Haloperidol and apomorphine differentially affect neuropeptidase activity.

In addition to their well characterized effects at dopamine receptors, neuroleptic drugs have been shown to affect the level and in vitro metabolism of neuropeptides. In the present study, the effect of acute and subchronic administration of the neuroleptic haloperidol and the nonselective, dopamine agonist apomorphine on neuropeptidase activity was determined in regional, rat brain P2 membranes. Subchronic administration of haloperidol decreased the activity of aminopeptidase N in the frontal cortex and caudate-putamen. In contrast, subchronic administration of apomorphine increased aminopeptidase N activity in the frontal cortex and caudate-putamen. Neutral endopeptidase 24.11 also was affected differentially in the caudate-putamen, but both subchronic haloperidol and apomorphine decreased neutral endopeptidase 24.11 activity in the frontal cortex. Metalloendopeptidase 24.15 activity was decreased in the caudate-putamen after acute haloperidol and increased in the frontal cortex after acute apomorphine administration; however, no effect was noted after subchronic administration of either drug. Angiotensin converting enzyme was not affected by any treatment. Therefore, neuroleptic-induced alterations in aminopeptidase N, neutral endopeptidase 24.11 and metalloendopeptidase 24.15 activity may account for previously reported alterations in neuropeptide degradation. In view of the interaction between mesocorticolimbic dopamine neurons and neuropeptides, e.g., substance P, neurotensin and enkephalins, neuroleptic-induced alterations in the activities of neuropeptidases, and thus neuropeptide metabolism can, in turn, play a role in modulating midbrain dopaminergic activity.

Subchronic haloperidol administration decreases aminopeptidase N activity and [Met5]enkephalin metabolism in rat striatum and cortex.

Previously we have shown that subchronic intraperitoneal (i.p.) administration of haloperidol decreases the degradation of [Met5]enkephalin by regional brain slices (Waters et al., 1995, J. Pharmacol. Exp. Ther. 274, 783). In the present study, subchronic (7-day i.p.) administration of haloperidol (1 mg/kg) decreased the accumulation of aminopeptidase-derived fragments Tyr and Gly-Gly-Phe-Met on cortical and striatal slices. The accumulation of Tyr-Gly-Gly, however, was not altered by haloperidol treatment on slices from either region. Further, aminopeptidase N activity was decreased in P2 membranes isolated from either the cortex or striatum of haloperidol-treated animals. These data suggest that the haloperidol-induced decrease in [Met5]enkephalin metabolism results, at least in part, from a reduction in the activity of aminopeptidase N.

Regional metabolism of Met-enkephalin and cholecystokinin on intact ratbrain slices: characterization of specific peptidases.

The metabolism of Met-enkephalin and cholecystokinin (CCK) 8-(sulfated) by intact microslices was studied in rat brain regions. Incubation of brain slices with Met-enkephalin (400 microM) resulted in a linear rate of disappearance of parent peptide and appearance of metabolic fragments whose rate of accumulation was specific to brain region. The degradative rate (pmol/min/mg of protein) of Met-enkephalin was high in caudate-putamen (5,160 +/- 120) and lower in nucleus accumbens (3,630 +/- 110) and frontal cortex (3,180 +/- 120). Inhibition of aminopeptidases decreased Met-enkephalin degradation (50-97% vs. control) in frontal cortex but was less effective in caudate-putamen (20-34%). Tyr-Gly-Gly and Phe-Met were recovered in caudate-putamen and nucleus accumbens, whereas negligible quantities of these fragments were recovered from frontal cortex. Phosphoramidon, an inhibitor of neutral endopeptidase 24.11, decreased Met-enkephalin degradation in caudate-putamen (14%) but had no effect on that in frontal cortex. A cocktail of bestatin or leuhistin (inhibitors of aminopeptidases), phosphoramidon, and captopril (an inhibitor of angiotensin converting enzyme) protected Met-enkephalin from degradation (recovery > 95%) in caudate-putamen. CCK 8-(sulfated) degradation on slices from caudate-putamen, nucleus accumbens, and frontal cortex was not altered by inhibitors of neutral endopeptidase 24.11, metalloendopeptidase 24.15, angiotensin converting enzyme, or thiol proteases. Inhibitors of either aminopeptidases or serine proteases produced small reductions (13-30%) in CCK degradation in each region. These data provide evidence for regional and structural specificity in terminating the actions of neuropeptides.

Chronic treatment with neuroleptics alters neutral endopeptidase 24.11 activity in rat brain regions.

Chronic administration of neuroleptics has been shown to affect the endogenous levels, mRNA, posttranslational processing, and metabolism of neuropeptides in specific regions of rat brain. Neutral endopeptidase 24.11 (NEP) is known to metabolize a variety of neuropeptide substrates, including the enkephalins and neurotensin, thus modifying or terminating the bioactivity of such peptides. In the present study, chronic treatment with haloperidol (1 mg/kg/day, 12 days) increased NEP activity in nucleus accumbens, and chronic treatment with chlorpromazine (4 mg/kg/day, 12 days) increased NEP activity in caudate putamen. Higher dosages with either compound did not significantly alter NEP activity, and none of the treatments altered NEP activity in the hypothalamus. Chronic treatment with apomorphine (5 mg/kg/day, 12 days) decreased NEP activity in both nucleus accumbens and caudate putamen. These data suggest that chronic treatment with neuroleptic drugs may lead to regionally specific alterations in the metabolism of neuropeptides.

Opioid and cannabinoid receptor inhibition of adenylyl cyclase in brain.

Both opioids and cannabinoids bind to G-protein-coupled receptors to inhibit adenylyl cyclase in neurons. These reactions were assayed in brain membranes, where maximal inhibitory activity occurred in the following regions: mu-opioid inhibition in rat thalamus, delta-opioid inhibition in rat striatum, kappa-opioid inhibition in guinea pig cerebellum, and cannabinoid inhibition in cerebellum. The inhibition of adenylyl cyclase by both cannabinoid and opioid agonists was typical of G-protein-linked receptors: they required GTP, they were not supported by non-hydrolyzable GTP analogs, and they were abolished (in primary neuronal cell culture) by pertussis toxin treatment. The immediate targets of this system were determined by assaying protein phosphorylation in the presence of receptor agonists and App(NH)p, a substrate for adenylyl cyclase. In striatal membranes, opioid agonists inhibited the phosphorylation of at least two bands of MW 85 and 63 kDa, which may be synapsins I and II, respectively. Other experiments determined the long-term effects of this second messenger system. In primary neuronal cultures, opioid-inhibited adenylyl cyclase attenuated forskolin-stimulated pro-enkephalin mRNA levels, thus providing a feedback regulation of opioid synthesis. Finally, in cerebellar granule cells, both cannabinoid and opioid receptors may exist on the same cells. In these cells, agonists which bind to different receptor types may produce similar biological responses.