In the present studies, a novel form of highly purified cytochrome P-450 (cytochrome P-452) isolated from the hepatic microsomes of clofibrate-pretreated rats has been compared to the major isozymes isolated from the hepatic microsomes of rats pretreated with phenobarbital (cytochrome P-450) and 2-naphthoflavone (cytochrome P-447) using a number of biochemical criteria.The results show that these three isozymes exhibit marked structural differences from each other as judged by a complete lack of immunochemical cross-reactivity between the isozymes and the heterologous rabbit serum antibodies using Ouchterlony double diffusion, and non-identity between the limited proteolytic digestion maps of the three isozymes obtained in the presence of chymotrypsin, papain and Staphylococcus aureus V, proteases. Furthermore, the three isozymes exhibited clear differences in their monomeric molecular weights determined on calibrated sodium dodecyl sulphate/polyacrylamide gel electrophoresis in gels of varying acrylamide concentration. Substantial differences were also observed in the substrate specificities of the isozymes, which were reflected in differences in the turnover rates and positional selectivities of the hemoproteins for some model substrates. In addition, the isozymes differed in their substrate binding affinities and their ability to interact with purified hepatic microsomal cytochrome b,, as judged using difference spectrophotometry. Finally, subtle differences were detected in the ultraviolet visible absorbance spectra of the hemoproteins in the ferric, ferrous, and carbonmonoxyferrous states.Taken collectively, the above data provides compelling evidence that fundamental differences exist between these cytochrome P-450 isozymes, further establishing the uniqueness of the major form of cytochrome P-450 induced by clofibrate pretreatment.Cytochrome P-450 is the terminal hemoprotein component of the hepatic microsomal electron-transfer chain which functions in the oxidation of a structurally diverse number of xenobiotics and endogenous compounds [I]. Considerable evidence has been presented in support of the existence of Much confusion exists regarding the terminology of cytochrome P-450 isozymes primarily because there is no uniform classification system which has been adopted to make unequivocal assignments of the similarity between isozymes isolated in different laboratories. Accordingly, the above three isozymes described in this paper have been classified according to the wavelength maxima of the ferrouscarbonmonoxy adducts. In the current paper, limited use is made of the term cytochrome P-450 in a general sense to collectively indicate all the cytochrome P-450 isozymes. Where appropriate, we make extended use of the term cytochrome P-450 to indicate that major isozyme induced by phenobarbital pretreatment in rat liver. This is regarded as necessary because to make a distinction for this latter isozyme and rename it would add further confusion to the literature. Similarly, throughout this paper, the terms c...
Release of excitatory amino acids and dopamine plays a central role in neuronal damage after cerebral ischaemia. In the present study, we used an in vitro model of ischaemia to investigate the effects of sevoflurane on dopamine, glutamate and aspartate efflux from rat corticostriatal slices. Slices were superfused with artificial cerebrospinal fluid at 34 degrees C and episodes of 'ischaemia' were mimicked by removal of oxygen and reduction in glucose concentration from 4 to 2 mmol litre(-1) for < or = 30 min. Dopamine efflux was monitored in situ by voltammetry while glutamate and aspartate concentrations in samples of the superfusate were measured by HPLC with fluorescence detection. Neurotransmitter outflow from slices was measured in the absence or presence of sevoflurane (4%). After induction of ischaemia in control slices, there was a mean (SEM) delay of 166 (7) s (n = 5) before sudden efflux of dopamine which reached a maximum extracellular concentration of 77.0 (15.2) micromol litre(-1). Sevoflurane (4%) reduced the rate of dopamine efflux during ischaemia (6.90 (1.5) and 4.73 (1.76) micromol litre(-1) s(-1) in controls and sevoflurane-treated slices, respectively; P<0.05), without affecting its onset or magnitude. Excitatory amino acid efflux was much slower. lschaemia-induced glutamate efflux had not reached maximum after 30 min of ischaemia. Basal (pre-ischaemic) glutamate and aspartate efflux per slice was 94.8 (24.8) and 69.3 (31.5) nmol litre(-1) superfusate (n = 4) and was not significantly reduced by 4% sevoflurane. lschaemia greatly increased glutamate and aspartate efflux (to a maximum of 919 (244)% and 974 (489)% of control, respectively). However, ischaemia-induced efflux of both glutamate and aspartate was significantly reduced by 4% sevoflurane (P < 0.001 for glutamate, P < 0.01 for aspartate). In summary, sevoflurane may owe part of its reported neuroprotective effect to a reduction of ischaemia-induced efflux of excitatory amino acids and, to a lesser extent, dopamine.
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