Isoproterenol increases peripheral [Ca2+]i and decreases inner [Ca2+]i in single airway smooth muscle cells

Abstract

The effects of isoproterenol (Iso) or photolysis of caged adenosine 3',5'-cyclic monophosphate (cAMP) on intracellular Ca2+ concentration ([Ca2+]i) were studied in single airway smooth muscle cells. Changes in [Ca2+]i were measured ratiometrically. In cells loaded with 10 microM fura 2-acetoxymethyl ester (AM), superfusion of Iso (10 microM) increased [Ca2+]i in 20 of 22 cells from 153.2 +/- 21.3 to 252.8 +/- 38.3 nM, and this increase depended on extracellular Ca2+ and was blocked by ryanodine (50 microM). Photolysis of intracellular caged cAMP increased [Ca2+]i by 104.0 +/- 17.3 nM in 20 cells and decreased [Ca2+]i by 49.3 +/- 9.4 nM in 10 cells. With modified confocal microscopy, peripheral [Ca2+]i was increased and inner cytosolic [Ca2+]i was decreased during stimulation with Iso. Iso-induced decreases in [Ca2+]i were also observed with conventional optics in cells loaded with 0.5 microM fura 2-AM. However, in these same cells, Iso increased [Ca2+]i in the presence of low concentrations of ryanodine (1-20 microM). We concluded that Iso decreased [Ca2+]i in the inner cytosol but increased [Ca2+]i in the peripheral cytosol by mechanisms that depended on both extracellular and sarcoplasmic reticulum Ca2+ release channels. Our study suggests that fluorescence from a peripheral cytosol with high [Ca2+]i can sometimes confound the measurement of “cytosolic” [Ca2+]i with conventional optics.