific inhibitor of vacuolar H+-ATPase, or 50 mM 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.

Original languageEnglish
Pages (from-to)698-704
Number of pages7
JournalJournal of Neurochemistry
Volume82
Issue number3
DOIs
Publication statusPublished - Aug 12 2002

Keywords

  • Bafilomycin A
  • DAMP
  • Pineal gland
  • Synaptic vesicles
  • Synaptic-like microvesicles
  • Vacuolar H-ATPase

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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