Inhibition by calcium of beta adrenoceptor mediated cAMP responses in isolated bovine corneal epithelial cells

Abstract

The increases in adenosine 3′,5′ monophosphate (cAMP) content were measured in isolated bovine corneal epithelial cells in response to either adrenergic agonists or adenylate cyclase stimulation. The beta selective adrenergic agonist, isoproterenol, and the adrenergic agonists, norepinephrine as well as epinephrine elicited large increases in cAMP accumulation. At their maximum effective concentrations, the respective increases were 16-fold, 6.6-fold and 4.7-fold. These stimulatory effects were completely inhibited by the beta selective adrenergic antagonist, propranolol. Similarly forskolin increased cAMP content more than 3-fold. These increases and the previous identification of β adrenoceptors in fresh intact bovine corneas as well as cells in culture indicate that this enzymatic dissociation procedure does not affect the cAMP responses to either adrenergic agonists or forskolin. The relationship was considered between increases in Ca²⁺ concentration and the effects of either isoproterenol or forskolin, on cAMP accumulation. There were no changes in any of the cAMP responses at bathing solution Ca²⁺ concentrations between 0.01 μM and 1 μM. However, in cells permeabilized to Ca²⁺ with 10 μM ionomycin, increases within this concentration range depressed the baseline levels of cAMP content. Furthermore, the stimulatory effects of both forskolin and isoproterenol on cAMP accumulation were significantly blunted in this concentration range. These blunting effects by Ca²⁺ were not the result of any measurable decrease in ATP content. This negative relationship between increases in Ca²⁺ concentration and increases in cAMP content indicates that changes in intracellular Ca²⁺ concentration could modulate the second messenger function of cAMP linked to these agents.