Extracellular ATP Induces Contraction of Bovine Trabecular Meshwork

Abstract

Abstract: : Purpose: An increase in the contractility of the trabecular meshwork (TM) is usually accompanied by an increase in resistance to aqueous humor outflow. In this study, we have investigated changes in the contractility of bovine TM and ciliary muscles (CM) in response to ATP and UTP, endogenous agonists for G_αq/11 coupled P2Y2 receptors. Methods: TM and CM strips were excised from freshly isolated bovine eyes and mounted for measurement of isometric tension. Under perfusion with a HCO₃^––free ([Ca²⁺] = 1.2 mM) Ringers, TM/CM strips were exposed to carbachol (1 µM) and ATP/UTP (50 µM) sequentially for short periods. Response to ATP/UTP was calculated as a % of carbachol–induced contraction. Contraction was also characterized at the level of myosin light chain (i.e., regulatory light chain of myosin II) phosphorylation by exposing bovine TM cells in culture to extracellular ATP. As controls, TM cells were exposed to thrombin (1 U/mL; known to elicit MLC phoshorylation through activation of PAR–1 receptors) and Y–27632 (inhibitor of rho kinase). Protein extracts were assayed for MLC phosphorylation using a protocol involving urea–glycerol gel electrophoresis and immunoblotting. Results: Acute exposure of TM/CM strips to ATP led to rapid contraction similar to that of carbachol but the extent of contraction was lower (45.92+17.47% in TM and 37.43+7.12% in CM; n=7 each; with carbachol–induced contraction set to 100%). UTP did not yield contractions in TM or CM. TM strips pre–contracted by exposure to carbachol showed relaxation upon exposure to Y–27632 (7.2 + 2.9% at 1 µM; n = 9 and 25.6 + 5.5% at 5 µM; n = 7). In TM cells, exposure to thrombin induced MLC phosphorylation (13%) which could be inhibited significantly by Y–27632 (30%; n=2). Exposure of TM cells to ATP (100 µM; 18 min; n=2) did not show significant effect on MLC phosphorylation. Conclusions: (1) In vascular endothelium, extracellular ATP leads to MLC dephosphorylation (Noll et al, AJP Cell, 2000). This is not apparent in TM cells as contractions of strips were noted and moreover, MLC dephosphorylation was not found in response to ATP. (2) Thrombin–induced phosphorylation and its inhibition by Y–27632 are consistent with regulation of MLC phosphorylation by rho kinase mediated inhibition of MLC phosphatase. (3) Since both TM and CM underwent contractions, the effect of ATP on intraocular pressure may be similar to that by pilocarpine.

Keywords: outflow: trabecular meshwork • outflow: ciliary muscle • pharmacology