Predominant localization in glial cells of freel -arginine. Immunocytochemical evidence

Hram Researcii, ",47 (1991) i~J( l')z (C) 1991 Elsevier Science Publishers B.V tJ(106-8993/91/$03.51) 4 D()NIS 000689930116537 \

190

BRES 16537

Predominant localization in glial cells of free L-arginine. Immunocytochemical evidence Eiko Aoki 1, Reiji Semba 1, Katsuhiko Mikoshiba z and Shigeo Kashiwamata 1 t Department of Perinatology, Institute for Developmental Research, Aichi Prefecture Colony, Kasugai, Aichi 480-03 (Japan), 2Division of Regulation of Macromolecular Function, Institute for Protein Research, Osaka University, Osaka (Japan) (Accepted 13 November 1990)

Key words: Arginine; Nitric oxide; Endothelium-derived relaxing factor; Astroglia; Bergmann glia; Vascular endothel; Rat brain; Immunocytochemistry

Nitric oxide has been recently identified as an endogenous activator of the soluble guanylate cyclase in the brain as well as in vascular endothelial cells and macrophages. In the present study, we determined the localization of free arginine in the brain because nitric Oxide was formed from the terminal guanido group of L-arginine. Anti-arginine antiserum was raised in guinea pigs by repeated injection of L-arginine covalently conjugated to guinea pig serum albumin via glutaraldehyde. Specific anti-arginine antibody was purified from the antiserum by using an affinity gel coupled with L-arginine. Arginine-like immunoreactivity in the rat brain and spinal cord was found concentrated mainly in astrocytes including Bergmann glial cells in the cerebellum and processes of astrocytes around blood Vessels. The present results suggest that glial cells, particularly astrocytes, are the main locus of L-arginine, a nitric oxide precursor, in the brain.

INTRODUCTION Nitric oxide has b e e n r e c e n t l y identified as an e n d o g e n o u s activator of the soluble g u a n y l a t e c y d a s e in the b r a i n as well as in vascular e n d o t h e l i a l cells a n d m a c r o p h a g e s 1°'14. Nitric oxide is t h o u g h t to be identical to e n d o t h e l i u m - d e r i v e d relaxing factor ( E D R F ) in terms of biological activity 14. Nitric oxide was s h o w n to be f o r m e d from the t e r m i n a l g u a n i d o n i t r o g e n atom(s) of La r g i n i n e 13. In the b r a i n , nitric oxide synthase 3 has b e e n r e p o r t e d to occur p r i m a r i l y in n e u r o n s a n d in the e n d o t h e l i u m of b l o o d vessels with n o glial localization ~7. H o w e v e r , the cellular d i s t r i b u t i o n of L-arginine, a prec u r s o r of nitric oxide, has n o t b e e n r e p o r t e d so far. In this c o n t e x t , we focused o u r a t t e n t i o n on the localization of free a r g i n i n e in the CNS. T h e p r e s e n t p a p e r deals with the first visualization of free a r g i n i n e by using antia r g i n i n e a n t i b o d y raised in g u i n e a pigs a n d its i m m u n o c y t o c h e m i c a l d i s t r i b u t i o n in the rat b r a i n a n d spinal cord. MATERIALS AND METHODS L-Arginine was a product of Ajinomoto Co., and guinea pig serum albumin was obtained from Seikagaku Kogyo, Japan. Glutaraldehyde was purchased from TAAB Laboratories, U.K., and anti-guinea pig immunoglobulin G (goat) and peroxidase-

antiperoxidase complex (guinea pig) were from Miles-Yeda, Israel. Guinea pigs, Slc:Hartley strain, were supplied from SLC, Japan. All other chemicals were of the purest grade commercially available. Anti-arginine antibody was prepared as follows. L-Arginine (100 gmol) was dissolved in 1 ml of 0.1 M sodium phosphate buffer, pH 7.4, and formaldehyde (120 gmol) was mixed with the solution to block about 60% of the amino groups of L-arginine. After the mixture was kept standing for 30 rain. glutaraldehyde (GAL: 50 gmol) was coupled with the residual amino groups. Thirty minutes later, 12 mg of guinea pig serum albumin (GPSA) dissolved in 1 ml of the above buffer was added to initiate the reaction to produce an L-arginine-GPSA complex conjugated via GAL and was incubated for 3 h. The reaction was terminated by adding 0.1 ml of sodium borohydride solution (4 mg/ml). The L-arginine-GAL-GPSA conjugate was dialyzed for 48 h against 2 l of 0. l M sodium phosphate buffer, pH 7.4, and emulsified with an equal volume of complete Freund's adjuvant. All operations were carried out at room temperature (25-27 °C). The emulsion was repeatedly injected intracutaneously into the multiple sites on the back of the guinea pig. Production of anti-arginine antibody was examined by the Ouchterlony double diffusion test on 1% agar plates. Anti-arginine antibody was purified by affinity chromatography with L-arginine immobilized on formyl ceUulofine (Seikagaku Kogyo, Japan). Specificity of the antibody was checked by a dot-immunobinding assay using a nitrocellulose membrane (Schleicher & Schiill. Germany)9. Reactivity of the antibody was studied against Larginine-, L-ornithine-, L-aspartate-. L-glutamate-. GABA-. glycine-. taurine-, L-leucine-, L-isoleucine-. L-valine-. fl-alanine-, histamine-. L-histidine-, L-cysteic acid- and L-cystathionine-albumin complexes produced as described previously1. The purified anti-arginine antibody was found to be specific to the L-arginine-GAL-GPSA complex. Rats of Sprague-Dawley strain of 2 months old were perfused via

Correspondence: E. Aoki, Department of Perinatology, Institute for Developmental Research, Aichi Prefecture Colony, Kasugai, Aichi 480-03, Japan.

191 the heart with a mixture of 1% GAL, 4% formaldehyde, 0.2% picric acid and 2% sucrose in 0.l M sodium acetate buffer, pH 6.0 (ref. 19). Each brain was sectioned in slices of about 3 mm thickness, fixed in the above mixture for 4-5 h, and rinsed several times with 50 mM Tris-HCl buffer, pH 7.6. Thirty-/~m-thick sections were serially cut on a Vibratome, and sections were processed for immunocytochemistry. Sections were incubated with the purified antibody (dilution, 1:500), and left overnight at room temperature. The location of arginine was visualized by the peroxidase-antiperoxidase method 2° with diaminobenzidine as chromogen. Control sections incubated with non-immune guinea pig serum showed no positive staining.

RESULTS Arginine-like immunoreactivity was shown mainly in glial cells. In the cerebral cortex, corpus callosum, internal capsule, thalamus, brainstem, cerebral peduncle, c e r e b e l l u m and spinal cord, a n u m b e r of astrocytes were i m m u n o s t a i n e d with the anti-arginine antibody. Bergmann glial cells in the cerebellum and astrocytes around vascular endothelial cells were intensely stained (Fig. l a,b). Studies on the p a i r e d surface of adjacent sections from 3- and 10-day-old rat brains disclosed that oligodendrocytes, as identified with anti-myelin basic protein a n t i b o d y s, were not i m m u n o r e a c t i v e to the anti-arginine antibody. I m m u n o p o s i t i v e neurons were also found in some nuclei including the deep cerebellar nuclei and vestibular nuclei. Stainings of neuronal s o m a t a were not very strong. H o w e v e r , labelings of some fibers in the brainstem and spinal cord were p r o m i n e n t (Fig. 2). DISCUSSION We showed here that free arginine is selectively c o n c e n t r a t e d in certain cell types in the rat brain. A r g i n i n e - l i k e immunoreactivity was observed mainly in glial cells and also in some neurons. The synthesis of arginine from arginosuccinate by arginosuccinase and the d e g r a d a t i o n of arginine to ornithine and urea by arginase have been shown in the brain 4'5A6. It was also r e p o r t e d that arginosuccinase was m o r e c o n c e n t r a t e d in glial cells lhan in neurons and also that arginase activity was found in n e u r i n o m a and glioma cellsZk These biochemical d a t a support the present findings on the localization of arginine in gliai cells. C e r e b e l l a r B e r g m a n n glial cells were clearly stained coinciding with the r e p o r t that the free arginine level was highest in the cerebellum ~5"18. Some years ago, L-arginine was identified as an e n d o g e n o u s activator for the soluble guar~ylate cyclase in the rat brain 6'7. M o r e recently, nitric oxide was evidenced Io be f o r m e d from the terminal guanido nitrogen atom(s) of free L-arginine or peptides containing an L-arginine residue at the N- or C-terminal position through the reaction by nitric oxide synthase, a 150-kDa calmodulin-

Fig. 1. Photograph showing the distribution of arginine-like immunoreactivity in a 2-month-old rat cerebellum. Bergmann glial cells (a) and astrocytes wrapping vascular endothelial cells in the granule cell layer (b) are intensely stained. E, endplate of Bergmann glial cells; P, Purkinje cells; B, blood vessels. (a) Bar = 30 ~m. (b) Bar = 20 pm.

Fig. 2. Photograph showing arginine-like immunoreactive fibers in the brainstem. Bar = 200/~m.

192 requiring enzyme, in the presence of Ca 2+ and NADPH3"~', Nitric oxide thus formed is known to stimulate the soluble guanylate cyclase and hence to elevate cGMP levels in the brain as well as in vascular endothelial cells and macrophages m. However, the cellular distribution of L-arginine, a precursor of nitric oxide, has not been reported so far. The present immunocytochemical study revealed a p r e d o m i n a n t distribution of free arginine in glial cells, particularly in astrocytes. It is well known that the capillary endothelium in the brain is surrounded by the sheath of astrocyte foot processes. Endothelial cells in the vascular system have been shown to synthesize endothelium-derived relaxing factor ( E D R F ) , i.e. nitric oxide, from L-arginine3, and nitric oxide synthase has been also proved in the endothelium of blood vessels 17. Thus, it seems likely that free L-arginine may be released

mechanism may operate m the cerebellar cortex: arginine is released from B e r g m a n n glial cells and taken up by adjacent basket cells and their processes to synthesize nitric oxide, which diffuses locally across the cell m e m b r a n e s to activate guanylate cyclase in adjacent Purkinje cells. Such a hypothesis would be strengthened by the fact that of all cerebellar cells Purkinje cells possessed the highest levels of guanylate cyclasc 1: and cyclic G M P 2. Recently, an astrocyte-derived vasorelaxing factor with properties similar to those of nitric oxide has been also reported lb. Thus, the formation of nitric oxide in the brain is thought to occur in various types of cells including endothelial cells, neurons and astrocytes. Considering these, the present results suggest that gliai cells play a significant role in controlling the neural activity through L-arginine and nitric oxide.

from astrocyte processes, incorporated into capillary endothelial cells in the brain and cleaved to t'orm nitric oxide. Since nitric oxide synthase-like immunoreactivity in the cerebellum has been reported to be located in basket cells and their horizontal axonal processes that make synapses onto the Purkinje cell bodies ~7. a similar

REFERENCES 1 Aoki, E., Semba, R. and Kashiwamata, S., Purification of specific antibody against aspartate and immunocytochemical localization of aspartergic neurons in the rat brain, Neuroscience, 21 (1987) 755-765. 2 Bredt, D.S. and Snyder, S.H., Nitric oxide mediates glutamatelinked enhancement of cGMP levels in the cerebellum, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 9030-9033. 3 Bredt, D.S. and Snyder, S.H., Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 682-685. 4 Buniatian, H.C., The urea cycle. In F.A. Lajtha (Ed.), Handbook of Neurochemistry, Vol. 5, Plenum, London, 1971, pp. 235-247. 5 Buniatian, H.C. and Davtian, M.A., Urea synthesis in brain, J. Neurochem., 13 (1966) 743-753. 6 Deguchi, T. and Yoshioka, M., L-Arginine identified as an endogenous activator for soluble guanylate cyclase from neuroblastoma cells, J. Biol. Chem., 257 (1982) 10147-10151. 7 Garthwaite, J., Charles, S.L. and Chess-Williams, R., Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intracellular messenger in the brain, Nature, 336 (1988) 385-387. 8 Hartman, B.K., Agrawal, HC., Agrawal, D. and Katmbach, S., Development and maturation of central nervous system myelin: comparison of immunohistochemical localization of proteolipid protein and basic protein in myelin and oligodendrocytes, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 4217-4220. 9 Hawkes, R., Niday, E. and Gordon, J., A dot-immunobinding assay for monoclonal and other antibodies, Anal. Biochem., 119 (1982) 142-147. l0 Knowles, R.G., Palacios, M., Palmer, R.M.J. and Moncada, S., Formation of nitric oxide from e-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase, Proc. Natl. Acad. Sci. U.S.A.. 86 (1989)

Acknowledgements. A part of this research was supported by Grant 63A-2 from the National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare and also by a Grand-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

5159-5162. 11 Murphy, S., Minor, R.L., Welk, G. and Harrison, D.G., Evidence for an astrocyte-derived vasorelaxing factor with properties similar to nitric oxide, J. Neurochem., 55 (1990) 349- 351. 12 Nakane, M., lchikawa, M. and Deguchi, T , Light and electron microscopic demonstration of guanylate cyelase in rat brain, Brain Research, 273 (1983) 9-15. 13 Palmer, R.M.J., Ashton, D.S. and Moncada, S., Vascular endothelial cells synthesize nitric oxide from ~-arginine, Nature, 333 (1988) 664-666. 14 Palmer, R.M.J., Ferrige, A.G. and Moncada, S., Nitric oxide release accounts for the biological activity of endotheliumderived relaxing factor, Nature, 327 (1987) 524-526. 15 Perry, T.L., Berry, K., Diamond, S. and Mok, C., Regional distribution of amino acids in human brain obtained at autopsy, J. Neurochem., 18 (1971) 513-519. 16 Ratner, S., Morell, H. and Carvalho, E., Enzymes of arginine metabolism in brain, Arch. Biochem. Biophys., 91 (1960) 280-289. 17 Ross, C.A., Bredt, D. and Snyder, S.H., Messenger molecules in the cerebellum, Trends' Neurosci., 13 (1990) 216-222. 18 Sadasivudu, B. and Rao, T.I., Studies on functional and metabolic role of urea cycle intermediates in brain, J. Neurochem., 27 (1976) 785-794. 19 Schmechel, D.E., Brightman, M.W. and Marangos, P.J., Neurons switch from non-neuronal enolase to neuron-specific enolase during differentiation, Brain Research, 190 (1980) t95-214. 2(I Sternberger, L.A., The unlabeled antibody enzyme method. In L.A. Sternberger (Ed.), Immunocvtochemistry, Prentice-Hall, Englewood Cliffs, 1974, pp. 129-171. 21 Trapeznikova, S.S., Navasadaryantz, D.G., Levchenko, L.I., Khalansky, A.S. and Khokhlov, A.P., Activity of arginase isoenzymes in human brain tumors and in the tumors of experimental animals, Vopr. Med. Khim., 32 (1986) 29-34.