Mechanism of sodium hyperabsorption in cultured cystic fibrosis nasal epithelium: a patch-clamp study.

Transepithelial Na+ absorption is increased two to three times in cystic fibrosis (CF) compared with normal (NL) airway epithelia. This increase has been associated with a higher Na+ permeability of the apical membrane of airway epithelial cells. Because Na+ absorption is electrogenic and abolished by amiloride, Na+ channels are thought to dominate the apical membrane Na+ permeability. Three Na+ channel-related mechanisms may explain the increase in apical Na+ permeability in CF cells: increased number of channels, increased single-channel conductance, and increased single-channel open probability. We compared the properties of Na(+)-permeable channels in the apical membrane of confluent preparations of human NL and CF nasal epithelial cells cultured on permeable supports. Na(+)-permeable channels were studied using the patch-clamp technique in the excised inside-out and cell-attached configurations. The same types of Na(+)-permeable channels were recorded in CF and NL cells. In excised patches, nonselective (Na+/K+) cation channels were recorded, and no differences between CF and NL were found in the properties, incidence, single-channel conductance, and single-channel open probability. In cell-attached patches, channels with a higher Na+ vs. K+ selectivity dominated. There was no difference between CF and NL cells in the incidence (18.8 vs. 21.4%, respectively) and conductance (17.2 +/- 2.8 vs. 21.4 +/- 1.5 pS, respectively) of Na(+)-permeable channels. However, the open probability was higher in CF cells compared with NL cells (30.0 +/- 3.4%, n = 6, vs. 15.0 +/- 3.9%, n = 13; P < 0.05). We conclude that, in CF nasal epithelial cells, the increase in Na+ permeability of the apical membrane results from an increase in the open probability of Na(+)-permeable channels in the apical membrane.

Sodium-permeable channels in the apical membrane of human nasal epithelial cells.

We used patch-clamp techniques to study the channels that underlie the Na+ conductance of the apical membrane of human normal nasal epithelial cells. Cells were cultured on permeable supports and studied after confluence. In 172 of 334 (52%) excised membrane patches, we observed 20-pS Na(+)-permeable channels that do not discriminate between Na+ and K+ (pNa/pK = 1.33). These nonselective cation channels contained subpopulations that differed by dependence of open probability on voltage and bath Ca2+ activity, suggesting two or more channel types with similar electrical properties. In the presence of 10(-4) M amiloride in the pipette, the proportion of excised patches with nonselective cation channels decreased to 52 of 139 patches (37%), but the decrease was spread across all subpopulations of nonselective cation channels in excised patches. Thus no distinctive Na(+)-selective amiloride-sensitive channels were identified in excised patches. In cell-attached patches, Na(+)-permeable channels were recorded in 56 of 262 patches (21%). Their conductance was 21.4 +/- 1.5 pS (n = 25), and most were selective for Na+ over K+ (pNa/pK > 6). In the presence of amiloride (10(-4) M) in the pipette, the frequency of lambda Na(+)-permeable channels in cell-attached patches decreased to 8 of 134 patches (6%), revealing a population of Na(+)-selective channels recorded in cell-attached patches that was inhibited by amiloride. We conclude that, in excised patches, Na(+)-permeable channels are nonselective for Na+ over K+ and < 30% appear to be amiloride sensitive. In contrast, in cell-attached patches, most channels that conduct sodium are 1) selective for Na+ over K+ and 2) amiloride sensitive. Although we have not discovered the explanation for the discrepancy between cell-attached and excised patch data, we speculate that the channels recognized on cell account for the amiloride-sensitive Na+ conductance of the apical membrane, whereas the excision process alters the properties of the Na(+)-permeable channels and/or activate new channels.

Regulation of Cl- channels in normal and cystic fibrosis airway epithelial cells by extracellular ATP.

The rate of Cl- secretion by human airway epithelium is determined, in part, by apical cell membrane Cl- conductance. In cystic fibrosis airway epithelia, defective regulation of Cl- conductance decreases the capability to secrete Cl-. Here we report that extracytosolic ATP in the luminal bath of cultured human airway epithelia increased transepithelial Cl- secretion and apical membrane Cl- permeability. Single-channel studies in excised membrane patches revealed that ATP increased the open probability of outward rectifying Cl- channels. The latter effect occurs through a receptor mechanism that requires no identified soluble second messengers and is insensitive to probes of G protein function. These results demonstrate a mode of regulation of anion channels by binding ATP at the extracellular surface. Regulation of Cl- conductance by external ATP is preserved in cystic fibrosis airway epithelia.

Frequency dependence of effective nasal resistance.

Forced random noise techniques were used to characterize the effective resistance of the nasal passages in normal subjects and subjects who were candidates for surgical correction of nasal obstruction. The slope of the effective resistance curve was characterized by the average resistances over 3 to 5 Hz and 13 to 15 Hz. Subjects with nasal obstruction had effective nasal resistance curves which were more frequency-dependent than those of normal subjects (p less than 0.05). A mathematical model consisting of two series resistance-inertance elements in parallel was used to simulate the nasal passages. With this model we were able to generate effective resistance curves similar to those measured in both normal and obstructed subjects. The forced random noise technique may be a convenient, noninvasive technique for studying the fluid mechanical properties of airflow in the nasal passages and the effects of pharmacological and surgical interventions on nasal resistance.

Pulmonary impedance in dogs measured by forced random noise with a retrograde catheter.

Retrograde catheter and forced random noise techniques were combined to study the distribution of resistance and compliance in dogs following the inhalation of aerosols containing 2.5 and 5.0 mg/ml of histamine. Mean base-line peripheral resistance was 0.367 cmH2O . l-1 . s, agreeing with previous estimates. After correction for the endotracheal tube, the mean central airway resistance was 0.040 cmH2O . l-1 . s, considerably lower than previous estimates. This discrepancy was attributed to an overcorrection for the endotracheal tube resistance. The lower histamine dose caused a substantial increase in peripheral resistance, a relatively small increase in central resistance, and substantial decreases in total and peripheral compliance. After the higher histamine dose, changes in peripheral resistance and both compliances were similar to those obtained with the lower dose; however, the increase in central resistance was much larger than at the lower dose. The difference between total and peripheral compliance yielded estimates of airway compliance of 0.00306 l/cmH2O before and 0.00104 l/cmH2O after 2.5 mg/ml of histamine.

Correlation of airway resistance with forced random noise resistance parameters.

The correlation between airway resistance (Raw) measured in a plethysmograph and three respiratory resistance parameters measured by forced random noise was evaluated. Forced random noise resistance parameters were the average resistance between 5 and 9 hz (R5-9), the average resistance between 20 and 24 Hz (R20-24), and the extrapolated resistance at 1 Hz (R1). We studied 22 healthy, nonsmoking subjects, 10 of whom had a history suggesting childhood asthma. Serial measurements were made after inhalation of aerosols containing increasing concentrations of methacholine of aerosols containing increasing concentrations of methacholine to obtain data in various stages of bronchoconstriction. There was a broad range of responsiveness to methacholine; the ratio of the peak to base-line values of Raw ranged from 1.1 to 6.3. R1, R5-9, and R20-24 were related to Raw by linear regression analysis. R1 and R5-9 showed excellent correlation with Raw (r = 093 and 4=0.91, respectively); R20-24 showed much poorer correlation (r = 0.62). The slopes of the regression equations for R1 and R5-9 were 1.56 and 0.51, respectively; thus neither provided an exact measure of Raw. The data appear to be consistent with a model in which most of the methacholine-induced increase in resistance occurred in the peripheral airways and only a small fraction in the central airway.

Detection of respiratory mechanical dysfunction by forced random noise impedance parameters.

Respiratory mechanical parameters were computed from forced random noise impedance data in normal adults (group 1), asymptomatic smokers (group 2), and patients with obstructive pulmonary disease (group 3). Mean values for all derived parameters were significantly different (p less than 0.025) for group 3. Mean values of resonant frequency, the ratio of low-frequency (5 to 9 Hz) resistance to high-frequency resistance (15 to 19 Hz), and conductance normalized by height, were significantly different (p less than 0.05) between groups 1 and 2. This approach appears to yield respiratory mechanical parameters that are sensitive to mechanical alterations induced by early pulmonary disease. The rapid, noninvasive, and effort-independent nature of this approach make it especially useful for epidemiologic studies and for studies of "noncooperative" subjects.

Fractionating respiratory resistance in young children.

Total respiratory resistance (Rrs) and the fraction of this resistance located peripherally, (Fp) were measured in a group of children (3--5 yr old). The technique used forced random noise to characterize the frequency dependence of effective resistance and an algorithm to compute Rrs and Fp from these data. The overall mean value +/- SD for Fp was 0.534 +/- 0.070; the mean value of 0.464 for the six children near 5 yr of age was significantly different (P less than 0.001) from the mean value of 0.576 found in the younger children. The overall mean value +/- SD for Rrs was 16.9 +/- 5.8 cmH2O.l-1.s; individual values appeared to be correlated with height. These results agree with previous reports indicating that children have a larger fraction of respiratory resistance in the periphery than do adults.

Respiratory impedance and derived parameters in young children by forced random noise.

The frequency dependence of total respiratory impedance during spontaneous breathing was measured repeatedly in 16 children (3--5 yr old) over a 3-mo period using forced random noise and spectral analysis. Total respiratory resistance, compliance, and inertance, which were calculated fromthe impedance data using regression analysis with a second-order model, had overall mean values +/- SD of 5.61 +/- 0.49 cmH2O.l-1.s, 403 +/- 1.04 ml.cmH2O-1, and 0.0120 +/- 0.0024 cmH2O.l-1.s2, respectively. Linear regression analysis showed that resistance and compliance correlated with either height or forced vital capacity with r values in the range 0.77--0.89. Analysis of measurement variability suggested that resistance measurements had, on the average, a coefficient of variation of 15%; corresponding values for compliance and inertance measurements were 25%.