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Nitrergic vasodilator nerves running in the parasympathetic outflow innervate the retinal and ciliary vascular beds in a wide range of mammalian species, including humans (for reviews see Koss, 1999; Toda and Okamura, 2003). In the bovine intraocular long posterior ciliary artery, electrical field stimulation (EFS) evokes biphasic vasodilatation, consisting of an initial component peaking at ∼10 s, which decays rapidly, but is followed by a slower component peaking at 50 s. The initial rapid component is abolished by the nitric oxide synthase (NOS) inhibitors, NG-nitro-L-arginine (L-NOARG) and NG-nitro-L-arginine methyl ester (L-NAME), and by the inhibitor of soluble guanylate cyclase, ODQ, and is clearly nitrergic, but the identity of the neurotransmitter mediating the slower component is the subject of debate (Wiencke et al., 1994; Overend et al., 2005).
Previous work has revealed that NOS inhibitors that are NG-substituted analogues of L-arginine do not all uniformly block nitrergic neurotransmission in bovine tissues. For example, in the bovine retractor penis muscle, nitrergic transmission is blocked by L-NOARG and L-NAME, but not by L-NMMA (Liu et al., 1991; Martin et al., 1993). Indeed, in this tissue, NG-monomethyl-L-arginine (L-NMMA) appears to act as an alternative substrate for nitric oxide (NO) production as, like the endogenous substrate, L-arginine, it can both prevent the onset of and reverse already established blockade of nitrergic transmission induced by L-NOARG or L-NAME. Interestingly, in the bovine penile artery, L-NMMA also fails to inhibit nitrergic transmission, but shares the ability of L-NOARG to block endothelium-dependent, NO-mediated vasodilatation (Liu et al., 1991). Thus, in the bovine penile artery, L-NMMA selectively inhibits vasodilatation mediated by endothelial nitric oxide synthase (eNOS), while acting as an alternative substrate for dilatation occurring through neuronal nitric oxide synthase (nNOS).
The aim of this study was to determine if L-NMMA has the ability to selectively block vasodilatation induced by endothelium-derived NO but not nitrergic nerves in the bovine ciliary artery, as it does in the penile artery. Furthermore, the selectivity of two more recently introduced putative inhibitors of nNOS, N-(4S)-4-amino-5-(aminoethyl)aminopentyl-N′-nitroguanidine (AAAN) and NG-propyl-L-arginine (L-NPA) (Zhang et al., 1997b; Hah et al., 2001) was investigated by comparing their actions on vasodilatation produced by endothelium-derived NO and nitrergic nerves in the bovine ciliary artery.
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The major new observations made in this study are that the NOS inhibitor, L-NMMA, does not block nitrergic vasodilatation in the bovine ciliary artery, but acts like the endogenous substrate, L-arginine, to prevent the inhibition induced by L-NAME. This occurs despite the ability of L-NMMA to inhibit endothelium-dependent, NO-mediated dilatation in the same tissue. Furthermore, two putative nNOS-selective inhibitors, AAAN and L-NPA (Zhang et al., 1997b; Hah et al., 2001) did not behave as expected in the bovine ciliary artery: AAAN failed to inhibit vasodilatation mediated by either the nitrergic nerves or endothelium-derived nitric oxide (EDNO) and L-NPA inhibited both, but with equal potency.
A large number of NG-substituted analogues of L-arginine have been introduced as inhibitors of NOS (Hibbs et al., 1987; Rees et al., 1989, 1990; Moore et al., 1990). The most commonly used agents, L-NOARG and L-NAME, are not isoform selective and reliably inhibit responses mediated by the L-arginine-NO system in a wide range of species. Although L-NMMA is also commonly used as a non-isoform-selective NOS inhibitor, many studies demonstrate anomalous findings with this agent. For example, L-NMMA does not block nitrergic transmission in the bovine retractor penis muscle, but acts like the endogenous substrate, L-arginine, to inhibit blockade by L-NOARG or L-NAME (Liu et al., 1991; Martin et al., 1993). Furthermore, in the bovine penile artery, L-NMMA also fails to block nitrergic transmission, despite being able to inhibit the endothelium-dependent, NO-mediated dilatation induced by acetylcholine (Liu et al., 1991). In addition, in the rat aorta and pulmonary artery, L-NMMA enhances rather than inhibits the production of NO, assessed by chemiluminescence detection, and inhibits the basal but not agonist-stimulated, endothelium-dependent dilatation produced by NO (Archer and Hampl, 1992; Frew et al., 1993).
The above anomalous results with L-NMMA have been found despite the general observation that it inhibits all three isoforms of NOS in standard enzyme assays (Moore et al., 1996). Nevertheless, more detailed biochemical analysis of murine macrophage inducible nitric oxide synthase (iNOS) has revealed that L-NMMA does not behave as a simple competitive inhibitor, but as an alternative substrate and mechanism-based inhibitor of the enzyme (Olken and Marletta, 1993). According to this scheme, L-NMMA is initially metabolized to NG-hydroxy-NG-methyl-L-arginine, and finally to NO and L-citrulline, but with the intermediate production of a ‘suicide inhibitor’ that slowly and irreversibly blocks the enzyme. These findings may, therefore, account for the seemingly anomalous ability of L-NMMA to augment NO production at some sites while blocking it at others. However, why L-NMMA fails to block nitrergic transmission at sites like the bovine ciliary artery is not entirely clear. One possible explanation is that at these sites the rate of formation of the suicide inhibitor is exceedingly slow. This seems unlikely because, although most of our experiments ran for 1–2 h, we failed to see any inhibitory effects even after prolonged treatment for up to 6 h (data not shown). It is, therefore, possible that at these sites, the particular isoforms of NOS either do not produce the suicide inhibitor or are insensitive to it. Further work is required to clarify this point.
According to present findings, L-NMMA fails to block nitrergic transmission in the bovine ciliary artery, but acts like L-arginine in inhibiting blockade by L-NAME, which are therefore in keeping with previous results on other nitrergically-innervated tissues (retractor penis muscle and penile artery) from this species (Liu et al., 1991; Martin et al., 1993). The inability of L-NMMA to block nitrergic transmission may, therefore, be a general feature of bovine tissues.
Our examination of the actions of L-NMMA on endothelium-dependent, NO-mediated dilatation in the bovine ciliary artery was initially confounded because of the simultaneous activation of other endothelium-derived factors. At sites where EDHF and NO are both active, it is, however, well established that blockade of either component alone may produce no effect or only a small reduction in the magnitude of the vasodilatation observed (Mügge et al., 1991; Tare et al., 2000; McNeish et al., 2003). The remaining vasodilatation will then be mediated solely by the other component and therefore be sensitive to inhibitors of that pathway. In keeping with this, we found that, following blockade of EDHF with apamin and charybdotoxin (Waldron and Garland, 1994; Zygmunt and Högestätt, 1996) and cyclooxygenase with indomethacin, vasodilator responses mediated solely by endothelium-derived NO could be elicited with bradykinin. These dilator responses, unlike those produced by the nitrergic nerves in the ciliary artery, were inhibited by L-NMMA. They were also inhibited by L-NAME. Thus, our findings in the bovine ciliary artery that L-NMMA inhibits endothelium-dependent, NO-mediated dilatation but not that produced by its nitrergic nerves parallels earlier findings on the bovine penile artery (Liu et al., 1991) and suggests that this may be a general property in this species.
Since the discovery of NO, great effort has been expended in developing isoform-selective inhibitors of NOS, both as investigational tools and as potential therapeutic agents (Fukuto and Chaudhuri, 1995; Hobbs et al., 1999; Li and Poulos, 2005). We, therefore, compared the effects of L-NMMA and L-NAME on bovine ciliary artery with those of some more recently introduced nNOS-specific inhibitors. The two agents we examined were AAAN, which has a ∼2500-fold greater selectivity for rat nNOS over bovine eNOS (Hah et al., 2001) and L-NPA, which is ∼150-fold more selective for bovine nNOS than eNOS (Zhang et al., 1997a, 1997b). We found that in the bovine ciliary artery, AAAN at concentrations up to 100 μM failed to affect vasodilatation induced either by the nitrergic nerves or endothelium-derived NO. A lack of effect on endothelium-dependent vasodilatation might have been expected, given its poor Ki for bovine eNOS (314 μM, Hah et al., 2001). Nevertheless, the failure to block nitrergic transmission was surprising, in view of its Ki of 0.12 μM for rat nNOS. Whether the failure of AAAN to block nitrergic transmission in the bovine ciliary artery results from poor tissue penetration, a major difference in Ki values for bovine and rat nNOS, or some other factor, remains to be established. Equally disappointing were the actions of L-NPA (Zhang et al., 1997a, 1997b) on the bovine ciliary artery. Although this agent did inhibit nitrergic vasodilatation, it failed to exhibit the ∼150-fold selectivity for nNOS over eNOS seen in enzyme assays (Zhang et al., 1997b). Indeed, L-NPA blocked endothelium-dependent, NO-mediated vasodilatation over the same concentration range as for blockade of nitrergic transmission. Our findings with AAAN and L-NPA therefore serve as a reminder that it is not always possible to extrapolate findings in biochemical assays to functional responses in intact tissues.
In conclusion, in the bovine ciliary artery, L-NMMA acts as a selective blocker of endothelium-dependent, NO-mediated vasodilatation, but has no inhibitory effect on dilatation produced by nitrergic nerves. In fact, L-NMMA behaves similarly to L-arginine in protecting nitrergic vasodilatation against blockade by L-NAME. No agent was identified that selectively inhibits vasodilatation mediated by the nitrergic nerves. Of the two putative nNOS inhibitors tested, AAAN failed to inhibit vasodilatation induced either by nitrergic nerves or endothelium-derived NO, and L-NPA inhibited both, but with no evidence of selectivity.