Reliable method for high quality His-tagged and untagged E. coli phosphoribosyl phosphate synthase (Prs) purification.

Prs (phosphoribosyl pyrophosphate synthase) is a broadly conserved protein that synthesises 5-phosphoribosyl 1-pyrophospate (PRPP); a substrate for biosynthesis of at least 10 enzymatic pathways including biosynthesis of DNA building blocks - purines and pyrimidines. In Escherichia coli, it is a protein of homo-hexameric quaternary structure, which can be challenging to work with, due to frequent aggregation and activity loss. Several studies showed brief purification protocols for various bacterial PRPP synthases, in most cases involving ammonium sulfate precipitation. Here, we provide a protocol for expression of E. coli Prs protein in Rosetta (DE3) and BL21 (DE3) pLysE strains and a detailed method for His-Prs and untagged Prs purification on nickel affinity chromatography columns. This protocol allows purification of proteins with high yield, purity and activity. We report here N-terminally His-tagged protein fusions, stable and active, providing that the temperature around 20 °C is maintained at all stages, including centrifugation. Moreover, we successfully applied this method to purify two enzyme variants with K194A and G9S alterations. The K194A mutation in conserved lysine residue results in protein variant unable to synthetize PRPP, while the G9S alteration originates from prs-2 allele variant which was previously related to thermo-sensitive growth. His-PrsG9S protein purified here, exhibited comparable activity as previously observed in-vivo suggesting the proteins purified with our protocol resemble their physiological state. The protocol for Prs purification showed here indicates guidance to improve stability and quality of the protein and to ensure more reliable results in further assays in-vitro.

The NHR1-1 of Prs1 and the pentameric motif 284KKCPK288 of Prs3 permit multi-functionality of the PRPP synthetase in Saccharomyces cerevisiae.

The five-membered PRS gene family of Saccharomyces cerevisiae is an example of gene duplication allowing the acquisition of novel functions. Each of the five Prs polypeptides is theoretically capable of synthesising PRPP but at least one of the following heterodimers is required for survival: Prs1/Prs3, Prs2/Prs5 and Prs4/Prs5. Prs3 contains a pentameric motif 284KKCPK288 found only in nuclear proteins. Deletion of 284KKCPK288 destabilises the Prs1/Prs3 complex resulting in a cascade of events, including reduction in PRPP synthetase activity and altered cell wall integrity (CWI) as measured by caffeine sensitivity and Rlm1 expression. Prs3 also interacts with the kinetochore-associated protein, Nuf2. Following the possibility of 284KKCPK288-mediated transport of the Prs1/Prs3 complex to the nucleus, it may interact with Nuf2 and phosphorylated Slt2 permitting activation of Rlm1. This scenario explains the breakdown of CWI encountered in mutants lacking PRS3 or deleted for 284KKCPK288. However, removal of NHR1-1 from Prs1 does not disrupt the Prs1/Prs3 interaction as shown by increased PRPP synthetase activity. This is evidence for the separation of the two metabolic functions of the PRPP-synthesising machinery: provision of PRPP and maintenance of CWI and is an example of evolutionary development when multiple copies of a gene were present in the ancestral organism.

The function of the three phosphoribosyl pyrophosphate synthetase (Prs) genes in hyphal growth and conidiation in Aspergillus nidulans.

Phosphoribosyl pyrophosphate synthetase, which is encoded by the Prs gene, catalyses the reaction of ribose-5-phosphate and adenine ribonucleotide triphosphate (ATP) and has central importance in cellular metabolism. However, knowledge about how Prs family members function and contribute to total 5-phosphoribosyl-α-1-pyrophosphate (PRPP) synthetase activity is limited. In this study, we identified that the filamentous fungus Aspergillus nidulans genome contains three PRPP synthase-homologous genes (AnprsA, AnprsB and AnprsC), among which AnprsB and AnprsC but not AnprsA are auxotrophic genes. Transcriptional expression profiles revealed that the mRNA levels of AnprsA, AnprsB and AnprsC are dynamic during germination, hyphal growth and sporulation and that they all showed abundant expression during the vigorous hyphal growth time point. Inhibiting the expression of AnprsB or AnprsC in conditional strains produced more effects on the total PRPP synthetase activity than did inhibiting AnprsA, thus indicating that different AnPrs proteins are unequal in their contributions to Prs enzyme activity. In addition, the constitutive overexpression of AnprsA or AnprsC could significantly rescue the defective phenotype of the AnprsB-absent strain, suggesting that the function of AnprsB is not a specific consequence of this auxotrophic gene but instead comes from the contribution of Prs proteins to PRPP synthetase activity.

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance.

Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.

Akt phosphorylation and regulation of transketolase is a nodal point for amino acid control of purine synthesis.

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway integrates environmental clues to regulate cell growth and survival. We showed previously that depriving cells of a single essential amino acid rapidly and reversibly arrests purine synthesis. Here we demonstrate that amino acids via mammalian target of rapamycin 2 and IκB kinase regulate Akt activity and Akt association and phosphorylation of transketolase (TKT), a key enzyme of the nonoxidative pentose phosphate pathway (PPP). Akt phosphorylates TKT on Thr382, markedly enhancing enzyme activity and increasing carbon flow through the nonoxidative PPP, thereby increasing purine synthesis. Mice fed a lysine-deficient diet for 2 days show decreased Akt activity, TKT activity, and purine synthesis in multiple organs. These results provide a mechanism whereby Akt coordinates amino acid availability with glucose utilization, purine synthesis, and RNA and DNA synthesis.

In Saccharomyces cerevisiae, impaired PRPP synthesis is accompanied by valproate and Li+ sensitivity.

The biosynthetic intermediate PRPP (phosphoribosylpyrophosphate) has a central role in cellular biochemistry since it links carbon and nitrogen metabolism. Its importance may be reflected in the fact that, in the Saccharomyces cerevisiae (yeast) genome, there are five unlinked genes, PRS1-PRS5, each of which is theoretically capable of encoding the enzyme synthesizing PRPP. Interference with the complement of PRS genes in S. cerevisiae has far-reaching consequences for yeast physiology and has uncovered unexpected metabolic links including cell wall integrity and phospholipid metabolism.

Ramifications of impaired PRPP synthesis in Saccharomyces cerevisiae.

The model eukaryote Saccharomyces cerevisiae is well suited to investigate the causes of metabolic disturbance. PRPP [5-phospho-D-ribosyl-1(alpha)-pyrophosphate] may be regarded as a junction of carbon and nitrogen metabolism. As a result of this central position, perturbations in its synthesis can give rise to many unexpected cellular events, such as impaired cell integrity. We have taken advantage of S. cerevisiae's genetic tractability to investigate the metabolic links responsible for connecting the biochemical intermediate PRPP to apparently unrelated cellular functions. This approach provides insight into the co-ordination of different biological processes.

Impaired PRPP-synthesizing capacity compromises cell integrity signalling in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, PRS genes comprise a family of five paralogous genes. Previously, it has been shown that in the cell the gene products are organized into two interacting complexes, one of which is a heterodimer and the other a heterotrimer. Here, it has been demonstrated that in addition to supplying the cell with the key metabolic intermediate PRPP [5-phospho-D-ribosyl-1(alpha)-pyrophosphate], the gene products contribute to the maintenance of cell integrity. Specifically, the phosphorylation of Rlm1, one of the end points of the cell integrity signalling pathway, is significantly impaired following deletion of any one of the PRS genes, in particular PRS1 and PRS3. This is reflected in changes in the expression of the alternative 1,3-beta-glucan synthase catalytic subunit, Fks2, as measured by its promoter activity. Yeast two-hybrid analysis has shown that Prs1, specifically the non-homologous region, NHR1-1 and Prs3, and to a lesser extent Prs2 and Prs4, interact with the MAPK (mitogen-activated protein kinase) of the cell integrity pathway, Slt2. When PRS1 is lacking, the basal level of phosphorylation of Slt2 is increased. Furthermore, prs1Delta and prs3Delta strains have an increased chitin content under normal growth conditions. alpha-Factor sensitivity and Calcofluor White resistance associated with the lack of Prs1 and Prs3 corroborate the involvement of these two gene products in cell integrity signalling. It is postulated that Prs polypeptides play a significant role in the remodelling of the cell wall and may have a direct involvement in cell integrity signalling.

Escherichia coli phnN, encoding ribose 1,5-bisphosphokinase activity (phosphoribosyl diphosphate forming): dual role in phosphonate degradation and NAD biosynthesis pathways.

An enzymatic pathway for synthesis of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) without the participation of PRPP synthase was analyzed in Escherichia coli. This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene, the gene encoding PRPP synthase (B. Hove-Jensen, J. Bacteriol. 178:714-722, 1996). The new pathway requires three enzymes: phosphopentomutase, ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase. The latter activity is encoded by phnN; the product of this gene is required for phosphonate degradation, but its enzymatic activity has not been determined previously. The reaction sequence is ribose 5-phosphate --> ribose 1-phosphate --> ribose 1,5-bisphosphate --> PRPP. Alternatively, the synthesis of ribose 1-phosphate in the first step, catalyzed by phosphopentomutase, can proceed via phosphorolysis of a nucleoside, as follows: guanosine + P(i) --> guanine + ribose 1-phosphate. The ribose 1,5-bisphosphokinase-catalyzed phosphorylation of ribose 1,5-bisphosphate is a novel reaction and represents the first assignment of a specific chemical reaction to a polypeptide required for cleavage of a carbon-phosphorus (C-P) bond by a C-P lyase. The phnN gene was manipulated in vitro to encode a variant of ribose 1,5-bisphosphokinase with a tail consisting of six histidine residues at the carboxy-terminal end. PhnN was purified almost to homogeneity and characterized. The enzyme accepted ATP but not GTP as a phosphoryl donor, and it used ribose 1,5-bisphosphate but not ribose, ribose 1-phosphate, or ribose 5-phosphate as a phosphoryl acceptor. The identity of the reaction product as PRPP was confirmed by coupling the ribose 1,5-bisphosphokinase activity to the activity of xanthine phosphoribosyltransferase in the presence of xanthine, which resulted in the formation of 5'-XMP, and by cochromatography of the reaction product with authentic PRPP.

Phosphoribosylpyrophosphate synthetase and the regulation of phosphoribosylpyrophosphate production in human cells.

between purine nucleoside diphosphate inhibition and inorganic phosphate (Pi) activation; and intracellular concentration of the PRS1 isoform. The operation of additional determinants of rates of PRPP synthesis in human cells is suggested by: (1) multiple PRS isoforms with distinctive physical and kinetic properties; (2) nearly immediate activation of intracellular PRPP synthesis in response to mitogens, growth-promoters, and increased intracellular Mg2+ concentrations; (3) tissue-specific differences in PRS1 and PRS2 transcript and isoform expression; and (4) reversible association of PRS subunits with one another and/or with PRS-associated proteins (PAPs), as a result of which the catalytic and perhaps regulatory properties of PRS isoforms are modified.

Genetic analysis and enzyme activity suggest the existence of more than one minimal functional unit capable of synthesizing phosphoribosyl pyrophosphate in Saccharomyces cerevisiae.

The PRS gene family in Saccharomyces cerevisiae consists of five genes each capable of encoding a 5-phosphoribosyl-1(alpha)-pyrophosphate synthetase polypeptide. To gain insight into the functional organization of this gene family we have constructed a collection of strains containing all possible combinations of disruptions in the five PRS genes. Phenotypically these deletant strains can be classified into three groups: (i) a lethal phenotype that corresponds to strains containing a double disruption in PRS2 and PRS4 in combination with a disruption in either PRS1 or PRS3; simultaneous deletion of PRS1 and PRS5 or PRS3 and PRS5 are also lethal combinations; (ii) a second phenotype that is encountered in strains containing disruptions in PRS1 and PRS3 together or in combination with any of the other PRS genes manifests itself as a reduction in growth rate, enzyme activity, and nucleotide content; (iii) a third phenotype that corresponds to strains that, although affected in their phosphoribosyl pyrophosphate-synthesizing ability, are unimpaired for growth and have nucleotide profiles virtually the same as the wild type. Deletions of PRS2, PRS4, and PRS5 or combinations thereof cause this phenotype. These results suggest that the polypeptides encoded by the members of the PRS gene family may be organized into two functional entities. Evidence that these polypeptides interact with each other in vivo was obtained using the yeast two-hybrid system. Specifically PRS1 and PRS3 polypeptides interact strongly with each other, and there are significant interactions between the PRS5 polypeptide and either the PRS2 or PRS4 polypeptides. These data suggest that yeast phosphoribosyl pyrophosphate synthetase exists in vivo as multimeric complex(es).

Fructose 3-phosphate and 5-phosphoribosyl-1-pyrophosphate formation in perfused human erythrocytes: 31P NMR studies.

31P NMR was used to study the formation of fructose 3-phosphate (F3P) and 5-phosphoribosyl-1-pyrophosphate (PRPP) in perfused human erythrocytes, in the presence of 10 different combinations and concentrations of glucose, inosine, pyruvate, fructose, and inorganic phosphate (Pi). (1) The cells were immobilized in alginate-coated agarose threads and perfused with a medium containing fructose, and the level of F3P increased continuously over more than 10 h. The net rate of F3P formation was independent of the concentration of 2,3-bisphosphoglycerate (2,3-DPG) present in the cells. (2) PRPP was formed in high concentrations, relative to normal, in immobilized cells when they were perfused with a medium containing Pi at a low pH (6.6). (3) The 2,3-DPG level decreased simultaneously when the sample was perfused with a medium containing fructose, but without inosine or pyruvate. The measured intracellular pH and free Mg2+ concentration were constant in these experiments. (4) The experiments confirmed the presence of fructose-3-phosphokinase (E.C. 2.7.1.-) and ribose-phosphate pyrophosphokinase (E.C. 2.7.6.1) activity in the human erythrocytes and that the biosynthetic pathways are active in immobilized cells at 37 degrees C. (5) The rates of accumulation of 2,3-DPG and phosphomonoesters (PME) appeared to be strongly correlated.

The importance of methylthio-IMP for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity in Molt F4 human malignant T-lymphoblasts.

The importance of methyl-thioIMP (Me-tIMP) formation for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity was studied in Molt F4 cells. Cytotoxicity of Me-MPR is caused by Me-tIMP formation with concomitant inhibition of purine de novo synthesis. Inhibition of purine de novo synthesis resulted in decreased purine nucleotide levels and enhanced 5-phosphoribosyl-1-pyrophosphate (PRPP) levels, with concurrent increased pyrimidine nucleotide levels. The Me-tIMP concentration increased proportionally with the concentration of Me-MPR. High Me-tIMP concentration also caused inhibition of PRPP synthesis. Maximal accumulation of PRPP thus occurred at low Me-MPR concentrations. As little as 0.2 microM Me-MPR resulted already after 2 h in maximal inhibition of formation of adenine and guanine nucleotides, caused by inhibition of purine de novo synthesis by Me-tIMP. Under these circumstances increased intracellular PRPP concentrations could be demonstrated, resulting in increased levels of pyrimidine nucleotides. So, in Molt F4 cells, formation of Me-tIMP from Me-MPR results in cytotoxicity by inhibition of purine de novo synthesis.

Fibroblast growth factor-dependent metabolism of hypoxanthine via the salvage pathway for purine synthesis in porcine aortic endothelial cells.

In this study we examined the metabolism of hypoxanthine in fibroblast growth factor (FGF)-stimulated porcine aortic endothelial cells (PAEC). Our previous report indicated that hypoxanthine in fetal bovine serum (FBS) was an essential component for both basal and FGF-dependent growth of PAEC (Hayashi et al., Exp Cell Res 185: 217-228, 1989). Besides hypoxanthine, the addition of various purine bases and purine nucleosides, but not xanthine, xanthosine or any pyrimidine metabolites, restored the limited growth of PAEC cultured in medium containing 10% dialyzed FBS in the presence or absence of FGF. The metabolism of [14C]hypoxanthine was compared in PAEC treated with and without FGF. Treatment of PAEC with FGF for 24 hr enhanced the radioactivity incorporation from [14C]hypoxanthine into both the acid-soluble and -insoluble fractions approximately 2-fold. Upon chromatographic analyses of hypoxanthine metabolites in the acid-soluble nucleotide fraction, it was found that in control PAEC hypoxanthine was largely metabolized to IMP, adenine nucleotides and uric acid, whereas in FGF-treated cells it was converted to ATP, ADP, GTP, xanthine and uric acid. The radioactivity of IMP was lowered in FGF-stimulated cells. The addition of FGF to PAEC increased phosphoribosyl pyrophosphate (PRPP) synthetase activity by approximately 8-fold and the PRPP content by approximately 2-fold, but it did not increase hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity or hypoxanthine transport. On the other hand, methotrexate, an inhibitor of de novo synthesis of purine, did not affect the growth of PAEC. Analyses of the rate of [14C]formate incorporation into total purine compounds showed that PAEC had a low capacity to synthesize purines de novo, which was not stimulated by FGF. These data indicate that FGF stimulates the synthesis of PRPP necessary for the salvage synthesis of purine nucleotides in conjunction with purine bases, e.g. hypoxanthine.

Acceleration of purine synthesis in mouse liver by glycogenolytic hormones.

Administration (ip) into fed mice of glucagon, epinephrine, vasopressin, oxytocin, angiotensin II, and dibutyryl cyclic AMP (dbcAMP) resulted in a rapid (within 2.5 to 15 min) elevation of PRPP content (two- to threefold) and in acceleration of the rate of de novo purine synthesis (twofold). Inhibition of the epinephrine-stimulated glycogenolysis by 2,5-anhydromannitol diminished markedly the acceleration effect of the hormone on the rate of purine synthesis. Administration of the hormones caused a rapid rise in the liver content of glucose 6-phosphate (G6P) by 15-70% but did not increase the ribose 5-phosphate (R5P) content. Liver ATP content was not affected. The hormones did not cause direct activation of PRPP synthetase, as gauged by the specific activity of the enzyme, its Km for substrates R5P and ATP, and its sensitivity to inhibition by ADP and GDP. The hormones did not increase the liver content of the enzyme activators Pi and Mg2+. The results suggest that the glycogenolytic hormones accelerate purine synthesis by a metabolic mechanism associated with the enhancement of glycogenolysis. PRPP synthesis is probably enhanced by the glycogenolysis-induced alterations in the cellular content of some metabolites other than R5P.

Stimulation of ribose-5-phosphate and 5-phosphoribosyl-1-pyrophosphate generation by pyrroline-5-carboxylate in mouse liver in vivo: evidence for a regulatory role of ribose-5-phosphate availability in nucleotide synthesis.

Pyrroline-5-carboxylase (P5C), a physiological stimulator of hexose-monophosphate-pentose pathway activity, was found before to increase 5-phosphoribosyl-1-pyrophosphate (PRPP) generation and nucleotide synthesis in human erythrocytes and cultured fibroblasts. We now report the stimulation of PRPP generation by P5C also in mouse liver in vivo. In addition we demonstrated a simultaneous elevation in ribose-5-phosphate (R5P) concentration, which was relatively smaller and transient. The demonstrated effect of P5C on liver R5P and PRPP content in vivo provides strong evidence for the physiological role of R5P availability in the regulation of PRPP and purine production.

Inhibition of phosphoribosylpyrophosphate synthetase by 4-methoxy-(MRPP) and 4-amino-8-(D-ribofuranosylamino) pyrimido[5,4-d]pyrimidine (ARPP).

The basis for the antitumor activities of the exocyclic amino nucleosides 4-amino-(ARPP) and 4-methoxy-8-(D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (MRPP) was investigated. The primary target of these nucleosides appeared to be 5-phospho-alpha-D-ribofuranose-1-pyrophosphate (PRPP) synthetase. MRPP-5'-monophosphate was a competitive inhibitor (Ki = 40 microM) of the activation of this enzyme by the cofactor inorganic phosphate (K alpha = 2.2 mM). Consequently, ARPP and MRPP treatment of WI-L2 cultures rapidly inhibited both de novo pyrimidine and purine synthesis as well as the nucleotide salvage reactions dependent on PRPP, ARPP or MRPP treatment completely prevented [14C]bicarbonate incorporation into acid-soluble pyrimidine and purine nucleotides. The rate of salvage of [8-14C]hypoxanthine to form IMP was decreased by 85%. Treatment of cells with these agents caused a 50% reduction in the steady-state level of PRPP. When the capacity of the treated cells for sustained synthesis of PRPP was examined by adenine incorporation, the rate of adenine uptake was inhibited by greater than 50%. In vivo treatment of BDF1 mice with a single dose of ARPP (173 mg/kg) or MRPP (62 mg/kg) extended the mean life span of the mice, which had been inoculated intraperitoneally 1 day earlier with 1 x 10(6) L1210 murine leukemia cells, by 62 and 82% respectively. These studies indicate that MRPP and ARPP inhibit PRPP synthetase, and that PRPP synthetase may be a viable target in the development of certain antitumor agents.

Changes in pathways of pentose phosphate formation in relation to phosphoribosyl pyrophosphate synthesis in the developing rat kidney. Effects of glucose concentration and electron acceptors.

Phosphoribosyl pyrophosphate (PPRibP), required in nucleotide synthesis, increases 2-fold in rat kidney from 1 day post partum to adult stage; there is no accompanying increase in PPRibP synthetase activity measured in vitro. Ribose 5-phosphate is a key factor in the regulation of PPRibP synthesis. The activity and regulation of 3 routes of ribose 5-phosphate formation have been measured in renal growth: (i) the flux through the oxidative pentose phosphate pathway was high in the neonatal period but increased only +50% thereafter; (ii) the non-oxidative pentose phosphate pathway, including transketolase, increased by +145%; (iii) the rate-limiting enzymes of the glucuronate-xylulose route increased +200% from 1 day to the adult stage. The importance of systems reoxidizing NADPH was shown by: (i) the stimulation of renal PPRibP formation from glucose by phenazine methosulphate; (ii) the early involvement of the oxidative pentose phosphate pathway at the stage where NADPH is used for biosynthetic routes; (iii) the increasing involvement of the glucuronate-xylulose route, which acts as a transhydrogenase producing NADP+ in addition to pentose phosphate formation and (iv) the correlation between renal PPRibP content and the activity of aldose reductase, which, by utilization of NADPH, stimulates ribose 5-phosphate formation via the oxidative pentose phosphate pathway. Evidence is adduced that the contribution of the 3 routes of ribose 5-phosphate formation in the kidney varies at different stages of development.

Functional and metabolic effects of ribose in combination with prazosin, verapamil and metoprolol in rats in vivo.

Ribose improves the function of the rat heart in various pathological conditions through its effects on cardiac energy metabolism, while having no direct haemodynamic actions. We therefore studied its functional and metabolic effects in closed chest rats when given in combination with prazosin, verapamil or metoprolol, all of which have direct effects on the circulation. Ribose administration for 24 h at 200 mg.kg-1.h-1 did not affect heart function but increased the available pool of 5-phosphoribosyl-1-pyrophosphate in heart (four fold) and skeletal muscle (1.7-fold), as assessed by the incorporation of 14C-adenine into the adenine nucleotides. The utilisation of adenine for adenine nucleotide synthesis, expressed as the ratio of adenine nucleotide radioactivity to tissue extract radioactivity, was 70% in heart and 20% in skeletal muscle under control conditions, and 97% and 88% after 24 h of ribose administration. Ribose decreased the 14C-adenine incorporation into the adenine nucleotides in kidney, lungs and liver. After 24 h infusion of prazosin (100 micrograms.kg-1.h-1), heart rate and LVdP/dtmax were not changed, but LVSP (-20%), mean aortic pressure (-16%) and peripheral resistance (-40%) were decreased. Cardiac output was enhanced (+40%). Verapamil (2mg.kg-1.h-1) and metoprolol (2mg.kg-1.h-1) infused for 24 h decreased the pressure-rate and pressure-volume product of the left ventricle to the same extent (-40%). Verapamil had no influence on cardiac output, while metoprolol depressed it (-30%). Simultaneous administration of prazosin, verapamil or metoprolol with ribose did not affect the ribose induced increase in the myocardial 5-phosphoribosyl-1-pyrophosphate pool.(ABSTRACT TRUNCATED AT 250 WORDS)