Abstract
Abstract: :
Purpose: To determine if the Na,K-ATPase is regulated by acetylcholine and nitric oxide in bovine trabecular meshwork and the possible second messengers involved. The trabecular meshwork is enriched with cholinergic nerves and nitric oxide synthase, the nitric oxide (NO) synthetic enzyme. The mechanisms by which these neurotransmitters regulate down stream targets are not clearly understood. Methods: Bovine eyes were obtained from an abattoir and the trabecular meshwork was dissected from the anterior segment. Tissue slice preparations were exposed to either carbachol or sodium nitroprusside (SNP, an NO donor). Na,K-ATPase activity was determined by the colorimetric ATPase assay: ATP was hydrolyzed and the released Pi was measured by forming a complex with molybdate. Activity was measured as the difference between ouabain treated and ouabain untreated samples. cGMP was measured by enzyme immunoassay (EIA). Results: Exposure of bovine trabecular meshwork tissue slices to carbachol and SNP resulted in a bi-directional regulation of the Na,K-ATPase. SNP inhibited ouabain-sensitive Na,K-ATPase activity. This inhibition involved activation of soluble guanylate cyclase and cGMP. However, carbachol caused an increase in ouabain-sensitive Na,K-ATPase activity. Activities measured in carbachol treated tissue slices alone, or slices treated with carbachol in the presence of the non-specific phosphodiesterase inhibitor, IBMX, were similar. Conclusions: The activity of the Na,K-ATPase is regulated not only by intracellular Na+ concentrations, but by certain hormones including carbachol and NO and intracellular messengers that require the actions of protein kinases and protein phosphatases. The SNP-induced decrease in Na,K-ATPase activity with concomitant increases in cGMP, and the carbachol-induced increase in Na,K-ATPase activity which is unaffected by IBMX suggest two antagonistic pathways in the regulation of the Na,K-ATPase. This is unlike our previously identified convergent cholinergic/NO pathways in ciliary process epithelium (Ellis et al. IOVS, 2001). The precise functions of the Na,K-ATPase in trabecular meshwork are unknown. The Na,K-ATPase catalyzes the transfer of 2 K+ from the extracellular space into the cell and the extrusion of 3 Na+ while hydrolyzing ATP to ADP and Pi. The resulting electrochemical gradient is harnessed and used by other transporters and co-transporters involved in regulating cell volume and muscle contraction.
Keywords: second messengers: pharmacology/physiology • trabecular meshwork • NaK ATPase