Characterization of soluble guanylate cyclase in NO-induced increases in aqueous humor outflow facility and in the trabecular meshwork.
OBJECTIVE
Nitric oxide (NO) increases the rate at which aqueous humor exits the eye; however, the involvement of soluble guanylate cyclase (sGC) is unknown. This study investigated the role of sGC in mediating the NO-induced increases in outflow facility.


METHODS
Outflow facility was measured in porcine eyes using the anterior segment organ culture perfusion system. sGC activity was assessed by cGMP production in low-passage porcine and human trabecular meshwork (TM) cells and transformed human TM cells, as measured by enzyme immunoassay. sGCalpha and sGCbeta isoform expression were determined using Western blot analysis.


RESULTS
Activation of sGC is necessary for the NO-induced increases in outflow facility (0.3215 microL/min per mm Hg [baseline outflow facility]+/-0.0837 [SEM]). NO resulted in increased sGC activity that was abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-1 (ODQ). Western blot analysis of total protein demonstrated an equivalent ratio of sGCalpha and sGCbeta subunit expression. In transformed cell fractions, however, the level of cytoplasmic sGCalpha subunit expression was decreased compared with low-passage human TM cells.


CONCLUSIONS
Activation of sGC is involved in the NO-induced increases in outflow facility. The expression of alpha and beta sGC subunits in an equivalent ratio would suggest a functional sGC heterodimer because DETA-NO increased cGMP levels in low-passage human and porcine TM cells. However, the inability of DETA-NO to cause increases in cGMP levels in transformed TM cells suggests that though the sGC heterodimer is necessary, it is not sufficient and may require other factors not present in transformed cells.
Ellis
Dorette Z
DZ
Department of Pharmacodynamics, University of Florida, Gainesville, Florida 32610, USA. ellis@cop.ufl.edu
Dismuke
William M
WM
Chokshi
Binna M
BM
D000328:Adult; D000369:Aged, 80 and over; D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism*; D015153:Blotting, Western; D018929:Cell Culture Techniques; D006152:Cyclic GMP / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000502:physiology*; D006801:Humans; D007527:Isoenzymes / Q000502:physiology; D009924:Organ Culture Techniques; D010069:Oxadiazoles / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000502:physiology*; D000071756:Soluble Guanylyl Cyclase; D013552:Swine; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism; D014226:Triazenes / Q000494:pharmacology*
C105351:1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene; C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010069:Oxadiazoles; D011810:Quinoxalines; D018160:Receptors, Cytoplasmic and Nuclear; D014226:Triazenes; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP