Mycoplasma hyopneumoniae Increases Intracellular Calcium Release in Porcine Ciliated Tracheal Cells

We investigated the effects of intact pathogenic Mycoplasma hyopneumoniae, nonpathogenic M. hyopneumoniae, and Mycoplasma flocculare on intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in porcine ciliated tracheal epithelial cells. The ciliated epithelial cells had basal [Ca(2+)](i) of 103 ± 3 nM (n = 217 cells). The [Ca(2+)](i) increased by 250 ± 19 nM (n = 47 cells) from the basal level within 100 s of the addition of pathogenic M. hyopneumoniae strain 91-3 (300 μg/ml), and this increase lasted ∼60 s. In contrast, nonpathogenic M. hyopneumoniae and M. flocculare at concentrations of 300 μg/ml failed to increase [Ca(2+)](i). In Ca(2+)-free medium, pathogenic M. hyopneumoniae still increased [Ca(2+)](i) in tracheal cells. Pretreatment with thapsigargin (1 μM for 30 min), which depleted the Ca(2+) 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 μM 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 [Ca(2+)](i) in ciliated tracheal cells. These results suggest that pathogenic M. hyopneumoniae activates receptors that are coupled to G(i) or G(o), which in turn activates a phospholipase C pathway, thereby releasing Ca(2+) from the endoplasmic reticulum. Thus, an increase in Ca(2+) may serve as a signal for the pathogenesis of M. hyopneumoniae.