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 (V_m 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 V_m 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 V_m 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.