Dataset Information

Retinal transfer of nicotinate by H+ -monocarboxylate transporter at the inner blood-retinal barrier.

ABSTRACT: Nicotinic acid is a constituent of the coenzymes NAD and NADP. It also serves as an agonist for the G-protein-coupled receptor GPR109A. Nicotinic acid is widely used at high doses as a lipid-lowering drug, which is associated with an ocular side effect known as niacin maculopathy. Here we investigated the mechanism by which nicotinate is transferred into retina across the inner blood-retinal barrier (BRB). In vivo the blood-to-retina transport of [(3)H]-nicotinate was studied using the carotid artery injection technique. The characteristics of nicotinate transport at the inner BRB were examined in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2), an in vitro model of inner BRB. The expression of transporters in TR-iBRB2 cells was determined by reverse transcription-polymerase chain reaction. In vivo [(3)H]-nicotinate uptake by the retina was 5.4-fold greater than that of [(14)C]-sucrose, a BRB impermeable vascular space marker. Excess amounts of unlabeled nicotinate and salicylate significantly decreased the in vivo retinal uptake of [(3)H]-nicotinate. [(3)H]-Nicotinate was taken up by TR-iBRB2 cells via an H(+)-dependent saturable process with a Michaelis constant of ~7 mM. Na(+) had minimal effect on the uptake. The H(+)-dependent uptake was significantly inhibited by endogenous monocarboxylates such as lactate and pyruvate, and monocarboxylic drugs such as valproate, salicylate, and ibuprofen. These characteristics are consistent with those of H(+)-coupled monocarboxylate transporters (MCTs). MCT1, MCT2, and MCT4 mRNAs were expressed in TR-iBRB2 cells. The Na(+)-dependent monocarboxylate transporters SMCT1 and SMCT2 were not expressed in these cells. In conclusion, transfer of nicotinate from blood to retina across the inner BRB occurs primarily via H(+)-coupled monocarboxylate transporters.

SUBMITTER: Tachikawa M

PROVIDER: S-EPMC3225029 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Retinal transfer of nicotinate by H+ -monocarboxylate transporter at the inner blood-retinal barrier.

Tachikawa Masanori M Murakami Koji K Martin Pamela M PM Hosoya Ken-ichi K Ganapathy Vadivel V

Microvascular research 20110629 3

Nicotinic acid is a constituent of the coenzymes NAD and NADP. It also serves as an agonist for the G-protein-coupled receptor GPR109A. Nicotinic acid is widely used at high doses as a lipid-lowering drug, which is associated with an ocular side effect known as niacin maculopathy. Here we investigated the mechanism by which nicotinate is transferred into retina across the inner blood-retinal barrier (BRB). In vivo the blood-to-retina transport of [(3)H]-nicotinate was studied using the carotid a ...[more]

PMID: 21741392

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Project description:BackgroundThe blood-brain barrier (BBB) is pivotal for the maintenance of brain homeostasis and it strictly regulates the cerebral transport of a wide range of endogenous compounds and drugs. While fasting is increasingly recognized as a potential therapeutic intervention in neurology and psychiatry, its impact upon the BBB has not been studied. This study was designed to assess the global impact of fasting upon the repertoire of BBB transporters.MethodsWe used a combination of in vivo and in vitro experiments to assess the response of the brain endothelium in male rats that were fed ad libitum or fasted for one to three days. Brain endothelial cells were acutely purified and transcriptionaly profiled using RNA-Seq. Isolated brain microvessels were used to assess the protein expression of selected BBB transporters through western blot. The molecular mechanisms involved in the adaptation to fasting were investigated in primary cultured rat brain endothelial cells. MCT1 activity was probed by in situ brain perfusion.ResultsFasting did not change the expression of the main drug efflux ATP-binding cassette transporters or P-glycoprotein activity at the BBB but modulated a restrictive set of solute carrier transporters. These included the ketone bodies transporter MCT1, which is pivotal for the brain adaptation to fasting. Our findings in vivo suggested that PPAR δ, a major lipid sensor, was selectively activated in brain endothelial cells in response to fasting. This was confirmed in vitro where pharmacological agonists and free fatty acids selectively activated PPAR δ, resulting in the upregulation of MCT1 expression. Moreover, dosing rats with a specific PPAR δ antagonist blocked the upregulation of MCT1 expression and activity induced by fasting.ConclusionsAltogether, our study shows that fasting affects a selected set of BBB transporters which does not include the main drug efflux transporters. Moreover, we describe a previously unknown selective adaptive response of the brain vasculature to fasting which involves PPAR δ and is responsible for the up-regulation of MCT1 expression and activity. Our study opens new perspectives for the metabolic manipulation of the BBB in the healthy or diseased brain.

Project description:BackgroundReduced folate carrier 1 (RFC1; SLC19a1) is the main responsible transporter for the B9 family of vitamins named folates, which are essential for normal tissue growth and development. While folate deficiency resulted in retinal vasculopathy, the expression and the role of RFC1 in blood-retinal barrier (BRB) are not well known.MethodsWe used whole mount retinas and trypsin digested microvessel samples of adult mice. To knockdown RFC1, we delivered RFC1-targeted short interfering RNA (RFC1-siRNA) intravitreally; while, to upregulate RFC1 we delivered lentiviral vector overexpressing RFC1. Retinal ischemia was induced 1-h by applying FeCl3 to central retinal artery. We used RT-qPCR and Western blotting to determine RFC1. Endothelium (CD31), pericytes (PDGFR-beta, CD13, NG2), tight-junctions (Occludin, Claudin-5 and ZO-1), main basal membrane protein (Collagen-4), endogenous IgG and RFC1 were determined immunohistochemically.ResultsOur analyses on whole mount retinas and trypsin digested microvessel samples of adult mice revealed the presence of RFC1 in the inner BRB and colocalization with endothelial cells and pericytes. Knocking down RFC1 expression via siRNA delivery resulted in the disintegration of tight junction proteins and collagen-4 in twenty-four hours, which was accompanied by significant endogenous IgG extravasation. This indicated the impairment of BRB integrity after an abrupt RFC1 decrease. Furthermore, lentiviral vector-mediated RFC1 overexpression resulted in increased tight junction proteins and collagen-4, confirming the structural role of RFC1 in the inner BRB. Acute retinal ischemia decreased collagen-4 and occludin levels and led to an increase in RFC1. Besides, the pre-ischemic overexpression of RFC1 partially rescued collagen-4 and occludin levels which would be decreased after ischemia.ConclusionIn conclusion, our study clarifies the presence of RFC1 protein in the inner BRB, which has recently been defined as hypoxia-immune-related gene in other tissues and offers a novel perspective of retinal RFC1. Hence, other than being a folate carrier, RFC1 is an acute regulator of the inner BRB in healthy and ischemic retinas.

Dataset Information

Retinal transfer of nicotinate by H+ -monocarboxylate transporter at the inner blood-retinal barrier.

Publications

Retinal transfer of nicotinate by H+ -monocarboxylate transporter at the inner blood-retinal barrier.

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