Peptidase inhibitors improve recovery of substance P and calcitonin gene-related peptide release from rat spinal cord slices

doi:10.1016/0196-9781(95)02091-8

Peptides

Volume 17, Issue 1, 1996, Pages 31-37

https://doi.org/10.1016/0196-9781(95)02091-8 Get rights and content

Abstract

The purpose of present study was to determine whether peptidase activity affects the release of substance P (SP) and calcitonin gene-related peptide (CGRP) from spinal cord slices. When slices were exposed to various inhibitors of endopeptidase 24.11, the resting and capsaicin-stimulated release of SP were less than 0.04% and 0.20% total content per minute, respectively. Resting CGRP release was approximately 0.10% and stimulated release was 0.40%. The combination of 20 μM bacitracin, 100 μM phenylalanylalanine (Phe-Ala), and 50 μM p-chloromercuriphenylsulfonic acid (PCMS) significantly increased both resting and stimulated release of SP and CGRP at least two- or threefold. Doubling the concentration of PCMS and Phe-Ala did not further improve peptide release. These results demonstrate that recovery of peptides released from spinal cord slices is dependent in part on activity of multiple peptidases in the tissues.

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Expression of CGRP was observed over laminae I and outer part of lamina II. Synaptic terminals could be discerned containing CGRP. Following incision, the expression decreased abruptly at 2 h. However, at 12 h, the expression had increased. Between days 1–5, the expression decreased again towards basal levels. The antinociceptive effect of BIBN was comparatively less than morphine, which robustly inhibited all three pain parameters at 2 h after incision.
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The amount of iCGRP released in each incubation was measured by a radioimmunoassay. The minimum amount of iCGRP detected by the radioimmunoassay is 5 fmol with a 95% confidence interval (29). After the release protocol, the remaining peptide content in each well was determined by exposing the cells to 2 n acetic acid for 10 min.
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They were then incubated for successive 10 min intervals with 0.4 ml of HEPES buffer alone (to measure resting release), with buffer containing 30 nM capsaicin (to measure stimulated release), then with buffer alone (to measure recovery). After each incubation, the buffer was removed and the amount of CGRP in each sample was measured using radioimmunoassay as previously described [29]. After the release experiment, the cells in each well were scraped and sonicated in 0.4 M HCl and an aliquot taken to measure total CGRP content in the cultures using radioimmunoassay.
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ArticlePeptidase inhibitors improve recovery of substance P and calcitonin gene-related peptide release from rat spinal cord slices

Abstract

Brain Res.

Neuropeptides

Neurosci. Lett.

Peptides

Biochem. Biophys. Res. Commun.

Brain Res.

J. Biol. Chem.

Peptides

Regul. Pept.

Neurochem. Int.

Neurosci. Lett.

Neuropharmacology

Brain Res.

Neurosci. Lett.

Brain Res.

Trends Pharmacol. Sci.

Brain Res.

Article
Peptidase inhibitors improve recovery of substance P and calcitonin gene-related peptide release from rat spinal cord slices