Abstract
Purpose: The G-protein-coupled receptor/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway is one of the most common and versatile signal pathways in eukaryotic cells. The aim of this study was to characterize subtypes of adrenergic G-protein-coupled receptors and their influence on cAMP concentration and PKA activity in bovine corneal epithelial and endothelial cells. Procedures: Adrenergic receptors and PKA were studied using polyclonal antibodies. cAMP concentration was determined with an enzyme immunoassay, and PKA activity was estimated by the kinases consumption of adenosine triphosphate. Results: In bovine corneal epithelial and endothelial cells, immunocytochemistry and Western blot were positive for α<sub>1</sub>-, α<sub>2A</sub>-, β<sub>1</sub>- and β<sub>2</sub>-adrenergic receptors. Stimulation of corneal epithelial and endothelial β-adrenoceptors with isoprenaline led to a dose-dependent increase in cAMP concentration and activation of PKA. Stimulation of corneal α<sub>2A</sub>-adrenoceptors with brimonidine resulted in a dose-dependent decrease in cAMP concentration and the inhibition of PKA activity. Conclusions: In corneal epithelial and endothelial cells, β-adrenergic stimulation leads to activation of PKA via stimulation of adenylyl cyclase, and α<sub>2A</sub>-adrenoceptor stimulation inhibits PKA activity via inhibition of adenylyl cyclase. Stimulation and inhibition of the corneal cAMP-PKA pathway may play a role in important corneal functions such as wound healing or homeostasis. Long-term therapy with α<sub>2A</sub>-agonists or β-antagonists may influence these functions in a currently unknown way.
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