Aequorin luminescence, myosin phosphorylation, and active stress in tracheal smooth muscle

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Abstract
During muscarinic activation of canine tracheal smooth muscle with carbachol, myosin phosphorylation is significantly more sensitive than stress to the external Ca2+ concentration ([Ca2+]o) [W. T. Gerthoffer. Am. J. Physiol. 250 (Cell Physiol. 19): C597-C604, 1986]. To determine whether the intracellular Ca2+ concentration ([Ca2+]i) correlated more closely with changes in phosphorylation or force, we measured isometric force and light emitted by the luminescent intracellular Ca2+ indicator aequorin as [Ca2+]o was increased in the presence of 1 microM carbachol or 60 mM K+. Myosin phosphorylation was measured using an immunoblot assay in a second set of muscle strips treated identically. Stimulation with carbachol increased aequorin luminescence slightly in strips incubated in Ca2+-free solution. Active stress and aequorin luminescence subsequently increased in parallel as [Ca2+]o was increased. Myosin phosphorylation at 0.05 mM [Ca2+]o (0.30 +/- 0.04 mol Pi/mol light chain) was significantly higher than phosphorylation in Ca2+-free solution with no carbachol (0.12 +/- 0.048 mol Pi/mol light chain) and increased to a maximum of 0.56 +/- 0.03 mol Pi/mol light chain at 1.6 mM [Ca2+]o. In contrast, active stress and aequorin luminescence remained low at low [Ca2+]o and reached a maximum at 2.4 mM [Ca2+]o. Stimulation with carbachol produced greater increases in myosin phosphorylation and active stress for a given change in aequorin luminescence than did K+ depolarization. Stimulation with carbachol also produced a different phosphorylation-stress relationship than did K+ depolarization. These observations are consistent with the possibility that carbachol induces increases in the Ca2+ sensitivity of contractile proteins in tracheal smooth muscle.