Endogenous regulation of human Schlemm's canal cell volume by nitric oxide signaling.
OBJECTIVE
There is a time-course correlation between nitric oxide (NO)-induced decreases in trabecular meshwork (TM) cell volume and NO-induced increases in outflow facility. The Schlemm's canal (SC) cells may also provide resistance to aqueous humor outflow; therefore, this study tests the involvement of the nitric oxide synthase (NOS) and NO signaling pathway and the BK(Ca)-channel in mediating SC cell volume decreases.


METHODS
Cell volume was measured in low-passage human SC cells using calcein AM fluorescent dye; images were captured with a confocal microscope, and data were quantified using NIH ImageJ software.


RESULTS
Inhibition of endogenous NOS resulted in a 7% increase in SC cell volume. Exposure of SC cells to DETA-NO resulted in a 12% to 16% decrease in cell volume that was abolished by the soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (5 μM), the protein kinase G (PKG) inhibitor (RP)-8-Br-PET-cGMP-S (50 μM), and the high-conductance calcium-activated potassium channel (BK(Ca) channel) inhibitor iberiotoxin (50 nM). Hypertonic media significantly decreased SC cell volume by 14%, whereas hypotonic media significantly increased cell volume by 11.2%.


CONCLUSIONS
These data suggest that endogenous NOS regulates steady state cell volume and the involvement of the NOS/NO/sGC/cGMP/PKG pathway and the BK(Ca)-channel in mediating NO-induced reductions in SC cell volume. These decreases in cell volume correlated with the time-course for NO-induced increases in outflow facility, suggesting that the NO-induced reduction in SC cell volume may also influence outflow facility.
Ellis
Dorette Z
DZ
Department of Pharmacodynamics, University of Florida College of Pharmacy, Gainesville, Florida 32610, USA. ellis@cop.ufl.edu
Sharif
Najam A
NA
Dismuke
William M
WM
D048429:Cell Size; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000502:physiology*; D009994:Osmolar Concentration; D015398:Signal Transduction / Q000502:physiology*; D014019:Tissue Donors; D014129:Trabecular Meshwork / Q000166:cytology*; D014226:Triazenes / Q000494:pharmacology
C105351:1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D051036:Large-Conductance Calcium-Activated Potassium Channels; D014226:Triazenes; C085925:calcein AM; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D019331:NG-Nitroarginine Methyl Ester