Native small airways secrete bicarbonate

Abstract

Since the discovery of Cl(-) impermeability in cystic fibrosis (CF) and the cloning of the responsible channel, CF pathology has been widely attributed to a defect in epithelial Cl(-) transport. However, loss of bicarbonate (HCO3(-)) transport also plays a major, possibly more critical role in CF pathogenesis. Even though HCO3(-) transport is severely affected in the native pancreas, liver, and intestines in CF, we know very little about HCO3(-) secretion in small airways, the principle site of morbidity in CF. We used a novel, mini-Ussing chamber system to investigate the properties of HCO3(-) transport in native porcine small airways (∼ 1 mm φ). We assayed HCO3(-) transport across small airway epithelia as reflected by the transepithelial voltage, conductance, and equivalent short-circuit current with bilateral 25-mM HCO3(-) plus 125-mM NaGlu Ringer's solution in the presence of luminal amiloride (10 μM). Under these conditions, because no major transportable anions other than HCO3(-) were present, we took the equivalent short-circuit current to be a direct measure of active HCO3(-) secretion. Applying selective agonists and inhibitors, we show constitutive HCO3(-) secretion in small airways, which can be stimulated significantly by β-adrenergic- (cAMP) and purinergic (Ca(2+)) -mediated agonists, independently. These results indicate that two separate components for HCO3(-) secretion, likely via CFTR- and calcium-activated chloride channel-dependent processes, are physiologically regulated for likely roles in mucus clearance and antimicrobial innate defenses of small airways.

Figure 1.

Anion selectivity of small airways. Small airways show significant conductance to Cl⁻ and bicarbonate (HCO₃⁻), as indicated by changes in transepithelial potential (V_t) and transepithelial conductance (G_t) after anion substitution with 150 mM gluconate in the presence of 3-isobutyl-1-methylxanthine (IBMX)/forskolin (Fsk) and amiloride in the luminal bath. Representative electrical trace (A) of V_t responses to substitutions of 150 mM gluconate (black) on the lumen of a small airway with equimolar HCO₃⁻ (gray) or Cl⁻ (white) while maintaining 150 mM NaCl on the serosal surface. Summary of results of anion replacements on V_t (B) and G_t (C). Representative trace (D) of constitutive electrical properties in bilateral 150 mM NaGlu with responses to substitution of 25 mM luminal Glu with either equimolar Cl⁻ or HCO₃⁻, which significantly depolarized the electrogenic Na⁺-dependent component of V_t. Note that Cl⁻-induced depolarization was more than three times that due to HCO₃⁻, indicating that HCO₃⁻ conductance is significantly greater than the impermeant gluconate, but much less than Cl⁻ in shunting the Na⁺ absorptive potential. Summary of V_t (E) and G_t (F) responses to replacements of 25 mM Glu⁻ (black) with equimolar HCO₃⁻ (gray) or Cl⁻ (white). 25HCO₃⁻, 25 mM NaHCO₃⁻; 25NaCl, 25 mM NaCl. *P < 0.05, **P < 0.001; data presented are means ± SEM.

Figure 2.

Effect of cAMP agonist Fsk/IBMX and inhibitors on HCO₃⁻ transport. (A) Representative trace of electrogenic HCO₃⁻ transport properties in native small airways with 25 mM HCO₃⁻ in both basolateral and luminal baths (Cl⁻ free). In the presence of luminal amiloride (Amil), mucosal addition of Fsk/IBMX significantly hyperpolarized V_t and stimulated equivalent short-circuit current (I_sc^eq). Luminal addition of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) inhibitor, GlyH-101 blocked the I_sc^eq response. Furthermore, addition of serosal 4,4′diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) plus acetazolamide reduced the I_sc^eq to significantly less than the initial, unstimulated constitutive current (white). Summary of the effects of Fsk/IBMX (black), GlyH-101 (gray), and DIDS + acetazolamide (stippled) on V_t (B), G_t (C), and I_sc^eq (D). *P < 0.05; data presented are means ± SEM.

Figure 3.

Effects of inhibitor GlyH-101on isoproterenol (IPR) stimulation of HCO₃⁻ transport. (A) Representative trace of changes in electrogenic HCO₃⁻ transport properties with addition of IPR (10 μM), which significantly hyperpolarized V_t and stimulated I_sc^eq (P < 0.05; n = 3). Luminal addition of inhibitor, GlyH-101, blocked stimulated HCO₃⁻ secretion. Summary constitutive properties (white) and of the effects of IPR (black) and GlyH-101 (gray) on V_t (B), G_t (C), and I_sc^eq (D). *P < 0.05; data presented are means ± SEM.

Figure 4.

Effect of the inhibitors 4,4′-dinitrostilbene-2,2′-disulphonic acid (DNDS) and GlyH-101 on Fsk/IBMX stimulation. (A) Representative trace of electrogenic HCO₃⁻ transport properties after luminal addition of Fsk/IBMX (black), which significantly hyperpolarized V_t, increased G_t, and stimulated I_sc^eq in the continuous presence of luminal amiloride (white). Subsequent contraluminal addition of the inhibitor DNDS (gray) significantly blocked V_t, G_t, and I_sc^eq response (P < 0.05; n = 3). Further luminal addition of GlyH-101 (stippled) reduced the remaining I_sc^eq to significantly less than the initial constitutive current. Summary of the effects of Fsk/IBMX (black), DNDS (gray), and GlyH-101 (stippled) on V_t (B), G_t (C), and I_sc^eq (D). *P < 0.05; data presented are means ± SEM.

Figure 5.

Effects of inhibitors, GlyH-101 and niflumic acid (NFA), on prostaglandin E (PGE) -2 stimulation. (A) Representative trace of electrogenic properties of HCO₃⁻ transport stimulated by luminal PGE2 (black; 1 μM). V_t hyperpolarized and I_sc^eq increased significantly (P < 0.05; n = 4). Luminal addition of inhibitor, GlyH-101 (gray), significantly decreased the HCO₃⁻ secretion, and further addition of the Ca²⁺-activated chloride channel (CaCC) antagonist, NFA (stippled), further depressed the HCO₃⁻ secretion to levels significantly below the initial constitutive value. Summary of the effects of PGE2 (black), GlyH-101 (gray), and NFA (stippled) on V_t (B), G_t (C), and I_sc^eq (D). *P < 0.05; data presented are means ± SEM.

Figure 6.

Effects of inhibitor, NFA, on UTP stimulation. (A) Representative trace of changes in electrical properties reflecting HCO₃⁻ transport induced by luminal addition of UTP (black; 100 μM), which significantly hyperpolarized V_t, increased G_t, and stimulated I_sc^eq (P < 0.05; n = 5). Luminal addition of the CACC inhibitor, NFA (gray), decreased the HCO₃⁻ secretion I_sc^eq to values significantly less than initial constitutive secretion in the continuous presence of luminal amiloride (white). Summary of the effects of UTP (black) and NFA (gray) on V_t (B), G_t (C), and I_sc^eq (D). *P < 0.05; data presented are means ± SEM.

Figure 7.

Additive effects of Fsk/IBMX and UTP stimulation. (A) Representative trace of electrical properties reflecting HCO₃⁻ transport when stimulated by Fsk/IBMX, which significantly hyperpolarized V_t, and increased G_t and I_sc^eq (P < 0.05; n = 4) in the presence of luminal amiloride. Subsequent addition of UTP to Fsk/IBMX hyperpolarized V_t further, and increased G_t and I_sc^eq even further (P < 0.05; n = 4). Summary of the additive effects of Fsk/IBMX (black) and UTP (gray) on V_t (B), G_t (C), and I_sc^eq (D) relative to control (white). *P < 0.05; data presented are means ± SEM.