Korean Red Ginseng aqueous extract improves markers of mucociliary clearance by stimulating chloride secretion.

BACKGROUND: Abnormal chloride (Cl-) transport has a detrimental impact on mucociliary clearance in both cystic fibrosis (CF) and non-CF chronic rhinosinusitis. Ginseng is a medicinal plant noted to have anti-inflammatory and antimicrobial properties. The present study aims to assess the capability of red ginseng aqueous extract (RGAE) to promote transepithelial Cl- secretion in nasal epithelium. METHODS: Primary murine nasal septal epithelial (MNSE) [wild-type (WT) and transgenic CFTR-/-], fisher-rat-thyroid (FRT) cells expressing human WT CFTR, and TMEM16A-expressing human embryonic kidney cultures were utilized for the present experiments. Ciliary beat frequency (CBF) and airway surface liquid (ASL) depth measurements were performed using micro-optical coherence tomography (µOCT). Mechanisms underlying transepithelial Cl- transport were determined using pharmacologic manipulation in Ussing chambers and whole-cell patch clamp analysis. RESULTS: RGAE (at 30µg/mL of ginsenosides) significantly increased Cl- transport [measured as change in short-circuit current (ΔISC = µA/cm2)] when compared with control in WT and CFTR-/- MNSE (WT vs control = 49.8±2.6 vs 0.1+/-0.2, CFTR-/- = 33.5±1.5 vs 0.2±0.3, p < 0.0001). In FRT cells, the CFTR-mediated ΔISC attributed to RGAE was small (6.8 ± 2.5 vs control, 0.03 ± 0.01, p < 0.05). In patch clamp, TMEM16A-mediated currents were markedly improved with co-administration of RGAE and uridine 5-triphosphate (8406.3 +/- 807.7 pA) over uridine 5-triphosphate (3524.1 +/- 292.4 pA) or RGAE alone (465.2 +/- 90.7 pA) (p < 0.0001). ASL and CBF were significantly greater with RGAE (6.2+/-0.3 µm vs control, 3.9+/-0.09 µm; 10.4+/-0.3 Hz vs control, 7.3 ± 0.2 Hz; p < 0.0001) in MNSE. CONCLUSION: RGAE augments ASL depth and CBF by stimulating Cl- secretion through CaCC, which suggests therapeutic potential in both CF and non-CF chronic rhinosinusitis.

Upregulation of airway smooth muscle calcium-sensing receptor by low-molecular-weight hyaluronan.

We investigated the mechanisms involved in the development of airway hyperresponsiveness (AHR) following exposure of mice to halogens. Male mice (C57BL/6; 20-25 g) exposed to either bromine (Br2) or Cl2 (600 or 400 ppm, respectively, for 30 min) developed AHR 24 h after exposure. Nifedipine (5 mg/kg body wt; an L-type calcium channel blocker), administered subcutaneously after Br2 or Cl2 exposure, produced higher AHR compared with Br2 or Cl2 alone. In contrast, diltiazem (5 mg/kg body wt; a nondihydropyridine L-type calcium channel blocker) decreased AHR to control (air) values. Exposure of immortalized human airway smooth muscle cells (hASMC) to Br2 resulted in membrane potential depolarization (Vm Air: 62 ± 3 mV; 3 h post Br2:-45 ± 5 mV; means ± 1 SE; P < 0.001), increased intracellular [Ca2+]i, and increased expression of the calcium-sensing receptor (Ca-SR) protein. Treatment of hASMC with a siRNA against Ca-SR significantly inhibited the Br2 and nifedipine-induced Vm depolarization and [Ca2+]i increase. Intranasal administration of an antagonist to Ca-SR in mice postexposure to Br2 reversed the effects of Br2 and nifedipine on AHR. Incubation of hASMC with low-molecular-weight hyaluronan (LMW-HA), generated by exposing high-molecular-weight hyaluronan (HMW-HA) to Br2, caused Vm depolarization, [Ca2+]i increase, and Ca-SR expression to a similar extent as exposure to Br2 and Cl2. The addition of HMW-HA to cells or mice exposed to Br2, Cl2, or LMW-HA reversed these effects in vitro and improved AHR in vivo. We conclude that detrimental effects of halogen exposure on AHR are mediated via activation of the Ca-SR by LMW-HA.

Sinupret activates CFTR and TMEM16A-dependent transepithelial chloride transport and improves indicators of mucociliary clearance.

INTRODUCTION: We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl-) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid. METHODS: Primary murine nasal septal epithelial (MNSE) [wild type (WT) and transgenic CFTR(-/-)], human sinonasal epithelial (HSNE), WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl- transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium [Ca(2+)]i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis. RESULTS: Sinupret-mediated Cl- secretion [ΔISC(µA/cm(2))] was pronounced in WT MNSE (20.7+/-0.9 vs. 5.6+/-0.9(control), p<0.05), CFTR(-/-) MNSE (10.1+/-1.0 vs. 0.9+/-0.3(control), p<0.05) and HSNE (20.7+/-0.3 vs. 6.4+/-0.9(control), p<0.05). The formulation activated Ca(2+) signaling and TMEM16A channels, but also increased CFTR channel open probability (Po) without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl- secretion. Sinupret also enhanced CBF and ASL depth. CONCLUSION: Sinupret stimulates CBF, promotes transepithelial Cl- secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca(2+)-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl- secretagogue based therapies as an emerging treatment modality for common respiratory diseases of MCC including acute and chronic bronchitis and CRS.