Inosine pools

Adenosine is not active orally, but adrninistered as an iv bolus dmg adenosine rapidly eliminates supraventricular tachycardias within 1—2 min after dosing. The dmg slows conduction through the AV node. Adenosine is rapidly removed from the circulation by uptake into red blood ceUs and vascular endothehal ceUs. Thus the plasma half-life is less than 10 s. Adenosine is rapidly metabolized to inosine or adenosine monophosphate and becomes part of the body pool for synthesis of adenosine-triphosphate. 

Until 2001, it was thought that the mechanism of action of ribavirin involved a decrease in cellular guanosine triphosphate (GTP) pools resulting from inhibition of inosine monophosphate dehydrogenase by ribavirin monophosphate. More recently, the mechanism of action for ribavirin has been expanded to include lethal mutagenesis of the viral genome as a result of ribavirin triphosphate utilization by the error-prone viral RNA-dependent RNA polymerase, and incorporation of ribavirin into viral RNA. 

MECHANISMS OE ACTION AND RESISTANCE Ribavirin alters cellular nucleotide pools and inhibits viral mRNA synthesis. Intracellular phosphorylation to the mono-, di-, and triphosphate derivatives is mediated by host cell enzymes. In both uninfected and RSV-infected cells, the predominant derivative is the triphosphate, which has an intracellular of <2 hours. Ribavirin monophosphate competitively inhibits cellular inosine-5 -phosphate dehydrogenase and interferes with the synthesis of GTP and thus nucleic acid synthesis. Ribavirin triphosphate also competitively inhibits the GTP-dependent 5 capping of viral messenger RNA and specifically influenza virus transcriptase activity. Ribavirin has multiple sites of action, and some of these e.g., inhibition of GTP synthesis) may potentiate others e.g., inhibition of GTP-dependent enzymes). Ribavirin also may enhance viral mutagenesis such that some viruses may be inhibited in effective replication, so-caUed lethal mutagenesis. 

The accumulation of fructose 1-phosphate also substantially depletes the phosphate pools. The fructokinase reaction uses ATP at a rapid rate such that the mitochondria regenerate ATP rapidly, which leads to a drop in free phosphate levels. The low levels of phosphate release inhibition of AMP deaminase, which converts AMP to inosine monophosphate (IMP). The nitrogenous base of IMP (hypoxanthine) is degraded to uric acid. The lack of phosphate and depletion of adenine nucleotides lead to a loss of ATP, further contributing to the inhibition of biosynthetic pathways, including gluconeogenesis. 

At very low values of EC, when AMP is elevated it is deaminated via AMP deaminase to inosine monophosphate (IMP). This further displaces the adenylate kinase reaction in the direction of ATP synthesis. The IMP is dephosphorylated by nucleotide phosphatase, and the inosine is phosphorylyzed via purine nucleotide phosphorylase, releasing hypoxanthine and ribose 1-phosphate. The latter is metabolized via the pentose phosphate pathway, and most of the carbon atoms enter glycolysis. Because this course of events depletes the overall adenine nucleotide pool, and hence the scope for ATP production in the longer term, it represents a metabolic last ditch stand by the cell to extract energy even from the energy currency itself ... 

Much of the inosinate that is not synthesized de novo is formed via adenosine, as outlined above. Some inosinate is produced via adenylate deaminase. The significance of this enzyme in priming the Krebs cycle has been emphasized by Setlow and Lowenstein (S6). The IMP formed can be reconverted to either AMP or GMP. The deaminase is under control of ATP and GTP (B20). Thus this route may be of importance in maintaining the A G ratio in the cellular nucleotide pool. 

The radioactivity lost from the adenine nucleotide pool is recovered in various purine derivatives. There is a transient increase in radioactive inosine and hypoxanthine, and a nearly constant accumulation of radioactive allantoin (Fig. IB). There is also a transient accumulation of radioactive adenosine and IMP (data not shown). However, the extent of that accumulation depends upon the moment that the cells are exposed to glycerol. When glycerol is added at the beginning of the incubation, about 8% of the total radioactivity is found both in adenosine and in IMP. However, when glycerol is added after a 15 min incubation, 17% of the total radioactivity is found in IMP and only 3% in adenosine. All these changes are similar to those induced by fructose under identical conditions (data not shown). 

These data demonstrate a marked abnormality in the ability of intact uremic RBC to metabolize adenosine (deoxyadenosine) to inosine (deoxyinosine) and hypoxanthine. The equivalent labelling by radioactive adenosine and deoxyadenosine of ATP pools suggests that the adenylate kinase pathway in uremic RBC is normal despite markedly elevated ATP levels. The latter may reflect decreased utilization of ATP in uremic RBC or an effect of high inorganic phosphate on ATP turnover (8). 

Fig. 29.2 Caffeine is produced from xanthosine derived from four routes (1) inosine-5 -monophosphate (IMP) originating from de novo purine synthesis (de novo route), (2) adenosine released from the 5-adenosyhnethionine (SAM) cycle, (3) the cellular adenosine nucleotide pool (AMP route), and (4) the guanine nucleotide pool (GMP route). Enzymes AMPDA AMP deaminase, APRT adenine phosphorihosyltransferase, AK adenosine kinase, ARN adenosine nucleosidase, GRD guanine deaminase, IMPDH IMP dehydrogenase, 5 NT 5 -nucleotidase...

Fig. 8. The efiect of P5C on inosine monophosphate formation. Erythrocytes were incubated with 25 /iM [ C]hypoxanthine for 1 hour and nucleotides were separated using high-pressure liquid chromatography. The concentration of P5C was 0.5 mM. IMP pools in P5C-treated ( ) and control (O) cells as well as the incorporation of [ C]hypoxanthine into IMP in P5C-treated (A) and control (A) cells are shown. Taken from ref. (118).

The distribution and specific radioactivities of the purine compounds found in perchloric acid extracts prepared from the labeled livers, after the 20 minute carrier perfusion, were then determined. The data of Table 1 confirm the rapid uptake of labeled purine and conversion to nucleotide, presumably via the phosphoribosyltransferases of the cell. The specific activity data also support the initial labeling of the nucleotides and the values are consistent with the relatively large pool of nucleotides within the cell. In each experiment, significant labeling of adenosine, inosine... 

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