title: NO-induced regulation of human trabecular meshwork cell volume and aqueous humor outflow facility involve the BKCa ion channel.
abstract: 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.
authors.affiliation: University of Florida, College of Pharmacy, Department of Pharmacodynamics, Gainesville, FL 32610, USA.
authors.forename: William M
authors.initials: WM
authors.lastname: Dismuke
authors.forename: Chigozirim C
authors.initials: CC
authors.lastname: Mbadugha
authors.forename: Dorette Z
authors.initials: DZ
authors.lastname: Ellis
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism*; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007429:Intraocular Pressure / Q000187:drug effects; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018613:Microscopy, Confocal; D008875:Middle Aged; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009994:Osmolar Concentration; D010069:Oxadiazoles / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D010477:Perfusion; D026902:Potassium Channel Blockers / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000378:metabolism; D000071756:Soluble Guanylyl Cyclase; D013552:Swine; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors; D046509:Tissue Culture Techniques; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*
chemical_list: C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D020030:Nitric Oxide Donors; D010069:Oxadiazoles; D010455:Peptides; D026902:Potassium Channel Blockers; D011810:Quinoxalines; D018160:Receptors, Cytoplasmic and Nuclear; D013873:Thionucleotides; C085925:calcein AM; C098740:8-bromo-beta-phenyl-1,N(2)-ethenoguanosine 3',5'-cyclic monophosphorothioate; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; C064719:iberiotoxin; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP