Regio-Selectivity of Purified Forms of Rabbit Liver Microsomal Cytochrome P-450 in the Metabolism of Benzo(a)pyrene,n-Hexane and 7-Ethoxyresorufin

Abstract: The specificity of electrophoretically homogeneous preparations of rabbit liver microsomal cytochrome P-450lm _2–4 towards oxygenation of n-hexane, 7-ethoxyresorufin and benzo(a)pyrene was examined using a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine. Epoxide hydrase was included when benzo(a)pyrene was used as substrate. Cytochrome P-450lm ₂ was most active in n-hexane and benzo(a)pyrene oxygenation especially with regard to the formation of 2-hexanol, B(a)P-4,5-dihydrodiol and B(a)P-phenol metabolites. 7-Ethoxyresorufin was, however, a very poor substrate for cytochrome P-450lm ₂. Cytochrome P-450lm ₃ had less activity towards the investigated substrates while cytochrome P-450lm ₄ preferentially formed 2- and 3-hexanol, resorufin and B(a)P-9,10-dihydrodiol. Cytochrome P-450lm ₄ isolated after pretreatment with 3-methylcholanthrene or pheno-barbital showed roughly the same characteristics except in the formation of 1-hexanol where cytochrome P-450lm ₄ isolated after phenobarbital treatment was the most effective. The formation of B(a)P-4,5- and ?9,10-dihydrodiols was greatly increased by incorporation of epoxide hydrase. Our results indicate a certain specificity of the different forms of cytochrome P-450 in the liver microsomes although some overlap in activities was observed.     

Thrombin induces thromboplastin synthesis in cultured vascular endothelial cells

Cultured human umbilical vein endothelial cells responded to thrombin (10(-2) - 10 NIH u/ml) with a 2-5 fold increase in thromboplastin activity. The maximum response was reached after 4 hr in serum-free medium. The effect of thrombin was fully inhibited by the presence of 50% (v/v) fetal calf serum or more in the medium, by preincubation of thrombin with hirudin or by treatment of thrombin with N-bromosuccinimide or phenylmethylsulfonyl fluoride. The thrombin-induced thromboplastin activity was inhibited by incubation of the cells with cycloheximide (2 micrograms/ml) or actinomycin D (2 micrograms/ml) showing that the response depended on de novo protein and RNA synthesis. It was also suppressed by exposure of the cells to two different phosphodiesterase inhibitors, 3-butyl-1-methyl-xanthine (5 X 10(-4) M) and rac-4 (3-butoxy-4-methoxybenzyl)-2-imidazole (5 X 10(-4) M), to the transmethylation inhibitors 3-deazaadenosine (10(-5) M) and 1-homocysteine thiolactone (2 X 10(-5) M) in combination and to the intracellular calcium antagonist 8-(N,N-diethylamino)-octyl 3,4,5,-tri-methoxybenzoate hydrochloride (8 X 10(-5) M). Our results suggest that small amounts of thrombin can induce thromboplastin synthesis in endothelial cells in vitro and that this synthesis probably is regulated by the intracellular level of cAMP, by cytoplasmic Ca2+ and possibly also by transmethylation reactions.     

Systemic lupus erythematosus and avascular bone necrosis.

Two cases of systemic lupus erythematosus (SLE) developing avascular bone necrosis are described. One case had eight separate areas involved (shouldres, hips, knees and both distal tibiae), which is the largest number in a single case of SLE yet reported. In both cases, at the onset of symptoms of necrosis in a new area, the antinuclear factor increased markedly compared to the preceding level. This indicates that the onset of the pathological process leading to necrosis is due to increased activity of the SLE rather than the steroid treatment.     

Prolonged elimination of midazolam after i. m. administration

The elimination pharmacokinetics of midazolam after i.m. administration was compared with combined i.m. and i.v. administration in a randomized study of 55 gynaecological patients in outpatient general anaesthesia. Group 1 (n = 40) received midazolam 0.1 mg/kg i.m. as premedication 45 min before induction of general anaesthesia with midazolam 0.3 mg/kg i.v. Group 2 (n = 15) received midazolam 0.1 mg/kg i.m. as premedication 45 min before induction of general anaesthesia with thiopentone 4 mg/kg. Serum midazolam concentration measurements were performed regularly post-induction for 7 h in each patient. The elimination half-life of midazolam after i.m. administration (Group 2) was 6.6 +/- 1.2 h (mean +/- s.e. mean), which was significantly longer (P less than 0.05) than the 3.9 +/- 0.3 h observed after the combined i.m. and i.v. administration of midazolam (Group 1), and significantly longer than 2.9 h obtained from a calculated i.v. administration curve. We postulate a slow i.m. depot release of midazolam, representing the rate-limiting step in the elimination of midazolam after i.m. administration.     

The in vivo effects of human urotensin II in the rabbit and rat pulmonary circulation: effects of experimental pulmonary hypertension

In vivo haemodynamic responses to human urotensin-II were determined in two models of pulmonary hypertension: rabbits with left ventricular dysfunction following coronary artery ligation and the hypoxic rat. Effects were also examined in the presence of the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). Human urotensin-II increased pulmonary arterial pressure to a greater extent in ligated rabbits than their controls and L-NAME increased pulmonary pressure without significantly affecting these responses to human urotensin-II. Human urotensin-II raised right ventricular pressure slightly in control rats but not in hypoxic rats. Human urotensin-II did not constrict control rat isolated small pulmonary arteries and only induced a small constriction of these vessels in hypoxic rats. In conclusion, exogenous human urotensin-II exerts pulmonary pressor responses in vivo in rabbits and also induced small pulmonary pressor responses in control rats. Pulmonary pressor responses to urotensin-II were increased by pulmonary hypertension in rabbits but not in rats.     

The effect of thrombin on fibronectin in cultured human endothelial cells

Cultures of human endothelial cells (EC) incubated for periods up to 24 h with highly purified thrombin (2 NIH u/ml) contained considerably less cell-associated fibronectin fibrils than corresponding controls. The loss of fibronectin fibrils was evident after 4 h and was accompanied by a 2-3 fold increase in the concentration of fibronectin in the incubation medium. Hirudin inhibited the effects of thrombin. Thrombin also induced characteristic shape changes of EC. These shape changes were reversible within a 4-6 h period and could not be reinvoked by new additions of thrombin. Thus, structural refractoriness to thrombin coincided temporally with a period when EC-associated fibronectin fibrils were markedly reduced.     

In situ characterization of cell infiltrates in human dental periapical granulomas. 1. Demonstration of receptors for the Fc region of IgG

The aims of the present study were to demonstrate FcR activity of dental periapical granulomas and to correlate the activity with the degree of lymphoreticular cell infiltration. Cryostat sections of 46 out of 51 granulomas adsorbed sheep erythrocytes(E) sensitized with rabbit IgG antibodies (A) (EA). No adsorption occurred using erythrocytes sensitized with F(ab')2 fragments of IgG. IgG and Fc fragments of human of rabbit IgG inhibited the binding of EA, whereas F(ab')2 fragments, human IgA, IgM or albumin did not, indicating the presence of receptors for the Fc region of IgG. Periodate, neutral formaldehyde and phospholipase C abolished the FcR activity whereas neuraminidase had no effect. Comparison of sections binding EA and adjacent sections stained with haematoxylin and eosin showed that EA adhered to areas infiltrated with mononuclear cells. The degree of binding of EA coincided with the density of mononuclear cell infiltration. Point attachments between the tissue sections and the adsorbed EA could be demonstrated by scanning electron microscopy. Sections with no infiltrates did not bind EA.