A fixed 20:1 combination of cafedrine/theodrenaline increases cytosolic Ca(2+) concentration in human tracheal epithelial cells via ryanodine receptor-mediated Ca(2+) release

Mucociliary clearance is a pivotal physiological mechanism that protects the lung by cleaning the airways from pollution and colonization, thereby preventing infection. Ciliary function is influenced by various signal transduction cascades, and Ca(2+) represents a key second messenger. A fixed 20:1 combination of cafedrine and theodrenaline has been widely used to treat perioperative hypotension and emergency hypotensive states since the 1960s; however, its effect on the intracellular Ca(2+) concentration ([Ca(2+)](i)) of respiratory epithelium remains unknown. Therefore, human tracheal epithelial cells were exposed to the clinically applied 20:1 mixture of cafedrine/theodrenaline and the individual substances separately. [Ca(2+)](i) was assessed by FURA-2 340/380 fluorescence ratio. Pharmacological inhibitors were applied to elucidate relevant signal transduction cascades, and reverse transcription polymerase chain reaction (RT-PCR) was performed on murine tracheal epithelium to analyze ryanodine receptor (RyR) subtype expression. All three pharmacological preparations instantaneously induced a steep increase in [Ca(2+)](i) that quickly returned to its baseline value despite the persistence of each substance. Peak [Ca(2+)](i) following the administration of 20:1 cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone increased in a dose-dependent manner, with median effective concentrations of 0.35 mM (7.32 mM cafedrine and 0.35 mM theodrenaline), 3.14 mM, and 3.45 mM, respectively. When extracellular Ca(2+) influx was inhibited using a Ca(2+)-free buffer solution, the peak [Ca(2+)](i) following the administration of cafedrine alone and theodrenaline alone were reduced but not abolished. No alteration in [Ca(2+)](i) compared with baseline [Ca(2+)](i) was observed during β-adrenergic receptor inhibition. Depletion of caffeine-sensitive stores and inhibition of RyR, but not IP(3) receptors, completely abolished any increase in [Ca(2+)](i). However, [Ca(2+)](i) still increased following the depletion of mitochondrial Ca(2+) stores using 2,4-dinitrophenol. RT-PCR revealed RyR-2 and RyR-3 expression on murine tracheal epithelium. Although our experiments showed that cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone release Ca(2+) from intracellular stores through mechanisms that are exclusively triggered by β-adrenergic receptor stimulation, which most probably lead to RyR activation, clinical plasma concentrations are considerably lower than those used in our experiments to elicit an increase in [Ca(2+)](i); therefore, further studies are needed to evaluate the ability of cafedrine/theodrenaline to alter mucociliary clearance in clinical practice.