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Astrocytic glucose-6-phosphatase and the permeability of brain microsomes to glucose 6-phosphate.

ABSTRACT: Cells from primary rat astrocyte cultures express a 36.5 kDa protein that cross-reacts with polyclonal antibodies to the catalytic subunit of rat hepatic glucose-6-phosphatase on Western blotting. Glucose-6-phosphate-hydrolysing activity of the order of 10 nmol/min per mg of total cellular protein can be demonstrated in cell homogenates. This activity shows latency, and is localized to the microsomal fraction. Kinetic analysis shows a Km of 15 mM and a Vmax. of 30 nmol/min per mg of microsomal protein in disrupted microsomes. Approx. 40% of the total phosphohydrolase activity is specific glucose-6-phosphatase, as judged by sensitivity to exposure to pH 5 at 37 degrees C. Previous reports that the brain microsomal glucose-6-phosphatase system does not distinguish glucose 6-phosphate and mannose 6-phosphate are confirmed in astrocyte microsomes. However, we demonstrate significant phosphomannose isomerase activity in brain microsomes, allowing for ready interconversion between mannose 6-phosphate and glucose 6-phosphate (Vmax. 15 nmol/min per mg of microsomal protein; apparent Km < 1 mM; pH optimum 5-6 for the two-step conversion). This finding invalidates the past inference from the failure of brain microsomes to distinguish mannose 6-phosphate and glucose 6-phosphate that the cerebral glucose-6-phosphatase system lacks a 'glucose 6-phosphate translocase' [Fishman and Karnovsky (1986) J. Neurochem. 46, 371-378]. Furthermore, light-scattering experiments confirm that a proportion of whole brain microsomes is readily permeable to glucose 6-phosphate.

SUBMITTER: Forsyth RJ

PROVIDER: S-EPMC1134577 | biostudies-other | 1993 Aug

REPOSITORIES: biostudies-other

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Project description:Astrocytes play diverse roles in the central nervous system (CNS) in both physiological and pathological conditions. Previous studies have identified many markers of astrocytes to analyze their complicated roles. Recently, closure of the critical period by mature astrocytes has been revealed, and the need for finding mature astrocyte-specific markers has been growing. We previously found that Ethanolamine phosphate phospholyase (Etnppl) was almost not expressed in the developing neonatal spinal cord, and its expression level slightly decreased after pyramidotomy in adult mice, which showed weak axonal sprouting, suggesting that its expression level negatively correlates with axonal elongation. Although the expression of Etnppl in astrocytes in adult is known, its utility as an astrocytic marker has not yet been investigated in detail. Here, we showed that Etnppl was selectively expressed in astrocytes in adult. Re-analyses using published RNA-sequencing datasets revealed changes in Etnppl expression in spinal cord injury, stroke, or systemic inflammation models. We produced high-quality monoclonal antibodies against ETNPPL and characterized ETNPPL localization in neonatal and adult mice. Expression of ETNPPL was very weak in neonatal mice, except in the ventricular and subventricular zones, and it was heterogeneously expressed in adult mice, with the highest expression in the cerebellum, olfactory bulb, and hypothalamus and the lowest in white matter. Subcellular localization of ETNPPL was dominant in the nuclei with weak expression in the cytosol in the minor population. Using the antibody, astrocytes in adult were selectively labeled in the cerebral cortex or spinal cord, and changes in astrocytes were detected in the spinal cord after pyramidotomy. ETNPPL is expressed in a subset of Gjb6 + astrocytes in the spinal cord. The monoclonal antibodies we created, as well as fundamental knowledge characterized in this study, will be valuable resources in the scientific community and will expand our understanding of astrocytes and their complicated responses in many pathological conditions in future analyses.

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Astrocytic glucose-6-phosphatase and the permeability of brain microsomes to glucose 6-phosphate.

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