The mechanism that removes homovanillic acid (HVA), an end metabolite of dopamine, from the brain is still poorly understood. The purpose of this study is to identify and characterize the brain-to-blood HVA efflux transporter at the rat blood-brain barrier (BBB). Using the Brain Efflux Index method, the apparent in vivo efflux rate constant of [3H]HVA from the brain, k(eff), was determined to be 1.69 x 10(-2) minute(-1). This elimination was significantly inhibited by para-aminohippuric acid (PAH), benzylpenicillin, indoxyl sulfate, and cimetidine, suggesting the involvement of rat organic anion transporter 3 (rOAT3). rOAT3-expressing oocytes exhibited [3H]HVA uptake (K(m) = 274 micromol/L), which was inhibited by several organic anions, such as PAH, indoxyl sulfate, octanoic acid, and metabolites of monoamine neurotransmitters. Neurotransmitters themselves did not affect the uptake. Furthermore, immunohistochemical analysis suggested that rOAT3 is localized at the abluminal membrane of brain capillary endothelial cells. These results provide the first evidence that rOAT3 is expressed at the abluminal membrane of the rat BBB and is involved in the brain-to-blood transport of HVA. This HVA efflux transport system is likely to play an important role in controlling the level of HVA in the CNS.
Taurine is the abundant sulfur-containing b-amino acid in brain where it exerts a neuroprotective effect. Although it is known that the blood-brain barrier (BBB) mediates taurine transport, the regulation of taurine transport have not been clarified yet. A conditionally immortalized rat brain capillary endothelial cells (TR-BBB13), an in vitro model of the BBB, exhibited [3 H]taurine uptake, which was dependent on both Na + and Cl -, and inhibited by b-alanine. Taurine transporter (TAUT) mRNA was detected in TR-BBB13 cells, and TAUT protein was also expressed at 70 kDa. TR-BBB13 cells exposed to 20 ng/mL TNF-a and under hypertonic conditions showed a 1.7-fold and 3.2-fold increase in [ 3 H]taurine uptake, respectively. In contrast, lipopolysaccharide and diethyl maleate did not significantly affect taurine uptake. The taurine uptake was reduced by pre-treatment with excess taurine (50 mM). The mRNA level of the TAUT in TNF-a and following hypertonic treatment was greater than that in control cells, whereas that under excess taurine conditions was lower than in controls. Therefore, taurine transport activity at the BBB appears to be regulated at the transcriptional level by cell damage, osmolality and taurine in the brain. Keywords: blood-brain barrier, immortalized brain capillary endothelial cell line, mRNA expression, osmoregulation, taurine transport, tumor necrosis factor-a. Taurine (2-aminoethanesulfonic acid) is one of the abundant free sulfur-containing b-amino acids in the CNS and is thought to play a role as an osmoregulator (Tuz et al. 2001) and a neuromodulator (Oja and Saransaari 1996). Taurine is also known to exert a neuroprotective effect against excitotoxic agents (French et al. 1986) and oxidative stress (Boldyrev et al. 1999). The taurine level in brain interstitial fluid is elevated in ischemia (Uchiyama-Tsuyuki et al. 1994;Matsumoto et al. 1996;Nakane et al. 1998), indicating that the brain controls its taurine level in response to cell damage for protecting neurons. The release of taurine from neuronal cells could be one of the regulating mechanisms. The release of taurine from hippocampal slices is increased under hypoglycemic and ischemic conditions, and in the presence of 2,4-dinitrophenol or media inducing free radical production by H 2 O 2 (Saransaari and Oja 2000). Another regulatory mechanism could involve the transport system at the blood-brain barrier (BBB). The BBB, which is formed by a complex of tight junctions of brain capillary endothelial cells, possesses a transport system for amino acids including taurine. The blood-to-brain influx Received July 16, 2002; revised manuscript received August 27, 2002; accepted September 11, 2002. Address correspondence and reprint requests to Professor Tetsuya Terasaki, Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan. E-mail: terasaki@mail.pharm.tohoku.ac.jp Abbreviations used: BBB, blood-brain barrier; DEM, diethyl maleat...
An in vivo model of chronic hypoglycemia was used to investigate changes in blood-brain barrier (BBB) glucose transport activity and changes in the expression of GLUT1 mRNA and protein in brain microvasculature occurring as an adaptive response to low circulating glucose levels. Chronic hypoglycemia was induced in rats by constant infusion of insulin via osmotic minipumps; control animals received infusions of saline. The criterion for chronic hypoglycemia was an average blood glucose concentration of < 2.3 mmol/l (42 mg/dl) after 5 days. The average blood glucose concentration at the end of the experimental period in the rats selected for study was 2.0 +/- 0.1 mmol/l (36 +/- 1 mg/dl) vs. 4.9 +/- 0.1 mmol/l (88 +/- 1 mg/dl) in the controls. Internal carotid artery perfusion studies demonstrated an increase in the BBB permeability-surface area (PS) product of 40% (P < 0.0005) in the chronically hypoglycemic animals as compared with controls. Western blotting of solubilized isolated brain capillaries demonstrated a 51% increase (P < 0.05) in immunoreactive BBB GLUT1 in the chronically hypoglycemic rats, and Northern blotting of whole-brain poly(A+) mRNA revealed a 50% increase in the GLUT1-to-actin ratio in the insulin-treated group (P < 0.05). Northern blotting analysis of microvessel-depleted total brain poly(A+) showed that the increase in GLUT1 mRNA in the chronically hypoglycemic rats was restricted to the BBB. The present study demonstrates increased expression of GLUT1 mRNA and protein at the BBB in chronic hypoglycemia and suggests that this increase is responsible for the compensatory increase in BBB glucose transport activity that occurs with chronically low circulating blood glucose levels.
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