Mutation screening of the muscarinic M(2) and M(3) receptor genes in normal and asthmatic subjects.

1. Muscarinic receptors are important in the development of airway hyperresponsiveness, and dysfunction of these receptors has been suggested to be present in asthma. 2. The human muscarinic M(2) and M(3) receptor genes were screened for polymorphic variation using single-stranded conformation polymorphism (SSCP) analysis, complemented by direct fluorescent sequencing. Forty-six random DNA samples and 46 respiratory physician diagnosed asthmatic samples were used as a template for analysis. 3. Within the muscarinic M(2) receptor gene, we identified two degenerate single base substitutions (1197T-->C, Thr-->Thr and 976A-->C, Arg-->Arg) in one random and one asthmatic sample respectively. Analysis of the 3' UTR region revealed an additional 'A' at bp 1793 (c.f. ATG). This was present in all of 49 samples analysed by sequencing or BsmI digest, suggesting that the published sequence (GenBank Accession NO: M16404) is incorrect. A common 3' UTR polymorphism (T-->A) was found at bp 1696 (c.f. ATG) (allelic frequency=65%, n=60), but this does not alter transcription factor recognition sites. 4. We were unable to identify any polymorphic variation within the muscarinic M(3) coding region or the flanking regions investigated, using the methods described. 5. The coding regions for the human muscarinic M(2) and M(3) receptor genes are both highly conserved. These data suggest that polymorphic variation within these coding sequences is unlikely to account for inter-individual variability in response to methacholine or anticholinergic therapy. The potential functional significance of the muscarinic M(2) receptor 3' UTR polymorphism (bp 1696) remains to be determined.

Selenium, glutathione peroxidase and superoxide dismutase in maltese asthmatic patients: effect of glucocorticoid administration.

Oxidative processes, mediated by free radical chemistry, are recognized to contribute significantly to the inflammatory pathology of bronchial asthma. This study analysed the degree of defence against reactive oxygen species in Maltese, asthmatic patients and in normal individuals, by measuring plasma selenium concentration, erythrocyte glutathione peroxidase (GSH-Px) activity and erythrocyte superoxide dismutase (SOD) activity, in order to determine their antioxidant status. The effect of glucocorticoids on the status of these antioxidants in patients was also investigated. The measurement of antioxidant status was carried out both in mild (n = 22) and severe (n = 37) asthmatics, as well as in healthy controls (n = 49). The same antioxidant profile was then investigated in a group of 16 severe asthmatics following treatment for 4 weeks with inhaled beclomethasone dipropionate (750 micrograms twice daily), and in a second group of 16 patients suffering from severe asthma, following 2-weeks treatment with oral prednisolone (15 mg daily during the first week and 10 mg daily during the second). No statistically significant difference was found in the plasma selenium concentrations and erythrocyte glutathione peroxidase activities between patients and controls. Both mild and severe asthmatics, however, exhibited a statistically significant lower erythrocyte superoxide dismutase activity than normal subjects (mild asthmatics: 62.9 (2.9) SOD 525 U/ml, severe asthmatics: 60.6 (1.9) SOD 525 U/ml, normal: 68.5 (1.1) SOD 525 U/ml, P < 0.01). Inhaled beclomethasone dipropionate exerted no effect on this antioxidant profile, while prednisolone caused a significant increase in plasma selenium concentration over pretreatment values (pretreatment: 118.3 (4.4) ng/ml, post-treatment: 138.1 (4.6 ng/ml, P < 0.01). It is thus suggested that asthmatic patients in Malta might be more susceptible to superoxide-induced damage than normal individuals. The reason for the prednisolone-induced augmentation of plasma selenium could not be determined from this study. It is postulated that the drug may decrease the excretion rate of the element, and may thus exert a positive antioxidant effect in individuals of established low selenium status.