BBB transport and P-glycoprotein functionality using MDR1A (-/-) and wild-type mice. Total brain versus microdialysis concentration profiles of rhodamine-123

PURPOSE: The effect of P-glycoprotein (Pgp) on brain distribution using mdr1a (-/-) mice was investigated. METHODS: Fluorescein (Flu) and FD-4 were used to check whether blood-brain barrier (BBB) integrity was maintained in mdr1a (-/-) mice. The Pgp substrate rhodamine-123 (R123) was infused and total brain, blood and brain microdialysate concentrations in mdr1a (-/-) mice and wild-type mice were compared. RESULTS: Maintenance of BBB integrity was indicated by equal total brain/blood ratios of Flu and FD-4 in both mice types. R123 concentrations in brain after i.v. infusion were about 4-fold higher in mdr1a (-/-) than in wild-type mice (P < 0.05), without changes in blood levels. After microdialysis experiments the same results were found, excluding artifacts in the interpretation of Pgp functionality by the use of this technique. However the 4-fold ratio in brain was not reflected in corresponding microdialysates. No local differences of R123 in the brain were found. By the no-net-flux method in vivo recovery appeared to 4.6-fold lower in mdrla (-/-) mice compared with wild-type mice. CONCLUSIONS: Pgp plays an important role in R123 distribution into the brain. Using intracerebral microdialysis, changes in in vivo recovery by the absence or inhibition of Pgp (or active efflux in general) need to be considered carefully.     

Multidrug resistance-related transport proteins in isolated human brain microvessels and in cells cultured from these isolates

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.     

Expression, purification, and characterization of a recombinant 5-lipoxygenase from potato tuber

The involvement of mdr1a P-glycoprotein (P-gP) on the tissue distribution of the multidrug resistance-reversing agent SDZ PSC 833 was assessed by use of mdr1a (-/-) mice. The mdr1a (-/-) and wild-type mdr1a (+/+) mice received a 4-h constantrate i.v. infusion (2 micrograms/min) of [14C]SDZ PSC 833. Mice were sacrificed at 0, 0.5, 1, 2 and 4 h during infusion and at 0.5, 1, 3, 8 and 24 h after stopping the infusion. Blood and tissues were analyzed on total (14C) and parental SDZ PSC 833 concentrations. Mdr1a (-/-) mice exhibited increased SDZ PSC 833 accumulation in cerebrum, cerebellum and somewhat in testes and small intestine compared with the wild-type mice. The difference between mdr1a (-/-) and (+/+) brain (cerebrum and cerebellum) penetration depended on SDZ PSC 833 blood concentrations, because this cyclosporin analog apparently governs its own brain penetration by inhibiting the P-glycoprotein pump in mdr1a (+/+) mice. Thus the mdr1a (-/-)/(+/+) ratio of brain concentrations tended to decrease and increase at high and low blood concentrations, respectively. These findings clearly demonstrate the interaction of SDZ PSC 833 with the P-glycoprotein present at the blood-brain barrier. The SDZ PSC 833 distribution in other mdr1a P-glycoprotein-expressed tissues, as well as its metabolism and elimination, was not affected by the mdr1a gene disruption. This suggests that factors other than mdr1a P-gP are involved in the disposition of this multidrug resistance-reversing agent.     

Tissue distribution of the multidrug-resistance gene product P-glycoprotein and its physiological function]

In normal tissues, P-glycoprotein(P-gp), which is expressed in various tumor cells, is found on the luminal surface of epithelia of the kidney proximal tubule, small intestine and colon and bile canalicular face of hepatocytes, as well as, in the adrenal and capillary endothelial cells in the brain and testis. The physiological function of P-gp remains unclear but growing amounts of information suggest that it can play a important role in the absorption from intestine, elimination from liver and kidney and distribution into brain across the blood-brain barrier for many cancer chemotherapeutic agents as well as other drugs which reverse multidrug resistance. The competition for the P-gp-mediated transports in tissues and organs, which express multidrug-transporter, might lead to unsuspected drug interactions among these drugs.     

Progressive systemic sclerosis: intrathecal pain management

PURPOSE: To determine if lucanthone crossed the blood-brain barrier in experimental animals; and to determine accelerated tumor regression of human brain metastases treated jointly with lucanthone and whole brain radiation. METHODS AND MATERIALS: The organ distribution of 3H lucanthone in mice and 125I lucanthone in rats was determined to learn if lucanthone crossed the blood-brain barrier. Size determinations were made of patients' brain metastases from magnetic resonance images or by computed tomography before and after treatment with 30 Gy whole brain radiation alone or with lucanthone. RESULTS: The time course of lucanthone's distribution in brain was identical to that in muscle and heart after intraperitoneal or intravenous administration in experimental animals. Lucanthone, therefore, readily crossed the blood-brain barrier in experimental animals. CONCLUSION: Compared with radiation alone, the tumor regression in patients with brain metastases treated with lucanthone and radiation was accelerated, approaching significance using a permutation test at p = 0.0536.     

Changes in doxorubicin distribution and toxicity in mice pretreated with the cyclosporin analogue SDZ PSC 833

SDZ PSC 833 (PSC 833) is a cyclosporin A analogue that is under clinical investigation in combination with doxorubicin (Dx) or other anticancer agents as a type-1 multidrug resistance (MDR-1)-reversing agent. The present study was focused on the effects of PSC 833 on the distribution and toxicity of Dx in non-tumor-bearing CDF1 male mice. Mice were given PSC 833 i.p. at 30 min before i.v. Dx treatment. Dx levels were determined by a high-performance liquid chromatography (HPLC) assay at different times during a 72-h period following Dx treatment in the serum, heart, intestine, liver, kidney, and adrenals of mice. In all tissues, Dx area under the concentration-time curve (AUC) values were much greater in mice receiving 10 mg/kg Dx in combination with 12.5 or 25 mg/kg PSC 833 than in mice receiving Dx alone. The highest increase in Dx concentrations was found in the intestine, liver, kidney, and adrenals. Lower, albeit significant, differences were found in the heart. PSC 833 did not appear to influence either urinary or fecal Dx elimination or Dx metabolism to a great extent. Doses of PSC 833 devoid of any toxicity potentiated the acute and delayed toxicity of Dx dramatically. The mechanism responsible for this enhanced toxicity has not yet been elucidated but is likely to be related to an increased tissue retention of Dx due to inhibition of the P-glycoprotein (Pgp) pump by PSC 833, as has recently been proposed for cyclosporin A.     

Diurnal uptake of circulating interleukin-1alpha by brain, spinal cord, testis and muscle

The effects, synthesis, and release of cytokines show diurnal patterns. We used recombinant human interleukin-1alpha radioactively labeled with 125I (I-IL) to determine whether its uptake by brain, spinal cord, testis and muscle showed a diurnal rhythm when tested every 4 h in mice. Each tissue showed statistically significant diurnal variation in their uptakes of I-IL ranging from a nearly 10-fold difference for the spinal cord to less than a 2-fold difference for muscle. All nadirs occurred at either 04.00 or 24.00 h and all peaks at 08.00 or 12.00 h. The pharmacokinetics of intravenously injected I-IL did not show any significant variations in blood. Recombinant human tumor necrosis factor-alpha, which does not cross the blood-brain barrier, did not show a diurnal rhythm in its uptake by any of these tissues. This diurnal variation in the rate of uptake of cytokines by tissues could underlie differences in potency when given at different times of the day.     

Increasing incidence of childhood class V lupus nephritis

1. Homozygously mdr1a gene disrupted mice (mdr1a(-/-) mice) and wild type mice (mdr1a(+/+) mice) were used to develop a method for P-glycoprotein (P-gp) function imaging non-invasively and to study the effect of a P-gp reversal agent on its function in vivo. 2. [11C]verapamil (0.1 mg/kg) was administered and the changes in tissue concentrations were determined ex vivo by organ extirpation and in vivo with PET. To block P-gp function, cyclosporin A was administered. 3. Biodistribution studies revealed 9.5-fold (P < 0.001) and 3.4-fold (P < 0.001) higher [11C]verapamil in the brain and testes of mdr1a(-/-) mice than in mdr1a(+/+) mice. Cyclosporin A (25 mg/kg) increased [11C]verapamil levels in the brain and testes of mdr1a(+/+) mice in both cases 3.3-fold (P < 0.01 (brain); P < 0.001 (testes)). Fifty mg/kg cyclosporin A increased [11C]verapamil in the brain 10.6-fold (P < 0.01) and in the testes 4.1-fold (P < 0.001). No increases were found in the mdr1a(-/-) mice. This indicates complete inhibition of P-gp mediated [11C]verapamil efflux. 4. Positron camera data showed lower [11C]verapamil levels in the brain of mdr1a(+/+) mice compared to those in mdr1a(-/-) mice. [11C]verapamil accumulation in the brain of mdr1a(+/+) mice was increased by cyclosporin A to levels comparable with those in mdr1a(-/-) mice, indicating that reversal of P-gp mediated efflux can be monitored by PET. 5. We conclude that cyclosporin A can fully block the P-gp function in the blood brain barrier and the testes and that PET enables the in vivo measurement of P-gp function and reversal of its function non-invasively.     

Expression of cell membrane complement regulatory glycoproteins along the normal and diseased human gastrointestinal tract

The viral coat of the HIV-1 virus, gp120, has been shown to cross the blood-brain barrier (BBB) in lectin-like fashion by inducing adsorptive endocytosis (AE), a vesicular mechanism that could provide pathways into and across brain endothelial cells for virus and infected immune cells. Here, we extended those findings to show that gp120 slowly crossed the BBB with about 0.15% of an intravenously injected dose entering the brain after about 2 hr. The plant lectin glycoprotein wheat germ agglutinin (WGA) greatly enhanced gp120 crossing without disrupting the BBB. WGA enhanced the uptake of gp120 into all peripheral tissues studied, but the greatest percent increase occurred for brain, whereas another barrier tissue, the testis, had the least increase. Five other plant lectins tested had little or no effect on gp120 uptake by brain, suggesting a key role for sialic acid and N-acetyl-beta-D-glucosaminyl acid, the sugars to which WGA binds, in the uptake of gp120 by brain endothelial cells. WGA did not enhance the uptake of nonglycosylated gp120 and the uptake of gp120 was not self-inhibitable or altered by pretreatment of mice with aluminum. In conclusion, these studies show that gp120 crosses the BBB by a lectin-like mechanism resembling AE that is likely mediated by binding to specific sugar moieties and is rather selective for brain.     

Comparison of human immunodeficiency virus 1 DNA polymerase chain reaction and qualitative and quantitative RNA polymerase chain reaction in human immunodeficiency virus 1-exposed infants

The endothelial lining of the blood-brain barrier tightly controls the distribution of peptide hormones between the central nervous system and the circulation. By using primary cultures of brain microvessel endothelial cells, an in vitro model of the blood-brain barrier, we report here the uptake and transport of the octapeptide angiotensin II by a specific receptor population. With the angiotensin II antagonists losartan (AT1 specific) and PD 123,319 (AT2 specific), we showed that both the uptake and transport of angiotensin II were mediated by the AT1 receptor. Western blot analysis confirmed the existence of the AT1 receptor in our cell-culture model. Rhodamine 123 studies also suggested that both angiotensin II antagonists, but not angiotensin II, were substrates for the P-glycoprotein efflux system, thus restricting the transport of these compounds. These results suggest an AT1 receptor mediates uptake and transport of angiotensin II at the blood-brain barrier and may contribute to the regulation of cerebrovascular levels of the peptide.     

The effect of drugs on the mortality of mice inoculated with Friend leukaemia virus or toxoplasma gondii

P-glycoprotein (P-gp), a product of the multidrug-resistant (mdr) genes, is expressed in the endothelial cells of the blood-brain barrier (BBB). Effects of glial factors and retinoic acid (RA) on P-gp activity and level were investigated in the immortalized rat brain endothelial cell line RBE4, which expressed immunodetectable P-gp associated with a decrease in accumulation of the P-gp substrates, vinblastine and colchicine. When RBE4 cells were cultured either in the presence of C6-conditioned medium or on C6- or astrocyte-extracellular matrix, intracellular vinblastine and colchicine concentrations were decreased. When the cells were treated with RA, increases in P-gp activities were correlated with increases in P-gp levels. Effects of simultaneous treatments with glial factors and RA were studied in RBE4 cells cultured on astrocyte-extracellular matrix and were shown to be additive on P-gp activity and level. RBE4 cells may serve as a useful in vitro model for basic research on P-gp regulation at the level of the BBB.     

Activation of protein kinase C does not participate in disruption of the blood-brain barrier to albumin during acute hypertension

The blood-brain barrier minimizes the entry of macromolecules into brain tissue. During acute increases in arterial blood pressure, disruption of the blood-brain barrier occurs primarily in cerebral venules and veins. Mechanisms by which increases in cerebral venous pressure produce disruption of the blood-brain barrier during acute hypertension are not clear. The goal of this study was to determine the role of activation of protein kinase C in disruption of the blood-brain barrier during acute hypertension. We examined the microcirculation of the cerebrum in vivo. Permeability of the blood-brain barrier was quantitated by the formation of venular leaky sites and clearance of fluorescent-labeled albumin (FITC-albumin) before and during phenylephrine-induced acute hypertension. In addition, we examined changes in pial arteriolar and pial venular pressure before and during phenylephrine-induced acute hypertension. We compared responses of the blood-brain barrier to acute hypertension in control (untreated) rats and in rats treated with inhibitors of protein kinase C; calphostin C (0.1 microM) or sphingosine (1.0 microM). Under control conditions, no venular leaky sites were visible and clearance of FITC-albumin was minimal in all groups. Phenylephrine infusion increased systemic arterial, pial arteriolar and pial venular pressures, and increased the formation of venular leaky sites and clearance of FITC-albumin by a similar magnitude in all groups. The findings of the present study suggest that inhibition of protein kinase C does not significantly alter the formation of venular leaky sites and/or clearance of FITC-albumin during acute hypertension. Thus, disruption of the blood-brain barrier during acute hypertension does not appear to be influenced by activation of protein kinase C.     

Role of P-glycoprotein in colchicine and vinblastine cellular kinetics in an immortalized rat brain microvessel endothelial cell line

Uptake and efflux of colchicine and vinblastine, whose effects are related to their high-affinity binding to tubulin, were studied in the immortalized rat brain microvessel endothelial cell line RBE4. At 10 nM extracellular drug concentration, uptake equilibrium was approached at 45 hr for colchicine, but at only 3.5 hr for vinblastine. After 1 hr preincubation with 200 nM colchicine or vinblastine, drug efflux fitted biexponential kinetics with an initial fast phase (half-life = 2.2 min and 9.6 min, respectively) and a later slow phase (half-life = 3.6 hr and 1.8 hr, respectively). After 6 hr preincubation with 200 nM colchicine, only the slow phase (half-life = 3.6 hr) could be observed. The colchicine and vinblastine uptake rate was increased by cyclosporin A, an inhibitor of the drug efflux pump P-glycoprotein, which is expressed at the blood-brain barrier. Whereas cyclosporin A decreased vinblastine efflux, its effect on colchicine efflux was apparent after only 13 hr washout and was associated with the re-uptake by cells of colchicine molecules. Differences in uptake kinetics of colchicine and vinblastine could be related to differences in their lipid solubility, and mainly in their binding affinities to tubulin. Differences in efflux kinetics could in addition be explained by the involvement of P-glycoprotein in the efflux of vinblastine, whereas efflux of colchicine was not influenced by this pump. Indeed, binding of colchicine to tubulin would imply that most intracellular colchicine may be inaccessible to P-glycoprotein. In the case of a cytotoxic drug such as colchicine, which is tightly bound to intracellular receptors, the role of P-glycoprotein within the blood-brain barrier would be more to protect the brain against entry of this drug than to detoxify the brain by its extraction.     

Evaluation of sport injuries in children and adolescents

Dengue type 2 virus (DV)-induced cytotoxic factor (CF) is capable of reproducing various pathological lesions in mice that are seen in human dengue. The present study was undertaken to investigate the protective effect of active immunization of mice with CF. Mice were immunized with 5 microgram of CF and prevention of CF-induced increase in capillary permeability and damage to the blood-brain barrier were studied at weekly intervals, up to 48 weeks, by challenging with 3 microgram of CF. Maximum protection against increase in capillary permeability and damage to the blood-brain barrier was observed in week 4 after immunization. A breakthrough in the protection occurred with higher doses of CF in a dose-dependent manner. Challenge with a lethal intracerebral (i.c.) dose of DV showed significantly prolonged mean survival time and delayed onset of symptoms of sickness in the immunized mice compared with the normal mice, but the titre of the virus in the brain was similar in the two groups. On i.p. challenge with the virus the protection against damage to the blood-brain barrier was 86 +/- 7% at week 4 and 17 +/- 4% at week 26 after immunization. Sera obtained from the immunized mice showed the presence of CF-specific antibodies by ELISA, Western blot, and by neutralization of the cytotoxic activity of CF in vitro. The present study describes successful prevention of a cytokine-induced pathology by specific active immunization.     

A unique pulmonary venous abnormality: with associated pulmonary arterial anomaly and arteriovenous malformation

Lead and cadmium were administered intraperitoneally, singly and jointly, to the mice. The levels of cadmium, copper, manganese, lead and zinc were determined in liver, kidney and brain by atomic absorption spectrophotometric technique and delta-aminolevulinic acid dehydratase (ALA-D) activity was determined in erythrocytes. The tissue levels of some of these metals were found significantly altered by cadmium and lead both, but cadmium was found to have no effect on blood on ALA-D activity.     

Role of P-glycoprotein in the blood-brain transport of colchicine and vinblastine

Classically, drug penetration through the blood-brain barrier depends on the lipid solubility of the substance, except for some highly lipophilic drugs, like colchicine and vinblastine, both substrates of P-glycoprotein, a drug efflux pump present at the luminal surface of the brain capillary endothelial cells. Colchicine and vinblastine uptake into the brain was studied in the rat using the in situ brain perfusion technique and two inhibitors of P-glycoprotein, verapamil and SDZ PSC-833. When rats were pretreated with PSC-833 (10 mg/kg, intravenous bolus), colchicine and vinblastine uptake was enhanced 8.42- and 9.08-fold, respectively, in all the gray areas of the rat brain studied. The mean colchicine distribution volume was increased from 0.67 +/- 0.41 to 5.64 +/- 0.70 microliters/g and vinblastine distribution volume from 2.74 +/- 1.15 to 24.88 +/- 4.03 microliters/g. When rats were pretreated with verapamil (1 mg/kg, intravenous bolus), colchicine distribution volume was increased 3.70-fold. The increase in colchicine and vinblastine did not differ between the eight brain gray areas. PSC-833 and verapamil pretreatment had no influence on the distribution volume of either drug in the choroid plexus. Nevertheless, distribution volumes remained small, considering the highly lipophilic nature of the substances. We suggest that P-glycoprotein is either only partially inhibited (difficulty of fully saturating P-glycoprotein, especially under in vivo conditions) or not the only barrier to these two drugs.     

Telomere length regulation during postnatal development and ageing in Mus spretus

Telomere shortening has been causally implicated in replicative senescence in humans. To examine the relationship between telomere length and ageing in mice, we have utilized Mus spretus as a model species because it has telomere lengths of approximately the same length as humans. Telomere length and telomerase were analyzed from liver, kidney, spleen, brain and testis from >180 M.spretus male and female mice of different ages. Although telomere lengths for each tissue were heterogeneous, significant changes in telomere lengths were found in spleen and brain, but not in liver, testis or kidney. Telomerase activity was abundant in liver and testis, but weak to non-detectable in spleen, kidney and brain. Gender differences in mean terminal restriction fragment length were discovered in tissues from M.spretus and from M.spretus xC57BL/6 F1 mice, in which a M. spretus -sized telomeric smear could be measured. The comparison of the rank order of tissue telomere lengths within individual M. spretus showed that certain tissues tended to be longer than the others, and this ranking also extended to tissues of the M.spretus xC57BL/6 F1 mice. These data suggest that telomere lengths within individual tissues are regulated independently and are genetically controlled.     

Functional expression of P-glycoprotein in an immortalised cell line of rat brain endothelial cells, RBE4

The presence of P-glycoprotein in the cell plasma membrane limits the penetration of many cytotoxic substances into cells that express the gene product. There is considerable evidence also to indicate that P-glycoprotein is expressed as part of the normal blood-brain barrier in the luminal membranes of the cerebral capillary endothelial cells, where it presumably performs a protective function for the brain. This report describes the functional expression of P-glycoprotein in an immortalised cell line, RBE4, derived from rat cerebral capillary endothelial cells. The expression of P-glycoprotein is demonstrated by western immunoblotting and by immunogold and fluorescent staining with monoclonal antibodies. The cellular accumulation of [3H]colchicine and [3H]-vinblastine is investigated and shown to be enhanced by the presence of azidothymidine, chlorpromazine, verapamil, cyclosporin A, and PSC 833 ([3'-keto-Bmt1]-[Val2]-cyclosporin) at 50 or 100 microM concentration. It is concluded that the RBE4 cell line is a valuable tool for investigating the mechanisms of P-glycoprotein activity both in the blood-brain barrier and in multidrug resistance in general.     

Localization of cellular retinoic acid-binding protein (CRABP) II and CRABP in developing rat testis

Retinoic acid (RA) has been implicated as a signaling molecule for the morphogenesis of some tissues and organs. The morphogenesis of the rat testis occurs relatively late in development, culminating in puberty. Two members of the superfamily of small intracellular carrier proteins for lipophilic compounds are cellular Ra-binding protein (CRABP) and cellular RA-binding protein II (CRABP-II). Both CRABP and CRABP-II are present at various sites in the developing mouse embryo. Here we report the developmental expression and localization of CRABP and CRABP-II in rat testis. Northern blot analysis of CRABP-II demonstrated the highest messenger RNA expression on day 4 (the earliest time point assayed by this technique), decreasing thereafter until day 20, when it became undetectable. Western blot analysis, begun on day 19 of fetal development, indicated that high levels of protein expression in the testis already existed at that time. CRABP messenger RNA expression reached its highest levels between postnatal days 16-20 and decreased thereafter. Immunolocalization revealed that CRABP-II was confined to the fetal population of Leydig and Sertoli cells. We observed that CRABP-II was expressed in certain cells that synthesized retinoic acid in the uterus and ovary (unpublished). The expression of CRABP-II in Sertoli cells and fetal Leydig cells suggested that these cells may well be the site of RA synthesis in the developing testis. CRABP was localized to gonocytes in earlier stages and spermatogonia later, where it was clearly excluded from the nucleus, indicating that the role of CRABP may be to protect these cells from the effects of RA. The reported expression of CRABP-II in embryonal tissues, which are RA responsive and undergoing morphogenesis, coupled with CRABP-II expression in the testis at a critical morphogenic stage suggest that RA may play a prominent role in the morphogenesis of the testis.