Sandra L. Kuentzel

Hexose uptake in primary cultures of bovine brain microvessel endothetial 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)

Kinetic and morphological evidence for endocytosis of mammalian cell integrin receptors by using an anti-fibronectin receptor β subunit monoclonal antibody

Monoclonal antibody (mAb) 7E2.2, which recognizes the beta subunit of the hamster fibronectin receptor (FnR) (Brown, P.J., and Juliano, R. L. (1988) Exp. Cell Res. 177. 303), was used to examine the distribution of and to quantify the internalization of the FnR and possibly related integrins on adherent fibroblasts. Purified 7E2.2 IgG was iodinated and used in binding and internalization studies. Binding to Chinese hamster ovary cells was saturable with a Km of 0.3 nM and an estimated total number of cell surface beta subunits at 2 x 10(5) per cell. The FnR colocalized with fibronectin at cell adhesion contact sites and also was distributed evenly over the dorsal cell surface as discrete clusters. By using a direct immunocolloidal gold approach, the FnR was not associated with coated pits at 4 degrees C until internalization followed warming of the labeled cells to 37 degrees C. A proportion of the FnRs were endocytosed with a half-time of 6.5 min and, consistent with clathrin-mediated uptake, this was sensitive to hypertonic conditions. Receptor-immunocomplexes rapidly became localized within coated pits, small diameter tubules, and peripheral endosomes but the majority remained at the cell surface. At subsaturating concentrations of bound 7E2.2, approximately one-fourth of the total cell receptor population resided intracellularly at any one moment following steady-state; however, appreciable degradation of the iodinated mAb was not detected following accumulation for 4 h at 37 degrees C. These data showed that at least a portion of the FnR are endocytosed via a receptor-mediated pathway and suggested that these receptors do not immediately enter a degradative compartment.

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.

Studies of the biochemical pharmacology of the fermentation-derived antitumor agent, (αS, 5S) -α-amino-3-chloro-4, 5-dihydro-5-isoxazoleacetic acid (at-125)

Abstract (αS, 5S)-α-Amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) isolated from fermentation broths of Streptomyces sviceus was found to have significant activity against a number of tumors in experimental animals. It is currently being developed by the U.S. National Cancer Institute for clinical evaluation. AT-125 was shown to be a potent inhibitor of growth of L1210 and KB cells in culture. These effects were markedly dependent on the time of exposure to the agent. Similar results were observed when survival (residual proliferative capacity) of L1210 cells was measured. With L1210 cells (in culture and in vivo) AT-125 inhibited the incorporation of 3H-TdR into macromolecules to a greater extent than was observed when 3H-UR was the precursor. The activity of AT-125 in crude fermentation broths was detected in an in vitro antimicrobial prescreen designed to specifically detect materials with antimetabolite activity. Its activity in such a system was rather specifically antagonized by the amino acid, L-histidine. Such was not the case, however, in mammalian cells; L-histidine was without effect on AT-125 activity. In mammalian cells, the effects of AT-125 on growth inhibition or inhibition of radioactive precursors into macromolecules were rather specifically antagonized by L-glutamine. AT-125 was found to have significantly greater toxicity towards female than male mice, and sensitivity appeared to decrease with animal age. Further studies showed that AT-125 toxicity could be alleviated by coadministration of testosterone. In this respect, the biological activity of AT-125 was quite similar to that of a nucleoside antitumor agent, deaza UR. The primary locus of activity of deaza UR (after conversion to the triphosphate) has been shown to be inhibition of CTP synthetase. In studies with rat liver homogenates, AT-125 has been shown to inhibit CTP synthetase as well as other enzymes which catalyze the transfer of the amido group of L-glutamine. Further investigation of the effects of AT-125 have shown it to be a very potent inhibitor (Ki = 2 × 10−6 M) of purified rat liver CTP synthetase. AT-125 also causes effects on ribonucleotide pools which are consistent with inhibition of this enzyme and with the inhibition of another L-glutamine-dependent enzyme, XMP aminase. The importance of these two enzymes as loci for the biochemical action of AT-125 was supported when it was found that a combination of CR and GR significantly antagonized the growth inhibitory activity of AT-125.

Detection and assay of antitumor antibiotics.

Cell culture techniques and antimicrobial systems can be used as detection systems for new antibiotic structures. Antimicrobial systems by virtue of their speed, economy, ease of use, and adaptation to chromatographic (bioautographic) techniques are definitely superior for assay and for dereplication purposes. A prescreen assay system which combines the advantages and minimizes the disadvantages of the two approaches is described.

Overexpression of a COOH-terminal fragment of β-amyloid precursor protein in HeLa cells results in accumulation in a pre-Golgi compartment and generation of an Aβ-like fragment

The COOH-terminal 103-amino acid segment of human β-amyloid precursor protein (AβPP) was transfected in HeLa cells to study the effects of its overexpression on its subcellular distribution, accumulation and processing. This AβPP segment contains the intact Aβ sequence near its NH2 terminus. Butyrate-induced overexpression resulted in (1) the production and secretion of a 4.5 kDa Aβ-related peptide that was not detected in media conditioned by the corresponding untransfected cells; (2) the formation of large, cytoplasmic inclusions within the early secretory pathway (pre-Golgi) that were immunoreactive with antibodies to the AβPP COOH-terminal region and/or to the NH2-terminal region of the Aβ domain; and (3) the intracellular accunntlation of M, 14 and 16 kDa polypeptides that were shown to contain the saute AβPP epitopes as the intracellular inclusions. This study shows that a protein with an NH2-terminus similar to the. α-secretase product is a suitable a-secretase substrate and that the COON-terminal ...