Depolarization decreases the [Ca 2+ ] i sensitivity of myosin light‐chain kinase in arterial smooth muscle: comparison of aequorin and fura 2 [Ca 2+ ] i estimates

Histamine stimulation of swine arterial smooth muscle is associated with a high [Ca 2 +] i sensitivity for increases in myosin light‐chain phosphorylation. In contrast, KCl depolarization produces a relatively lower [Ca 2+ ] i sensitivity (i.e., similar increases in [Ca 2+ ] i induce less myosin phosphorylation). We evaluated whether 1 ) artifacts in the methodology for measuring [Ca 2+ ] i or 2 ) true alterations in the [Ca 2+ ] i sensitivity of myosin light‐chain kinase were responsible for these apparent changes in the [Ca 2+ ] i sensitivity of phosphorylation. The [Ca 2+ ] i sensitivity of phosphorylation was higher with histamine stimulation regardless of whether the [Ca 2+ ] i indicator was aequorin (which was loaded intracellularly by reversible hyperpermeabilization) or Fura 2 (which was loaded intracellularly by incubation of the tissues in Fura 2 AM). Aequorin and Fura 2 appeared to detect qualitatively similar stimulus‐induced changes in [Ca 2+ ] i with the exception that the initial response to histamine stimulation was different (histamine initially induced a large aequorin light transient and a relatively smaller increase in Fura 2 fluorescence). The [Ca 2 +] i sensitivity of myosin light‐chain kinase extracted from KCl depolarized tissues was lower than the [Ca 2+ ] i sensitivity of myosin light‐chain kinase extracted from unstimulated or histamine stimulated tissues. These results suggest that depolarization specifically modifies myosin light‐chain kinase to decrease its [Ca 2+ ] i sensitivity. Changes in the [Ca 2+ ] i sensitivity of myosin light‐chain phosphorylation are not an artifact of the [Ca 2+ ] i measurement technique.—Gilbert, E. K.; Weaver, B. A.; Rembold, C. M. Depolarization decreases the [Ca 2+ ] sensitivity of myosin light‐chain kinase in arterial smooth muscle: comparison of aequorin and Fura 2 [Ca 2+ ] estimates. FASEB J. 5: 2593‐2599; 1991.