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K.Y. Allen, X.C. Sun, J.A. Bonanno; P1 Purinergic Receptors in Bovine Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4724.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the subtype expression of adenosine receptors (ARs) in native and cultured bovine corneal endothelial cells (CBCE). The A1 and A3 adenosine receptors decrease [cAMP]i while the A2a and A2b increase [cAMP]i. Methods: We screened for A1, A2a, A2b and A3 adenosine receptors using RT-PCR. [cAMP]i was measured by Elisa. Western Blot (WB) and immunofluorescence(IF) using specific A2b antibody were also performed. Results: Our RT-PCR results showed the presence of A1 and A2a PCR products. A2a PCR products showed a correct bandsize of 795 bp for CBCE and positive controls (bovine cerebellum and cortex). The expected 556 bp A1 PCR bands were obtained for CBCE using bovine brain as positive control. A2b and A3 screening were negative. Previous pharmacological studies suggested A2b expression, so further screening was done by WB and IF. Confocal microscopy of A2b immunostained with A2b specific antibody on cultured cells showed predominant apical protein expression. Our preliminary Western Blot for A2b from NHS-sulfo biotin membrane preparation showed a single 50 kDa band from fresh bovine corneal endothelial cells. cAMP assays with 10 µM adenosine yielded a three to five-fold range increase in [cAMP]i over control samples in the presence of rolipram, a phosphodiesterase inhibitor, and AMPCP, a 5' nucleotidase inhibitor. The same Elisa assay done in the absence of rolipram showed an approximate two-fold [cAMP]i increase. The general A2 antagonist, DMPX, totally inhibited the effect of adenosine on [cAMP]i. Previous studies done on CBCE showed that adenosine or forskolin caused membrane depolarization, consistent with the presence of A2 receptors of either the A2a or A2b subtype. Conclusion: The above preliminary results support the presence of A1, A2a, and A2b ARs in bovine corneal endothelial cells. Our A2b immunofluorescence and Western blot results do indicate a need for new A2b primer designs for its RT-PCR detection. The large increase in [cAMP]i suggests that A2 receptor activation predominates.
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