کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
9341477 | 1602626 | 2005 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Regulation of Na-K-2Cl cotransport in cultured bovine corneal endothelial cells
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
ایمنی شناسی و میکروب شناسی
ایمونولوژی و میکروب شناسی (عمومی)
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![عکس صفحه اول مقاله: Regulation of Na-K-2Cl cotransport in cultured bovine corneal endothelial cells Regulation of Na-K-2Cl cotransport in cultured bovine corneal endothelial cells](/preview/png/9341477.png)
چکیده انگلیسی
We have previously demonstrated the presence of a Na+-K+-2Cl cotransporter in cultured bovine corneal endothelial cells (CBCEC) and determined that this cotransporter is located in the basolateral membrane. This transporter may contribute to volume regulation and transendothelial fluid transport. We have now investigated factors regulating the activity of the cotransporter. This activity was assessed by measuring the bumetanide-sensitive 86Rubidium (86Rb) uptake in 86Rb-containing solutions. Data were normalized to protein content determined with a Lowry protein assay. We investigated the regulation by extracellular and intracellular ion concentrations, by osmotic gradients, and by second messengers. Our results indicate that extracellular Na+ and K+ each are required for activation of the cotransporter and activate with first-order kinetics at half-maximally effective concentrations (k1/2) of 21·1 and 1·33 mm, respectively. Extracellular Clâ is also required for cotransport activation, but shows higher order kinetics; the k1/2 for Clâ is 28·1 mm and the Hill coefficient 2·1. HCO3â exerts a modulating effect on cotransporter activity; at 0 HCO3â the bumetanide-sensitive K+ uptake is reduced by 30% compared to that at 26 mmHCO3â. Manipulations of the intracellular [Clâ] by preincubation in Clâ-free solution or inhibition of Clâ efflux resulted in increased uptake at low [Clâ]i and decreased uptake at high [Clâ]i. To assess the role of protein kinases in the regulation of cotransport, we have determined the effect of protein kinase inhibitors. H-89 and KT5270, inhibitors of PKA, inhibit cotransport almost completely, while calphostin C, an inhibitor of PKC, produces a small activation of cotransport. The tyrosine kinase inhibitor genistein reduced K+ uptake while its inactive analog daidzein was without effect. The calmodulin kinase inhibitor KN-93 was without effect. We also investigated the effects of phosphatase inhibitors. Calyculin A (k1/2=21 nm) and okadaic acid (k1/2=915 nm) produced approximate doubling of K+ uptake, suggesting that phosphatase 1 is dominant. We also investigated the role of the cytoskeleton and its activation. Reduction of Cai2+ by preincubation in Ca2+-free medium as well as by exposure to W-7, an inhibitor of the binding of Ca2+ to calmodulin, reduced K+ uptake. Consistent with this, ML-7, a relatively specific inhibitor of the Ca2+-calmodulin activated myosin light chain kinase, inhibited cotransport by 40%. The Ca2+-calmodulin activated myosin light chain kinase contributes to the modulation of the cytoskeleton by regulating the actin-myosin interaction. Consistent with the above, disruption of the actin polymerization by cytochalasin D led to a decrease in K+ uptake. We conclude that extracellular Na+, K+ and Clâ are requirements for the function of the CBCEC Na+-K+-2Clâ cotransporter, while intracellular Clâ and extracellular HCO3â modulate its activity. Several protein kinases, including PKA, PKC, tyrosine kinase, and myosin light chain kinase, modulate the K+ uptake. Another modulating pathway for cotransport involves the state of the cytoskeleton.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Experimental Eye Research - Volume 80, Issue 6, June 2005, Pages 777-785
Journal: Experimental Eye Research - Volume 80, Issue 6, June 2005, Pages 777-785
نویسندگان
Friedrich P. Diecke, Quan Wen, Pavel Iserovich, Jianfeng Li, Kunyan Kuang, Jorge Fischbarg,