Smooth muscle hyperplasia/hypertrophy is a well recognized pathological change of asthma and may play an important role in the development of airway hyperreactivity, the hallmark of asthma. Lysosomal hydrolases, secretory products of inflammatory cells, are implicated in pathogenesis of allergen-induced asthma and ozone-induced muscarinic hyperreactivity. The proposed study is based on our preliminary findings of a potent mitogenic action of purified lysosomal hydrolases (hexosaminidases) on bovine tracheal airway myocyte by lysosomal hydrolases. The objective of this proposal is to investigate the mechanism(s) involved in airway myocyte proliferation induced by lysosomal hydrolases.
Specific aims are 1) To determine if a receptor-ligand interaction (specifically mannose receptor) is required for the mitogenic action of hydrolases. 2) To investigate whether arachidonic acid metabolites, particularly prostaglandin E2 (PGE2) is a mediator of lysosomal hydrolase induced mitogenesis. 3) To investigate whether or not mitogenic action of hydrolases is mediated through the activation of adenylate cyclase. 4) To explore a possible contribution of protein kinase C (PKC) and diacylglycerol (DAG) in the mitogenic effects of lysosomal hydrolases. 5) To establish the role cytosolic calcium in the mitogenic effects of lysosomal hydrolases. To accomplish specific aim 1: a) The presence of mannose receptor on bovine tracheal myocytes will be documented by antibody recognition and receptor type(s) will be characterized. Iodinated glycoproteins will be used for binding study and mannan and fructose-1-phosphate will be used for competition of binding. b) We will examine the effect of blocking receptor-ligand interaction. Receptor inhibitors and blockers will be utilized and their effect will be correlated to 3H-thymidine (TdR) incorporation induced by lysosomal hydrolases. c) Possible internalization of lysosomal hydrolases as a requirement in mitogenic action will be studied. Inhibitors of mannose receptor and agarose and sepharose bound hydrolases will be applied to inhibit internalization.
For aims 2, 3, 4, and 5, changes of PGE2 production, cAMP level, DAG level, PKC activity, cytosolic [Ca++] and inositol-triphosphate (IP3) levels; respectively will be studied in the presence and the absence of lysosomal hydrolases. Inhibitors and activators of second messenger systems and cyclooxygenase will be tested for their effects on mitogenesis induced by lysosomal hydrolases. Intracellular cAMP levels and PGE2 production will be measured by RIA and cytosolic calcium will be measured using Fura 2 fluorescent indicator. [32P] ATP, phosphatidyl serine and Histone IIIS will be used to assay PKC activity. Above measurements will be correlated to 3H-TdR incorporation. Understanding the receptor-dependent and the receptor- independent mechanism(s) of lysosomal hydrolase-induced airway myocyte proliferation would allow us to develop a method of prevention of smooth muscle hyperplasia/hypertrophy seen in asthma.
| Lew, D B; Brown, E R; Dempsey, B K et al. (1997) Contribution of PKC to beta-hexosaminidase-induced airway smooth muscle proliferation. Am J Physiol 272:L639-43 |
| Lew, D B; Songu-Mize, E; Pontow, S E et al. (1994) A mannose receptor mediates mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth muscle cells. J Clin Invest 94:1855-63 |
| Lew, D B; Nebigil, C; Malik, K U (1992) Dual regulation by cAMP of beta-hexosaminidase-induced mitogenesis in bovine tracheal myocytes. Am J Respir Cell Mol Biol 7:614-9 |
| Lew, D B; Rattazzi, M C (1991) Mitogenic effect of lysosomal hydrolases on bovine tracheal myocytes in culture. J Clin Invest 88:1969-75 |
