Sodium cyanide increases cytosolic free calcium: Evidence for activation of the reversed mode of the Na⁺/Ca²⁺ exchanger and Ca²⁺ mobilization from inositol trisphosphate-insensitive pools

This study characterized the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in NaCN-treated human A-431 cells. The resting [Ca²⁺]_i was 85 ± 8 nM (n = 141) in untreated cells at 37°C, determined with the fura-2 fluorescence probe, When cells were treated with NaCN, [Ca²⁺]_i increased in a time- and NaCN concentration-dependent manner. When cells were exposed to 10 mM NaCN for 10 min, [Ca²⁺]_i increased 278 ± 28% (n = 5) but returned to normal within 45 min after treatment. The [Ca²⁺]_i increase depended on the presence of external Ca²⁺. La³⁺ and Cd²⁺, but not verapamil or nifedipine, inhibited the NaCN-induced [Ca²⁺]_i increase. The NaCN-induced [Ca²⁺]_i increase also depended on external Na⁺ (K_{1 2} = 85 mM). The intracellular Na⁺ concentration, measured with the fluorescence probe SBFI, increased 267 ± 16% after NaCN treatment. The NaCN-induced [Ca²⁺]_i increase was modulated by treatment with ouabain or veratridine and was completely blocked by tetrodotoxin, amiloride (K_{1 2} = 5.4 μM), and dichlorobenzamil (K_{1 2} = 0.28 μM). These results suggest NaCN activates the Na⁺/Ca²⁺ exchange system. TMB-8 and ryanodine both partially blocked the increase in [Ca²⁺]_i in the presence of external Ca²⁺, indicating that Ca²⁺ release from intracellular pools also occurred after the initial Ca²⁺ influx. NaCN decreased inositol trisphosphates production. U-73122, bradykinin, or monensin did not prevent the NaCN-induced increase in [Ca²⁺]_i. However, the magnitude of the [Ca²⁺]_i increase caused by NaCN was abolished in ionomycin-treated cells, indicating that intracellular Ca²⁺ release induced by NaCN is derived from an ionomycin-sensitive Ca²⁺ pool. The results suggest that NaCN initially increased Na⁺ influx, which activated the reverse mode of a Na⁺/Ca²⁺ exchanger, leading to an increase in Ca²⁺ influx. The Ca²⁺ influx induced a Ca²⁺ mobilization from only an ionomycin-sensitive intracellular Ca²⁺ pool containing ryanodine receptors.