Timolol effects on erythrocyte deformability and nitric oxide metabolism

Issue title: Special Issue in Honour of the Editor-in-Chief Prof. Dr. Ing. Friedrich Jung on his 70th Birthday

Article type: Research Article

Authors: Saldanha, Carlota^{; *} | Freitas, Teresa | Silva-Herdade, Ana Santos

Affiliations: Institute of Biochemistry, Institute of Molecular Medicine, Faculty of Medicine University of Lisbon, Lisbon, Portugal

Correspondence: [*] Corresponding author: Carlota Saldanha, Institute of Biochemistry, Institute of Molecular Medicine, Faculty of Medicine University of Lisbon, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal. E-mail: carlotasaldanha@medicina.ulisboa.pt.

Abstract: Timolol maleate is a compound used in treatment for reducing increased intra-ocular pressure by limiting aqueous humor production. Decreased erythrocyte deformability (ED), increased activity of erythrocyte acetylcholinesterase (AChE), increased values of nitrosoglutathione (GSNO) and nitic oxide (NO) and decreased plasma levels of NO metabolites, were described in primary open angle glaucoma patients. In healthy human red blood cells (RBCs), timolol is an inhibitor of AChE and induces NO efflux and GSNO efflux from that blood component in lower concentration than those obtained in presence of the natural AChE substrate, acetylcholine (ACh). The signal transduction pathway in RBCs described for NO in dependence of AChE-ACh active complex involves Gi protein, protein tyrosine kinase (PTK like Syk and p53/56Lyn), protein tyrosine phosphatase (PTP) and adenylyl cyclase (AC). The aim of this in vitro study was to verify the effect of timolol maleate in ED, NO efflux and NO derivatives molecules (NOx) like nitrite (NO2–), nitrate (NO3–, peroxynitrite (–ONOO) and GSNO under the presence of PTK, PTP, AC and guanylyl cyclase (GC) enzyme proteins inhibitors. Blood samples from healthy donors were each one divided and were performed aliquots in absence (control aliquots) and presence of timolol or timolol plus each inhibitor and Gi protein uncoupling. No significant differences in erythrocyte NO efflux, GSNO, peroxynitrite, nitrite and nitrate concentrations in response to timolol when compared with the untreated blood samples aliquots were obtained. It was observed an increase in erythrocyte deformability at high shear stresses induced by the simultaneous presence of timolol and band 3 protein dephosphorylation by PTK syk inhibitor. No significant differences where verified in peroxynitrite levels in the blood aliquots in presence of timolol plus each enzyme inhibitor and Gi protein uncoupling in relation to the control aliquots. No variation of GSNO concentration occurs under the presence of timolol and AMGT (PTK lyn inhibitor) besides the significant higher values observed with each one of the other inhibitors. Nitrate concentration increases significantly in all aliquots with timolol plus each one of the inhibitors. The same was observe with nitrite levels with exception of the aliquots with timolol plus AMGT or timolol plus Gi protein uncoupling showing no significant values in relation to the control aliquots. Besides the changes in NO derivative molecules and NO efflux from RBCs obtained in this study with blood samples of healthy donors under the effect of timolol plus each inhibitor of the proteins participants in NO signal transduction mechanism, further analogue studies must be promoted with blood samples of patients with glaucoma or any other inflammatory vascular disease.

Keywords: Erythrocyte deformability, nitric oxide, acetylcholinesterase, timolol

DOI: 10.3233/CH-189110

Journal: Clinical Hemorheology and Microcirculation, vol. 69, no. 1-2, pp. 165-173, 2018