Focal increases in [Ca]i may account for apparent low Ca 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 in increases in [Ca²⁺]_i could impair such measures of [Ca²⁺]_i sensitivity. Stimulation of swine carotid artery with 10 μM 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 μM 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 uncharged 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.