Depolarization decreases the [Ca2+]i sensitivity of myosin light-chain kinase in arterial smooth muscle: comparison of aequorin and fura 2 [Ca2+]i estimates.
Histamine stimulation of swine arterial smooth muscle is associated with a high [Ca2+]i sensitivity for increases in myosin light-chain phosphorylation. In contrast, KCl depolarization produces a relatively lower [Ca2+]i sensitivity (i.e., similar increases in [Ca2+]i induce less myosin phosphorylation). We evaluated whether 1) artifacts in the methodology for measuring [Ca2+]i or 2) true alterations in the [Ca2+]i sensitivity of myosin light-chain kinase were responsible for these apparent changes in the [Ca2+]i sensitivity of phosphorylation. The [Ca2+]i sensitivity of phosphorylation was higher with histamine stimulation regardless of whether the [Ca2+]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 [Ca2+]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 [Ca2+]i sensitivity of myosin light-chain kinase extracted from KCl depolarized tissues was lower than the [Ca2+]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 [Ca2+]i sensitivity. Changes in the [Ca2+]i sensitivity of myosin light-chain phosphorylation are not an artifact of the [Ca2+]i measurement technique.
Gilbert
E K
EK
Department of Internal Medicine, University of Virginia Health Science Center, Charlottesville 22908.
Weaver
B A
BA
Rembold
C M
CM
D000331:Aequorin / Q000737:chemistry*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D016257:Fura-2 / Q000737:chemistry*; D006632:Histamine / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010766:Phosphorylation; D013050:Spectrometry, Fluorescence; D013552:Swine
D000331:Aequorin; D006632:Histamine; D009219:Myosin-Light-Chain Kinase; D002118:Calcium; D016257:Fura-2