Changes In Expression Of Transporters Research Articles

Signals Modulating p‐Glycoprotein Activity in Brain Capillaries

At the blood-brain barrier, p-glycoprotein, an ATP-driven efflux pump, limits drug entry into the brain and thus CNS pharmacotherapy. We have identified multiple extracellular and intracellular signals that regulate this transporter. Eight signaling pathways have been defined and partially mapped. Three pathways are triggered by elements of the brain’s innate immune response, one by reactive oxygen species, one by glutamate, one by drug-nuclear receptor (PXR) interactions and two by elevated beta-amyloid levels. Three work over the short-term (minutes) to reduce transport function with no change in transporter expression. Five work over the long-term (hours to days). Of these, four increase both function and expression and one decreases function and expression. Signaling is complex, with several pathways having common signaling elements (TNF-R1, ET-B receptor, protein kinase C, NO synthase), suggesting a regulatory network. Several pathways utilize autocrine/paracrine elements, involving release of the pro-inflammatory cytokine, TNF-alpha, and the polypeptide hormone, endothelin. Finally, several steps in signaling are potential therapeutic targets that could be used to modulate p-glycoprotein activity.

Synaptic Vesicles: Half Full or Half Empty?

The regulation of quantal size through pre- rather than postsynaptic mechanisms has recently received considerable attention as a potential mechanism for plasticity. Vesicular transporters catalyze the filling of synaptic vesicles with transmitter and are thus potential substrates for such presynaptic regulation. In this issue of Neuron, Prado et al. pursue this line of investigation and show that changes in transporter expression that alter quantal size can affect behavior.

Quantitative analysis of glutamate transporter mRNA expression in prefrontal and primary visual cortex in normal and schizophrenic brain

Abnormalities of the glutamatergic system in schizophrenia have been identified in numerous studies, but little is known about the role of glutamate transporters and their messenger RNA (mRNA) expression. In addition, the abundances of the two major isoforms of human excitatory amino acid transporter 2 ( EAAT2) or its rat ortholog, glutamate transporter 1, have never been compared in a quantitative manner. Using quantitative reverse transcription–polymerase chain reaction, we established that the expression of the EAAT1, EAAT2a, EAAT2b, and EAAT3 transcripts was not different in the dorsolateral prefrontal and primary visual cortices of persons with schizophrenia relative to matched controls. EAAT2a expression was about 25-fold and 10-fold higher than EAAT2b in human and rat brain, respectively. The data provided no evidence of an effect of antipsychotic medications on the mRNA expression of the glutamate transporters. However, because most of the schizophrenic subjects in the cohort had been treated with antipsychotics for many years, it is still possible that changes in transporter expression were masked by medication effects.

Fasting-induced upregulation of basolateral hepatic bile salt transporters in rats is mediated by the PGC–1alpha / HNF–4alpha pathway

Background & Aim: Fasting leads to extensive adaptive responses in the enterohepatic circulation of bile acids in rats including increased basolateral taurocholate-uptake in the presence of decreased intestinal absorption (Dumaswala AJP 1994). Since transport proteins may be involved in this regulatory process we studied expression and regulation of hepatic bile salt transporters. Methods: Rats (n=4) were fed with standard chow or starved for 48 hours. After RNA extraction, transporter expression of sodium dependent taurocholate cotransporting peptide (ntcp), organic anion transporting peptides (oatp) 1, 2 and 4, multidrug-resistance protein 2 (mrp2) and bile salt export pump (bsep) was assessed by Northern blotting. mRNA expression of PPARγ coactivator–1α (PGC–1α) and hepatocyte nuclear factor (HNF)–4α was quantified by RT-PCR. Results: mRNA of basolateral transporter was significantly upregulated in a coordinated fashion (Ntcp 155±10%, Oatp1 181±8%, Oatp2 316±18% and Oatp4 170±11% of fed controls, respectively, P<0.05). Canalicular transporters were differentially regulated (Bsep 165±24%, P<0.05; Mrp2 104±11%, P=n.s.). Changes in transporter expression appeared to be specific since Gapdh mRNA expression was unchanged. In response to fasting, expression of PGC–1α and HNF–4α was upregulated to 360±226% and 215±64%, respectively (P<0.05). Conclusions: Increased basolateral bile acid uptake during fasting is mediated by upregulated transporters of the SLC-family in the liver. This upregulation occurs as a result of PGC–1α-mediated activation of HNF–4α, which represents a common regulator of all basolateral transport systems. This pathway is not only involved in gluconeogenesis but obviously represents a common signalling cascade involved in a more global fasting response.

Cytokine-dependent regulation of hepatic organic anion transporter gene transactivators in mouse liver

Proinflammatory cytokines such as TNF-alpha and IL-1beta lead to downregulation of hepatic organic anion transporters in cholestasis. This adapted response is transcriptionally mediated by nuclear hormone receptors and liver-specific transcription factors. Because little is known in vivo about cytokine-dependent regulatory events, mice were treated with either TNF-alpha or IL-1beta for up to 16 h. Transporter mRNA expression was determined by Northern blot analysis, nuclear activity, and protein-expression of transactivators by EMSA and Western blotting. TNF-alpha induces a sustained decrease in Ntcp, Oatp1/Oatp1a1, and Bsep mRNA expression but exerts only transient [multidrug resistance-associated protein 2 (Mrp2)] or no effects (Mrp3) on Mrps. In addition to Ntcp and Oatp1/Oatp1a1, IL-1beta also downregulates Bsep, Mrp2, and Mrp3 mRNAs to some extent. To study transcriptional regulation, Ntcp and Bsep promoters were first cloned from mice revealing a new distal Ntcp hepatocyte nuclear factor 1 (HNF-1) element but otherwise show a conserved localization to known rat regulatory elements. Changes in transporter-expression are preceeded by a reduction in binding activities at IR-1, ER-8, DR-5, and HNF-1alpha sites after 4 h by either cytokine, which remained more sustained by TNF-alpha in the case of nuclear receptors. Nuclear protein levels of retinoid X receptor (RXR)-alpha are significantly decreased by TNF-alpha but only transiently affected by IL-1beta. Minor reductions of retinoic acid receptor, farnesoid X receptor, pregnane X receptor, and constitutive androstane receptor nuclear proteins are restricted to 4 h after cytokine application and paralleled by a decrease in mRNA levels. Basolateral and canalicular transporter systems are downregulated by both cytokines, TNF-alpha and IL-1beta. Activity of HNF-1alpha as regulator of mNtcp is suppressed by both cytokines. Decreased binding activities of nuclear receptor heterodimers may be explained by a reduction of the ubiquitous heterodimerization partner RXR-alpha.

Reply

We thank Dr. Ostrow and Dr. Tiribelli for their comments and we appreciate the opportunity to answer some aspects of the discussion on the regulation of transport proteins in diseased human livers. Ostrow and Tiribelli may be correct that variation in age may affect the expression of transporters in liver, however, this is a hypothetical comment which cannot be tested because of ethical reasons which do not allow to perform liver biopsies in healthy children. As already pointed out in our paper1 administration of ursodeoxycholic acid (UDCA) might contribute to changes in transporter expression (abstracts by Marschall et al.2 and by Trauner et al.3).In the paper by Marschall et al.4, which appeared after publication of our manuscript, protein levels of transporters were determined before and after UDCA treatment of apparently normal adults. An increase of MRP4 protein of only 1.6-fold was reported. This increase in response to UDCA is too low to explain the 5- to 9-fold increase of MRP4 in UDCA-treated PFIC children.1 Therefore, factors other than UDCA are likely to cause the upregulation of MRP4 in PFIC. On the basis of some of our data, Ostrow and Tiribelli speculate on potential mechanisms of jaundice in PFIC. However, we did not observe any significant correlation between bilirubin levels and protein expression of single transporters (R2 values were below 0.10 for MRP2, OATP1B1 and OATP1B3 and high bilirubin levels were associated with low expression of MRP4). Besides the concerted regulation of multiple transporters, other mechanisms, such as substrate competition, should be considered as potential causes of jaundice in PFIC. Ostrow and Tiribelli refer to the small increase in MRP1 mRNA expression in our study and mention that MRP1 exports unconjugated bilirubin from cells. We would like to stress that mRNA levels in liver tissue do not necessarily indicate the presence of MRP1 protein in hepatocytes. Indeed, we have not been able to detect MRP1 protein in human hepatocytes.5 Ostrow and Tiribelli point out that Mrp3/MRP3 upregulation occurs in Gunn rats and in patients with the Dubin–Johnson Syndrome (DJ), with PBC or with obstructive cholestasis. It is not surprising, that MRP3 was not elevated in PFIC because of the completely different genetic and pathophysiological background. The early event in PFIC is a defect in bile salt secretion while in Gunn rats, DJ, PBC or obstructive cholestasis an increase in unconjugated or conjugated bilirubin might represent the major effector of transporter regulation. Verena Keitel*, Martin Burdelski , Dietrich Keppler , Dieter Häussinger*, Ralf Kubitz* , * Klinik für Gastroenterologie, Hepatologie und Infektiologie, Heinrich Heine Universität, Düsseldorf, Germany, Pädiatrische Gastroenterologie und Hepatologie, Universitätsklinikum Hamburg-Eppendorf, Germany, Abteilung Tumorbiochemie, Deutsches Krebsforschungszentrum Heidelberg, Germany.

Role of nuclear bile acid receptor, FXR, in adaptive ABC transporter regulation by cholic and ursodeoxycholic acid in mouse liver, kidney and intestine

Background/Aims: Adaptive changes in transporter expression in liver and kidney provide alternative excretory pathways for biliary constituents during cholestasis and may thus attenuate liver injury. Whether adaptive changes in ATP-binding cassette (ABC) transporter expression are stimulated by bile acids and their nuclear receptor FXR is unknown. Methods: Hepatic, renal and intestinal ABC transporter expression was compared in cholic acid (CA)- and ursodeoxycholic acid (UDCA)-fed wild-type (FXR +/+) and FXR knock-out mice (FXR −/−). Expression was assessed by reverse transcription–polymerase chain reaction, immunoblotting and immunofluorescence microscopy. Results: CA feeding stimulated hepatic Mrp2, Mrp3, Bsep and renal Mrp2 as well as intestinal Mrp2 and Mrp3 expression. Lack of Bsep induction by CA in FXR −/− was associated with disseminated hepatocyte necrosis which was not prevented by compensatory induction of Mrp2 and Mrp3. With the exception of Bsep, UDCA stimulated expression of hepatic, renal and intestinal ABC transporters independent of FXR without inducing liver toxicity. Conclusions: Toxic CA and non-toxic UDCA induce adaptive ABC transporter expression, independent of FXR with the exception of Bsep. Stimulation of hepatic Mrp3 as well as intestinal and renal Mrp2 by UDCA may contribute to its therapeutic effects by inducing alternative excretory routes for bile acids and other cholephiles.

Reduced tubular cation transport in diabetes: Prevented by ACE inhibition

The renal clearance of organic cations is important for the homeostasis of a number of exogenous and endogenous compounds. The organic cation transporters (OCTs) situated on the basolateral surface of proximal tubular cells mediate active cation excretion. Alterations of cation transport may occur in diabetes, although the role of the OCTs has not been previously assessed. Experimental diabetes was induced in rats with streptozotocin (55 mg/kg) and animals were randomly assigned to receive ramipril (3 mg/mL) in drinking water for 24 weeks. In a second protocol, rats were infused with angiotensin II (Ang II) at a dose of 58.3 ng/kg/min for 2 weeks via an implanted osmotic pump. Expression of the OCTs and renal clearance of the endogenous cation N-methyl-nicotinamide (NMN) was assessed. Diabetes was associated with a reduction in gene and protein expression of both OCT-1 and OCT-2 and a reduction in NMN clearance. These effects were prevented by ramipril, associated with the prevention of albuminuria and tubular injury as demonstrated by the expression of osteopontin and glutathione peroxidase 3 (GPX-3). An infusion of Ang II also reduced NMN clearance but without altering the renal expression of OCTs. We hypothesize that reduced expression of OCTs in diabetes may be a marker of tubular injury. However, Ang II may also directly augment renal cation clearance independent of changes in transporter expression. Together these effects may provide additional mechanism to explain treatment-related improvements in creatinine clearance and renoprotection in diabetes following blockade of the renin-angiotensin system (RAS).

A Light and Electron Microscopic Study of Divalent Metal Transporter-1 Distribution in the Rat Hippocampus, after Kainate-Induced Neuronal Injury

An accumulation of iron occurs in the hippocampus of rats injected with kainate over time, but thus far whether this accumulation is associated with any changes in expression of iron transporters is not known. The present study was therefore carried out using an antibody to the divalent metal transporter-1 (DMT-1) and immunoblot and immunocytochemical analyses to elucidate possible changes in expression of the transporter in the rat hippocampus after kainate injections. A significant increase in density ratios of DMT-1/β-actin bands was observed in Western blots in the 1-week, 1-month, and 2-months post-kainate-injected hippocampus, compared to uninjected and 1-day post-kainate-injected hippocampus. The increase in DMT-1 protein was paralleled by an increase in DMT-1 immunoreactivity in astrocytes. Light staining for DMT-1 was observed in the uninjected, saline-injected, and 1-day post-kainate-injected rat hippocampus. In contrast, an upregulation of DMT-1 was observed in reactive glial cells at 1 week, 1 month, and 2 months post-kainate injection. Electron microscopy confirmed that the glial cells had morphological features of astrocytes. DMT-1 is a cellular iron transporter responsible for transport of metal ions from the plasma membrane to endosomes. The observation that DMT-1 is present on astrocytic end feet in contact with blood vessels suggests that these cells may be involved in uptake of iron from endothelial cells.

Physiological leukocytosis during pregnancy is associated with changes in glucose transporter expression of maternal peripheral blood granulocytes and monocytes.

The scarce data on glucose transporter expression of leukocytes are contradictory and nothing is known about changes accompanying physiological leukocytosis during pregnancy, which imposes acute metabolic demands on the cells. Cytospin preparations of intravascular leukocytes were searched immunocytochemically for the high affinity glucose transporters GLUT1, 3 and 4. Pregnancy-associated quantitative changes in transporter expression were assessed by flow cytometry. Granulocytes and monocytes stained for GLUT1, 3 and 4. Major changes in cell surface transporter expression during pregnancy were a 36% (P < 0.05) down-regulation of granulocyte GLUT1 at term, and an increase in monocyte GLUT3 levels to 137% (P < 0.05), paralleled by a 24% (P < 0.05) decrease in GLUT4 content in second trimester. Apart from a minor subpopulation, lymphocytes were negative for these carriers. GLUT1, 3 and 4 are abundantly expressed in granulocytes and monocytes. The particular isoforms are differentially regulated during pregnancy, suggesting an individual functional significance.

Antagonists of protein kinase C inhibit rat retinal glutamate transport activity in situ.

Neuronal and glial high-affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by following the fate of the non-metabolisable glutamate transporter substrate, d-aspartate. In the rat retina, pan-isoform PKC inhibition with chelerythrine suppressed glutamate uptake by GLAST (glutamate/aspartate transporter), the dominant excitatory amino acid transporter localized to the glial Müller cells. This effect was mimicked by rottlerin but not by Gö6976, suggesting the involvement of the PKCdelta isoform, but not PKCalpha, beta or gamma. Western blotting and immunohistochemical labeling revealed that the suppression of glutamate transport was not due to a change in transporter expression. Inhibition of PKCdelta selectively suppressed GLAST but not neuronal glutamate transporter activity. These data suggest that the targeting of specific glutamate transporters with isoform-specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis, e.g. glaucoma and amyotrophic lateral sclerosis.

Sustained Hypoglycemia Affects Glucose Transporter Expression of Human Blood Leukocytes

ABSTRACTThe scarce data available on leukocyte glucose transporter expression are contradictory and nothing is known about its regulation by glycemic state. Therefore, cytospin preparations of blood leukocytes were searched immunocytochemically for the high-affinity glucose transporters GLUT1, 3, and 4. Hypoglycemia-associated quantitative changes in transporter expression were assessed by flow cytometry. Granulocytes and monocytes stained for GLUT1, 3, and 4. Granulocyte GLUT4 levels were increased by 73% (P < 0.05) under hypoglycemic conditions, which was paralleled by a reduction in GLUT1 and a rise in GLUT3. In monocytes, GLUT3 was elevated by 134% (P < 0.05), whereas GLUT1 and GLUT4 remained unaffected upon hypoglycemia. Apart from a minor subpopulation, lymphocytes were negative for these carriers. In conclusion, GLUT1, 3, and 4 are abundantly expressed in granulocytes and monocytes. The differential response of individual isoforms to hypoglycemia may represent a mechanism to protect the cells from the stress of glucose deprivation.

Regulation of jejunal glucose transporter expression by forskolin

We have investigated the effects of forskolin on enterocyte membrane expression of the glucose transporters, SGLT1 and GLUT2, which are thought to be the main entry and efflux pathways for glucose, respectively. Forskolin treatment increased SGLT1 but decreased GLUT2 expression in mid and lower villus enterocytes. No change in transporter expression was noted in upper villus cells. Likewise, cyclic AMP levels were raised in mid and lower but not upper villus cells. The implications of these data for glucose transport are discussed.

Cholestatic expression pattern of sinusoidal and canalicular organic anion transport systems in primary cultured rat hepatocytes

To maintain ongoing vectorial bile secretion, hepatocytes localize distinct transport systems at their basolateral and canalicular membrane domains. Here we compare the expression of the basolateral Na+-taurocholate cotransporter (Ntcp) and organic anion transporting polypetides 1 and 2 (Oatp1, Oatp2) and the canalicular bile salt export pump (Bsep) and multidrug resistance–associated protein 2 (Mrp2) in primary cultured rat hepatocytes. During 72 hours of culturing time the messenger RNA (mRNA) and protein levels of Ntcp and Oatp1 decreased in parallel to 2% to 7% of initial values at 3 hours. Although Oatp2 mRNA exhibited a similar down-regulation to 4%, Oatp2 protein and function were maintained at 25% to 47% of initial values. Furthermore, Bsep and Mrp2 protein levels were maintained at about 50%, while the Mrp2 mRNA showed a transient up-regulation to 154% at 24 and 48 hours. Also, induction of Mrp1 mRNA and protein was observed starting after 24 hours. These results indicate transcriptional down-regulation or decreased mRNA stability of Ntcp and Oatp1, transcriptional and posttranslational regulation of Oatp2, Bsep, and Mrp2 and transcriptional up-regulation of Mrp1 in primary cultured hepatocytes. Furthermore, and most importantly, the observed changes in transporter expression closely resemble the altered transporter phenotypes of cholestatic and proliferating hepatocytes in vivo, thus indicating that primary cultured hepatocytes acquire a cholestatic phenotype, and that the transporter expression might be a suitable differentiation marker for maintenance of hepatocytes in vitro. (HEPATOLOGY 2001;33:776-782.)

INTERLEUKIN 1βINCREASES ARGININE ACCUMULATION AND ACTIVATES THE CITRULLINE–NO CYCLE IN RAT PANCREATICβCELLS

Nitric oxide (NO) may contribute to pancreatic β cell damage during the development of type 1 diabetes. Its formation can be triggered by cytokines which induce the expression of the inducible form of nitric oxide synthase (iNOS) in pancreatic islets. In the iNOS-catalyzed reaction, arginine is converted into citrulline and NO. Cellular NO formation may be regulated by the availability of arginine. Arginine can be provided extracellularly, entering the cell mainly through the cationic amino acid transporter system y+CAT, and intracellularly, by protein degradation or synthesis from citrulline (the citrulline–NO cycle). This study demonstrates for the first time that the citrulline–NO cycle is induced in FACS-purified rat βcells exposed to interleukin-1β(IL-1β) and that extracellular arginine or citrulline is required for NO production byβcells. Moreover, the accumulation of arginine was higher in IL-1β-treatedβcells than in control cells.β cells expressed mRNAs for the two y+CAT transporters CAT-2A and CAT-2B with no change in transporter expression after exposure to IL-1β. It is concluded that the activation of the citrulline–NO cycle and an increase in arginine accumulation may be adaptive responses in cytokine-exposed β-cells to assure an adequate arginine supply for continuous NO production in the presence of low extracellular arginine levels which may prevail during insulitis.

The hemopoietic growth factor, interleukin-3, promotes glucose transport by increasing the specific activity and maintaining the affinity for glucose of plasma membrane glucose transporters.

Most mammalian cells rely on an external supply of glucose for survival, proliferation, and function. Glucose enters cells through specific transporter molecules at the plasma membrane by a facilitative process that does not expend energy. Regulation of glucose transport into cells is thought to occur largely through transporter expression at the cell surface, but the extent to which the intrinsic properties of glucose transporters are regulated is at present controversial. Using a bone marrow-derived cell line that responds to the hemopoietic growth factor, interleukin-3 (IL-3), we investigated IL-3 regulation of glucose transport. IL-3 significantly increased 2-deoxyglucose (2-DOG) uptake within 1 h (26 +/- 8.0%, n = 11) with a maximum 73% increase after 6 h. Withdrawal of IL-3 resulted in decreased uptake within 1 h and this continued to decline to 43% of initial uptake by 16 h. To determine whether these changes in 2-DOG uptake were associated with corresponding changes in glucose transporter expression, subtype-specific antisera against Glut-1 and Glut-3 were used. Little change in membrane expression of these transporters was observed prior to 16 h. Fractionation of cell membranes on Nycodenz gradients showed that the majority of each transporter subtype was associated with the plasma membrane (63-93%) and that transporter distribution did not change markedly in response to addition or withdrawal of IL-3. These results demonstrate that IL-3 regulates glucose uptake by modulating the intrinsic transporting ability of glucose transporters. Decreased transporter affinity for 2-DOG and 3-O-methylglucose was observed following IL-3 withdrawal. Similar affinity changes were observed with 2-DOG following exposure of IL-3-stimulated cells to the protein kinase inhibitors, genistein and staurosporine. In contrast, the tyrosine phosphatase inhibitor, vanadate, acted like IL-3 to increase transporter affinity for glucose. Together these results demonstrate that IL-3 acts to maintain the intrinsic transport properties of glucose transporters without markedly affecting their expression or translocation.

Glucose transport correlates with GLUT2 abundance in rat liver during altered thyroid status

Glucose transport activity ([ 3H] d-glucose uptake) in liver sinusoidal membrane vesicles (SMVs) from hyperthyroid rats was significantly higher than that from euthyroid controls (2.1-times increase in V max with K m unchanged at approximately 18 mM), associated with increased GLUT2 expression. In contrast, glucose transport V max into SMVs from hypothyroid rats was reduced to 0.75-times that of euthyroid controls, associated with a reduced GLUT2 abundance. GLUT1 expression in SMVs was unaffected by changes in thyroid status. GLUT2, but not GLUTl abundance on the blood-facing membrane of liver cells is sensitive to changes in thyroid status and these changes in transporter expression directly correlate ( r=0.96) with altered glucose transport activity.