Marked increases in mucociliary clearance produced by synergistic secretory agonists or inhibition of the epithelial sodium channel.
ABSTRACT: Mucociliary clearance (MCC) is a critical host innate defense mechanism in airways, and it is impaired in cystic fibrosis (CF) and other obstructive lung diseases. Epithelial fluid secretion and absorption modify MCC velocity (MCCV). We tested the hypotheses that inhibiting fluid absorption accelerates MCCV, whereas inhibiting fluid secretion decelerates it. In airways, ENaC is mainly responsible for fluid absorption, while anion channels, including CFTR and Ca2+-activated chloride channels mediate anion/fluid secretion. MCCV was increased by the cAMP-elevating agonists, forskolin or isoproterenol (10??M) and by the Ca2+-elevating agonist, carbachol (0.3??M). The CFTR-selective inhibitor, CFTRinh-172, modestly reduced MCCV-increases induced by forskolin or isoproterenol but not increases induced by carbachol. The ENaC inhibitor benzamil increased basal MCCV as well as MCCV increases produced by forskolin or carbachol. MCC velocity was most dramatically accelerated by the synergistic combination of forskolin and carbachol, which produced near-maximal clearance rates regardless of prior treatment with CFTR or ENaC inhibitors. In CF airways, where CFTR-mediated secretion (and possibly synergistic MCC) is lost, ENaC inhibition via exogenous agents may provide therapeutic benefit, as has long been proposed.
Project description:In upper airways airway surface liquid (ASL) depth and clearance rates are both increased by fluid secretion. Secretion is opposed by fluid absorption, mainly via the epithelial sodium channel, ENaC. In static systems, increased fluid depth activates ENaC and decreased depth inhibits it, suggesting that secretion indirectly activates ENaC to reduce ASL depth. We propose an alternate mechanism in which cholinergic input, which causes copious airway gland secretion, also inhibits ENaC-mediated absorption. The conjoint action accelerates clearance, and the increased transport of mucus out of the airways restores ASL depth while cleansing the airways. We were intrigued by early reports of cholinergic inhibition of absorption by airways in some species. To reinvestigate this phenomenon, we studied inward short-circuit currents (Isc) in tracheal mucosa from human, sheep, pig, ferret, and rabbit and in two types of cultured cells. Basal Isc was inhibited 20-70% by the ENaC inhibitor, benzamil. Long-lasting inhibition of ENaC-dependent Isc was also produced by basolateral carbachol in all preparations except rabbit and the H441 cell line. Atropine inhibition produced a slow recovery or prevented inhibition if added before carbachol. The mechanism for inhibition was not determined and is most likely multi-factorial. However, its physiological significance is expected to be increased mucus clearance rates in cholinergically stimulated airways.
Project description:<h4>Background</h4>Cystic fibrosis (CF), caused by reduced CFTR function, includes severe sinonasal disease which may predispose to lung disease. Newly developed CF pigs provide models to study the onset of CF pathophysiology. We asked if glands from pig nasal turbinates have secretory responses similar to those of tracheal glands and if CF nasal glands show reduced fluid secretion.<h4>Methodology/principal findings</h4>Unexpectedly, we found that nasal glands differed from tracheal glands in five ways, being smaller, more numerous (density per airway surface area), more sensitive to carbachol, more sensitive to forskolin, and nonresponsive to Substance P (a potent agonist for pig tracheal glands). Nasal gland fluid secretion from newborn piglets (12 CF and 12 controls) in response to agonists was measured using digital imaging of mucus bubbles formed under oil. Secretion rates were significantly reduced in all conditions tested. Fluid secretory rates (Controls vs. CF, in pl/min/gland) were as follows: 3 µM forskolin: 9.2±2.2 vs. 0.6±0.3; 1 µM carbachol: 143.5±35.5 vs. 52.2±10.3; 3 µM forskolin + 0.1 µM carbachol: 25.8±5.8 vs. CF 4.5±0.9. We also compared CF(?F508/?F508) with CFTR(-/-) piglets and found significantly greater forskolin-stimulated secretion rates in the ?F508 vs. the null piglets (1.4±0.8, n?=?4 vs. 0.2±0.1, n?=?7). An unexpected age effect was also discovered: the ratio of secretion to 3 µM forskolin vs. 1 µM carbachol was ?4 times greater in adult than in neonatal nasal glands.<h4>Conclusions/significance</h4>These findings reveal differences between nasal and tracheal glands, show defective fluid secretion in nasal glands of CF pigs, reveal some spared function in the ?F508 vs. null piglets, and show unexpected age-dependent differences. Reduced nasal gland fluid secretion may predispose to sinonasal and lung infections.
Project description:The role of cystic fibrosis transmembrane conductance regulator (CFTR) in lacrimal gland (LG) function has only recently received some attention, mainly from our group. In the present study, we investigated the potential changes of LG pathology, tear secretion, ocular surface integrity, and fluid secretion in isolated LG ducts from CFTR knockout (KO) mice.Tear production and ocular surface integrity were investigated in anesthetized wild-type (WT) and KO mice using cotton threads and fluorescein staining, respectively. Immunofluorescence was used to localize CFTR protein in the LGs. Ductal fluid secretions evoked by forskolin (10 ?M); cell-permeable cAMP analogue (8-bromo cAMP, 100 ?M); or carbachol (100 ?M) were measured in isolated LG ducts using video-microscopy. Intracellular Ca2+ homeostasis underlying carbachol stimulation was investigated with microfluorometry.Significant decrease in tear secretion and impaired ocular surface integrity were observed in KO mice. Immunofluorescence demonstrated the predominant presence of CFTR protein in the apical membranes of the duct cells from WT mice. Continuous fluid secretion was evoked by forskolin and 8-bromo cAMP in LG ducts from WT mice, while no secretory response was observed in ducts from KO mice. Carbachol caused similar secretory responses in ducts from WT and KO animals without significant differences in cytosolic Ca2+ signaling.Our results suggest the important role of CFTR in LG ductal secretion and in the maintenance of ocular surface integrity, suggesting that CFTR may be a promising target of novel therapeutic approaches in the treatment of dry eye.
Project description:Chronic bacterial airway infections are the major cause of mortality in cystic fibrosis (CF). Normal airway defenses include reflex stimulation of submucosal gland mucus secretion by sensory neurons that release substance P (SubP). CFTR is an anion channel involved in fluid secretion and mutated in CF; the role of CFTR in secretions stimulated by SubP is unknown. We used optical methods to measure SubP-mediated secretion from human submucosal glands in lung transplant tissue. Glands from control but not CF subjects responded to mucosal chili oil. Similarly, serosal SubP stimulated secretion in more than 60% of control glands but only 4% of CF glands. Secretion triggered by SubP was synergistic with vasoactive intestinal peptide and/or forskolin but not with carbachol; synergy was absent in CF glands. Pig glands demonstrated a nearly 10-fold greater response to SubP. In 10 of 11 control glands isolated by fine dissection, SubP caused cell volume loss, lumen expansion, and mucus flow, but in 3 of 4 CF glands, it induced lumen narrowing. Thus, in CF, the reduced ability of mucosal irritants to stimulate airway gland secretion via SubP may be another factor that predisposes the airways to infections.
Project description:Mucus secretion from individual tracheal glands in adult ferrets was studied with time-lapse optical imaging of mucus droplets under an oil layer. Density of functional glands (determined by responses to 1 muM carbachol) was 1.5 +/- 0.3 per mm(2) (n = 6). Secretion rates (in pl.min(-1).gland(-1)) were as follows: 4.1 +/- 0.7 basal (unstimulated; n = 27, 669 glands), 338 +/- 70 to 10 microM forskolin (n = 8, 90 glands), 234 +/- 13 to 1 microM VIP (n = 6, 57 glands), 183 +/- 92 to 10 microM isoproterenol (n = 3, 33 glands), 978 +/- 145 to 1 microM carbachol (n = 11, 131 glands), and 1,348 +/- 325 to 10 muM phenylephrine (n = 7, 74 glands). The potency (EC(50), in microM) and efficacy (V(max), in pl x min(-1) x gland(-1)) were 7.6 (EC(50)) and 338 +/- 16 (V(max)) to forskolin, 1.0 (EC(50)) and 479 +/- 19 (V(max)) to VIP, 0.6 (EC(50)) and 1,817 +/- 268 (V(max)) to carbachol, and 3.7 (EC(50)) and 1,801 +/- 95 (V(max)) to phenylephrine. Although carbachol and phenylephrine were equally effective secretagogues, only carbachol caused contractions of the trachealis muscle. Synergy was demonstrated between 300 nM isoproterenol and 100 nM carbachol, which, when combined, produced a secretion rate almost fourfold greater than predicted from their additive effect. The dependence of fluid secretion on Cl(-) and HCO(3)(-) varied depending on the mode of stimulation. Secretion stimulated by VIP or forskolin was reduced by approximately 60% by blocking either anion, while carbachol-stimulated secretion was blocked 68% by bumetanide and only 32% by HEPES replacement of HCO(3)(-). These results provide parametric data for comparison with fluid secretion from glands in ferrets lacking CFTR.
Project description:Transport of water and electrolytes in airway epithelia involves chloride-selective ion channels, which are controlled either by cytosolic Ca<sup>2+</sup> or by cAMP The contributions of the two pathways to chloride transport differ among vertebrate species. Because rats are becoming more important as animal model for cystic fibrosis, we have examined how Ca<sup>2+</sup>- dependent and cAMP- dependent Cl<sup>-</sup> secretion is organized in the rat tracheal epithelium. We examined the expression of the Ca<sup>2+</sup>-gated Cl<sup>-</sup> channel anoctamin 1 (ANO1), the cystic fibrosis transmembrane conductance regulator (CFTR) Cl<sup>-</sup> channel, the epithelial Na<sup>+</sup> channel ENaC, and the water channel aquaporin 5 (AQP5) in rat tracheal epithelium. The contribution of ANO1 channels to nucleotide-stimulated Cl<sup>-</sup> secretion was determined using the channel blocker <i>Ani9</i> in short-circuit current recordings obtained from primary cultures of rat tracheal epithelial cells in Ussing chambers. We found that ANO1, CFTR and AQP5 proteins were expressed in nonciliated cells of the tracheal epithelium, whereas ENaC was expressed in ciliated cells. Among nonciliated cells, ANO1 occurred together with CFTR and Muc5b and, in addition, in a different cell type without CFTR and Muc5b. Bioelectrical studies with the ANO1-blocker <i>Ani9</i> indicated that ANO1 mediated the secretory response to the nucleotide uridine-5'-triphosphate. Our data demonstrate that, in rat tracheal epithelium, Cl<sup>-</sup> secretion and Na<sup>+</sup> absorption are routed through different cell types, and that ANO1 channels form the molecular basis of Ca<sup>2+</sup>-dependent Cl<sup>-</sup> secretion in this tissue. These characteristic features of Cl<sup>-</sup>-dependent secretion reveal similarities and distinct differences to secretory processes in human airways.
Project description:Cystic fibrosis (CF) is a monogenic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR dysfunction is characterized by abnormal mucociliary transport due to a dehydrated airway surface liquid (ASL) and hyperviscous mucus, among other pathologies of host defense. ASL depletion is caused by the absence of CFTR mediated chloride secretion along with continued activity of the epithelial sodium channel (ENaC) activity, which can also be affected by CFTR mediated anion conductance. Therefore, ENaC has been proposed as a therapeutic target to ameliorate ASL dehydration and improve mucus transport. Inhibition of ENaC has been shown to restore ASL hydration and enhance mucociliary transport in induced models of CF lung disease. To date, no therapy inhibiting ENaC has successfully translated to clinical efficacy, in part due to concerns regarding off-target effects, systemic exposure, durability of effect, and adverse effects. Recent efforts have been made to develop novel, rationally designed therapeutics to produce-specific, long-lasting inhibition of ENaC activity in the airways while simultaneously minimizing off target fluid transport effects, systemic exposure and side effects. Such approaches comprise next-generation small molecule direct inhibitors, indirect channel-activating protease inhibitors, synthetic peptide analogs, and oligonucleotide-based therapies. These novel therapeutics represent an exciting step forward in the development of ENaC-directed therapies for CF.
Project description:An imbalance of chloride and sodium ion transport in several epithelia is a feature of cystic fibrosis (CF), an inherited disease that is a consequence of mutations in the cftr gene. The cftr gene codes for a Cl(-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Some mutations in this gene cause the balance between Cl(-) secretion and Na(+) absorption to be disturbed in the airways; Cl(-) secretion is impaired, whereas Na(+) absorption is elevated. Enhanced Na(+) absorption through the epithelial sodium channel (ENaC) is attributed to the failure of mutated CFTR to restrict ENaC-mediated Na(+) transport. The mechanism of this regulation is controversial. Recently, we have found evidence for a close association of wild type (WT) CFTR and WT ENaC, further underscoring the role of ENaC along with CFTR in the pathophysiology of CF airway disease. In this study, we have examined the association of ENaC subunits with mutated ?F508-CFTR, the most common mutation in CF. Deletion of phenylalanine at position 508 (?F508) prevents proper processing and targeting of CFTR to the plasma membrane. When ?F508-CFTR and ENaC subunits were co-expressed in HEK293T cells, we found that individual ENaC subunits could be co-immunoprecipitated with ?F508-CFTR, much like WT CFTR. However, when we evaluated the ?F508-CFTR and ENaC association using fluorescence resonance energy transfer (FRET), FRET efficiencies were not significantly different from negative controls, suggesting that ?F508-CFTR and ENaC are not in close proximity to each other under basal conditions. However, with partial correction of ?F508-CFTR misprocessing by low temperature and chemical rescue, leading to surface expression as assessed by total internal reflection fluorescence (TIRF) microscopy, we observed a positive FRET signal. Our findings suggest that the ?F508 mutation alters the close association of CFTR and ENaC.
Project description:BACKGROUND AND PURPOSE: The protoberberine alkaloid berberine has been reported to inhibit colonic Cl(-) secretion. However, it is not known if other protoberberine alkaloids share these effects. We have therefore selected another protoberberine alkaloid, palmatine, to assess its effects on active ion transport across rat colonic epithelium. EXPERIMENTAL APPROACH: Rat colonic mucosa was mounted in Ussing chambers and short circuit current (I (SC)), apical Cl(-) current and basolateral K(+) current were recorded. Intracellular cAMP content was determined by an enzyme immunoassay. Intracellular Ca(2+) concentration was measured with Fura-2 AM. KEY RESULTS: Palmatine inhibited carbachol-induced Ca(2+)-activated Cl(-) secretion and the carbachol-induced increase of intracellular Ca(2+) concentration. Palmatine also inhibited cAMP-activated Cl(-) secretion induced by prostaglandin E(2) (PGE(2)) or forskolin. Palmatine prevented the elevation of intracellular cAMP by forskolin. Determination of apical Cl(-) currents showed that palmatine suppressed the forskolin-stimulated, apical cAMP-activated Cl(-) current but not the carbachol-stimulated apical Ca(2+)-activated Cl(-) current. Following permeabilization of apical membranes with nystatin, we found that palmatine inhibited a carbachol-stimulated basolateral K(+) current that was sensitive to charybdotoxin and resistant to chromanol 293B. However, the forskolin-stimulated basolateral K(+) current inhibited by palmatine was specifically blocked by chromanol 293B and not by charybdotoxin. CONCLUSIONS AND IMPLICATIONS: Palmatine attenuated Ca(2+)-activated Cl(-) secretion through inhibiting basolateral charybdotoxin-sensitive, SK4 K(+) channels, whereas it inhibited cAMP-activated Cl(-) secretion by inhibiting apical CFTR Cl(-) channels and basolateral chromanol 293B-sensitive, KvLQT1 K(+) channels.
Project description:Alveolar fluid clearance driven by active epithelial Na(+) and secondary Cl(-) absorption counteracts edema formation in the intact lung. Recently, we showed that impairment of alveolar fluid clearance because of inhibition of epithelial Na(+) channels (ENaCs) promotes cardiogenic lung edema. Concomitantly, we observed a reversal of alveolar fluid clearance, suggesting that reversed transepithelial ion transport may promote lung edema by driving active alveolar fluid secretion. We, therefore, hypothesized that alveolar ion and fluid secretion may constitute a pathomechanism in lung edema and aimed to identify underlying molecular pathways. In isolated perfused lungs, alveolar fluid clearance and secretion were determined by a double-indicator dilution technique. Transepithelial Cl(-) secretion and alveolar Cl(-) influx were quantified by radionuclide tracing and alveolar Cl(-) imaging, respectively. Elevated hydrostatic pressure induced ouabain-sensitive alveolar fluid secretion that coincided with transepithelial Cl(-) secretion and alveolar Cl(-) influx. Inhibition of either cystic fibrosis transmembrane conductance regulator (CFTR) or Na(+)-K(+)-Cl(-) cotransporters (NKCC) blocked alveolar fluid secretion, and lungs of CFTR(-/-) mice were protected from hydrostatic edema. Inhibition of ENaC by amiloride reproduced alveolar fluid and Cl(-) secretion that were again CFTR-, NKCC-, and Na(+)-K(+)-ATPase-dependent. Our findings show a reversal of transepithelial Cl(-) and fluid flux from absorptive to secretory mode at hydrostatic stress. Alveolar Cl(-) and fluid secretion are triggered by ENaC inhibition and mediated by NKCC and CFTR. Our results characterize an innovative mechanism of cardiogenic edema formation and identify NKCC1 as a unique therapeutic target in cardiogenic lung edema.