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Effects of cationic proteins on electrolyte transport depend on the specific channels and electrochemical driving forces expressed by epithelia. The bioelectric responses of canine tracheal and bronchial epithelia (CTE and CBE, respectively) to a polycationic protein, protamine, were therefore compared. CTE exhibited a brief transient inhibition of shortcircuit current (I(SC)) followed by a prolonged increase of 18 microA/cm2. The apical membrane transiently hyperpolarized and then depolarized by 11 mV. The increase in I(SC) was inhibited by bumetanide. Adenosine 3',5'-cyclic monophosphate, ionomycin, and thapsigargin attenuated the response whereas indomethacin or hypotonic solution had no effect, indicating that latent cystic fibrosis transmembrane regulator Cl- channels were activated. CBE preparations exhibited a 4-microA/cm2 decrease in I(SC), 2 mV hyperpolarization of the apical membrane, and an increase in fractional resistance of the apical membrane on exposure to protamine. These results were consistent with inhibition of the Na+ conductance in the apical membrane of CBE and confirmed that polycationic proteins exert differential effects on Cl- secretory and Na+ absorptive airways.

Polycationic proteins alter electrolyte transport by epithelium and endothelium, and in asthma are thought to disrupt the airway epithelium and contribute to hyperresponsiveness and airway plugging. In the present study, we used primary cultures of human nasal epithelial cells to investigate the response of respiratory tract epithelium to luminal presentation of a polycationic protein, protamine. Protamine (100 micrograms/ml) in the apical bathing solution had no significant effect on basal transepithelial resistance (Rt) but decreased short-circuit current (Isc) and hyperpolarized the apical membrane, indicating that Na+ absorption had been inhibited. Pretreating with amiloride inverted the response to protamine, resulting in an increase in Isc, depolarization of the apical membrane, and decrease in the fractional resistance of the apical membrane (fRa). The increase in Isc was inhibited by pretreatment with bumetanide. These results indicated that protamine augmented amiloride-induced Cl- secretion. Induction of Cl- secretion by bathing the apical surface in 3 mM Cl(-)-Ringer solution similarly resulted in protamine-induced depolarization of the apical membrane. Heparin precipitated protamine from solution and reversed the Isc responses. In summary, low concentrations of polycationic protein can alter electrolyte transport by human airway epithelium without desquamation, and the response is dependent on the secretory state of the tissue.

Previous studies demonstrated that elevation of intracellular calcium concentration ([Ca2+]i) increased electrogenic anion transport by bronchiolar epithelia. Extracellular nucleotides were shown to elevate [Ca2+]i and transepithelial short-circuit current (Isc) in proximal airways epithelia. In this study purine and pyrimidine nucleotides were investigated for their ability to regulate ion transport by rabbit nonciliated bronchiolar epithelial (Clara) cells in culture. ATP in the apical bath induced a concentration-dependent transient increase in [Ca2+]i and Isc. Mean effective doses (ED50) of the responses were 10(-7) M and 10(-6) M, respectively. Transepithelial resistance (Rt) decreased. The peak changes in Isc and Rt were 7.8 +/- 1.2 microA/cm2 and -59 +/- 14 omega.cm2 (n = 26, basal Isc = 47.4 +/- 4.3 microA/cm2 and Rt = 428 +/- 40 omega.cm2). Some preparations exhibited a small residual increase in Isc after the initial response, but the change was not statistically significant (delta Isc = 1.7 +/- 1.2 microA/cm2, n = 18). Addition of ATP to the basolateral bath had no detectable effects. Purinoceptor agonists were used to characterize the receptors mediating the change in Isc. UTP and ATP gamma S increased Isc and inhibited subsequent stimulation by ATP. ADP, ADP beta S, 2-methylthio-ATP, and alpha, beta-methylene-ATP had negligible effects on the peak delta Isc and subsequent stimulation by ATP. The ionic mechanism underlying the ATP-induced increase in Isc was investigated with the use of specific ion-transport inhibitors and by ion substitution.(ABSTRACT TRUNCATED AT 250 WORDS)

The utility of a transgenic murine model of cystic fibrosis (CF) lung disease will likely depend on whether the mouse's proximal airway epithelium is characterized by Na(+)- and Cl(-)-conductive pathways comparable to those found in human airways. Therefore, the electrophysiological properties of primary cultures of mouse tracheal epithelium (MTE) were investigated using double-barreled, Cl(-)-selective microelectrodes. Epithelial cells isolated from freshly excised mouse tracheae formed confluent polarized monolayers on permeable collagen supports and developed significant transepithelial potential differences (approximately -10 mV) within 5-6 days postseeding. Under basal conditions, the MTE monolayers had an equivalent short-circuit current (Ieq) of -21.1 +/- 2.1 microA/cm2 and a transepithelial resistance of 424 +/- 49 omega.cm2. Intracellular measurements indicated that the apical (Va) and basolateral (Vb) membrane potential differences were -16.9 +/- 1.5 and -25.4 +/- 1.5 mV, respectively; apical membrane fractional resistance was 0.36 +/- 0.03; and intracellular Cl- activity was 56.1 +/- 2.3 mM. The presence of an apical Na+ conductance was demonstrated by luminal amiloride application (10(-4)M), which decreased Ieq, hyperpolarized Va, and increased the fractional resistance of the apical membrane. The presence of an apical Cl- conductance was demonstrated by substitution of Cl- with gluconate in the luminal bath, which decreased intracellular Cl- activity and increased the fractional resistance of the apical membrane. Luminal application of ATP (10(-4) M was also found to increase the rate of Cl- secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated whether Ca2+ was involved in regulation of ion transport across rabbit distal airway epithelial cells by studying the effects that elevation of intracellular Ca2+ (Cai) had on the bioelectric properties of nonciliated bronchiolar (Clara) cell epithelia in culture. Exposure of Clara cells to 5 x 10(-7) M ionomycin increased Cai concentration and transepithelial short-circuit current (Isc). Changing extracellular Ca2+ concentration in the presence of ionomycin demonstrated that changes in Isc paralleled changes in Cai. Another ionophore, 4-bromo-A23187, also increased Cai and Isc. Ionomycin-induced changes in Isc were insensitive to amiloride and were inhibited greater than 50% by pretreating the cells with bumetanide or substituting gluconate for Cl- in the bathing solution. Bradykinin and carbachol, which increased Cai and caused an increase in Isc across tracheal cell cultures, had no effect on Cai or Isc in Clara cell preparations. These results support the hypothesis that changes in Cai are linked to regulation of Cl- secretion across bronchiolar epithelial cells, but physiological regulators of Cai in Clara cells remain to be defined.

Sonicates of freshly isolated Clara cells produced thromboxane B2 (TxB2), prostaglandin (PG) D2, PGE2, PGF2 alpha, hydroxyheptadecatrienoic acid (HHT), and 12-hydroxyeicosatetraenoic acid (12-HETE) as detected using high-performance liquid chromatography (HPLC). Sonicates of Clara cells cultured on collagen matrices produced the same metabolites. Rates of [3H]arachidonic acid metabolism increased in culture, but the changes were not associated with changes in cell number. Sonicates of freshly isolated tracheal cells produced mainly 12-HETE. Cyclooxygenase products were not produced consistently. Sonicates of tracheal cultures produced significant quantities of TxB2, PGD2, PGE2, PGF2 alpha, and HHT, but 12-HETE remained the major metabolite. Equivalent short-circuit current (Ieq) across cultured Clara cell epithelia was unaffected by bilateral exposure to TxB2, PGD2, PGE2, PGF2 alpha, HHT, or 12-HETE. A minor (1%) decrease in transepithelial resistance (Rt) followed exposure to PGD2. Indomethacin had no significant effect on Rt or Ieq, but exposure of indomethacin-pretreated preparations to PGE2 revealed a minor (2%) increase in Ieq. In contrast, tracheal cell epithelia exhibited significant changes in Rt and Ieq in response to PGF2 alpha, PGE2, and HHT. These results indicate that Clara cells metabolize arachidonic acid to biologically active eicosanoids, but the resulting products do not play a major role in regulation of transepithelial ion transport by this cell type.

Insulin (INS), endothelial cell growth supplement (ECGS), transferrin (TF), cholera toxin (CT), hydrocortisone (HC), triiodothyronine (T3), and epidermal growth factor (EGF) were systematically examined for their effects on proliferation, ion transport activity, and morphological differentiation of canine tracheal epithelial (CTE) cells in culture. INS, ECGS, TF, and CT increase proliferation of CTE cells cultured on plastic Petri dishes but exhibit no acute effect on the bioelectric activity of freshly excised CTE. CT increases amiloride-insensitive transepithelial ion transport across both freshly excised canine trachea and CTE cultures. EGF has no effect on proliferation of CTE cells on plastic but induces hyperplasia of CTE cells on collagen matrices. EGF does not alter basal transepithelial ion transport across cultures but increases cellular responsiveness to beta-adrenergic stimulation. HC and T3 have no effect on proliferation or transepithelial bioelectric properties of CTE cells but improve morphological differentiation yielding cultures of complex epithelia containing cuboidal ciliated and nonciliated cells. These results demonstrate that growth and function of cultured CTE cells are affected by specific growth factors.

Radioisotopic flux measurements were performed on rabbit Clara cell epithelium cultured in serum-free hormone-supplemented medium to identify the major ion transport pathways in the cell type. Clara cells cultured in serum-free hormone-supplemented medium exhibit a large short-circuit current compared with cells maintained in serum-containing medium (45 microA/cm2 vs. 15 microA/cm2). The responses to amiloride and isoproterenol, however, are similar for cells grown in the two media. A net amiloride-sensitive movement of Na+ in the mucosal (M)-to-serosal (S) direction undershort- and open-circuit conditions is detected (1.48 and 0.67 mueq.h-1.cm-2, respectively). No statistically significant difference in the unidirectional fluxes of Cl- is apparent in the basal state, but a net flux of Cl- in the S-to-M direction is observed after exposure of the apical membrane to amiloride (0.93 mueq.h-1.cm-2). The partial ionic conductances for Na+ and Cl- estimated from the fluxes measured in the passive directions (JNaS----M, JClM----S) exceed the total tissue conductance by 20%. Ussing flux ratio analyses of Cl- movements at clamped potentials between -60 and +20 mV show that Cl- movements are not strictly through passive conductive pathways at negative potentials. The movement of Cl- can be modeled by passive diffusion combined with Cl- -Cl- exchange equal to 20% of total passive fluxes of Na+ and Cl-. These observations indicate that 1) Na+ absorption is the major active ion transport pathway across cultured Clara cells, 2) active Cl- secretion is minimal in the basal state, and 3) approximately 20% of the unidirectional Cl- fluxes occur via nonconductive pathways.