Arginine-related guanidino compounds and nitric oxide synthase in the brain of ornithine transcarbamylase deficient spf mutant mouse: effect of metabolic arginine deficiency

ELSEVIER

Neuroscience Letters 215 (1996) 153-156

N[UROSCI[HC[ LETTERS

Arginine-related guanidino compounds and nitric oxide synthase in the brain of ornithine transcarbamylase deficient spf mutant mouse: effect of metabolic arginine deficiency L. Ratnakumari a, I.A. Qureshi a'*, R.F. Butterworth b, B. Marescau c, P.P. D e D e y n c aDivision of Medical Genetics, Sainte-Justine Hospital, 3175 Chemin Cote-Ste-Catherine, Montreal, Qudbec H3T 1C5, Canada bNeuroscience Research Unit, Saint-Luc Hospital, Montreal, Canada CLaboratory of Neurochemistry and Behavior, Born-Bunge Foundation, University of Antwerp, U.1.A., 2610 Antwerp, Belgium

Received 3 January 1996; revised version received 29 July 1996; accepted 31 July 1996

Abstract

The sparse-fur (spf) mouse, with an X-linked hepatic ornithine transcarbamylase (OTC, E.C.2.1.3.?" deficiency, exhibits significantly lower levels of arginine in the brain as compared to normal controls. In the present study, the effe~ t of a sustained lower metabolic arginine was studied by measuring the levels of several arginine-related guanidino compounds ir~ brain. The concentrations of ~,guanidinobutyric acid (3,-GBA), N-~-acetylarginine (N-~-AA), argininic acid (Arg-A), guanidinoacO c acid (GAA), and creatine were significantly lower in spf mice as compared to controls. Since arginine is the precursor for nitric oxide we also measured the activity of nitric oxide synthase which was significantly reduced in cerebellum, striatum, hippocampus and erebral cortex of spf mice. The changes seen in cerebral guanidino compound and nitric oxide metabolism of spf mice could be due to a sustained deficiency of arginine, caused by a metabolic block in the urea cycle. Keywords: Guanidines; Brain; Congenital hyperammonemia; Nitric oxide synthase; Metabolic arginine deficiency

The guanidino derivatives accumulate significantly in certain metabolic disorders such as uremia [2,3] and hyperargininemia [ 10] which contributes to the pathophysiology of these disorders. Arginine, the conditionallyessential amino acid, plays an important role in the guanidino compound metabolism [20] by providing the guanidino group. The c~-keto-6-guanidinovaleric acid (ot-k-6GVA), N-cc-acetylarginine (N-c~-AA), argininic acid (Arg-A) and 3,-guanidinobutyric acid (3,-GBA) are the catabolic products of arginine resulting from the action of a transaminase, an acetylase, a dehydrogenase and a transamidinase. The sparse-fur (spf) mutant mouse, with an X-chromosomal defect of hepatic ornithine transcarbamylase (OTC), serves as a useful animal model to study the metabolic consequences of congenital urea cycle disorders [12,13].

* Corresponding author. Tel.: +1 514 3454931, ext. 3587; fax: +1 514 3454766.

With less than 10% of normal liver OTC activity and a significantly increased urinary orotate excretion, the spf mutant mouse very closely resembles the congenital h y p e r a m m o n e m i a type II syndrome with metabolic arginine deficiency seen in children [8]. The serum and cerebral arginine levels are significantly lower in spf mutant mice as compared to control mice [5,14,19]. In the present study, we investigated the effect of sustained lower arginine levels on the concentration of arginine-related guanidino compounds in OTC deficiency. The cerebral levels of several of these guanidino compounds were significantly decreased in spf mutant mouse as compared to normal controls. Decreased levels of cerebral arginine in OTC deficiency might hypothetically result in decreased levels of nitric oxide (NO). NO is produced by nitric oxide synthase (NOS) from arginine through oxidation of its guanidino nitrogen [6]. To assess the role of sustained low levels of arginine on the cerebral NO production, we have also measured the activity of NOS in four different brain

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regions of OTC deficient spf mice and observed significantly lower levels. Male spf mice used in this study were the progeny of matings of homozygous affected spf/spf females with spf males [12]. CD-1 strain mice obtained from Canadian Breeding Farms, St. Constant, Qu6bec, Canada were used as normal controls. All animals were kept in a controlled environment (12 h dark/12 h light) with free access to water and food (Purina mouse chow; Ralston Purina, St. Louis, MO, USA) at the animal house of the Saint-Justine Hospital. The animals were kept and experimented upon according to the guidelines of the Canadian Council on Animal Care (Guide to the care and use of experimental animals, Vol. 2, 1984). [h-3H]Arginine (specific activity, 58 Ci/mmol) was purchased from Amersham. (6R)-5,6,7,8-tetrahydro-h-biopterin dihydrocfiloride, nicotinamide adenine dinucleotide phosphate (NADPH), calmodulin and N-c0-nitro-h-arginine were obtained from Sigma Chemical Co., MO, USA. Dowex 50WX (hydrogen form) was obtained from Sigma Chemical Co., and later converted to the Na + form. Brain ammonia levels were estimated by an ionexchange method followed by colorimetric measurement of isolated ammonia nitrogen with the Berthelot phenatehypochlorite reaction [15,16]. Liver OTC activity was measured as described previously [12] to verify the mutant status of the spf animals. Cerebral amino acids were separated over a cation exchange column using lithium citrate buffers and were detected with the colorimetric ninhydrin method [11] using a Biotronik LC 6001 (Biotronik, Maintal, Germany) amino acid analyzer. The guanidino compounds were separated over a cation exchange column using sodium citrate buffers and were detected with the fluorescence ninhydrin method ]11] using a Biotronic LC 5001 amino acid analyzer. The NOS activity was determined as formation of [LPH]citrulline from [L-3H]arginine [ 1]. Protein concentrations were determined by the method

OTC

NH3 Orn Cit Arg -120

-80

-40

0

40

80

120

Percent change over control

Fig. h Changes in hepatic ornithine transcarbamylase (OTC) activity, cerebral ammonia (NH3) and amino acid levels in the spf mutant mouse. n = 4. *P < 0.01. **P < 0.001 compared to control (Student's t-test).

Table 1 Cerebral levels of arginine-related guanidino compounds in OTC deficient spf mutant mouse Compound

Control

spf/Y

% Change over control

Arginine ct-k-6- GVA 7-GBA N-a-AA Arg-A GAA CT CTN GSA

128 -+ 3.1 0.65 -+ 0.05 1.14 -+ 0.02 0.7 -+ 0.11 1.36 _+0.04 6.91 -+ 0.49 8006 4- 199 226 -+ 11 0.24 -+ 0.02

78 + 7.5** 0.59 _+0.04 0.82 + 0.03* 0.08 -+ 0.04** 0.9 + 0.01"* 4.53 + 0.73** 7224 _+ 180" 210 _+ 15 0.19 _+0.01"

-40 -9 -25 -89 -34 -34 -10 -7 21

Each value is the mean _+SE of four animals. Units, nmol/g, c~-k-6-GVA, ~-keto-6-guanidinovaleric acid; 7-GBA, guanidinobutyric acid; N-o~AA, N-acetylarginine; Arg-A, argininic acid; GAA, guanidinoacetic acid; CT. creatine; CTN, creatinine; GSA, guanidinosuccinic acid. of Lowry et al. [9]. Statistical analysis was done by the Student's t-test. All the data are expressed as mean _+ SE. The brain ammonia levels were significantly elevated in spf mutant mice (2.85 + 0.2 versus 1.4 + 0.05/xmol/g tissue) as compared to control mice (Fig. 1). The hepatic OTC activity levels of spf mutant mice were approximately 10% of the activity levels of control mice (6.23 + 0.8 versus 64.2 _+ 4 ~mol/min per mg citrulline) (Fig. 1). The cerebral levels of all three amino acids i.e. arginine (128 _+ 3.1 versus 78 _+ 7.5 nmol/g), omithine (12 _+ 3 versus 7 + 0.7 nmol/g) and citrulline (12 _+ 0.3 versus 8 + 0.1nmol/g) were significantly lower in spf mice as compared to normal controls (Fig. 1). The levels of 3,-GBA, N-c~-AA, Arg-A, guanidinoacetic acid (GAA), creatine, and guanidinosuccinic acid were significantly decreased in spf mutant mice as compared to normal controls (Table 1). Cerebral levels of homoarginine and/3-guanidinopropionic acid were below the level of detection in both control and spf mutant mice (not reported in Table 1). NOS activities were significantly decreased in the cerebral cortex (-22%), striatum (-22%), cerebellum ( - 4 2 % ) and hippocampus ( - 1 8 % ) of t h e spf mutant mice as compared to controls (Fig. 2). Results of the present study show that spf mice with congenital OTC deficiency are hyperammonemic (Fig. 1), exhibiting only 1/10th of the control hepatic OTC activity (Fig. 1) which agrees well with our earlier reports [15,16]. The decrease observed in the cerebral arginine, citrulline and ornithine levels of spf mutant mice as compared to normal controls may be the consequence of decreased levels of these amino acids in the serum [14]. Several catabolic products of arginine, like 3,-GBA, N-uA A and Arg-A were significantly decreased in the spf mutant mouse brain (Table 1) which probably is caused by the lower levels of cerebral arginine. G A A was also decreased along with creatine (Table 1). In another urea cycle disorder, arginase deficiency, which is associated with excessive arginine in cerebrospinal fluid (CSF),

L. Ratnakumari et al. / Neuroscience Letters 215 (1996) 153-156

[] •

control spf/Y

.=

c-

.:cc~ ..?-<-~ .5cc~

O

:i:i:il 'J,2"J4 :!:!:il

E e.

'5,?,?4

0

CC

CB

ST

HC

Brain regions

Fig. 2. Activity levels of nitric oxide synthase in the cerebral cortex (CC), cerebellum (CB), striatum (ST) and hippocampus (HC) of control and OTC deficient spf mutant mice. Each value is mean + SE from five different animals. *P < 0.01, **P < 0.001 compared to control (Student's t-test). plasma and other body fluids, a significant increase in most of the arginine-related guanidino compounds like, creatine, c¢-k-6-GVA, 3,-GBA, G A A , N - ~ - A A , A r g - A and homoarginine has been reported [10]. A comparison of the pathobiochemistry of these two metabolic disorders would demonstrate the role of arginine levels on the concentration of arginine-related guanidino compounds. Although a totally functional urea cycle is present only in liver, it has been shown that arginine synthesis takes place in brain [20]. However, some arginine enters the brain through the b l o o d - b r a i n barrier by an active transport system. The arginine synthesis in the brain depends on circulating citrulline levels [4] which mainly comes from the intestinal mucosa [23]. The exact regulatory mechanism for guanidino compound synthesis in brain is not yet clear. The studies of Watanabe et al. [22] suggested a different regulatory mechanism for G A A in the brain. It is possible that some of the changes seen in the cerebral levels of guanidino compounds in the present study might be the result of altered arginine, ornithine and citrulline metabolism of other organs like liver, intestine and kidney. Arginine has the unique property of needing to be synthesized endogenously from circulating citrulline arising from its intestinal synthesis. As already mentioned, this metabolic capacity of endogenous arginine synthesis is compromised in the spf mutant mice due to a deficiency of intestinal mucosal OTC [13], which is necessary for the synthesis of circulating citrulline [23]. The normal +/Y mice used as controls in our studies had an adequate capacity to synthesize metabolic arginine due to normal intestinal OTC activity which would contribute normal levels of citrulline [13]. Since NO is derived from the guanidino group of arginine by NOS, we studied the levels of NOS in different brain regions of spf mouse. The results demonstrate a significant decrease in the activities of the constitutive NOS in cerebellum, striatum, hippocampus and cerebral cortex of spf mice when compared to normal controls. It has been

155

suggested that one way of inhibiting NOS in biological systems is to modulate the supply of one of its substrates or cofactors [6]. Exogenous administration of L-arginine has been shown to enhance NO production in brain slices [21]. In spf mice the chronic and congenital deficiency of arginine has been observed at plasma, hepatic and cerebral levels but most of these studies were done using adult animals. However, it is important to study the developmental changes in NOS activity levels along with the arginine and citrulline levels to understand the possible mechanism underlying the downregulation of NOS activities. Recently, we also demonstrated a downregulation of N-methyl-D-aspartate ( N M D A ) receptors [ 17] and upregulation of muscarinic cholinergic receptors [18], in several brain regions of adult spf mice. It has been reported that inhibition of NOS could cause decreased acetylcholine release in medial pontine reticular formation [7]. In conclusion, the results of the present study would indicate that during congenital OTC deficiency, the sustained low levels of cerebral arginine would result in a decreased production of several guanidino compounds including creatine and NO. Since NO serves as an important messenger molecule in the regulation of cell function and also as an endogenous anti-convulsant, the observed decrease in NOS could have important pathophysiological implications in seizures and cerebral atrophy seen in this metabolic disorder. The studies described were funded by The Medical Research Council of Canada (MT-9124), U.I.A., N F W O (grants 3.0044.92 and 3.0064,93), Born-Bunge Foundation, the United Fund of Belgium and the O C M W Medical Research Foundation. W e would like to thank Mr. Michel Leblanc for his help in breeding the spf mutant mice and Ms. Micheline Patenaude for her secretarial assistance. Part of this work was presented at the IVth International symposium on Guanidines, Montreal, 1994. [1] Bredt, D.S. and Snyder, S.H., Isolation of nitric oxide synthetase, a calmodulin requiring enzyme, Proc. Natl. Acad. Sci. USA, 87 (1990) 682-685. [2] Cohen, B.D., Stein, I.M. and Bonas, J.E., Guanidinosuccinic aciduria in ureamia. A possible alternate pathway for urea synthesis, Am. J. Med., 45 (1968) 63-68. [3] De Deyn, P.P., Marescau, B., Lornoy, W., Becaus, I. and Lowenthal, A., Guanidino compounds in ureamic dialysed patients, Clin. Chim. Acta, 157 (1986) 143-150. [4] Featherston, W.R., Rogers. Q.R. and Freedland, R.A., Relative importance of kidney and liver in synthesis of arginine, Am. J. Physiol., 224 (1973) 127-129. [5] Inoue, I., Gushiken, T., Kobayashi, K. and Saheki, T., Accumulation of large neutral amino acids in the brain of sparse-fur mice at hyperammonemic state, Biochem. Med. Metab. Biol., 38 (1987) 378-386. [6] Knowles, R.G. and Moncada, S., Nitric oxide synthases in mammals, Biochem. J., 298 (1994) 249-258. [7] Leonard, T.O. and Lydic, R., Nitric oxide synthase inhibition

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