Effect of IgG1 and its fragments on resting membrane potential of isolated tracheal myocytes

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Abstract
We enzymatically isolated airway smooth muscle cells from the trachea of guinea pigs (400–600 g body wt). After removal of connective tissue, strips of trachealis muscle were cleaned under a dissecting microscope and incubated with collagenase (type I, 1 mg/ml) and elastase (type I, 15 U/ml) for 20 min. Cells were resuspended in Dulbecco's modified Eagle's medium and seeded on 1.5% gelatin-coated Petri dishes (35 mm). The viability of cells was assessed by trypan blue exclusion. Individual myocytes were impaled with glass microelectrodes (input resistance 90–100 M omega). Resting membrane potential (Em) was determined before and after administration of 1) immune serum, 2) highly purified specific immunoglobulin G1 (IgG1), and 3) enzymatically prepared fragments of IgG1-F(ab')2 and Fc. We found that 1) Em of isolated tracheal myocytes is -60.5 +/- 0.5 mV; 2) some myocytes exhibit spontaneous electrical rhythm with mean frequency of 16.9 +/- 12 min-1 and mean amplitude of 3.7 +/- 0.6 mV; 3) immune serum, IgG1, and Fc fragments induced a biphasic change in Em: the initial mean depolarization (-51.5 +/- 0.8 mV) was followed by a steady-state hyperpolarization (-68.3 +/- 0.6 mV); and 4) pretreatment of myocytes with amiloride (10(-5) M) or exposure of myocytes to a low-sodium environment prevented changes in Em induced by the passive in vitro sensitization. It is likely that airway smooth muscle cells have a low-affinity Fc receptor, the occupancy of which leads to activation of amiloride-sensitive sodium influx.

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