Biosynthesis of agmatine in isolated mitochondria and perfused rat liver: studies with 15N-labelled arginine

Abstract

An important but unresolved question is whether mammalian mitochondria metabolize arginine to agmatine by the ADC (arginine decarboxylase) reaction. 15N-labelled arginine was used as a precursor to address this question and to determine the flux through the ADC reaction in isolated mitochondria obtained from rat liver. In addition, liver perfusion system was used to examine a possible action of insulin, glucagon or cAMP on a flux through the ADC reaction. In mitochondria and liver perfusion, 15N-labelled agmatine was generated from external 15N-labelled arginine. The production of 15N-labelled agmatine was time- and dose-dependent. The time-course of [U-15N4]agmatine formation from 2 mM [U-15N4]arginine was best fitted to a one-phase exponential curve with a production rate of approx. 29 pmol x min(-1) x (mg of protein)(-1). Experiments with an increasing concentration (0- 40 mM) of [guanidino-15N2]arginine showed a Michaelis constant Km for arginine of 46 mM and a Vmax of 3.7 nmol x min(-1) x (mg of protein)(-1) for flux through the ADC reaction. Experiments with broken mitochondria showed little changes in Vmax or Km values, suggesting that mitochondrial arginine uptake had little effect on the observed Vmax or Km values. Experiments with liver perfusion demonstrated that over 95% of the effluent agmatine was derived from perfusate [guanidino-15N2]arginine regardless of the experimental condition. However, the output of 15N-labelled agmatine (nmol x min(-1) x g(-1)) increased by approx. 2-fold (P<0.05) in perfusions with cAMP. The findings of the present study provide compelling evidence that mitochondrial ADC is present in the rat liver, and suggest that cAMP may stimulate flux through this pathway.

Figure 1. A representative GC–MS chromatogram of single ion monitoring of the HFAA derivative of agmatine

(A) A freeze-clamped liver extract from an overnight fasted rat. The chromatogram corresponds to approx. 2 pmol of agmatine (m/z 284). (B) Same extract shown in (A) after spiking with [guanidino-¹⁵N₂]agmatine (2 nmol/g). (C) Mitochondrial extract after incubation for 7 min with 10 mM [guanidino-¹⁵N₂]arginine. Peak corresponds to approx. 4 pmol of [guanidino-¹⁵N₂]agmatine (m/z 286). (D) Same extract shown in (C) after the addition of unlabelled agmatine (∼1.2 nmol/3 mg of protein). The m/z ratio 286/284 was used to determine the isotopic enrichment as well as the concentration of agmatine by isotope dilution.

Figure 2. Time-course of ¹⁵N-labelled agmatine production during the incubation of whole mitochondria with [U-¹⁵N₄]arginine

Experiments were performed with [U-¹⁵N₄]arginine (2 mM) and other metabolites as indicated under the Materials and methods section. The line shows the best fit (r²=0.87) of a single-phase exponential association [Y=Y_max*(1−e^(−kt))]. The straight line corresponds to the linear stage of the reaction (r²=0.97). ¹⁵N-labelled agmatine was determined by the product of ¹⁵N enrichment (at.% excess/100) times total amount (pmol/mg of protein). Results are expressed as means±S.D. for 4–6 independent experiments.

Figure 3. Saturation curves of ¹⁵N-labelled agmatine production from [guanidino-¹⁵N₂]arginine

(A) Experiments with intact mitochondria and (B) experiments with broken mitochondria. Insets represent a Lineweaver–Burk plot of these data. Results were obtained after 7 min incubation of whole or broken mitochondria with increasing [arginine] as indicated. Results are the means (n=3–5) for the combined results obtained from experiments with the addition of Mn²⁺ (0.1 mM) and no EDTA or with EDTA (2 mM) and without the addition of Mn²⁺. The lines represent the best fit (r²>0.90). Results are expressed as means±S.D.

Figure 4. Production and output of ¹⁵N-labelled agmatine from livers perfused with [guanidino-¹⁵N₂]arginine

(A) ¹⁵N enrichment (at.% excess) in effluent agmatine. (B) Output (nmol·min⁻¹·g⁻¹) of ¹⁵N-labelled agmatine as determined by the product of ¹⁵N enrichment (at.% excess/100) times total output (nmol·min⁻¹·g⁻¹). Data were obtained from measurements done at the steady-state (i.e. 25–40 min after start of 0.5 mM [guanidino-¹⁵N₂]arginine infusion and other nutrients as indicated under the Materials and methods section without (CON, control) or with the addition of 10⁻⁷ M insulin (+INS), glucagon (+GLU) or 10⁻⁴ M dibutyryl-cAMP. Results are expressed as means±S.D. for three livers. **P<0.05.