Relative expression of mRNAS coding for glutaminase isoforms in CNS tissues and CNS tumors.

Glutaminase (GA) in mammalian tissues occurs in three isoforms: LGA (liver-type), KGA (kidney-type) and GAC (a KGA variant). Our previous study showed that human malignant gliomas (WHO grades III and IV) lack expression of LGA mRNA but are enriched in GAC mRNA relative to KGA mRNA. Here we analyzed the expression of mRNAs coding for the three isoforms in the biopsy material derived from other central nervous system tumors of WHO grades I-III. Non-neoplastic resective epileptic surgery samples served as control, as did cultured rat astrocytes and neurons. The GAC mRNA/KGA mRNA expression ratio was as a rule higher in the neoplastic than in control tissues, irrespective of the cell type dominating in the tumor or tumor malignancy. LGA mRNA expression was relatively very low in cultured astrocytes, and very low to absent in astrocytoma pilocyticum, ependymoma and subependymal giant cell astrocytoma (SEGA), tumors of astrocytic origin. LGA mRNA expression was almost as high as that of KGA and GAC mRNA in cultured neurons and epileptic surgery samples which were enriched in neurons. LGA mRNA was also relatively high in ganglioglioma which contains a discernable proportion of neuronal cells, and in oligodendroglioma. The results show that low expression of LGA mRNA is a feature common to normal astrocytes and astroglia-derived tumor cells or ependymomas and can be considered as a cell-type, rather than a malignancy marker.

Regulation of pH in the mammalian central nervous system under normal and pathological conditions: facts and hypotheses.

The maintenance of pH homeostasis in the CNS is of key importance for proper execution and regulation of neurotransmission, and deviations from this homeostasis are a crucial factor in the mechanism underlying a spectrum of pathological conditions. The first few sections of the review are devoted to the brain operating under normal conditions. The article commences with an overview of how extrinsic factors modelling the brain at work: neurotransmitters, depolarising stimuli (potassium and voltage changes) and cyclic nucleotides as major signal transducing vehicles affect pH in the CNS. Further, consequences of pH alterations on the major aspects of CNS function and metabolism are outlined. Next, the major cellular events involved in the transport, sequestration, metabolic production and buffering of protons that are common to all the mammalian cells, including the CNS cells. Since CNS function reflects tight interaction between astrocytes and neurons, the pH regulatory events pertinent to either cell type are discussed: overwhelming evidence implicates astrocytes as a key player in pH homeostasis in the brain. The different classes of membrane proteins involved in proton shuttling are listed and their mechanisms of action are given. These include: the Na+/H+ exchanger, different classes of bicarbonate transporters acting in a sodium-dependent- or -independent mode, monocarboxylic acid transporters and the vacuolar-type proton ATPase. A separate section is devoted to carbonic anhydrase, which is represented by multiple isoenzymes capable of pH buffering both in the cell interior and in the extracellular space. Next, impairment of pH regulation and compensatory responses occurring in brain affected by different pathologies: hypoxia/ischemia, epilepsy, hyperammonemic encephalopathies, cerebral tumours and HIV will be described. The review is limited to facts and plausible hypotheses pertaining to phenomena directly involved in pH regulation: changes in pH that accompany metabolic stress but have no distinct implications for the pH regulatory mechanisms are not dealt with. In most cases, the vast body of knowledge derived from in vitro studies remains to be verified in in vivo settings.

Selective decrease of SN1(SNAT3) mRNA expression in human and rat glioma cells adapted to grow in acidic medium.

The system N glutamine (Gln) transporter SN1(SNAT3) is overexpressed in human malignant glioma cells in situ as compared to the adjacent brain tissue or metastases from different organs [Sidoryk, M., Matyja, E., Dybel, A., Zielinska, M., Bogucki, J., Jaskólski, D.J., Liberski, P.P., Kowalczyk, P., Albrecht, J., 2004]. Increased expression of a glutamine transporter SNAT3 is a marker of malignant gliomas. NeuroReport 15, 575-578], but its role in tumor growth as compared to the other Gln transporters is unknown. One of the profound, growth-promoting effects of glial tumor in situ is acidification of the extracellular space. In the kidney SN1(SNAT3) mRNA participates in the adaptation to acidosis. In this study therefore, expression of mRNAs coding for SN1(SNAT3) and other Gln transporters was measured in human (T98G) and rat (C6) glioma cells incubated for 4h in an acidic medium (AI) (pH 6.5). MTT assay revealed no cell loss in AI cells, and intracellular pH (pHi) as measured by a fluorescent probe (BCECF-AM) was slightly alkaline in C6 and T98G cells, indicating that the cells have adapted to AI. AI significantly decreased the SN1(SNAT3) mRNA expression in C6 (a 60% decrease) and T98G cells (a 50% decrease). The decrease retreated in C6 cells 4h after transferring them back to the neutral medium. The expression of ASCT2 mRNA (system ASC), ATA1 mRNA (system A) and SN2(SNAT5) mRNA (system N) were not affected by AI in either of the cell lines. [(3)H]Gln uptake in C6 or T98G cells grown in neutral medium was mainly mediated by system ASCT2: system N contributed to only approximately 7% of the uptake. AI did not affect the total Gln uptake, and only slightly decreased the system N-mediated component of the uptake. Hence, SN1(SNAT3) does not seem to be involved in the adaptation of cultured glioma cells to acidic millieu.