Nitric oxide synthase in cat brain: Cofactors—enzyme-substrate interaction

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Free Radical Biology & Medicine,Vol. 21, No. 1, pp. 109-115, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/96 $15.00 + .00

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SSDI 0891-5849(95)02208-2

ELSEVIER

Brief Communication NITRIC COFACTORS

OXIDE

SYNTHASE

--ENZYME-

IN CAT

SUBSTRATE

BRAIN:

INTERACTION

JEAN-FRAN(~OIS COTI~ and ANDRI~E G. ROBERGE INRS-Sant6, Universit6 du Qu6bec, 245 Boulevard Hymus, Pointe-Claire, Qu6bec, Canada H9R 1G6

(Received 25 July 1995; Revised 1 November 1995; Accepted 6 November 1995)

Abstract--Nitric oxide, derived from L-arginine by the enzyme nitric oxide synthase, is an activator of the soluble guanylate cyclase and a cellular messenger. This work demonstrates that, in cat brain, the neuronal constitutive nitric oxide synthase activity is a) NADPH/calcium dependent, b) independent upon exogenous calmodulin in crude brain supernatant, c) significantly enhanced by exogenous FAD and tetrahydrobiopterin (Vmax: 118 instead of 59.4 pmol of citrulline formed "mg of prot- -t min -1, d) inhibited by calcium chelators and calmodulin antagonist, and e) present in several neuroanatomical structures. Moreover, the K~ value for L-arginine was of 11 #M instead of 41 #M in the presence of FAD and tetrahydrobiopterin in the incubation mixture, thus demonstrating that these cofactors are able to stabilize the enzyme-substrate interactions. Keywords--Nitric oxide synthase, Cat brain, FAD, FMN, Tetrahydrobiopterin, Free radicals

ogous to cytochrome P-450 reductase, 21 a flavoprotein binding both FAD and FMN. The purpose of the present work was to study a) the kinetic properties and the biochemical parameters of the constitutive nNOS enzyme in cat brain and b) its regional distribution based on neuroanatomical pathways.

INTRODUCTION

Tissues and cells types such as macrophages and endothelial cells, ~ neutrophils, 2 brain tissue, 3 and adrenals 4 are able to oxidize L-arginine to yield nitric oxide ( N O ) and L-citrulline ( C I T ) , as a coproduct. Such a cytosolic enzyme conversion is yielded by NO synthase (NOS; EC 1.14.13) that exists in three isoforms. 5'6 Two of the three isoforms are C a 2 + / c a l m o d u lin (CaM)-dependent enzymes and are constitutively expressed in neuronal (nNOS) and endothelial cells. The other enzyme isoform is known as an inducible NOS, whose expression is regulated by substances such as cytokines 7 or by tumour necrosis factor and LPS. 8 Kinetic parameters have been studied in rat forebrain 9 and cerebellum 3 using a semipurified enzyme source and a purified cerebellum enzyme in rat, ~°A1 porcine, 12 bovine, 13 and human. 14 The distribution of the constitutive nNOS has been described in rat brain 15.16 and identified by immunochemical mapping in rat, 17 human, TM and mouse brain) 9'2° The role of r a v i n in NO synthesis was first taken into account when NOS was cloned and demonstrated to be homol-

MATERIALS AND METHODS

Twelve adult mongrel cats of both sexes weighing 2.8 _+ 0.2 kg were adapted to environmental conditions for at least 3 weeks and were judged healthy by a veterinarian. They were housed in individual stainless steel cages in a room controlled at constant temperature (21 °C ) and humidity (55 % ), with fluorescent light and background music from 0600 to 1800 h. They were fed with a casein diet 22 and had water ad lib. Cats were guillotined and brains were rapidly set aside on ice. The following structures were dissected out according to the method of Glowinski and Iversen: 23 the frontal cortex, hippocampus, striatum, thalamus, hypothalamus, mesencephalon and pons raphe nuclei, mesencephalon, medulla, and pons. These structures were rapidly stored at - 8 0 ° C until biochemical determina-

Address correspondence to: Andr6e G. Roberge, INRS-Sant6, 245 Hymus Blvd, Pointe Claire, Qu6bec Canada H9R 1G6. 109

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NADPH (raM) Fig. 1. Effect of NADPH concentrations on specific NOS enzyme activity in cat brain supematant. Each point on the graph represents a triplicate obtained from a pool of cat brain and expressed as mean ± SEM.

tions were performed. P o o l e d brains were u s e d to determine the kinetic properties and b i o c h e m i c a l p a r a m e ters. Tissues were w e i g h e d and h o m o g e n i z e d in 10 vol o f buffer ( 2 0 m M H E P E S at p H 7.5, 0.4 m M E D T A , 1 m M DTT, 0.32 M s u c r o s e ) using a p o t t e r - E l v e h j e m h o m o g e n i z e r . The h o m o g e n a t e was centrifuged for 20 m i n at 20,000 × g at 4°C using a Sorvall R C 5C e q u i p p e d with a S S - 3 4 rotor. To r e m o v e e n d o g e n o u s arginine, the supernatant was p a s s e d through m i c r o c o lumns containing a p r e e q u i l i b r a t e d 2 ml ion e x c h a n g e

resin A G 50 W X 8 2 0 0 - 4 0 0 . Proteins were m e a s u r e d b y the m e t h o d o f B r a d f o r d 24 using b o v i n e serum albumin as a standard. The supernatant was stored at - 2 0 ° C until e n z y m e assays were carried out.

Enzyme assays N O S e n z y m e activity was e s s a y e d b y m e a s u r i n g the c o n v e r s i o n o f [ 3H ] arginine to [ 3H ] citrulline ( C I T ) according to Bredt and Snyder. 1° T h e incubation me-

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CALCIUM (mM) Fig. 2. Effect of Ca 2+ concentrations on specific NOS enzyme activity in cat brain supernatant with ( e ) or without (©) CaM and in the presence of 5 mM EDTA ( 1 ) . Each point on the graph represents a triplicate obtained from a pool of cat brain and expressed as mean __+SEM.

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prepared in the same manner as the samples except that the enzyme source was replaced by buffer. Two sets of internal standards were prepared, one set containing [3H] arginine (0.5 #Ci/ml) and the other [ 14C] CIT (13 nCi/ml). External standards contained [3H] arginine (0.5 /zCi/ml) and [~4C] CIT (13 nCi/ml). The results are expressed in pmol of CIT formed • mg of prot. -~ min -I .

Materials

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L-[2,3 3H] arginine (35.0 Ci/mmol), L-dithiothreitol, EDTA, EGTA, FMN, and FAD were obtained from Sigma Company (St. Louis, MO). Calmodulin, NADPH (reduced form), W-7 [N-(6-aminohexyl)-5chloro 1-naphtalenesulfonamide], arginine, and HEPES buffer were obtained from Calbiochem (San Diego, CA). Ion exchange resin AG50 WX8 200400 mesh was purchased from BIO-RAD (Ontario, Canada) and L-[ureido-~4C]-citrulline (55.6 mCi/ mmol) was obtained from NEN/DuPont (Ontario, Canada). Tetrahydrobiopterin (HaB) was purchased from Dr. B. Schirks Laboratories (Jona, Switzerland).

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RESULTS AND DISCUSSION 20 0

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W 7 (pM) Fig. 3. (A) Effect of calcium chelators EDTA (O) and EGTA (©) on specific NOS enzyme activity in cat brain supernatant. (B) Effect of CaM antagonist W-7. The IC50 is 218 /~M. Each point on the graph represents a triplicate obtained from a pool of cat brain and expressed as mean +__SEM.

dium contained: 300/zl of enzyme source ( ~ 450/zg of prot) and 120 /zl of 20 mM HEPES buffer containing (in final concentration): 200 #M L-arginine ( 0.5 #Ci/ml; dilution factor 1:14000 "hot" vs. "cold" L-arginine), 30 nM calmodulin, 0.3 mM CaC12, 10 ~M FAD, FMN, and tetrahydrobiopterin (H4B), and 1.6 mM NADPH in a final volume of 420 lzl. The incubation was carried out at 37°C for 60 min in a shaking water bath. The enzyme reaction was started with the enzyme source and stopped by the addition of iced water (500/A). The CIT formed was separated from arginine by decanting the incubation medium on microcolumns containing a preequilibrated 2 ml ion exchange resin AG50 WX8 200-400 mesh. The [3H] CIT was eluted with 1 ml of water and counted in a LKB scintillation counter. Boiled enzyme (5 min at 100°C) was used as a blank. Internal standards were

The citrulline (CIT) formed was proportional to the protein concentration used in the incubation mixture ranging from 0 to 2.8 mg/ml of supernatant (data not shown). A supernatant containing 1.6 mg prot/ml was usually retained. For optimal pH, the specific NOS enzyme activity was determined in a pool of cat brain using a range of pH from 6.6 to 8 done in triplicate (data not shown). The enzyme activity reached a maximum at pH 7.5, and this pH was retained for all further experiments. The incubation time was tested for periods up to 60 min and the formation of CIT was found linear. The incubations were carried out at pH 7.5 for 60 min.

NADPH dependence In Fig. 1, the effects of NADPH (0-2.6 mM) are demonstrated with 200 #M arginine concentration in the incubation mixture. A progressive and significant increased enzyme NOS activity was noted followed by a plateau reached at 1 mM NADPH and maintained till 2.6 mM. Without NADPH in the incubation mixture, the NOS enzyme activity was reduced to 7% of the control value (1.6 mM NADPH; p < .001). For all experiments, the NADPH concentration used was 1.6 mM. The present results demonstrate that, in cat brain, NADPH is an essential cofactor of the reaction involving the oxidation of arginine to CIT by nNOS

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ARGININE (pM) Fig. 4. Effect of arginine concentration on specific NOS activity in cat brain supernatant with (O) or without (B) 10 #M FAD and H4B. Each point on the graph represents a triplicate obtained from a pool of cat brain and expressed as mean ___SEM. Kinetic parameters of NOS enzyme in cat brain supernatant were calculated from plots of substrate concentration according to Lineweaver-Burk (25). Km and Vm~xvalues were respectively of 41 #M and 59.4 pmol of CIT formed • nag prot. ~ rain-~ without FAD and H.B and 11 #M and 118 pmol of CIT formed .mg prot. -~ rnin-~ with FAD and H4B in the incubation medium.

enzyme as also observed in rat brain, 9'1° porcine cerebellum, 12 and mouse neuroblastoma cells. 25

Ca2+/CaM relationship Figure 2 shows the effects of calcium ranging from 0 to 1.3 m M in the incubation mixture in the presence ( • ) or absence ( O ) of exogenous 30 nM CaM. With calcium concentrations up to 0.3 raM, a progressive and significant increased enzyme activity was observed followed by a plateau maintained till 1.3 m M (p < .001 ). In crude brain supernatant, exogenous CaM did not enhance the cat NOS enzyme activity as previously demonstrated in rat brain, m However, in the presence of W-7, a CaM antagonist, ranging from 10 to 1000 #M, a significant and progressive inhibition in the enzyme activity was observed with a IC50 of ~ 220 # M (p < .001 ) (Fig. 3b). Since at 1 m M W-7, no enzyme activity was observed, it thus suggests that cat brain nNOS enzyme activity is dependent upon endogenous CaM content. In Fig. 3a, the effects of added E D T A ( • ) and E G T A ( O ) in the incubation mixture with concentrations ranging from 0 to 2 m M are shown on nNOS enzyme activity. A progressive and significant de-

crease in the enzyme activity was observed with both EDTA and E G T A (p < .05; p < .01). At 2 mM E D T A and EGTA, the nNOS enzyme activity was, respectively, 47 and 63% of the control values, whereas at 5 m M EDTA (Fig. 2, I I ) , the enzyme activity was resumed to 26% of the control values. In rat brain, no enzyme activity was noted in the presence of a calcium chelator ( E G T A ) , 9 thus suggesting that the remaining enzyme activity observed with 5 mM EDTA, in cat brain, might be related to the endogenous CaM, which could be more tightly bound to the active site of the enzyme, thus conferring to it a greater stability in the absence of calcium.

Kinetic parameters and FAD/H4b involvement In Fig. 4, the effects of arginine ( 0 - 2 0 0 # M ) in the incubation mixture are described in the absence (11) or presence ( O ) of 10 # M FAD and I-LB. The enzyme activity significantly increased in respect to arginine concentrations till a plateau was reached at 200 # M (11) and 100 # M ( O ) , respectively. The kinetic parameters were calculated according to Lineweaver and Burk 26 according to arginine concentrations ( 0 - 2 0 0 # M ) in the incubation medium. Without

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Moreover, its homology with cytochrome P-450 reductase indicates similar mechanisms for electrons transport because these two enzymes are the only proteins known to be functionally related to FAD and FMN. 7'21'27 However, in spite of the fact that the requirements for H4B were specific to NOS,28 its role is still obscure, 29 probably being involved in the hydroxylation of arginine by mechanisms similar to other hydroxylases such as tyrosine and tryptophan hydroxylase) ° In the present study, it was clearly demonstrated that the presence of FAD and HnB in the incubation mixture had significantly enhanced the nNOS in cat brain thus confirming the NOS enzyme as a biopterinand ravin-enzyme containing multifunctions.

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In Fig. 5, the effects of L-Nw-nitroarginine (LNNA) and L-Nu)-monomethylarginine ( L - N M M A ) added in the incubation mixture with the following concentrations 0.2 #M, 0.5 #M and 1 #M for L-NNA (Fig. 5a) and 10 #M and 20 #M for L - N M M A (Fig. 5b) are described. The kinetic parameters calculated for each arginine analog gave the following results: the Ki values were for L-NNA 0.041 +_ 0.009 #M and for L-NMMA 0.31 + 0.5 #M. In rat cerebellum, LNMMA was found as a potent inhibitor of the L-arginine:NO pathway with a Ki value of 1.5 #M 3 and 1.6 #M, ~ respectively. Similar results were observed in bovine adrenal glands. 4 In cat brain, a dose-response curve using L-NMMA has demonstrated the absence of enzyme activity with 500 #M L-NMMA, whereas

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FAD and H4B, the apparent Km and V m a x values for arginine were 41.1 #M and 59.4 pmol of CIT formed • mg of prot- -~ min -~, respectively, whereas in the presence of these cofactors, the Km and Vm~x values were 11 #M and 118 pmol of CIT formed . m g of prot- -~ min -~ , respectively. A greater affinity of nNOS for its substrate arginine noted in the presence of FAD and HaB suggests their involvement in the protein structure thus facilitating the e n z y m e - s u b strate interactions. According to the litterature, the Km and V max values observed in cat brain for nNOS are in the same order of magnitude that the one noted in a semipurified rat enzyme source 3'9 in the presence of FAD and H4B in the medium. The role of NADPH as essential cofactor was first associated to FAD and FMN when nNOS was cloned. 2~

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Fig. 6. Regional distribution of NOS enzyme activity in cat brain. The enzymeactivitywas measured in 10 neuroanatomicalstructures: mesencephalon raphe nuclei, thalamus, hypothalamus, striatum, pons, hippocampus, medulla, pons raphe nuclei (pons r.n), cortex, and mesencephalon (mes). Twelve cat brains were used and each value represents the mean + SEM of 12 determinations done in triplicate.

114

J.-F. (~O'tE and A. G. ROBI~RGE

in rat brain 3 higher concentrations (2000 #M) are needed to completely inhibit the enzyme activity thus suggesting a greater sensitivity of cat brain nNOS enzyme activity to arginine analogs inhibitors.

2.

3.

Enzyme distribution Figure 6 shows the regional distribution of nNOS enzyme activity measured in 10 different brain structures obtained from 12 cat brains. The highest value was observed in the mesencephalon raphe nuclei and the lowest one in the frontal cortex and mesencephalon (rues). Based on neuroanatomical pathways, the structures belonging to the mesolimbic system showed a greater nNOS enzyme activity than the structures related to the motor system particularly with a low activity in the frontal cortex. The present study done in cat brain confirms that, through the central nervous system, nNOS enzyme activity is widely distributed as already described in extensive studies relating the localization of NADPH-diaphorase staining to NOS enzyme activity. 16"31-33Moreover, in respect to catecholamines content within the cat brain, the dopamine fi-hydroxylase enzyme activity corresponds, in its distribution, to some extent to nNOS because the highest activity was observed in the hypothalamus, the raphe nuclei areas and piriform lobe and the lowest one in the structures belonging to the motor system. 34 In this respect, NOS and dopamine fi-hydroxylase enzyme activities were found t o b e colocalized in bovine superior cervical ganglion cells. 35 The present study describes for the first time the characteristics and kinetic parameters of the constitutive nNOS enzyme in cat brain and demonstrates that nNOS enzyme activity in crude brain supernatant is a) NADPH/calcium dependent, b) independent upon exogenous CaM, c) significantly enhanced by FAD and H4B added in the incubation medium and, d) widely distributed within the brain, its activity being related to neuroanatomical pathways. Moreover, a greater affinity for its substrate arginine was noted in the presence of FAD and H4B, thus suggesting their involvement in the protein structure facilitating the enzyme-substrate interactions and in the electrons transport mechanisms. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). The authors want to acknowledge Michel Charest for his special technical assistance training tO Jean-Franqois crt6 and Dr. JoE1 De la Notie, Director of GREREBA at Laval University, for its particular comprehensiveness.

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5. 6. 7.

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12. 13. 14. 15.

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17. 18. 19.

Acknowledgements - -

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