The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.     

A novel, convenient assay of lanosterol 14α-demethylase

A rapid and sensitive kinetic assay of lanosterol 14α-demethylation has been developed and analyzed. Three substrates, [32-³H]-24,25-dihydrolanosterol, [32-³H]lanost-8-en-3β,32-diol, and [32-³H]lanost-7-en-3β-32-diol, were studied. In all cases, the rate of tritium released into aqueous solution provided a simple and direct assay of 14α-demethylase activity. The kinetic parameters of K_m and V_max for each substrate have been determined in a reconstituted system from rat liver. The percentage of turnover monitored by the novel tritium release assay was comparable to that observed by conventional GC methods. Separation of unreacted sterol from tritiated formate and water via reverse-phase chromatography permitted several samples to be analyzed at once.     

A sensitive and specific tritium release assay for dopamine-beta-hydroxylase (DbetaH) in serum.

The release of tritium from [7-³H₂]dopamine was investigated as a possible procedure for the assay for dopamine-β-hydroxylase (DβH) in rat and human serum. The release was found to have the same characteristics as those deseribed previously for DβH in serum; for example, an optimum rate of reaction at pH 5.0 or an enhancement of release with agents such as Cu²⁺ ions and N-ethylmaleimide which are known to inactivate endogenous inhibitors of DβH in serum. Tritium release was blocked by the DβH inhibitor fusaric acid but not by inhibitors of other dopamine-metabolizing enzymes in serum. Incubation of ¹⁴C-labeled dopamine along with [7-³H₂]dopamine revealed that, under the standard assay conditions, the formation of [¹⁴C]norepinephrine was accompanied by release of one of the two tritium atoms on the 7-carbon. It was concluded that the procedure provided a simple and sensitive assay of DβH activity in serum.     

Effect of ibogaine on serotonergic and dopaminergic interactions in striatum from mice and rats

The effect of ibogaine (Endabuse, NIH 10567) on serotonin uptake and release, and on serotonergic modulation of dopamine release, was measured in striatal tissue from rats and mice. Two hours after treatment in vivo with ibogaine (40 mg/kg i.p.), the uptake of labeled [³H]serotonin and [³H]dopamine uptake in striatal tissue was similar in the ibogaine-treated animal to that in the control. The 5HT_1B agonist CGS-12066A (10^–5 M) had no effect on stimulation-evoked tritium release from mouse or rat striatal tissue preloaded with [³H]serotonin; however, it elevated tritium efflux from striatal tissue preloaded with [³H]dopamine. This increase was not seen in mice treated with ibogaine 2 or 18 hours previously, or in rats treated 2 hours before. Dopamine autoreceptor responses were not affected by ibogaine pretreatment in either mouse or rat striatal tissue; sulpiride increased stimulation-evoked release of tritium from tissue preloaded with [³H]dopamine. The long-lasting effect of ibogaine on serotonergic functioning, in particular, its blocking of the 5HT_1B agonist-mediated increase in dopamine efflux, may have significance in the mediation of its anti-addictive properties.Special issue dedicated to Dr. Sidney Ochs.     

Synthesis of [H]- and -[C]-labelled sulprostone (CP-34,089/ZK-57,671)

The synthesis of N-methanesulfonyl 16-phenoxy-ω-tetranor PGE₂ carboxamide (sulprostone — CP-34,089/ZK-57,671) labeled with tritium and carbon-14 is described. Sulprostone labeled with tritium in the phenoxy moiety by means of catalytic hydrogenolysis was obtained in a 17% radiochemical yield with a specific activity of 1.0 Ci/mmol. The methanesulfonamide-¹⁴C derivative of sulprostone was prepared from methyl-¹⁴C iodide in an 11.8% radiochemical yield having a specific activity of 18.8 mCi/mmol.     

VITAMIN B6 TRANSPORT IN THE CENTRAL NERVOUS SYSTEM: IN VITRO STUDIES

Abstract— The transport into and release of tritium labeled vitamin B₆ ([³H]B₆) from rabbit brain slices and isolated choroid plexuses were studied. In vitro, both brain slices and choroid plexus concentrated [³H]B₆ by an energy dependent uptake system when [³H]pyridoxine (PIN) was added to the incubation medium. Most of the [³H] within the tissues was phosphorylated [³H]B₆. In each tissue, the nonphosphorylated vitamers inhibited the uptake of [³H]PIN from the medium significantly more than the phosphorylated vitamers. The concentrations of the nonphosphorylated B₆ vitamers necessary to inhibit brain and choroid plexus uptake of [³H]PIN from the medium by 50% were approx 0.4 μm and 5–10μm respectively after a 30 min incubation. Both brain slices and choroid plexus readily released (46 and 56% respectively in 30 min) previously accumulated [³H]B₆ into artificial CSF. However, brain slices released only nonphosphorylated [³H]B₆, whereas the choroid plexus released predominantly phosphorylated [³H]B₆. Addition of unlabeled PIN to the release media significantly increased the percentage of [³H]B₆ released by both brain slices and choroid plexus. The results of these in vitro studies provide evidence that: (1) both brain slices and chloroid plexus possess specific uptake and release mechanisms for B₆, and (2) these mechanisms tend to regulate intracellular B₆ levels. These studies also suggest that the choroid plexus serves as a locus for the transfer of B₆ from blood to CSF and is the source of most of the phosphorylated B₆ in CSF.     

Synthesis of [3H]- and -[14C]-labelled sulprostone (CP-34,089/ZK-57,671)

The synthesis of N-methanesulfonyl 16-phenoxy-ω-tetranor PGE₂ carboxamide (sulprostone — CP-34,089/ZK-57,671) labeled with tritium and carbon-14 is described. Sulprostone labeled with tritium in the phenoxy moiety by means of catalytic hydrogenolysis was obtained in a 17% radiochemical yield with a specific activity of 1.0 Ci/mmol. The methanesulfonamide-¹⁴C derivative of sulprostone was prepared from methyl-¹⁴C iodide in an 11.8% radiochemical yield having a specific activity of 18.8 mCi/mmol.     

N-type calcium channels are involved in the dopamine releasing effect of nicotine

Mouse striatum was incubated with [³H]dopamine ([³H]DA) and superfused with and the tritium efflux induced by nicotine, electrical stimulation, or simultaneous nicotine and electrical stimulation was measured, to characterize the role of different Ca²⁺ channels in the transmitter release. Nicotine stimulation and electrical stimulation exerted additive effects on tritium efflux. Separation of the released radioactivity on alumina columns indicated that nicotine or electrical stimulation increases the release of [³H]DA and that the outflow of³H-labeled metabolites was similar with the two different stimulation procedures. Removal of Ca²⁺ from the superfusate resulted in a marked reduction in the tritium release evoked by nicotine, whereas the electrical stimulation-evoked tritium release was completely dependent on external Ca²⁺. The L-and N-type calcium channel blockers omega-conotoxin GVIA and Cd²⁺ inhibited the tritium release from the striatum evoked by either nicotine or electrical stimulation, whereas the L-type and T-type channel blockers diltiazem and Ni²⁺ did not alter release of [³H]DA. We conclude that N-type voltage-sensitive Ca²⁺ channels participate in striatal dopamine release, and we speculate that nicotinic receptor-operated ion channels permeable to cations such as Ca²⁺ and N-type voltage-sensitive calcium channels may simultaneously open up, and they additively increase free intracellular Ca²⁺ concentration.     

Histochemical demonstration of transmitter release from noradrenaline,dopamine and 5-hydroxytryptamine nerve terminals in field stimulated rat brain slices

Summary The effect of electrical field stimulation on noradrenaline (NA), dopamine (DA) and 5-hydroxytryptamine (5-HT) nerve terminals in rat brain slicesin vitro was investigated. Slices prepared from the cerebral cortex or the neostriatum were incubated in physiologic buffer for 30 min and then superfused by buffer and stimulated by an electrical field (biphasic pulses, 10 Hz, 12 mA, 2 ms) for various time periods. The effect of the stimulation was studied at the cellular level with the histochemical fluorescence technique of Falck and Hillarp. The transmitter overflow into the superfusing buffer caused by the stimulation was studied with isotope technique. Cerebral Cortex NA Nerve Terminals. Stimulation caused release of NA from cortical NA nerve terminals. Already after 2 min stimulation a slight decrease of the fluorescence intensity of the nerve terminals could be found. Stimulation for 15 to 30 min greatly reduced the fluorescence intensity. In slices preincubated with³H-NA the stimulation-induced overflow of tritium during 2 min stimulation was about 15% (i.e. 15% of the tissue tritium content was overflowing into the superfusing buffer in response to stimulation for 2 min). During prolonged stimulation there was a considerable decline of the tritium efflux. Cerebral Cortex 5-HT Nerve Terminals. The 5-HT-analogue 6-hydroxytryptamine (6-HT) which is readily taken up into 5-HT nerve terminals was used to permit good visualization of these nerve terminals. Uptake of 6-HT into cortical NA nerve terminals was prevented by preincubation with 6-hydroxydopamine (6-OH-DA) or protriptyline. Stimulation for 15 to 30 min reduced the fluorescence intensity of the 5-HT nerve terminals. In slices preincubated with³H-5-HT the stimulation-induced overflow of tritium during 2 min stimulation was about 5%. The tritium efflux slowly decreased during continuous stimulation. Neostriatal DA Nerve Terminals. In slices frozen directly after preparation an intense diffuse fluorescence could be seen. After incubation in drug-free buffer at 37° C the fluorescence was localized in the varicosities of the DA nerve terminals. The central parts of the slices almost completely lacked specific fluorescence, while the outer zones were brightly fluorescent. No clear reduction of the fluorescence intensity of the DA nerve terminals in the outer zone could be observed after stimulation for 30 min. In slices preincubated with³H-DA the stimulation-induced overflow of tritium during 2 min stimulation was about 2%. The tritium efflux slowly decreased during continuous stimulation.It is suggested that the differences in release between the various nerve terminal systems foundin vitro reflect differences in transmitter release occurringin vivo. The comparably high release of NA per impulse from the cortical NA nerve terminals implicate that the discharge rate of these neuronsin vivo is very low.This investigation has been supported by grants from the Swedish Medical Research Council (B72-14X-2330-05A) and Magnus Bergwall's Foundation.The author is greatly indebted to Mrs. Annika Hamberger for her skillful technical assistance. For generous supplies of drugs thanks are due to Hässle, Göteborg, Sweden, through Dr. H. Corrodi (6-HT, 6-OH-DA and H44/68).     

Excitatory and Inhibitory Effects of A1 and A2A Adenosine Receptor Activation on the Electrically Evoked [3H]Acetylcholine Release from Different Areas of the Rat Hippocampus

Abstract: The modulation by adenosine analogues and endogenous adenosine of the electrically evoked release of [³H]acetylcholine ([³H]ACh) was compared in subslices of the three areas of the rat hippocampus (CA1, CA3, and dentate gyrus). The mixed A₁/A₂ agonist 2-chloroadenosine (CADO; 2–10 µM) inhibited, in a concentration-dependent manner, the release of [³H]ACh from the three hippocampal areas, being more potent in the CA1 and CA3 areas than in the dentate gyrus. The inhibitory effect of CADO (5 µM) on [³H]ACh release was prevented by the A₁ antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM) in the three hippocampal areas and was converted in an excitatory effect in the CA3 and dentate gyrus areas. The A_2A agonist CGS-21680 (30 nM) produced a greater increase of the evoked release of [³H]ACh in the CA3 than in the dentate gyrus areas, whereas no consistent effect was found in the CA1 area or in the whole hippocampal slice. The excitatory effect of CGS-21680 (30 nM) in the CA3 area was prevented by the adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 µM). Both adenosine deaminase (2 U/ml) and DPCPX (250 nM) increased the evoked release of [³H]ACh in the CA1 and CA3 areas but not in the dentate gyrus. The amplitude of the effect of DPCPX and adenosine deaminase was similar in the CA1 area, but in the CA3 area DPCPX produced a greater effect than adenosine deaminase. It is concluded that the electrically evoked release of [³H]ACh in the three areas of the rat hippocampus can be differentially modulated by adenosine. In the CA1 area, only A₁ inhibitory receptors modulate ACh release, whereas in the CA3 area, both A_2A excitatory and A₁ inhibitory adenosine receptors modulate ACh release. In the dentate gyrus, both A₁ inhibitory and A_2A excitatory adenosine receptors are present, but endogenous adenosine does not activate them.     

Tritium release assay for oleic acid desaturation

The “tritium release” assay for the enzymic conversion of stearic acid into oleic acid introduced by Talamo and Bloch in 1969 (1) represented a major advance in the measurement of enzymic fatty acid desaturation. By measuring the release of tritium from the 9- and 10-positions of erythro-[9,10³H₂]stearic acid into water instead of isolating the oleic acid chromatographically, the processing time can be reduced from about 2–3 hr to 5–10 min per assay. Recently, Johnson and Gurr (2) showed that the release of tritium does not represent an absolute measure of enzymic activity due to a primary kinetic isotope effect discriminating against tritium. In that paper we recorded the magnitude of the isotope effect, described the synthesis and use of threo-[9,10³H₂]stearic acid, which increases the sensitivity of the assay, and demonstrated that release of tritium from this substrate was proportional to the formation of oleic acid.     

Effect of Isoprostanes on Sympathetic Neurotransmission in the Human Isolated Iris-Ciliary Body

Isoprostanes (IsoP's) are prostaglandin-like compounds that are derived from free-radical catalyzed peroxidation of arachidonic acid independent of the cyclcooxygenase enzyme. In the present study, we investigated the effect of IsoP's on norepinephrine (NE) release from human isolated iris-ciliary bodies. Isolated human iris-ciliary bodies were prepared for studies of [³H]NE release using the superfusion method. Both 8-iso-prostaglandin F₂ (F₂-IsoP) and the thromboxane (Tx) receptor agonist, U46619 enhanced field-stimulated [³H]NE release from isolated, superfused human iris-ciliary bodies without affecting basal tritium efflux. On the other hand, an equimolar concentration (10 M) of 8-iso-prostaglandin E₂ (E₂-IsoP) inhibited evoked [³H]NE overflow. The Tx-receptor antagonist, SQ 29548 blocked the enhancements of electrically-evoked [³H]NE release induced by F₂-IsoP and U46619. However, the inhibitory responses elicited by E₂-IsoP was not antagonized by SQ 29548. We conclude that IsoP's can produce both excitatory and inhibitory effects on sympathetic neurotransmission in human isolated iris-ciliary bodies. The stimulatory effects of IsoP' on NE release may be mediated by Tx-receptors.     

Charcoal-catalyzed transfer of tritium into 3H2O from regiospecifically-labeled 2-hydroxyestradiol in the presence of thiols

Charcoal was found to catalyze the release of ³H₂O from [1-³H]2-hydroxyestradiol-17β ([1-³H]2-OHE₂) or [4-³H]2-hydroxy-estradiol-17β ([4-³H]2-OHE₂) and this effect was shown to occur in the presence of glutathione or other thiols and to depend on the concentration of free steroid. The radiometric assay for measuring the formation of ³H₂O was not affected significantly by subsequent treatment of the incubation mixture with charcoal if the ratio of steroid to tissue (rat brain or liver microsomes) was low and only initial rates of ³H release were measured. 2-Hydroxyestradiol did not show the charcoal effect in the presence of tyrosinase, either when it was generated from its parent estrogen or added to the enzyme. The formation of ³H₂O from [4-³H]2-OHE₂ in the presence of glutathione was inhibited by ascorbic acid but the addition of dextran or albumin did not protect the catechol estrogen from the charcoal-catalyzed loss of tritium. The reaction with glutathione and charcoal occurred even at 4°C but other adsorbants such as alumina, silica or hydroxylapatite were without effect.     

The Synthesis of (3H)-Gibberellin A9 with High Specific Activity

The synthesis of 2,3-(³H)-gibberellin A₉ (GA₉) with a specific activity of 47 Ci mmole^?1 is described. △^2,3GA₉ methyl ester epoxide was converted to (³H)-GA₉ methyl ester epoxide using carrier-free tritium gas. This product was de-epoxidized then hydrolysed to yield (³H)-GA₉.     

Depolarization-induced release of [3H]histamine by high potassium concentrations,electrical stimulation and veratrine from rat brain slices after incubation with the radiolabelled amine

Brain slices obtained from neocortex, hypothalamus or hippocampus were incubated with [³H]histamine and subsequently superfused and exposed to different depolarizing stimuli, viz. high K⁺-concentrations, electrical field stimulation and veratrine. K⁺-induced release of tritium was completely calcium-dependent and its magnitude depended on the K⁺-concentration, with maximal release being reached at 56 mM K⁺. Electrically-evoked release of tritium increased with increasing frequencies and reached its maximum at about 20 Hz. The electrically-evoked release appeared to be totally calcium-dependent and it was strongly inhibited by tetrodotoxin. Veratrine (5–100 μM) also induced a release of tritium; maximal release was obtained at 100 μM veratrine. Veratrine-induced release was partially calcium-dependent and was strongly reduced by tetrodotoxin.Taken together the data indicate that the depolarization-induced release of tritium from brain slices pre-labelled with [³H]histamine, represents [³H]histamine release from neurons and not from either mast cells or glial cells. It remains to be established whether these neurons are specifically histaminergic.     

Einfluß des Rohproteinniveaus in Maisfutterrationen bei verschiedenen Harnstoffgaben in der Jungbullenmast

Mycotoxins are fungal secondary metabolites that elicit a wide spectrum of toxicological effects, including the alteration of normal immune function. In the present study we investigated the independent effect of four mycotoxins, aflatoxin B₁ (AFB₁), fumonisin B₁ (FB₁), deoxynivalenol (DON) and nivalenol (NIV), on lymphocyte proliferation using human and porcine lymphocytes. Human and porcine peripheral blood mononuclear cells and porcine splenocytes were cultured with increasing concentrations of mycotoxins for 72 hours and labelled in the last 24 hours with [methyl-³H]-thymidine. The results showed that increased concentrations of AFB₁, DON and NIV affected the [methyl-³H]-thymidine cellular proliferation following mitogen stimulation in both species and cell types. Lower concentrations of mycotoxins enhanced cellular proliferation, which was more pronounced in human than in porcine cells, while higher concentrations caused a dose-dependent decrease. DON and NIV were the most potent mycotoxin in both species and both cell types. Based on the results of this in vitro study, high correlations were found between proliferation of human and porcine lymphocytes after mycotoxin exposure, especially for DON and NIV.     

Effects of External pH Variations on Brain Presynaptic Sodium and Calcium Channels; Repercussion on the Evoked Release of Amino Acid Neurotransmitters

The effects of external pH (pH _out) variations on the Na⁺ and on the Ca²⁺ dependent fractions of the evoked amino acid neurotransmitter release were separately investigated, using GABA as a model transmitter. In [³H]GABA loaded mouse brain synaptosomes, the external acidification (pH _out6.0) markedly decreased the Na⁺ dependent fraction of [³H]GABA release evoked by veratridine (10 M) in the absence of external Ca²⁺, as well as the Ca²⁺ dependent fraction of [³H]GABA release evoked by high (20 mM) K⁺ in the absence of external Na⁺. The depolarization-induced elevation of [Na _i ] (monitored in synaptosomes loaded with the Na⁺ indicator dye, SBFI) and the depolarization-induced elevation of [Ca _i ] (monitored in synaptosomes loaded with the Ca²⁺ indicator dye fura-2) were also markedly decreased at pH _out 6. On the contrary, the external alkalinization (pH _out 8) facilitated all the above responses. A slight increase of the baseline release of the [³H]GABA was observed when pH _out was changed from 7.4 to 8. This effect was only observed in the presence of Ca²⁺. pH _out changes from 7.4 to 6 or to 7 did not modify the baseline release of the transmitter. All the effects of pH _out variations on [³H]GABA release were independent on the presence of HCO-₃. It is concluded that external H⁺ regulate amino acid neurotransmitter release by their actions on presynaptic Na⁺ channels, as well as on presynaptic Ca²⁺ channels.     

The synthesis of [3H]gibberellin A3 and [3H]gibberellin A1 by the palladium-catalyzed actions of carrier-free tritium on gibberellin A3

Reaction of gibberellin A₃ (GA₃) with carrier-free tritium gas and 5% palladium on calcium carbonate as catalyst gave a complex mixture of products, several of which were isolated and identified. Three of the purified products are the radioactive forms of naturally occurring gibberellins: [³H]GA₃ (1), [³H]GA₁ (2) and [³H]tetrahydro GA₃ (4). Another substance was isolated and tentatively identified as [³H]16,17-dihydro GA₃ (3). GLC was used to determine the specific activities of 1 and 2. [³H]GA₃ likely arises from palladium catalysed nonspecific exchange of GA₃ alkane hydrogen atoms with tritium. [³H]GA₁ is also exchange labeled but most of its radioactivity is due to tritium addition to the C-1,2 olefinic bond of GA₃.     

The glycollate pathway during the photoassimilation of acetate by Chlorella

Summary When Chlorella pyrenoidosa photoassimilates ³H–¹⁴C-acetate glycollic acid rapidly becomes labelled with both tritium and ¹⁴C. The ³H/¹⁴C ratio was 10 in glycollate, (compared with 4 in the acetate added) and the only other intermediates showing similar ³H/¹⁴C ratios to glycollate were glycerate and serine. This suggests a glycollate pathway for the formation of serine was operating in Chlorella pyrenoidosa during the photoassimilation of acetate. When Chlorella pyrenoidosa assimilated ³H–¹⁴C-acetate in the dark glycollate was not labelled with either ¹⁴C or tritium. Although glycerate and serine both became labelled with ¹⁴C and tritium in the dark they did not show the high ³H/¹⁴C ratios recorded in the light. When cells were aerated with unlabelled 5% CO₂ during the photoassimilation of ³H–¹⁴C-acetate, the ³H/¹⁴C ratios of glycollate, glycerate and serine were slightly decreased. Similarly, under anaerobic conditions in the light the ³H/¹⁴C ratio was decreased compared with aerobic conditions.     

A highly sensitive radiometric assay for zoxazolamine hydroxylation by liver microsomal cytochrome P-450 and P-448: properties of the membrane-bound and purified reconstituted system.

A radiometric assay for the in vitro metabolism of zoxazolamine has been developed which combines high sensitivity and rapid determination of product. [4,6-³H]zoxazolamine was metabolized to 6-hydroxyzoxazolamine, and the tritium released as ³H₂O was determined after treating the incubation mixture with activated charcoal. This treatment efficiently removes labeled substrate (99.98%), permitting enzymatically released tritium to be measured directly in the aqueous medium. Since the preponderant in vitro product of zoxazolamine metabolism by rat liver microsomes and the purified reconstituted mixed function oxidase system is 6-hydroxyzoxazolamine, and since this aryl hydroxylation occurs without significant NIH shift, the subsequent release of tritium from the 6-position accurately represents metabolism of the molecule. The use of [4,6-³H]zoxazolamine for a tritium release assay of mixed function oxidase activity is ideal since this compound shows no significant isotope effect or NIH shift during metabolic conversion to 6-hydroxyzoxazolamine. 3-Methylcholanthrene treatment of rats resulted in a fourfold induction of zoxazolamine hydroxylation while phenobarbital or pregnenolone 16α-carbonitrile pretreatment caused only a 20–50% increase in zoxazolamine metabolism. The use of a purified reconstituted system revealed that cytochrome P-448 from 3-methylcholanthrene-treated rats was approximately 10- to 15-fold more efficient than cytochrome P-450 from phenobarbital-treated rats in catalyzing the hydroxylation of zoxazolamine.