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Comparative Study
. 2011 Aug;80(2):304-13.
doi: 10.1124/mol.111.072090. Epub 2011 May 3.

Regulation of Neuropeptide Processing Enzymes by Catecholamines in Endocrine Cells

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Free PMC article
Comparative Study

Regulation of Neuropeptide Processing Enzymes by Catecholamines in Endocrine Cells

Michael Helwig et al. Mol Pharmacol. .
Free PMC article

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 V(max), with no change in the K(m) 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 (IC(50) 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.

Figures

Fig. 1.
Fig. 1.
Effect of reserpine treatment on CPE, PC1/3, and PC2 activities in adrenal chromaffin cells. A, Reserpine treatment increases the enzymatic activity of CPE after 72 h but does not affect the activity of another carboxypeptidase (CP), or of N-acetylglucosaminidase. For CPE and other CPs, n = 18 for the controls, n = 6 for 24-h, n = 3 for 48-h, and n = 9 for 72-h groups. For N-acetylglucosaminidase, n = 6 for control, and n = 3 for treated groups. *, statistically different from control (P < 0.01) using Student's t test. Error bars show S.E.M. B, lack of effect of reserpine treatment on levels of CPE immunoreactivity and mRNA. CPE protein levels were assessed by Western blotting, and CPE mRNA was determined using slot blots, as described in Materials and Methods. Error bars show S.E.M.; n = 3 for all groups. C, the velocity of the CPE reaction increases in the presence of reserpine (Lineweaver-Burk analysis). V is expressed in nanomoles of product per hour per 2 × 104 cells. Error bars show the S.D. (n = 6). CPE activity is calculated from the difference between carboxypeptidase activity measured in the presence or absence of GEMSA (without added CoCl2). D, reserpine treatment affects CPE activity and not the related carboxypeptidase D. Control and reserpine-treated chromaffin cell extracts were purified on a benzoyl-arginine Sepharose affinity column, which can be used to physically separate CPE and CPD, allowing for their selective measurement. CPE activity elutes from the column at pH 8.0 (elute 1) and shows a difference between reserpine-treated and nontreated control extracts. CPD remains bound under this condition but can be eluted with a combination of pH 8.0 and arginine. Note the similar levels of CPD activity between the control and reserpine-treated extracts. Error bars show S.E.M. for triplicate determinations. E and F, reserpine increases the enzymatic activity of PC1/3 and PC2 present in chromaffin cell extracts. Cells were incubated with reserpine or vehicle for 2 days. Results are presented as the mean ± S.E.M., as the percentage of control rate (PC1/3 control rate = 22 pmol AMC/min; PC2 control rate = 24 pmol/min; n = 3 per group).
Fig. 2.
Fig. 2.
Reserpine treatment of adrenal PC12 cells increases endogenous CPE and PC activity but has no effect on protein expression. A to D, bar charts showing enzyme activity of CPE, PC1/3, and PC2 in PC12 cells treated with reserpine or vehicle; quantification of enzyme protein levels is presented in the lower portion of each figure. Quantitative analysis of enzyme protein is presented after normalization of enzyme immunoreactivity for β-actin immunoreactivity within the same samples and is expressed as arbitrary units (AU) and as a percentage of nontreated controls. Values represent the mean ± S.E.M. as a percentage of the control rate. Left, PC1/3-overexpressing PC12 cells treated with reserpine for 48h. A, CPE activity increases in the presence of 5 μM reserpine (n = 3 per group), whereas CPE protein expression remains unchanged. C, PC1/3 activity increases after treatment of cells with 1 μM and 10 μM reserpine, whereas protein levels are only slightly increased (control rate = 32 pmol AMC/min). Right, PC2-expressing PC12 cells treated with reserpine for 48 h. B, CPE activity increases in PC12-PC2 cells in the presence of 5 μM reserpine (n = 3/group). CPE protein levels remain unchanged. D, PC2 activity is increased after treatment with either 1 or 10 μM reserpine; PC2 protein levels are unaltered (control rate = 54 pmol AMC/min).
Fig. 3.
Fig. 3.
Catecholamines affect purified CPE activity. Reactions were preincubated with different concentrations of catecholamines for 2 h. Increasing concentrations of catecholamines result in decreased CPE activity (n = 6/group). A, open bars, dopamine. B, light gray bars, dopamine quinone. C, dark gray bars, norepinephrine; D, black bars, epinephrine. E, time-dependence of the catecholaminergic inhibition of CPE over 12 h. Whereas dopamine quinone in relatively low concentrations lead to a near complete inhibition of CPE activity after 6 h, dopamine, norepinephrine, and epinephrine inhibited enzyme activity after 12 h. Results are presented as the mean ± S.D., as a percentage of control rate (n = 3 per time point). **, P < 0.01 and *, P < 0.05; statistically different from CPE activity in the nontreated group using ANOVA.
Fig. 4.
Fig. 4.
Catecholamines decrease the activity of purified PC1/3 (left) and PC2 (right). A to D, increasing concentrations of dopamine and dopamine quinone decrease PC1/3 and PC2 enzymatic activity in reactions incubated for 2 h. E to H, norepinephrine and epinephrine significantly inhibit enzyme activity of both convertases. Values are presented as means ± S.D., percentage of controls (n = 3/group). **, P < 0.01 and *, P < 0.05; statistically different from PC1/3 (control rate = 3 pmol AMC/min) and PC2 (control rate = 8.6 pmol AMC/min) activity in the nontreated control group using ANOVA.
Fig. 5.
Fig. 5.
l-DOPA treatment of PC12 cells decreases endogenous CPE activity. CPE activity in PC12 cells decreases after 24 h in the presence of 30 μM l-DOPA in the medium (n = 3/group). CPE activity increases when PC12 cells are preincubated for 24 h with 5 μM reserpine before the addition of l-DOPA for another 24 h (n = 3/group). ***, P < 0.001; **, P < 0.01; and *, P < 0.05; statistically different from control (control rate = 29 pmol AMC/min) using ANOVA.
Fig. 6.
Fig. 6.
Catecholaminergic inhibition of CPE by dopamine, norepinephrine, and epinephrine is reversible, whereas inhibition by dopamine quinone is irreversible. CPE was incubated for 6 h at 37°C; reactions were then sequentially diluted and the specific activity [relative fluorescence units per minute per milligram] of untreated CPE was compared with diluted catecholamine-treated enzyme (n = 3/group). Dopamine quinone inhibits CPE activity regardless of the dilution factor. Whereas treatment of CPE with dopamine, norepinephrine, and epinephrine decreases enzymatic activity initially, enzyme activity can be restored by dilution to levels comparable with those of untreated CPE. Results are presented as mean ± S.D. **, P < 0.01, and *, P < 0.05; statistically different from enzyme activity in the nontreated group using ANOVA.

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