Tachyphylaxis to the inhibitory effect of L-type channel blockers on ACh-induced [Ca2+]i oscillations in porcine tracheal myocytes

Summary Discrepancies about the role of L-type voltage-gated calcium channels (VGCC) in acetylcholine (ACh)-induced [Ca²⁺]_i oscillations in tracheal smooth muscle cells (TSMCs) have been seen in recent reports. We demonstrate here that ACh-induced [Ca²⁺]_i oscillations in TMCS were reversibly inhibited by three VGCC blockers, nicardipine, nifedipine and verapamil. Prolonged (several minutes) application of VGCC blockers, led to tachyphylaxis; that is, [Ca²⁺]_i oscillations resumed, but at a lower frequency. Brief (15–30 s) removal of VGCC blockers re-sensitized [Ca²⁺]_i oscillations to inhibition by the agents. Calcium oscillations tolerant to VGCC blockers were abolished by KB-R7943, an inhibitor of the reverse mode of Na⁺/Ca²⁺ exchanger (NCX). KB-R7943 alone also abolished ACh-induced [Ca²⁺]_i oscillations. Enhancement of the reverse mode of NCX via removing extracellular Na⁺ reversed inhibition of ACh-induced [Ca²⁺]_i oscillations by VGCC blockers. Inhibition of non-selective cation channels using Gd³⁺ slightly reduced the frequency of ACh-induced [Ca²⁺]_i oscillations, but did not prevent the occurrence of tachyphylaxis. Altogether, these results suggest that VGCC and the reverse mode of NCX are two primary Ca²⁺ entry pathways for maintaining ACh-induced [Ca²⁺]_i oscillations in TSMCs. The two pathways complement each other, and may account for tachyphylaxis of ACh-induced [Ca²⁺]_i oscillations to VGCC blockers.