TY - JOUR
T1 - An excised patch membrane sensor for arachidonic acid released in mouse hippocampal slices under stimulation of L-glutamate
AU - Saitoh, Hiroto
AU - Namatame, Yuko
AU - Hirano, Ayumi
AU - Sugawara, Masao
N1 - Funding Information:
This work was supported by Grant-in-Aid for Scientific Research by the Ministry of Education, Science and Culture, Japan, and Nihon University Research Grant for General Individual Research Grant.
PY - 2004/6/15
Y1 - 2004/6/15
N2 - 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.
AB - 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.
KW - Arachidonic acid
KW - Channel-type current
KW - L-Glutamate-stimulated release
KW - Mouse hippocampal slice
KW - Patch membrane sensor
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U2 - 10.1016/j.ab.2004.03.060
DO - 10.1016/j.ab.2004.03.060
M3 - Article
C2 - 15158474
AN - SCOPUS:2542439969
VL - 329
SP - 163
EP - 172
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -