Myosin phosphorylation and calcium in tonic and phasic contractions of colonic smooth muscle.

The time dependence of lightly loaded shortening velocity, myosin phosphorylation, and changes in myoplasmic Ca2+ concentration ([Ca2+]i) were measured during tonic and phasic contractions of circular smooth muscle from the proximal colon of the dog. Shortening velocity was measured by quick release to a 10% afterload. Myosin phosphorylation was measured by an immunoblot method, and changes in [Ca2+]i were estimated by measuring fluorescence intensity at 550 nm in muscle strips loaded with fluo-3. During tonic contractions induced by 60 mM K+, phosphorylation increased monotonically from 0.11 +/- 0.011 to 0.29 +/- 0.015 mol Pi/mol light chain at 10 min. In contrast, lightly loaded shortening velocity increased rapidly within 10 s to 0.042 +/- 0.003 lengths/s and decreased exponentially to 0.013 +/- 0.001 lengths/s at 15 min. During transient contractions induced by 100 microM acetylcholine, phosphorylation increased from 0.16 +/- 0.03 to 0.30 +/- 0.06 mol Pi/mol light chain at 19 s. In contrast, shortening velocity increased to 0.068 +/- 0.015 lengths/s within 2.4 s and decreased significantly to 0.027 +/- 0.009 lengths/s at 22 s. Fluo-3 fluorescence increased in parallel with force during both tonic and transient contractions. In a smooth muscle that is able to contract both tonically and phasically we observed transient increases in shortening velocity without concurrent phosphorylation or [Ca2+]i transients. Therefore, there are factors in addition to myosin phosphorylation or changes in [Ca2+]i that regulate cross-bridge cycling rates in both tonic and phasic contractions.

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

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.

Inhibition of tracheal smooth muscle contraction and myosin phosphorylation by ryanodine.

Previous studies have shown that muscarinic activation of airway smooth muscle in low Ca++ solutions increases myosin phosphorylation without increasing tension. Blocking Ca++ influx reduced phosphorylation, but not to basal levels. It was proposed that release of intracellular Ca++ contributed to dissociation of phosphorylation and contraction. To test this hypothesis the effects of ryanodine were studied under similar conditions. Ryanodine (10(-7) to 10(-5) M) antagonized caffeine-induced contraction of canine tracheal smooth muscle. Ryanodine also reduced carbachol-induced contractions and carbachol-induced myosin phosphorylation. The effect of ryanodine on potassium and serotonin-induced contractions was also investigated to test for a nonspecific inhibitory effect. In contrast to the effect on carbachol responses, ryanodine (10(-5) M) potentiated the contractile response to low concentrations of serotonin and potassium, but had no effect on the maximum response to either stimulant. Carbachol (10(-6) M) and ryanodine (10(-5) M) both significantly decreased 45Ca++ content of tracheal muscle. The effect of ryanodine and carbachol together on 45Ca++ content was not greater than either drug alone suggesting that ryanodine reduces the caffeine and carbachol responses by depleting releaseable Ca++ stores. Ryanodine significantly reduced Ca++-induced contraction and myosin phosphorylation in carbachol-stimulated muscle, suggesting that some of the Ca++ responsible for elevated phosphorylation is released from the sarcoplasmic reticulum.