Presence of norepinephrine and related enzymes in isolated brain microvessels.

Abstract

Norepinephrine and the enzymes involved in its synthesis and degradation were found to be associated with isolated brain microvessels. The significance of these results are discussed with respect to adrenergic innervation of the cerebral microvessels and thereby neural regulation of the cerebral microcirculation. The cerebral vasculature consists of two systems of blood vessels: intracerebral and extracerebral vessels. Physiological and pharmacological studies of the brain blood vessels have generally been performed on isolated extracerebral arteries, such as pial arteries and basilar artery (1, 2), probably due to the difficulty in isolating the intracerebral vessels. Studies on the intracerebral vessels have usually been confined to histochemical and morphological procedures (1, 3).We were able to isolate microvessels (arterioles, capillaries, and venules) from the rat brain with a modification of the method used by Brendel et al. (4). In the present study, we report on the presence of norepinephrine in these microvessels as well as the enzymes necessary for both its biosynthesis and catabolism. was loosely fitted to the tube (0.5 mm clearance). The homogenates were sieved over a 153-,um nylon fabric, and the filtrate was collected in a beaker immersed in an ice bath. This has been designated as brain filtrate. The brain tissues retained on the nylon cloth were transferred to another test tube and gently homogenized and sieved as before. A relatively homogeneous preparation of microvessels could be obtained by carrying out the homogenization and sieving procedure four times. Homogeneity of the microvessels was examined under a microscope during the course of isolation. The yield of cerebral microvessels obtained from one rat brain was approximately 2 mg wet weight. The combined brain filtrates were centrifuged at 150 X g for 10 min. The precipitate was then resuspended in suitable buffer for determination of enzyme activities. Tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase(3-hydroxylating); EC 1.14.16.2] was determined by a modification of the method of Nagatsu et al. (5). All tissues were homogenized in phosphate buffer (67 mM; pH 6.2): whole brain in 20 volumes, mesenteric arteries in 10 volumes, and microvessels (from four to eight brains) in 80,.ul. The reaction mixture consisted of 0.1 mM L-tyrosine containing about 2 X 105 cpm of L-[3,5-3H]tyrosine; 0.74 mM 2-amino-6-methyl-5,6,7,8,-tetrahydropteridine; 140 mM 2-mercaptoethanol; 200 mM sodium acetate buffer (1 M; pH 6.0); 1000 units of catalase; and tissue homogenates in a total volume of 500 ,ul for brain tissue and 100,u1 for blood vessels. The mixture was incubated for 15 min at 370, and the reaction was then terminated by adding 100 ,u1 of 12.5% trichloroacetic acid for brain, and 500 ,ul of 2.5% trichloroacetic acid for vessels. After centrifugation of the sample, the supernatant was passed over a Dowex column (+H, 200-400 mesh). The effluent and 1.4 ml of water, used to wash the column,...

Developmental changes in metabolism and transport properties of capillaries isolated from rat brain.

“…The enzymes L-DOPA decarboxylase and monoamine oxidase are also known to be enriched in brain capillaries (Lai et al 1975). These enzymes are considered responsible for a metabolic BBB since they limit brain capillary permeability to substrates like L-DOPA (Bertler, Falck, Owman & Rosengren, 1966).…”

“…In recent years, several laboratories have developed methods for isolation of brain capillaries. These preparations are enriched in enzymatic markers for brain capillary endothelial cells (Orlowski, Sessa & Green, 1974;Goldstein, Wolinsky, Csejtey & Diamond, 1975;Lai, Udenfriend & Spector, 1975;Betz, Firth & Goldstein, 1980), are metabolically active 0022-3751/81/3940-0911 $07.50 ©) 1981 The Physiological Society A. L. BETZ AND G. W. GOLDSTEIN (Brendel, Meezan & Carlson, 1974;Goldstein, 1979a) and exhibit transport properties similar to those described at the BBB in vivo (Betz & Goldstein, 1978; Hjelle, Baird-Lambert, Cardinale, Spector &JUdenfriend, 1978; Csejtey & Eisenberg & Suddith, 1979). Thus, they provide a convenient model system for studying transport and metabolic aspects of BBB function.…”

Monoamine Oxidase Activity in the Cerebral Vasculature: Comparison between Fresh Microvessels from Different Structures and Cell Cultures Derived from Microvessels

“…The preparation of microvessels, based on the tech niques published by Goldstein et al (1975) and Lai et al (1975), has been recently described in detail (Lasbennes et aI. , 1983).…”

Correlation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity with blood-brain barrier monoamine oxidase activity.

“…This suggests that the rat's resistance to systemic MPTP may be a property of the blood-brain barrier and other organs that can metabolize MPTP and prevent it from reaching its brain targets in sufficient concentrations. Because MPTP is a known substrate for enzymatic oxidation by MAO-B (6,16), and since brain capillaries possess MAO activity (17)(18)(19)(20)(21)(22), we reasoned that MAO activity in brain microvessels from different species may correlate invrsely with their susceptibility to systemic MPTP neurotoxicity.…”