Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-42803
Ruderisch, N. Amino acid transport across the murine blood-brain barrier. 2010, University of Zurich, Faculty of Science.
The brain microvascular endothelial cells (BMECs) of the blood-brain barrier (BBB) provide a diffusion barrier between blood and brain interstitial fluid (ISF). The BBB plays a role in protecting the brain from changes in the blood. Amino acid transport through the BBB is crucial for establishing and maintaining an asymmetry in amino acid concentration between blood and brain and for the distribution of drugs and diagnostic markers. Interestingly, the cerebrospinal fluid (CSF) amino acid concentration is only about 10% that of plasma, except for glutamine .
Characterizing the expression and localization of solute carrier (SLC) transporters is one of the first steps in understanding the mechanisms regulating BBB transendothelial transport. Our aim is to understand how the amino acid gradient is maintained by BMEC amino acid transporters.
To address this aim, we first examined which (amino acid) transporters are expressed in total at the murine BBB on the mRNA level and how their expression is affected by a short culture step . By mRNA microarray analysis using freshly isolated as well as single-cultured (5 days) and co-cultured (with glial cells in non-contact) BMECs, we have shown that as much as 60% of the known amino acid transporters are expressed in BMECs. In freshly isolated, noncultured, BMECs Lat1-4F2hc is most prominently expressed, followed by the sodium-dependent amino acid transporters Taut, Snat2, Snat5, and Eaat3. Although glial coculture is often used to mimic in vitro the BBB in vivo, levels of 73% of the amino acid transporter mRNAs were strongly altered by culture. In particular, for 78% of the transporters highly expressed in noncultured BMECs down-regulation was verified by qPCR (Lat1-4F2hc, Taut, Cat-1, Xpct, Snat3, and Snat5). In contrast, y+Lat2, xCT, and Snat1 are expressed at low levels in noncultured BMECs and are upregulated by culture.
We hypothesize that down-regulation of transporter mRNA during culture is characteristic of transporters, such as Lat1-4F2hc, mediating transendothelial amino acid transport in vivo. Conversely, we postulate that transporter mRNA upregulated by culture do so to serve increased cellular amino acid demands for growth.
To understand the functional organization of the BBB and the role of transporters in physiological and (neuro)pathophysiological states, many of them characterized by impaired brain amino acid concentrations, it is necessary to not only determine at the mRNA level which transporters are expressed at the BBB, but also whether the actual transporters are expressed at the luminal and / or abluminal membranes of BMECs, and how they are regulated.
Therefore, we studied the expression of Lat1-4F2hc (Slc7a5-Slc3a2), Snat1 (Slc38a1), and Snat3 (Slc38a3) in vivo on mouse brain tissue sections and additionally by in vivo biotinylation of the mouse brain vascular lumen with subsequent Western blot analysis. By combining the results obtained by the two different approaches, we, for the first time, could show that the sodium-dependent amino acid transporter Snat3, along with the exchanger Lat1-4F2hc, is localized to both the abluminal and the luminal membranes of BMECs. Furthermore, they are subject to posttranslational modifications. For example, Snat3 expressed in the luminal membrane has been found to be highly glycosylated, suggesting the possibility that glycosylation might play a role for its luminal membrane insertion.
The finding of Snat3 present on the luminal membrane of the BBB challenges the current hypothesis that sodium-dependent amino acid transport is an exclusive feature of the abluminal membrane [1, 3-9], in a position to protect the brain from neurotoxic levels of amino acids.
We also found Snat1, another sodium-dependent transporter, localized to the luminal membrane of vascular endothelial cells, with higher expression in the bigger vessels compared to brain microvessels. Therefore, we conclude that Snat1 seems not to play a pivotal role in the differentiated endothelial cells of the BBB, which stands in agreement with the finding that Snat1-mRNA shows very low expression in noncultured BMECs .
Taken together, we showed expression of a high number of not characterized amino acid transporters at the BBB, as well as their differing modulation by culture, implying functional differences. We also showed the unexpected localization of secondary active transport mechanisms at the luminal membrane, facing the blood with its high amino acid levels. Therefore, to be able to better understand the BBB transportome and its role in brain amino acid homeostasis, further studies and re-thinking of current hypotheses are needed.
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|Other titles:||Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr.sc.nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich|
|Referees:||Verrey F, Fritschy J M, Engelhardt B|
|Communities & Collections:||04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
04 Faculty of Medicine > Center for Integrative Human Physiology
|DDC:||570 Life sciences; biology
610 Medicine & health
|Deposited On:||03 Feb 2011 12:52|
|Last Modified:||17 Oct 2012 20:51|
|Number of Pages:||165|
|Additional Information:||Amino acid transport across the murine blood-brain barrier / von Nadine Ruderisch. - Zürich, 2010|
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