calcein-am and iberiotoxin

Inhibition of the BK(Ca) channel attenuated the nitric oxide-induced increase in outflow facility and decrease in trabecular meshwork (TM) cell volume suggesting the involvement of the BK(Ca) channel in TM cell function. This study examined the effects of activation of the BK(Ca) channel on outflow facility and TM cell volume and determined if the effects of NO and BK(Ca) channel activation on TM cell volume were additive.. Porcine eyes were used to measure outflow facility using the anterior segment organ culture perfusion system. Cell volume was measured using Calcein AM fluorescent dye, detected by confocal microscopy, and quantified using NIH ImageJ software.. NS1619 increased outflow facility 86% over baseline. Additionally, there was a concentration-dependent decrease in TM cell volume in response to NS1619, which was abolished by iberiotoxin (IBTX). While NS1619 alone and DETA-NO alone decreased TM cell volume, together their effects were not additive. The time course for NS1619-induced increases in outflow facility correlated with the time course for NS1619-induced decreases in cell volume.. BK(Ca) channel activation increases outflow facility and decreases cell volume suggesting that K(+) efflux regulates TM cell function.

Topics: Animals; Benzimidazoles; Cell Line; Cell Size; Dose-Response Relationship, Drug; Fluoresceins; Fluorescent Dyes; Humans; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Microscopy, Confocal; Nitric Oxide; Peptides; Swine; Time Factors; Trabecular Meshwork; Triazenes

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