Margery A. Barrand

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

Movement of substrates between blood and brain is known to be influenced by P-glycoprotein (P-gp) at the luminal surface of the endothelium lining brain microvessels and by multidrug resistance associated protein 1 (MRP1) at the basolateral surface of the choroid plexus epithelium. Here, using RT-PCR and Western blotting, we investigate other ABC transporters in both normal and tumour human brain tissue and demonstrate the presence of breast cancer resistance protein (BCRP). Immunofluorescence confocal microscopy demonstrates that BCRP is located at the blood–brain barrier, mainly at the luminal surface of microvessel endothelium. This localization closely resembles that of P-gp. BCRP has several substrates in common with P-gp and may pose an additional barrier to drug access to the brain.

Mechanisms of fluid movement into, through and out of the brain: evaluation of the evidence

Interstitial fluid (ISF) surrounds the parenchymal cells of the brain and spinal cord while cerebrospinal fluid (CSF) fills the larger spaces within and around the CNS. Regulation of the composition and volume of these fluids is important for effective functioning of brain cells and is achieved by barriers that prevent free exchange between CNS and blood and by mechanisms that secrete fluid of controlled composition into the brain and distribute and reabsorb it. Structures associated with this regular fluid turnover include the choroid plexuses, brain capillaries comprising the blood-brain barrier, arachnoid villi and perineural spaces penetrating the cribriform plate. ISF flow, estimated from rates of removal of markers from the brain, has been thought to reflect rates of fluid secretion across the blood-brain barrier, although this has been questioned because measurements were made under barbiturate anaesthesia possibly affecting secretion and flow and because CSF influx to the parenchyma via perivascular routes may deliver fluid independently of blood-brain barrier secretion. Fluid secretion at the blood-brain barrier is provided by specific transporters that generate solute fluxes so creating osmotic gradients that force water to follow. Any flow due to hydrostatic pressures driving water across the barrier soon ceases unless accompanied by solute transport because water movements modify solute concentrations. CSF is thought to be derived primarily from secretion by the choroid plexuses. Flow rates measured using phase contrast magnetic resonance imaging reveal CSF movements to be more rapid and variable than previously supposed, even implying that under some circumstances net flow through the cerebral aqueduct may be reversed with net flow into the third and lateral ventricles. Such reversed flow requires there to be alternative sites for both generation and removal of CSF. Fluorescent tracer analysis has shown that fluid flow can occur from CSF into parenchyma along periarterial spaces. Whether this represents net fluid flow and whether there is subsequent flow through the interstitium and net flow out of the cortex via perivenous routes, described as glymphatic circulation, remains to be established. Modern techniques have revealed complex fluid movements within the brain. This review provides a critical evaluation of the data.

Multidrug Resistance‐Related Transport Proteins in Isolated Human Brain Microvessels and in Cells Cultured from These Isolates

Abstract: The multidrug transporter, P‐glycoprotein (Pgp), at the blood‐brain barrier is thought to be important for limiting access of toxic agents to the brain, but controversy surrounds its cellular location, whether on endothelium or on adjacent astrocyte foot processes. In the present study, the distribution of protein and mRNA for Pgp and for another transporter, multidrug resistance‐associated protein (MRP), is compared with that for the endothelial marker, platelet‐endothelial cell adhesion molecule‐1 (PECAM‐1) and for the astrocyte‐derived glial fibrillary acidic protein (GFAP) in microvessels isolated from human brain and in cells grown from these microvessels. Activities of the multidrug transporters are assessed in the cultured cells from the effects of transport inhibitors on intracellular [3H]vincristine accumulation. The isolated microvessels show strong immunocytochemical staining for Pgp and PECAM‐1 and little or no staining for GFAP and MRP, and they contain mRNAs detectable by RT‐PCR encoding only Pgp and PECAM‐1, but not GFAP or MRP. Thus, Pgp may well be synthesised and expressed on cells within the microvessels rather than on adherent astrocyte foot processes. In cells grown from the microvessels, although PECAM‐1 remains, Pgp expression decreases and MRP appears. Evidence suggests these multidrug transporters are functionally active in the cultured cells.

Modulatory effects of plant phenols on human multidrug-resistance proteins 1, 4 and 5 (ABCC1, 4 and 5).

Plant flavonoids are polyphenolic compounds, commonly found in vegetables, fruits and many food sources that form a significant portion of our diet. These compounds have been shown to interact with several ATP‐binding cassette transporters that are linked with anticancer and antiviral drug resistance and, as such, may be beneficial in modulating drug resistance. This study investigates the interactions of six common polyphenols; quercetin, silymarin, resveratrol, naringenin, daidzein and hesperetin with the multidrug‐resistance‐associated proteins, MRP1, MRP4 and MRP5. At nontoxic concentrations, several of the polyphenols were able to modulate MRP1‐, MRP4‐ and MRP5‐mediated drug resistance, though to varying extents. The polyphenols also reversed resistance to NSC251820, a compound that appears to be a good substrate for MRP4, as predicted by data‐mining studies. Furthermore, most of the polyphenols showed direct inhibition of MRP1‐mediated [3H]dinitrophenyl S‐glutathione and MRP4‐mediated [3H]cGMP transport in inside‐out vesicles prepared from human erythrocytes. Also, both quercetin and silymarin were found to inhibit MRP1‐, MRP4‐ and MRP5‐mediated transport from intact cells with high affinity. They also had significant effects on the ATPase activity of MRP1 and MRP4 without having any effect on [32P]8‐azidoATP[αP] binding to these proteins. This suggests that these flavonoids most likely interact at the transporters substrate‐binding sites. Collectively, these results suggest that dietary flavonoids such as quercetin and silymarin can modulate transport activities of MRP1, ‐4 and ‐5. Such interactions could influence bioavailability of anticancer and antiviral drugs in vivo and thus, should be considered for increasing efficacy in drug therapies.

Activation of β-catenin signalling by GSK-3 inhibition increases p-glycoprotein expression in brain endothelial cells

This study investigates involvement of β‐catenin signalling in regulation of p‐glycoprotein (p‐gp) expression in endothelial cells derived from brain vasculature. Pharmacological interventions that enhance or that block β‐catenin signalling were applied to primary rat brain endothelial cells and to immortalized human brain endothelial cells, hCMEC/D3, nuclear translocation of β‐catenin being determined by immunocytochemistry and by western blot analysis to confirm effectiveness of the manipulations. Using the specific glycogen synthase kinase‐3 (GSK‐3) inhibitor 6‐bromoindirubin‐3′‐oxime enhanced β‐catenin and increased p‐gp expression including activating the MDR1 promoter. These increases were accompanied by increases in p‐gp‐mediated efflux capability as observed from alterations in intracellular fluorescent calcein accumulation detected by flow cytometry. Similar increases in p‐gp expression were noted with other GSK‐3 inhibitors, i.e. 1‐azakenpaullone or LiCl. Application of Wnt agonist [2‐amino‐4‐(3,4‐(methylenedioxy) benzylamino)‐6‐(3‐methoxyphenyl)pyrimidine] also enhanced β‐catenin and increased transcript and protein levels of p‐gp. By contrast, down‐regulating the pathway using Dickkopf‐1 or quercetin decreased p‐gp expression. Similar changes were observed with multidrug resistance protein 4 and breast cancer resistance protein, both known to be present at the blood–brain barrier. These results suggest that regulation of p‐gp and other multidrug efflux transporters in brain vasculature can be influenced by β‐catenin signalling.

P‐glycoprotein expression in rat brain endothelial cells: evidence for regulation by transient oxidative stress

During ischaemia/reperfusion, cells of the blood–brain barrier are subjected to oxidative stress. This study uses primary cultured rat brain endothelial cells to examine the effect of such stresses on expression of multidrug transporters. H2O2 up to 500 µm applied to cell monolayers caused a concentration‐dependent increase in expression of P‐glycoprotein (Pgp) but not of multidrug resistance‐associated protein (Mrp1). Concentrations > 250 µm H2O2 decreased cell viability. Application of 100 µm H2O2 caused a significant increase after 48 h in Pgp functional activity, as assessed from [3H]vincristine accumulation experiments. At this concentration, H2O2 produced a transient increase within 10 min followed by a sustained decrease in levels of intracellular reactive oxygen species (iROS), detectable by flow cytometry. Reoxygenation of cell monolayers after 6 h hypoxia gave rise to a similar transient increase in iROS and this also led to increased Pgp expression by 24 h. Increases were also observed within 4 h after both H2O2 and hypoxia/reoxygenation treatments in mdr1a and mdr1b mRNA. Evidence suggests this was due to enhanced transcription rather than mRNA stabilization. Therefore, oxidative stress, by changing Pgp expression, may affect movement of Pgp substrates in and out of the brain.

Comparisons of P-glycoprotein expression in isolated rat brain microvessels and in primary cultures of endothelial cells derived from microvasculature of rat brain, epididymal fat pad and from aorta

In vivo expression of P‐glycoprotein in isolated rat brain microvessels is compared with that in vitro in primary cultures of brain endothelial cells. More P‐glycoprotein is detected by Western immunoblotting in microvessels than in cultured endothelium. RT‐PCR with isoform‐specific primers and immunoblotting with a mdr1b‐specific antibody reveals only mdr1a in vivo but both mdr1a and mdr1b in vitro. Thus mdr1a decreases whereas mdr1b increases during culture. P‐Glycoprotein activity is evident in vitro, with resistance modulators, e.g. verapamil, producing increases in intracellular [3H]vincristine accumulation. Endothelial cells cultured from epididymal fat pad microvasculature and aorta contain little or no P‐glycoprotein. Here, resistance modulators are less effective.

Monoamine oxidase activities in brown adipose tissue of the rat: Some properties and subcellular distribution

Amine oxidase activity towards 5-hydroxytryptamine (5HT), tyramine (TYR), 2-phenylethylamine (PEA) and benzylamine (BZ) was studied in homogenates of interscapular brown adipose tissue of the rat. By the use of clorgyline, an irreversible inhibitor of MAO, it was established that 5HT was deaminated solely by MAO-A, and TYR and PEA mainly by MAO-A and clorgyline-resistant semicarbazide-sensitive amine oxidase (CRAO). BZ appeared to be oxidized almost entirely by CRAO. A very small amount of MAO-B activity was detectable with PEA and BZ as substrates. A variety of amines, amino acids and known amine oxidase inhibitors were tested for their ability to inhibit the deamination of BZ by CRAO. BZ metabolism by the enzyme was not affected by any secondary amines, unlike enzymes of the flavin type, but it was inhibited by carbonyl reagents, like the pyridoxal phosphate and copper-dependent amine oxidases described in plasma and connective tissue. Unlike these enzymes, however, CRAO in brown adipose tissue was resistant to KCN and unaffected by the amines, histamine, mescaline and some polyamines but it was inhibited by cuprizone. It was found to have a low Km (<5 μM) for BZ and showed the greatest similarity to a clorgyline-resistant enzyme described in rat blood vessels. Cell fractionation studies revealed that CRAO, being associated with the particulate fractions, was mainly membrane-bound. The distribution of CRAO activity between various cell fractions was different from that of the mitochondrial enzymes assayed and was more like that of either the plasma membrane or microsomal enzymes. When microsomal and plasma membrane vesicles were separated CRAO activity appeared distributed equally between the two fractions, suggesting that the enzyme may have a dual location within the cell. The specific activity of CRAO was higher in brown adipose tissue from obese animals than in tissue from lean animals. The significance of these findings is discussed in relation to the possible physiological function of this enzyme.

Functional and molecular characterization of a volume‐sensitive chloride current in rat brain endothelial cells

1 Volume‐activated chloride currents in cultured rat brain endothelial cells were investigated on a functional level using the whole‐cell voltage‐clamp technique and on a molecular level using the reverse transcriptase‐polymerase chain reaction (RT‐PCR). 2 Exposure to a hypotonic solution caused the activation of a large, outward rectifying current, which exhibited a slight time‐dependent decrease at strong depolarizing potentials. The anion permeability of the induced current was I− (1.7) > Br− (1.2) > Cl− (1.0) > F− (0.7) > gluconate (0.18). 3 The chloride channel blocker 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB, 100 μM) rapidly and reversibly inhibited both inward and outward currents. The chloride transport blocker 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS, 100 μM) also blocked the hypotonicity‐induced current in a reversible manner. In this case, the outward current was more effectively suppressed than the inward current. The volume‐activated current was also inhibited by the antioestrogen tamoxifen (10 μM). 4 The current was dependent on intracellular ATP and independent of intracellular Ca2+. 5 Activation of protein kinase C by phorbol 12,13‐dibutyrate (PDBu, 100 nM) inhibited the increase in current normally observed following hypotonic challenge. 5 Extracellular ATP (10 mM) inhibited the current with a more pronounced effect on the outward than the inward current. 6 Verapamil (100 μM) decreased both the inward and the outward hypotonicity‐activated chloride current. 7 RT‐PCR analysis was used to determine possible molecular candidates for the volume‐sensitive current. Expression of the ClC‐2, ClC‐3 and ClC‐5 chloride channels, as well as pICln, could be shown at the mRNA level. 8 We conclude that rat brain endothelial cells express chloride channels which are activated by osmotic swelling. The biophysical and pharmacological properties of the current show strong similarities to those of ClC‐3 channel currents as described in other cell types.

Polarized distribution of nucleoside transporters in rat brain endothelial and choroid plexus epithelial cells

This study investigated mRNA expression and protein localization of equilibrative and concentrative nucleoside transporters (ENTs, CNTs) in primary cultures of rat brain endothelial cells (RBEC) and rat choroid plexus epithelial cells (RCPEC). Reverse transcriptase PCR analysis revealed that RBEC and RCPEC contained mRNA for rENT1, rENT2 and rCNT2 and for rENT1, rENT2, rCNT2 and rCNT3, respectively. Immunoblotting of membrane fractions of RBEC, fresh RCPEC and primary cultures of RCPEC revealed the presence of rENT1, rENT2 and rCNT2 proteins in all samples. Measurement of [14C]adenosine uptake into cells grown as monolayers on permeable plastic supports revealed a polarized distribution of Na+‐dependent adenosine uptake in that CNT activity was associated exclusively in membranes of RBEC facing the lower chamber (which corresponds to the surface facing the interstitial fluid in situ) and in membranes of RCPEC facing the upper chamber (which corresponds to the surface facing the cerebrospinal fluid in situ). In both RBEC and RCPEC, adenosine uptake from the opposite chambers was Na+‐independent and partially inhibited by nitrobenzylthioinosine, indicating the presence of the equilibrative sensitive transporter rENT1.