Real-Time Direct Measurement of Nitric Oxide in Bovine Perfused Eye Trabecular Meshwork Using a Clark-Type Electrode

Abstract

NO was detected in bovine trabecular meshwork (TM). Bovine eyes were perfused (posterior ciliary artery). In some eyes (operated eyes) a NO electrode was inserted adjacent to the TM (scleral flap). Vascular perfusion/intraocular pressures (VPP/IOP) were recorded.

In operated eyes, epinephrine (1 nM-100 μM) increased NO (maximally 979.9 ± 117.6 nM, mean ± SDM). Timolol (1 mM) shifted the epinephrine-NO concentration-response curve rightward (2.94 log units) without significantly changing the maximal response (903.0 ± 67.7 nM, mean ± SDM). The non-selective NO synthase (NOS) inhibitor L-NMMA (100 μM) virtually abolished the NO response to epinephrine. L-NMMA alone (1 μM-100 μM) significantly reduced tonic NO generation (maximally 109.5 ± 24.9 nM, mean ± SDM), whereas timolol alone (1 μM-1 mM) had no effect.

In unoperated eyes, epinephrine (1 nM-100 μM) reduced IOP (maximally 2.56 ± 0.64 mmHg, mean ± SDM). Epinephrine (100 μM) mildly increased VPP (4.6 ± 1.3 mmHg, mean ± SDM). Baseline aqueous humor formation rate (11.5 ± 3.2 μl/min, mean ± SDM) was unaffected.

Effluent perfusate (effusate) total NO₂^- was determined by enzymatically reducing all NO₃^- to NO₂^-, then assessing resultant NO₂^- (Griess assay). Epinephrine (1 nM-1μM) increased effusate NO₂^- (maximally 15.8 ± 4.9 μM, mean ± SDM). Timolol (1 mM) reduced, and L-NMMA (100 μM) virtually abolished effusate NO₂^- response to epinephrine. L-NMMA alone (1 μM-100 μM) reduced tonic effusate NO₂^- (maximally from 5.8 ± 1.6 μM to 1.1 ± 0.9 μM, mean ± SDM), whereas timolol alone (1 μM-1 mM) had no effect.

NO is generated tonically in bovine TM and increases in response to epinephrine.