|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1991 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1990 : ¥1,300,000 (Direct Cost : ¥1,300,000)
By using canine tracheal epithelium in culture, electrical properties (short-circuit current, transepithelial potential difference, cell conductance) and single ion channel current were estimated through Ussings short-circuited technique and patch clamp method. Tracheal epithelial cells were seeded on polycarbonate filters and incubated for seven days in a CO_2 incubator, which were then mounted between Lucite half-Chambers and bioelectric properties were continuously measured.
In patch clamp studies, single cannel activity was observed in both cell-attached and inside-out patch configurations. However, these channels were more activated in excised status, inside-out patches (cytoplasmic side of the patch membrane was facing 0.9 mM Ca in the bath). In inside-out parches, nonselective cation chamels (NS channels) and K selective channels were detected. NS channels were active at both positive and negative membrane potentials. Similar channel currents were observed in a normal Ringer solu
tion (140 NaCl) whether patch-pipette solution contained 140 NaCl, 130 Nagluconate/10 NaCl, or 70 NaCl/75 KC1. The current voltage (I-V) relationship was linear and intercepted the X-axis at near Vm (membrane potential) being 0 mV. Single Cl channel current was also detected and activated by application of neuropeptides including substance P and neurokinin B, which was abolished by the Cl channel blocker diphenylamine-2-carboxylate.
Tracheal epithelial short-circuit current was increased by application of substance P, endothelin, and platelet-activating factor. These effects were concentration-dependent, and attenuated by pretreatment of cells by diphenylamine-2-carboxylate, the Cl transport inhibitor furosemide or bumetanide, and substitution of Cl in the medium by iodide that cannot be transported across airway epithelium. Transepithelial potential difference and cell conductance were also increased. The increase in short-circuit current induced by substance P was greatly potentiated by pretreatment of cells with phosphoramidon, a specific inhibitor of neutral endopeptidasse, whereas it was not altered by captopril, leupeptin, or bestatin. The stimulatory effect of substance P on the epithelial electrical properties was accompanied by the increase in intracellular cyclic AMP levels, as detected by radioimmunoassay. These results indicate the various neuropeptides can sellectively stimulate Cl secretion across airway epithelium through activation of intracellular signal transduction systems, and may play a role in regulating mucociliary transport function in the respiratory tract. Less