- Email: [email protected]
Identification of nitric oxide synthases in isolated bovine brain vessels
MEUROSCgENCE RESEARCH ELSEVIER
Neuroscience Research 25 (1996) 195-199
Rapid communication
Identification of nitric oxide synthases in isolated bovine brain vessels R.E. Catalfin a'*, A.M. Martinez b, M.D. Aragon6s b, F. Hern/mdez a ~Departamento de Biologia Molecular, Centro de Biologia Molecular "Severo Ochoa" (CSIC-UAM), Universidad Aut6noma de Madrid, E-28049 Madrid, Spain bDepartamento de Bioquimica y Biologla Molecular I, Facultad de Ciencias Quimicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain
Received 5 January 1996; accepted 18 March 1996
Abstract
We have studied the presence of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) in parenchymal and pial bovine cerebral vessels by using western blot analysis. The different vessel structures were analysed by microscopic observation and their biochemical features determined by using ?-glutamiltranspeptidase (y-GTP) as an endothelial marker and ~-smooth muscle actin (c¢-SMA) as a smooth muscle cell marker, nNOS could not be found in parenchymal vessels, being present only in pial vessels; eNOS was present in all vessel-fractions studied, albeit a different distribution was found. Thus, the eNOS is more abundant in the parenchymal vessels which are associated with smooth muscle cells whereas both pial vessels and brain microvessels, almost devoid of actin, show extremely low levels. Two main conclusions can be obtained: first, nNOS is exclusively restricted to pial vessels and, second, eNOS is present in endothelial cells which are in association with smooth muscle cells. Keywords: Blood-brain barrier; Endothelial cells; Microvessels; Nitric oxide; Nitric oxide synthase
Brain microvessels are the major components of the blood-brain barrier (BBB), which is involved in the maintenance of brain interstitial fluid. In addition to these microvessels, composed basically by endothelial cells, there exists in the brain the pial vessels and other intraparenchymal blood vessels, venules/arterioles, which have smooth muscle cells together with the endothelial ones. The development of methods for isolating brain vessels of different caliber offers the opportunity for the comparative study of these vessels as well as the characterization of the unique features of the BBB 0 o 6 , 1985, 1992). To date, there is evidence that nitric oxide (NO) plays an important role in the regulation of the cerebral blood flow as it does in other vascular beds (Moncada et al., 1991). However, the source(s) of N O remains to be elucidated. The presence of nitric oxide synthase (NOS) in the brain vessels has been demonstrated *Corresponding author. Tel.: +34 1 3974869; fax: +34 1 3974870.
(Bredt et al., 1990; Iadecola et al., 1993). However, most of the studies have been focused on the histological observation of vessels and on the detection of N A D P H - d i a p h o r a s e activity, an unspecific marker of NOS isoforms. Thus, there is no biochemical study on isolated vessels showing the distribution of endothelial and neuronal isoforms of NOS. Furthermore, it is difficult to know whether the different brain vessels showed differences in this distribution since they are different from a functional point of view. In the last few years, our interest has been focused on the biochemical characterization and the study of the transduction signal systems of the BBB (Catalfin et al., 1989, 1990, 1992, 1993a,b, 1995). Taking into account the importance of N O in the transduction signal systems and more importantly, the unsolved localization and role of NOS isoforms in brain vessels (as stated above), the aim of the present work was to clear this point by using western blot ffnalysis in an attempt to deepen the study of the functioning of the BBB.
0168-0102/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PH SO168-0102(96)01038-3
196
R.E. Catal(m et al. / Neuroscience Research 25 (1996) 195 199
.o,"
": C
,,,.
• ,~.fllI
Fig. 1. P h o t o m i c r o g r a p h s of p a r e n c h y m a l vessels, toluidine blue-O staining. (I) " > 600 p m fraction', (I1) '600-200 p m fraction'and (111) '200-50 p m fraction'. In (I) bar = 250 p m , (II) bar = 200 p m and (Ill) bar = 50 ~ m .
Brain vessels were isolated by a modification of methods described previously (Catakin et al., 1993b; Hardebo et al., 1980). Briefly, bovine brains were obtained from a nearby slaughterhouse. The meninges were carefully removed and pial vessels were collected and kept in cold buffer A (mM): 103 NaCI, 4.7 KC1, 1.2 KH2PO4, 1.2 MgSO4, 15 Hepes, pH 7.4, 10 Glucose and 25 NaHCO3. Grey matter was minced and homogenized in 5 volumes of buffer A with a Teflon-glass homogenizer using a slow speed motor driven-pestle. Six to ten upwards and downwards strokes were applied during the homogenization. The homogenates were centrifuged at 3500 × g, 10 min at 4°C. The pellets were suspended in 15% dextran (MW = 79 500). Samples were then centrifuged again for 15 min at 5 000 x g at 4°C. The pellets were pooled in 50 ml of buffer A and passed over a 600 /zm-pore size nylon mesh. The tissue was forced to pass through the mesh using a end-cutted syringe which was filled with the mesh. The material not trapped was refiltered through the same 600/tm-mesh. The process was repeated twice with the material not retained but using sequentially 200 and 50 /~m nylon meshes. The material retained on the first mesh (600 /~m) was named ' > 600 /tm fraction', the tissue trapped on the second mesh (200/~m) was named '600-200 /~m fraction'. The material passing through the 200 /tm-pore size mesh was collected on a 50 /am-pore size mesh and was named '200-50 /Lm fraction'. The purified brain vessels were pooled by centrifugation. The purity of each vessel preparation was assessed by both morphological and biochemical criteria (Catalan et al., 1990). Immunoblotting was performed as described previously (Catal~in et al., 1993b). Proteins were separated by 6% SDS/PAGE. Then, proteins were transferred from gel to a nitrocellulose membrane (0.2 /~m). The non-specific binding sites on the nitrocellulose were
blocked in 0.1 M phosphate buffered saline containing 0.05% Tween-20 (solution B) with 3% skimmed milk for 60 min. The nitrocellulose filters were then incubated for 180 min at room temperature with monoclonal anti-endothelial NOS (1:500 dilution;raised in mouse against a synthetic peptide of human endothelial NOS), policlonal anti-neural NOS (1:500 dilution; raised in rabbit against a synthetic peptide of human neuronal NOS), both from Affiniti (Nottingham, UK) or monoclonal anti-a-smooth muscle actin (c~-SMA, 1:1000 dilution) from SIGMA (USA) in solution B with 3% skimmed milk and 0.1% timerosal. The nitrocellulose filters were then washed 3 x 10 min in solution B before incubation with the second antibody (from Affiniti, Nottingham, UK). The nitrocellulose filters were incubated overnight at room temperature with 1:2000 dilution of either polyclonal anti-mousehorseradish peroxidase (for detection of eNOS and 7-SMA) or 1:2000 dilution of polyclonal anti-rabbithorseradish peroxidase (for the detection of nNOS) in solution B with 3% skimmed milk. The second antibody was removed by 4 x 10 min washes in solution B. The specific binding was developed with the western blotting detection ECL system (Amersham, Buckinghamshire, UK). ~-Glutamyl transpeptidase (~-GTP) was determined with L-~-glutamyl-p-nitro-analide as substrate according to the procedure of Wahlefeld and Bergmeyer (1983). The method of Lowry et al. (1951) was used for protein determination. Fig. 1 shows the photomicrographs of the isolated parenchymal fractions. The ' > 600 /~m fraction' was formed by large arterial/venous segments (vessels with smooth muscle cells) with microvessels (vessels without smooth muscle cells) associated in networks. The '600200 /~m fraction' is primarily formed by small arterioles/venules and microvessels associated in networks.
198
R.E. Cataldn et al. / Neuroscience Research 25 (1996) 195-199
addition, Katusic (1992) has suggested that, compared with large cerebral vessels, vascular tone in smaller vessels is less dependent on production of NO due to the apparent smaller basal activity of eNOS in pial arteries. We also demonstrate the presence of eNOS in both pial and parenchymal brain vessels, and it seems that a relationship between the level of eNOS and ~-SMA-immunoreactivity may exist. It has been reported that cultured endothelial cells from brain microvessels (20050 ~m fraction in our study) do not express NOS (Frelin et al., 1992; Durieu-Trautmann et al., 1993). Our results are in keeping with this assumption, since it is interesting to note that in parenchymal vessels the eNOS-immunoreactivity appears to be less intense in small cerebral vessels paralleled with e-SMA-immunoreactivity, maintaining unchanged the amount of endothelial cells (measured as y-GTP activity). Therefore, an attractive hypothesis could be that smooth muscle cells induce eNOS in adjacent endothelial cells. However, the possibility that endothelial cells from brain microvessels show different properties than those from other vascular beds, cannot be excluded: In this regard, we must point out that important differences between aortic endothelial cells and brain capillary endothelial cells have been observed (Vigne et al., 1990). An increase of eNOS in brain microvessel in Alzheimer's disease has been reported recently (Dorheim et al., 1994). The presence of NOS in human brain microvessels appears to be discrepant with the results reported here, but the authors do not rule out that a percentage of smooth muscle cells or glial processes could contribute to the NOS identification. In addition, these discrepancies could also be attributed to differences between the species. In this regard, it has been reported that the relative contribution of constitutive NOS on the maintenance of brain vascular tone differs depending on the animal species, the brain region, and, in the case of eNOS, the segment of the cerebral vasculature (Iadecola et al., 1994). From this study it could be suggested that microvessels do not produce NO. However, these cells are targets for NO as they have soluble guanylyl cyclase (Karnushina et al., 1980), and NO induces accumulation of cyclic GMP (Marsault and Frelin, 1992). In addition, cGMP causes a decrease in the electrical resistance of the BBB endothelial monolayer (Rubin et al., 1991). Two main conclusions can be obtained: first, nNOS is exclusively restricted to pial vessels and, second, eNOS is present in endothelial cells that are in association with the smooth muscle cell. In any case, many questions remain to be elucidated in order to know the exact role of NO and NOS isoforms as well as the possible cross-talk with other pathways.
Acknowledgements The authors also wish to thank Mrs M.V. Mora-Gil for technical assistance. This work was supported in part by grants from the Fundacidn Ramdn Areces, FIS, CAM and DGICYT.
References Bredt, D.S., Hwang, P.M. and Snyder, S.H. (1990) Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature, 347: 768-770. Catalan, R.E., Martinez, A.M., Aragon,s, M.D. and Fernandez, I. (1989) Substance P stimulates protein kinase C in brain microvessels. Biochem. Biophys. Res. Commun., 164:595 600. Catalan, R.E., Martinez, A.M., Aragon,s, M.D. and Hernandez F. (1990) Molecular forms of butyrylcholinesterase in rat brain microvessels. Neurosci. Lett., 120: 46-49. Catalan, R.E., Martinez, A.M., Aragon,s, M.D. and Hernandez, F. (1992) Phorbol esters stimulate phosphoinositide phosphorylation and phosphatidylcholine metabolism in brain microvessels. Biochem. Int., 27: 231-242. Catalan, R.E., Martinez, A.M., Aragon,s, M.D., Garde, E. and Diaz, G. (1993a) Platelet-activating factor stimulates protein kinase C translocation in cerebral microvessels. Biochem. Biophys. Res. Commun., 192: 446-451. Catalan, R.E., Martinez, A.M., Aragon6s, M.D. and Diaz, G. (1993b) Identification of GTP binding proteins in brain microvessels and their role in phosphoinositide turnover. Biochem. Biophys. Res. Commun., 195: 952-957. Catalan, R.E., Martinez, A.M., Aragon,s, M.D., Fernandez, I. and Hernandez, F. (1995) Involvement of calcium in phosphoinositide metabolism in blood-brain barrier. Cell. Signal., 7: 261-267. Dorheim, M-A., Tracey, W.R., Pollock, J.S. and Grammas, P. (1994) Nitric oxide synthase activity is elevated in brain microvessels in Alzheimer's disease. Biochem. Biophys. Res. Commun., 205: 659665. Durieu-Trautmann, O., F6d6rici, C., Cr6minon, C., FoignantChaverot, N., Roux, F., Claire, M., Strosberg, A.D. and Couraud, P.O. (1993) Nitric oxide and endothelin secretion by brain microvessel endothelial cells: regulation by cyclic nucleotides. J. Cell. Physiol., 155: 104-111 Frelin, C., Marsault, R. and Vigne, P. (1992) Endothelial cells from brain microvessels are target cells for neuronal NO. In: S. Moncada, M.A. Marietta, J.B. Hibbs and E.A. Higgs (Eds.), The Biology of Nitric Oxide, Portland Press, London, pp. 81-83. Hardebo, J.E., Emson, P.C., Falck, B., Owman, Ch. and Rosengren, E. (1980) Enzymes related to monoamine transmitter metabolism in brain microvessels. J. Neurocbem., 35:1388 1393. Iadecola, C., Beitz, A.J., Renno, W., Xu, X., Mayer, B. and Zhang, F. (1993) Nitric oxide synthase-containing neural processes on large cerebral arteries and cerebral microvessels. Brain Res., 606: 148-155. Iadecola, C., Pelligrino, D.A., Moskowitz, M.A. and Lassen, N.A. (1994) Nitric oxide synthase inhibition and cerebrovascular regulation. J. Cereb. Blood Flow Metab., 14: 175-192. Jo6, F. (1985) The blood-brain barrier in vitro: ten years of research on microvessels isolated from the brain. Neurochem. Int., 7: 1-25.
Jo6, F. (1992) The cerebral microvessels in culture, an update. J. Neurochem., 58: 1-7. Karnusbina, I., Toth, 1., Dux, E. and Jo6, F. (1980) Presence of the soluble guanylate cyclase in brain capillaries: histochemical and biochemical evidence. Brain Res., 189: 588-592.
R.E. Catalgm et al./ Neuroscience Research 25 (1996) 195-199
197
2500
e-NOS 40C 4a
3o0
el 1oo
A
n-NOS
B
d
1 (; ±
6
A
~
6000
~
5000
c ml
?.. 4 0 0 0
.la
B
tl
C
D
3000 2000
a~-SMA
,o
1000
A
B
C
D
0
A
B
C
D
- - I 1 '
A B
6
Fig. 2. Presence of nNOS, eNOS and c~-SMA in bovine brain vessels. Left: Western blots demonstrating eNOS, nNOS and ~-SMA in pial vessels (A, 250 p g of protein per well), ' > 600 p m fraction' (B, 169 g g of protein), '600-200 p m fraction' (C, 154 # g of protein) and, '200-50 # m fraction' (D, 216 #g o f protein). The total absorbance (data from I) in each of the samples is shown in right side. 7-GTP activity is also shown. Experimental conditions are described in detail in the text. The blots and the data are representative of three similar and independent experiments. 7-GTP activity is expressed as units/lane (1 unit is the amount of protein that is able to generate 1 #mol of 3-carboxy-4-nitroaniline per minute).
The '200-50 #m fraction' was formed almost exclusively by short segments of microvessels together with microvessels associated in networks. These observations were confirmed, identifying specific proteins by western blot analysis and enzymatic marker determinations. Results are shown in Fig. 2. Isolated pial vessels showed abundant ~-SMA-immunoreactivity. In contrast, parenchymal vessels showed a level of 7-SMAimmunoreactivity which decreased as the size of the vessel decreased. To study the distribution of NOS isoforms among the different parenchymal fractions, we loaded the polyacrilamide gels with the same amount of 7-GTP (Fig. 2). We were interested in loading the same amount of endothelial protein per well, albeit the total protein amount was similar. 7-GTP has been widely used as a marker for brain microvessels and differentiated properties of brain endothelial cells (Mischeck et al., 1989; Meyer et al., 1990). Results shown in Fig. 2 indicated that nNOS-immunoreactivity (155 KDa) could not be detected in parenchymal vessels, being present only in pial vessels. By contrast, eNOS-immunoreactivity (140 KDa) was present in all vessel fractions analyzed (Fig. 2). It is noteworthy that as it occurs with ~-SMA-immunoreactivity (Fig. 2), the level of eNOS in parenchymal vessels decreased as the caliber of the vessel decreased. Thus, the amount of eNOS
present in the '200-50 #m fraction' is abotit a 25% of the ' > 600 #m fraction'. The present work is the first evidence focused on the distribution of two constitutive isoforms of NOS (nNOS and eNOS) in pial vessels and parenchymal vessels including microvessels, which are the main components of BBB. In regard to nNOS, our data showing the presence of nNOS in pial vessels are in agreement with results previously described using a different antibody against nNOS (Bredt et al., 1990; Tomimoto et al., 1994). The detection of nNOS in larger vessels might be explained by the fact that pial and parenchymal vessels are innervated in a different manner (Iadecola et al., 1993, 1994). Thus, NOS-containing nerves of peripheral origin innervate large cerebral arteries while NOS-containing neural processes of central origin are closely associated with cerebral arterioles and capillaries. In this respect, we must point out that the influence of NO on large cerebral arteries is likely to be different from that exerted on intraparenchymal microvessels. Thus, in large arteries with a muscular_ coat NO-mediated guanylyl cyclase activity may produce smooth muscle relaxation and modulate global changes in cerebral blood flow (Iadecola et al., 1993). In contrast, in cerebral microvessels NO-induced elevation in cyclic GMP may influence microvascular contractility and endothelial permeability (Kelly et al., 1987). In
R.E. Catal~n et al. / Neuroscience Research 25 (1996) 195-199
Katusic, Z.S. (1992) Endothelial L-arginine pathway and regional cerebral arterial reactivity to vasopressin. Am. J. Physiol., 262: H1557-HI562. Kelly, C., D'Amore, P., Hechtman, H.B. and Shepro, D. (1987) Microvascular pericyte contractility in vitro: comparison with other cells of the vascular cells of the vascular wall. J. Cell Biol., 104: 483-490. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Marsault, R. and Frelin, C. (1992) Activation by nitric oxide of guanylate cyclase in endothelial cells from brain capillaries. J. Neurochem., 59:942 945. Meyer, J., Mischeck, U., Veyhl, M., Henzel, K. and Galla, H.-J. (1990) Blood-brain barrier characteristic enzymatic-properties in cultured brain capillary endothelial-cells. Brain Res., 514: 305309. Mischeck, U., Meyer, J. and Galla, H.-J (1989) Characterization of -glutamyl transpeptidase activity of cultured endothelial cells from porcine brain capillaries. Cell Tissue Res., 256:221 226.
199
Moncada, S., Palmer, R.M.J. and Higgs, E.A. (1991) Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol. Rev., 43: 109-142. Rubin, L.L., Hall, D.E., Porte, S., Barbu, K., Cannon, C., Horner, H.C., Janatpour, N., Liaw, C.W., Manning, K., Morales, J., Tanner, L.I., Tommaselli, K.J. and Bard, F. (1991) A cell culture model of the blood-brain barrier. J. Cell. Biol., 115:1725 1735. Vigne, P., Marsault, R., Breittmayer, J.P. and Frelin, C. (1990) Endothelin stimulates phosphatidylinositol hydrolysis and DNA synthesis in brain capillary endothelial cells. Biochem. J., 266: 415-420. Tomimoto, H., Nishimura, M., Suenaga, T., Nakamura, S., Akiguchi, I., Wakita, H., Kimura, J. and Mayer, B. (1994) Distribution of nitric oxide synthase in the human cerebral blood vessels and brain tissues. J. Cereb. Blood Flow Metab., 14: 930-938. Wahlefeld, A.W. and Bergmeyer, H.V. (1983) ~-glutamyltransferase, routine method, In: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Vol. III, 3rd edn., Verlag Chemie, Winheim, pp. 352-356.
Neuroscience Research 25 (1996) 195-199
Rapid communication
Identification of nitric oxide synthases in isolated bovine brain vessels R.E. Catalfin a'*, A.M. Martinez b, M.D. Aragon6s b, F. Hern/mdez a ~Departamento de Biologia Molecular, Centro de Biologia Molecular "Severo Ochoa" (CSIC-UAM), Universidad Aut6noma de Madrid, E-28049 Madrid, Spain bDepartamento de Bioquimica y Biologla Molecular I, Facultad de Ciencias Quimicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain
Received 5 January 1996; accepted 18 March 1996
Abstract
We have studied the presence of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) in parenchymal and pial bovine cerebral vessels by using western blot analysis. The different vessel structures were analysed by microscopic observation and their biochemical features determined by using ?-glutamiltranspeptidase (y-GTP) as an endothelial marker and ~-smooth muscle actin (c¢-SMA) as a smooth muscle cell marker, nNOS could not be found in parenchymal vessels, being present only in pial vessels; eNOS was present in all vessel-fractions studied, albeit a different distribution was found. Thus, the eNOS is more abundant in the parenchymal vessels which are associated with smooth muscle cells whereas both pial vessels and brain microvessels, almost devoid of actin, show extremely low levels. Two main conclusions can be obtained: first, nNOS is exclusively restricted to pial vessels and, second, eNOS is present in endothelial cells which are in association with smooth muscle cells. Keywords: Blood-brain barrier; Endothelial cells; Microvessels; Nitric oxide; Nitric oxide synthase
Brain microvessels are the major components of the blood-brain barrier (BBB), which is involved in the maintenance of brain interstitial fluid. In addition to these microvessels, composed basically by endothelial cells, there exists in the brain the pial vessels and other intraparenchymal blood vessels, venules/arterioles, which have smooth muscle cells together with the endothelial ones. The development of methods for isolating brain vessels of different caliber offers the opportunity for the comparative study of these vessels as well as the characterization of the unique features of the BBB 0 o 6 , 1985, 1992). To date, there is evidence that nitric oxide (NO) plays an important role in the regulation of the cerebral blood flow as it does in other vascular beds (Moncada et al., 1991). However, the source(s) of N O remains to be elucidated. The presence of nitric oxide synthase (NOS) in the brain vessels has been demonstrated *Corresponding author. Tel.: +34 1 3974869; fax: +34 1 3974870.
(Bredt et al., 1990; Iadecola et al., 1993). However, most of the studies have been focused on the histological observation of vessels and on the detection of N A D P H - d i a p h o r a s e activity, an unspecific marker of NOS isoforms. Thus, there is no biochemical study on isolated vessels showing the distribution of endothelial and neuronal isoforms of NOS. Furthermore, it is difficult to know whether the different brain vessels showed differences in this distribution since they are different from a functional point of view. In the last few years, our interest has been focused on the biochemical characterization and the study of the transduction signal systems of the BBB (Catalfin et al., 1989, 1990, 1992, 1993a,b, 1995). Taking into account the importance of N O in the transduction signal systems and more importantly, the unsolved localization and role of NOS isoforms in brain vessels (as stated above), the aim of the present work was to clear this point by using western blot ffnalysis in an attempt to deepen the study of the functioning of the BBB.
0168-0102/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PH SO168-0102(96)01038-3
196
R.E. Catal(m et al. / Neuroscience Research 25 (1996) 195 199
.o,"
": C
,,,.
• ,~.fllI
Fig. 1. P h o t o m i c r o g r a p h s of p a r e n c h y m a l vessels, toluidine blue-O staining. (I) " > 600 p m fraction', (I1) '600-200 p m fraction'and (111) '200-50 p m fraction'. In (I) bar = 250 p m , (II) bar = 200 p m and (Ill) bar = 50 ~ m .
Brain vessels were isolated by a modification of methods described previously (Catakin et al., 1993b; Hardebo et al., 1980). Briefly, bovine brains were obtained from a nearby slaughterhouse. The meninges were carefully removed and pial vessels were collected and kept in cold buffer A (mM): 103 NaCI, 4.7 KC1, 1.2 KH2PO4, 1.2 MgSO4, 15 Hepes, pH 7.4, 10 Glucose and 25 NaHCO3. Grey matter was minced and homogenized in 5 volumes of buffer A with a Teflon-glass homogenizer using a slow speed motor driven-pestle. Six to ten upwards and downwards strokes were applied during the homogenization. The homogenates were centrifuged at 3500 × g, 10 min at 4°C. The pellets were suspended in 15% dextran (MW = 79 500). Samples were then centrifuged again for 15 min at 5 000 x g at 4°C. The pellets were pooled in 50 ml of buffer A and passed over a 600 /zm-pore size nylon mesh. The tissue was forced to pass through the mesh using a end-cutted syringe which was filled with the mesh. The material not trapped was refiltered through the same 600/tm-mesh. The process was repeated twice with the material not retained but using sequentially 200 and 50 /~m nylon meshes. The material retained on the first mesh (600 /~m) was named ' > 600 /tm fraction', the tissue trapped on the second mesh (200/~m) was named '600-200 /~m fraction'. The material passing through the 200 /tm-pore size mesh was collected on a 50 /am-pore size mesh and was named '200-50 /Lm fraction'. The purified brain vessels were pooled by centrifugation. The purity of each vessel preparation was assessed by both morphological and biochemical criteria (Catalan et al., 1990). Immunoblotting was performed as described previously (Catal~in et al., 1993b). Proteins were separated by 6% SDS/PAGE. Then, proteins were transferred from gel to a nitrocellulose membrane (0.2 /~m). The non-specific binding sites on the nitrocellulose were
blocked in 0.1 M phosphate buffered saline containing 0.05% Tween-20 (solution B) with 3% skimmed milk for 60 min. The nitrocellulose filters were then incubated for 180 min at room temperature with monoclonal anti-endothelial NOS (1:500 dilution;raised in mouse against a synthetic peptide of human endothelial NOS), policlonal anti-neural NOS (1:500 dilution; raised in rabbit against a synthetic peptide of human neuronal NOS), both from Affiniti (Nottingham, UK) or monoclonal anti-a-smooth muscle actin (c~-SMA, 1:1000 dilution) from SIGMA (USA) in solution B with 3% skimmed milk and 0.1% timerosal. The nitrocellulose filters were then washed 3 x 10 min in solution B before incubation with the second antibody (from Affiniti, Nottingham, UK). The nitrocellulose filters were incubated overnight at room temperature with 1:2000 dilution of either polyclonal anti-mousehorseradish peroxidase (for detection of eNOS and 7-SMA) or 1:2000 dilution of polyclonal anti-rabbithorseradish peroxidase (for the detection of nNOS) in solution B with 3% skimmed milk. The second antibody was removed by 4 x 10 min washes in solution B. The specific binding was developed with the western blotting detection ECL system (Amersham, Buckinghamshire, UK). ~-Glutamyl transpeptidase (~-GTP) was determined with L-~-glutamyl-p-nitro-analide as substrate according to the procedure of Wahlefeld and Bergmeyer (1983). The method of Lowry et al. (1951) was used for protein determination. Fig. 1 shows the photomicrographs of the isolated parenchymal fractions. The ' > 600 /~m fraction' was formed by large arterial/venous segments (vessels with smooth muscle cells) with microvessels (vessels without smooth muscle cells) associated in networks. The '600200 /~m fraction' is primarily formed by small arterioles/venules and microvessels associated in networks.
198
R.E. Cataldn et al. / Neuroscience Research 25 (1996) 195-199
addition, Katusic (1992) has suggested that, compared with large cerebral vessels, vascular tone in smaller vessels is less dependent on production of NO due to the apparent smaller basal activity of eNOS in pial arteries. We also demonstrate the presence of eNOS in both pial and parenchymal brain vessels, and it seems that a relationship between the level of eNOS and ~-SMA-immunoreactivity may exist. It has been reported that cultured endothelial cells from brain microvessels (20050 ~m fraction in our study) do not express NOS (Frelin et al., 1992; Durieu-Trautmann et al., 1993). Our results are in keeping with this assumption, since it is interesting to note that in parenchymal vessels the eNOS-immunoreactivity appears to be less intense in small cerebral vessels paralleled with e-SMA-immunoreactivity, maintaining unchanged the amount of endothelial cells (measured as y-GTP activity). Therefore, an attractive hypothesis could be that smooth muscle cells induce eNOS in adjacent endothelial cells. However, the possibility that endothelial cells from brain microvessels show different properties than those from other vascular beds, cannot be excluded: In this regard, we must point out that important differences between aortic endothelial cells and brain capillary endothelial cells have been observed (Vigne et al., 1990). An increase of eNOS in brain microvessel in Alzheimer's disease has been reported recently (Dorheim et al., 1994). The presence of NOS in human brain microvessels appears to be discrepant with the results reported here, but the authors do not rule out that a percentage of smooth muscle cells or glial processes could contribute to the NOS identification. In addition, these discrepancies could also be attributed to differences between the species. In this regard, it has been reported that the relative contribution of constitutive NOS on the maintenance of brain vascular tone differs depending on the animal species, the brain region, and, in the case of eNOS, the segment of the cerebral vasculature (Iadecola et al., 1994). From this study it could be suggested that microvessels do not produce NO. However, these cells are targets for NO as they have soluble guanylyl cyclase (Karnushina et al., 1980), and NO induces accumulation of cyclic GMP (Marsault and Frelin, 1992). In addition, cGMP causes a decrease in the electrical resistance of the BBB endothelial monolayer (Rubin et al., 1991). Two main conclusions can be obtained: first, nNOS is exclusively restricted to pial vessels and, second, eNOS is present in endothelial cells that are in association with the smooth muscle cell. In any case, many questions remain to be elucidated in order to know the exact role of NO and NOS isoforms as well as the possible cross-talk with other pathways.
Acknowledgements The authors also wish to thank Mrs M.V. Mora-Gil for technical assistance. This work was supported in part by grants from the Fundacidn Ramdn Areces, FIS, CAM and DGICYT.
References Bredt, D.S., Hwang, P.M. and Snyder, S.H. (1990) Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature, 347: 768-770. Catalan, R.E., Martinez, A.M., Aragon,s, M.D. and Fernandez, I. (1989) Substance P stimulates protein kinase C in brain microvessels. Biochem. Biophys. Res. Commun., 164:595 600. Catalan, R.E., Martinez, A.M., Aragon,s, M.D. and Hernandez F. (1990) Molecular forms of butyrylcholinesterase in rat brain microvessels. Neurosci. Lett., 120: 46-49. Catalan, R.E., Martinez, A.M., Aragon,s, M.D. and Hernandez, F. (1992) Phorbol esters stimulate phosphoinositide phosphorylation and phosphatidylcholine metabolism in brain microvessels. Biochem. Int., 27: 231-242. Catalan, R.E., Martinez, A.M., Aragon,s, M.D., Garde, E. and Diaz, G. (1993a) Platelet-activating factor stimulates protein kinase C translocation in cerebral microvessels. Biochem. Biophys. Res. Commun., 192: 446-451. Catalan, R.E., Martinez, A.M., Aragon6s, M.D. and Diaz, G. (1993b) Identification of GTP binding proteins in brain microvessels and their role in phosphoinositide turnover. Biochem. Biophys. Res. Commun., 195: 952-957. Catalan, R.E., Martinez, A.M., Aragon,s, M.D., Fernandez, I. and Hernandez, F. (1995) Involvement of calcium in phosphoinositide metabolism in blood-brain barrier. Cell. Signal., 7: 261-267. Dorheim, M-A., Tracey, W.R., Pollock, J.S. and Grammas, P. (1994) Nitric oxide synthase activity is elevated in brain microvessels in Alzheimer's disease. Biochem. Biophys. Res. Commun., 205: 659665. Durieu-Trautmann, O., F6d6rici, C., Cr6minon, C., FoignantChaverot, N., Roux, F., Claire, M., Strosberg, A.D. and Couraud, P.O. (1993) Nitric oxide and endothelin secretion by brain microvessel endothelial cells: regulation by cyclic nucleotides. J. Cell. Physiol., 155: 104-111 Frelin, C., Marsault, R. and Vigne, P. (1992) Endothelial cells from brain microvessels are target cells for neuronal NO. In: S. Moncada, M.A. Marietta, J.B. Hibbs and E.A. Higgs (Eds.), The Biology of Nitric Oxide, Portland Press, London, pp. 81-83. Hardebo, J.E., Emson, P.C., Falck, B., Owman, Ch. and Rosengren, E. (1980) Enzymes related to monoamine transmitter metabolism in brain microvessels. J. Neurocbem., 35:1388 1393. Iadecola, C., Beitz, A.J., Renno, W., Xu, X., Mayer, B. and Zhang, F. (1993) Nitric oxide synthase-containing neural processes on large cerebral arteries and cerebral microvessels. Brain Res., 606: 148-155. Iadecola, C., Pelligrino, D.A., Moskowitz, M.A. and Lassen, N.A. (1994) Nitric oxide synthase inhibition and cerebrovascular regulation. J. Cereb. Blood Flow Metab., 14: 175-192. Jo6, F. (1985) The blood-brain barrier in vitro: ten years of research on microvessels isolated from the brain. Neurochem. Int., 7: 1-25.
Jo6, F. (1992) The cerebral microvessels in culture, an update. J. Neurochem., 58: 1-7. Karnusbina, I., Toth, 1., Dux, E. and Jo6, F. (1980) Presence of the soluble guanylate cyclase in brain capillaries: histochemical and biochemical evidence. Brain Res., 189: 588-592.
R.E. Catalgm et al./ Neuroscience Research 25 (1996) 195-199
197
2500
e-NOS 40C 4a
3o0
el 1oo
A
n-NOS
B
d
1 (; ±
6
A
~
6000
~
5000
c ml
?.. 4 0 0 0
.la
B
tl
C
D
3000 2000
a~-SMA
,o
1000
A
B
C
D
0
A
B
C
D
- - I 1 '
A B
6
Fig. 2. Presence of nNOS, eNOS and c~-SMA in bovine brain vessels. Left: Western blots demonstrating eNOS, nNOS and ~-SMA in pial vessels (A, 250 p g of protein per well), ' > 600 p m fraction' (B, 169 g g of protein), '600-200 p m fraction' (C, 154 # g of protein) and, '200-50 # m fraction' (D, 216 #g o f protein). The total absorbance (data from I) in each of the samples is shown in right side. 7-GTP activity is also shown. Experimental conditions are described in detail in the text. The blots and the data are representative of three similar and independent experiments. 7-GTP activity is expressed as units/lane (1 unit is the amount of protein that is able to generate 1 #mol of 3-carboxy-4-nitroaniline per minute).
The '200-50 #m fraction' was formed almost exclusively by short segments of microvessels together with microvessels associated in networks. These observations were confirmed, identifying specific proteins by western blot analysis and enzymatic marker determinations. Results are shown in Fig. 2. Isolated pial vessels showed abundant ~-SMA-immunoreactivity. In contrast, parenchymal vessels showed a level of 7-SMAimmunoreactivity which decreased as the size of the vessel decreased. To study the distribution of NOS isoforms among the different parenchymal fractions, we loaded the polyacrilamide gels with the same amount of 7-GTP (Fig. 2). We were interested in loading the same amount of endothelial protein per well, albeit the total protein amount was similar. 7-GTP has been widely used as a marker for brain microvessels and differentiated properties of brain endothelial cells (Mischeck et al., 1989; Meyer et al., 1990). Results shown in Fig. 2 indicated that nNOS-immunoreactivity (155 KDa) could not be detected in parenchymal vessels, being present only in pial vessels. By contrast, eNOS-immunoreactivity (140 KDa) was present in all vessel fractions analyzed (Fig. 2). It is noteworthy that as it occurs with ~-SMA-immunoreactivity (Fig. 2), the level of eNOS in parenchymal vessels decreased as the caliber of the vessel decreased. Thus, the amount of eNOS
present in the '200-50 #m fraction' is abotit a 25% of the ' > 600 #m fraction'. The present work is the first evidence focused on the distribution of two constitutive isoforms of NOS (nNOS and eNOS) in pial vessels and parenchymal vessels including microvessels, which are the main components of BBB. In regard to nNOS, our data showing the presence of nNOS in pial vessels are in agreement with results previously described using a different antibody against nNOS (Bredt et al., 1990; Tomimoto et al., 1994). The detection of nNOS in larger vessels might be explained by the fact that pial and parenchymal vessels are innervated in a different manner (Iadecola et al., 1993, 1994). Thus, NOS-containing nerves of peripheral origin innervate large cerebral arteries while NOS-containing neural processes of central origin are closely associated with cerebral arterioles and capillaries. In this respect, we must point out that the influence of NO on large cerebral arteries is likely to be different from that exerted on intraparenchymal microvessels. Thus, in large arteries with a muscular_ coat NO-mediated guanylyl cyclase activity may produce smooth muscle relaxation and modulate global changes in cerebral blood flow (Iadecola et al., 1993). In contrast, in cerebral microvessels NO-induced elevation in cyclic GMP may influence microvascular contractility and endothelial permeability (Kelly et al., 1987). In
R.E. Catal~n et al. / Neuroscience Research 25 (1996) 195-199
Katusic, Z.S. (1992) Endothelial L-arginine pathway and regional cerebral arterial reactivity to vasopressin. Am. J. Physiol., 262: H1557-HI562. Kelly, C., D'Amore, P., Hechtman, H.B. and Shepro, D. (1987) Microvascular pericyte contractility in vitro: comparison with other cells of the vascular cells of the vascular wall. J. Cell Biol., 104: 483-490. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Marsault, R. and Frelin, C. (1992) Activation by nitric oxide of guanylate cyclase in endothelial cells from brain capillaries. J. Neurochem., 59:942 945. Meyer, J., Mischeck, U., Veyhl, M., Henzel, K. and Galla, H.-J. (1990) Blood-brain barrier characteristic enzymatic-properties in cultured brain capillary endothelial-cells. Brain Res., 514: 305309. Mischeck, U., Meyer, J. and Galla, H.-J (1989) Characterization of -glutamyl transpeptidase activity of cultured endothelial cells from porcine brain capillaries. Cell Tissue Res., 256:221 226.
199
Moncada, S., Palmer, R.M.J. and Higgs, E.A. (1991) Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol. Rev., 43: 109-142. Rubin, L.L., Hall, D.E., Porte, S., Barbu, K., Cannon, C., Horner, H.C., Janatpour, N., Liaw, C.W., Manning, K., Morales, J., Tanner, L.I., Tommaselli, K.J. and Bard, F. (1991) A cell culture model of the blood-brain barrier. J. Cell. Biol., 115:1725 1735. Vigne, P., Marsault, R., Breittmayer, J.P. and Frelin, C. (1990) Endothelin stimulates phosphatidylinositol hydrolysis and DNA synthesis in brain capillary endothelial cells. Biochem. J., 266: 415-420. Tomimoto, H., Nishimura, M., Suenaga, T., Nakamura, S., Akiguchi, I., Wakita, H., Kimura, J. and Mayer, B. (1994) Distribution of nitric oxide synthase in the human cerebral blood vessels and brain tissues. J. Cereb. Blood Flow Metab., 14: 930-938. Wahlefeld, A.W. and Bergmeyer, H.V. (1983) ~-glutamyltransferase, routine method, In: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Vol. III, 3rd edn., Verlag Chemie, Winheim, pp. 352-356.