The cytochrome P450 2D‐mediated formation of serotonin from 5‐methoxytryptamine in the brain in vivo: a microdialysis study

The cytochrome P450 2D (CYP2D) mediates synthesis of serotonin from 5‐methoxytryptamine (5‐MT), shown in vitro for cDNA‐expressed CYP2D‐isoforms and liver and brain microsomes. We aimed to demonstrate this synthesis in the brain in vivo. We measured serotonin tissue content in brain regions after 5‐MT injection into the raphe nuclei (Model‐A), and its extracellular concentration in rat frontal cortex and striatum using an in vivo microdialysis (Model‐B) in male Wistar rats. Naïve rats served as control animals. 5‐MT injection into the raphe nuclei of PCPA‐(tryptophan hydroxylase inhibitor)‐pretreated rats increased the tissue concentration of serotonin (from 40 to 90% of the control value, respectively, in the striatum), while the CYP2D inhibitor quinine diminished serotonin level in some brain structures of those animals (Model‐A). 5‐MT given locally through a microdialysis probe markedly increased extracellular serotonin concentration in the frontal cortex and striatum (to 800 and 1000% of the basal level, respectively) and changed dopamine concentration (Model‐B). Quinine alone had no effect on serotonin concentration; however, given jointly with 5‐MT, it prevented the 5‐MT‐induced increase in cortical serotonin in naïve rats and in striatal serotonin in PCPA‐treated animals. These results indicate that the CYP2D‐catalyzed alternative pathway of serotonin synthesis from 5‐MT is relevant in the brain in vivo, and set a new target for the action of psychotropics.

     

Influence of local administration of apomorphine on citrulline extracellular level in the striatum: participation of the dopamine D1 and D2 receptors

By means of in vivo microdialysis combined with HPLC analysis, we have shown that local infusions of 1 mM N-nitro-L-arginine (NO-synthase inhibitors) in the rat striatum reduced, and infusions of 100 microM apomorphine (agonists of the dopamine receptors) increased the level of citrulline (a NO co-product) in extracellular space of this structure. The apomorphine-induced increase in citrulline extracellular levels in the striatum was completely prevented by infusions of N-nitro-L-arginine in this structure, and 10 microM raclopride (dopamine D2 receptor blocker), but not by infusions of 50 microM SCH-23390 (dopamine D1 receptor blocker). The data obtained suggest that the increase in citrulline extracellular levels in striatum resulted from local activation of NO-synthase, and this effect is mediated by D2 rather than D1 dopamine receptors.     

CYP11A1 stimulates the hydroxylase activity of CYP11B1 in mitochondria of recombinant yeast in vivo and in vitro.

In mammals, hydrocortisone synthesis from cholesterol is catalyzed by a set of five specialized enzymes, four of them belonging to the superfamily of cytochrome P-450 monooxygenases. A recombinant yeast expression system was recently developed for the CYP11B1 (P45011beta) enzyme, which performs the 11beta hydroxylation of steroids such as 11-deoxycortisol into hydrocortisone, one of the three mitochondrial cytochrome P-450 proteins involved in steroidogenesis in mammals. This heterologous system was used to test the potential interaction between CYP11B1 and CYP11A1 (P450scc), the mitochondrial cytochrome P-450 enzyme responsible for the side chain cleaving of cholesterol. Recombinant CYP11B1 and CYP11A1 were targeted to Saccharomyces cerevisiae mitochondria using the yeast cytochrome oxidase subunit 6 mitochondrial presequence fused to the mature form of the two proteins. In yeast, the presence of CYP11A1 appears to improve 11beta hydroxylase activity of CYP11B1 in vivo and in vitro. Fractionation experiments indicate the presence of the two proteins in the same membrane fractions, i.e. inner membrane and contact sites of mitochondria. Thus, yeast mitochondria provide interesting insights to study some molecular and cellular aspects of mammalian steroid synthesis. In particular, recombinant yeast should permit a better understanding of the mechanism permitting the synthesis of steroids (sex steroids, mineralocorticoids and glucocorticoids) with a minimal set of enzymes at physiological level, thus avoiding disease states.     

Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes.

Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 microM and 0.74 nM x min(-1) per mg microsomal protein vs. 0.11 nM x min(-1) per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC50 in hepatocytes than in microsomes. The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.     

Expression,structure-function,and molecular modeling of vitamin D P450s

Although vitamin D(3) is a natural product of a sunlight-mediated process in the skin, the secosteroid's biological function is dependent upon specific cytochrome P450 enzymes that mediate the parent vitamin's bioactivation and inactivation. Cytochrome P450C1 (CYP27B1) is the regulatory rate-limiting enzyme that directs the bioactivation process through introduction of a C-1alpha hydroxyl group. The resultant 1,25-dihydroxyvitamin D(3) (1,25D) is the biologically active secosteroid hormone that directs the multitude of vitamin D-dependent actions involved with calcium homeostasis, cellular differentiation and growth, and the immune response. The circulating and cellular level of 1,25D is regulated through a coordinated process involving the hormone's synthesis and degradation. Central to the degradation and turnover of 1,25D is the regulatory multi-catalytic cytochrome P450C24 (CYP24) enzyme that directs the introduction of C-24R groups onto targeted 25-hydroxy substrates. Discussed in this article is the action of the rat CYP24 to catalyze the side-chain oxidation and cleavage of 25-hydroxylated vitamin D metabolites. Expression and characterization of purified recombinant rat CYP24 is discussed in light of mutations directed at the enzyme's active site.     

Effect of Quinine on Autoreceptor-Regulated Dopamine Release in the Rat Striatum

In vivo brain microdialysis was used to examine the role of potassium channel activation in dopamine (DA) autoreceptor function in the striatum of freely moving rats. Local application of the D2 receptor agonists quinpirole or N-0437 through the dialysis probe significantly reduced extracellular concentrations of DA. Local application of the D2 antagonist (-)-sulpiride produced significant increases in DA. Local perfusion with quinine, a K+ channel blocker, completely blocked the (-)-sulpiride-induced increases in DA but did not affect the DA agonist-induced decreases. (-)-Sulpiride completely blocked the effect of quinpirole on DA both in control and in quinine-treated animals. At the highest dose used, quinine caused a large transient increase in extracellular DA. Local application of tetrodotoxin or infusion of Mg2+ in the absence of Ca2+ did not prevent this quinine-induced transient increase in extracellular DA. These results demonstrate that DA autoreceptors in the striatum regulate DA release in awake, behaving animals. Local application of (-)-sulpiride increases DA levels by blocking the tonic activation of autoreceptors by endogenous DA. Quinine blocks the neuroleptic-induced increase in DA, perhaps by preventing the K+ channel opening that would normally accompany endogenous autoreceptor activation. The fact that exogenously applied DA receptor agonists can decrease extracellular DA levels in the presence of quinine suggests that they may be acting at extrasynaptic autoreceptors that are not tonically active in vivo. The effect of DA agonists on this site is via a DA receptor because it is blocked by (-)-sulpiride. However, this receptor does not appear to be coupled to a quinine-sensitive potassium channel.     

Expression and localization of the CYP2B subfamily predominantly in neurones of rat brain

Despite the very small amounts of cytochrome P450 (P450, CYP) enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450s is determined by inconsistent interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the constitutive expression of CYP2B1 and CYP2B2 mRNAs in rat brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed the same expression pattern throughout the brain predominantly in neuronal populations, but to some extent in astrocytes of corpus callosum and olfactory bulb. The well known testosterone-metabolizing capacity and the presence of CYP2B isoforms shown in steroid hormone-sensitive areas and neurones (e.g. hippocampus) clarify the significance of isoforms like CYP2B1 and CYP2B2 for impairment of steroid hormone actions by P450 inducing environmental substances. We argue that cerebral P450 isoforms which are induced by xenobiotics and are able to metabolize these as well as endogenous substrates help us to understand fundamental aspects of brain's functioning.     

Expression, microsomal and mitochondrial activities of cytochrome P450 enzymes in brain regions from control and phenobarbital-treated rabbits

Expression and monooxygenase activity of various cytochrome P450 (CYP) enzymes along with constitutive androstane (CAR) and the pregnane X (PXR) receptors were investigated in the brain of control and phenobarbital-treated rabbits (80 mg/kg for 4 days). RT-PCR analysis, using specific primers, demonstrated that in control rabbits mRNAs of CYP 2A10, 2B4/5 and 3A6 were expressed, though to a different extent, in the liver, as well as in brain cortex, midbrain, cerebellum, striatum, hippocampus and hypothalamus, whilst CYP2A11 and 4B1 were not expressed in the hypothalamus. CAR was expressed in liver and all the brain regions examined, whereas the PXR was expressed only in liver and cortex. Real time RT-PCR analysis demonstrated that in vivo treatment with phenobarbital, in contrast with what happened in liver, did not induce the expression of CYP 2B4/5 mRNA in cortex, midbrain and cerebellum. NADPH cytochrome c reductase and some other enzymatic activities markers of CYP 2A, 2B, 3A and 4B activities were studied in liver microsomes as well as in microsomes and mitochondria of brain cortex, midbrain and cerebellum of control and phenobarbital-treated rabbits. In contrast to what was observed in liver, phenobarbital treatment did not induce the aforementioned monooxygenase activities in brain. However, we cannot exclude that a longer phenobarbital treatment may lead to a significant induction of CYP activities in brain. These findings indicated that brain CYPs, despite the presence of CAR, were resistant to phenobarbital induction, indicating a possible different regulation of these enzymes between brain and liver.     

Induction of rat hepatic drug metabolizing enzymes by dimethylcyclosiloxanes

Low molecular weight dimethylcyclosiloxanes (DMCS) are important precursors in the synthesis of polydimethysiloxane polymers widely used in industry, and in medical and personal care products. The objective of this study was to characterize the ability of two DMCS, octamethylcyclosiloxane (D4) and decamethylcyclopentasiloxane (D5) to induce drug metabolizing enzymes in rats. Male and female Sprague-Dawley rats were administered 1, 5, 20, or 100 mg/kg D4 or D5 in corn oil daily by gavage for 4 days. Changes in the levels of activity and/or immunoreactivity of CYP1A1/2, CYP2B1/2, CYP3A1/2 and NADPH cytochrome P450 reductase in liver microsomes were examined. Significant increases were observed in the liver to body weight ratio in female rats administered either D4 or D5 at doses > or = 20 mg/kg. Increases in the liver to body weight ratio were observed in male rats treated with > or = 100 mg/kg D5 but not with D4. Relatively large increases in CYP2B1/2 enzymatic activity and immunoreactive protein were observed with increasing concentrations of both D4 and D5. Significant increases in 7-pentoxyresorufin O-depentylase (PROD) activity were also detected in male and female rats given D4 at doses > or = 5 mg/kg. D5 increased PROD activity in male rats at doses > or = 20 mg/kg and in female rats at doses > or = 5 mg/kg. 7-Ethoxyresorufin O-deethylase (EROD) activity was increased in both male and female rats receiving > or = 20 mg/kg D4 or > or = 5 mg/kg D5; however, no changes were detected in CYP1A1/2 immunoreactive protein in rats of either sex. D4 and D5 caused significant increases in CYP3A1/2 immunoreactive protein in only male rats treated with 100 mg/kg of either compound. However, significant increases were detected in CYP3A1/2 immunoreactive protein in female rats at D4 doses > or = 20 mg/kg and D5 doses > or = 5 mg/kg. Induction of NADPH cytochrome P-450 reductase immunoreactive protein was observed with D4 in female rats and in both male and female rats with D5. Induction of CYP2B/1/2, CYP3A1/2 and NADPH cytochrome P450 reductase was observed in rats treated with 50 mg/kg phenobarbital by intraperitoneal injection. Maximal CYP2B induction detected with D4 was approximately 50% of the increase observed with phenobarbital. In summary, D4 and D5 induced CYP2B1/2 in adult rat liver in a manner similar to that observed with phenobarbital; however, differences were observed between D4 and D5 in their ability to induce CYP3A1/2 and NADPH cytochrome P450 reductase. Female rats were more sensitive to the inductive properties of low doses of both DMCS than male rats whereas male rats were more responsive to phenobarbital induction.     

Differences in the expression and inducibility of cytochrome P450 2B isoenzymes in cultured rat brain neuronal and glial cells

Studies initiated to investigate the distribution of cytochrome P450 2B (CYP2B) isoenzymes in rat brain cells revealed significant activity of CYP2B-dependent 7-pentoxyresorufin-O-dealkylase (PROD) in microsomes prepared from both, cultured rat brain neuronal and glial cells. Neuronal cells exhibited 2-fold higher activity of PROD than the glial cells. RT-PCR and immunocytochemical studies demonstrated significant constitutive mRNA and protein expression of CYP2B in cultured neuronal and glial cells. Induction studies with phenobarbital (PB), a known CYP2B inducer, revealed significant concentration dependent increase in the activity of PROD in cultured brain cells with glial cells exhibiting greater magnitude of induction than the neuronal cells. This difference in the increase in enzyme activity was also observed with RT-PCR and immunocytochemical studies indicating differences in the induction of CYP2B1 and 2B2 mRNA as well as protein expression in the cultured brain cells. Furthermore, a greater magnitude of induction was observed in CYP2B2 than CYP2B1 in the brain cells. Our data indicating differences in the expression and sensitivity of the CYP2B isoenzymes in cultured rat brain cells will help in identifying and distinguishing xenobiotic metabolizing capability of these cells and understanding the vulnerability of the specific cell types toward neurotoxins.     

Production of cytochrome P450 reductase yeast-rat hybrid proteins in Saccharomyces cerevisiae.

We present a novel strategy for increasing the level of functional mammalian cytochrome P450 (Cyt.P450) and NADPH:cytochrome P450 reductase enzymes produced in yeast. A cDNA encoding the rat P450 reductase was modified by the addition of a sequence coding for the N-terminal region of P450 reductase from Saccharomyces cerevisiae. The addition of this hydrophobic tail greatly increased the apparent stability of the reductase protein produced in S. cerevisiae, as compared to the unmodified rat P450 reductase. When the rat hybrid reductase was produced simultaneously with one of two mammalian Cyt.P450s, the rat CYP2B1 or the human CYP2A6, there was a significant increase in the specific activity of each of the Cyt.P450s. The optimization of this approach and its extrapolation to other organisms should lead to a marked improvement in our ability to study and exploit the P450 system.     

Voltammetric study of the control of striatal dopamine release by glutamate

The central dopamine systems are involved in several aspects of normal brain function and are implicated in a number of human disorders. Hence, it is important to understand the mechanisms that control dopamine release in the brain. The striatum of the rat receives both dopaminergic and glutamatergic projections that synaptically target striatal neurons but not each other. Nevertheless, these afferents do form frequent appositional contacts, which has engendered interest in the question of whether they communicate with each other despite the absence of a direct synaptic connection. In this study, we used voltammetry in conjunction with carbon fiber microelectrodes in anesthetized rats to further examine the effect of the ionotropic glutamate antagonist, kynurenate, on extracellular dopamine levels in the striatum. Intrastriatal infusions of kynurenate decreased extracellular dopamine levels, suggesting that glutamate acts locally within the striatum via ionotropic receptors to regulate the basal extracellular dopamine concentration. Infusion of tetrodotoxin into the medial forebrain bundle or the striatum did not alter the voltammetric response to the intrastriatal kynurenate infusions, suggesting that glutamate receptors control a non-vesicular release process that contributes to the basal extracellular dopamine level. However, systemic administration of the dopamine uptake inhibitor, nomifensine (20 mg/kg i.p.), markedly decreased the amplitude of the response to kynurenate infusions, suggesting that the dopamine transporter mediates non-vesicular dopamine release. Collectively, these findings are consistent with the idea that endogenous glutamate acts locally within the striatum via ionotropic receptors to control a tonic, impulse-independent, transporter-mediated mode of dopamine release. Although numerous prior in vitro studies had suggested that such a process might exist, it has not previously been clearly demonstrated in an in vivo experiment.     

Immunochemical characterization and toxicological significance of P-450HFLb purified from human fetal livers.

Immunochemical properties of P-450HFLb purified from human fetal livers were investigated. P-450HFLb cross-reacted with antibodies to rat P-4501A1 but not with antibodies to CYP2A6, CYP2C9, CYP3A7 (P-450HFLa) and rat CYP2B1. In addition, P-450HFLb also cross-reacted with both monospecific antibodies to rat CYP1A1 and CYP1A2. However, P-450HFLb was shown to be an immunochemically distinct form of cytochrome P-450 from P-450PA (human CYP1A2). Immunoblot analysis of human fetal livers with the antibodies to P-450HFLb showed that P-450HFLb was expressed in all fetal livers studied although there appeared to be individual differences in the amounts of P-450HFLb expressed in fetal livers. The formation of mutagens from IQ (but not from AFB1) in fetal liver homogenates was inhibited by the antibodies to P-450HFLb in a dose dependent manner. These results suggest that P-450HFLb may be a form of human cytochrome P-450 classified into CYP1 gene family, and that the cytochrome P-450 is, in part, responsible for the mutagenic activation of IQ in human fetal livers as well as CYP3A7 (P-450HFLa).     

Effect of direct and indirect dopamine agonists on brain extracellular ascorbate levels in the striatum and nucleus accumbens of awake rats

Systemic administration of direct and indirect dopamine agonists resulted in increased extracellular ascorbic acid levels in the striatum and, to a lesser degree, in the nucleus accumbens as measured by in vivo voltammetry. Intraperitoneal d-amphetamine sulfate (5mg/kg) increased ascorbate concentrations in striatal extracellular fluid. Amphetamine also increased extracellular ascorbate levels in the nucleus accumbens although more gradually and to a lesser extent. Intraperitoneal phenethylamine hydrochloride (20 mg/kg) following pargyline hydrochloride pretreatment (20 mg/kg) increased extracellular ascorbate levels in the striatum significantly above the small increase seen in the nucleus accumbens. The direct acting dopamine agonists Ly-141865 and Ly-163502 when given i.p. at 1 mg/kg, resulted in increased extracellular ascorbate concentrations in both brain areas, again with a significantly greater effect in the striatum. These results indicate that brain extracellular ascorbate levels can be modulated by dopaminergic neuro-transmission and that this modulation is quantitatively different in different dopamine-containing brain structures.     

Cytochrome P450s in the synthesis of cholesterol and bile acids--from mouse models to human diseases

The present review describes the transgenic mouse models that have been designed to evaluate the functions of the cytochrome P450s involved in cholesterol and bile acid synthesis, as well as their link with disease. The knockout of cholesterogenic Cyp51 is embrionally lethal, with symptoms of Antley-Bixler syndrome occurring in mice, whereas the evidence for this association is conflicting in humans. Disruption of Cyp7a1 from classic bile acid synthesis in mice leads to either increased postnatal death or a milder phenotype with elevated serum cholesterol. The latter is similar to the case in humans, where CYP7A1 mutations associate with high plasma low-density lipoprotein and hepatic cholesterol content, as well as deficient bile acid excretion. Disruption of Cyp8b1 from an alternative bile acid pathway results in the absence of cholic acid and a reduced absorption of dietary lipids; however, the human CYP8B1 polymorphism fails to explain differences in bile acid composition. Unexpectedly, apparently normal Cyp27a1(-/-) mice still synthesize bile acids that originate from the compensatory pathway. In humans, CYP27A1 mutations cause cerebrotendinous xanthomatosis, suggesting that only mice can compensate for the loss of alternative bile acid synthesis. In line with this, Cyp7b1 knockouts are also apparently normal, whereas human CYP7B1 mutations lead to a congenital bile acid synthesis defect in children or spastic paraplegia in adults. Mouse knockouts of the brain-specific Cyp46a1 have reduced brain cholesterol excretion, whereas, in humans, CYP46A1 polymorphisms associate with cognitive impairment. At present, cytochrome P450 family 39 is poorly characterized. Despite important physiological differences between humans and mice, mouse models prove to be an invaluable tool for understanding the multifactorial facets of cholesterol and bile acid-related disorders.