Differential regulation of transport proteins in the periinfarct region following reversible middle cerebral artery occlusion in rats

doi:10.1016/j.neuroscience.2006.07.056

Neuroscience

Volume 142, Issue 4, 3 November 2006, Pages 1071-1079

https://doi.org/10.1016/j.neuroscience.2006.07.056 Get rights and content

Abstract

Members of various transport protein families including ATP-binding cassette transporters and solute carriers were shown to be expressed in brain capillaries, choroid plexus, astrocytes or neurons, controlling drug and metabolite distribution to and from the brain. However, data are currently very limited on how the expression of these transport systems is affected by damage to the brain such as stroke. Therefore we studied the expression of four selected transporters, P-glycoprotein (Mdr1a/b; Abcb1a/b), Mrp5 (Abcc5), Bcrp (Abcg2), and Oatp2 (Slc21a5) in a rat model for stroke. Transporter expression was analyzed by real-time polymerase chain reaction in the periinfarcted region and protein localization and cellular phenotyping were done by immunohistochemistry and confocal immunofluorescence microscopy. After stroke, P-glycoprotein staining was detected in endothelial cells of disintegrated capillaries and by day 14 in newly generated blood vessels. There was no significant difference, however, in the Mdr1a mRNA amount in the periinfarcted region compared with the contralateral site. For Bcrp, a significant mRNA up-regulation was observed from days 3–14. This up-regulation was followed by the protein as confirmed by quantitative immunohistochemistry. Oatp2, located in the vascular endothelium, was also up-regulated at day 14. For Mrp5, an up-regulation was observed in neurons in the periinfarcted region (day 14).

In conclusion, after stroke the transport proteins were up-regulated with a maximum at day 14, a time point that coincides with behavioral recuperation. The study further suggests Bcrp as a pronounced marker for the regenerative process and a possible functional role of Mrp5 in surviving neurons.

Section snippets

Animals

Eighteen hours prior to surgery, male 3-month-old Sprague–Dawley rats were deprived of food to minimize variability in ischemic damage that can result from varying plasma glucose levels. The experiments reported in this study were conducted in accordance with the statement regarding the care and use of animals and were approved by a federal animal care committee. Every effort was made to minimize the number of animals used and their suffering.

Reversible occlusion of the middle cerebral artery

Blood flow through the middle cerebral artery was

Time course of the P-gp expression

Focal cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery in 3-month-old rats. After 1, 3, 14 and 28 days of reperfusion, brains were removed, cut into 2 mm thick slices and the periinfarcted area was dissected after TTC staining as indicated in Fig. 1a (A). Using NeuN, a sensitive marker of neuronal degeneration, we found that the areas most affected by stroke were the parietal cortex and, to a lesser extent, the posterior frontal cortex (Fig. 1a (B)).

Discussion

The integrity of the BBB and the functionality of the transport systems localized at the BBB are crucial to the proper functioning of the CNS. Little is known about the expression of transport systems following damage to the BBB, such as stroke. In this study, we analyzed the impact of focal cerebral ischemia on the expression and localization of selected transport proteins possibly involved in the maintenance of the BBB as well as in neuronal function. Overall, the results show that after

Acknowledgments

This work was supported by the German Federal Ministry for Education and Research (NBL3 program, reference 01 ZZ 0103). The anti-Oatp2 serum was kindly provided by Dr. B. Stieger, Division of Clinical Pharmacology and Toxicology, Zürich, Switzerland. The anti-MRP5 serum AMF was provided by Prof. D. Keppler, Deutsches Krebsforschungszentrum, Heidelberg, Germany.

References (43)

H. Clayton et al.
Growth and differentiation of progenitor/stem cells derived from the human mammary gland
Exp Cell Res
(2004)
P. Dazert et al.
Expression and localization of the multidrug resistance protein 5 (MRP5/ABCC5), a cellular export pump for cyclic nucleotides, in human heart
Am J Pathol
(2003)
T. Eisenblatter et al.
Characterisation of the brain multidrug resistance protein (BMDP/ABCG2/BCRP) expressed at the blood-brain barrier
Brain Res
(2003)
G. Jedlitschky et al.
The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides
J Biol Chem
(2000)
M. Kakyo et al.
Immunohistochemical distribution and functional characterization of an organic anion transporting polypeptide 2 (oatp2)
FEBS Lett
(1999)
J. Konig et al.
Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance
Biochim Biophys Acta
(1999)
C.M. Martin et al.
Persistent expression of the ATP-binding cassette transporter, Abcg2, identifies cardiac SP cells in the developing and adult heart
Dev Biol
(2004)
H. Meyer zu Schwabedissen et al.
Expression, localization and function of MRP5 (ABCC5), a transporter for cyclic nucleotides, in human placenta and cultured human trophoblasts: effects of gestational age and cellular differentiation
Am J Pathol
(2005)
A. Nies et al.
Expression and immunolocalization of the multidrug resistance proteins, MRP1-MRP6 (ABCC1-ABCC6), in human brain
Neuroscience
(2004)
A.T. Nies et al.
Immunolocalization of multidrug resistance protein 5 in the human genitourinary system
J Urol
(2002)

T. Ohnishi et al.

In vivo and in vitro evidence for ATP-dependency of P-glycoprotein-mediated efflux of doxorubicin at the blood-brain barrier

Biochem Pharmacol

(1995)

C. Reichel et al.

Localization and function of the organic anion-transporting polypeptide Oatp2 in rat liver

Gastroenterology

(1999)

A. Sakata et al.

In vivo evidence for ATP-dependent and P-glycoprotein-mediated transport of cyclosporin A at the blood-brain barrier

Biochem Pharmacol

(1994)

E. Schroeder et al.

Neurofilament expression in the rat brain after cerebral infarction: effect of age

Neurobiol Aging

(2003)

H. Sun et al.

Drug efflux transporters in the CNS

Adv Drug Deliv Rev

(2003)

I. Badan et al.

Accelerated accumulation of N- and C-terminal beta APP fragments and delayed recovery of microtubule-associated protein 1B expression following stroke in aged rats

Eur J Neurosci

(2004)

I. Badan et al.

Temporal dynamics of degenerative and regenerative events associated with cerebral ischemia in aged rats

Gerontology

(2003)

S. Choudhuri et al.

Constitutive expression of various xenobiotic and endobiotic transporter mRNAs in the choroid plexus of rats

Drug Metab Dispos

(2003)

S. Cisternino et al.

Expression, up-regulation, and transport activity of the multidrug-resistance protein Abcg2 at the mouse blood-brain barrier

Cancer Res

(2004)

H.C. Cooray et al.

Localisation of breast cancer resistance protein in microvessel endothelium of human brain

Neuroreport

(2002)

J.M. Croop et al.

The three mouse multidrug resistance (mdr) genes are expressed in a tissue-specific manner in normal mouse tissues

Mol Cell Biol

(1989)

Cited by (59)

RNA sequencing uncover crucial genes mediating progression of large-artery atherosclerotic and small-artery occlusion ischemic stroke
2022, Brain Research
The goal of our study is to uncover the pathogenesis of large-artery atherosclerotic ischemic stroke (LAAIS) and small-artery occlusion ischemic stroke (SAOIS) and analyze their difference using RNA sequencing.
RNA sequencing was used to filtrate differentially expressed mRNAs (DEmRNAs) and differentially expressed lncRNAs (DElncRNAs) in LAAIS and SAOIS. Specific DEmRNAs and DElncRNAs in LAAIS and SAOIS were further found. Functional annotation and DElncRNA-DEmRNA co-expression network were built to reveal biological function of DEmRNAs.
A total of 832 DEmRNAs and 96 DElncRNAs were identified in LAAIS vs normal controls. 587 DEmRNAs and 105 DElncRNAs were identified in SAOIS vs normal controls. In LAAIS vs SAOIS, 636 DEmRNAs and 112 DElncRNAs were identified. Among which, 571 DEmRNAs and 61 DElncRNAs were LAAIS specific DEmRNAs and DElncRNAs, respectively. 325 DEmRNAs and 66 DElncRNAs were respectively SAOIS specific DEmRNAs and DElncRNAs. We also obtained 3086 LAAIS specific DElncRNA-DEmRNA co-expression pairs and 661 SAOIS specific DElncRNA-DEmRNA co-expression pairs. Oxidative phosphorylation and Alzheimer's disease were significantly enriched pathways in both LAAIS specific DEmRNAs and DEmRNAs in LAAIS specific DElncRNA-DEmRNA co-expression network. ECM-receptor interaction, hypertrophic cardiomyopathy and dilated cardiomyopathy were significantly enriched pathways in both SAOIS specific DEmRNAs and DEmRNAs in SAOIS specific DElncRNA-DEmRNA co-expression network.
This finding may help to understand the mechanisms of LAAIS and SAOIS and offer novel clues for finding specific biomarkers for LAAIS and SAOIS.
Targeting organic cation transporters at the blood-brain barrier to treat ischemic stroke in rats
2022, Experimental Neurology
Drug discovery and development for stroke is challenging as evidenced by few drugs that have advanced beyond a Phase III clinical trial. Memantine is a N-methyl-d-aspartate (NMDA) receptor antagonist that has been shown to be neuroprotective in various preclinical studies. We have identified an endogenous BBB uptake transport system for memantine: organic cation transporters 1 and 2 (Oct1/Oct2). Our goal was to evaluate Oct1/Oct2 as a required BBB mechanism for memantine neuroprotective effects. Male Sprague-Dawley rats (200–250 g) were subjected to middle cerebral artery occlusion (MCAO) for 90 min followed by reperfusion. Memantine (5 mg/kg, i.v.) was administered 2 h following intraluminal suture removal. Specificity of Oct-mediated transport was evaluated using cimetidine (15 mg/kg, i.v.), a competitive Oct1/Oct2 inhibitor. At 2 h post-MCAO, [³H]memantine uptake was increased in ischemic brain tissue. Cimetidine inhibited blood-to-brain uptake of [³H]memantine, which confirmed involvement of an Oct-mediated transport mechanism. Memantine reduced post-MCAO infarction and brain edema progression as well as improved neurological outcomes during post-stroke recovery. All positive effects of memantine were attenuated by co-administration of cimetidine, which demonstrates that Oct1/Oct2 transport is required for memantine to exert neuroprotective effects in ischemic stroke. Furthermore, Oct1/Oct2-mediated transport was shown to be the dominant mechanism for memantine brain uptake in the MCAO model despite a concurrent increase in paracellular “leak.” These novel and translational findings provide mechanistic evidence for the critical role of BBB transporters in CNS delivery of stroke therapeutics, information that can help such drugs advance in clinical trials.
Dysfunction of ABC transporters at the blood-brain barrier: Role in neurological disorders
2020, Pharmacology and Therapeutics
ABC (ATP-binding cassette) transporters represent one of the largest and most diverse superfamily of proteins in living species, playing an important role in many biological processes such as cell homeostasis, cell signaling, drug metabolism and nutrient uptake. Moreover, using the energy generated from ATP hydrolysis, they mediate the efflux of endogenous and exogenous substrates from inside the cells, thereby reducing their intracellular accumulation. At present, 48 ABC transporters have been identified in humans, which were classified into 7 different subfamilies (A to G) according to their phylogenetic analysis. Nevertheless, the most studied members with importance in drug therapeutic efficacy and toxicity include P-glycoprotein (P-gp), a member of the ABCB subfamily, the multidrug-associated proteins (MPRs), members of the ABCC subfamily, and breast cancer resistance protein (BCRP), a member of the ABCG subfamily. They exhibit ubiquitous expression throughout the human body, with a special relevance in barrier tissues like the blood-brain barrier (BBB). At this level, they play a physiological function in tissue protection by reducing or limiting the brain accumulation of neurotoxins. Furthermore, dysfunction of ABC transporters, at expression and/or activity level, has been associated with many neurological diseases, including epilepsy, multiple sclerosis, Alzheimer's disease, and amyotrophic lateral sclerosis. Additionally, these transporters are strikingly associated with the pharmacoresistance to central nervous system (CNS) acting drugs, because they contribute to the decrease in drug bioavailability. This article reviews the signaling pathways that regulate the expression and activity of P-gp, BCRP and MRPs subfamilies of transporters, with particular attention at the BBB level, and their mis-regulation in neurological disorders.
Repression of adenosine triphosphate–binding cassette transporter ABCG2 by estrogen increases intracellular glutathione in brain endothelial cells following ischemic reperfusion injury
2018, Neurobiology of Aging
The adenosine triphosphate–binding cassette efflux transporter ABCG2, which is located in the blood-brain barrier limits the entry of endogenous compounds and xenobiotics into the brain, and its expression and activity are regulated by estrogen. This study was aimed to define the role of ABCG2 in estrogen-mediated neuroprotection against ischemic injury. ABCG2 protein levels before and after ischemic stroke were increased in the brain of female mice by ovariectomy, which were reversed by estrogen replacement. In brain endothelial cell line bEnd.3, estrogen reduced the basal ABCG2 protein level and efflux activity and protected cells from ischemic injury without inducing ABCG2 expression. When bEnd.3 cells were transfected with ABCG2 small interfering RNA, ischemia-induced cell death was reduced, and the intracellular concentration of glutathione, an antioxidant that is transported by ABCG2, was increased. In addition, after ischemic stroke in ovariectomized mice, estrogen prevented the reduction of intracellular glutathione level in brain microvessels. These data suggested that the suppression of ABCG2 by estrogen is involved in neuroprotection against ischemic injury by increasing intracellular glutathione, and that the modulation of ABCG2 activity offers a therapeutic target for brain diseases in estrogen-deficient aged women.
Stroke and Drug Delivery - In Vitro Models of the Ischemic Blood-Brain Barrier
2016, Journal of Pharmaceutical Sciences
Citation Excerpt :
The organic anion transporting polypeptide Oatp1a4 (named Oatp2 in the reference) was found to be rapidly upregulated in vivo.70,71 This transporter is of special interest because it seems to be involved in the transport of opioid peptides such as 2,5-Pen-enkephalin and deltorphin II from blood to the brain.70 Oatp1a4 is also mediating the transendothelial transport of atorvastatin, a commonly prescribed statin which has been shown to have neuroprotective properties.71,76
Stroke is a major cause of death and disability worldwide. Both cerebral hypoperfusion and focal cerebral infarcts are caused by a reduction of blood flow to the brain, leading to stroke and subsequent brain damage. At present, only few medical treatments of stroke are available, with the Food and Drug Administration–approved tissue plasminogen activator for treatment of acute ischemic stroke being the most prominent example. A large number of potential drug candidates for treatment of ischemic brain tissue have been developed and subsequently failed in clinical trials. A deeper understanding of permeation pathways across the barrier in ischemic and postischemic brain endothelium is important for development of new medical treatments. The blood-brain barrier, that is, the endothelial monolayer lining the brain capillaries, changes properties during an ischemic event. In vitro models of the blood-brain barrier are useful tools to investigate the effects of induced ischemia under controlled conditions. In the present mini review, we aim to give a brief overview of the in vitro models of ischemia. Special focus is given to the expression of uptake and efflux transport pathways in the ischemic and postischemic endothelium. Finally, we will point toward future challenges within the field of in vitro models of brain ischemia.
ABCB1 and ABCG2 Regulation at the Blood-Brain Barrier: Potential New Targets to Improve Brain Drug Delivery
2023, Pharmacological Reviews

View all citing articles on Scopus

¹: The first two authors have contributed equally to this work.

View full text

Cellular neuroscienceDifferential regulation of transport proteins in the periinfarct region following reversible middle cerebral artery occlusion in rats

Abstract

Section snippets

Animals

Reversible occlusion of the middle cerebral artery

Time course of the P-gp expression

Discussion

Acknowledgments

Exp Cell Res

Am J Pathol

Brain Res

J Biol Chem

FEBS Lett

Biochim Biophys Acta

Dev Biol

Am J Pathol

Neuroscience

J Urol

Biochem Pharmacol

Gastroenterology

Biochem Pharmacol

Neurobiol Aging

Adv Drug Deliv Rev

Accelerated accumulation of N- and C-terminal beta APP fragments and delayed recovery of microtubule-associated protein 1B expression following stroke in aged rats

Eur J Neurosci

Temporal dynamics of degenerative and regenerative events associated with cerebral ischemia in aged rats

Gerontology

Constitutive expression of various xenobiotic and endobiotic transporter mRNAs in the choroid plexus of rats

Drug Metab Dispos

Expression, up-regulation, and transport activity of the multidrug-resistance protein Abcg2 at the mouse blood-brain barrier

Cancer Res

Localisation of breast cancer resistance protein in microvessel endothelium of human brain

Neuroreport

The three mouse multidrug resistance (mdr) genes are expressed in a tissue-specific manner in normal mouse tissues

Mol Cell Biol

Cellular neuroscience
Differential regulation of transport proteins in the periinfarct region following reversible middle cerebral artery occlusion in rats