8-bromo-beta-phenyl-1-n(2)-ethenoguanosine-3--5--cyclic-monophosphorothioate and iberiotoxin

Nitric oxide (NO) donors decrease intraocular pressure (IOP) by increasing aqueous outflow facility in the trabecular meshwork (TM) and/or Schlemm's canal. However, the cellular mechanisms are unknown. Cellular mechanisms known to regulate outflow facility include changes in cell volume and cellular contractility. In this study, we investigated the effects of NO donors on outflow facility and NO-induced effects on TM cell volume. We tested the involvement of soluble guanylate cyclase (sGC), cGMP, PKG, and the large-conductance Ca2+-activated K+ (BKCa) channel using inhibitors and activators. Cell volume was measured using calcein AM fluorescent dye, detected by confocal microscopy, and quantified using NIH ImageJ software. An anterior segment organ perfusion system measured outflow facility. NO increased outflow facility in porcine eye anterior segments (0.4884-1.3956 microl.min(-1).mmHg(-1)) over baseline (0.2373-0.5220 microl.min(-1).mmHg(-1)) within 10 min of drug application. These NO-induced increases in outflow facility were inhibited by the the BKCa channel inhibitor IBTX. Exposure of TM cells to NO resulted in a 10% decrease in cell volume, and these decreases were abolished by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and IBTX, suggesting the involvement of sGC and K+ eflux, respectively. NO-induced decreases in cell volume were mimicked by 8-Br-cGMP and abolished by the PKG inhibitor (RP)-8-Br-PET-cGMP-S, suggesting the involvement cGMP and PKG. Additionally, the time course for NO-induced decreases in TM cell volume correlated with NO-induced increases in outflow facility, suggesting that the NO-induced alterations in cell volume may influence outflow facility.

Topics: Adult; Aged; Aged, 80 and over; Animals; Aqueous Humor; Cell Line; Cell Size; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fluoresceins; Fluorescent Dyes; Guanylate Cyclase; Humans; Intraocular Pressure; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Microscopy, Confocal; Middle Aged; Nitric Oxide; Nitric Oxide Donors; Osmolar Concentration; Oxadiazoles; Peptides; Perfusion; Potassium Channel Blockers; Quinoxalines; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Swine; Thionucleotides; Time Factors; Tissue Culture Techniques; Trabecular Meshwork

The aim of the present study was to evaluate the role of K+ channels in the vasorelaxant effect of the phosphodiesterase 5 inhibitor, sildenafil, in isolated horse penile resistance arteries mounted in microvascular myographs. In phenylephrine-precontracted arteries, sildenafil elicited potent relaxations which were markedly reduced by raising extracellular K+, by the non-selective blocker of Ca2+-activated K+ channels (KCa), tetraethylammonium and by the blocker of large- and intermediate-conductance KCa channels, charybdotoxin. Sildenafil relaxant responses were also reduced by the selective inhibitor of large conductance KCa (BK(Ca)) channels iberiotoxin, but not by the blocker of small conductance KCa channels apamin. The inhibitor of the cGMP-dependent protein kinase (PKG), Rp-8-Br-PET-cGMPS, reduced the relaxations elicited by sildenafil but combined treatment with iberiotoxin and Rp-8-Br-PET-cGMPS did not further inhibit these relaxations, compared to the effect of either blocker alone. Iberiotoxin also shifted to the right the relaxations elicited by both the NO donor, S-nitrosoacetyl-D,L-penicillamine (SNAP) and the adenylate cyclase activator forskolin; treatment with both iberiotoxin and Rp-8-Br-PET-cGMPS did cause an additional inhibition. The present results demonstrate that the relaxant effect of sildenafil and NO in penile resistance arteries is due in part to activation of BK(Ca) channels through a PKG-dependent mechanism.

Topics: Animals; Arteries; Charybdotoxin; Colforsin; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Horses; In Vitro Techniques; Male; Nitric Oxide Donors; Penis; Peptides; Piperazines; Potassium; Potassium Channels, Calcium-Activated; Purines; S-Nitroso-N-Acetylpenicillamine; Sildenafil Citrate; Sulfones; Tetraethylammonium; Thionucleotides; Vascular Resistance; Vasodilation; Vasodilator Agents