Reductions in stored mucin and airway responses to secretogogues in a guinea pig model of chronic asthma

E John, A Jackson and K J Broadley, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3XF and Novartis, Horsham, RH12 4HE, UK.

Excessive mucus production is an important feature of asthma, which may lead to mucus plugging and contribute to airway obstruction ( Jackson AD 2001). Although there is intense investigation into the mechanisms behind the development of a mucus hypersecretory phenotype, no published data has directly linked this phenotype to a reduction in lung function. In sensitised guinea pigs, chronic allergen challenge stimulates increased goblet cell-stored mucin (John E et al 2005) and histamine and UTP have been identified as mucin secretogogues ( Jackson AD 2001). In this study we investigate the relationship between mucus secretion and changes in lung function.

Male Dunkin-Hartley guinea pigs (200-250g) were sensitised with 100µg ovalbumin (OA) and 100mg AlOH3 i.p. injection on days 1 and 5. Guinea pigs were challenged with nebulised OA (0.01%/60mins) on day 15, and (0.1%/60mins) on days 17, 19, 21, 23, 25, 27 and 29, with mepyramine maleate (MEP) (30 mg.kg-1 i.p.) 1hr prior to OA challenge on days 17-27. On day 30, 9 groups of guinea pigs (n=6) received one of 3 secretogogue exposure protocols: nebulised UTP (1mM/15mins) or vehicle, 30mins following an i.p. injection of suramin hexasodium salt (60 mg/kg) or vehicle; nebulised histamine (10mM/30mins) or vehicle, 30mins following an i.p. injection of MEP (30 mg.kg-1) and vehicle or MEP (10 mg.kg-1) and ranitidine (10 mg.kg-1); nebulised UTP (1mM/15mins) and histamine (10mM/30mins) exposure, 30mins following i.p. injection of MEP (30 mg.kg-1). Whole body plethysmography measured specific airway conductance (sGaw) before and up to 1hr following secretogogue exposure. Guinea pigs received sodium pentobarbitone 24hrs post OA challenge, lungs removed and inflated with formaldehyde (1ml.100g-1). Fixed tissues were embedded in paraffin wax, sectioned (3μm) using a Leica microtome and stained with alcian blue/periodic acid-Schiff (AB/PAS). Total epithelial area and area of AB/PAS-stained epithelium were calculated using SigmaScan Image Analysis computer program and total mucus content of the bronchial epithelia was expressed as % AB/PAS-positive epithelial area.

UTP or histamine exposures revealed a gradual decline in sGaw, reaching nadir at 45mins (-28.5 ± 11.0 and -27.8 ± 7.2% respectively) and a 50% reduction in the AB/PAS-positive epithelial area compared to vehicle-exposed animals. Pre-treatment with suramin inhibited UTP-mediated mucin secretion and converted the UTP-associated decline in lung function to an increase in sGaw. Responses following histamine exposure were attenuated following ranitidine pre-treatment. Combined secretogogue exposures revealed a gradual decline in sGaw, (-22.1 ± 11.1% at 45mins), suggesting that these effects are not additive.

The reduction in goblet cell-associated mucin following UTP and histamine exposures suggests mucin secretion by these secretogogues. This was associated with a slowly developing reduction in lung function. Both responses were attenuated by suramin and ranitidine suggesting that mucus secretion is mediated via P2 (possibly P2Y2) and H2 receptors respectively and that airway function changes are a consequence of mucus secretion.

Jackson AD (2001) Trends Pharmacol Sci. 22, 38-45
John E, Jackson AD and Broadley KJ (2005). Proc Brit Pharm Soc. 3, 119P

Acknowledgement: MRC, Novartis, June Giddings, Gareth Jones and Francis Schindler.