Temporal aspects of excitation-contraction coupling in airway smooth muscle

In airway smooth muscle (ASM), ACh induces propagating intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations (5-30 Hz). We hypothesized that, in ASM, coupling of elevations and reductions in [Ca²⁺]_i to force generation and relaxation (excitation-contraction coupling) is slower than ACh-induced [Ca²⁺]_i oscillations, leading to stable force generation. When we used real-time confocal imaging, the delay between elevated [Ca²⁺]_i and contraction in intact porcine ASM cells was found to be ∼450 ms. In β-escin-permeabilized ASM strips, photolytic release of caged Ca²⁺ resulted in force generation after ∼800 ms. When calmodulin (CaM) was added, this delay was shortened to ∼500 ms. In the presence of exogenous CaM and 100 μM Ca²⁺, photolytic release of caged ATP led to force generation after ∼80 ms. These results indicated significant delays due to CaM mobilization and Ca²⁺-CaM activation of myosin light chain kinase but much shorter delays introduced by myosin light chain kinase-induced phosphorylation of the regulatory myosin light chain MLC₂₀ and cross-bridge recruitment. This was confirmed by prior thiophosphorylation of MLC₂₀, in which force generation occurred ∼50 ms after photolytic release of caged ATP, approximating the delay introduced by cross-bridge recruitment alone. The time required to reach maximum steady-state force was >15 s. Rapid chelation of [Ca²⁺]_i after photolytic release of caged diazo-2 resulted in relaxation after a delay of ∼1.2 s and 50% reduction in force after ∼57 s. We conclude that in ASM cells agonist-induced [Ca²⁺]_i oscillations are temporally and spatially integrated during excitation-contraction coupling, resulting in stable force production.