Abstract
The biosynthetic enzyme peptidylglycine alpha-amidating monooxygenase catalyzes the formation of a variety of biologically active alpha-amidated peptides from respective COOH-terminal glycine-extended peptide precursors. Peptidylglycine alpha-amidating monooxygenase activity is dependent on copper, ascorbate, and molecular oxygen and is inhibited by the relatively selective copper chelator N,N-diethyldithiocarbamate or its disulfide dimer disulfiram (Antabuse). In the present study, chronic disulfiram treatment (100 mg/kg/day, for 12-25 days) resulted in significant changes in several neurochemical parameters in the mouse central nervous system, including levels of substance P-like, unamidated substance P-Gly-like, and protease-generated substance P-Gly-Lys-like immunoreactivities (SP-LI, SP-G-LI, and SP-G-K-LI, respectively). Combined high performance liquid chromatography/radioimmunoassay analyses of the extracted SP-LI, SP-G-LI, and SP-G-K-LI species indicated very similar chromatographic and immunochemical behavior as demonstrated for chemically authentic peptide standards. Additionally, changes in levels of monoamines and their metabolites were observed after drug administration. Complementary immunohistochemical analyses using affinity-purified anti-SP-G sera localized these drug-induced changes in levels of immunoreactive unamidated precursor to neural elements that normally express SP. As a functional corollary to alterations in neurochemical parameters, we observed significant disulfiram-induced increases in pain thresholds, potentiated by capsaicin treatment. Overall, our results indicate that the observed changes in steady state levels of immunoreactive SP and of the immature COOH-terminal extended forms of SP may reflect compensatory biosynthetic and posttranslational processing events in SP-containing neural systems after pharmacological challenge.
Highlights
The biosynthetic enzyme peptidylglycine cY-amidating monooxygenase catalyzes the formation of a variety of biologically active cY-amidated peptides from respective COOH-terminal glycine-extended peptide precursors
It has been recently demonstrated that administration of the relatively selective copper chelator iV,N-diethyldithiocarbamate or its disulfide dimer, disulfiram (Antabuse), produces a dose-dependent increase in glycine-extended immature forms of Lu-melanocyte-stimulating hormone and of joining peptide in the intermediate pituitary and in cultured mouse corticotrophic tumor cells, via inhibition of peptidylglycine cy-amidating monooxygenase activity [12]
We have examined the effects of disulfiram administration on several neurochemical parameters in mouse CNS, including levels of Substance P (SP)-LI, SP-G-LI
Summary
Analyses-Levels of biogenic amines were quantified by HPLC-ECD and levels of peptides by RIA from the same tissue samples using the extraction procedure, as described here. The acidified upper layer was reserved for peptide RIAs as described above By this extraction and reconstitution procedure, recoveries of peptides as monitored by liquid scintillation counting of exoaenouslv added HPLC-nurified tritium-labeled. The injection volume was 1 ml, and 0.5-ml fractions were collected and stored for peptide RIAs. Blank runs consisting of injections of 1 ml of 40 mM potassium phosphate, pH 3.5, were performed between sample runs and after calibration of standard retentions times. RIA standard curves were generated in the presence of equivalent aliquots of lyophilized HPLC mobile phase
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
