Excitation-Contraction Coupling in Airway Smooth Muscle

Wanglei Du,Timothy J Mcmahon,Zhu-Shan Zhang,Jonathan A Stiber,Gerhard Meissner,Jerry P Eu

doi:10.1074/jbc.m606541200

Abstract

Excitation-contraction (EC) coupling in striated muscles is mediated by the cardiac or skeletal muscle isoform of voltage-dependent L-type Ca(2+) channel (Ca(v)1.2 and Ca(v)1.1, respectively) that senses a depolarization of the cell membrane, and in response, activates its corresponding isoform of intracellular Ca(2+) release channel/ryanodine receptor (RyR) to release stored Ca(2+), thereby initiating muscle contraction. Specifically, in cardiac muscle following cell membrane depolarization, Ca(v)1.2 activates cardiac RyR (RyR2) through an influx of extracellular Ca(2+). In contrast, in skeletal muscle, Ca(v)1.1 activates skeletal muscle RyR (RyR1) through a direct physical coupling that negates the need for extracellular Ca(2+). Since airway smooth muscle (ASM) expresses Ca(v)1.2 and all three RyR isoforms, we examined whether a cardiac muscle type of EC coupling also mediates contraction in this tissue. We found that the sustained contractions of rat ASM preparations induced by depolarization with KCl were indeed partially reversed ( approximately 40%) by 200 mum ryanodine, thus indicating a functional coupling of L-type channels and RyRs in ASM. However, KCl still caused transient ASM contractions and stored Ca(2+) release in cultured ASM cells without extracellular Ca(2+). Further analyses of rat ASM indicated that this tissue expresses as many as four L-type channel isoforms, including Ca(v)1.1. Moreover, Ca(v)1.1 and RyR1 in rat ASM cells have a similar distribution near the cell membrane in rat ASM cells and thus may be directly coupled as in skeletal muscle. Collectively, our data implicate that EC-coupling mechanisms in striated muscles may also broadly transduce diverse smooth muscle functions.

Highlights

In cardiac muscle and in skeletal muscle, depolarization of the cell membrane causes voltage-dependent L-type Ca2ϩ channels localized to the cell membrane to activate the juxtaposing intracellular Ca2ϩ release channels known as ryanodine receptors (RyRs)2 [1, 2]
Since airway smooth muscle (ASM) expresses RyR1 and RyR2 (Figs. 4 and 6) [7], ASM possesses all of the Ca2ϩ channels involved in the EC coupling of striated muscles [30]
The reversal of depolarization-induced rat ASM contractions by RyR inhibition (200 ␮M ryanodine [1, 23]), as shown in Fig. 1, indicates that the depolarizing signals in the cell membrane of ASM are transmitted to the sarcoplasmic reticulum via L-type channels, thereby leading to stored Ca2ϩ release by RyRs (i.e. EC coupling in ASM)

Summary

EXPERIMENTAL PROCEDURES

Materials—The mouse monoclonal anti-Cav1.1␣1 antibody and an anti-RyR antibody that recognizes RyR2 were obtained from Affinity BioReagents Inc (Golden, CO). RT-PCR Analyses of ␣1 Subunit of L-type Channel (Cav1␣1) and RyR Isoforms in Rat ASM—After removing adventitial and epithelial tissues, airway smooth muscle layers from trachea and large bronchi of each animal were pooled, and total RNA was extracted using the Qiagen RNeasy mini kit (Qiagen Inc., Carlsbad, CA) using a method described previously [7, 26]. Immunofluorescence Study of RyR1 and Cav1.1 in Cultured Rat ASM Cells—To determine whether RyR1 and Cav1.1 are expressed in close proximity in rat ASM cells and could be physically coupled as in skeletal muscle, immunofluorescence studies using the rabbit polyclonal anti-RyR1 (1:100 dilution) and the mouse monoclonal anti-Cav1.1␣1 (1 ng/␮l) were performed on non-overlapping, elongated rat ASM cells (ϳ8 days old, third passage) using a method described previously [7]. A p value of Ͻ 0.05 was considered statistically significant

RESULTS

Nucleotide position

DISCUSSION