One role for nitric oxide in the anterior segment may be to modulate outflow resistance either directly at the level of the trabecular meshwork, Schlemm's canal, and collecting channels, or indirectly through alteration in the tone of the longitudinal ciliary muscle. Nitrovasodilators were shown to relax bovine trabecular meshwork1
as well as bovine and monkey ciliary muscle1,2,24
strips precontracted with carbachol in vitro. Small increases in OF are produced by modulation of the nitric oxide system in organ-cultured human anterior segments.25
Nitric oxide decreases cell volume in the trabecular meshwork and increases OF in porcine eye anterior segments, suggesting that the nitric oxide–induced alterations in cell volume may regulate outflow resistance.26
IOP and AHF are decreased in isolated pig eyes perfused with nitrovasodilators,27
suggesting mechanisms independent of ocular vasculature. Systemic administration of L-NAME to rabbits resulted in an increase in MAP, but a dramatic reduction in IOP, possibly due to a reduction in ciliary blood flow sufficient to impair aqueous production.28,29
Early studies reported conflicting results of the effects of nitrovasodilators on aqueous humor dynamics in the living primate eye. Nitroglycerin and hydralazine increased OF in monkey eyes by 92% and 28%, respectively, but only after intracameral bolus injection of 10−3
Nitroglycerin may or may not decrease IOP when applied topically to monkey eyes.12,13
Topical administration of hydralazine to ocular normotensive humans resulted in an increase in IOP accompanied by conjunctival hyperemia.14
Intravenous administration of L-arginine lowered IOP in healthy human volunteers and increased nitrite levels in the aqueous humor of rabbits compared with controls, presumably as a result of conversion to nitric oxide by NOS.30
In a different study, no effect of intravenous L-arginine on IOP in humans was detected.31
Study ethnicities, design, and analysis may account for some of the differences in findings between these two studies.
Caution is advised in comparing results from different nitric oxide donor compounds delivered via different routes to different species since there may be confounding effects of these compounds other than those resulting from nitric oxide production alone. Some additional mechanisms need to be verified in ocular tissues.
Hydralazine-induced vasodilatation is associated with powerful stimulation of the sympathetic nervous system, which results in increased heart rate and contractility, increased plasma renin activity, and fluid retention; all of these effects counteract the antihypertensive effect of hydralazine. Although most of the sympathetic activity is due to a baroreceptor-mediated reflex, hydralazine may stimulate the release of norepinephrine from sympathetic nerve terminals and augment myocardial contractility directly.32
Nitroglycerin is extensively metabolized, yielding dinitrates, mononitrates, nitric oxide, and inorganic nitric oxide. A number of enzymes/proteins have been reported to metabolize nitroglycerin, including glutathione S-transferase, cytochrome P-450, and an uncharacterized vascular microsomal enzyme. Nonproteinous thiols can also react with nitroglycerin to generate nitric oxide or S-nitrosothiol. Nitroglycerin administration can lead to increased vascular oxidative stress resulting in multiple changes in signal transduction and gene regulation. Studies have shown that other nitric oxide donors such as SNP, S-nitrosothiols, and organic nitrites, either do not induce or are less effective in inducing vascular tolerance when compared with organic nitrates.33
SNP is not without its confounding issues as well. SNP was originally thought to spontaneously release nitric oxide. However, a membrane-bound nicotine adenine dinucleotide oxidoreductase appears to contribute to the release of nitric oxide from nitroprusside, but not nitroglycerin, in calf pulmonary artery.34
In addition, SNP is reported to degrade to cyanide in vivo and with light exposure. Maximal degradation of SNP under our subdued room light conditions is calculated to be 10% for topical IOP experiments for the protocol giving the maximum IOP response (500 μg at 30-minute intervals for 2 hours). Maximal degradation during OF measurements would be 10% for a 2 hour experiment (1 dose of SNP) or 20% for a 2-dose, 3 hour experiment. As stated in Arnold et al,35
this degradation would not be expected to alter the extent of nitric oxide available for producing the physiologic response. Cyanide production under these circumstances would be expected to be less than 0.01% of the SNP dose. It is possible that cyanide toxicity may have contributed to the increased incidence of hemorrhage into the anterior chamber upon removing the needles from eyes perfused with the highest dose of SNP (10−3
In the current studies, the nitric oxide donor SNP was effective in lowering IOP following repeated topical administration of a high dose at short intervals. Thus far, our data suggest that the mechanism for this IOP lowering response is, at least partly, due to an increase in OF, when a very high dose of 10−3
M SNP was utilized intracamerally. This intracameral concentration is comparable to what would be expected following topical delivery of 2 mg SNP (4×500 μg) assuming 1% corneal penetration36
and no loss over the 1.5 hours of delivery (30-minute intervals). This high concentration requirement may be due to a low rate of penetration into the target tissues, or decreased concentration of the drug at the target sites due to the diverse biologic roles of nitric oxide and its short half-life (T1/2
≤ 10 minutes at 37°C) in living tissues.37,38
As a reference point, in humans with congestive heart failure and life-threatening high blood pressure, the maximum recommended dose for intravenous administration of SNP (see package insert for Nitropress; Hospira, Inc., Lake Forest, IL) is 100 μg/kg delivered over a 10-minute period (for a 70-kg human, this would amount to 7 mg).
Additional support for an effect of nitric oxide on outflow was recently reported. Transgenic mice over expressing endothelial NOS39
were found to have lower IOP15
compared with wild-type mice. However, both mean aortic pressure and pulmonary artery pressure are also reduced by 30% in the transgenic mice.39
In situ, transgenic mice eyes (n
= 4) demonstrated increased pressure-dependent drainage compared with wild-type mice.15
This effect on pressure-dependent drainage was reversed with the NOS inhibitor L-NAME.
Thus, the nitric oxide system may be a signal/transduction arm that mediates response to stressors such as mechanical deformation of the trabecular meshwork caused by stress–strain, shear–stress, and other pressure-related phenomena, or biochemical, hormonal, or metabolic alterations in the surrounding milieu.40
Enhancement of uveoscleral outflow, may be another component involved in the IOP lowering mechanism, as suggested by our previous studies on ciliary muscle relaxation in vitro,2
we did not measure the effects of SNP on uveoscleral outflow in living animals, nor did we measure episcleral venous pressure in the current study. It could be beneficial to study the effect of SNP on those parameters in nonhuman primates in the future.
Results from the current study reporting a decrease in IOP in monkeys in response to the nitrovasodilator SNP are in contrast to reports of the opposite effect on IOP in rabbits.8,9,11
These conflicting results may be due to species differences. They also illustrate that the nitric oxide story is complicated and that its actions should be described and interpreted cautiously. Another possible species-related response difference in our study was the lack of an IOP response to topical SNAP in our monkeys (n
= 4) as compared with the purported longer acting and greater IOP-lowering response obtained in rabbit studies.10
Additional studies in monkeys were not carried out with SNAP since the superior IOP-lowering response to SNP was chosen for mechanistic studies.
The NOS inhibitor L-NAME did not have an effect on IOP in the current study in contrast to the reduction in IOP produced by L-NAME in rabbit studies.9,28,29
However, effects of L-NAME may be limited by the amount of NOS in the eye at the time of drug administration, unintended reactions in the eye, and/or the ability of the compound to localize to the regions of the eye with the highest NOS.2,6,9
L-NAME has been shown to be a muscarinic antagonist, which could result in a reduction of ciliary muscle tension on the trabecular meshwork leading to a decrease in OF.41
In the current studies, we did not detect any changes in PD or Rfx in response to L-NAME treatment that would suggest a muscarinic antagonist effect. Additional studies are warranted before conclusions can be made regarding the effect of NOS inhibition on ocular physiology and the responses to other classes of pharmaceuticals in nonhuman primates.
The nitric oxide system could potentially be targeted to enhance aqueous outflow and lower IOP in glaucoma. Since NOS levels appear to be diminished in glaucomatous eyes,5,6
pharmacotherapy would have to bypass this part of the nitric oxide pathway. A nitric oxide–releasing prostaglandin analog was shown to produce a larger IOP reduction compared with latanoprost alone in ocular hypertensive rabbits, dogs, and monkeys.42
Gene therapy in targeted sites where pharmacologic delivery of nitric oxide donors may not reach sufficient concentrations for therapeutic purposes may be a viable option as well.43
Endothelial NOS would be a likely candidate.39
However, caution is advised since manipulating nitric oxide homeostasis could be fraught with potential problems (e.g., elimination of vascular autoregulation).