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Pharmacology of α-7 Nicotinic Acetylcholine Receptors (α-7 nAChR) Agonists in Cystic Fibrosis
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A3882 - Pharmacology of α-7 Nicotinic Acetylcholine Receptors (α-7 nAChR) Agonists in Cystic Fibrosis
Author Block: L. W. Rasmussen1, J. LaFontaine1, D. Stanford2, K. Patel1, L. Tang1, M. Mazur1, D. Wang1, S. M. Rowe2, S. Raju2; 1University of Alabama Birmingham, Birmingham, AL, United States, 2University of Alabama at Birmingham, Birmingham, AL, United States.
INTRODUCTION: Cystic fibrosis (CF) is caused by genetic mutations in CFTR, an epithelial channel that conducts anions to regulate airway surface hydration and mucociliary clearance (MCC). Previous reports documented decreased CFTR function in transgenic mice lacking α-7 nAChR expression. Based on that, we tested α-7 nAChR specific agonist GTS-21 in wild type (WT) ferret airways and found augmented CFTR-activity and mucociliary transport (MCT). Here, we have investigated the expression of non-neuronal α-7 nAChR in CF lungs and tested whether α-7 nAChR specific agonist GTS-21 can rescue function of mutated CFTR channels.
METHODS: Primary human bronchial epithelial (HBE) cells were isolated from CF patients homozygous for F508del CFTR and healthy donors. CFTR activity was measured in short-circuit current (Isc) units in HBE cells using modified Ussing chambers and by nasal potential difference (NPD) measurements in vivo in CFTR-/- and WT rats. α-7 nAChR expression was assessed using immunohistochemistry (IHC) in human and rat lungs. Cellular cAMP levels were measured using a commercial calorimetric kit.
RESULTS: Analysis of airway tissues from CF patients and CFTR knockout (KO, CFTR-/-) rats identified prominent distribution of α-7nAChR on the conducting airway epithelium. GTS-21 infusion increased CFTR activity by NPD in WT but not in CFTR-/- rats (ΔGTS21 in mV, -3.8 ± 0.8 WT vs -0.5 ± 0.6 CFTR-/-, P≤0.01), indicating CFTR specificity. In CF HBE cells, acute addition of GTS-21 activated mutated CFTR (Isc in µA/cm2, ΔGTS21: 7.35 vs ΔVeh: -0.03, P≤0.001) that paralleled a 3-fold increase in cAMP. Pretreatment with GTS21 for 30 min resulted in no apparent sustained elevations in cAMP but still, markedly enhanced CFTR activity following subsequent stimulation with forskolin (ΔForskolin Isc in µA/cm2, Veh-treated: 1.04 vs GTS-treated: 4.5, P≤0.01), suggesting additional cAMP-independent unknown mechanism. Since CF airways are prone to acidic pH, we tested GTS-21 in non-CF HBE cells under variable pH conditions and found CFTR responses to be highly pH-dependent and were inhibitory under acidic conditions (ΔGTS21 Isc in µA/cm2, pH-7.4: 7.12 vs pH-6.4: 3.16, P≤0.01).
CONCLUSIONS: α-7 nicotinic acetylcholine receptors are widely distributed in CF airways and their activation results in partial rescue of CFTR function. Benefits of α-7 nAChR agonists on epithelial ion transport are highly specific to CFTR and are pH-dependent. These data assign a novel regulatory role for α-7 nAChR in CFTR physiology and warrant additional studies to determine the mechanisms and potential applications of α-7 nAChR agonists in CF therapy.
Author Block: L. W. Rasmussen1, J. LaFontaine1, D. Stanford2, K. Patel1, L. Tang1, M. Mazur1, D. Wang1, S. M. Rowe2, S. Raju2; 1University of Alabama Birmingham, Birmingham, AL, United States, 2University of Alabama at Birmingham, Birmingham, AL, United States.
INTRODUCTION: Cystic fibrosis (CF) is caused by genetic mutations in CFTR, an epithelial channel that conducts anions to regulate airway surface hydration and mucociliary clearance (MCC). Previous reports documented decreased CFTR function in transgenic mice lacking α-7 nAChR expression. Based on that, we tested α-7 nAChR specific agonist GTS-21 in wild type (WT) ferret airways and found augmented CFTR-activity and mucociliary transport (MCT). Here, we have investigated the expression of non-neuronal α-7 nAChR in CF lungs and tested whether α-7 nAChR specific agonist GTS-21 can rescue function of mutated CFTR channels.
METHODS: Primary human bronchial epithelial (HBE) cells were isolated from CF patients homozygous for F508del CFTR and healthy donors. CFTR activity was measured in short-circuit current (Isc) units in HBE cells using modified Ussing chambers and by nasal potential difference (NPD) measurements in vivo in CFTR-/- and WT rats. α-7 nAChR expression was assessed using immunohistochemistry (IHC) in human and rat lungs. Cellular cAMP levels were measured using a commercial calorimetric kit.
RESULTS: Analysis of airway tissues from CF patients and CFTR knockout (KO, CFTR-/-) rats identified prominent distribution of α-7nAChR on the conducting airway epithelium. GTS-21 infusion increased CFTR activity by NPD in WT but not in CFTR-/- rats (ΔGTS21 in mV, -3.8 ± 0.8 WT vs -0.5 ± 0.6 CFTR-/-, P≤0.01), indicating CFTR specificity. In CF HBE cells, acute addition of GTS-21 activated mutated CFTR (Isc in µA/cm2, ΔGTS21: 7.35 vs ΔVeh: -0.03, P≤0.001) that paralleled a 3-fold increase in cAMP. Pretreatment with GTS21 for 30 min resulted in no apparent sustained elevations in cAMP but still, markedly enhanced CFTR activity following subsequent stimulation with forskolin (ΔForskolin Isc in µA/cm2, Veh-treated: 1.04 vs GTS-treated: 4.5, P≤0.01), suggesting additional cAMP-independent unknown mechanism. Since CF airways are prone to acidic pH, we tested GTS-21 in non-CF HBE cells under variable pH conditions and found CFTR responses to be highly pH-dependent and were inhibitory under acidic conditions (ΔGTS21 Isc in µA/cm2, pH-7.4: 7.12 vs pH-6.4: 3.16, P≤0.01).
CONCLUSIONS: α-7 nicotinic acetylcholine receptors are widely distributed in CF airways and their activation results in partial rescue of CFTR function. Benefits of α-7 nAChR agonists on epithelial ion transport are highly specific to CFTR and are pH-dependent. These data assign a novel regulatory role for α-7 nAChR in CFTR physiology and warrant additional studies to determine the mechanisms and potential applications of α-7 nAChR agonists in CF therapy.
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