Ribose-enhanced synthesis of UTP, CTP, and GTP from parent nucleosides in cardiac myocytes.

The participation of ribose and its metabolites in some nucleoside salvage reactions is well established. Isolated adult rat cardiac myocytes were used as a model system to determine whether ribose acts as a general stimulant of salvage reactions in cardiac muscle, or whether only certain classes of nucleosides are affected by ribose. Myocytes were incubated with [3H]-adenosine, [3H]-cytidine, [3H]-guanosine, [3H]-thymidine, or [3H]-uridine for 30 or 60 min in the presence or absence of 5 mM ribose. The cells were extracted and the extracts assayed for [3H]-nucleoside and [3H]-nucleotide products. Salvage synthesis of cytosine, guanine and uracil nucleotides from the parent nucleosides was stimulated by ribose. Guanosine and uridine salvage appeared saturated at 50 microM external nucleoside (the dose response of cytidine salvage was not examined). Adenosine salvage was unaffected by ribose addition; the response to increasing external adenosine concentration was non-Michaelis-Menten, showing a peak of activity at 25 microM external nucleoside. Thymidine salvage was also unaffected by ribose, and was saturated at 50 microM external thymidine. These data suggest that adenosine and thymidine are metabolized to their respective nucleotide monophosphates by kinase activity. Cytidine, guanosine, and uridine salvage are stimulated by ribose, and must therefore be metabolized in part by nucleoside phosphorylase and phosphoribosyltransferase activity.

Effect of anoxia on cyclic nucleotides and inositol phosphate turnover in cardiac myocytes.

The loss of 5'-nucleotides (especially ATP and GTP) from cardiac muscle cells is a distinguishing feature of myocardial ischemia. Isolated adult rat cardiac myocytes were used as a model system to determine whether GTP depletion could affect (1) the ability of the myocytes to synthesize cyclic GMP (cGMP), or (2) the ability of the myocytes to respond to alpha-adrenergic challenge. Myocytes were made anoxic for 30- or 60-min periods, then challenged with either 1 mM sodium nitroprusside (NaNP) for 1 min or 40 microM norepinephrine (NE) for 20 min. The cells were extracted and the extracts assayed for cyclic GMP (NaNP challenge) or phosphoinositides (NE challenge). When challenged with NaNP, anoxic myocytes made up to five-fold more cGMP than aerobic controls (1401 +/- 353 fmol cGMP/mg cell protein in anoxic cells v 121 +/- 23 fmol/mg in aerobic controls). Phosphoinositide turnover was reduced in anoxic cells v aerobic controls. Stimulation of this pathway by NE was reduced two-fold after 30 min of anoxia, and abolished after 60 min of anoxia. Similar results were obtained with 30 microM and 60 microM phenylephrine. The authors concluded that nucleotide depletion under anoxic conditions has no effect on the production of cyclic GMP, but may interfere with the linkage of alpha-adrenergic receptors to phosphatidylinositol breakdown.

Guanosine metabolism in adult rat cardiac myocytes: inhibition by acyclovir and analysis of a metabolic pathway.

The metabolic fate of transported guanosine was examined in adult rat cardiac myocytes. Freshly isolated cells were incubated with 50 microM 8-[3H]-guanosine and the purine nucleoside phosphorylase (PNP) inhibitor acyclovir, and the nucleotide products extracted and examined for radiolabel distribution. Acyclovir inhibited guanosine incorporation into the 5'-nucleotide pool up to 66%. The drug did not inhibit guanosine transport. Other experiments using 5'-[3H]-guanosine and 8-[14C]-guanosine in concert as metabolic tracers showed both tritium and radiocarbon in the guanine nucleotide products. We concluded from this study that both a kinase (probably adenosine kinase) and the enzyme pair purine nucleoside phosphorylase/hypoxanthine-guanine phosphoribosyltransferase are responsible for guanosine salvage in heart cells.