Mycoplasma hyopneumoniae increases intracellular calcium release in porcine ciliated tracheal cells

We investigated the effects of intact pathogenic Mycoplasma hylopneumoniae, nonpathogenic M. hyopneumoniae, and Mycoplasma flocculare on intracellular free Ca2+ concentrations ([Ca2+](i)) in porcine ciliated tracheal epithelial cells. The ciliated epithelial cells had basal [Ca2+](i) of 103 +/- 3 nM (n = 217 cells). The [Ca2+](i) increased by 250 +/- 19 nM (n = 47 cells) from the basal level within 100 s of the addition of pathogenic M. hylopneumoniae strain 91-3 (300 mug/ml), and this increase lasted similar to 60 s. In contrast, nonpathogenic M. hyopneumoniae and M. flocculare at concentrations of 300 mug/ml failed to increase [Ca2+](i). In Ca2+-free medium, pathogenic M. hylopneumoniae still increased [Ca2+](i) in tracheal cells. Pretreatment with thapsigargin (1 muM for 30 min), which depleted the Ca2+ store in the endoplasmic reticulum, abolished the effect of M. hyoneumoniae. Pretreatment with pertussis toxin (100 ng/ml for 3 h) or U-73122 (2 muM for 100 s), an inhibitor of phospholipase C, also abolished the effect of M. hyopneumoniae. The administration of mastoparan 7, an activator of pertussis toxin-sensitive proteins G(i) and G(o), increased [Ca2+](i) in ciliated tracheal cells. These results suggest that pathogenic M. hylopneumoniae activates receptors that are coupled to G(i) or G(o), which in turn activates a phospholipase C pathway, thereby releasing Ca2+ from the endoplasmic reticulum. Thus, an increase in Ca2+ may serve as a signal for the pathogenesis of M. hyopneumoniae.