Adenosine Receptors and the Nucleoside Transporter in Human Brain Vasculature

Abstract

Summary: Evidence suggests that adenosine modulates neuronal and cerebral vascular functions by interacting with specific receptors on brain cells and blood vessels. Adenosine and other nucleosides are also transported across the blood-brain barrier via a saturable, carrier mediated mechanism. Using direct ligand binding methods, we studied the two adenosine receptor sub types, Al and A2 and the nucleoside transporter moiety in human brain ' microvessels, pial vessels, choroid plexus, and cerebral cortex membranes. The following specific tritiated ligands were used: cyclohexyladenosine (CHA) for Al receptors; 5'-N-ethylcarboxamide adeno sine (NECA) for A2 receptors; nitrobenzylthioinosine (NBMPR) and dipyridamole (DPY) for nucleoside trans porters. We find that cerebral microvessels, pial vessels, and choroid plexus have few, if any, Al receptors, in con tradistinction to cerebral membranes, which have a 1O-20-fold higher density of Al receptor sites. SpecificIt is generally accepted that adenosine modulates central nervous system functions via its interaction with adenosine I (AI) and adenosine2 (A2) receptors located on cell membranes (Phillis and Wu, 198 1; Daly et aI., 1983). Al receptor agonists inhibit cy clic adenosine monophosphate formation, whereas A2 receptor stimulation increases adenylate cyclase activity and cAMP synthesis (Daly et aI., 1981). Marked changes in extracellular adenosine concen trations were described in a variety of conditions 32high-affinity NECA binding to A2 receptors in cerebral microvessels, pial vessels, and choroid plexus was satu rable and was equivalent to that of cerebral cortical membranes. The Bmax and Kd of the high-affinity NECA binding to vessel preparations were � 1.3 pmol/mg pro tein and �250 nM, respectively, which is similar to our previous findings in the rat and pig. NBMPR and DPY binding were also saturable and were consistent with a single class of high-affinity binding sites. The density of nucleoside transporters was �four-fold higher in cerebral microvessels than in cerebral cortex, pial vessels, and choroid plexus. These results suggest that human cere bral microvessels have A2 but not Al receptors and are particularly enriched with the adenosine transporter moiety. Key Words: Adenosine receptors-Blood-brain barrier-capillary endothelium-Cerebral blood vessels -Nucleoside transporter.that perturb brain functions, such as ischemia, hyp oxia, hypoglycemia, and status epilepticus. Also, there is evidence suggesting that adenosine and its analogs dilate cerebral blood vessels and increase brain perfusion (Winn et aI., 1981;Berne et al., 1983). On the other hand, it is also known that adenosine and other nucleosides (guanosine, ino sine, and uridine) traverse cell membranes, in cluding the blood-brain barrier (BBB), by a car rier-mediated, saturable, nonconcentrative, and non-energy-requiring facilitated diffusion mecha nism (Cornford and Oldendorf, 1975;Young and Jarvis, 1983).We have previously demonstrated by specific li gand binding methods the existence o...

Metabolic Effects of R-Phenylisopropyladenosine during Reversible Forebrain Ischemia Studied by in vivo 31P Nuclear Magnetic Resonance Spectroscopy

“…This protection may be due to any one of the numerous effects of adenosine on the central nervous system. Nevertheless, it is unlikely that a vas cular effect is responsible for these metabolic ef fects since R-PIA is one of the most selective Al agonists (Daly, 1982); cerebral vessels have A 2 but not Al receptors (Kalaria and Harik, 1988), and it has been shown that these doses of R-PIA have no cerebrovascular effects (Nehlig et aI., 1990).…”

Purinergic Signaling and Blood Vessels in Health and Disease

“…A 2 receptors were identified, mediating relaxation of cat cerebral arteries (Edvinsson and Fredholm, 1983) and rabbit cerebral microvessels (Li and Fredholm, 1985). It has been claimed that human cerebral microvessels express A 2 , but not A 1 , receptors (Kalaria and Harik, 1988). A 2 receptors have also been described in porcine basilar arteries (McBean et al, 1988), goat cerebral vessels (Torregrosa et al, 1990b), and rat pial arterioles (Ibayashi et al, 1991).…”

Identification of a stable fragment of the Alzheimer amyloid precursor containing the beta-protein in brain microvessels.

“…Microvessels were purified at 40C by a modification of the method of Tsuji et al (9). Cerebral gray matter (usually 5-10 g of wet weight) was minced into -2 mm3 blocks, suspended in 5-10 vol of Tris-buffered saline (TBS) (50 mM Tris/150 mM sodium chloride/2 mM EDTA/0.1 mM diisopropyl fluorophosphate/ leupeptin at 1 Ag/ml/pepstatin at 0.1 ug/ml/7-amino-1-chloro-3-tosylamide-2-heptanone at 1 tig/ml, pH 7.6), and homogenized with a motor-driven, loosely fitting Teflon pestle (5)(6)(7)(8)(9)(10) up-and-down strokes). The homogenate was sieved through 73-,.m nylon mesh.…”

“…After centrifugation at 58,000 x g in an SW 28 rotor for 120 min, the microvessel fraction was recovered as a pellet from the bottom of the tube and resuspended in 1-2 ml of TBS. Some microvessel fractions were prepared from fresh brain tissue by an alternate but comparable purification method described by Kalaria and Harik (10).…”