Elevated cAMP opposes (TNF-alpha)-induced loss in the barrier integrity of corneal endothelium.
OBJECTIVE
Elevated cyclic adenosine monophosphate (cAMP) enhances the barrier integrity of the corneal endothelium and thereby facilitates stromal hydration control, which is necessary for corneal transparency. This study investigates whether elevated cAMP is effective against the tumor necrosis factor-alpha (TNF-alpha)-induced loss of barrier integrity in monolayers of bovine corneal endothelial cells (BCEC).


METHODS
BCEC in primary culture were used for the study. Trans-endothelial electrical resistance (TER), a measure of barrier integrity, was determined by electrical cell-substrate impedance sensing. The changes were also ascertained by measuring paracellular permeability to fluorescein isothiocyanate (FITC)-dextran (10 kDa) across cells grown on porous culture inserts, and by immunofluorescence imaging of the apical junctional complex (AJC). The activation of p38 MAP kinase was assessed using western blotting.


RESULTS
Co-treatment with forskolin, which activates adenylate cyclase, and rolipram, which inhibits cAMP-dependent phosphodiesterase PDE4, reduced the TNF-alpha-induced increase in the flux of FITC-dextran. Similar co-treatment also prevented the TNF-alpha-induced disorganization of zona occludens-1 (ZO-1) and cadherins at the AJC. Co-treatment, as well pre-treatment, with forskolin plus rolipram prevented the TNF-alpha-induced decrease in TER. The influence of the agents was significant after 12 h of exposure to the cytokine. This effect was also mimicked by A2B agonists, adenosine and 5'-N-ethylcarboxamidoadenosine (NECA), which are known to mobilize cAMP in BCEC. Elevated cAMP also inhibited the cytokine-induced activation of p38 MAP kinase, and further blocked the disassembly of microtubules as well as the disruption of the PAMR (peri-junctional actomyosin ring) at the AJC.


CONCLUSIONS
These results suggest that elevated cAMP opposes the TNF-alpha-induced loss in barrier integrity of the corneal endothelium. This effect follows inhibition of the cytokine-induced activation of p38 MAP kinase and its downstream signaling involved in the disruption of AJC and PAMR, as well as the disassembly of microtubules.
Shivanna
Mahesh
M
School of Optometry, Indiana University, Bloomington, IN 47405, USA.
Srinivas
Sangly P
SP
D000205:Actomyosin / Q000378:metabolism; D000241:Adenosine / Q000494:pharmacology; D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology; D000818:Animals; D001813:Blood-Retinal Barrier / Q000187:drug effects* / Q000473:pathology*; D015820:Cadherins / Q000378:metabolism; D002417:Cattle; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D017097:Electric Impedance; D004728:Endothelium, Corneal / Q000187:drug effects* / Q000473:pathology*; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D007365:Intercellular Junctions / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D010750:Phosphoproteins / Q000378:metabolism; D020889:Rolipram / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D062826:Zonula Occludens-1 Protein; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D015820:Cadherins; D008565:Membrane Proteins; D010750:Phosphoproteins; D014409:Tumor Necrosis Factor-alpha; D062826:Zonula Occludens-1 Protein; D005576:Colforsin; D019830:Adenosine-5'-(N-ethylcarboxamide); D000205:Actomyosin; D000242:Cyclic AMP; D048051:p38 Mitogen-Activated Protein Kinases; D020889:Rolipram; D000241:Adenosine
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