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 (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.