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Characterization of adenosine receptors in bovine corneal endothelium.

ABSTRACT: Previous studies indicated that adenosine can increase [cAMP](i) and stimulate fluid transport by corneal endothelium. The purpose of this study was to determine which adenosine receptor subtype(s) are expressed and to examine their functional roles in modulating [cAMP](i), [Ca(2+)](i) and effects on Cl(-) permeability in corneal endothelium. We screened bovine corneal endothelium (BCE) for adenosine receptor subtypes by RT-PCR and immunoblotting, and examined the effects of pharmacological agents on adenosine stimulated Cl(-) transport, [cAMP](i) and [Ca(2+)](i). RT-PCR indicated the presence of A(1) and A(2b) adenosine receptors, while A(2a) and A(3) were negative. Western blot (WB) confirmed the presence of A(2b) ( approximately 50 kDa) and A(1) ( approximately 40 kDa) in fresh and cultured BCE. Ten micromolar adenosine increased [cAMP](i) by 2.7-fold over control and this was inhibited 66% by 10 microm alloxazine, a specific A(2b) blocker. A(1) activation with 1 micromN(6)-CPA (a specific A(1) agonist) or 100 nm adenosine decreased [cAMP](i) by 23 and 6%, respectively. Adenosine had no effect on [Ca(2+)](i) mobilization. Indirect immunofluorescence localized A(2b) receptors to the lateral membrane and A(1) to the apical surface in cultured BCE. Adenosine significantly increased apical Cl(-) permeability by 2.2 times and this effect was nearly abolished by DMPX (10 microm), a general A(2) blocker. Adenosine-induced membrane depolarization was also inhibited by 33% (n=6) in the presence of alloxazine. Bovine corneal endothelium expresses functional A(1) and A(2b) adenosine receptors. A(1), preferentially activated at <1 microm adenosine, acts to decrease [cAMP](i) and A(2b), activated at >1 microm adenosine, increase [cAMP](i).

SUBMITTER: Tan-Allen KY

PROVIDER: S-EPMC4113554 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

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Characterization of adenosine receptors in bovine corneal endothelium.

Tan-Allen Kah Y KY Sun Xing Cai XC Bonanno Joseph A JA

Experimental eye research 20050501 5

Previous studies indicated that adenosine can increase [cAMP](i) and stimulate fluid transport by corneal endothelium. The purpose of this study was to determine which adenosine receptor subtype(s) are expressed and to examine their functional roles in modulating [cAMP](i), [Ca(2+)](i) and effects on Cl(-) permeability in corneal endothelium. We screened bovine corneal endothelium (BCE) for adenosine receptor subtypes by RT-PCR and immunoblotting, and examined the effects of pharmacological agen ...[more]

PMID: 15862176

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Project description:Increased actomyosin contraction of the dense band of actin cytoskeleton at the apical junctional complex (perijunctional actomyosin ring, PAMR) breaks down the barrier integrity of corneal endothelium. This study has investigated the efficacy of statins, which inhibit activation of RhoA, in opposing the thrombin-induced loss of barrier integrity of monolayers of cultured bovine corneal endothelium.Myosin light chain (MLC) phosphorylation, a biochemical measure of actomyosin contraction, was assayed by urea-glycerol gel electrophoresis, followed by western blot analysis. The locus of MLC phosphorylation and changes in the organization of the PAMR were visualized by immunostaining. Phosphorylation of MYPT1, a regulatory subunit of myosin light-chain phosphatase (MLCP), was assessed by Western blot analysis to determine down-regulation of RhoA. The barrier integrity was assessed in terms of trans-endothelial electrical resistance (TER), and further confirmed by determining permeability to FITC dextran (10 kDa) and distribution of ZO-1, a marker of tight junctional assembly.Lovastatin, a prototype of lipophilic statins, induced MLC dephosphorylation under basal conditions. It opposed increase in phosphorylation of MLC and MYPT1 in response to thrombin and nocodazole, agents known to activate RhoA in the endothelium. Pretreatment with the statin opposed the thrombin- and nocodazole-induced disruption of the PAMR and the thrombin-induced decline in TER. Lovastatin also opposed the thrombin- and nocodazole-induced increase in permeability to FITC dextran and redistribution of ZO-1. However, upon supplementation with GGPP (geranylgeranyl pyrophosphate), lovastatin failed to oppose the effects of thrombin and nocodazole on the PAMR, ppMLC, and ZO-1 distribution.Lovastatin attenuates RhoA activation in the corneal endothelium presumably by reducing its isoprenylation. This underlies the suppression of the thrombin-induced loss in barrier integrity of the corneal endothelium.

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Characterization of adenosine receptors in bovine corneal endothelium.

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Characterization of adenosine receptors in bovine corneal endothelium.

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