title: Elevated cAMP opposes (TNF-alpha)-induced loss in the barrier integrity of corneal endothelium.
abstract: 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.
authors.affiliation: School of Optometry, Indiana University, Bloomington, IN 47405, USA.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Shivanna
authors.forename: Sangly P
authors.initials: SP
authors.lastname: Srinivas
mesh_terms: 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
chemical_list: 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
references.citation: Exp Eye Res. 2009 Dec;89(6):950-9
references.pmid: 19695246
references.citation: Exp Eye Res. 2005 Aug;81(2):183-92
references.pmid: 16080912
references.citation: Invest Ophthalmol Vis Sci. 2008 Nov;49(11):4816-27
references.pmid: 18614806
references.citation: Invest Ophthalmol Vis Sci. 2007 Apr;48(4):1518-27
references.pmid: 17389480
references.citation: Exp Eye Res. 2004 Oct;79(4):477-86
references.pmid: 15381032
references.citation: Prog Retin Eye Res. 2003 Jan;22(1):69-94
references.pmid: 12597924
references.citation: Invest Ophthalmol Vis Sci. 2005 Jan;46(1):104-13
references.pmid: 15623761
references.citation: Am J Physiol Lung Cell Mol Physiol. 2004 Jul;287(1):L86-93
references.pmid: 15003930
references.citation: J Invest Dermatol. 2010 Aug;130(8):2049-56
references.pmid: 20357815
references.citation: Br J Pharmacol. 2001 Jan;132(1):270-6
references.pmid: 11156586
references.citation: Invest Ophthalmol Vis Sci. 1996 Aug;37(9):1924-9
references.pmid: 8759364
references.citation: Curr Eye Res. 2007 Dec;32(12):1005-16
references.pmid: 18085464
references.citation: Am J Physiol Lung Cell Mol Physiol. 2004 Jan;286(1):L37-48
references.pmid: 12807699
references.citation: Exp Cell Res. 2007 Jul 1;313(11):2504-20
references.pmid: 17493609
references.citation: Exp Eye Res. 2009 Sep;89(3):333-43
references.pmid: 19345211
references.citation: Invest Ophthalmol Vis Sci. 2004 Aug;45(8):2674-81
references.pmid: 15277491
references.citation: J Cell Physiol. 2007 Oct;213(1):221-8
references.pmid: 17476691
references.citation: Am J Physiol. 1998 Jan;274(1):F1-9
references.pmid: 9458817
references.citation: Br J Ophthalmol. 2005 May;89(5):533-6
references.pmid: 15834077
references.citation: Curr Eye Res. 2009 Jun;34(6):454-65
references.pmid: 19899980
references.citation: Exp Eye Res. 2005 May;80(5):687-96
references.pmid: 15862176
references.citation: Invest Ophthalmol Vis Sci. 1998 Oct;39(11):2076-84
references.pmid: 9761286
references.citation: Semin Cell Dev Biol. 2000 Aug;11(4):315-24
references.pmid: 10966866
references.citation: Am J Physiol Lung Cell Mol Physiol. 2005 Jul;289(1):L75-84
references.pmid: 15778245
references.citation: Invest Ophthalmol Vis Sci. 1996 Jan;37(1):1-10
references.pmid: 8550312
references.citation: Optom Vis Sci. 2010 Apr;87(4):E239-54
references.pmid: 20142793
references.citation: Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7919-23
references.pmid: 1518814
references.citation: Am J Physiol. 1993 Apr;264(4 Pt 1):C894-905
references.pmid: 8476021
references.citation: Invest Ophthalmol Vis Sci. 2007 Jan;48(1):120-33
references.pmid: 17197525
references.citation: Invest Ophthalmol Vis Sci. 2010 Mar;51(3):1575-82
references.pmid: 19797215
references.citation: J Physiol. 1972 Feb;221(1):29-41
references.pmid: 4259586
references.citation: Am J Physiol Lung Cell Mol Physiol. 2004 Nov;287(5):L1017-24
references.pmid: 15246972
references.citation: Arterioscler Thromb Vasc Biol. 2008 May;28(5):899-905
references.pmid: 18292388
references.citation: Exp Eye Res. 2004 Oct;79(4):543-51
references.pmid: 15381038
references.citation: Cornea. 2000 May;19(3):263-73
references.pmid: 10832681
references.citation: CNS Drug Rev. 2001 Winter;7(4):387-98
references.pmid: 11830756
references.citation: FASEB J. 2003 Mar;17(3):407-16
references.pmid: 12631580
references.citation: Am J Physiol Lung Cell Mol Physiol. 2006 Apr;290(4):L674-L684
references.pmid: 16284212
references.citation: Invest Ophthalmol Vis Sci. 2005 Apr;46(4):1208-18
references.pmid: 15790881
references.citation: Mol Cell Biol. 2006 Feb;26(4):1223-34
references.pmid: 16449637
references.citation: Invest Ophthalmol Vis Sci. 2006 May;47(5):1754-67
references.pmid: 16638979
references.citation: Clin Exp Immunol. 2000 Oct;122(1):109-16
references.pmid: 11012626
references.citation: Trans Am Ophthalmol Soc. 2007;105:330-43
references.pmid: 18427620
references.citation: Am J Physiol Lung Cell Mol Physiol. 2005 Feb;288(2):L359-69
references.pmid: 15516490
references.citation: J Cell Physiol. 2008 Jun;215(3):715-24
references.pmid: 18064650
references.citation: Pflugers Arch. 2000 Oct;440(6):866-70
references.pmid: 11041552
references.citation: Exp Eye Res. 2006 Nov;83(5):1225-37
references.pmid: 16938292
references.citation: Am J Physiol Lung Cell Mol Physiol. 2001 Jun;280(6):L1168-78
references.pmid: 11350795
references.citation: Invest Ophthalmol Vis Sci. 2010 Apr;51(4):2139-48
references.pmid: 20019371
references.citation: Exp Eye Res. 2004 Mar;78(3):537-41
references.pmid: 15106932
references.citation: Am J Physiol. 1995 May;268(5 Pt 1):C1104-13
references.pmid: 7762602
references.citation: Am J Respir Cell Mol Biol. 2003 May;28(5):574-81
references.pmid: 12707013
references.citation: Invest Ophthalmol Vis Sci. 2006 Sep;47(9):4011-8
references.pmid: 16936117
references.citation: J Ocul Pharmacol Ther. 2010 Feb;26(1):1-10
references.pmid: 20148651
references.citation: Blood. 2005 May 1;105(9):3569-76
references.pmid: 15650061