Adrenergic regulation of cAMP/protein kinase A pathway in corneal epithelium and endothelium.
OBJECTIVE
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.


METHODS
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 alpha(1)-, alpha(2A)-, beta(1)- and beta(2)-adrenergic receptors. Stimulation of corneal epithelial and endothelial beta-adrenoceptors with isoprenaline led to a dose-dependent increase in cAMP concentration and activation of PKA. Stimulation of corneal alpha(2A)-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, beta-adrenergic stimulation leads to activation of PKA via stimulation of adenylyl cyclase, and alpha(2A)-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 alpha(2A)-agonists or beta-antagonists may influence these functions in a currently unknown way.
Grueb
Matthias
M
University of Tubingen, Department of Ophthalmology, Tubingen, Germany. matthias_grueb@yahoo.de
Bartz-Schmidt
Karl Ulrich
KU
Rohrbach
Jens Martin
JM
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D000068438:Brimonidine Tartrate; D002417:Cattle; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004728:Endothelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D011810:Quinoxalines / Q000494:pharmacology; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*
D000316:Adrenergic alpha-Agonists; D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D011810:Quinoxalines; D018341:Receptors, Adrenergic, alpha-2; D011943:Receptors, Adrenergic, beta; D000068438:Brimonidine Tartrate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases