NG-Nitro-l -arginine methyl ester and α-methyl-l -ornithine inhibit kyotorphin synthetase from rat brain

Peptides, Vol. 16, No. 7. pp. 1317-1319, 1995 Copyright 0 1995 Elsevier Science Inc. Printed in the USA. All rights reserved 0 196.978 l/95 $9.50 + 00

Pergamon 0196-9781(95)02017-Q

BRIEF COMMUNICATION

NG-Nitro-L-Arginine Methyl Ester and a-Methyl-L-Ornithine Inhibit Kyotorphin Synthetase From Rat Brain ATSUFUMI Department

KAWABATA,

MAKI TANAKA,

of Pharmacology,

HITOMI MUGURUMA

Faculty of Pharmaceutical Sciences, Higashi-Osaka 577, Japan

AND HIROSHI TAKAGI’

Kinki University,

3-4-l

Kowakae,

Received 3 February 1995 KAWABATA, A., Nl. TANAKA, H. MUGURUMA AND H. TAKAGI. N”-Nifro-L-arginine methyl ester and cr-methyl-L-omithine inhibit kyoforphin synthetase from rat brain. PEPTIDES 16(7) 13 17- 13 19, 1995.-Kyotorphin (KTP), an antinociceptive dipeptide (Tyr-Arg), is formed by KTP synthetase from L-Tyr and L-Arg in the brain. We examined the effects of various L-Arg analogues on immunoreactive KTP (IKTP) formation by KTP synthetase purified partially from rat brain. The NO synthase inhibitor NC-nitro-L-arginine methyl ester (L-NAME), but not NC-nitro-L-arginine and N-iminoethyl-L-omithine, suppressediKTP formation by KTP synthetase from 1 mM of L-Arg and L-Tyr, the lCso value being 2.33 mM. Similarly, a-methyl-L-omithine (aMO) inhibited KTP synthetase. the IC,,, value being 2.51 mM. D-Arg at high concentrations also exhibited a weak inhibitory effect. Kinetic experiments indicated that the inhibition by L-NAME and &-MO of KTP synthetase is competitive. Thus, these LArg analogues appex to act as the competitive inhibitor of KTP synthetase. Kyotorphin o-Arginine

Antinociceptive

dipeptide

NG-Nitro-L-arginine

KYOTORPHIN

(KTP), an endogenous [ Met5]enkephalin-re( Tyr-Arg:), plays a role in pain modulation in the mammalian CNS ( 16,17), and is formed by KTP synthetase (KTP-S) from its constituent amino acids, L-Tyr and L-Arg, in the presence of ATP and Mg2+ in the brain ( 18). Exogenously applied L-Arg acts as an effective precursor for KTP, resulting in antinociception in rats and mice (57,9), because the K, value for L-Arg of KTP-S is much higher than its concentration in the mammalian brain under ph;rsiological conditions ( 18). L-Arg is also a substrate for nitric oxide (NO) synthase (NOS). However, exogenously applied L-Arg is incapable of enhancing NO formation in the brain, because NOS is saturated by L-Arg under physiological conditions in the brain (3,7,10). NO appears to play a pro-nociceptive role in the brain (7,10), although some argument still remains at present ( 11). NOS inhibitors including L-NC-nitro-L-arginine methyl ester (LNAME), administered ICV or SC, exhibit antinociceptive activity (7,13). This effect of L-NAME is resistant to L-Arg alone, but reversed by L-Arg in combination with the KTP receptor antagonist L-leucyl-L-arginme ( Leu-Arg ) (7). Thus, L-Arg is considered to be antinociceptive via the KTP pathway, but noleasing

for reprints

should be addressed

a-Methyl-L-omithine

L-Arginine

ciceptive via the NO pathway, based on the in vivo study employing L-NAME (7,10). A number of L-Arg analogue inhibitors of NOS are currently available, but their specificity is not necessarily well established. L-NAME at high concentrations acts as a competitive antagonist of muscarinic receptors (2). NC-Nitro-L-arginine ( L-NOARG) is a weak inhibitor of arginase from rat liver ( 15). Such nonspecific actions of NOS inhibitors may lead to misinterpretation of the results obtained. The present study examined the effects of L-Arg analogue NOS inhibitors on immunoreactive KTP (iKTP) formation by KTP-S from L-Arg and L-Tyr. In addition, we also tested the effect of the L-omithine analogue cy-methyl+omithine ((Y-MO), an inhibitor of omithine decarboxylase, because KTPS may utilize L-omithine as a substrate alternative to t-Arg (8). The results described here indicate that L-NAME and cu-MO act as the competitive inhibitor of KTP-S.

dipeptide

’ Requests

methyl ester

METHOD

KTP-S activity was determined by the method of Ueda et al. ( 18). Male Wistar rats weighing 200-250 g (Japan SLC. Inc.)

to Hiroshi Takagi.

1317

1318

KAWABATA

by ANOVA followed by Newman-Keuls’ < 0.05 level.

ET AL.

test, and was set at p

RESULTS

Concentratton of L-arginine analogs (mM)

6

b

; ;2 Ef

o.oa0.05 -

‘G

8 2 0.04 2 h !$ E 0.03 ‘E 1 g g 0.02 &a = - 0.01 z E

-:

0

’

-’

_a

_

0

0.5 l/[L-Arg] (mM-1)

1

FIG. 1. (a) Effects of L-Arg analogues on the activity of KTP-S from rat brain. L-NAME (O), L-NOARG (Cl), L-NIO (A), a-MO (W), or o-Arg (A) at various concentrations was added to the enzyme reaction mixture containing 1 mM of L-Arg and L-Tyr, 4 mM of ATP and MgCI,, 10 @4 bestatin, and 30 @fp-chloromercuribenzoate. The activity of KTP-S was determined as the amount of iKTP formed for l-h incubation. Results show percent of the control activity and are means from four experiments. (b) Kinetic analysis of the inhibitory effect of L-NAME and (Y-MO on KTP-S. The L-Arg concentration dependence of KTP-S was examined in the absence (0) and presence of 3 mA4 L-NAME (0) or (YMO (W). Results show Lineweaver-Burk plots of representative data.

were killed by decapitation. The diencephalon and brain stem were removed, weighed, and homogenized in 5 vol. of 50 mM Tris-HCl buffer (pH 8.0). The homogenate was centrifuged at 100,000 X g for 1 h at 4”C, and the supematant was concentrated to 0.5-l ml by ultrafiltration with Centriprep-30 (Amicon, USA). This soluble extract was applied to a gel filtration chromatography column, Sephacryl S-300 ( 1 X 90 cm, Pharmacia, Sweden), for partial purification of KTP-S, and the active fraction of the eluate was collected and used as an enzyme sample, because further purifications result in poor recovery and instability of KTP-S ( 18). An aliquot ( 100 ~1) of the enzyme sample was incubated at 37°C with 1 mM of L-Arg and L-Tyr, 4 mM of ATP and MgCl*, 10 PM bestatin, and 30 @4 p-chloromercuribenzoate. The incubation mixture also contained L-NAME (Sigma), L-NOARG (Aldrich), or N-iminoethyl-L-omithine (LNIO; Peptide Institute, Japan), NOS inhibitors, or a-MO (Sigma), an omithine decarboxylase inhibitor, or D-arginine (DArg; Nacalai Tesque, Japan) at various concentrations. After 1 h, the reaction was stopped by boiling for 2 min, and the KTP formed was determined by the radioimmunoassay. The activity and specificity of the anti-KTP serum (a gift from Drs. Shiomi and Nakamura, Fukuyama Univ., Japan) have been tested and reported (9). In kinetic experiments, the L-Arg concentration dependence of KTP-S was determined in the absence and presence of 3 mM L-NAME or a-MO. The results are expressed as the mean with or without SEM. Statistical significance was analyzed

The partially purified KTP-S formed 65.8 pmol/mg protein of iKTP from 1 mM L-Arg and 1 mM L-Tyr for l-h incubation. The NOS inhibitor L-NAME at 0.3-30 mM inhibited the formation of iKTP from 1 mM L-Arg by KTP-S in a concentrationdependent manner [Fig. 1 (a)], the It& value being 2.33 mM (Table 1). By contrast, L-NIO at O.l- 10 mM and L-NOARG at 0.1-3 mM did not significantly affect the activity of KTP-S {Fig. 1 (a)]. The L-Arg concentration dependence of KTP-S in the absence and presence of 3 mM L-NAME as double-reciprocal plots from representative data indicates a competitive form of inhibition by L-NAME of KTP-S [Fig. 1 (b)] . In the absence and presence of L-NAME, the Km values (mM) for L-Arg of KTP-S were 3.32 ? 0.82 and 10.44 2 0.86, and the V,,,,, values (pmol/ mg protein/h) were 186.9 2 19.2 and 190.9 ? 13.5, respectively (n = 4). L-NAME at 3 mM significantly (p < 0.01) elevated the K,,, , but not the V,,,,, . cr-MO inhibited iKTP formation in a concentration-dependent manner [Fig. 1 (a)], the I& value being 2.5 1 mM (Table 1) The kinetic analysis of L-Arg concentration dependence in the absence and presence of 3 mM a-MO indicates a competitive inhibition by tr-MO of KTP-S [Fig. 1 (b)] . The Km value for LArg and V,,, value of KTP-S in the presence of (Y-MO were 9.50 ? 1.46 mM and 220.1 t- 13.8 pmol/mg protein/h, respectively (n = 4). a-MO significantly (p < 0.01) elevated the K,,, , but not the V,,,,,. D-Arg also notably inhibited KTP-S activity, although it failed to produce complete inhibition of iKTP formation, even at 100 mM. DISCUSSION

This study revealed that the NOS inhibitor L-NAME and the omithine decarboxylase inhibitor (Y-MO competitively inhibit iKTP formation by KTP-S from the rat brain at relatively high concentrations, although it is not clear at present if they act as substrates alternative to L-Arg for KTP. In the mammalian brain, KTP binds to its specific receptors and activates phospholipase C in a G-protein-coupled manner ( 19), playing a role in pain modulation ( 17). Leu-Arg, a synthetic dipeptide, is considered a KTP receptor antagonist, because it binds to KTP receptors but does not activate phospholipase C ( 19). Actually, Leu-Arg, administered ICV, completely antag-

TABLE

1

COMPARISONOF INHIBITORYEFFECTS

OF L-NAME AND a-MO ON KTP-S AND NOS OR ORNITHINE DECARBOXYLASE IC,,, or K, Value (mM)

Enzymes

I(, Value for the Substrate(n&f)

KTP-S

0.93-3.32*t (for L-A@

NOS

0.002-0.008~ (for L-Arg)

Omithine decarboxylase

0.047t (for Lomithine)

* Values obtained from the present study. t From (1,10,12.18).

L-NAME

a-MO

2.51* 2.33* (ICsO in the presence of 1 mM L-Arg) 0.0016t (ICsO in the presence of 0.02 m&f L-Arg) 0.02-t (K, value)

L-ARG ANALOGUES

AND KYOTORPHIN

onizes the antinociception induced by KTP and by L-Arg (6). We have previously demonstrated that the antinociceptive effect of ICV administration of L-NAME at 10 pg/mouse was resistant to an antinociceptive dose (30 pg/mouse) of ICV L-Arg, but was reversed by the same dose of ICV L-Arg when the antinociceptive activity of L-Arg itself was blocked by coadministration of the KTP receptor antagonist Leu-Arg, thereby suggesting opposing roles of NO and KTP in supraspinal nociceptive processing (7). Thus,

L-Arg

plays

a dual

role

in pain

modulation

1319

SYNTHETASE

in the brain,

being pro-nociceptive via NO formation and antinociceptive via KTP formation (7,10). The inhibitory effect of L-NAME on KTP-S in the present study may complicate the interpretation of the results from the above in vivo experiments (7). However, LNAME at 10 pg/mouse does not appear to effectively suppress increased KTP formation by L-Arg at 30 pg/mouse, considering its weak inhibitory effect on KTP-S. In addition, in vivo inhibition by L-NAME of formation of the antinociceptive KTP, if any, would rather promote nociception. In support of the above notion, we actually confirmed antinociceptive activity of L-NIO (3 pg/mouse, ICV) ( 11 ), anotlher NOS inhibitor that did not affect KTP-S in the present study. the effect being reversed by L-Arg (30 pg/mouse, ICV) in combination with Leu-Arg ( 10). In addition, the novel type of NOS inhibitor 7-nitro-indazole and other indazole derivatives also produce potent antinociception ( 14). As shown in Table 1, the Km value for L-Arg of NOS is much lower than that of KTP-S, suggesting that the former enzyme but not the latter are saturated by L-Arg in the brain under physiological conditions (7,10). NOS is very sensitive to L-NAME in vitro, the I&, value being I .6 pM in the presence of 20 pM LArg (Table 1) ( 12). Nevertheless, extremely large doses of LNAME have been often employed in vivo (4,13 ). The effect of

L-NAME on KTP-S should be taken into consideration, if it is employed at doses larger than those of L-Arg. Surprisingly, L-NOARG (0.1-3 mM) did not notably alter the activity of KTP-S, differing from L-NAME, its alkyl ester. The L-Arg-omitted reaction mixture including 3 mM of LNOARG, L-NAME or L-NIO did not generate any immunoreactive substances for l-h incubation (data not shown). Therefore, it is unlikely that L-NOARG inhibits KTP formation but forms an unexpected immunoreactive dipeptide (for instance, L-Tyr-LNOARG) by acting as a substrate alternative to L-Arg for KTPS, apparently resulting in no inhibition. The reason for the difference between L-NAME and L-NOARG in inhibiting KTP-S remains to be investigated. D-Arg, when administered SC or ICV in combination with SC or ICV L-Arg, abolishes the antinociceptive activity of L-Arg in rats and mice (5,6). This phenomenon may be explained, in part, by the inhibitory effect of D-Arg on KTP-S, although other mechanisms also seem involved, considering its weak inhibitory effect. a-MO may be used as a more potent inhibitor of KTP-S, considering the lCso value in the present study (Table 1) . In our preliminary experiments, ICV a-MO, which itself produced no effect, actually inhibited L-Arg (lCV)-induced antinociception in mice (data not shown). Collectively, the inhibitory effects of L-NAME, a-MO, and D-Arg on KTP-S should be considered, if they are employed in pain research. ACKNOWLEDGEMENTS We are grateful to Drs. H. Shiomi and A. Nakamura (Fukuyama Univ., Japan) for the generous gift of anti-KTP serum. This paper is the eighth on “pain modulation

by neuroactive

amino acids.”

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