Adenosine induces dephosphorylation of myosin II regulatory light chain in cultured bovine corneal endothelial cells

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] Western blot (WB) confirmed the presence of A 2b (∼50 kDa) and A 1 (∼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 μM alloxazine, a specific A 2b blocker. A 1 activation with 1 μM N 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 μM), 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 μM adenosine, acts to decrease [cAMP] i and A 2b , activated at >1 μM adenosine, increase [cAMP] i .

Section: Discussionmentioning

confidence: 63%

Characterization of adenosine receptors in bovine corneal endothelium

Tan-Allen

Sun

Bonanno

2005

“…increased anion flux and increased transepithelial resistance (Srinivas et al 2004)) occurs are not known. We have hypothesized that endothelial stress, which leads to ATP release and subsequent conversion to adenosine, would activate A 2b receptors and increase [cAMP] in an attempt to counteract the negative effects of the stress.…”

Section: Discussionmentioning

confidence: 99%

“…A role for Cl -in stimulated transport however, has not been tested. Interestingly, increasing [cAMP] and PKA activity also increases paracellular resistance (Srinivas et al 2004), through a myosin light chain kinase mediated process. This leads to a small increase in endothelial electrical resistance, rather than a decrease that would be predicted based solely on apical anion permeability.…”

Section: Hcomentioning

confidence: 99%

Dependence of cAMP meditated increases in Cl− and HCO3− permeability on CFTR in bovine corneal endothelial cells

Allen

Sun

et al. 2008

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is present on the apical membrane of corneal endothelial cells. Increasing intracellular [cAMP] with forskolin stimulates an NPPB and glibenclamide-inhibitable apical Cl -and HCO 3 -permeability. To definitively determine that the increased permeability is dependent on CFTR, we used an siRNA knockdown approach. Apical Cl -and HCO 3 -permeability and steady-state HCO 3 -flux were measured in the presence or absence of forskolin using cultured bovine corneal endothelial cells that were transfected with CFTR siRNA or a scrambled sequence control. CFTR protein expression was reduced by ∼80% in CFTR siRNA treated cultures. Forskolin (10 μM) increased apical chloride permeability by 7 fold, which was reduced to control level in siRNA treated cells. CFTR siRNA treatment had no effect on baseline apical chloride permeability. Apical HCO 3 -permeability was increased 2 fold by 10 μM forskolin, which was reduced to control level in siRNA treated cultures. Similarly, there was no effect on baseline apical HCO 3 -permeability by knocking down CFTR expression. The steady-state apicalbasolateral pH gradient (ΔpH) at four hours in control cultures was increased ∼2.5 fold by forskolin. In CFTR siRNA treated cells, the baseline ΔpH was similar to control, however forskolin did not have a significant effect. We conclude that forskolin induced increases in apical HCO 3 -permeability in bovine corneal endothelium requires CFTR. However, CFTR does not have a major role in determining baseline apical chloride or HCO 3 -permeability.The corneal endothelium is responsible for maintaining the hydration and transparency of the cornea by counteracting the fluid imbibition properties of the glycosaminoglycan rich stroma. Numerous studies have shown that this process is dependent on the presence of HCO 3 -and slowed by inhibition of carbonic anhydrase activity (Hodson 1971;Barfort and Maurice 1974;Fischbarg and Lim 1974;Hodson 1974;Kuang et al. 1990;Riley et al. 1995). The endothelium develops a small apical side negative transendothelial potential (0.5 mV) that is significantly reduced by HCO 3 -withdrawal or by carbonic anhydrase inhibitors (Fischbarg 1972;Barfort and Maurice 1974;Fischbarg and Lim 1974;Hodson et al. 1977). Corneal swelling is also induced by the Na + /K + ATPase inhibitor ouabain, indicating that the endothelial pump involves active transport.HCO 3 -uptake at the basolateral (stromal) membrane has been identified as being predominantly via the 1Na + :2HCO 3 -cotransporter (NBCe1) (Sun et al. 2000;Sun and Bonanno 2003). A recent study showed that siRNA knockdown of NBC significantly reduced basolateral HCO 3 -permeability and eliminated net basolateral to apical fluxes (Li et al. 2005). Interestingly, studies using cultured cells indicate that basolateral HCO 3 -permeability is 3-4 times greater than apical permeability (Bonanno et al. 1999). The low HCO 3 -permeability of the apical membrane suggests that it is rate limiting for HCO 3 -fluxes and tha...

“…Moreover, ATP released by endothelium is converted to adenosine, which acitivates A2 B receptors leading to increased [cAMP] (Tan-Allen et al, 2005). Activation of PKA increases apical CFTR permeability and dephosphorylates MLC leading to increased barrier resistance (Srinivas et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

Molecular expression and functional involvement of the bovine calcium-activated chloride channel 1 (bCLCA1) in apical HCO3− permeability of bovine corneal endothelium

Zhang

Xie

et al. 2006

Corneal endothelium secretes from basolateral (stroma) to apical (anterior chamber) compartments. Apical permeability can be enhanced by increasing [Ca 2+ ] i . We hypothesized that the bovine calcium-activated chloride channel 1 (bCLCA1), shown previously by PCR screening to be expressed in corneal endothelium, is involved in Ca 2+ activated apical permeability. bCLCA1 expression in cultured bovine corneal endothelial cells (CBCEC) was examined by in situ hybridization analysis, immunoblotting, immunofluorescence and confocal microscopy. Rabbit polyclonal antibodies were generated using a 14 aa polypeptide (417-430) from the predicted sequence of bCLCA1. The small interference RNA (siRNA) knock down technique was used to evaluate the functional involvement of bCLCA1 in apical permeability. In situ hybridization confirmed prominent bCLCA1-specific mRNA expression in CBCEC. bCLCA1 antiserum detected the heterologously expressed bCLCA1 in HEK293 cells and a 90 kDa band in CBCEC, which was absent when using the pre-immune serum or antigen absorption of serum. Immunofluoresence staining with anti-bCLCA1 antibody and confocal microscopy indicates an apical membrane location in CBCEC. In CBCEC transfected with bCLCA1 specific siRNA, bCLCA1 expression was reduced by 80%, while transfection with siControl scrambled sequence had no effect. Increasing [ ] by application of ATPγS or cyclopiazonic acid (CPA) increased apical permeability in siControl transfected CBCEC, while having no effect on apical permeability in bCLCA1 specific siRNA transfected cells. Baseline permeability, however, was not different between controls and siRNA treated cells. We conclude that the calcium-activated chloride channel (bCLCA1) is expressed in bovine corneal endothelial cells and can contribute to Ca 2+ dependent apical permeability, but not resting permeability, across the corneal endothelium.