Browsing by Author "Djuricic, Bogdan (7004603010)"

Sheep choroid plexus epithelium expresses equilibrative nucleoside transporters (ENT) 1 and 2 and concentrative nucleoside transporter 2 at the transcript level. This study aimed to explore the kinetics and functional role of these transporters at the basolateral side of the sheep choroid plexus epithelium perfused in situ. The cellular uptake of [3H]adenosine and [3H]uridine was insensitive to 1 μm nitrobenzylthioinosine (NBTI), and the uptake of [3H]adenosine was reduced significantly when 10 μm NBTI was present in low-Na+ Ringer solution. This might suggest that ENT2, a transporter sensitive to micromolar NBTI, is functionally active at the basolateral side of the choroid plexus epithelium while ENT1, a transporter sensitive to nanomolar NBTI, is not active. When low-Na+ Ringer solution was used for the in situ perfusion, the Na+ concentration in the venous effluent decreased to 14 mm; under these conditions the maximal uptake (Umax) of [3H]adenosine and [ 3H]uridine did not change significantly when compared with the U max obtained when Ringer solution that contained 145 mm Na + was used. Kinetic analysis revealed apparent Michaelis-Menten constants (Km,app) for cellular uptake of [3H]adenosine, [3H]inosine and [3H]thymidine of 1.2 ± 0.2, 15.7 ± 2.6 and 3.8 ± 0.9 μm, respectively. The HPLC and HPLC-fluorometric analysis of the sheep plasma and cerebrospinal fluid revealed nanomolar concentrations of adenosine and thymidine and micromolar levels of inosine and nucleobases. Considering the estimated Km,app values, it appears that under normal conditions inosine is the more important nucleoside substrate for uptake by the basolateral membrane of the choroid plexus epithelium than other nucleosides. © 2008 The Authors.

Sheep choroid plexus epithelium expresses equilibrative nucleoside transporters (ENT) 1 and 2 and concentrative nucleoside transporter 2 at the transcript level. This study aimed to explore the kinetics and functional role of these transporters at the basolateral side of the sheep choroid plexus epithelium perfused in situ. The cellular uptake of [3H]adenosine and [3H]uridine was insensitive to 1 μm nitrobenzylthioinosine (NBTI), and the uptake of [3H]adenosine was reduced significantly when 10 μm NBTI was present in low-Na+ Ringer solution. This might suggest that ENT2, a transporter sensitive to micromolar NBTI, is functionally active at the basolateral side of the choroid plexus epithelium while ENT1, a transporter sensitive to nanomolar NBTI, is not active. When low-Na+ Ringer solution was used for the in situ perfusion, the Na+ concentration in the venous effluent decreased to 14 mm; under these conditions the maximal uptake (Umax) of [3H]adenosine and [ 3H]uridine did not change significantly when compared with the U max obtained when Ringer solution that contained 145 mm Na + was used. Kinetic analysis revealed apparent Michaelis-Menten constants (Km,app) for cellular uptake of [3H]adenosine, [3H]inosine and [3H]thymidine of 1.2 ± 0.2, 15.7 ± 2.6 and 3.8 ± 0.9 μm, respectively. The HPLC and HPLC-fluorometric analysis of the sheep plasma and cerebrospinal fluid revealed nanomolar concentrations of adenosine and thymidine and micromolar levels of inosine and nucleobases. Considering the estimated Km,app values, it appears that under normal conditions inosine is the more important nucleoside substrate for uptake by the basolateral membrane of the choroid plexus epithelium than other nucleosides. © 2008 The Authors.

Severe forebrain ischemia was produced in rats by occluding both carotid and vertebral arteries. Following 30 min ischemia brains were recirculated for 8 or 24 h. Twelve animals subjected to 8 or 24 h recirculation (n = 6, each group) were given α-difluoromethylornithine (DFMO; injected intraperitoneally) immediately before samples were taken from the cerebellum, cortex, caudatoputamen and hippocampus. Samples from the left hemisphere were used for measuring ornithine decarboxylase (ODC) activity, and those from the right hemisphere for determining putrescine profiles. During recirculation ODC activity increased markedly in all brain structures, the most pronounced change being in the caudatoputamen after 8 h recirculation. Putrescine increased drastically after 8 h and even more after 24 h recirculation. DFMO-treatment significantly reduced ODC activity after 8 h recirculation and following 24 h recirculation. Putrescine, however, was significantly reduced following 24 h but not after 8 h recirculation. The discrepancy between reduction in ODC activity and putrescine levels in DFMO-treated animals was not prominent in the hippocampus after 8 h recirculation: here DFMO reduced ODC activity to control values without affecting putrescine levels. The results suggest that the observed overshoot in putrescine formation following ischemia is only partly caused by activation of ODC. © 1988.

Severe forebrain ischemia was produced in rats by occluding both carotid and vertebral arteries. Following 30 min ischemia brains were recirculated for 8 or 24 h. Twelve animals subjected to 8 or 24 h recirculation (n = 6, each group) were given α-difluoromethylornithine (DFMO; injected intraperitoneally) immediately before samples were taken from the cerebellum, cortex, caudatoputamen and hippocampus. Samples from the left hemisphere were used for measuring ornithine decarboxylase (ODC) activity, and those from the right hemisphere for determining putrescine profiles. During recirculation ODC activity increased markedly in all brain structures, the most pronounced change being in the caudatoputamen after 8 h recirculation. Putrescine increased drastically after 8 h and even more after 24 h recirculation. DFMO-treatment significantly reduced ODC activity after 8 h recirculation and following 24 h recirculation. Putrescine, however, was significantly reduced following 24 h but not after 8 h recirculation. The discrepancy between reduction in ODC activity and putrescine levels in DFMO-treated animals was not prominent in the hippocampus after 8 h recirculation: here DFMO reduced ODC activity to control values without affecting putrescine levels. The results suggest that the observed overshoot in putrescine formation following ischemia is only partly caused by activation of ODC. © 1988.

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 CA, 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 CA, 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.

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 CA, 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 CA, 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.