Heart mitochondrial TTP synthesis and the compartmentalization of TMP

Abstract

The primary pathway of TTP synthesis in the heart requires thymidine salvage by mitochondrial thymidine kinase 2 (TK2). However, the compartmentalization of this pathway and the transport of thymidine nucleotides are not well understood. We investigated the metabolism of [(3)H]thymidine or [(3)H]TMP as precursors of [(3)H]TTP in isolated intact or broken mitochondria from the rat heart. The results demonstrated that [(3)H]thymidine was readily metabolized by the mitochondrial salvage enzymes to TTP in intact mitochondria. The equivalent addition of [(3)H]TMP produced far less [(3)H]TTP than the amount observed with [(3)H]thymidine as the precursor. Using zidovudine to inhibit TK2, the synthesis of [(3)H]TTP from [(3)H]TMP was effectively blocked, demonstrating that synthesis of [(3)H]TTP from [(3)H]TMP arose solely from the dephosphorysynthase pathway that includes deoxyuridine triphosphatelation of [(3)H]TMP to [(3)H]thymidine. To determine the role of the membrane in TMP metabolism, mitochondrial membranes were disrupted by freezing and thawing. In broken mitochondria, [(3)H]thymidine was readily converted to [(3)H]TMP, but further phosphorylation was prevented even though the energy charge was well maintained by addition of oligomycin A, phosphocreatine, and creatine phosphokinase. The failure to synthesize TTP in broken mitochondria was not related to a loss of membrane potential or inhibition of the electron transport chain, as confirmed by addition of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone and potassium cyanide, respectively, in intact mitochondria. In summary, these data, taken together, suggest that the thymidine salvage pathway is compartmentalized so that TMP kinase prefers TMP synthesized by TK2 over medium TMP and that this is disrupted in broken mitochondria.

Keywords: Mitochondria; Mitochondrial Metabolism; Nucleoside/Nucleotide Biosynthesis; Nucleoside/Nucleotide Metabolism; Thymidine Kinase 2.

FIGURE 1.

Model of thymidine metabolism in the cytosol and mitochondrial matrix. Blue pathways represent potential contributions to TMP synthesis from non-thymidine precursors. Black pathways represent potential contributions to TTP synthesis from thymidine. The green pathway represents thymidine breakdown. The thymidylate synthase pathway is generally known to be cytosolic. However, recent evidence has suggested its presence in liver mitochondria. Previous work from our laboratory has been unable to detect either a cytosolic or mitochondrial thymidylate synthase pathway in the perfused heart or in isolated heart mitochondria. TP, thymidine phosphorylase; dUTPase, deoxyuridine pyrophosphorylase; CDA, cytidine deaminase; cdN, cytosolic deoxynucleotidase; mdN, mitochondrial deoxynucleotidase; DHFR, dihydrofolate reductase; TYMS, thymidylate synthase; dCK, deoxycytidine kinase; TMPK, cytosolic thymidine monophosphate kinase; ENT1/2, equilibrative nucleoside transporter 1/2; methylene-THF, methylene-tetrahydrofolate; DHF, dihydrofolate.

FIGURE 2.

The metabolism of [³H]thymidine and [³H]TMP in isolated intact rat heart mitochondria. Freshly isolated mitochondria (4 mg of protein/ml) were incubated at various time points in incubation medium at 30 °C containing 100 nm of either [³H]thymidine or [³H]TMP (∼2200 dpm/pmol) and processed as described under “Experimental Procedures.” The ³H products formed were detected and quantified using UPLC and an inline liquid scintillation counter. The amount of ³H products formed were expressed as picomole of product per milligram of mitochondrial protein and plotted against time. All data points represent the mean and S.E. of three independent determinations from three individual rat tissue mitochondrial isolates.

FIGURE 3.

The effect of unlabeled TMP on [³H]thymidine metabolism in isolated intact rat heart mitochondria. Mitochondria were incubated and processed and data were plotted as described under “Experimental Procedures” and in the legend for Fig. 2, except that 10 μm of unlabeled TMP was added to the incubations. Dashed lines represent results from Fig. 2 obtained in the absence of 10 μm unlabeled TMP for comparison. All data points represent the mean and S.E. of three independent determinations from three individual rat tissue mitochondrial isolates.

FIGURE 4.

Energy charge in the mitochondrial incubation systems. The distribution (percent) of AMP/ADP/ATP in the intact and broken mitochondrial systems after 2 h of incubation is shown. A, intact mitochondria (no additions). B, broken mitochondria (no additions). C, intact mitochondria + oligomycin A (50 nm), phosphocreatine (PC, 12 mm), and creatine phosphokinase (CPK, 50 units/ml). D, broken mitochondria + oligomycin A (50 nm), phosphocreatine (12 mm), and creatine phosphokinase (50 units/ml). E, intact mitochondria as in C + FCCP (3 μm). F, intact mitochondria as in C + KCN (6 mm). All data points represent the mean and S.E. of three independent determinations from three individual rat tissue mitochondrial isolates.

FIGURE 5.

Metabolism of [³H]thymidine and [³H]TMP in broken rat heart mitochondria. Broken mitochondria were prepared, incubated, processed, and plotted as described under “Experimental Procedures” and in the legend for Fig. 2. The energy charge of the experiments was maintained as described in Fig. 4D. All data points represent the mean and S.E. of three independent determinations from three individual rat tissue mitochondrial isolates.

FIGURE 6.

The effect of AZT on [³H]TMP metabolism in intact rat heart mitochondria. A, mitochondria were prepared, incubated, processed, and plotted as described under “Experimental Procedures” and in the legend for Fig. 2 except for the presence and absence of 200 μm AZT. Because AZT inhibits the conversion of [³H]thymidine to [³H]TMP, [³H]thymidine arising from the dephosphorylation of [³H]TMP is trapped as [³H]thymidine. B, the data in A was used to calculate the difference in [³H]thymidine and [³H]TMP levels observed in the presence and absence of 200 μm AZT (see text for details). All data points represent the mean and S.E. of three independent determinations from three individual rat tissue mitochondrial isolates.

FIGURE 7.

The effect of KCN and FCCP on [³H]thymidine metabolism in intact rat heart mitochondria. Mitochondria were prepared, incubated, processed, and plotted as described in the legend for Fig. 4D except that KCN and FCCP were added as indicated. Shown is the [³H]thymidine metabolism in the presence of KCN (6 mm) and FCCP (3 μm), respectively after 2 h of incubation. The energy charge of these incubations are shown in Fig. 4, C, E, and F. *, p < 0.005; **, p < 0.001. All data points represent the mean and S.E. of three independent determinations from three individual rat tissue mitochondrial isolates.

FIGURE 8.

Proposed model of thymidine and TMP salvage in rat heart mitochondria. Shown is the proposed model to account for TMP compartmentalization. cdN, cytosolic deoxynucleotidase; mdN, mitochondrial deoxynucleotidase.