Focal increases in [Ca2+]i may account for apparent low Ca2+ sensitivity in swine carotid artery.
Estimates of [Ca2+]i sensitivity in intact smooth muscle are frequently obtained by measuring [Ca2+]i with indicators such as aequorin or Fura-2. We investigated whether focal increases in [Ca2+]i could impair such measures of [Ca2+]i sensitivity. Stimulation of swine carotid artery with 10 microM histamine increased aequorin estimated [Ca2+]i, Fura-2 estimated [Ca2+]i and Ca2+ sensitivity without significantly altering the aequorin/Fura-2 ratio (an estimate of [Ca2+]i homogeneity). Subsequent inhibition of Na+/Ca2+ exchange by replacement of Na+ in the PSS with choline+ significantly increased aequorin-estimated [Ca2+]i but only minimally increased Fura-2 estimated [Ca2+]i, myosin light chain (MLC) phosphorylation and force. This resulted in a large increase in the aequorin/Fura-2 ratio, suggesting an increase in [Ca2+] inhomogeneity. Addition of 100 microM histamine to tissues in the choline+ buffer initially increased both aequorin and Fura-2 estimated [Ca2+]i, but after 10 min exposure both of the [Ca2+]i estimates declined to pre-histamine levels. Histamine addition significantly increased MLC phosphorylation and force, indicating increased Ca2+ sensitivity, but the aequorin/Fura-2 ratio remained elevated and unchanged from pre-histamine values. These data show that under certain conditions, aequorin and Fura-2 can yield widely differing estimates of [Ca2+]i and thus can cause misleading assessments of Ca2+ sensitization mechanisms. These discrepancies may arise from inhomogeneous or focal increases in [Ca2+]i which can be evaluated with the aequorin/Fura-2 ratio.
Van Riper
D A
DA
Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Chen
X L
XL
Gould
E M
EM
Rembold
C M
CM
D000331:Aequorin / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D002339:Carotid Arteries / Q000378:metabolism; D002794:Choline / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D016257:Fura-2 / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D008163:Luminescent Measurements; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D012680:Sensitivity and Specificity; D013268:Stimulation, Chemical; D013552:Swine
D005456:Fluorescent Dyes; D018994:Myosin Light Chains; D000331:Aequorin; D006632:Histamine; D002794:Choline; D002118:Calcium; D016257:Fura-2