Data from both cynomolgus and rhesus ciliary muscle strips were utilized. The number of cynomolgus samples was small (7) relative to the number of rhesus samples (56), so it is difficult to evaluate any species differences that may be present. However, the CARB contraction forces for cynomolgus ciliary muscles were within the same range of values as for rhesus ciliary muscles (LONG, 22.5–147.5 mg; CIRC, 12.5–72.5 mg). In vivo, physiologic responses such as accommodation and outflow facility of rhesus and cynomolgus eyes to pilocarpine are robust and similar in magnitude (citations for examples include (Gabelt et al., 1991
, Gabelt and Kaufman, 1992
; Kiland et al., 1997b
, Okka et al., 2004
, Takagi et al., 2004
)), so there is no reason to suspect that the relative ciliary muscle responses might be different although this needs to be investigated.
Nitric oxide generating compounds were effective in relaxing CARB precontracted monkey ciliary muscle in vitro. The most effective agent was the nonnitrate, SNP, which produced nearly complete relaxation. The spontaneous release of nitric oxide from SNP may contribute to the magnitude of its effect as compared to the other agents investigated. Incomplete inhibition of SNP-induced relaxation following methylene blue treatment led us to utilize the more specific inhibitor, ODQ. However, incomplete inhibition of the initial relaxation response in the presence of ODQ suggests that other mechanisms may contribute to the SNP-induced relaxation (see below). Continued incubation with ODQ did result in nearly complete attenuation (not shown) of the relaxation produced by SNP, indicating that sustained relaxation was the result of cGMP production. The contributions of calcium channel blockade, K+ channel activation cAMP- or prostanoid (Mollace et al., 2005
) -mediated mechanisms were not investigated in the current study. Alternatively, differential sensitivity to ODQ inhibition of relaxation by nitric oxide donors has been reported (Tseng et al., 2000
). Organic nitrates such as ISDN require bio-activation for the release of nitric oxide, which could limit the levels of nitric oxide available as compared to SNP [reviewed in (Mollace et al., 2005
The endogenous substrate, L-arg, produced approximately half the relaxation response compared to SNP, suggesting that the capacity for stimulating relaxation via this pathway is limited by the amount of nitric oxide synthase present. Inhibition of phosphodiesterase with IBMX did not further enhance the initial relaxation response but may have contributed to sustaining the relaxation upon continued incubation. This lack of enhancement of the L-arg relaxation response in the presence of IBMX also supports hypothesis that nitric oxide synthesis, and not cGMP hydrolysis, may be the limiting factor in the magnitude of the relaxation response to L-arg.
However, another nitric oxide signaling pathway that may confound all of the nitric oxide responses described thus far is S-nitrosylation of cysteine groups in proteins (Torta et al., 2008
). S-nitrosylation has been reported to desensitize soluble guanylyl cyclase (Sayed et al., 2007
) and to decrease G-actin polymerization (Dalle-Donne et al., 2000
). These additional actions by nitric oxide could possibly contribute to the magnitude of the relaxation with SNP (actin depolymerization contribution), the transient relaxation responses with ISDN and L-arg as well as the lack of enhancement of relaxation by IBMX (contribution of guanylyl cyclase desensitization). However, these speculations need to be further investigated and will also need to be considered when interpreting data from other ocular physiology studies involving nitric oxide generating agents.
The lesser magnitude of response following 8-Br cGMP as compared to SNP suggests that either cGMP is not the only mediator of the relaxation response, or that 8-Br cGMP does not penetrate the cells as well as nitric oxide.
Nonspecific inhibition of nitric oxide synthase with L-NAME produced an initial relaxation response. The enhancement of CARB contraction in the LONG vector with continued incubation in the presence of L-NAME indicates that there is endogenous production of nitric oxide that modulates the contraction response to CARB. However, alkyl esters of arginine, such as L-NAME, have been reported to also act as muscarinic antagonists (Buxton et al., 1993
). Therefore the biphasic response of monkey ciliary muscle contraction in the presence of L-NAME can be explained by both the competitive antagonism of CARB contraction which could be responsible for the apparent initial relaxation and, subsequently, by the inhibition of nitric oxide synthase causing a reduction in nitric oxide levels. The reduction in nitric oxide levels when combined with the continued presence of CARB and competition at the muscarinic receptor, may result in enhanced contraction, at least in the LONG vector. These differential results in CIRC and LONG vectors in response to L-NAME possibly suggests that CIRC vector fibers may contain less nitric oxide synthase or that the number or affinity of muscarinic receptors may be different from those in LONG muscle fibers. Initial contraction responses to CARB were generally less in CIRC than LONG vectors (see legends in and ). Differential accommodative and outflow facility responses in vivo to cholinomimetic drugs has been reported in humans (Fechner et al., 1975
, Keren and Treister, 1980
; Lieberman and Leopold, 1967
) and in monkeys (Erickson-Lamy and Schroeder, 1990
) but are not likely due to differences in muscarinic receptor subtypes (Gabelt and Kaufman, 1992
; Poyer et al., 1994
Relaxation responses in CARB-contracted ciliary muscle were similar in the LONG compared to the CIRC vectors with SNP and with L-arg. There were tendencies toward greater relaxation in the LONG vector with ISDN and in the CIRC vector with 8-Br cGMP. However, previous studies in intact monkey eyes, did not detect regional differences in NADPH-diaphorase staining, representative of nitric oxide synthase activity (Chen et al., 1998
Bovine ciliary muscle strips at resting tension were reported to relax further in the presence of nitric oxide donors (Wiederholt et al., 1994
). No further relaxation of resting tension was produced by nitric oxide donors in cat ciliary muscle (Goh et al., 1995
). Relaxation responses in resting monkey ciliary muscle were not investigated in the current study. However, in vivo, monkey ciliary muscle may exist in a partially contracted state even at accommodative rest (night or anesthetic myopia, tonic accommodation, etc.) (Kiland et al., 1997a
) so that modulation by enhanced nitric oxide generation could be effective in fine tuning outflow through both ciliary muscle- and trabecular meshwork-associated pathways. Alternatively, due to the intimate association of LONG ciliary muscle fibers with the trabecular meshwork, (Rohen et al., 1967
) relaxation of the ciliary muscle could potentially decrease outflow facility. However, the effects of nitric oxide on trabecular meshwork relaxation may be the predominating response (see below). The magnitude of an IOP response to ciliary muscle relaxation in vivo may be small and of short duration.
Stimulating ocular nitric oxide/cGMP production decreased IOP and also increased outflow facility in monkeys in vivo (Schuman et al., 1994
) and in human organ-cultured anterior segments (Schneemann et al., 2002
). Also, in monkeys in vivo, intracameral 8-Br cGMP increased outflow facility and intravitreal dosing decreased aqueous humor flow rate (Kee et al., 1994
). Nitric oxide modulators produced greater relaxation in bovine trabecular meshwork compared to ciliary muscle in vitro (Wiederholt et al., 1994
). This suggests that the trabecular meshwork, as compared to the ciliary muscle, may be the more likely therapeutic target for nitric oxide-induced IOP effects. IOP was significantly reduced in ocular normotensive humans during i.v. L-arginine infusion (10 min) but recovered rapidly after infusion ceased. No changes in pupil diameter or accommodative amplitude were found (Chuman et al., 2000
). This would be in keeping with the finding that nitric oxide synthase activity is more prevalent in the LONG portion of the ciliary muscle in humans. Conversely, no reduction in IOP was found after topical nitroglycerin administration to normal and glaucomatous monkeys (Wang and Podos, 1995
The nitric oxide system could potentially be targeted to enhance of aqueous outflow and lower IOP in glaucoma. Since nitric oxide synthase levels appear to be diminished in glaucomatous eyes, (Chen et al., 1998
, Nathanson and McKee, 1995a
) pharmacotherapy would have to bypass this part of the nitric oxide pathway. Recently, a nitric oxide -releasing prostaglandin analog was shown to produce a larger IOP reduction compared to latanoprost alone in ocular hypertensive rabbits, dogs and monkeys (Borghi et al., 2010
). Long-term nitric oxide therapy might also contribute to enhanced neuroprotective properties as a result of nitric oxide -mediated inhibition of oxidative stress, pro-inflammatory mediators, and cytokine production (Neufeld, 1999
). Alternatively, gene therapy (Kaufman et al., 2000
) could potentially be used to restore or elevate nitric oxide synthase levels in target tissues.
Relaxation of the CIRC vector by the nitric oxide compounds used in the current study, also supports the use of these types of compounds to prevent myopia (Beauregard et al., 2001
). Intrinsic nitric oxide synthase -positive nerve cells concentrated in the inner parts of the human ciliary muscle indicate a physiological role of nitric oxide for disaccommodation or fine adjustment of focus during accommodation (Tamm et al., 1995
). Most recently, in human ciliary muscle, an intrinsic network of proprioceptive nerve terminals was identified that, in part, surrounds the nitrergic neurons indicating that contraction of the CIRC muscle can be modulated locally via a self-contained reflex arc (Flugel-Koch et al., 2009
The current study demonstrates the large capacity of the ciliary muscle to respond to nitric oxide regulation which may potentially be utilized in glaucoma and presbyopia therapy.