Dorzolamide-induced relaxation of porcine retinal arterioles in vitro depends on nitric oxide but not on acidosis in vascular smooth muscle cells A. El-Galaly a, * , C. Aalkjaer b , S.K. Kringelholt a , M.W. Misfeldt a , T. Bek a a Department of Ophthalmology, Aarhus University Hospital, 8000 Aarhus C, Denmark b Institute of Biomedicine (Physiology), Aarhus University, 8000 Aarhus C, Denmark article info Article history: Received 27 February 2014 Received in revised form 15 September 2014 Accepted in revised form 19 September 2014 Available online 20 September 2014 Keywords: Carbonic anhydrase inhibitors Dorzolamide Retina Relaxation Nitric oxide Intracellular acidosis abstract The carbonic anhydrase inhibitor dorzolamide can induce relaxation of retinal arterioles with a conse- quent increase in blood flow and oxygenation of the retina. It has been shown that the mechanisms underlying this relaxation are independent of extracellular acidosis and CO 2 . The purpose of the present study was to investigate the possible involvement of nitric oxide (NO) and intracellular acidosis in dorzolamide-induced relaxation of retinal arterioles. Porcine retinal arterioles were mounted in a wire myograph and dorzolamide induced relaxation was studied after 1) the addition of the NO synthase inhibitor L-NAME (3  10 4 M) or the guanylyl cyclase inhibitor ODQ (3  10 6 M), and 2) after loading the smooth muscle cells with the pH sensitive fluorophore SNARF-1-AM and studying changes in vascular tone and intracellular fluorescence after the induction of hypoxia, addition of lactate (10 2 M), and extracellular acidification (pH ¼ 7.0) alone and in the presence of dorzolamide (10 3 M). Dorzola- mide significantly relaxed retinal arterioles (p < 0.03), and the effect was significantly higher in the presence of perivascular tissue than in isolated vessels at the highest concentration (p < 0.01). In the presence of perivascular tissue dorzolamide-induced relaxation could be reduced by NO inhibition (p < 0.02). Dorzolamide increased intracellular acidification (p < 0.02) during extracellular acidosis, but there was no relation between relaxation and intracellular acidosis. In conclusion, dorzolamide-induced vasorelaxation depends on NO and the perivascular retinal tissue, but is independent of acidification in the extracellular and the intracellular space of retinal vascular smooth muscle cells. Other factors than NO and acidification are involved in dorzolamide-induced relaxation of retinal arterioles. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Carbonic anhydrase inhibitors (CAIs) can lower the intraocular pressure in primary open angle glaucoma (Costagliola et al., 2009). The pressure lowering effect is due to a reduction in the production of aqueous humour, but CAI may also induce vasorelaxation that increases blood flow and tissue oxygenation in the retina and the optic nerve which may protect these tissues from glaucoma dam- age (Stefansson et al., 1999; la Cour et al., 2000; Pedersen et al., 2005). The results of studies on ciliary arteries and vessels outside the eye suggest that CAI-induced vasorelaxation may depend on nitric oxide (NO) synthesized in the vascular endothelium (Aamand et al., 2009; Kringelholt et al., 2012). Relaxation of retinal arterioles can also be induced by extracellular acidosis and lactate (Brazitikos et al., 1993; Hessellund et al., 2006), but CAI-induced relaxation of retinal arterioles has been shown to be independent of the in- hibition of carbonic anhydrase (Kehler et al., 2007; Torring et al., 2009). However, the role of NO in CAI-induced relaxation of retinal arterioles has not been studied, and it is unknown whether the relaxing effect of CAI is related to intracellular changes in pH and the buffering of these changes induced by carbonic anhydrase (Josefsson et al., 2004). Therefore, the purpose of the present study was to investigate the involvement of NO and of intracellular pH in vascular smooth muscle cells on CAI-induced vasorelaxation. Porcine retinal arteri- oles were mounted in myographs and the effect of the CAI * Corresponding author. Department of Ophthalmology, Aarhus University Hos- pital, DK-8000 Aarhus C, Denmark. E-mail address: galaly@me.com (A. El-Galaly). Contents lists available at ScienceDirect Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer http://dx.doi.org/10.1016/j.exer.2014.09.006 0014-4835/© 2014 Elsevier Ltd. All rights reserved. Experimental Eye Research 128 (2014) 67e72