Effects of volatile anesthetics on cytoplasmic Ca2+ signaling and transmitter release in a neural cell line

To provide new insights into the effects of volatile agents on the basic regulatory events involved in cytoplasmic free Ca2+ ([Ca2+]i) and stimulus-secretion coupling, the well-characterized clonal rat pheochromocytoma cell line PC12 was chosen as an experimental model. This cell line possesses nicotinic and muscarinic receptors, L-type voltage-operated channels (VOCs), and receptor-operated Ca2+ channels (ROCs). A PC12 variant, defective in nicotinic response, made it possible to study the influx-independent inositol trisphosphate-mediated intracellular Ca2+ release that is triggered by muscarinic receptor stimulation. [Ca2+]i was measured with the fluorescent Ca2+ indicator fura-2. Dopamine and norepinephrine secretion were determined by high-performance liquid chromatography. High K+ and nicotinic-receptor-induced [Ca2+]i increase and catecholamine secretion were inhibited by halothane, enflurane, isoflurane, and methoxyflurane in a dose-dependent manner; half-maximal inhibition (IC50) occurred within the clinically relevant concentration range. The inhibition was reversible after wash-out of anesthetic; was not restricted to dihydropyridine-sensitive L-type VOCs; and could not be overcome by increasing extracellular Ca2+. The inhibitory mechanisms of volatile anesthetics therefore differed from those of classical organic Ca2(+)-channel blockers, a difference also reflected by the differing Hill coefficients found for both substance groups. In contrast, the muscarinic-receptor-evoked internal Ca2+ release remained unimpaired, and secretion even increased under anesthetic exposure. In conclusion, the current study provides evidence that volatile anesthetics depress the Ca2+ influx through at least two independent Ca2+ channels, one of which proved insensitive to the dihydropyridine Ca2(+)-channel blocker nifedipine. This is particularly noteworthy, since dihydropyridine-insensitive N-type VOCs, so far found exclusively in neurons, are assumed to play a dominant role in synaptic transmission, which, although resistant to dihydropyridine inhibition, is effectively blocked by volatile anesthetics.