Abstract
Treatment of cultured bovine adrenal chromaffin cells with the catecholamine transport blocker reserpine was shown previously to increase enkephalin levels severalfold. To explore the biochemical mechanism of this effect, we examined the effect of reserpine treatment on the activities of three different peptide precursor processing enzymes: carboxypeptidase E (CPE) and the prohormone convertases (PCs) PC1/3 and PC2. Reserpine treatment increased both CPE and PC activity in extracts of cultured chromaffin cells; total protein levels were unaltered for any enzyme. Further analysis showed that the increase in CPE activity was due to an elevated Vmax, with no change in the Km for substrate hydrolysis or the levels of CPE mRNA. Reserpine activation of endogenous processing enzymes was also observed in extracts prepared from PC12 cells stably expressing PC1/3 or PC2. In vitro experiments using purified enzymes showed that catecholamines inhibited CPE, PC1/3, and PC2, with dopamine quinone the most potent inhibitor (IC50 values of ∼50–500 μM); dopamine, norepinephrine, and epinephrine exhibited inhibition in the micromolar range. The inhibition of purified CPE with catecholamines was time-dependent and, for dopamine quinone, dilution-independent, suggesting covalent modification of the protein by the catecholamine. Because the catecholamine concentrations found to be inhibitory to PC1/3, PC2, and CPE are well within the physiological range found in chromaffin granules, we conclude that catecholaminergic transmitter systems have the potential to exert considerable dynamic influence over peptidergic transmitter synthesis by altering the activity of peptide processing enzymes.
Footnotes
This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants R01-DA04494, R01-DA05084]; and the German Academy of Sciences Leopoldina [Grant LPDS 2009-33].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.072090.
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ABBREVIATIONS:
- PC1/3
- prohormone convertases 1 and 3
- PC2
- prohormone convertase 2
- AMC
- 7-amino-4-methylcoumarin
- CPE
- carboxypeptidase E
- DA
- dopamine
- DAQ
- dopamine quinone
- E-64
- trans-epoxysuccinic acid
- E
- epinephrine
- GEMSA
- guanidinoethylmercaptosuccinic acid
- NE
- norepinephrine
- PC12
- pheochromocytoma 12
- TBS
- Tris-buffered saline
- ANOVA
- analysis of variance.
- Received March 3, 2011.
- Accepted May 3, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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