Research reportInorganic phosphate enhances phosphonucleotide concentrations in cultured fetal rat cortical neurons
Introduction
Intracellular inorganic phosphate (Pi) serves as a substrate for ATP synthesis in most, if not all, cells. Maintaining intracellular ATP levels is especially important in neurons due to their relatively high metabolic rate and absence of significant fuel reserves. Ischemic or excitotoxic insults acutely increase neuronal cytosolic calcium concentrations and disrupt oxidative phosphorylation 10, 12, 32. Reestablishment of ionic homeostasis, however, requires ATP 38, 44. Under conditions of intensified high-energy phosphate utilization and decreased production, a critical metabolic imbalance may develop, resulting in acute or delayed cell death [21]. Maintenance of high-energy phosphate compounds is associated with reduced tissue damage and facilitated recovery from anoxic and/or excitotoxic insults 27, 35, 50, 51.
Adequate Pi stores are also needed for the synthesis of NADPH, a principal participant in many anabolic processes. One of the most crucial of these is the generation of reduced glutathione (GSH) from the oxidized form (GSSG) by glutathione reductase, a reaction requiring NADPH as the electron donor. GSH is one of the most important intracellular antioxidants and is present in high concentrations in rat brain [15]. Reduced intracellular [NADPH] can contribute to a fall in GSH levels, which in turn potentiates oxidative injury. By contrast, enhancement of GSH stores can protect cells against damage due to ischemia and reperfusion [43]. Depletion of GSH or malfunction of GSH-linked enzymes has been implicated in oxidative stress-related disorders such as amyotrophic lateral sclerosis [36]and cataracts [43].
Oxidative and excitotoxic stresses are thought to be final common events underlying neurodegeneration from diverse causes 8, 21. Augmentation of high-energy phosphate and antioxidant status enhances neuronal survival under excitotoxic conditions. Therefore, understanding the factors which regulate ATP and NADPH synthesis in neurons may have important therapeutic implications.
Our laboratory has recently isolated and cloned a cDNA that encodes a brain- and neuron-specific Na+-dependent Pi cotransporter which is constitutively expressed in rat cerebral cortex, hippocampus and cerebellum [34]. Transcription of the corresponding mRNA is increased in cerebellar granule cells which are resistant to glutamate toxicity 9, 34, suggesting that Pi uptake may be important to neuronal survival under excitotoxic conditions. We have also demonstrated [34]and characterized saturable Na+-dependent Pi import into fetal rat cortical neurons and shown that a substantial fraction of the Pi so accumulated is incorporated into ATP [20].
It has long been known that Pi has regulatory effects on energy metabolism in whole cells [24]. Extracellular Pi increases glucose utilization 4, 40, and ATP and lactate production in erythrocytes [31], HeLa cells [47]and ascites tumor cells 48, 49. Decreased Pi and glucose transport are associated with significant ATP depletion in renal [42]and LLC-PK1 cells [1], whereas ATP hydrolysis is prevented by intracellular Pi in E. coli[22]. In vivo, the plasma Pi concentration correlates positively with erythrocyte ATP and 2,3-diphosphoglycerate content [6]. Although it is reasonable to assume that Pi has similar regulatory effects on energy metabolism in neurons, very little is known of Pi utilization in these cells. We therefore investigated the effects of varying [Pi]e on the ATP and NADPH content of cultured fetal rat cortical neurons. Our data demonstrate that import of Pi indeed raises the ATP and NADPH concentration in neurons and suggest that these effects improve survival after excitotoxic insults.
Section snippets
Preparation of primary neurons
Primary cultures of fetal rat cerebral cortical neurons were prepared from embryonic day 18 rats as described previously [20]. Briefly, cortical tissue was dissociated by sequential immersion at 37°C in 0.05% trypsin/0.53 mM EDTA, and 50 μg/ml DNase (Sigma Chemical Co., St. Louis, MO, USA). After collection by centrifugation at 1000×g, neurons were resuspended to a final concentration of 6–8×105 cells/ml in culture medium consisting of 10% fetal bovine serum and 90% Dulbecco's Modified Eagle's
Pi import into cortical neurons
Na+-dependent Pi import into primary cultures of fetal rat cortical neurons displays an apparent Km of approximately 54 μM [20]. The [Pi] of plasma and cerebrospinal fluid, however, are 1.1–1.5 mM [19], suggesting that uptake is saturated under physiological conditions. Fig. 1 confirms this hypothesis. 33Pi import into cortical neurons displayed a hyperbolic dependence upon [Pi]e, reaching a maximum at 100–250 μM and remaining constant up to 1 mM. As expected, the total free intracellular Pi
Discussion
In the present study, we have confirmed that Pi transport into cortical neurons is fully saturated at physiological [Pi]e. We have also demonstrated that both the ATP and NADPH contents of cultured fetal cortical neurons are highly correlated with [Pi]i, and that [ATP], [NADPH] and [Pi]i are dependent upon [Pi]e. Although previous studies have shown a relationship between extra- and intracellular Pi, glucose utilization and ATP content in erythrocytes and cultured cell lines 1, 4, 22, 31, 40, 42
Acknowledgements
We would like to thank Drs. Paul Hyslop and Lee Phebus for their helpful comments.
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