Effect of Cl-, Na+, and amiloride on the electrical properties of dog tracheal epithelium, in vitro.

We investigated the effects on the cellular electrical properties of changes in the chemical gradient of Cl- and Na- across the luminal membrane of dog tracheal cells. We used standard microelectrode techniques to measure the electrical profile of these cells during Cl- and Na+ substitution in the luminal solution and during the addition of 10(-4) M amiloride, a Na+ conductance blocker, to the luminal solution. In 111 cell penetrations, we observed an apparent bimodal distribution of luminal membrane potential. About 75% of the cells constituted the lower mode, and the greatest number of penetrations in this mode had luminal potentials of -10 to -15 mV. The greatest number of penetrations in the higher mode had luminal potentials of -40 to -45 mV. In 16 penetrations, Cl- substitution changed the luminal membrane potential (+/- SE) by +12.3 +/- 0.6 mV and transepithelial potential by +11.6 +/- 0.9 mV. Both Na+ substitution and amiloride addition resulted in a minimal hyperpolarization of the luminal membrane potential with only a slight change in the submucosal potential. These results suggested the Cl- conductance contributed to a greater extent than that of Na+ to the maintenance of the luminal membrane potential difference and supported the model recently reviewed by Frizzell and coworkers (5) proposing Na+-dependent electrogenic CL- secretion in dog tracheal cells.

Effect of calcium on sulfated mucous glycoprotein secretion in dog trachea.

In this experiment utilizing dog trachea we determined the effect of calcium concentration in the bathing media on the kinetics of secretion of sulfate incorporated into mucous glycoprotein. The results showed that in eight tracheas the secretion rate of 269 +/- 65 pmol SO4 . cm-2 . hr-1 (mean +/- SE) of tissues bathed in solution lacking calcium throughout the experiment was significantly less than the rate of 526 +/- 87 pmol SO4 . cm-2 . hr-1 of tissues bathed in solution containing 1.9 mM Ca2+. When tissues bathed in 1.9 mM CA2+ were incubated with 35SO4 to a constant secretion rate and then placed in 0 mM Ca2+ solution, the secretion of incorporated sulfate was unchanged relative to tissues bathed 1.9 mM Ca2+ throughout the experiment. Pool size of 727 +/- 150 pmol SO4/cm2 in tissues incubated in 0 mM Ca2+ throughout the experiment was significantly less than the pool size of 1,069 +/- 190 pmol SO4/cm2 in 1.9 mM Ca2+ solutions. This study showed that either the uptake of sulfate and/or biosynthetic pool size of sulfate incorporated into mucous glycoproteins was decreased in solution lacking calcium. However, release of sulfate incorporated into mucous glycoprotein in the lumen was independent of calcium in the external bathing media.

Kinetics of sulfated mucous glycoprotein secretion in dog trachea in vitro.

Previous studies on mucous glycoprotein secretion in respiratory epithelium most often provided qualitative, rather than quantitative, data. This study describes a new technique to measure the secretion rate, pool size, and turnover time of the pool of sulfated mucous glycoproteins of dog tracheal epithelium. The technique involved interposing dissected tracheal epithelium between the halves of an Ussing-type chamber, incubating the submucosal side of the tissue with 35SO4, and measuring the rate of appearance of nondialyzable 35SO4 on the luminal side of the chamber of both during the labeling and "washout" of the mucous pool. By analyzing the pattern of elimination, we showed that 1) a steady secretion rate of labeled mucous glycoprotein occurs within 3-4 h when free 35 SO4 is present on te submucosal side of the tissue, 2) secretion is consistent with first-order kinetics from a single pool, 3) puromycin decreases the rate of secretion of labeled mucous glycoprotein, 4) secretion rate is greater in medium 199 than in modified Krebs-Henseleit solution, and 5) 1.2 mM SO4 supports maximal baseline secretion. In six tracheas, bathed in Krebs-Henseleit solution, the secretion rate was (mean +/- SE) 467 +/- 74 pmol SO4 x cm-2 x h-1, pool size, 964 +/- 144 pmol SO4/cm2, and turnover time, 2.12 +/- 0.16 h. This technique provides a quantitative method to characterize kinetics of sulfated mucous glycoprotein secretion.