Ouabain-induced changes of calcium and potassium in slices of hippocampus of the rat: comparison to hypoxia and effect of R 56865.

Simple and reliable in vitro models of cerebral ischaemia are important for the identification of antiischaemic/antihypoxic compounds. Alterations of the concentrations of potassium and calcium were recorded in slices of hippocampus of the rat. The slices were subjected to hypoxia in the presence and absence of intoxication with glucose or ouabain (1 mmol/l). Normoxic slices of hippocampus showed an extracellular space of 57% and a tissue concentration of potassium of 45 mmol/kg wet wt. A cellular concentration of potassium of 92 mmol/kg was calculated. Hypoxia, in the presence of glucose, only slightly reduced tissue concentrations of potassium and did not influence concentrations of calcium. Omission of glucose during hypoxia led to tissue concentrations of potassium below 10 mmol/kg, within 10-30 min of hypoxia. Concentrations of calcium only increased from 3.3 to 3.5 mmol/kg after 30 min of hypoxia, without glucose. Intoxication with ouabain is proposed as alternative experimental model of ionic movements, associated with cerebral ischaemia/hypoxia. Tissue concentrations of potassium fell rapidly to values below 10 mmol/kg, within 5 min and concentrations of calcium rose to 5.2 mmol/kg, within 30 min of intoxication with ouabain. In quantitative terms, the model for cerebral ischaemia with intoxication with ouabain is suggested to be superior to the model based on hypoxia without glucose. To verify intoxication with ouabain as an experimental model for ischaemic/hypoxic insults, the effect of an investigational drug with antiischaemic/hypoxic properties (R 56865) was evaluated in the model. The drug R 56865 produced dose-dependent attenuation of the fall in tissue concentrations of potassium, between 3 x 10(-7) and 5 x 10(-6) mol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosomal assignment of human cytochrome P-450 (debrisoquine/sparteine type) to chromosome 22.

In order to determine on which chromosome the gene controlling human cytochrome P-450 (debrisoquine/sparteine type) is located a linkage study of polymorphic sparteine oxidation (PSO) to various polymorphic markers was carried out. Positive information for linkage between PSO and the P1 blood group was obtained with a maximal LOD-score of LOD = 3.35 for both male and female recombination fraction estimates of theta m = theta f = 0.0. The P1 blood group has been recently mapped to the long arm of chromosome 22. Thus it can be concluded that the gene controlling human cytochrome P-450 (debrisoquine/sparteine) is situated on the long arm of chromosome 22 in close vicinity to P1.

Evidence for altered catalytic properties of the cytochrome P-450 involved in sparteine oxidation in poor metabolizers.

Sparteine metabolism was studied in human liver microsomes from nine extensive ([EM] urinary metabolic ratio [MR] less than 20) and four poor metabolizers ([PM] MR greater than 20). The formation of 2- and 5-dehydrospartein displayed monophasic Michaelis-Menten kinetics. In livers from PMs the formation of the major sparteine metabolite 2-dehydrosparteine was characterized by more than a thirtyfold increase in Michaelis-Menten constant (Km)(1880 +/- 1044 mumol/L) as compared with EM subjects with an MR less than 1 (58.3 +/- 38.8 mumol/L). EM subjects with an MR greater than 3 who may constitute heterozygous metabolizers showed Km values in between (658 +/- 301 mumol/L). There was no difference in maximum rate of metabolism (Vmax) of 2-dehydrosparteine formation between EM and PM subjects (101 +/- 39 vs. 86 +/- 52 pmol/min/mg). The formation of 5-dehydrosparteine exhibited a Km of 100 +/- 123 mumol/L similar to the Km of 2-dehydrosparteine formation. The urinary MR correlated positively with the Km for 2-dehydrosparteine formation. The intrinsic clearance for 2-dehydrosparteine showed a highly significant negative correlation with the MR. The pronounced differences in Km together with the significant correlation between Km and MR with no marked differences in Vmax between phenotypes suggest that the impaired oxidation capacity in PM subjects is more likely the result of a P-450 isozyme with altered catalytic properties rather than a decreased amount of enzyme.

A sensitive capillary GC assay for the determination of sparteine oxidation products in microsomal fractions of human liver.

A sensitive method for the assay of sparteine oxidase activity in vitro by microsomal fractions of human liver is described. The activity of sparteine oxidase was assessed by the formation of 2- and 5-dehydrosparteines, which were estimated by capillary gas chromatography with N2-FID detection. The limit of detection of the two metabolites, 2- and 5-dehydrosparteine, was 10 pmol (2.3 ng) per sample. Sparteine oxidase activity was linear with microsomal protein concentration ranging from 25 to 200 ug and with incubation times between 5 and 60 minutes. Omission of NADPH on incubation under an atmosphere of carbon monoxide inhibited formation of both metabolites, thus indicating that aforementioned metabolites arise in reaction catalyzed by cytochrome P-450. In three liver samples from humans classified as extensive (EM) metabolizers the formation of 2- and 5-dehydrosparteines was observed, 2-dehydrosparteine being the major metabolite. In these samples sparteine oxidase activity was characterised by Vmax = 136 +/- 53 pmol/min/mg and Km = 44 +/- 12 microM for 2-dehydrosparteine formation. For 5-dehydrosparteine formation the following values were obtained: Vmax = 57 +/- 18 pmol/min/mg and Km = 42 +/- 26 microM. In a liver sample from a poor metabolizer (PM) only the formation of 2-dehydrosparteine was detected with the method of analysis used. In this sample a great increase in Km (Km PM = 3033 microM) was noted, while Vmax was very similar to those obtained for 2-dehydrosparteine formation in EM subjects (Vmax PM = 147 pmol min/mg).