Biomedical subjects

L R Drewes

Publications and source records attributed to L R Drewes.

At least 37 records · Page 2Linked to original sources

PubMed1993

Application of novel PCR strategies to amplify and sequence glucose transporters in canine brain.

We employ here a modified oligo(dT) primer called a "lock-docking" primer which enables the production of a cDNA template that can subsequently be amplified in the 3'-RACE PCR procedure to produce discrete, first-round products for 3'-cDNA ends of any reverse-transcribed poly(A) RNAs. An upstream consensus primer targeted to a highly conserved region (amino acid region 448-455 in human Glut1) among all glucose transporter isoforms was used in the 3'-RACE PCR procedure with lock-docked cDNA template. Sources of lock-docked cDNA template were canine intestine, kidney, brain cortex, and brain microvessels. This procedure made it possible to delineate, in one round of PCR (31 cycles), all of the more abundantly expressed glucose transporter isoforms in each of these tissues. Other as yet unaccounted for products were also obtained. These products are potentially alternately terminated transcripts of glucose transporter isoforms, alternately spliced transcripts, or as yet uncharacterized isoforms. After electrophoresis on an agarose gel and purification of the DNA, each of these PCR products is available for direct sequencing.

Animals↗

PubMed1992

Neurons and microvessels express the brain glucose transporter protein GLUT3.

To elucidate glucose transport mechanisms in brain and to demonstrate the cellular expression of the brain-type glucose transporter (GLUT3), antisera to a synthetic peptide corresponding to the C terminus were prepared and used as probes for this isoform of the facilitative glucose transporter family. Immunocytochemistry of frozen sections of dog and rat brain demonstrated GLUT3 antigen in pyramidal cell bodies and processes, in microvessels, and in intima pia or glia limitans. Immunoanalysis of Western blots identified a protein (Mr, 45,000) that was present in both neuron/neuropil and microvessel fractions. The presence of the GLUT3 message in brain was confirmed by Northern blot analysis and by amplifying and partially sequencing GLUT3 cDNA by PCR. These findings demonstrate a neuron glucose transporter in tissue and suggest that GLUT3 may play an important role in brain metabolism under physiological and pathophysiological conditions.

Amino Acid Sequence↗

PubMed1992

Vascular differentiation and glucose transporter expression in rat gliomas: effects of steroids.

The GLUT1 isoform of the glucose transporter is normally expressed at high levels in differentiated brain vessels that also express a permeability barrier. In contrast, malignant brain neoplasms have relatively undifferentiated vessels that are highly permeable, proliferate to high vascular densities, and often lose GLUT1 expression. Using the rat intracerebral 9L glioma model, we investigated whether dexamethasone-induced changes in permeability are associated with the appearance of other differentiated vascular properties. The percentage of vessels expressing immunohistochemically detectable GLUT1 (74.2 +/- 6.1%) and the tumor vessel density as assessed by laminin immunostaining (282 +/- 37 vessels/mm2) did not vary with control tumor size. Dexamethasone treatment resulted in an 83% reduction of vascular permeability to intravenous Evans blue, an increased percentage of vessels expressing GLUT1 (106.4 +/- 10.5%), lower vascular density (102 +/- 64 vessels/mm2), and smaller tumor size (control cross-sectional area, 17.0 +/- 3.4 mm2; treated, 4.6 +/- 1.0 mm2). Essentially all vessels became GLUT1-positive after dexamethasone treatment. Increased GLUT1 expression by glioma vessels in association with the appearance of other signs of differentiation (low vascular density, slow tumor growth) suggests that immunostaining for GLUT1 may identify neoplasms that are biologically less aggressive.

Animals↗

PubMed1992

A lock-docking oligo(dT) primer for 5' and 3' RACE PCR.

We describe a method that can be used to obtain and sequence 3' and 5' ends of cDNA transcripts directly from PCR products. The method employs a modified oligo(dT) primer that enables it to "lock-dock" at the junction of gene-specific cDNA sequence and a natural (3') or appended (5') poly(A) tail. As a result, discrete, first-round PCR products are obtained that are easily isolated and sequenced directly.

Animals↗

PubMed1992

Vascular expression of glucose transporter in experimental brain neoplasms.

Vascular abnormalities in brain neoplasms are important to tumor biology and therapy. Glucose transporter (GLUT1) expression is a differentiated property of normal cerebral microvessels typically associated with expression of the blood-brain barrier. We investigated the relationship of GLUT1 expression to other vascular characteristics in F98, 9L, and C6 gliomas and Walker 256 carcinomas implanted into adult rat brains. The percentages of microvessels with immunohistochemically detectable GLUT1 were 95.5 +/- 3.9 in F98, 60.9 +/- 3.9 in 9L, 45.4 +/- 5.6 in C6, and 1.2 +/- 0.3 in Walker 256 (mean +/- SEM). The percentage of GLUT1-positive vessels in F98 was not statistically different from that in normal brain. GLUT1 expression was not dependent on restricted permeability as all tumors were highly permeable to Evans blue. GLUT1 expression was unrelated to vascular density, vascular morphology, and parenchymal GFAP expression. The expression of GLUT1, a marker of cerebral endothelial differentiation, is a newly described property of glial tumor vessels that may have diagnostic and prognostic significance.

Animals↗

PubMed1991

Cross-talk between receptor-regulated phospholipase D and phospholipase C in brain.

Because receptors, G proteins, and phospholipases all exist within a membrane lipid environment, it is not unreasonable to assume that an enzyme capable of changing the lipid environment can affect the coupling relationship among these signal transducing components. Our previous study showed that a muscarinic acetylcholine receptor regulates phosphatidylcholine phospholipase D via a G protein in brain. We demonstrate here that phosphatidylinositol phospholipase C and phosphatidylcholine phospholipase D are simultaneously activated within 15 s by muscarine in the presence of 1 microM GTP gamma S. More important, inhibition of phospholipase D by zinc attenuated carbamylcholine-induced activation of phospholipase C by 30%. Our additional evidence strongly indicates that the receptor-regulated phospholipase D plays an important modulatory role in agonist-stimulated phosphatidylinositol breakdown. This modulatory effect may be achieved by changing the membrane microenvironment in which phospholipase C and phosphoinositol lipids reside, consequently amplifying the inositol phospholipid signaling process. Our results lead us to postulate that the potential interaction between two different signaling pathways may provide a cell with intracellular coordination and enable the cell to achieve functional responses.

Animals↗

PubMed1991

Formation of free choline in brain tissue during in vitro energy deprivation.

Free choline and ATP contents were measured in Mongolian gerbil hippocampal slices (tissue) and incubation media (media) during exposure to 30 min of aglycemia, high potassium, anoxia, or ischemia. Changes in choline levels reflected the degree of energy reduction, lower ATP levels being associated with high choline (4-fold increase during exposure to high potassium and anoxia, and 11-fold increase during ischemia). Media (extracellular) choline was particularly affected and increased about twofold during relatively mild energy depletion (e.g., aglycemia), but tissue choline content was less sensitive to energy reduction. A plot of choline vs. ATP levels indicated a nonlinear correlation, and the sharp increase in choline occurred when ATP values fell to about 2.5 nmol/mg of protein. Inhibition of acetylcholine sterase by 10 microM physostigmine during ischemia did not prevent an increase in choline contents but rather enhanced them, indicating that acetylcholine hydrolysis was not the source of free choline. Formation of free choline was Ca2+ independent. These findings suggest the involvement of phospholipase D and phosphatidylcholine hydrolysis in free choline formation during energy stress. The extent of choline formation may be an indicator of the degree of membranal damage, which in turn reflects damage to the metabolic machinery of the cell.

Acetylcholinesterase↗

PubMed1991

Quantitative immunocytochemistry (image analysis) of glucose transporters in the normal and postischemic rodent hippocampus.

The bilateral carotid occlusion model and a polyclonal antibody to the carboxyl terminus of the rat brain/human hepatoma glucose transporter were used to examine quantitatively changes in the transporter in gerbil hippocampal microvessels following 6-7.5 min of ischemia. The optical densities of immunocytochemically stained microvessels in the stratum lacunosum-moleculare (SLM) below the CA1 subfield were determined using image analysis of frozen sections from gerbils killed 2 h, 3 days, 6 days, 4 weeks, and 7 weeks after the ischemic episode. Microvessels were sparsely distributed in the stratum oriens, stratum pyramidale, and stratum radiatum. In contrast, the SLM was relatively well vascularized, and this distribution of microvessels persisted following ischemia. The SLM was identifiable based solely on microvessel distribution both in control gerbils and in gerbils that exhibited complete destruction of CA1 pyramidal cells. The abundance of the glucose transporter in SLM microvessels remained constant, suggesting that down-regulation of this protein cannot account for reported declines in brain glucose utilization and cell death following ischemia. Conversely, the presence and metabolic activity of CA1 pyramidal cells do not appear to be determinants of glucose transporter abundance in hippocampal microvessels. The brain/hepatoma glucose transporter was abundant in brain microvessels and the epithelial cells of the choroid plexus of gerbil and rat. Staining of hippocampal neuropil was less intense, poorly localized, and, at the light microscope level, not clearly associated with a particular cell type.

Animals↗

PubMed1990

A novel mechanism for acetylcholine to generate diacylglycerol in brain.

The classical scheme involving inositol phospholipid breakdown by phospholipase C as the sole source of diacylglycerol (DAG) has recently been challenged by evidence that phosphatidylcholine (PC) is an alternative source. In synaptic membranes of canine cerebral cortex, cholinergic agonists caused rapid accumulation of [3H]phosphatidic acid (PA) from [3H]PC within 15 s, whereas [3H]DAG formation showed a transient lag period before becoming elevated and then exceeding the amount of [3H]PA. Additional evidence shows that DAG is produced from PC by the action of phospholipase D to yield PA, which is further dephosphorylated to DAG by PA phosphatase. Our results indicate that this muscarinic acetylcholine receptor-regulated PC phospholipase D-PA phosphatase pathway may be a novel mechanism in cell signal transduction processes for activation of protein kinase C in brain.

Acetylcholine↗

PubMed1990

Glucose transporters at the blood-nerve barrier are associated with perineurial cells and endoneurial microvessels.

The blood-nerve barrier consists of continuous layers of cells linked by tight junctions and includes the endothelial cells which line the endoneurial capillaries and the perineurial cells which surround fascicles of nerve fibers. A facilitated transport carrier protein allows D-glucose to penetrate the barrier. To determine the specific cellular location of the transport system, an antiserum to a synthetic peptide corresponding to the carboxyl-terminus of the glucose transporter protein was used for light and electron immunocytochemical analyses. Glucose transporters were abundant both in endoneurial capillaries and the perineurial sheath. In perineurium, transporters were located in the plasma membranes and cytoplasm of the perineurial cells. Approximately two-thirds of the transporters associated with perineurial cells were localized in the plasma membranes. Perineurial cells are thus similar to cerebral endothelial cells in that they lack a large intracellular pool of transporters which might be sensitive to hormonal regulation. The presence of hexose carriers in perineurium suggests that glucose transport from epineurium to endoneurium may play a significant role in the metabolism of peripheral nerve fibers. These results support the concept that the blood-nerve barrier serves as an important nutrient delivery system.

Animals↗

PubMed1990

The glucose transporter and blood-brain barrier of human brain tumors.

The glucose transporter of the human brain has been localized to endothelial cells expressing the blood-brain barrier, but little is known regarding its mechanism of induction or whether its expression is exclusively linked with restricted vascular permeability. We investigated glucose transporter expression by vessels in human astrocytic tumors and pulmonary metastases to the brain using immunohistochemical techniques. Vessels in 9 of 10 low-grade astrocytomas and 8 of 10 anaplastic astrocytomas were positive for glucose transporter. Glioblastoma vessels were transporter-positive in only 2 of 10 specimens. Vessels in all three metastatic tumors were negative for the glucose transporter. The decrease in transporter expression observed in higher-grade tumors occurred independently of increases in vascular permeability. In low-grade astrocytomas and glioblastomas transporter expression and contrast enhancement were inversely related, but vessels in 6 of 9 anaplastic astrocytomas were transporter-positive despite contrast enhancement. These findings suggest that separate mechanisms induce the glucose transporter and the permeability restrictions of the human blood-brain barrier. They also have potential implications for the therapy and prognosis of astroglial neoplasms.

Adult↗

PubMed1990

Guanine nucleotide-binding protein regulation of microsomal phospholipase D activity of canine cerebral cortex.

The hydrolytic activity of microsomal phospholipase D from canine cerebral cortex was measured by a radiochemical assay using 1,2-dipalmitoyl-sn-glycerol-3-phosphoryl[3H]choline and 1-palmitoyl-2-[9,10(n)-3H]palmitoyl-sn-glycerol-3-phosphorylcholine as the exogenous substrates. Of several detergents tested, Triton X-100 was found to be the most effective in allowing expression of phospholipase D hydrolytic activity. The microsomal phospholipase D does not require any metal ion for its hydrolytic activity. Calcium and magnesium were slightly inhibitory between concentrations of 1 and 4 mM, but zinc was greatly inhibitory, causing a loss of greater than 90% activity at the 4 mM concentration. Non-hydrolyzable guanine nucleotide analogues such as guanosine 5'-(3-O-thio)triphosphate and guanyl-5'-yl-(beta, gamma-methylene)diphosphonate but not guanosine 5'-(2-thio)diphosphate were able persistently to stimulate phospholipase D hydrolytic activity at micromolar concentrations. Guanosine 5'-(2-thio)diphosphate was capable of partially blocking guanosine 5'-(3-O-thio)triphosphate stimulation of phospholipase D. Aluminum fluoride was able to cause a two- to threefold increase in hydrolytic activity of the phospholipase D. Cholera toxin had a stimulatory effect on the hydrolytic activity of phospholipase D, whereas islet-activating protein pertussis toxin had no effect. These results indicate that regulation of microsomal phosphatidylcholine phospholipase D activity by the guanine nucleotide-binding protein(s) in canine cerebral cortex may play an important role in signal transduction processes as well as in brain choline metabolism.

Aluminum↗

PubMed1989

Muscarinic acetylcholine receptor regulates phosphatidylcholine phospholipase D in canine brain.

The hydrolytic activity of phosphatidylcholine phospholipase D in the synaptosomes from canine brain was examined using a radiochemical assay with 1,2-dipalmitoyl-sn-glycerol-3-phosphoryl[3H]choline as the exogenous substrate. The involvement of G protein(s) in regulation of this enzyme was demonstrated by a 2- to 3-fold stimulation of the basal activity (4.81 +/- 0.44 nmol choline released/mg protein/h) with guanosine 5'-(3-O-thiol)triphosphate (GTP gamma S), guanyl-5'-yl-(beta, gamma-methylene)diphosphonate, aluminum fluoride, or cholera toxin. The stimulation of phospholipase D hydrolytic activity by GTP gamma S was inhibited by 2 mM guanosine 5'-(2-O-thiol)diphosphate. GTP gamma S at the maximum stimulatory concentration (10 microM) had an additive effect on the maximum cholera toxin stimulation of phospholipase D activity. However, the reverse was not true, thus indicating the possibility that more than one G protein may be involved. Furthermore, cholinergic agonists, including acetylcholine, carbachol, and muscarine, were able to increase the phospholipase D hydrolytic activity at low but not maximally stimulatory concentrations of guanine nucleotide. These cholinergic stimulations were antagonized by atropine, a muscarinic blocker. In addition, O-tetradecanoylphorbol 13-acetate, a protein kinase C activator, was able to stimulate the hydrolytic activity of phospholipase D more than 300% in the presence of 0.2 microM GTP gamma S. However, in the absence of GTP gamma S, stimulation was less than 60%. Our results not only indicate that the receptor-G protein-regulated phospholipase D may be directly responsible for the rapid accumulation of choline and phosphatidic acid in the central nervous system but also reveal that muscarinic acetylcholine receptor-G protein-regulated phospholipase D is a novel signal transduction process coupling the neuronal muscarinic receptor to cellular responses.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1989

Glucose transporter localization in brain using light and electron immunocytochemistry.

A polyclonal antibody to a synthetic 13 amino acidpeptide found at the carboxyl-terminal end of the glucose transporter protein was raised in rabbit and used in light and electron immunocytochemical studies of human and canine brain. This antibody identified a broad band of polypeptide of average Mr 55,000 on immunoblots (immunogold-silver stains) of electrophoresed membrane proteins from human red blood cells. A similar polypeptide band (Mr 45,000-60,000) was identified on immunoblots of microvessel membrane proteins isolated from canine cerebrum, suggesting that this antibody is a useful tool for studying the distribution and abundance of the glucose transporter protein in mammalian nervous tissue. Peroxidase antiperoxidase stains of cerebrum using this antibody demonstrated that transporters are abundant in the intima pia, in the endothelium of blood vessels in the subarachnoid space, and in the endothelium of arterioles, venules, and capillaries of gray and white matter. In cerebellum, reaction product was localized in the vessels of the subarachnoid space and in microvessels of the molecular layer, the granular layer, and the white matter. However, transporters were not found in the intima pia of cerebellum. In medulla oblongata, transporters were found in the intima pia, the endothelium of some subarachnoid vessels, and the microvessels of gray and white matter. In pituitary, microvessels in adenohypophysis contained no reaction product, but the antigen was detected in some microvessels in neurohypophysis. Electron microscopy of cerebral cortex using a protein A-gold technique demonstrated that glucose transporters are equally abundant on the luminal and abluminal membranes of microvessel endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Protection against acute hyperammonemia: the role of quaternary amines.

The quaternary amine L-carnitine is able to protect Swiss Albino mice from hyperammonemia when administered in high doses before ammonium acetate. This has been explained by its specific ability to shuttle fatty acids into mitochondria. The structure of L-carnitine resembles the chemical structure of other substances that have been described as being able to protect living cells against osmotic stress. We subjected Swiss Albino mice to hyperammonemia after pretreatment with L-carnitine or "osmoprotectants" such as the quaternary amines choline and betaine, and trimethylamine N-oxide. L-Carnitine proved to be the drug of choice to protect against acute hyperammonemia. Nevertheless, the other tested compounds appeared also to be effective, suggesting that osmoregulation plays a major role in protection against hyperammonemia.

Acetates↗

PubMed1988

Phorbol ester stimulates hexose uptake by brain microvessel endothelial cells.

Glucose uptake into cultured endothelial cells (EC) derived from brain microvessels was determined in the absence and presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), EGTA, the calcium ionophore A23187, and insulin. EC were obtained from dog and human (autopsy) brain microvessels and maintained in culture for up to four passages. Monolayers of EC were treated with TPA and other compounds immediately prior to harvesting for hexose uptake measurements using 3-O-[3H]methyl-D-glucose, 2-[3H]deoxy-D-glucose, or D-[3H]glucose. Typically, treatment with TPA (0.1-100 ng/ml) resulted in hexose uptake levels 2 to 3 times those of controls, although occasionally levels 5 to 10 times those of controls were observed. Similar stimulation was observed with all radiolabeled hexoses. Stimulation by TPA was greatest in primary or first passage cells and was greatly diminished in older cells. Neither chelation of extracellular calcium with EGTA nor the presence of both EGTA and A23187 in the culture medium prevented the stimulatory effect of TPA. Insulin (1200 ng/ml) failed to stimulate hexose uptake. Treatment with 100 ng/ml TPA did not alter the appearance of actin filaments in canine EC as visualized with rhodamine phalloidin. These results, in combination with other recent studies, suggest that blood-brain glucose transport may be regulated by phorbol ester-activated protein kinase C.

Animals↗

PubMed1988

Cultured human and canine endothelial cells from brain microvessels.

Cultures of endothelial cells (EC) derived from human (autopsy) and canine brain microvessels were characterized with respect to growth, morphology, and biochemical features. The endothelial nature of these cells was confirmed by analyses of angiotensin-converting enzyme activity, Factor VIII-related antigen, and ultrastructure. Human EC required coated substrates and tumor-conditioned medium to achieve rapid growth, and cells derived from human microvessels were morphologically diverse. In contrast, canine EC grew rapidly on plastic substrates and produced colonies of uniform morphology. Morphological variations of EC were associated with the use of heparin-containing medium and with the use of a commercially-prepared basement membrane extract (Matrigel). Lectin histochemistry demonstrated that human EC lack the abundant alpha-galactose residues characteristic of canine EC membranes and organelles and that canine EC lack the alpha-N-acetylgalactosamine residues which are associated with human EC. The lectin Ricinus communis agglutinin I may be useful for distinguishing canine EC from pericytes. Gel electrophoresis of membrane proteins revealed protein bands present in human EC at Mr 210,000 and 37,000-39,500 which were not present in canine EC. These proteins may be related to the presence of junctional complexes in cultures of human EC.

Animals↗

PubMed1988

Choline transport and metabolism in soman- or sarin-intoxicated brain.

The metabolism and blood-brain transport of choline (Ch) were investigated in perfused canine brain under control conditions and for 60 min after inhibition of brain cholinesterases by the organophosphorus (OP) compounds soman (pinacolylmethylphosphonofluoridate). Ch and acetylcholine (ACh) in blood and brain samples were analyzed using gas chromatography-mass spectrometry methods. Net transport of Ch was determined by Ch analysis in arterial and venous samples. Unidirectional transport of [3H]Ch was determined using the indicator dilution method. During control perfusion periods of 90 min, net efflux of brain Ch occurred at a rate of 1.6 +/- 0.4 nmol/g/min, and the Ch content of the recirculated perfusate increased 10-fold to approximately 8 microM. Brain Ch content increased in proportion to the increase in perfusate Ch level, but brain ACh was unaltered. Rapid administration of soman (100 micrograms) or sarin (400 micrograms) into the arterial perfusate after a 40-min control period resulted in a greater than 10-fold increase in ACh content in cerebral cortex, brainstem, and hippocampus. The ACh content of cerebellum increased only slightly. The Ch level in all four brain regions studied also increased two- to fourfold above control levels. Ch efflux from brain, however, decreased to 0.2 +/- 0.1 nmol/g/min during the 60 min after OP exposure. Unidirectional influx of [3H]Ch was 0.49 +/- 0.07 nmol/g/min before and did not change significantly 10 or 40 min after OP exposure, thus indicating that the Ch transporter of the brain endothelial cell is not directly inhibited.2+ Based on these results, it is proposed that (a) efflux of brain Ch occurs from the extracellular compartment, which becomes depleted when ACh breakdown is inhibited;(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗