An excised patch membrane sensor for arachidonic acid released in mouse hippocampal slices under stimulation of L-glutamate

Hiroto Saitoh, Yuko Namatame, Ayumi Hirano, Masao Sugawara

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

An excised patch membrane sensor for arachidonic acid (AA) is described, whose response stems from AA-induced channel-type transport of ions across the excised patch membrane. The patch membrane sensor was prepared in situ by excising mouse hippocampal cell membranes with patch pipets having a tip diameter of <0.5μm. The sensor responds to AA, giving rise to a channel-type current, and its magnitude (apparent conductance) increased with increasing AA concentration in the range from 10 to 30nM. The detection limit was 2.1nM (S/N=3). The induction of channel-type currents was selective to AA over fatty acids such as palmitic acid, stearic acid, oleic acid, γ-linolenic acid, and docosahexaenoic acid and AA metabolites such as 12-HETE, 5-HETE, and prostaglandin D2. The sensor was applied to quantification of AA released from various neuronal regions (CA1, CA3, and DG) of mouse hippocampus under stimulation of 100μM L-glutamate. The release of AA from each region was observed 1min after the stimulation and the concentration of AA 5min after the stimulation varied among the neuronal sites, i.e., 8±1 nM (n=5) for CA1, 15±3 nM (n=3) for CA3, and 6±2 nM (n=9) for DG. The L-glutamate-evoked release of AA was partly inhibited by ionotropic glutamate receptor antagonists (APV and DNQX) and completely blocked by phospholipase A2 (PLA2) inhibitor (MAFP), suggesting that the release of AA occurred by glutamate receptor-mediated activation of PLA2. The potential use of the present sensor for detecting local concentration of AA at various neuronal sites is discussed.

Original languageEnglish
Pages (from-to)163-172
Number of pages10
JournalAnalytical Biochemistry
Volume329
Issue number2
DOIs
Publication statusPublished - 2004 Jun 15
Externally publishedYes

Keywords

  • Arachidonic acid
  • Channel-type current
  • L-Glutamate-stimulated release
  • Mouse hippocampal slice
  • Patch membrane sensor

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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