Calcium does not act as a second messenger for adrenergic and cholinergic agonists in corneal epithelial cells

The role of changes in intracellular [Ca2+]i as a second messenger in response to either adrenergic or cholinergic agonists was determined in isolated bovine corneal epithelial cells. [Ca2+]i was measured in suspensions of cells loaded with either of the fluorescent indicators quin2 or indo-1, as well as in single cells loaded with fura-2. Fluorescence from the cell suspensions was measured in a spectrofluorometer while single cell fluorescence was measured using a modified fluorescence microscope with a photon counting photometer. Cells were loaded with these dyes by incubation in Ringer's (pH 8.1) containing 2-50 microM of the acetoxymethyl ester of the indicator. Fluorescence was measured before and after exposure to either, one of the adrenergic agonists isoproterenol, phenylephrine or epinephrine, or the cholinergic agonist carbachol. The resting [Ca2+]i level from the quin2 experiments was 115 nM +/- 41 nM (SEM) (n = 23) whereas with fura-2 it was 71 +/- 10 nM (n = 30). In no case did we see any change in [Ca2+]i within 15 min after addition of any agonist but we were able to observe increased calcium when 0.5 microM ionomycin was added to either the same or untreated cells. The disparity in the resting levels determined by the two methods may result from various calibration problems. Our results indicate that changes in [Ca2+]i have no second messenger role in response to these agonists.