Glutamine synthetase expression in liver, muscle, stomach and intestine of Bostrichthys sinensis in response to exposure to a high exogenous ammonia concentration.

A previous study provided evidence that the adaptive strategy used by the teleost fish Bostrichthys sinensis (sleeper) for detoxifying ammonia during extended periods of air exposure was to synthesize and store glutamine, primarily in the muscle, accompanied by an increase in glutamine synthetase (GSase) activity in liver. The aim of the present study was to assess the effect on GSase expression in various tissues of exposure of B. sinensis to exogenous ammonia. Exogenous ammonia increases internal ammonia concentrations in fish, mimicking environmental situations such as air exposure that preclude loss of ammonia across the gills, and thus triggering alternative mechanisms for ammonia detoxification. The results reveal relatively high levels of GSase activity, not only in liver but also, unexpectedly, in muscle, and even higher levels in intestine and, in particular, stomach. Exposure to ammonia results in significant increases in GSase activity, GSase protein and GSase mRNA levels in all of these tissues except stomach. The amino acid sequences of GSases from liver and stomach deduced from the cDNA sequences are essentially identical and are >97 % identical to the amino acid sequences of GSases from Gulf toadfish (Opsanus beta) and marble goby (Oxyeleotris marmoratus).

The FET4 gene encodes the low affinity Fe(II) transport protein of Saccharomyces cerevisiae.

Previous studies on Fe(II) uptake in Saccharomyces cerevisiae suggested the presence of two uptake systems with different affinities for this substrate. We demonstrate that the FET3 gene is required for high affinity uptake but not for the low affinity system. This requirement has enabled a characterization of the low affinity system. Low affinity uptake is time-, temperature-, and concentration-dependent and prefers Fe(II) over Fe(III) as substrate. We have isolated a new gene, FET4, that is required for low affinity uptake, and our results suggest that FET4 encodes an Fe(II) transporter protein. FET4's predicted amino acid sequence contains six potential transmembrane domains. Overexpressing FET4 increased low affinity uptake, whereas disrupting this gene eliminated that activity. In contrast, overexpressing FET4 decreased high affinity activity, while disrupting FET4 increased that activity. Therefore, the high affinity system may be regulated to compensate for alterations in low affinity activity. These analyses, and the analysis of the iron-dependent regulation of the plasma membrane Fe(III) reductase, demonstrate that the low affinity system is a biologically relevant mechanism of iron uptake in yeast. Furthermore, our results indicate that the high and low affinity systems are separate uptake pathways.

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.

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)

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.

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.