PubMed · 2074787
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D H Malinowska. 1990. Permeabilizing parietal cells.. https://doi.org/10.1016/0076-6879(90)92065-l
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The reaction of demethylation mediated by cytochrome P450 (CYP) leads to the equimolar production of demethylated metabolite and formaldehyde. From a 13C-substrate labeled on a carbon of the methyl moiety, [13C]formaldehyde (H13CHO) is liberated. A highly sensitive and specific assay involving the oxidation of H13CHO to 13CO(2) by a double-enzymatic-step reaction is reported. The 13CO(2) was quantified by the method of reverse isotopic dilution based on gas chromatography-isotope ratio mass spectrometry analysis. The method first involves the limiting step of the CYP-dependent reaction, which is stopped with a mixture of zinc sulfate 5 mM and trichloroacetic acid 100 mM. Then, the transformation of H13CHO to 13CO(2) is performed with the formaldehyde (0.2 unit) and the formate (0.2 unit) dehydrogenase NAD-dependent enzymes. The recovery of 13CO(2) from the incubation mixture was equal to 91.4 +/- 3.0%. The accuracy and the precision of the present method were within 12 and 10%, respectively. The limit of quantification was set to 25 pmol. The performance of the assay was validated on human liver microsomes with five probes: [13C]erythromycin, [1-13C]caffeine, [3-13C]caffeine, [7-13C]caffeine, and [13C(2)]aminopyrine. This method is useful for the rapid determination of N-demethylase activity of human liver microsomes from methyl-13C-substrates.
Extracts of the adult worms of both Schistosoma mansoni and Schistosoma haematobium can metabolise some typical P450 substrates but to differing degrees. S. mansoni worm extracts displayed a approximately 12-fold higher specific activity for an aminopyrine substrate than rat liver microsomes. At 4 mM substrate concentration the demethylation reaction with N-nitrosodimethylamine (NDMA) (5 nmol HCHO/mg protein/min) was only half that of rat liver microsomes, whereas in extracts of S. haematobium, no detectable activity was found towards NDMA. Using ethylmorphine as substrate the demethylation activity of S. mansoni extracts (1.82 nmol HCHO/mg protein/min) was 5.5-fold lower than that of rat liver microsomes. Benzphetamine demethylase activity was also readily detectable in S. mansoni worm extracts at 6.79 nmol HCHO/mg protein/min compared with 10.20 nmol HCHO/mg protein/min in the case of rat liver microsomes. When aniline was used as substrate, surprisingly, no activity was found in worm extracts of either S. mansoni or S. haematobium, whereas rat liver microsomes showed high activity towards this amine. The anti-P450 2E1 and 2B1/2 cross-reacted with both worm homogenates and gave a specific band corresponding to a protein of molecular weight of approximately 50.0 kDa. A study with anti-P450 IVA antibody revealed that while this protein was strongly expressed in S. haematobium worm extracts, no immunoreactivity was observed with extracts of S. mansoni. Immunoblotting analyses with anti-P450 IIIA and P450 1A1 did not detect immunoreactive protein in either S. mansoni or S. haematobium.
Abomasal parasites inhibit gastric acid secretion and reduce the number of acid secreting parietal cells either through physical contact with gastric tissue, the release of inhibitory excretory/secretory (ES) products or by initiating the host inflammatory response. To examine the role of parasite ES products, adult Haemonchus contortus were incubated in a medium based on Hank's balanced salt solution and three incubates were tested for the presence of inhibitors of acid secretion by dispersed rabbit gastric glands in vitro, using the intracellular accumulation of 14C-aminopyrine (AP) as an indirect measure of acid secretion. Acceptably sensitive rabbit preparations (80%) for testing ES products showed increased 14C-AP accumulation with either 10(-5) or 10(-4)M histamine. Sheep glands proved unsuitable as a test system as only two of six preparations responded weakly to histamine. Inhibitory activity was demonstrated in all the three parasite incubates, although responses varied quantitatively between tests, even with the same batch of incubate. In single tests, 16% ES products significantly reduced the 14C-AP ratio (P<0.05) of 10(-4)M histamine-stimulated glands (15 of 19 tests with Batch 1 and two of six with Batch 3). Even at 1 and 5%, ES products (Batch 2) were inhibitory for 10(-5)M histamine-stimulated glands: at 1% the mean reduction was 26.0% (range 7.8-54.8%, n=10), four of which were statistically significant and at 5% was 17.6% (range -15.3-53.8%, n=17), four of which were statistically significant. ES products were overall inhibitory (-12%, P<0.05), when tested on glands exposed to increasing histamine concentrations from 10(-6)M to 10(-3)M (which increased the 14C-AP ratio (P<0.001)). Responses by individual gland preparations differed (P<0.001). The active component(s) of the parasite ES products were less than 5000 molecular weight. Ammonium chloride, tested over the range of concentrations of ammonia present in worm incubates (0.2-1 mM, final concentrations in the gland incubations of 0.005-0.1 mM), had variable, but overall inhibitory effects on 10(-5)M histamine-stimulated glands (n=3). When tested with a range of histamine concentrations, 0.01 mM NH4Cl overall reduced the response by 8.6% (P<0.05)(n=4). The similarity of responses of rabbit glands to parasite ES products and to NH4Cl suggests that ammonia may be the small molecular weight ES product of adult H. contortus, which inhibited acid secretion in vitro.