Effects of methylene blue and LY83583 on neuronal nitric oxide synthase and NADPH-diaphorase

eji) ELSEVIER

European Journal of Pharmacology Molecular PharmacologySection 290 (1995) 247-251

molecular pharm~

Effects of methylene blue and LY83583 on neuronal nitric oxide synthase and NADPH-diaphorase Dasan Luo, Sheela Das, Steven R. Vincent ~ Di~'ision of Neurological Sciences, Department of Psychiat~', The UnicersiO' of British Columbia, Vancouuer. B.C. V6T IZ3, Canada

Received 20 April 1995; accepted4 May 1995

Abstract Methylene blue and 6-anilino-5,8-quinolinedione (LY83583) have often been used as 'selective' inhibitors of soluble guanylyl We report that in in vitro assays, both these compounds were potent inhibitors of rat cerebellar nitric oxide synthase activity. Me blue had an apparent K i of 2.7/xM, while for LY83583 the K~ was 15.8 IzM. Furthermore, methylene blue, but not LY83583, il the NADPH-diaphorase histochemical reaction associated with nitric oxide synthase. Our results indicate that many of the effects drugs which have been attributed to inhibition of guan'ylyl cyclase, may derive from their direct inhibition of nitric oxide synthase instead. Ken'word.s: Nitric oxide (NO) synthase; Methylene blue; LY83583; cGMP; NADPH-diaphorase

I. Introduction The enzyme nitric oxide (NO) synthase catalyzes the formation of nitric oxide from L-arginine via a unique mechanism combining a cytochrome P450 reductase-like domain with a heme-containing cytochrome P450-1ike domain (Klatt et al., 1992; McMillan et al., 1992; White and Marietta, 1992). Soluble guanylyi cyclase, which appears to be the physiological receptor for nitric oxide, also contains a heme moeity, with which nitric oxide interacts to activate the enzyme (Gerzer et al., 1981). Drugs such as metal protoporphyrins, which can displace this labile heme inhibit guanylyl cyclase, but are without effect on NO synthase (Ignarro et al., 1984; Luo and Vincent, 1994a). In contrast, nitric oxide, carbon monoxide, and other cornpounds that interact with ferrous iron can inhibit nitric oxide synthase and activate guanylyl cyclase (Briine et al., 1990). Other drugs which may interact with heme have been suggested as selective inhibitors of soluble guanylyl cyclase, including methylene blue and 6-anilino-5,8quinolinedione (LY83583). Methylene blue inhibits the ability of NO donors to activate soluble guanylyl cyclase (Katsuki et al., 1977).

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This observation has resulted in the widespread use compound as a selective inhibitor of guanylyl c However, evidence suggests that the inhibition ma3 through the cellular reduction of methylene blue at sequent generation of superoxide, and not via a inhibition of guanylyl cyclase (Brune et al., 1990, V~ al., 1990). In contrast, methylene blue was recentl) to inhibit ionomycin-stimulated formation of citrul cultured endothelial cells independent of superoxi mation (Shimizu et al., 1993). This suggested a inhibition of endothelial nitric oxide synthase by thi pound. A similar conclusion was reached by Ma) colleagues (1993)who found that methylene blue in endothelium-dependent relaxation and NO synthase. dent cGMP formation. This group also found that 1 cerebellar NO synthase was completely inhibited t centrations of methylene blue having very little ef guanylyl cyclase (Mayer et al., 1993). LY83583 was initially found to lower tissue le cGMP but not to inhibit soluble guanylyl cyclas guinea pig lung (Schmidt et al., 1985). Unfortl LY83583 has subsequently often been assumed to an inhibitor of soluble guanylyl cyclase (i.e. Lusti~ 1992; Watson et ai., 1990; Zhou et al., 1994). L' inhibited both endothelial-dependent and nitroprus: duced relaxation and cGMP increases in aorta rin amond, 1987; Malta et al., 1988). The block c~

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D. l, uo et al. / European Journal of Pharmacology - Molecular Pharmacology Section 290 119951 247-251

overcome by increasing concentrations of sodium nitroprusside, but not by increasing endothelium-dependent relaxants such as acetylcholine (Malta et al., 1988). MiJlsch et al. (1988), found that LY83583 could inhibit soluble guanylyl cyclase at high concentrations following its chemical reduction. This may be dependent upon the generation of superoxide anions following LY83583 metabolism (Cherry et al., 1990; Mi.ilsch et al., 1989). In contrast, this drug appeared to directly inhibit endotheiium-derived relaxing factor (EDRF) production from endothelial cells at concentrations at which guanylyl cyclase was unaffected (Miilsch et al., 1988). This suggests a direct inhibition of NO synthase by LY83583. In the present study, we have directly examined the actions of methylene bluc and LY83583 on neuronal NO synthase from rat cerebellum. We have also compared their abilities to inhibit the NADPH-diaphorase histochemical reaction associated with NO synthase (Hope et al., 1991).

2. Materials and methods

with counting scintillant and the amount of [~H]cit determined in a liquid scintillation counter. The results of the radiochemical assay were also pared with the chemiluminescence determination c using a Sievers Nitric Oxide Analyser (Menon 1991). The assays were run in parallel, with the exc that the radioactive tracer was left out for the cher~ nescence detection. After the incubation and stop, a] were injected into a refluxing solution of 1% pou iodide in acetic acid, from which NO was carried NO analyzer by a constant flow of helium. The ass~ calibrated by injection of standard solutions of 1 deoxygenated water. 2.3. NADPH-diaphorase histochemistry

The NADPH-diaphorase staining was done as ously described (Vincent and Kimura, 1992). Briefl mals were anesthetized with pentobarbital and pe

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2.1. Chemicals

LY83583 was purchased from Calbiochem, while methylene blue was from British Drug House (BDH). Bnicotinamideadeninedinucleotidephosphate, rcducedform (B-NADPH), calmodulin, and nitro blue tetrazolium were from Sigma. [3H]-L-arginine was from New England Nuclear,

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arginine by passage through a Dowex-50W cation exchange column (50 × 2-2110, Sigma), followed by elution with 2 ml of water. The pooled eluents (4 ml) were mixed

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Cerebella were removed from rats following decapitation, and homogenized in ice-cold 50 mM HEPES buffer, pH 7.4, containing 1 mM EDTA (10 / x l / m g wet weight). The homogenate was centrifuged at 10000 × g at 4°C for 40 min. The supernatant was then used as a NO synthase source. Protein was determined using the method of Lowry~ et al. (1951). Enzyme activity was measured by determining the conversion of tritiated arginine to citrulline (Bredt and Snyder, 1990), as previously described (Luo and Vincent, 1994a, Luo and Vincent, 1994b). Briefly, 25 tzl of L-arginine (at 3, 30 and 300 p.M), 25 ul of 44 nM [3H]-L-arginine (2.5 /xCi/ml, NEN), 25 p.I of drug to be examined, 25 /.tl of buffer containing 4 mM CaCi 2, 0.4 mM NADPH and 16110 units/ml of calmodulin in 50 mM Hepes buffer and 25 p.l enzyme preparation were mixed and incubated for 3.5 min at 37°C. The reaction was stoped by adding 2 ml of ice-cold buffer containing 20 mM Hepes, 2.0 mM EDTA, 0.2 mM EGTA, pH 5.5. Citrulline was separated from

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D. l.uo et a l . / European Journal of Pharmacolog3' - Molecular Pharmacology Section 290 (1995) 247-251

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Results The NO synthase activity in the cerebellar supern

w a s s a t u r a b l e ' w i t h a Vmax ° f l ' 7 n m ° l / m g p r ° t e i n ' and a K m with respect to arginine of 5.3 + 0.4 /zM 1A). Under these assay conditions, citrulline prod1 was stoichiometric with NO generation as determin chemiluminescence (Fig. I B). Thus this assay giv accurate assessment of NO synthase activity. Methylene blue was a potent inhibitor of NO syl activity in vitro, with an apparent K i of 2.7 + 0." (Fig. 2A). LY83583 was a somewhat less effectiv still potent inhibitor of neuronal NO synthase, with of 15.8 + 5.2 /xM (Fig. 2B). When added to tissue sections prior to and durit NADPH diaphorase reaction, methylene blue dose-c dently inhibited the histochemical reaction with cot blockade occuring between 0.1 and 1 mM (Fig. 31 inhibition of NADPH-diaphorase staining by metl blue was unaffected by the presence of 100 uni superoxide dismutase in the reaction mixture. As exl: methylene blue also produced a light staining of al ronal cell bodies (i.e. Nissl stain). In contrast, LY83583 did not inhibit the NADI aphorase histochemical reaction, although this was di tO see at the usual concentrations of nitro blue tetraz due to an additional staining of astroglial cells, Be glial cells and tanicytes, not seen in the abser

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through the ascending aorta with 4% paraformaidehyde in 0.1 M sodium phosphate buffer containing 0.9% saline. Following 2 h postfixation at 4°C, the brains were placed in 15% sucrose for 24 h. Sections were cut at 20 /.tm thickness on a freezing microtome and collected in phosphate buffered saline. The sections were incubated in buffer containing vehicle, methylene blue or LY83583 for 30 min. at room temperature, and then incubated in 50 mM sodium phosphate buffer containing the inhibitor plus 1.0 m g / m i B-NADPH and 0.1 m g / m l nitro blue tetrazolium with 0.3% Triton X-100 at 37°C for 30 min. The sections were rinsed in buffer, dried, rinsed in distilled water, dried, soaked in xylene and coverslips applied with Permount. An additional series of experiments was undertaken to examine the role of superoxide anions in the actions of methylene blue or LY83583. 100 U n i t s / m l of superoxide dismutase (Sigma) was included during the preincubation and NADPH-diaphorase hibitor,

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D. Luo et al. / European Journal of Pharmacology - Molecular Pharmacolog)., Section 200 (I995) 247-251

LY83583. This new staining reaction appeared to depend on the redox cycling of LY83583, required the presence of both nitro blue tetrazolium and B-NADPH and was blocked by dicumerol (Vincent and Das, in preparation). When the nitro blue tetrazolium concentration was diluted 8 fold, substantial NADPH-diaphorase staining was still apparent, which was not blocked by 1 mM LY83583.

of guanylyl cyclase (i.e. Lustig et al., 1992; Watson 1990; Zhou et al., 1994). The present results sugge they may instead be due to direct inhibition of N( thase. However, in some situations, high doses of r lene blue do not mimic the effects seen with oth, synthase inhibitors (i.e. Dawson et al., 1993). Thi reflect other actions of methylene blue, not shared w arginine analogues, such as the generation of supe anions.

4. Discussion The present results indicate that both methylene blue and LY83583 arc potent inhibitors of neuronal NO synthase. This agrees with results from other groups which found that micromolar concentrations of methylene blue can inhibit EDRF or citrulline formation in cultured endothelial cells (Palmer and Moncada, 1989: Shimizu et al., 1993), and using purified NO synthase from pig cerebellum (Mayer ct al., 1993). Likewise, Miilsch et al. (1988) have shown that LY83583 can inhibit EDRF production in cultured endothelial cells,

The action of methylene blue on NO synthasc may derive from its ability to oxidize ferrous iron, since NO synthase is known to contain a heme (Klatt et al., 1992: McMillan et al., 1992; White and Marietta, 1992). However, methylene blue is also very readily reduced by cytochrome P450 reductase, persumably by accepting electrons directly from the flavin moieties (Kelner et al., 1988). The reduction of nitro blue tetrazolium by NO synthase appears to derive from the ability of the tetrazolium to accept electrons from the flavins independent of the heine group (Klatt et al., 1992). Methylene blue was a potent inhibitor of the NADPH-diaphorase reaction as w e l l as the NO synthase reaction, suggesting that it shunts single electrons from the flavin-containing cytochrome P450 reductase-like domain of NO synthase. LY83583 does not appear to share this ability since it does not inhibit the NADPH-diaphorase reaction, although it c o n rains a quinone moiety which appears capable of one or two electron reduction. Miilsch et al. (1988) have suggested that LY83583 c a n be reduced in cells to a compound that is able to inhibit soluble guanylyl cyclase. This may correspond to the hydroquinone produced by the two electron reduction of LY83583. Alternatively, the one electron reduction of LY83583 might generate a semiquinone radical, which through redox cycling would lead to superoxide generation (Miilsch et al., 1989). A similar redox-cycling mechanism has been suggested to underlie the ability of methylene blue to inhibit this enzyme (Briine et al., 1990; Wolin et al., 1990). Such a mechanism is likely not involved in the action of methylene blue on NO synthase, since the inhibition of the NADPH-diaphorase reaction was unaffected by addition of superoxide dismutase. Some of the pharmacological effects of methylene blue and LY83583 have previously been attributed to blockade

Acknowledgements Supported by a grant from the Medical Research cil of Canada.

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