A rat neuroma cell line (B103 4C), deficient of hypoxanthine‐guanine phosphoribosyltransferase (HGPRT), was utilized as a model tissue in search for the biochemical basis of the Lesch‐Nyhan syndrome (LNS). The HGPRT‐deficient neurons exhibited the following properties: an almost complete absence of uptake of guanine and of hypoxanthine into intact cell nucleotides (0.92% and 0.69% of normal, respectively); a significant increase in the availability of 5′‐phosphoribosyl‐1‐pyrophosphate; a three‐ to fourfold acceleration of the rate of de novo nucleotide synthesis; a normal excretion of xanthine, but 15‐fold increase in the excretion of hypoxanthine into the culture media; a normal cellular purine nucleotide content, including the absence of 5‐amino‐4‐imidazole carboxamide nucleotides (Z‐nucleotides), but enhanced turnover of adenine nucleotides (loss of 86% of the radioactivity of the prelabeled pool in 24 h, in comparison to 73% in the normal line), and an elevated UTP content. The results suggest that, under physiological conditions, guanine salvage does not occur in the normal neurons, but that hypoxanthine salvage is of great importance in the homeostasis of the adenine nucleotide pool. The finding of the normal profile of purine nucleotides in the HGPRT‐deficient neurons indicates that the lack of hypoxanthine salvage is adequately compensated by the enhanced de novo nucleotide synthesis. These results did not furnish evidence in support of the possibility that GTP or ATP depletion, or Z‐nucleotide accumulation, occurs in HGPRT‐deficient neurons and that these are etiological factors causing the neurological abnormalities in LNS. On the other hand, the results point to the possibility that elevated hypoxanthine concentration in the brain may have an etiological role in the pathogenesis of LNS.

The present study was conducted in order to clarify the role of the glia in brain purine metabolism. This, in connection with the clarification of the etiology of the neurological manifestations associated with some of the inborn errors of purine metabolism in man. Purine nucleotide content, the capacity for de novo and salvage purine synthesis and the activity of several enzymes of purine nucleotide degradation, were assayed in primary cultures of rat astroglia in relation to culture age. The capacity of the intact cells to produce purine nucleotides de novo exhibited a marked decrease with the culture age, but the activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), catalyzing salvage nucleotide synthesis, increased. Aging was also associated with a marked increase in the activity of the degradation enzymes AMP deaminase, purine nucleoside phosphorylase (PNP) and guanine deaminase (guanase). The activity of adenosine deaminase and of AMP-5'-nucleotidase, increased markedly during the first 17 days in culture, but decreased thereafter. The results indicate that purine nucleotide metabolism in the cultured astroglia is changing with aging to allow the cells to maintain their nucleotide pool by reutilization of preformed hypoxanthine, rather than by de-novo production of new purines. Aging is also associated with increased capacity for operation of the adenine nucleotide cycle, contributing to the homeostasis of adenine nucleotides and to the energy charge of the cells. In principle, the age-related alterations in purine metabolism in the astroglia resemble those occurring in the maturating neurons, except for the capacity to produce purines de novo, which exhibited inverse trends in the two tissues. However, in comparison to the neurons, the cultured astroglia possess the capacity for a more intensive metabolism of purine nucleotides.

The activities (Vmax) of several enzymes of purine nucleotide metabolism were assayed in premature and mature primary rat neuronal cultures and in whole rat brains. In the neuronal cultures, representing 90% pure neurons, maturation (up to 14 days in culture) resulted in an increase in the activities of guanine deaminase (guanase), purine-nucleoside phosphorylase (PNP), IMP 5'-nucleotidase, adenine phosphoribosyltransferase (APRT), and AMP deaminase, but in no change in the activities of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenosine deaminase, adenosine kinase, and AMP 5'-nucleotidase. In whole brains in vivo, maturation (from 18 days of gestation to 14 days post partum) was associated with an increase in the activities of guanase, PNP, IMP 5'-nucleotidase, AMP deaminase, and HGPRT, a decrease in the activities of adenosine deaminase and IMP dehydrogenase, and no change in the activities of APRT, AMP 5'-nucleotidase, and adenosine kinase. The profound changes in purine metabolism, which occur with maturation of the neuronal cells in primary cultures in vitro and in whole brains in vivo, create an advantage for AMP degradation by deamination, rather than by dephosphorylation, and for guanine degradation to xanthine over its reutilization for synthesis of GMP. The physiological meaning of the maturational increase in these two ammonia-producing enzymes in the brain is not yet clear. The striking similarity in the alterations of enzyme activities in the two systems indicates that the primary culture system may serve as an appropriate model for the study of purine metabolism in brain.

The metabolic fate of guanine and of guanine ribonucleotides (GuRNs) in cultured rat neurons was studied using labeled guanine. 8-Aminoguanosine (8-AGuo), an inhibitor of purine nucleoside phosphorylase, was used to clarify the pathways of GMP degradation, and mycophenolic acid, an inhibitor of IMP dehydrogenase, was used to assess the flux from IMP to GMP and, indirectly, the activity of the guanine nucleotide cycle (GMP----IMP----XMP----GMP). The main metabolic fate of guanine in the neurons was deamination to xanthine, but significant incorporation of guanine into GuRNs, at a rate of approximately 8.5-13.1% of that of the deamination, was also demonstrated. The turnover rate of GuRNs was fast (loss of 80% of the radioactivity of the prelabeled pool in 22 h), reflecting synthesis of nucleic acids (32.8% of the loss in radioactivity) and degradation to xanthine, guanine, hypoxanthine, guanosine, and inosine (49.3, 4.3, 4.1, 1.1, and 0.5% of the loss, respectively). Of the radioactivity in GuRNs, 7.9% was shifted to adenine nucleotides. The accumulation of label in xanthine indicates (in the absence of xanthine oxidase) that the main degradative pathway from GMP is that to xanthine through guanosine and guanine. The use of 8-AGuo confirmed this pathway but indicated the operation of an additional, relatively slower degradative pathway, that from GMP through IMP to inosine and hypoxanthine. Hypoxanthine was incorporated mainly into adenine nucleotide (91.5%), but a significant proportion (6%) was found in GuRNs.(ABSTRACT TRUNCATED AT 250 WORDS)

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A B S T R A C T Human peripheral blood leukocytes were studied for the presence and the regulatory properties of the pathway of de novo synthesis of purine nucleotides. The cells were found to incorporate the labeled precursors formate and glycine into purines. The rate of [14C]-formate incorporation was decreased by several compounds known to inhibit purine synthesis by affecting the activity of glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase, the first committed enzyme in the pathway, either through decreasing the availability of PRPP, a substrate for this enzyme, or through exerting inhibition on this enzyme.PRPP availability in the leukocyte was found to be limiting for purine synthesis. Increased PRPP availability resulting from activation of PRPP synthetase by increasing inorganic phosphate (Pi) concentration caused acceleration of purine synthesis. On the other hand, no clear-cut evidence was obtained for the availability of ribose-5-phosphate in the leukocyte being rate limiting at physiological extracellular Pi concentration for PRPP generation, and thus for purine synthesis. However, the addition of methylene blue, which accelerates the oxidative pentose shunt that produces ribose-5-phosphate, resulted in acceleration of PRPP gen- eration and of purine synthesis only when PRPP synthetase was largely activated at high Pi concentration. These results may be taken to suggest that ribose-5-phosphate availability is indeed not limiting for PRPP generation under physiological conditions. Purine synthesis de novo was accelerated more than 13-fold in the leukocytes of two gouty patients affected with partial deficiency of hypoxanthine-guanine phosphoribosyltransferase, but was normal in the leukocytes of an obligate heterozygote for this enzyme abnormality.The results demonstrate in peripheral human leukocytes the presence of the complete pathway of de novo synthesis of purine nucleotides and the manifestation in these cells of the biochemical consequences of hypoxanthine-guanine phosphoribosyltransferase deficiency, i.e., increased availability of PRPP and acceleration of purine synthesis de novo. The results indicate the usefulness of leukocytes as a model tissue for the study of purine metabolism in man.

Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8.; HPRT) catalyzes the salvage synthesis of inosine-5'-monophosphate (IMP) and guanosine-5'-monophosphate (GMP) from the purine bases hypoxanthine and guanine, respectively. Complete deficiency of HPRT activity is associated with the Lesch-Nyhan syndrome (LNS), characterized by excessive purine production and severe neurological manifestations. The etiology of the metabolic consequences of HPRT deficiency is clarified, but that of the neurological manifestations is not yet understood. HPRT-deficient mice represent an experimental animal model of LNS. In search for a possible metabolic abnormality in LNS brains, connecting the neurological deficit to HPRT deficiency, the purine and pyrimidine nucleotide content of cultured neurons, prepared from HPRT-deficient transgenic mice, was now determined. The HPRT-deficient neuronal cultures exhibited a significantly elevated content of the pyrimidine nucleotides UTP (1.33-fold the normal level, p = 0.0002) and CTP (1.28-fold the normal level, p = 0.02), but normal content of the purine nucleotides ATP and GTP. This abnormality in neuronal pyrimidine nucleotide content may be associated with the pathophysiology of the neurological deficit in LNS.