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• W2080631661 abstract "In the brain, three isoforms of nitric oxide (NO) synthase (NOS), namely neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3), have been implicated in biological roles such as neurotransmission, neurotoxicity, immune function, and blood vessel regulation, each isoform exhibiting in part overlapping roles. Previous studies showed that iNOS is induced in the brain by systemic treatment with lipopolysaccharide (LPS), a Gram-negative bacteria-derived stimulant of the innate immune system. Here we found that eNOS mRNA is induced in the rat brain by intraperitoneal injection of LPS of a smaller amount than that required for induction of iNOS mRNA. The induction of eNOS mRNA was followed by an increase in eNOS protein. Immunohistochemical analysis revealed that eNOS is located in astrocytes of both gray and white matters as well as in blood vessels. Induction of eNOS in response to a low dose of LPS, together with its localization in major components of the blood-brain barrier, suggests that brain eNOS is involved in early pathophysiologic response against systemic infection before iNOS is induced with progression of the infection. In the brain, three isoforms of nitric oxide (NO) synthase (NOS), namely neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3), have been implicated in biological roles such as neurotransmission, neurotoxicity, immune function, and blood vessel regulation, each isoform exhibiting in part overlapping roles. Previous studies showed that iNOS is induced in the brain by systemic treatment with lipopolysaccharide (LPS), a Gram-negative bacteria-derived stimulant of the innate immune system. Here we found that eNOS mRNA is induced in the rat brain by intraperitoneal injection of LPS of a smaller amount than that required for induction of iNOS mRNA. The induction of eNOS mRNA was followed by an increase in eNOS protein. Immunohistochemical analysis revealed that eNOS is located in astrocytes of both gray and white matters as well as in blood vessels. Induction of eNOS in response to a low dose of LPS, together with its localization in major components of the blood-brain barrier, suggests that brain eNOS is involved in early pathophysiologic response against systemic infection before iNOS is induced with progression of the infection. nitric-oxide synthase neuronal NOS inducible NOS endothelial NOS lipopolysaccharide cornu ammonis glial fibrillary acidic protein Nitric oxide (NO) is a gaseous messenger molecule functioning mainly in vascular regulation, immunity, and neurotransmission (for recent reviews, see Refs. 1.Michel T. Feron O. J. Clin. Invest. 1997; 100: 2146-2152Crossref PubMed Scopus (853) Google Scholar and 2.Mayer B. Hemmens B. Trends Biochem. Sci. 1997; 22: 477-481Abstract Full Text PDF PubMed Scopus (512) Google Scholar). NO is formed froml-arginine by NO synthase (NOS).1 At least three isoforms of NOS have been identified, namely, neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3). In the brain, all three NOS isoforms are expressed constitutively or inducibly, and implicated in a number of physiologic and pathophysiologic functions. nNOS is expressed constitutively in specific neurons of various brain regions (3.Bredt D.S. Hwang P.M. Snyder S.H. Nature. 1990; 347: 768-770Crossref PubMed Scopus (2698) Google Scholar, 4.Rodrigo J. Springall D.R. Uttenthal O. Bentura M.L. Abadia-Molina F. Riveros-Moreno V. Martinez-Murillo R. Polak J.M. Moncada S. Phil. Trans. R. Soc. Lond. B. 1994; 345: 175-221Crossref PubMed Scopus (376) Google Scholar, 5.Iwase K. Iyama K. Akagi K. Yano S. Fukunaga K. Miyamoto E. Mori M. Takiguchi M. Mol. Brain Res. 1998; 53: 1-12Crossref PubMed Scopus (88) Google Scholar). Expression of iNOS is under a detectable level in the normal brain, but is induced in neurons, glias, and other cells in various pathological conditions such as ischemia (6.Endoh M. Maiese K. Wagner J. Brain Res. 1994; 651: 92-100Crossref PubMed Scopus (300) Google Scholar, 7.Iadecola C. Zhang F. Xu S. Casey R. Ross M.E. J. Cereb. Blood Flow Metab. 1995; 15: 378-384Crossref PubMed Scopus (457) Google Scholar), trauma (8.Clark R.S.B. Kochanek P.M. Schwarz M.A. Schiding J.K. Turner D.S. Chen M. Carlos T.M. Watkins S.C. Pediatr. Res. 1996; 39: 784-790Crossref PubMed Scopus (142) Google Scholar), multiple sclerosis (9.Mitrovic B. Ignarro L.J. Montestruque S. Smoll A. Merrill J.E. Neuroscience. 1994; 61: 575-585Crossref PubMed Scopus (247) Google Scholar, 10.Bagasra O. Michaels F.H. Zheng Y.M. Bobroski L.E. Spitsin S.V. Fu Z.F. Tawadros R. Koprowski H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12041-12045Crossref PubMed Scopus (436) Google Scholar), Parkinson's disease (11.Hunot S. Boissiere F. Faucheux B. Brugg B. Mouatt-Prigent A. Agid Y. Hirsch E.C. Neuroscience. 1996; 72: 355-363Crossref PubMed Scopus (527) Google Scholar), Alzheimer's disease (12.Vodovotz Y. Lucia M.S. Flanders K.C. Chesler L. Xie Q.-W. Smith T.W. Weidner J. Mumford R. Webber R. Nathan C. Roberts A.B. Lippa C.F. Sporn M.B. J. Exp. Med. 1996; 184: 1425-1433Crossref PubMed Scopus (263) Google Scholar), tumors (13.Hara E. Takahashi K. Tominaga T. Kumabe T. Kayama T. Suzuki H. Fujita H. Yoshimoto T. Shirato K. Shibahara S. Biochem. Biophys. Res. Commun. 1996; 224: 153-158Crossref PubMed Scopus (101) Google Scholar), acquired immunodeficiency syndrome encephalitis (14.Adamson D.C. Wildemann B. Sasaki M. Glass J.D. McArthur J.C. Christov V.I. Dawson T.M. Dawson V.L. Science. 1996; 274: 1917-1921Crossref PubMed Scopus (397) Google Scholar), infection (15.Koprowski H. Zheng Y.M. Heber-Katz E. Fraser N. Rorke L. Fu Z.F. Hanlon C. Dietzschold B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3024-3027Crossref PubMed Scopus (472) Google Scholar), and lipopolysaccharide (LPS) treatment (16.Hom G.J. Grant S.K. Wolfe G. Bach T.J. MacIntyre D.E. Hutchinson N.I. J. Pharmacol. Exp. Ther. 1995; 272: 452-459PubMed Google Scholar, 17.Liu S.F. Adcock I.M. Old R.W. Barnes P.J. Evans T.W. Crit. Care Med. 1996; 24: 1219-1225Crossref PubMed Scopus (123) Google Scholar, 18.Wong M.-L. Rettori V. Al-Shekhlee A. Bongiorno P.B. Canteros G. McCann S.M. Gold P.W. Licinio J. Nat. Med. 1996; 2: 581-584Crossref PubMed Scopus (260) Google Scholar). The locus of eNOS expression in the brain is controversial. In addition to blood vessels, eNOS-like protein was detected immunohistochemically in pyramidal neurons of the hippocampus, exhibiting profiles different between the rat (19.Dinerman J.L. Dawson T.M. Schell M.J. Snowman A. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4214-4218Crossref PubMed Scopus (615) Google Scholar) and human (20.Doyle C.A. Slater P. Neuroscience. 1997; 76: 387-395Crossref PubMed Scopus (99) Google Scholar). eNOS has been also described to be present in astrocytes (20.Doyle C.A. Slater P. Neuroscience. 1997; 76: 387-395Crossref PubMed Scopus (99) Google Scholar, 21.Barna M. Komatsu T. Reiss C.S. Virology. 1996; 223: 331-343Crossref PubMed Scopus (81) Google Scholar, 22.Gabbott P.L.A. Bacon S.J. Brain Res. 1996; 714: 135-144Crossref PubMed Scopus (49) Google Scholar), and several groups reported that eNOS was detected preferentially in astrocytes rather than in neurons (21.Barna M. Komatsu T. Reiss C.S. Virology. 1996; 223: 331-343Crossref PubMed Scopus (81) Google Scholar, 22.Gabbott P.L.A. Bacon S.J. Brain Res. 1996; 714: 135-144Crossref PubMed Scopus (49) Google Scholar). Gene disruption studies have revealed that the three NOS isoforms in the brain play interdigitated biomedical roles, functioning cooperatively or antagonistically. While mice deficient in nNOS displayed aggressive behavior and inappropriate sexual behavior (23.Nelson R.J. Demas G.E. Huang P.L. Fishman M.C. Dawson V.L. Dawson T.M. Snyder S.H. Nature. 1995; 378: 383-386Crossref PubMed Scopus (547) Google Scholar), long term potentiation in the hippocampal cornu ammonis (CA) 1 region of the mutant mice was almost normal (24.O'Dell T.J. Huang P.L. Dawson T.M. Dinerman J.L. Snyder S.H. Kandel E.R. Fishman M.C. Science. 1994; 265: 542-546Crossref PubMed Scopus (369) Google Scholar), which was also the case for eNOS-deficient mice (25.Son H. Hawkins R.D. Martin K. Kiebler M. Huang P.L. Fishman M.C. Kandel E.R. Cell. 1996; 87: 1015-1023Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). On the other hand, mice doubly deficient in nNOS and eNOS showed a significant reduction of long term potentiation in the stratum radiatum of the CA1 region, suggesting a reciprocally compensatory role for nNOS and eNOS (25.Son H. Hawkins R.D. Martin K. Kiebler M. Huang P.L. Fishman M.C. Kandel E.R. Cell. 1996; 87: 1015-1023Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). In brain ischemia following occlusion of the middle cerebral artery, infarct volume was reduced in mice deficient in nNOS (26.Shimizu-Sasamata M. Bosque-Hamilton P. Huang P.L. Moskowitz M.A. Lo E.H. J. Neurosci. 1998; 18: 9564-9571Crossref PubMed Google Scholar) or iNOS (27.Iadecola C. Zhang F. Casey R. Nagayama M. Ross M.E. J. Neurosci. 1997; 17: 9157-9164Crossref PubMed Google Scholar). In contrast, eNOS-deficient mice showed an increase in infarct volume (28.Huang Z. Huang P.L. Ma J. Meng W. Ayata C. Fishman M.C. Moskowitz M.A. J. Cereb. Blood Flow Metab. 1996; 16: 981-987Crossref PubMed Scopus (613) Google Scholar). Apparently, nNOS- and iNOS-derived NO is toxic, while eNOS-derived NO is protective, to the ischemic brain. In order to clarify roles of NOS isoforms in the nervous system, we previously examined precise distribution of nNOS mRNA in the rat brain, by using high resolution non-radioisotopic in situhybridization (5.Iwase K. Iyama K. Akagi K. Yano S. Fukunaga K. Miyamoto E. Mori M. Takiguchi M. Mol. Brain Res. 1998; 53: 1-12Crossref PubMed Scopus (88) Google Scholar). We have also studied regulation of the iNOS gene in response to LPS in peripheral organs, being concerned with coordination with genes for l-arginine-metabolizing enzymes (29.Nagasaki A. Gotoh T. Takeya M., Yu, Y. Takiguchi M. Matsuzaki H. Takatsuki K. Mori M. J. Biol. Chem. 1996; 271: 2658-2662Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 30.Sonoki T. Nagasaki A. Gotoh T. Takiguchi M. Takeya M. Matsuzaki H. Mori M. J. Biol. Chem. 1997; 272: 3689-3693Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). In the course of examining the effects of LPS on expression of the three NOS isoforms, we by chance found that a relatively low dose of LPS induces mRNA for eNOS, but not for iNOS, in the rat brain. Specific pathogen-free male Wistar rats (5–6 weeks old) were injected intraperitoneally withEscherichia coli LPS (serotype 0127:B8, Sigma), and after appropriate times brain and other organs were excised from the rats anesthetized in ether. Regional dissection of the rat brain was done essentially according to the division of Glowinski and Iversen (31.Glowinski J. Iversen L.L. J. Neurochem. 1966; 13: 655-669Crossref PubMed Scopus (5041) Google Scholar). Total RNA from rat tissues was prepared by the guanidinium thiocyanate-phenol-chloroform extraction procedure (32.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). After electrophoresis in formaldehyde-containing agarose gels, RNA was transferred to nylon membranes. The digoxigenin-labeled antisense RNA probe was synthesized using the DIG-RNA labeling kit (Roche Molecular Biochemicals, Mannheim, Germany) with cDNA templates for rat eNOS (corresponding to nucleotides 44–1164 of the human sequence of Ref. 33.Janssens S.P. Shimouchi A. Quertermous T. Bloch D.B. Bloch K.D. J. Biol. Chem. 1992; 267: 14519-14522Abstract Full Text PDF PubMed Google Scholar), rat iNOS (nucleotides 2529–3211 of Ref.34.Geng Y.-J. Almqvist M. Hansson G.K. Biochim. Biophys. Acta. 1994; 1218: 421-424Crossref PubMed Scopus (63) Google Scholar), and rat nNOS (nucleotides 296–2150 of Ref. 35.Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2173) Google Scholar). eNOS and iNOS cDNAs were amplified using reverse transcription-polymerase chain reaction, and cloned into the EcoRV site of pcDNAII and the HincII site of pGEM3Zf(+), respectively. nNOS cDNA was isolated as described previously (5.Iwase K. Iyama K. Akagi K. Yano S. Fukunaga K. Miyamoto E. Mori M. Takiguchi M. Mol. Brain Res. 1998; 53: 1-12Crossref PubMed Scopus (88) Google Scholar). After hybridization and washing as recommended by Roche Molecular Biochemicals, chemiluminescent detection of hybridized probes on x-ray films was done using the alkaline phosphatase-conjugated DIG antibody (Roche Molecular Biochemicals) and CDP-Star™ (Tropix Inc., Bedford, MA). Densitometric quantification was performed using the MacBas bioimage analyzer (Fuji Photo Film Co., Tokyo, Japan). Rat tissues were homogenized in nine volumes of 20 mm potassium HEPES buffer (pH 7.4) containing 1 mm dithiothreitol, 50 μm antipain, 50 μm leupeptin, 50 μm chymostatin, and 50 μm pepstatin. The homogenate was centrifuged at 25,000 × g for 30 min at 4 °C, and supernatant was used as tissue extract. Protein in the tissue extract was determined with the protein assay reagent (Bio-Rad) using bovine serum albumin as standard. The tissue extracts (15 μg of protein/lane) were subjected to SDS-6% polyacrylamide gel electrophoresis, and protein was electrotransferred to nitrocellulose membranes. As a primary antibody, a rabbit polyclonal anti-human eNOS antibody (1:50 dilution; N30030; Transduction Laboratories, Lexington, KY) in Fig. 3 or a monoclonal anti-human eNOS antibody (1 μg/ml IgG; N30020; Transduction Laboratories) in Fig. 4 was incubated with the membranes. Immunodetection was performed using the ECL kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). Chemiluminescent signals were detected on x-ray films and quantified using the MacBas bioimage analyzer.Figure 4Distribution of NOS isoforms in the rat brain regions. A, Northern blot analysis for detection of mRNAs for nNOS, iNOS, and eNOS was performed with total RNAs from various brain regions: Ob, olfactory bulb; Cx, cerebral cortex; St, striatum; Hp, hippocampus;Hy, hypothalamus; MT, midbrain and thalamus;Cb, cerebellum; PM, pons and medulla oblongata. As positive controls (P.C.) for iNOS and eNOS mRNAs, total RNAs prepared from the LPS-treated rat lung and the normal rat heart, respectively, were coelectrophoresed. Below, ethidium bromide staining of 28 and 18 S rRNAs is presented. B, Western blot analysis. Distribution of eNOS protein was examined for tissue extracts from the brain regions. As a positive control (P.C.), a human endothelial cell lysate was coelectrophoresed.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The tissue extracts were prepared as described above. NOS catalytic activity was assayed by determining the conversion of l-[3H]arginine tol-[3H]citrulline essentially as described (36.Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9030-9033Crossref PubMed Scopus (1750) Google Scholar, 37.Hevel J.M. Marletta M.A. Methods Enzymol. 1994; 233: 250-258Crossref PubMed Scopus (424) Google Scholar). Ca2+-dependent NOS activity was measured by incubating 50 μg of extract protein in a mixture (200 μl) containing 50 mm potassium HEPES (pH 7.5), 1 mm dithiothreitol, 1 mm CaCl2, 0.1 mm (6R)-5,6,7,8-tetrahydrobiopterin, 1 mm NADPH, 10 μm FAD, 10 μm FMN, 0.5 μm calmodulin, and 0.1 μml-[3H]arginine (49.0 Ci/mmol, Amersham Pharmacia Biotech) at 37 °C for 30 min. In the Ca2+-independent NOS assay, EGTA (1 mm) was substituted for CaCl2. The reaction was stopped by adding 100 μl of 3% trichloroacetic acid. After 30 min on ice, 250 μl of 1.5 m potassium HEPES (pH 7.5) were added, and the precipitates were removed by centrifugation at 15,800 ×g for 5 min at 4 °C. The supernatants were applied to 1-ml columns of Dowex 50W-X8 (Na+ form), and elutedl-[3H]citrulline was measured by liquid scintillation counting. Enzyme activity was expressed as picomoles ofl-[3H]citrulline produced/mg of protein in 1 min. Data are represented as mean ± range. Excised rat brains were embedded in OCT Compound™ (Miles, Elkhart, IN) and frozen in the liquid nitrogen. Ten-μm sections were cut, air-dried, washed in Dulbecco's phosphate-buffered saline, and fixed with methanol for 10 min on ice. After inhibition of endogenous peroxidase activity by the method of Isobe et al. (38.Isobe Y. Chen S.-T. Nakane P.K. Brown W.R. Acta Histochem. Cytochem. 1977; 10: 161-171Crossref Scopus (229) Google Scholar), the sections were subjected to incubation with the primary antibody diluted with phosphate-buffered saline containing 0.5% bovine serum albumin (fraction V) overnight at 4 °C. A primary antibody used was a rabbit polyclonal anti-human eNOS antibody (1:200–5000 dilution) or a mouse monoclonal anti-human glial fibrillary acidic protein (GFAP) antibody (28 μg/ml IgG; Dako Corp., Carpinteria, CA). After incubation with the biotinylated secondary antibody (1:200; Vector Laboratories Inc., Burlingame, CA) for 2 h, the sections were further incubated with a mixture of avidin and horseradish peroxidase-conjugated biotin for 1 h. Peroxidase activity was visualized using 3,3′-diaminobenzidine as a substrate. Double-staining analysis with the rabbit polyclonal anti-eNOS antibody and the mouse monoclonal anti-GFAP antibody was performed by the peroxidase-antiperoxidase and alkaline phosphatase-antialkaline phosphatase procedure using the Doublestain kit (Dako Corp.). For control, sections were incubated with rabbit serum or non-immunized mouse IgG1 instead of each specific primary antibody. Sections were slightly counterstained with hematoxylin, and the slides were mounted in Aquatex (E. Merck, Darmstadt, Germany). Rats were injected intraperitoneally withEscherichia coli LPS at 2.5 μg/g body weight, and 12 h later eNOS mRNA levels in various organs were examined by RNA blot analysis (Fig. 1 A). In the heart and kidney, eNOS mRNA levels were lowered by the LPS treatment, concordant with a previous report (17.Liu S.F. Adcock I.M. Old R.W. Barnes P.J. Evans T.W. Crit. Care Med. 1996; 24: 1219-1225Crossref PubMed Scopus (123) Google Scholar). On the other hand, in the brain, eNOS mRNA was increased in response to LPS. No apparent induction of iNOS mRNA was observed in the brain with this amount of LPS, while the induction was obvious in the lung (Fig.1 B). The LPS treatment had no effect on nNOS mRNA levels in the brain (Fig. 1 C). Previously, it has been reported that mRNA for iNOS, rather than eNOS, is induced in the brain as well as in other organs by LPS treatment (16.Hom G.J. Grant S.K. Wolfe G. Bach T.J. MacIntyre D.E. Hutchinson N.I. J. Pharmacol. Exp. Ther. 1995; 272: 452-459PubMed Google Scholar, 17.Liu S.F. Adcock I.M. Old R.W. Barnes P.J. Evans T.W. Crit. Care Med. 1996; 24: 1219-1225Crossref PubMed Scopus (123) Google Scholar, 18.Wong M.-L. Rettori V. Al-Shekhlee A. Bongiorno P.B. Canteros G. McCann S.M. Gold P.W. Licinio J. Nat. Med. 1996; 2: 581-584Crossref PubMed Scopus (260) Google Scholar). Since the discrepancy can result from differences of experimental conditions, we examined changes in eNOS and iNOS mRNA levels with various amounts of LPS (Fig.2). eNOS mRNA was substantially induced with LPS at 2.5 μg/g, and reached a plateau level at 10 μg/g. On the other hand, only a slight increase in iNOS mRNA was detected at 10 μg/g, and a maximum increase at 50 μg/g. In previous studies (16.Hom G.J. Grant S.K. Wolfe G. Bach T.J. MacIntyre D.E. Hutchinson N.I. J. Pharmacol. Exp. Ther. 1995; 272: 452-459PubMed Google Scholar, 17.Liu S.F. Adcock I.M. Old R.W. Barnes P.J. Evans T.W. Crit. Care Med. 1996; 24: 1219-1225Crossref PubMed Scopus (123) Google Scholar, 18.Wong M.-L. Rettori V. Al-Shekhlee A. Bongiorno P.B. Canteros G. McCann S.M. Gold P.W. Licinio J. Nat. Med. 1996; 2: 581-584Crossref PubMed Scopus (260) Google Scholar), induction of iNOS mRNA in the brain has been detected with comparable amounts of LPS (15–50 μg/g). mRNA levels for both eNOS and iNOS decreased with LPS at 100 μg/g, compared with at 50 μg/g. The exact reason for these decreases is not known, but a possible cause is the nonspecific catastrophic damage of the brain, since the LPS treatment at this concentration occasionally resulted in death of the animals. In conclusion, in the pathophysiologic dose range, higher amounts of LPS are required for induction of iNOS mRNA than in that for eNOS mRNA. Time course of changes in mRNA and protein levels for eNOS in the brain after treatment with LPS (2.5 μg/g) are shown in Fig.3. eNOS mRNA levels were raised 6 h after the treatment, and reached a maximum at 12 h. A delayed increase in eNOS protein levels was apparent at 12 h, and an additional increase at 24 h. Therefore, induction of eNOS mRNA in response to LPS leads to accumulation of eNOS protein. We also examined changes in NOS catalytic activity in the brain 24 h after treatment with LPS (2.5 μg/g). Total Ca2+-dependent NOS activity, which is derived mainly from nNOS and eNOS isoforms, slightly increased to 5.28 ± 0.22 pmol/mg/min compared with 4.50 ± 0.25 pmol/mg/min for the control (mean ± range, n = 2). On the other hand, iNOS-derived Ca2+-independent activity was below the detectable level in both control and LPS-treated rats. We further tried to measure eNOS activity specifically, by using nNOS inhibitors such asN ω-propyl-l-arginine (39.Zhang H.Q. Fast W. Marletta M.A. Martasek P. Silverman R.B. J. Med. Chem. 1997; 40: 3869-3870Crossref PubMed Scopus (190) Google Scholar) and 1-(2-trifluoromethylphenyl)imidazole (40.Handy R.L.C. Wallace P. Gaffen Z.A. Whitehead K.J. Moore P.K. Br. J. Pharmacol. 1995; 116: 2349-2350Crossref PubMed Scopus (91) Google Scholar), and by using antibodies against eNOS and nNOS (for each isoform, two commercially available antibodies) for immunoselection of eNOS and immunodepletion of nNOS, respectively. However, these attempts were unsuccessful, presumably at least in part because of relatively high nNOS activity that amounts to more than 90% of total NOS activity in the normal rodent brain (41.Huang P.L. Dawson T.M. Bredt D.S. Snyder S.H. Fishman M.C. Cell. 1993; 75: 1273-1286Abstract Full Text PDF PubMed Scopus (1147) Google Scholar). Distribution of NOS isoforms in various regions of the rat brain was examined by RNA blot analysis (Fig. 4 A). nNOS mRNA was detected in all brain regions examined, exhibiting higher levels in the olfactory bulb and cerebellum than in other regions, concordant with previous reports (5.Iwase K. Iyama K. Akagi K. Yano S. Fukunaga K. Miyamoto E. Mori M. Takiguchi M. Mol. Brain Res. 1998; 53: 1-12Crossref PubMed Scopus (88) Google Scholar, 35.Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2173) Google Scholar). iNOS mRNA was under the detectable level in any region of the normal rat brain. eNOS mRNA was detected in all brain regions, and showed more equal distribution than nNOS mRNA. By using immunoblot analysis (Fig.4 B), the eNOS protein of about 135 kDa was also detected in all brain regions, and showed rather uniform distribution resembling that of eNOS mRNA. To determine the precise localization of the eNOS protein, immunohistochemical staining was performed on brain sections (Fig.5, A–H). As expected, blood vessels showed strong eNOS immunoreactivity in almost all brain regions examined (Fig. 5, A and D). No apparent signal was detected with a control non-immune antibody (data not shown). Strong staining was also detected in cells morphologically resembling astrocytes in the white matter of the striatum (Fig. 5 A), anterior commissure (Fig. 5 B), corpus callosum, fimbria of the hippocampus, internal capsule (Fig. 5 C), and cerebellum (Fig. 5 H). Less intense but obvious staining was detected in astrocyte-like cells in the gray matter of the cerebral cortex (Fig. 5,D and E), hippocampus (Fig. 5, F andG), and cerebellum (Fig. 5 H). Some astrocyte-like cells were seen to radiate the processes coming in contact with blood vessels (Fig. 5 A; see also Fig. 5 M). In the hippocampus, immunolabeling was located in the stratum oriens and stratum radiatum (Fig. 5 G). On the other hand, in contrast to the previous reports (19.Dinerman J.L. Dawson T.M. Schell M.J. Snowman A. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4214-4218Crossref PubMed Scopus (615) Google Scholar, 24.O'Dell T.J. Huang P.L. Dawson T.M. Dinerman J.L. Snyder S.H. Kandel E.R. Fishman M.C. Science. 1994; 265: 542-546Crossref PubMed Scopus (369) Google Scholar), no obvious signal was detected in pyramidal neurons under the present condition. In the cerebellum (Fig.5 H), the deep white matter showed strong staining. Moderate and weak signals were detected in the granular layer and molecular layer, respectively, while no apparent signal in the Purkinje cell layer. Expression of eNOS protein in astrocytes was confirmed by examining colocalization with GFAP as a marker for astrocytes (Fig. 5,I–K). Immunostaining of serial sections with the anti-eNOS antibody (Fig. 5 I) or anti-GFAP antibody (Fig.5 J) exhibited a similar pattern of distribution of positive cells. Furthermore, double staining for eNOS and GFAP (Fig.5 K) detected overlapping immunoreactive cells, verifying colocalization of these two proteins. The effect of LPS treatment on eNOS immunostaining was examined (Fig.5, L and M). Concordant with the results of protein blotting analysis (Fig. 3), the strength of immunolabeling increased by the LPS treatment for 24 h, while precise quantitative analysis on increase in the number of positive astrocytes and/or in the intensity of labeling in each cell remains to be performed. This study showed that intraperitoneal injection of LPS causes an increase in eNOS expression specifically in the brain, making a sharp contrast to decreases in the heart and kidney (Fig. 1 A). This can be at least in part because of the difference of cells expressing eNOS in the brain and other organs. As reported previously (20.Doyle C.A. Slater P. Neuroscience. 1997; 76: 387-395Crossref PubMed Scopus (99) Google Scholar, 21.Barna M. Komatsu T. Reiss C.S. Virology. 1996; 223: 331-343Crossref PubMed Scopus (81) Google Scholar, 22.Gabbott P.L.A. Bacon S.J. Brain Res. 1996; 714: 135-144Crossref PubMed Scopus (49) Google Scholar) and confirmed here (Fig. 5), astrocytes as well as endothelial cells are a major source of eNOS in the brain. It remains to be examined if the increase in eNOS expression in astrocytes is directly triggered by LPS itself or mediated by LPS-induced agents such as cytokines. The latter possibility is especially noteworthy, taking the blood-brain barrier into account. Astrocytes themselves constitute the outer component of the blood-brain barrier, and are separated from blood flow by tightly connected endothelial cells and the basement membrane. Therefore, it should be carefully examined if LPS of low concentrations in blood flow can directly reach astrocytes. Possible involvement of the blood-brain barrier in the LPS response of the brain was also suggested by the fact that higher amounts of LPS were necessary for iNOS induction in the brain than in the lung (Figs.1 B and 2). What are the roles of astroglial eNOS? Barna et al. (21.Barna M. Komatsu T. Reiss C.S. Virology. 1996; 223: 331-343Crossref PubMed Scopus (81) Google Scholar) reported that infection of the mouse brain by vesicular stomatitis virus led to increases in eNOS protein in astrocytes, implying a role of astroglial eNOS in inhibition of local brain infection. On the other hand, the present demonstration that astroglial eNOS was induced in response to intraperitoneal injection of LPS suggests involvement of astroglial eNOS in protection of the brain against systemic bacterial infection. Localization of eNOS in endothelial cells and astrocytes, namely two major components of the blood-brain barrier that is the defensive front of the brain, is also concordant with this notion. The amount of LPS required for induction of eNOS was smaller than that required for induction of iNOS, suggesting that eNOS can be induced in relatively early stages following bacterial infection. In summary, astroglial eNOS is likely to be involved in early precaution and defense against systemic infection. Studies with eNOS knockout mice revealed that eNOS is involved in protection of neurons on brain ischemia (28.Huang Z. Huang P.L. Ma J. Meng W. Ayata C. Fishman M.C. Moskowitz M.A. J. Cereb. Blood Flow Metab. 1996; 16: 981-987Crossref PubMed Scopus (613) Google Scholar), in addition to regulation of systemic blood pressure (42.Huang P.L. Huang Z. Mashimo H. Bloch K.D. Moskowitz M.A. Bevan J.A. Fishman M.C. Nature. 1995; 377: 239-242Crossref PubMed Scopus (1788) Google Scholar, 43.Shesely E.G. Maeda N. Kim H.-S. Desai K.M. Krege J.H. Laubach V.E. Sherman P.A. Sessa W.C. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13176-13181Crossref PubMed Scopus (791) Google Scholar). It remains to be determined whether eNOS with this protective effect is attributable to astrocytes or endothelial cells. Gene knockout and related studies have also revealed that eNOS is involved in neuronal functions such as long term potentiation in the hippocampus (24.O'Dell T.J. Huang P.L. Dawson T.M. Dinerman J.L. Snyder S.H. Kandel E.R. Fishman M.C. Science. 1994; 265: 542-546Crossref PubMed Scopus (369) Google Scholar, 25.Son H. Hawkins R.D. Martin K. Kiebler M. Huang P.L. Fishman M.C. Kandel E.R. Cell. 1996; 87: 1015-1023Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar) andN-methyl-d-aspartate-stimulated γ-aminobutyric acid release in various brain regions (44.Kano T. Shimizu-Sasamata M. Huang P.L. Moskowitz M.A. Lo E.H. Neuroscience. 1998; 86: 695-699Crossref PubMed Scopus (96) Google Scholar). These functions of eNOS have been attributed to the enzyme located in neurons, on the assumption that brain eNOS is mainly distributed in neurons (19.Dinerman J.L. Dawson T.M. Schell M.J. Snowman A. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4214-4218Crossref PubMed Scopus (615) Google Scholar). However, since the present and other studies (21.Barna M. Komatsu T. Reiss C.S. Virology. 1996; 223: 331-343Crossref PubMed Scopus (81) Google Scholar, 22.Gabbott P.L.A. Bacon S.J. Brain Res. 1996; 714: 135-144Crossref PubMed Scopus (49) Google Scholar) demonstrated preferential distribution of eNOS in astrocytes rather than in neurons, it is tempting to speculate that NO derived from astroglial eNOS modulates these neuronal functions. Possible involvement of eNOS in neuronal functions suggests that a part of anti-microbial effects of astroglial eNOS can be mediated also through neuronal activities such as driving the autonomic nervous system that controls both brain and peripheral organs. Conditional astrocyte-specific disruption of the eNOS gene will provide useful tools to evaluate these intriguing roles of astroglial eNOS. We thank S. Fujimura for encouragement and S. Goto, K. Iyama, K. Sugahara, M. Takeya, T. Hiwasa, and colleagues for suggestions and discussions." @default.
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