Effect of IgG1 and its fragments on resting membrane potential of isolated tracheal myocytes.
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.
John B. Pierce Laboratory, Yale University School of Medicine, New Haven, Connecticut 06519.
M
M
Souhrada
J F
JF
Souhrada
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D006168:Guinea Pigs; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D007141:Immunoglobulin Fc Fragments / Q000494:pharmacology; D007074:Immunoglobulin G / Q000494:pharmacology*; D066298:In Vitro Techniques; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000276:immunology; D009130:Muscle, Smooth / Q000276:immunology / Q000502:physiology; D010047:Ovalbumin / Q000276:immunology; D014132:Trachea / Q000276:immunology* / Q000502:physiology
D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D000584:Amiloride; D010047:Ovalbumin