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

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.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.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.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

Sinupret raises cytosolic Ca²⁺ concentrations in human sinonasal epithelium.

<p>Representative tracings of Fura-2 Ca²⁺ imaging (A). Stimulation of intracellular Ca²⁺ was demonstrated following the addition of Sinupret to HSNE cells. UTP (positive control) also led to increased intracellular Ca²⁺.</p

Activation of TMEM16A channels expressed in HEK-293 cells with Sinupret.

<p>Control whole cell current was recorded in the presence of DMSO vehicle. DMSO was without any effect on TMEM16A expressed at the surface of HEK cells (A). Perfusion with the solution containing 2.5 mg/ml (B). Sinupret induced the activation of a Cl⁻ current that was inhibited by 200 µM tannic acid (<b>C</b>). IV curves are illustrated during the activation of the Cl⁻ current with Sinupret and inhibition by tannic acid when voltage steps were varied from −80 mV to 100 mV in 20 mV increments for 500 ms (D). The cell membrane potential was held at 0 mV throughout the experiment. Control current recorded before cells were treated with UTP - a known activator of TMEM16A channels (E). TMEM16A response to 200 µM UTP in the perfusing solution (F). Inhibition of the UTP activated current with 200 µM tannic acid (G). IV curves (H) showing the activation of the current at a range of voltage stimulation as in (D).</p

Sinupret is a robust activator of CFTR-mediated and calcium-activated Cl^- secretion.

<p>Representative Ussing chamber tracings demonstrate pharmacologic manipulation of ion transport with activation of Cl⁻ secretion (Agonist = Sinupret or DMSO control vehicle) in murine nasal septal epithelial (MNSE, wild type and CFTR^−/−) and human sinonasal epithelial (HSNE) cell cultures following blockade of epithelial sodium channels by amiloride. Strong inhibition of short-circuit current (I_SC) by the specific CFTR inhibitor INH-172 indicates activation of CFTR-mediated pathways in WT MNSE (A) and HSNE (B). CFTR^−/− MNSE tested with forskolin confirm absence of cAMP-dependent CFTR-dependent transport, but retained strong stimulation of anion secretion by Sinupret (C). Summary data for Sinupret-stimulated ΔI_SC in WT MNSE, CFTR^−/− MNSE and HSNE (D). Sinupret significantly increased ΔI_SC (in µA/cm²)when compared to controls in both WT (20.7+/−0.9 vs. 5.6+/−0.9, p<0.05) and CFTR^−/− MNSE (ΔI_SC in µA/cm², 10.1+/−1.0 vs. 0.9+/−0.3, p<0.05). HSNE cultures also exhibited robust Cl⁻ secretion when compared to controls (ΔI_SC, 20.7+/−0.3 vs. 6.4+/−0.9, p<0.05). There were significant decreases in I_SC when cultures were treated with CFTR inhibitor INH-172 in WT MNSE (−16.1+/−1.3 vs. −6.5+/−0.7, p<0.05) and HSNE cells (−17.0+/−1.6 vs. −11.1+/−1.7, p<0.05).</p

Stimulation of CBF.

<p>CBF (fold-change/baseline) was significantly increased when Sinupret was compared to control (vehicle) following apical (2.05+/−0.15 vs. 1.52+/−0.10), basal (1.37+/−0.09 vs. 0.99+/−0.04), or apical + basolateral exposures (2.17+/−0.12 vs. 1.53+/−0.09). (*p<0.05).</p

Activation of chloride secretion occurs through a mechanism independent of PKA and R-Domain phosphorylation of CFTR.

<p>MNSE cells were exposed to forskolin (20 µM, positive control, P<0.05), vehicle (DMSO), or graded concentrations of Sinupret for 10 minutes prior to assay. Sinupret did not induce a mean increase in cAMP compared to vehicle control, while forskolin led to a marked elevation of cAMP (A). Sinupret does not induce R-domain phosphorylation (B). The recombinant CFTR R-Domain was expressed in stably transfected NIH-3T3 cells and detected by immunoblotting with an antibody to the HA tag (black arrow). A 2–4 kD mobility shift is indicative of R-Domain phosphorylation (white arrow) following 5 minute treatment with forskolin (20 µM, positive control). Sinupret had no detectable effect on R- domain phosphorylation at a maximum concentration of 2.5 mg/ml.</p

Elimination of TMEM16A-mediated I_SC with tannic acid suggests Sinupret stimulates CFTR-mediated Cl⁻ transport.

<p>Representative Ussing chamber tracings demonstrate administration of the TMEM16A blocker tannic acid abolishes agonist (Sinupret)-stimulated Cl⁻ secretion in CFTR^−/− cells (A). Pre-treatment of WT MNSE shows persistent activation despite elimination of TMEM16A contributions to the I_SC (B). Summary data (C) for Sinupret-stimulated ΔI_SC in WT MNSE [µA/cm², 12.8+/−2.2 vs. 3.6+/−0.9 (control), p<0.05] following tannic acid block of TMEM16A-mediated I_SC) and CFTR blockade with INH-172 (−11.5+/−1.7 vs. −9.0+/−1.5, p<0.05).</p