ADENINE-NUCLEOTIDE METABOLISM IN PRIMARY RAT NEURONAL CULTURES

The metabolism of adenine nucleotides (AdRN) has been studied previous ly in whole brains, brain slices and brain extracts, containing mixed populations of neurons and glia. The availability of primary neuronal cultures enables us to study these pathways in almost pure neuronal pr eparations. The aim of the present study was to characterize the relat ive importance of the pathways of AdRN metabolism in the neurons. The metabolic fate of (8-C-14)adenine and of AdRN prelabeled with (8-C-14) adenine were studied in immature and mature primary rat neuronal cultu res. Specific inhibitors were used to clarify the various metabolic fl uxes, which were evaluated based on the time-related changes in the di stribution of label (tb cellular nucleotide content did not change dur ing incubation). The turnover rate of AdRN was found to reflect mainly conversion of label to acid insoluble derivatives (AID) and partly de gradation to hypoxanthine. The turnover was faster in the immature neu rons. The combined addition of 2'-deoxycoformycin (2'-dCF) and of 5'-a mino-5'-deoxyadenosine, inhibiting adenosine metabolism, resulted in b oth cultures in enhanced loss of label from AdRN, mainly to adenosine and adenine. This finding indicates the activity of the futile cycle A MP-->adenosine-->AMP. In both cultures, in the presence of these inhib itors, the ratio (hypoxanthine + inosine)/(adenine + adenosine) was 1. 1, indicating that the fluxes through AMP deamination and AMP dephosph orylation are about equal. Addition of L-alanosine, inhibiting the con version of IMP to AMP, resulted in both cultures, but especially in th e mature neurons, in enhanced loss of label from AdRN to hypoxanthine and inosine. This finding indicates the functioning of the adenine nuc leotide cycle (AMP-->IMP-->adenylosuccinic acid-->AMP). Under conditio ns of enhanced degradation of ATP (induced by iodoacetate and antimyci n A), addition of 2'-dCF resulted in the immature cultures in lowering the ratio (hypoxanthine + inosine + IMP)/(adenine + adenosine) to 0.6 2, indicating a shift in favor of AMP dephosphorylation.