Cultured brain microvessel endothelial cells as in vitro models of the blood-brain barrier.
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Animals↗
Biomedical subjects
Y Takakura
Publications and source records attributed to Y Takakura.
[The influence of lung volume and body position on pulmonary blood flow under hypoxic exposure].
We examined the changes in the electromagnetically measured left lower lobe blood flow (QLLL) when the left lower lobe (LLL) was exposed to alveolar hypoxia selectively under two different positions (supine and right lateral position in 18 dogs. QLLL/CO, Qs/Qt and other cardiopulmonary parameters were obtained during the following experimental sequence; 1) the whole lung ventilated with 100% O2 (control), 2) LLL collapsed and the remainder ventilated with 100% O2, 3) N2 expansion of LLL (N2 CPAP) and the remainder ventilated with 100% O2, 4) O2 expansion of LLL (O2 CPAP) and the remainder ventilated with 100% O2, and 5) The whole lung ventilated with 100% O2. In the supine position group (n = 9), both selective collapse and N2 CPAP of LLL caused QLLL/CO to decrease significantly (P less than 0.02) from control values (14.9 +/- 1.7%) to 10.6 +/- 0.9% and 11.9 +/- 1.6%, respectively. But there was no difference in QLLL/CO between collapse and N2 CPAP of LLL. In the right lateral position group (n = 9), hypoxic exposure of LLL caused no decrease in QLLL/CO from control values. But QLLL/CO during N2 CPAP (8.5 +/- 1.1%) decreased significantly than that during collapse (10.8 +/- 1.5%) (P less than 0.02). Qs/Qt during N2 CPAP (15.6 +/- 1.4%) also decreased significantly (P less than 0.05) from that during collapse (18.5 +/- 2.2%). We conclude that the difference of the changes in QLLL/CO under hypoxic exposure between supine and right lateral position was caused by hydrostatic pressure which influenced more during lateral position than during supine position.
Animals↗
Hexose uptake in primary cultures of bovine brain microvessel endothelial cells. I. Basic characteristics and effects of D-glucose and insulin.
The basic characteristics of hexose uptake and regulation of the glucose transporter (GLUT1) by D-glucose and insulin were studied in primary cultures of bovine brain microvessel endothelial cells (BMECs). A non-metabolizable glucose analog, 3-O-[3H]methyl-D-glucose [( 3H]3MG), was used as a model substrate, and the uptake was studied using BMECs grown in tissue culture plates. Uptake of [3H]3MG was equilibrative, temperature-dependent, and independent of sodium. The uptake also decreased gradually with culture age from 7 to 13 days. Saturation kinetics were observed for [3H]3MG uptake and the apparent Km and Vmax values were determined to be 13.2 mM and 169 nmol/mg per min, respectively. Pre-incubation with high concentrations of D-glucose and 3MG accelerated [3H]3MG uptake by BMECs by a counter-transport mechanism. D-Glucose, 2-deoxy-D-glucose, D-mannose, D-xylose, D-galactose and D-ribose showed significant competitive inhibition with [3H]3MG, whereas L-glucose, D-fructose, and sucrose did not affect [3H]3MG uptake by BMECs. [3H]3MG uptake was inhibited significantly by cytochalasin B and phloretin but not by phlorizin, 2,4-dinitrophenol, or ouabain. D-Glucose starvation of BMECs by incubation with D-glucose-free media for 24 h resulted in a significant increase (40-70%) in uptake of [3H]3MG compared with control conditions (7.3 mM D-glucose). Low D-glucose treatments (2.43 and 1.83 mM) for 7 days induced a slight but significant increase (20%) in [3H]3MG uptake, while long-term high glucose treatments (25 mM) showed no significant effect on [3H]3MG uptake irrespective of exposure time. The increase in [3H]3MG accumulation following D-glucose starvation was dependent upon starvation time (12 to 48 hr) and protein synthesis. Refeeding of D-glucose (7.3 mM) to D-glucose-starved BMECs resulted in a return of [3H]3MG uptake to control levels in 48 h. The D-glucose-starvation-induced increase in [3H]3MG uptake was shown to result from an increase in Vmax; the Km remained constant. In addition, D-glucose-starved BMECs were shown to have an increased level of GLUT1 using an antibody against human GLUT1 and an enzyme-linked immunosorbent assay (ELISA). The increased uptake following D-glucose starvation was not significantly affected by the presence of L-glucose, was partially impaired by the presence of D-galactose, D-fructose, and D-xylose, and was completely inhibited by the presence of D-mannose and 3MG. Furthermore, preincubation of BMECs with insulin (10 micrograms/ml) for 20 min did not affect the uptake of [3H]3MG or 2-deoxy-D-[3H]glucose ([3H]2DG).(ABSTRACT TRUNCATED AT 400 WORDS)
2,4-Dinitrophenol↗
Hexose uptake in primary cultures of bovine brain microvessel endothelial cells. II. Effects of conditioned media from astroglial and glioma cells.
Regulation of glucose uptake by an astroglial cell secreted factor(s) was studied in primary cultures of brain microvessel endothelial cells (BMECs). Uptake of a non-metabolizable glucose analog, 3-O-[3H]methyl-D-glucose ([3H]3MG), was measured after the BMECs were treated with media conditioned by primary cultures of rat astrocytes (Astrocyte Conditioned Media: ACM) or rat C6 glioma cells (Glioma Cell Conditioned Media: GCM). Uptake of [3H]3MG was significantly increased by ACM (30-50%) and GCM (60-200%) treatments, whereas conditioned medium from 3T3 fibroblasts (3T3) caused no significant effect. The elevation in [3H]3MG uptake increased with increasing time of exposure of BMECs to these conditioned media (CM), and the effect was shown to be reversible. Glucose depletion of CM was shown not to be a factor. The presence of cycloheximide, a protein synthesis inhibitor, during treatment of the BMECs with ACM and GCM blocked the increase in [3H]3MG uptake by the cells. These results suggested that ACM or GCM treatment elevated de novo synthesis of brain-type glucose transporter (GLUT1). Indeed, enhanced GLUT1 expression by these treatments in BMECs was demonstrated directly by enzyme-linked immunosorbent assay (ELISA) using antibodies against human GLUT1. After trypsinization of ACM and GCM, both conditioned media still induced significant stimulation of [3H]3MG uptake by BMECs. A significant increase in [3H]3MG uptake was also observed when ACM or GCM was exposed to BMECs through a dialysis membrane with a molecular weight cutoff of 1000. To examine whether the effects were specific to brain endothelial cells, [3H]3MG uptake experiments were performed employing aortic endothelial cells (AECs), pulmonary microvessel endothelial cells (PMECs), and 3T3 cells. ACM treatment did not alter 3MG uptake by these cells, suggesting that the ACM effect was specific to BMECs. On the other hand, [3H]3MG uptake by AECs and PMECs treated with GCM was significantly enhanced. The present study demonstrated that some factor(s) of relatively small molecular weight, which was released from astrocytes or glioma cells, stimulated glucose uptake by enhancing GLUT1 synthesis in BMECs.
3T3 Cells↗
Cloning and sequencing of human FSH receptor cDNA.
We have isolated and sequenced a cDNA encoding the follicle stimulating hormone (FSH) receptor. The deduced amino acid sequence (678 residues) containing seven putative transmembrane segments which displays sequence similarity to G protein-coupled receptors. The receptor consists of 359 residue extracellular domain which contains four N-linked glycosylation sites. While the protein is 89% identical overall with the previously cloned rat FSH receptor, the most highly conserved regions are the putative transmembrane segments (95% similarity).
Amino Acid Sequence↗
Hormonal regulation of gonadotropin receptor mRNA in rat ovary during follicular growth and luteinization.
To investigate the regulation of the follicle-stimulating hormone (FSH) and luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor genes by gonadotropins, we examined the effect of pregnant mare's serum gonadotropin (PMSG) or PMSG-hCG on the expression of FSH and LH/hCG receptors in rat ovaries. After administration of PMSG, Northern blot analysis using the FSH receptor cDNA probe revealed that a major band of 2400 nucleotides was detected which reached the maximal level on day 3. On the other hand, the level of LH/hCG receptor mRNA, a major mRNA of 5400 nucleotides and minor species of 7500, 3600, 2300 and 1200 nucleotides, increased progressively during 4 days. Treatment with hCG resulted in a decrease of FSH and LH/hCG receptor mRNA levels, and the level of FSH receptor mRNA was completely suppressed. Although the level of LH/hCG receptor mRNA was also suppressed from 3 h to an almost undetectable level at 24 h after hCG injection, it recovered to the control level by 48 h and exceeded this level several fold by 72 h. The reappearance of LH/hCG receptors following desensitization was preceded by an increase in mRNA levels. These studies demonstrate that hormonal regulation of gonadotropin receptor mRNAs on rat ovary reflects the changes in gonadotropin receptor levels.
Animals↗
Blood-brain barrier: transport studies in isolated brain capillaries and in cultured brain endothelial cells.
The development of in vitro BBB models consisting of isolated brain capillaries and cultured brain microvessel endothelial cells has made possible the study of BBB transport phenomena at the cellular level. Basic characteristics of BBB transport of endogenous and exogenous solutes and their biochemical, pharmacological, ontogenic, and pathological regulation mechanisms have been investigated. This information has led not only to a better understanding of BBB transport but also to the construction of strategies for improving drug delivery to the CNS for diagnosis and therapeutics. To elucidate the complexity of BBB transport, in vivo studies are always necessary at some point; however, in vitro systems can be useful complements to the in vivo systems. The tissue culture systems seem to be especially important in the clarification of cellular, biochemical and molecular features of BBB transport. Appropriate systems should be selected or combined, depending on the purpose of the investigation.
Animals↗
Hepatic disposition characteristics of electrically charged macromolecules in rat in vivo and in the perfused liver.
The effect of electric charge on the hepatic disposition of macromolecules was studied in the rat. Charged derivatives of dextran (T-70) and bovine serum albumin (BSA), mitomycin C-dextran conjugates (MMC-D), and lactosaminated BSA (Lac-BSA) were employed as model macromolecules. After intravenous injection, cationic macromolecules were rapidly eliminated from plasma because of their extensive hepatic uptake, while anionic and neutral macromolecules were slowly eliminated. Cationic macromolecules were recovered from parenchymal and nonparenchymal hepatic cells at a cellular uptake (per unit cell number) ratio of 1.4-3.2, while that of Lac-BSA was 14. During liver perfusion using a single-pass constant infusion mode, cationic macromolecules were continuously extracted by the liver, with extraction ratios at steady-state (Ess) ranging between 0.03 and 0.54, whereas anionic and neutral macromolecules were almost completely recovered in the outflow at steady state. The Ess for cationized BSA (Cat-BSA) and cationic MMC-Dcat were concentration dependent and decreased at low temperatures and in the presence of colchicine and cytochalasin B. The possible participation of the internalization process in the uptake of cationic macromolecules by hepatocytes was suggested.
Animals↗
Hepatic disposition characteristics of 111In-labeled lactosaminated bovine serum albumin in rats.
The hepatic disposition of lactosaminated bovine serum albumin (Lac-BSA) in rats was studied at the whole body, isolated liver, and isolated parenchymal cell levels. After intravenous injection, 111In-Lac-BSA (1 mg/kg) was rapidly eliminated from the plasma due to extensive uptake by liver parenchymal cells; however, a significant decrease in hepatic clearance was observed at high dose (50 mg/kg). In a single-pass, constant infusion experiment in the isolated liver, 111In-Lac-BSA was continuously extracted. The extraction ratio at steady state (Ess) for 111In-Lac-BSA was significantly decreased by coadministrating galactose, NH4Cl, or chloroquine, and at low temperature, suggesting that hepatic uptake of Lac-BSA proceeds via receptor-mediated endocytosis for asialoglycoprotein. Kinetic analysis of 111In-Lac-BSA binding with isolated parenchymal cells at 4 degrees C yielded a dissociation constant (Kd) of 2.5 x 10(-8) M and a value of 3.5 x 10(5) maximal binding sites/cell (Bmax). The internalization rate constant (kint) for 111In-Lac-BSA was calculated to be 0.46 min-1 in liver perfusion experiments using the EDTA-wash method.
Animals↗
Tumor localization and in vivo antitumor activity of the immunoconjugate composed of anti-human colon cancer monoclonal antibody and mitomycin C-dextran conjugate.
The tissue distribution and in vivo antitumor activity of a novel monoclonal antibody-mitomycin C conjugate (A7-MMCD) composed of anti-human MAb A7 and MMC-dextran conjugate were investigated using tumor-bearing mice. A7-MMCD was prepared via an anionic dextran intermediate for the purpose of keeping the non-specific uptake by the reticuloendothelial system to a minimum. 111In-labeled A7-MMCD showed about a 5-times-greater accumulation in SW1116 (targeted tumor) than in S180 (non-targeted tumor) 48 h after injection, and produced a tumor-to-blood ratio which was 3 times higher in SW1116-bearing mice than in S180-bearing mice 96 h after injection. Accumulations in the liver, spleen, and kidney were also observed to some extent. Pharmacokinetic analysis revealed that A7-MMCD had nearly the same properties in the body as MMCDan (MMCD with an anionic charge), i.e., those of a negatively charged macromolecule. Both A7-MMCD and MMCDan had relatively similar tissue uptake rate indices for the liver and spleen. The tumor uptake rate index for SW1116 was about 2.5 times greater than that for S180, and the total amount of 111In-A7-MMCD accumulated in SW1116 was calculated to be approximately 5 times greater than the amount in S180. These results indicated that A7-MMCD could achieve site-specific targeting in the body. Furthermore, in the therapeutic experiment using SW1116 implanted subcutaneously, A7-MMCD suppressed tumor growth significantly, compared to free MMC and MMCDan. These results suggest that in designing an monoclonal antibody-drug conjugate via an intermediary, the physicochemical properties of intermediate macromolecules must also be taken into consideration to obtain a high degree of efficacy in vivo.
Animals↗
Tissue distribution of 111In-labeled uricase conjugated with charged dextrans and polyethylene glycol.
Uricase (UC) was conjugated with dextran, cationic diethylaminoethyl-dextran (DEAED), and anionic carboxymethyl-dextran (CMD) giving a molecular weight of 10000 by the periodate oxidation method. Their disposition characteristics were studied after intravenous injection (i.v.) in mice. Disposition of the conjugate with activated polyethylene glycol (PEG2) with a similar molecular weight was also studied for comparison. Tissue distribution of the 111In-labeled UC in these conjugates was evaluated by a tissue uptake clearance index calculated in terms of clearance. After i.v. injection, 111In-UC was slowly eliminated from the circulation and gradually accumulated in the liver, spleen, and kidney. Conjugation with neutral dextran slightly enhanced the uptake of 111In-UC by the liver and spleen, while PEG2 conjugation decreased the tissue uptake and resulted in extremely long plasma retention. On the other hand, DEAED and CMD conjugation resulted in significant enhancement and reduction of hepatic uptake, respectively. These results demonstrated that the pharmacokinetic behaviour of UC can be widely controlled by chemical modification with macromolecules having adequate physiochemical properties.
Animals↗
Tarsal tunnel syndrome. Causes and results of operative treatment.
From 1975 to 1988, operative treatment was performed on 50 feet in 45 patients with tarsal tunnel syndrome. The causes of this syndrome were correlated with operative findings and included ganglia in 18, and a bony prominence from talocalcaneal coalition in 15. Five feet had sustained an injury, tumours were found in three and there was no obvious cause in nine. In most cases in need of operative treatment, there was a space-occupying lesion. Classifying the results according to causes, those with coalition or a tumour fared better, and idiopathic and traumatic cases had a worse outcome. In cases with a definite lesion, an excellent result can be expected from surgical treatment carried out soon after onset of the condition.
Adolescent↗
Symptomatic talocalcaneal coalition. Its clinical significance and treatment.
The clinical significance of symptomatic talocalcaneal coalition was described subjectively, objectively, and identified roentgenographically after examining 67 feet of 42 patients between 1977 and 1987. Computed tomographic examinations, conducted on 29 of 42 patients, were useful in recognizing the extent of coalition, the condition of union, and the postoperative results. Tarsal tunnel syndrome was present in 23 feet with the coalition. Thirty-one feet (24 patients) were treated conservatively and 36 feet (29 patients) surgically. Of the surgically treated feet, the coalition was excised in 33 and talocalcaneal fusion was performed in three. Follow-up examinations were performed at an average of 5.3 years (range, 2.25-11.2 years). In the excision group, the results were excellent in 24 feet, good in seven, and fair in two. The results were good in three feet treated by arthrodesis.
Adolescent↗
Cloning and sequencing of human LH/hCG receptor cDNA.
We have isolated and sequenced a cDNA encoding the human luteinizing hormone--choriogonadotropin (LH/hCG) receptor. The deduced amino acid sequence (699 residues) containing seven putative transmembrane segments displays sequence similarity to G protein-coupled receptors. The receptor consists of 335 residue extracellular domain which contains six N-linked glycosylation sites. While the protein is 85 and 87% identical overall with the previously cloned rat and porcine LH/hCG receptor respectively, the most highly conserved regions are the putative transmembrane segments (91 and 94% similarity, respectively).
Amino Acid Sequence↗
Regulation of LH/hCG receptor by gonadotropins in rat ovary.
To investigate the regulation of the LH/hCG receptor gene by gonadotropins, we examined the effect of PMSG and hCG on the expression of LH/hCG receptor in immature rat ovary. Northern blot analysis of ovarian RNA revealed a major mRNA of 5400 nucleotides and minor species of 7500, 3600, 2300, and 1200 nucleotides, and PMSG treatment slightly increased the intensity of all LH/hCG receptor messengers. Subsequently, hCG treatment decreased the number of LH/hCG receptor by day 2 and mRNA levels by 12h after injection. The level of mRNA recovered and increased 5-fold of control by day 6, then returned to control levels by day 10, followed by slower decline in LH/hCG receptor in plasma membrane. These studies demonstrate that the effects of PMSG and hCG on the number of LH/hCG receptor are closely related to the actions of these hormones on LH/hCG receptor messenger levels.
Aging↗
Disposition of anticancer drugs after bolus arterial administration in a tissue-isolated tumor perfusion system.
Disposition characteristics of various anticancer drugs in a tissue-isolated tumor preparation were studied in Walker 256 carcinosarcoma-bearing rats using an in situ single-pass vascular perfusion technique. Three anticancer drugs, 5-fluorouracil, mitomycin C, and Adriamycin, and two lipophilic prodrugs of mitomycin C were tested in the tumor preparation perfused with Tyrode's solution containing 4.7% bovine serum albumin. After bolus arterial injection of test drugs, their outflow concentration-time curves were analyzed based on statistical moment theory. In each tumor preparation, the injection of drug was paired with that of vascular reference substance, Evans' blue-labeled bovine serum albumin, and disposition parameters of the drug were corrected with those of vascular reference substance. From the mean transit time values of vascular reference substance, the average vascular volume of the tumor preparation was calculated to be 0.063 ml/g, which decreased with tumor growth. All drugs showed significant extraction by the tumor tissue, depending on their physicochemical properties. Distribution volumes of tested drugs were from 1.53 to 3.33 times larger than the vascular volume. Calculated intrinsic clearance values for the protein-unbound fractions increased as the lipophilicity of the drug increased. The potential increase in tumor uptake was observed in lipophilic prodrugs of mitomycin C. The present experimental system is thus suggested to be useful for analyzing drug disposition in tumor tissue.
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Disposition characteristics of macromolecules in tumor-bearing mice.
As part of the strategy for the design of macromolecular carriers for drug targeting, the disposition characteristics of macromolecules were studied in mice bearing tumors that served as target tissues. Eight kinds of macromolecules including four polysaccharides and four proteins with different molecular weights and electric charges were used; tissue distribution and tumor localization after intravenous injection were studied. Pharmacokinetic analysis revealed that the tissue radioactivity uptake rate index calculated in terms of clearance was different among the tested compounds; especially, the urinary radioactivity excretion clearances and the total hepatic radioactivity uptake clearances varied widely. Compounds with low molecular weights (approximately 10 kD) or positive charges showed lower tumor radioactivity accumulation; radioactivity was rapidly eliminated from the plasma via rapid urinary excretion or extensive hepatic uptake, respectively. On the other hand, large and negatively charged compounds, carboxymethyl dextran, bovine serum albumin, and mouse immunoglobulin G, showed higher radioactivity accumulation in the tumor (calculated total amounts were 15.6, 10.8, and 20.8% of the dose, respectively) and prolonged retention in the circulation. These results demonstrated that the total systemic exposure rather than the uptake rate index was correlated with total tumor uptake. Molecular weight and electric charge of the macromolecules significantly affected their disposition characteristics and, consequently, determined radioactivity accumulation in the tumor. It was concluded that a drug-carrier complex designed for systemic tumor targeting should be polyanionic in nature and larger than 70,000 in molecular weight.
Animals↗
[Antigenic recognition of rats infected with Gnathostoma hispidum: comparison of antigens derived from the advanced third stage larvae of Gnathostoma hispidum and Gnathostoma doloresi].
To increase understanding of immune response of host against G. hispidum, antigenic recognition of rodent gnathostomiasis were identified by the immunoblot method after PAGE of the advanced third stage larvae of G. hispidum and G. doloresi. The molecular weight of the major antigenic bands of G. hispidum extracts were 35 kDa and 16 kDa, and of G. doloresi were 48 kDa, 37 kDa and 35 kDa. Of these bands, the 35 kDa antigen of G. hispidum and G. doloresi was recognized with the sera of rats infected with G. hispidum 5 weeks after inoculation. Lecting staining with endoglycosidase-H or hydrolytic degradation on nitrocellulose strips revealed that the 35 kDa band of G. hispidum possessed the mannose-rich oligosaccharides. On the other hand, the band of G. doloresi had the hybrid-type glycopeptides. The larval extracts and excretory-secretory materials of G. hispidum were not antigenitically identical.
Animals↗