Serotonin (5-HT7) receptor-stimulated activation of cAMP-PKA pathway in bovine corneal epithelial and endothelial cells.
BACKGROUND
5-Hydroxytryptamine (5-HT; serotonin) is a major neurotransmitter, and its receptors are found throughout the whole body. The 5-HT7 receptor subtype was detected in human corneal epithelial and endothelial cells and found to be functionally active in a corneal epithelial cell line. The aim of the present study was to demonstrate that native bovine corneal epithelial and endothelial cells express a functional 5-HT7 receptor positively coupled to adenylyl cyclase and protein kinase A (PKA) formation.


METHODS
5-HT7 receptors were studied using polyclonal antibodies. cAMP concentration after 5-HT7 receptor stimulation with 5-carboxamidotryptamine (5-CT, a 5-HT7 agonist) was tested by enzyme immunoassay, PKA activity was estimated by kinase consumption of ATP.


RESULTS
Immunocytochemistry and immunofluorescence revealed the presence of 5-HT7 receptors in corneal epithelial and endothelial cells. Stimulation of corneal 5-HT7 receptors with 5-CT revealed a dose-dependent increase in intracellular cAMP concentration in corneal epithelium (0.01-0.34 pmol/ml) and endothelium (0.01-0.19 pmol/ml) between 10(-10) and 10(-7) mg/ml 5-CT (p = 0.001) with maximal stimulation from 10(-7) to 10(-3) mg/ml 5-CT (0.30 ± 0.03 and 0.18 ± 0.01 pmol/ml, respectively). Incubation with 10(-6) mg/ml SB269970 (a selective 5-HT7 antagonist) blocked 5-CT-induced cAMP increase in corneal epithelial (0.03 pmol/ml) and endothelial cells (0.02 pmol/ml; p = 0.001). Stimulation of corneal 5-HT7 receptors with 5-CT revealed a dose-dependent increase in PKA activity between 10(-10) and 10(-8) mg/ml 5-CT in corneal epithelium and endothelium (<1 to >99%; p = 0.013 and p = 0.017, respectively) with maximal stimulation from 10(-8) to 10(-4) mg/ml (>99%) 5-CT.


CONCLUSIONS
Our data demonstrate that native corneal epithelial and endothelial cells express a functional 5-HT7 receptor positively coupled to adenylyl cyclase and PKA formation. However, at the present time, the physiological role of 5-HT receptors and the cAMP-PKA pathway in the cornea remains a matter of speculation.
Department of Ophthalmology, University of Tübingen, Tübingen, Germany. matthias.grueb@web.de
Matthias
M
Grueb
Jens Martin
JM
Rohrbach
Torsten
T
Schlote
Joerg
J
Mielke
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004728:Endothelium, Corneal / Q000201:enzymology*; D004789:Enzyme Activation; D019573:Epithelium, Corneal / Q000201:enzymology*; D019084:Fluorescent Antibody Technique, Indirect; D007150:Immunohistochemistry; D010636:Phenols / Q000494:pharmacology; D011985:Receptors, Serotonin / Q000378:metabolism*; D012701:Serotonin / Q000031:analogs & derivatives / Q000494:pharmacology; D012702:Serotonin Antagonists / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology
D010636:Phenols; D011985:Receptors, Serotonin; C404922:SB 269970; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D013449:Sulfonamides; C104425:serotonin 7 receptor; D012701:Serotonin; C047087:5-carboxamidotryptamine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases