Concurrent absorption and secretion of airway surface liquids and bicarbonate secretion in human bronchioles

Abstract

Although small airways account for the largest fraction of the total conducting airway surfaces, the epithelial fluid and electrolyte transport in small, native airway epithelia has not been well characterized. Investigations have been limited, no doubt, by the complex tissue architecture as well as by its inaccessibility, small dimensions, and lack of applicable assays, especially in human tissues. To better understand how the critically thin layer of airway surface liquid (ASL) is maintained, we applied a "capillary"-Ussing chamber (area ≈1 mm²) to measure ion transport properties of bronchioles with diameters of ~2 mm isolated from resected specimens of excised human lungs. We found that the small human airway, constitutively and concurrently, secretes and absorbs fluid as observed in porcine small airways (50). We found that the human bronchiolar epithelium is also highly anion selective and constitutively secretes bicarbonate ( ${\text{HCO}}_3^{-}$ ), which can be enhanced pharmacologically by cAMP as well as Ca²⁺-mediated agonists. Concurrent secretion and absorption of surface liquid along with ${\text{HCO}}_3^{-}$ secretion help explain how the delicate volume of the fluid lining the human small airway is physiologically buffered and maintained in a steady state that avoids desiccating or flooding the small airway with ASL.

Keywords: ASL; CFTR; Cl transport; ENaC; airway surface liquid; transport.

Fig. 1.

Bi-ionic Cl⁻ diffusion potentials in human small airways. A: representative electrical trace of transepithelial potential (V_t) with constant current pulses (0.5 µA) on the effect of substituting luminal gluconate, an impermeant anion, for the permeable Cl⁻ anion on a dissected small airway mounted in a capillary-Ussing chamber. Replacing luminal Cl⁻ with gluconate hyperpolarized the V_t significantly, indicating a predominant Cl⁻ conductance that appears to be constitutively active. B: box plots showing median, upper, and lower quartiles and whiskers showing upper and lower extreme data points of the results for V_t from similar experiments. When 150 mM NaGlu replaced luminal 150 mM NaCl, V_t hyperpolarized markedly (P < 0.001; n = 21). C: box plots as in B for changes in transepithelial conductance (G_t). Substituting gluconate for luminal Cl⁻ significantly decreased G_t. *P < 0.001; n = 21.

Fig. 2.

Effect of inhibiting of absorption and secretion in human native small airway epithelia in bilateral 150 mM NaCl. A: representative trace of effects of luminal addition of amiloride (10 μM). Transepithelial potential (V_t) depolarized, transepithelial conductance (G_t), and equivalent short circuit current (I_sc^eq) decreased significantly. Subsequent addition of bumetanide (10 μM) to the serosal bath significantly decreased G_t and almost abolished V_t and I_sc^eq. B–D: box plots of data showing the effects of amiloride and bumetanide on V_t (B), G_t (C), and I_sc^eq (D). *P < 0.05; n = 3. Plot parameters as in Fig. 1.

Fig. 3.

Effects of agonists on transepithelial ion transport in human native small airway. A: a representative trace of transepithelial potential (V_t) showing effects of Ca²⁺-mediated agonist UTP, cAMP-mediated agonists forskolin (Fsk)/3-isobutyl-1-methylxanthine (IBMX), and inhibitor GlyH-101 in bilateral 150 mM NaCl Ringer solutions on the small airway. The purinergic agonist UTP significantly hyperpolarized V_t. B–D: on the other hand, cAMP agonist Fsk/IBMX significantly depolarized V_t. E–G: box plots of data showing the effects of Ca²⁺-mediated purinergic agonist UTP and cAMP-mediated agonists Fsk/IBMX in bilateral 150 mM NaCl on V_t (B and E), transepithelial conductance (G_t; C and F), and equivalent short circuit current (I_sc^eq; D and G). Purinergic (UTP) and β-adrenergic (cAMP) agonists evoke distinctly opposite effects on V_t and I_sc^eq (n = 5). Plot parameters as in Fig. 1. *P < 0.05; n = 8.

Fig. 4.

Effect of cAMP agonist forskolin (Fsk)/3-isobutyl-1-methylxanthine (IBMX) and inhibitor GlyH-101 on ${\text{HCO}}_3^{-}$ transport. A: representative trace of electrogenic properties ${\text{HCO}}_3^{-}$ transport in human native small airways in bilateral 25 mM ${\text{HCO}}_3^{-}$ Ringer solution (Cl⁻ free + luminal amiloride). Luminal addition of Fsk/IBMX significantly hyperpolarized transepithelial potential (V_t) and stimulated equivalent short circuit current (I_sc^eq). Luminal addition of CFTR inhibitor GlyH-101 blocked the cAMP response. B–D: box plots showing median, upper, and lower quartiles and whiskers showing upper and lower extreme data points of the effects of Fsk/IBMX, GlyH-101 on V_t (B), transepithelial conductance, G_t (C), and I_sc^eq (D). *P < 0.05; n = 8. Plot parameters as in Fig. 1.

Fig. 5.

Effect of Ca²⁺-mediated agonist UTP and inhibitor niflumic acid (NFA) on ${\text{HCO}}_3^{-}$ transport. A: representative trace of electrogenic properties of ${\text{HCO}}_3^{-}$ transport in human native small airways in bilateral 25 mM ${\text{HCO}}_3^{-}$ (Cl⁻ free + luminal amiloride). In the presence of luminal amiloride, luminal addition of UTP hyperpolarized transepithelial potential (V_t) and stimulated equivalent short circuit current (I_sc^eq). Luminal addition of Ca²⁺-mediated inhibitor NFA blocked the I_sc^eq response. B–D: box plots of data showing the effects of UTP and NFA on V_t (B), transepithelial conductance (G_t; C), and I_sc^eq (D). *P < 0.05; n = 6. Plot parameters as in Fig. 1.

Fig. 6.

Additive effects of cAMP and Ca²⁺-mediated agonists on ${\text{HCO}}_3^{-}$ transport. A: representative trace of electrogenic properties ${\text{HCO}}_3^{-}$ transport in human native small airways with 25 mM ${\text{HCO}}_3^{-}$ in both bath and lumen (Cl⁻ free + luminal amiloride). In the presence of luminal amiloride, luminal addition of cAMP-mediated agonists forskolin (Fsk)/3-isobutyl-1-methylxanthine (IBMX) hyperpolarized transepithelial potential (V_t) and stimulated equivalent short circuit current (I_sc^eq) Subsequent luminal addition of Ca²⁺-mediated agonist UTP hyperpolarized V_t further, and increased I_sc^eq. B–D: box plots of data showing the additive effects of Fsk/IBMX and UTP on V_t (B), G_t (C), and I_sc^eq (D). *P < 0.05; n = 3. Plot parameters as in Fig. 1.