Electroosmotic sampling. Application to determination of ectopeptidase activity in organotypic hippocampal slice cultures.
We hypothesize that peptide-containing solutions pulled through tissue should reveal the presence and activity of peptidases in the tissue. Using the natural zeta-potential in the organotypic hippocampal slice culture (OHSC), physiological fluids can be pulled through the tissue with an electric field. The hydrolysis of the peptides present in the fluid drawn through the tissue can be determined using capillary HPLC with electrochemical detection of the biuret complexes of the peptides following a postcolumn reaction. We have characterized this new sampling method by measuring the flow rate, examining the use of internal standards, and examining cell death caused by sampling. The sampling flow rate ranges from 60 to 150 nL/min with a 150 microm (ID) sampling capillary with an electric field (at the tip of the capillary) from 30 to 60 V/cm. Cell death can be negligible with controlled sampling conditions. Using this sampling approach, we have electroosmotically pulled Leu-enkephalin through OHSCs to identify ectopeptidase activity in the CA3 region. These studies show that a bestatin-sensitive aminopeptidase may be critical for the hydrolysis of exogenous Leu-enkephalin, a neuropeptide present in the CA3 region of OHSCs.
Xu
Hongjuan
H
Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, Pennsylvania 15260, USA.
Guy
Yifat
Y
Hamsher
Amy
A
Shi
Guoyue
G
Sandberg
Mats
M
Weber
Stephen G
SG
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001737:Biuret / Q000737:chemistry; D053841:Electroosmosis / Q000379:methods*; D004743:Enkephalin, Leucine / Q000378:metabolism*; D006624:Hippocampus / Q000201:enzymology*; D006868:Hydrolysis; D009924:Organ Culture Techniques; D010447:Peptide Hydrolases / Q000032:analysis*; D010455:Peptides / Q000737:chemistry; D051381:Rats
D010455:Peptides; D004743:Enkephalin, Leucine; D001737:Biuret; D010447:Peptide Hydrolases