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PubMed1999

CYP2D6 and CYP2C19 activity in a large population of Dutch healthy volunteers: indications for oral contraceptive-related gender differences.

OBJECTIVE: We examined a large database containing results on CYP2D6 and CYP2C19 activity of 4301 Dutch volunteers phenotyped in the context of various clinical pharmacology studies. METHODS: The subjects were given 22 mg dextromethorphan, 100 mg mephenytoin and 200 mg caffeine. For CYP2D6, the dextromethorphan/dextrorphan metabolic ratios in urine samples taken for a subsequent 8 h were used. Dextromethorphan and dextrorphan were quantified by reversed-phase high performance liquid chromatography. For CYP2C19 similarly obtained (R)-mephenytoin and (S)-mephenytoin ratios were used. (S)-mephenytoin and (R)-mephenytoin were analysed and quantified by enantioselective capillary gas chromatography. In addition, CYP2C19 poor metabolizer (PM) subjects were reanalysed after acidic pre-treatment of urine samples to confirm the PM status. RESULTS: The investigated population mainly comprised Caucasian (98.9%) males (68%). The age ranged from 18 to 82 years. For CYP2D6, it was found that 8.0% of the subjects were PMs. The average metabolic ratio was 0.014 (0.033) for subjects who showed extensive metabolizing activity (EM) and 5.4 (7.6) for PM subjects. For CYP2C19, it was found that 1.8% of the subjects were PMs. The metabolic ratio was 0.162 (0.124) for EM subjects and 1.076 (0.040) for PM subjects. Within the EM group the metabolic ratio in females was significantly lower for CYP2D6 (-20%) and significantly higher for CYP2C19 (+40%) compared with males. For PMs there was no such difference for CYP2D6 (P = 0.79) or CYP2C19 (P = 0.20). Oral contraceptive (OC) use significantly decreased the CYP2C19 activity by 68% for mephenytoin as compared to non-OC using females. CONCLUSIONS: For CYP2D6, the PM incidence (8.0%) is in accordance with literature data. The CYP2C19, PM incidence (1.8%) is low compared to reports from other European countries. For mephenytoin, the acidification procedure has been shown to be very important for the confirmation of CYP2C19 PMs. In EM females compared to EM males, CYP2D6 activity is increased and CYP2C19 activity is reduced. For CYP2C19 in particular this reduction is substantial and most pronounced in the age range from 18 to 40 years. For CYP2C19, the reduced activity is associated with the use of oral contraceptives.

Adult↗
PubMed2001

An optimized methodology for combined phenotyping and genotyping on CYP2D6 and CYP2C19.

A method for simultaneous phenotyping and genotyping for CYP2D6 and CYP2C19 was tested. Six healthy volunteers were selected (three extensive and three poor metabolisers for CYP2D6). CYP2D6 was probed with dextromethorphan and metoprolol and CYP2C19 was probed with omeprazole. Blood samples were collected and analysed for dextromethorphan, dextrorphan, metoprolol, alpha-hydroxymetoprol, omeprazole and 5-hydroxyomeprazole by HPLC. Genotyping was performed for both CYP2D6 and CYP2C19. Generally, plasma levels could be measured up to 8 h post-dose except for alpha-hydroxymetoprolol in poor metabolizers (PMs) and dextromethorphan in extensive metabolizers (EMs) (35% below quantification limit). The correlation between the metabolic ratio based on timed individual measurements and the metabolic ratio based on the AUC0-12 values was significant at 3 h post-dose for all probes. In conclusion, the following procedure is suggested: administer metoprolol (100 mg) and omeprazole (40 mg); after 3 h, take a blood sample to assess the genotype and the metabolic ratio for CYP2D6 (metoprolol over alpha-hydroxymetoprolol) and CYP2C19 (omeprazole over 5-hydroxyomeprazole) in plasma. With this procedure, all necessary information on the individual CYP2D6 and CYP2C19 metabolising capacity can be obtained in a practical, single-sample approach.

Adult↗
PubMed1987

High-performance liquid chromatographic assays for bufuralol 1'-hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan O-demethylase in microsomes and purified cytochrome P-450 isozymes of human liver.

Bufuralol, debrisoquine, and dextromethorphan are three prototype substrates of the common genetic deficiency of oxidative drug metabolism in man known as debrisoquine/sparteine-type polymorphism. We describe assays for the in vitro metabolism of (+)- and (-)-bufuralol, debrisoquine, and dextromethorphan in human liver microsomes and reconstituted purified cytochrome P-450 isozymes. These assays combine nonextractive sample preparation by precipitation of protein with perchloric acid with reversed-phase inorganic ion-pair HPLC and fluorescence detection. The minimal detectable levels of the major metabolites formed are 1'-hydroxybufuralol, 0.1 ng/ml; 4-hydroxydebrisoquine, 0.8 ng/ml; and dextrorphan, 0.1 ng/ml. Formation of these metabolites is linear for at least 45 min and between 1 and 100 micrograms of microsomal protein. Comparative kinetic analysis of the three monooxygenase reactions in human liver microsomes revealed an apparent biphasicity of (+)- and (-)-bufuralol 1'-hydroxylation and dextromethorphan O-demethylation but monophasic formation of 4-hydroxydebrisoquine in the substrate concentration range (less than 1 mM) studied. These data, in combination with those obtained by purified human cytochrome P-450 isozymes indicate the involvement of the same enzyme in the metabolism of all three substrates investigated. However, additional and distinct activities contribute to the metabolism of (+)- and (-)-bufuralol and dextromethorphan.

Chromatography, High Pressure Liquid↗
PubMed1976

Binding of the opiate-like pentapeptide methionine-enkephalin to a particulate fraction from rat brain.

The characteristics of stereospecific binding of [3H] met-enkephalin (15 Ci/mmole) were studied in a particulate fraction from rat brain. The binding assay was performed for 70 min at degrees C and the bound radioactivity separated by filtration through glass fiber filters (Whatman, GF/C). In the absence of sodium, binding of [3H] met-enkephalin could be described on the basis of two independent binding sites with apparent KDs of 2.1 and 53 nM, respectively. The data are also consistent with one class of binding sites showing negative cooperativity. In the presence of 100 mM NaCl, binding of [3H] met-enkephalin was 90-95% reduced, thus indicating the agonist properties of the peptide. The highly stereospecific binding of [3H] met-enkephalin was evidenced by the 10,000-fold greater potency of levorphanol than its analgesically inactive enantiomer dextrorphan to compete for [3H] met-enkephalin binding. Similar conclusions could be reached using levallorphan, (+)-3-hydro-N-allyl-morphinan, (-) methadone and (+) methadone. The apparent affinity of various opiate agonists and antagonists for the binding sites was closely correlated with their known pharmacological activity.

Animals↗
PubMed1984

Receptor-mediated stimulation of brain GTPase by opiates in normal and dependent rats.

In membranes from rat brain striatum, opiate agonists stimulated low-K GTPase. Half-maximal enhancement of enzyme activity was obtained with 0. 09m microM morphine and 3.8 microM levorphanol. This order of potency corresponded to that of the affinities of these compounds in binding to opiate receptor. The effect was inhibited by the antagonist naloxone. As shown by the use of the enantiomers levorphanol and dextrorphan, only the pharmacologically active stereoisomer stimulated GTPase. In membranes isolated from morphine-dependent rats, the activity of GTPase was reduced 20-40% relative to that in control rats. After the precipitation of morphine abstinence by naloxone, brain GTPase activity was intermediate between the respective values for naive and dependent animals.

Animals↗
PubMed1985

NMB: a human neuroblastoma cell line with specific opiate binding sites.

The human neuroblastoma cell line designated NMB (Brodeur et al., 1977, Cancer 40: 2256) has been shown to have specific opiate binding sites. These sites are highly stereospecific. Two characteristic delta specific peptides, D-Ala2-D-Leu5 enkephalin and D-Thr2-D-Thr6 enkephalin, have high affinity for the binding sites. Morphine binds specifically but with a much lower affinity. Dextrorphan and the mu specific peptide morphiceptin (Tyr-Pro-Phe-Pro-CO-NH2) do not bind to the site. The binding sites are heat and trypsin sensitive. Sodium ions specifically lower agonist binding to the sites. Approximately 14,000 binding sites per cell are found. The binding characteristics of these sites are very similar to those of the delta sites characterized on mouse neuroblastoma cell lines.

Binding Sites↗
PubMed1984

Benz(f)isoquinolines as excitatory amino acid antagonists: an indication of their mechanism of action?

Using the technique of microelectrophoresis on cat and rat spinal neurones, the bridged benz(f)isoquinoline, LY154045, like ketamine and dextrorphan, was found to be a selective antagonist of N-methylaspartate, an amino acid used for characterizing excitatory amino acid synaptic receptors. The unbridged analogue, LY154005, was inactive as an amino acid antagonist. This result correlates well with the ability of LY154045, but not LY154005, to displace phencyclidine from CNS tissue and to mimic phencyclidine in behavioural tests. The potential role of N-methylaspartate antagonism in the aetiology of some of the behavioural effects of LY154045, phencyclidine and related drugs is considered.

Acetylcholine↗
PubMed1988

In-vivo and in-vitro dextromethorphan metabolism in SD and DA rat. An animal model of the debrisoquine-type polymorphic oxidation in man.

The female dark Agouti (DA) rat is well established as an animal model for the debrisoquine poor metabolizer phenotype (PM), whereas the SD rat represents the extensive metabolizer (EM). It is not known, however, if the DA rat also is representative for the dextromethorphan (DEM) PM, a compound recently demonstrated to be subjected to the debrisoquine phenotype in man. Studies were performed, therefore, to evaluate in-vivo and in-vitro metabolism of DEM in DA and SD rats. After oral administration of 50 mg/kg of DEM, the DA rat excreted 25 +/- 6% of the dose in 72-hr urine as O-demethylated product (dextrorphan), whereas the SD excreted 40 +/- 9% (P less than 0.002). Metabolic ratio of O-demethylation was 0.46 +/- 0.11 in DA and 0.02 +/- 0.01 in SD (P less than 0.001). As a compensatory mechanism, N-demethylation was ninefold increased in DA compared to SD (8.0 +/- 3% of the dose excreted in urine of DA as methoxymorphinan vs 0.9 +/- 0.7% in SD) (P less than 0.001). Total plasma clearance of DEM was 95 +/- 20 ml/min/kg in SD and 45 +/- 13 ml/min/kg in DA (P less than 0.001). In vitro, microsomal affinity for DEM O-demethylation was greater than 50 times higher in SD than in DA rats (P less than 0.004), whereas Vmax did not differ statistically. Vmax for N-demethylation was 80% increased in DA (P less than 0.01), whereas corresponding Km values did not differ. It appears that the differences in DEM metabolism between DA and SD rats are qualitatively similar to human EM and PM phenotypes, respectively. Whether this is also true for the underlying mechanism(s) however, remains to be resolved.

Algorithms↗
PubMed1991

Cytochrome P450IID subfamily in non-human primates. Catalytical and immunological characterization.

Interindividual variations of debrisoquine metabolism was recently identified in non-human primates tested in vivo. The catalytical and immunological characterization of cytochrome P450IID subfamily was undertaken in hepatic microsomes from extensive metabolizer primates. The NADPH/O2 mediated metabolism of debrisoquine, dextromethorphan and bufuralol was similar to the kinetics reported in humans. The CuOOH mediated metabolism of bufuralol suggested that at least two enzymes are responsible for bufuralol 1'-hydroxylation. Eleven compounds were tested for their capacity to modify P450IID function in vitro. Eight competitive inhibitors of P450IID6 in man were all and exclusively competitive inhibitor of P450IID subfamily in non-human primates. Quinidine, which is the strongest competitive inhibitor in man, exhibited the higher inhibitory potency in monkey (Ki = 0.75 microM). Anti-LKM antibody against P450IID subfamily cross-reacted with two proteins of 49 and 47 kDa, and sera containing anti-LKM antibody against these two proteins inhibited dextrorphan formation in vitro. These data provide evidence for catalytical and immunological similarities between human and monkey microsomes and indicate that the primate system could be a model for enzymatic studies of P450IID.

Animals↗
PubMed1993

Primary and secondary oxidative metabolism of dextromethorphan. In vitro studies with female Sprague-Dawley and Dark Agouti rat liver microsomes.

The O-demethylation of dextromethorphan (DM) to dextrorphan (DR) is catalysed by the polymorphic CYP2D6 (cytochrome P4502D6) isozyme in man. DM is commonly used as a probe for phenotyping subjects as either poor or extensive metabolizers for the debrisoquine/sparteine oxidative polymorphism via CYP2D6. The enzyme kinetics of DM O- and N-demethylation, and the N- and O-demethylations of the primary metabolites DR and 3-methoxymorphinan (3MM), respectively, were studied in liver microsomes from female Dark Agouti (DA) rats, the poor metabolizer counterpart, and female Sprague-Dawley (SD) rats, the extensive metabolizer counterpart. The formation of metabolites was quantified by HPLC with fluorescence detection and kinetic parameters were calculated. The intrinsic clearance (Vmax/Km) of the O-demethylation of 3MM to 3-hydroxymorphinan (3OHM) was 180-fold lower in DA rats (0.11 vs 20.77 mL/hr/mg) due to a 60-fold higher Km (108.7 vs 1.76 microM) and 3-fold lower Vmax (11.5 vs 35.95 nmol/mg/hr). The kinetics for DR N-demethylation to 3OHM did not differ between rat strains. The Michaelis-Menten constant (Km) for DM N-demethylation to 3MM was similar between SD and DA rats (85.04 vs 68.99 microM); however, SD rats displayed a 2-fold higher Vmax (83.37 vs 35.49 nmol/mg/hr) and intrinsic clearance (0.96 vs 0.51 mL/hr/mg). The O-demethylation of DM to DR in SD rats showed a high and low affinity enzyme component, with the high affinity intrinsic clearance contributing 98% of the total intrinsic clearance in these rats. DM O-demethylation in DA rats was characterized by a single enzyme system. The high affinity O-demethylating enzyme in SD rats showed a 20-fold lower Km (2.5 vs 55.6 microM) and a three-fold higher Vmax (51.04 vs 16.84 nmol/mg/hr) resulting in a 66-fold higher intrinsic clearance (20.04 vs 0.31 mL/hr/mg) compared to DA rats. Quinine, dextropropoxyphene, (+/-)methadone and (+/-)propafenone were shown to be potent inhibitors of 3MM and DM O-demethylation but did not inhibit DR or DM N-demethylation at similar concentrations. SD and DA rats showed a clear strain difference in 3MM O-demethylation and DM O-demethylation. In contrast, DR N-demethylation and DM N-demethylation do not appear to be under genetic control in the female SD-DA rat model. Kinetic parameters and inhibition studies suggest that 3MM and DM O-demethylation pathways in the rat may be mediated by the same cytochrome P450 isozyme.

Animals↗
PubMed1979

Interaction of morphine, etorphine and enkephalins with dopamine-stimulated adenylate cyclase of monkey amygdala.

Adenylate cyclase activity (AC) of homogenates of monkey amygdaloid nucleus was approximately doubled in the presence of dopamine (10 micrometer). Morphine, etorphine, and several enkephalin analogs (met-enkephalin, D-ala2-met-enkephalin, and D-met2, pro5-enkephalinamide) were capable of inhibiting the stimulation of AC produced by dopamine (90-100% with etorphine or D-ala2-met-enkephalin). Unlike morphine and etorphine, the peptides exhibited bell-shaped dose-response curves for this inhibition with maximal effects at approximately 1 X 10(-7) M, but negligible effects at 1 X 10(-5) M. Under the conditions studied, only etorphine inhibited basal AC. Naloxone antagonized the inhibitory effects of each of the opioids tested, and dextrorphan, an inactive L-(+)-opiate, failed to inhibit the dopamine response. Together these data indicate that the effects were mediated via the classically described stereospecific opiate receptor. The relative order of potency (etorphine greater than enkephalins greater than morphine) was similar to that previously reported for the binding affinities of these drugs in rat brain homogenates. The influence of narcotic agents on dopamine stimulated AC was eliminated by either freezing the amygdaloid tissue or preincubating the homogenate at 4 degrees C; the dopamine responses, however, could still be elicited. The narcotic receptor interaction with the adenylate cyclase thus appears to be distinct from and more labile than that of the dopamine receptor. Gpp(NH)p-stimulated AC was not inhibited by morphine. It is postulated that the inhibition involves interaction of opiate receptors with catalytic units of dopamine-stimulated AC, but not with other cyclase species which may provide the major component of Gpp(NH)p-stimulated activity in amygdala.

Adenylyl Cyclases↗
PubMed1981

The effect of systemic morphine upon diffuse noxious inhibitory controls (DNIC) in the rat: evidence for a lifting of certain descending inhibitory controls of dorsal horn convergent neurones.

The effects of exogenous opiates upon diffuse noxious inhibitory controls (DNIC) was investigated in intact anaesthetized rats. 58 convergent neurones, responding to both noxious and innocuous stimuli applied to their cutaneous receptive fields, were recorded at the lumbar level. These cells received A- and C-peripheral fibre inputs as shown by electrical stimulation of their receptive fields and were mainly located in the medial part of the dorsal horn. The immersion of the distal two-thirds of the tail in hot water (52 degrees C) induced strong inhibition of the responses to both A-(23%) and C-(69%) fibres. Post-effects of long duration were commonly observed after cessation of the conditioning stimulus. While systemic injection of morphine at a low dose-range (0.1-1 mg/kg) did not significantly affect the unconditioned responses, the DNIC-mediated inhibitions were profoundly altered. (a) DNIC of responses to C fibres were dose-dependently (P less than 0.01) lifted by morphine: (b) the post-effects observed after cessation of conditioning stimuli were dose-dependently (P less than 0.01) diminished; (c) DNIC of responses to A-fibre were similarly altered but this effect was less significant (P less than 0.05); (d) DNIC of responses to sustained moderate pressure were greatly diminished by morphine (P less than 0.01); and (e) these effects were specific since they were antagonized by the opiate antagonist, naloxone. In addition, they were shown to be stereospecific since while the dextrogyre stereoisomer, dextrorphan, was ineffective the levogyre derivative, levorphanol, induced a significant lifting of DNIC. It is concluded that morphine decreases the supraspinal inhibitory controls of dorsal horn convergent neurones, at least when these controls are triggered by noxious stimuli. Assuming that a basic somatosensory background activity (noise) is transmitted to higher centres by dorsal horn convergent neurones, and that the pain-signalling message is the contrast between the activity of the segmental pool of neurones induced by the noxious stimulus and the DNIC-mediated silence of the remaining neuronal population, it is proposed that, by a reduction in DNIC, low-dose morphine could restore the initial level of background activity, the final result being analgesia.

Animals↗
PubMed1981

Opiate antagonism of glycine-evoked membrane polarizations in cultured mouse spinal cord neurons.

The effect of several opiate receptor agonists on the responses of spinal cord neurons to putative inhibitory and excitatory amino acids was studied using an in vitro model system, cultured fetal mouse spinal cord neurons, and bath application of opiates. Intracellular recordings were made from the cultured neurons with conventional voltage recording techniques or under voltage clamp conditions. The putative amino acid neurotransmitters were applied by iontophoresis or micropressure ejection. Our main finding is that the opiate agonists, morphine and levorphanol (5-100 microM), consistently depressed the responses evoked by the putative inhibitory amino acid neurotransmitters glycine and beta-alanine but not GABA. Dextrorphan, the inactive isomer of levorphanol, also depressed the glycine and beta-alanine responses, but higher concentrations were required. The excitatory glutamate response was unaltered by these opiates. Leucine enkephalin, an opioid peptide, had no effect on the amino acid responses in the neurons where it was also tested. The opiate antagonist naloxone (10-100 microM) did not reverse the morphine or levorphanol depressions of the amino acid responses making it unlikely that opiate receptors mediate this effect. Strychnine was considerably more effective than morphine as a glycine antagonist, producing depressions at nM concentrations compared to the microM concentrations required for morphine. Preliminary studies indicate that both morphine and strychnine act in a non-competitive manner. However, additional studies will be required before the sites of action for these agents can be identified. The possible pharmacological or toxicological significance of the present work remains to be determined. Considering the high doses of opiates (microM concentrations) required to depress the glycine and beta-alanine responses, it is unlikely that this action is relevant to normal therapeutic situations. However, such concentrations of opiates are often utilized in pharmacological studies and may be achieved when opiates are applied by iontophoresis. Our data indicate that consideration of the present opiate action should be made when microM concentrations or iontophoretic application of opiates are used for pharmacological studies of CNS tissue.

Amino Acids↗
PubMed1982

Drug reinforcement studied by the use of place conditioning in rat.

Rats display a preference for an environment in which they previously received morphine. The present report provides behavioral and pharmacological data for this simple model of reinforcement produced by opiates and describes an aversion in rats for an environment in which they previously received naloxone. Preferences were produced with intravenous (i.v.) morphine sulfate at doses of 0.08-15 mg/kg and durations of the pairing between environment and morphine of 10 min to 1.5 h. Preferences were also seen with other opiate agonists (etorphine-HCl and levorphanol-tartrate), another route of drug administration (subcutaneous), and after 1-4 administrations of morphine. Cocaine-HCl (i.v.), a non-narcotic drug, known to be self-administered by humans, also produced a place preference. Lithium chloride (i.v.), an agent found to be a punishing stimulus in other situations, produced a place aversion. There was no appreciable preference for an environment paired with dextrorphan-tartrate and naloxone-HCl (2 mg/kg, i.p.) blocked the production of the preference produced by i.v. morphine. In contrast to the effect produced by morphine, aversions were produced with (-)-naloxone-HCl alone at doses of 0.1-45 mg/kg (i.v.). The aversion was not produced at (+)-naloxone. Implantation of rats with a 75 mg morphine pellet 3 days prior to place conditioning potentiated the aversive effect of naloxone. It was concluded that place conditioning produced by morphine and naloxone is mediated by specific opiate receptors and that stimulating and decreasing activity of the endogenous opioid peptide system with systemically administered drugs is positively reinforcing and aversive, respectively. The discussion emphasizes application of the simple and sensitive place conditioning model to drug reinforcement research, including analyses of reinforcement produced by microinjection of opiates into the brain.

Animals↗
PubMed1986

Increased food intake after opioid microinjections into nucleus accumbens and ventral tegmental area of rat.

The nucleus accumbens (ACC) and ventral tegmental area (VTA), two areas believed to subserve reinforcement and increases in locomotor activity produced by opioid microinjections, were examined for their involvement in opioid-produced changes in ingestive behavior. Opioids were infused bilaterally, and food and water intakes were measured for 1 h thereafter. Different morphine doses were administered and, with placements in globus pallidus and lateral ventricles as controls for diffusion, it was found that only ACC and VTA microinjections (0.1-10 nmol) produced dose-related increases in food intake. In both the ACC and VTA low doses of morphine also produced increases in water intake while in ACC high doses produced a decrease. Administration of morphine and an enkephalin analogue (Tyr-D-Met-Gly-Phe(4-NO2)-Pro-NH2) at different depths in the ACC indicated that the increase in food intake occurred at a site separate from that of the decrease in water intake. Using levorphanol, dextrorphan and morphine mixed with naloxone, it was shown that the effects were due to activation of opioid receptors. Additional experiments demonstrated that food intake is increased by ACC morphine under different levels of deprivation, with different times of testing and with availabilities of various goal objects in addition to food. The effect also did not appear to undergo development of tolerance or sensitization. It was concluded that there are sites in the ACC and VTA where increased activity of endogenous opioid peptide systems reliably increase food intake and it was hypothesized that these sites may contribute to changes in ingestive behavior after systemic morphine administration. Also, together with other effects produced by opioid microinjections into the ACC and VTA, the present findings suggest that increased opioid activity in these areas produce a pattern of behaviors similar to that produced in normal animals by food conditioned stimuli.

Animals↗
PubMed1987

Electrophysiological correlates of presynaptic opiate receptor activation: reduction in norepinephrine-mediated inhibition from the locus coeruleus.

Inhibitory responses of rat cerebellar Purkinje cells to locus coeruleus (LC) stimulation and iontophoresis of norepinephrine (NE) were examined before and after administration of morphine to determine whether the inhibitory modulation of NE release by opiates results in a functional impairment in noradrenergic synaptic action. Administration of morphine systemically (0.2-1.2 mg/kg, i.v.) or by iontophoresis reduced inhibitions in Purkinje firing elicited by LC stimulation without affecting depressions in activity induced by postsynaptic applications of NE. This antagonistic effect of morphine on LC-induced inhibition was reversed or prevented by naloxone and mimicked by administration of levorphanol but not dextrorphan. Morphine increased the excitatory response of Purkinje cells to monosynaptic input from the parallel fibers, whereas it blocked gamma-aminobutyric acid-induced inhibitions in firing via a non-opiate receptor-mediated mechanism. These results demonstrate that morphine interferes with synaptic inhibition derived from the LC and suggest that this may occur via activation of presynaptic opiate receptors residing on noradrenergic nerve terminals.

Animals↗
PubMed1988

Modulation of adenylate cyclase activity of mouse spinal cord-ganglion explants by opioids, serotonin and pertussis toxin.

Organotypic cultures of fetal mouse spinal cord-ganglion explants (2-4 weeks in vitro) contain forskolin-stimulated adenylate cyclase (AC) activity that is inhibited by levorphanol and other opioid agonists in a dose-dependent manner. Inhibition by levorphanol no longer occurs if sodium is omitted from the incubation and the levorphanol inhibition is blocked by the opioid antagonist, naloxone. These findings together with the ineffectiveness of dextrorphan indicate that the opioid inhibition of forskolin-stimulated AC is receptor mediated. Both the delta- and kappa-receptor subtypes appear to be involved since the selective delta-opioid agonist, [D-Pen2, D-Pen5]enkephalin, and the selective kappa-opioid agonist, t-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)cyclohexyl]-benzene acetamide (U-50,488H) are both effective at nanomolar concentrations. In contrast, the selective mu-opioid agonist, Tyr-D-Ala-Gly-N-MePhe-Gly-ol, has no significant effect even at micromolar concentrations. Both cord and ganglion components of the explants contain opioid-sensitive AC. Forskolin-stimulated AC of the explants is also inhibited by serotonin and carbachol. The serotonin effect appears to be mediated by 5-HT1A receptors, based on relative agonist and antagonist selectivity. Chronic exposure of cultures to morphine results in enhanced basal and forskolin-stimulated AC as well as attenuation of opioid-inhibition of AC assayed in the presence of forskolin; treatment of explants with pertussis toxin causes similar changes in the AC system. The inhibitory effect of serotonin is also attenuated by the pertussis toxin treatment. Basal AC activity of the explants (assayed without forskolin present) is stimulated to a small but significant extent by opioids and by serotonin. The opioid stimulatory effect is markedly enhanced following either morphine or pertussis toxin treatment of the explants. The attenuation of opioid- and serotonin-inhibition of AC produced by chronic exposure to pertussis toxin and the attenuation of opioid inhibition produced by exposure to morphine are consonant with the attenuation of opioid and monoaminergic depression of sensory evoked dorsal horn network responses after similar chronic treatments. It is proposed that the inhibitory effects of opioids and serotonin on these neurons are mediated by receptors that are negatively coupled via a pertussis toxin sensitive Gi protein to AC. Furthermore, alterations of AC with chronic morphine treatment may be involved in the development of physiologic tolerance to opioids.

Adenylate Cyclase Toxin↗
PubMed1988

GC and GC-MS procedures for simultaneous phenotyping with dextromethorphan and mephenytoin.

A genetic deficiency in the metabolism of dextromethorphan and mephenytoin may be revealed by the excretion pattern of dextromethorphan and its metabolite dextrorphan, and mephenytoin, 4-OH-mephenytoin, respectively, after a single dose of the test drugs. Existing methods were modified for determining the compounds in 0.1-0.5 ml urine samples. No prior derivatization of the compounds was necessary before their gaschromatographic or mass-spectrometric analysis by using crosslinked 5% phenylmethyl silicone fused silica columns. Seven healthy volunteers were phenotyped at weekly intervals with either 25 mg dextromethorphan or 100 mg mephenytoin, or both drugs. One subject was a poor metabolizer of mephenytoin, while all subjects were extensive metabolizers of dextromethorphan. Neither a pharmacokinetic nor an analytical interference was observed when the results of the single test were compared with those of the combined test. The results of the mephenytoin test were also tentatively given in form of metabolic ratios. The GC-MS assay was designed for clinical studies so that patients treated with other drugs could be phenotyped.

Adult↗