ABSTRACT
In the present study we used scanning electron microscopy (SEM) to investigate morphological changes of non-transformed line of human bronchial smooth muscle cells (bSMC) induced by different agonists. Explants of normal bronchi were dissected and subcultured between 2 and 6 passage. In addition, smooth muscle actin content was assessed by SDS-PAGE electrophoresis, and its isoelectric point by IPG followed by immunoblotting. SMC were fixed by 2.0% paraformaldehyde and 2% glutaraldehyde and then were post-fixed by OSO4 and followed by dehydration and gold coating. Cytosolic free calcium was measured using adherent cells incubated with 500 microM Fura-2 acetoxymethylester and monitored by single excitation fluorimetry. Cultured cells possess predominantly charged actin isoforms with pI at 4.95; they respond to acetylcholine (100 microM), bradykinin (5 microM) and sulfidopeptide leukotriens (0.3-1.0 microM) with contraction, marked morphological lesions, such as widespread monolayer disorganization, extension of cell contacts. The number of microvilli on the cell surfaces was correlated with the degree of the alterations of the cellular morphology. Receptor antagonists antagonized these changes: atropine (0.3 microM), HOE 140 (1 microM) and MK 571 (1 microM). Acetylcholine and bradykinin induced a biphasic elevation of cytosolic calcium, which was antagonized by their receptor antagonists. Calcium changes in response to agonists were maintained over repetitive passages. Therefore, morphological changes seen in human bronchial SMC in culture with physiological response to various, structurally unrelated agonists can be future concern for the study the possible testing of the different pharmacological substances.
