?-Glutamyl Transpeptidase Activity in Brain Microvessels Exhibits Regional Heterogeneity

Journal of Neurochemistry Raven Press, Ltd.. New York 0 1992 International Society for Neurochemistry

y-Glutamyl Transpeptidase Activity in Brain Microvessels Exhibits Regional Heterogeneity *$Johannes E. A. Wolff, $Luisa Belloni-Olivi, *$Joseph P. Bressler, and *=/-$GaryW. Goldstein Departments of *Neurology and ?Pediatrics, Johns Hopkins School of Medicine, and $The Kennedy Research Institute, Baltimore, Murylund, U S A .

Abstract: Brain microvessels form a tight blood-tissue permeability bamer and express high levels of specific enzymes, including y-glutamyl transpeptidase (GGTP). This differentiation is thought to be induced by perivascular astrocytes. By using histochemical methods, we found that the percentage of GGTP-positive vessels vaned considerably in different areas of rat brain. Enzyme activity was not found in the pineal gland or the median eminence, where the blood-brain bamer is not expressed. In areas where the blood-brain bamer is expressed, the percentage of GGTP-positive vessels vaned from 8% in the optic nerve to 100%in the anterior commissure. The neocortex showed a lower percentage of GGTPpositive vessels (2- 15%) than anterior olfactory nucleus (42%), subiculum (70%), hippocampus (48%), and striatum (5058%). Alkaline phosphatase, another brain microvessel-en-

riched enzyme, did not show these marked regional differences. The morphometric histochemical results were verified by enzymatic assays in homogenates of different regions from rat and bovine brain and in microvessel preparations of bovine putamen and neocortex. During the postnatal development of rat brain, the difference between neocortex and striatum appeared after day 20. The regional heterogeneity of brain microvessels may be caused by astrocytic heterogeneity and reflect regional heterogeneity in microvascular function. Key Words: Blood-brain bamer-y-Glutamyl transpeptidase-Alkaline phosphatase-Microvessels. Wolff J. E. A. et al. y-Glutamyl transpeptidase activity in brain microvessels exhibits regional heterogeneity. J. Neurochem. 58,909-9 15 ( 1992).

Brain microvascular endothelial cells help regulate the neuronal environment. They differ from endothelial cells of other organs in that they form tight junctions (Muir and Peters, 1962) and contain few cytoplasmic vesicles. This results in a blood-tissue barrier (Reese and Karnovsky, 1967). Transport of water-soluble substances through that barrier requires specific transport systems. Brain endothelial cells also have a high density of mitochondria (Oldendorf and Brown, 1975) and express high levels of enzymes, such as y-glutamyl transpeptidase (GGTP; EC 2.3.2.2) (Albert et al., 1966; Ghandour et al., 1980; Papandrikopoulou et al., 1989), not expressed in other endothelial cells. This suggests additional metabolic regulatory functions that are not yet well understood. The factors inducing expression of the blood-brain barrier phenotype by brain endothelial cells appear to be derived in part from astroglial cells (Svengaard et al., 1975; Stewart and Wiley, 1981; Janzer and R a g 1987; Dehouck et al., 1990).

GGTP is a membrane-bound enzyme localized in brain endothelial cells and various epithelial cells, including the choroid plexus. By using immunohistochemistry at the electron-microscopic level, it was localized to the luminal membrane of cerebellar endothelial cells (Ghandour et al., 1980). In rat brain, it is known to increase during the first 3 weeks of postnatal development (Betz and Goldstein, 1981). The biological function of GGTP may relate to amino acid transport across the blood-brain barrier (Orlowski et al., 1974).The main clinical symptom of GGTP deficiency is mental retardation (Goodman et al., 1971). The anatomical organization of the brain is characterized by regional differences in neuronal morphology and function. Astrocyte distribution and differentiation also vary greatly in different brain regions (Weigert, 1985; Chamak et al., 1987; El-Etr et al., 1989). However, regionally specific endothelial cell differentiation is known only in respect to the presence

Received May 22, 1991; revised manuscript received July 19, 1991; accepted July 29, 1991. Address correspondence and reprint requests to Dr. J. P. Bressler at Kennedy Research Institute, Inc., 707 N. Broadway, Baltimore, MD 2 1205, U.S.A. Each of the listed authors (a) has contributed to the paper signif-

icantly, (b) has reviewed the manuscript, and (c) stands behind the parts within his or her own area of expertise. Abbreviations used: AP, alkaline phosphatase;BSA, bovine serum albumin; GGTP, y-glutamyl transpeptidase; HBSS, Hanks’ balanced salt solution.

909

J. E. A . WOLFF ET AL.

910

or absence of barrier properties. Because astroglial cells induce the blood-brain barrier phenotype, we hypothesized that regionally specific differentiation may also occur in endothelial cells. Therefore, we investigated the regional distribution of GGTP and alkaline phosphatase (AP; EC 3.1.3.1)activity, two enzymes that are highly enriched in brain endothelial cells. MATERIALS AND METHODS All chemicals were purchased from Sigma (St. Louis, MO, U.S.A.) unless otherwise noted.

Determination of microvessel density Rats were anesthetized by intrapentoneal injection (3 ml/ kg dose) of a stock solution containing 25 mg/ml ketamine hydrochloride, 2.5 mg/rnl xylazine, and 14% (vol/vol) ethanol in normal saline. After opening the thorax, the right atrium was incised and the left ventricle was canalized and perfused (60 ml/min, pump 503s; Whatson-Marlow, Falmouth, U.K.), first with 200 ml of Hanks’ balanced salt solution (HBSS) containing 1 g/L bovine serum albumin (BSA) and 600 units/L heparin, and then with 200 ml of a solution containing 90% filtered black India ink (Pelican, Frankfurt, F.R.G.), 10% 10 X HBSS, 1 g/L BSA, and 600 units/L heparin, at pH 7.4 adjusted with sodium bicarbonate. Brains were removed, fxozen immediately, and cut in 10-pm thick coronal sections using a cryostat (Lipshaw, Detroit, MI, U.S.A.). Microvessel density was counted in five to 10 fields per brain region using a k i t z Aristoplan microscope with 40X objective in 0.1 I-mm2 fields.

Histochemistry Histochemical stains were done without fixation. GGTP staining was done as described by Rutenburg et al. (1969) using y-glutamyl-4-methoxy-2-naphthylamide (Vega Co., Tucson, AZ, U.S.A.) as the enzyme substrate and diazotinized 4-amino-2,5-diethoxybenzanilide(fast blue) as chromogen. AP staining was done using Sigma procedure no. 85 following the method of Kaplow (1968). GGTP- and AP-positive vessels were counted as described above and enumerated as a percentage of vessels filled with ink.

and resuspended in lysis buffer for enzyme assays as described below.

Enzyme assays Enzyme assays were done after sonication in lysis buffer containing 2 mMTris-HC1, pH 7.4,50 mMD-mannitol, and 1% Triton X-100. GGTP was measured as described by Orlowski and Meister ( 1 963) with bovine kidney GGTP as the standard. The final concentration of the reaction mixture was I mM L-y-glutamyl-pnitroanilide, 20 mMglycylglycine, and 10 mM Tris-HC1, pH 8. Absorbance at 410 nm was determined for a 30-min reaction at 37°C. One unit of GGTP activity represents the formation of 1 pmol ofp-nitroadinel min. AP was measured with the method described by Bowers and McComb (1966) using 16 mMp-nitrophenyl phosphate as substrate (Sigma procedure no. 245) and bovine intestinal mucosa AP as standard. One unit of AP activity is defined as the production of I pmol of p-nitrophenollmin at 30°C.

RESULTS Enzyme assays in bovine tissues Both GGTP and AP levels in total tissue homogenates differed greatly in various regions of bovine brain (Table 1). Because both enzymes localize to brain microvessels (Leduc and Wislocki, 1952; Albert et al., 1966),the different enzyme activities noted in different brain regions may be explained by differences in vessel density. In that case, the ratio between the two vessel markers should be constant. This was not true. GGTP activity divided by AP activity was lower in cortex than in other regions (Table l), suggesting that the GGTP was more active or AP was less active in cortical microvessels. In order to determine which of the enzyme TABLE 1. Enzyme activities in bovine brain homoaenates GGTP (U/g of protein)

AP (U/P of protein)

GGTP/AP ~~

Preparation of brain regions Brain regions were prepared from rat or bovine brain. Rats (Harlan, Sprague-Dawley, Inc., Indianapolis, IN, U.S.A.) were killed by asphyxiation in 100% CO,; brains were removed and immediately placed on ice. Cows were obtained from a local slaughterhouse.

Parietal cortex Parietal white matter Putamen Optic tract Optic nerve Spinal cord p (Kruskal-Wallis H test)

31.6 (1.9) 15.1 (1.7) 33.8 (5.3) 14.4 (1.3) 14.3 (1.4) 14.9 (0.3) 0.0026 ~~

Microvessel preparation Microvessels were prepared from different bovine brain regions by using the method of Goldstein et al. (1975). Meninges and choroid plexus were carefully removed. Tissues were homogenized in 5 ml/g HBSS containing 1% BSA by 20 strokes of a Teflon/glass homogenizer (0.25mm clearance, 50-ml tubes). Dextran was added to a final concentration of 1370, and the homogenate was centrifuged at 5,000 g for 20 min. The pellet containing the microvessels was passed through a 118-pm nylon mesh (Tetko, Elmsford, NY, U.S.A.). The filtrate was filtered through 1.2-cm columns of glass beads to retain the microvessels. Microvessels were washed out of the beads with HBSS, checked for purity by phase-contrast microscopy, centrifuged for 20 min at 800 g,

J . Neurochem , Vol 58, No 3, 1992

78.4 (6.6) 8.5 (0.9) 26.6 (7.9) 16.8 (7.4) 33.3 (8.6) 18.6 (3.3) 0.001 1 ~

0.40 (0.03) 1.66 (0.30) 1.18 (0.28) 1.02 (0.43) 0.52 (0.09) 0.86 (0.15) 0.0038 ~~

Brain regions were prepared and homogenized, and the enzyme activities of GGTP and AP were measured as described in Materials and Methods. Averages of six bovine brains in the case of the regions parietal cortex, parietal white matter, optic nerve, and spinal cord, and three brains in the case of the other regions, are given as units per gram of protein with SE in parentheses. One unit ofGGTP activity represents the formation of 1 gmol of pnitroanilinelmin at 37°C. One unit of AP activity is defined as the production of 1 rmol ofpnitrophenol/min at 30°C. The ratio GGTP/AP was calculated for each sample and is given as means of SE of those single ratios. The Kruskal-Wallis Htest was used to determine ifthere were differences between the regions. In all cases, these p values were below 0.05. Fischer’sprotected least significant difference test was used to compare cortex and putamen and showed that AP and GGTP/AP were different ( p < 0.005). but not GGTP.

y-GLUTAMYL TRANSPEPTIDASE IN BRAIN CAPILLARIES TABLE 2. Enzyme activities in isolated bovine brain microvessels

Parietal cortex Putamen p ( t test)

GGTP (U/g of protein)

AP (UJg of protein)

GGTPJAP

166.8 (1 1.5) 34 1.3 (6 1.4) 0.0006

596.9 (103.2) 538.6 (59.7) 0.7406

0.344 (0.032) 0.605 (0.076) 0.00 16

Microvessels were prepared from parietal cortex and putamen of bovine brains as described in Materials and Methods. Microvessel preparations were lysed and sonicated and enzyme activities of AP and GGTP measured. Values are given as units per gram of protein with SE in parentheses. p values are given for the comparison of cortex and putamen using the two-tailed unpaired t test. n = 13 in the case of cortex; n = 5 in the case of putamen.

activities was different, we prepared microvessels from two regions. Given that gray matter results in a higher yield of microvessels, we chose to compare putamen and neocortex. As seen in Table 2, the specific activity of AP did not differ in the two microvessel preparations, whereas that of GGTP was higher in putamenal microvessels. Enzyme assays in rat brain In order to determine if regional heterogeneity of microvessel GGTP activity is a general phenomenon among mammals, we measured the enzyme activities in different regions of the adult rat brain. As can be seen in Table 3, differences in the enzyme activities of rat brain regions were comparable to those found in bovine brain. The ratio GGTP/AP was higher in putamen than in cortex. Region-specific microvessel preparations of rat brain were not possible, because the amount of tissue necessary is not readily available from rat brain. Histochemical morphometric quantifications of GGTP-positive vessels Histochemical methods allowed investigations of rat brain regions in more detail. We used the ink perfusion technique to determine total vessel densities (Fig. 1). GGTP- and AP-positive vessels were counted after histochemical staining and expressed as a percentage of total vessel density. The intensity of histochemical stains increased with incubation time. At 5 min, GGTP staining appeared in the entire choroid plexus epithelium, but only in a few vessels in the corpus callosum and the striatum. After longer incubations, microvessels of other regions became positive, but the intensity and the number of stained vessels differed among anatomical regions. Those differences were most obvious in microscopic fields showing borders between two regions (Fig. 2). Vessels in the median eminence and the pineal gland did not stain even after incubations of over 1 h. In addition to the choroid plexus, non-vessel-related staining was observed in ependyma, median eminence after 30 min of incubation, and some faint diffuse

911

staining of white matter was seen after 1 h of incubation. For counting vessel densities, 20-min incubations were used. These conditions appeared to maximize differences between brain regions. The morphometric data agreed with the quantitative data of the enzymatic assays. There were more GGTPpositive vessels in putamen than in neocortex, whereas the total number of positive vessels was higher in neocortex (Fig. 3). The parietal and the occipital cortex, as well as the cortical regions for the limbs, had relatively few positive vessels (