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

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 · min ^-1 · (mg of protein)^-1. Experiments with an increasing concentration (0-40 mM) of [guanidino-¹⁵N ₂]arginine showed a Michaelis constant K_m for arginine of 46 mM and a V_max of 3.7 nmol · min^-1 · (mg of protein)^-1 for flux through the ADC reaction. Experiments with broken mitochondria showed little changes in V_max or K_m values, suggesting that mitochondrial arginine uptake had little effect on the observed V_max or K_m 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 · min^-1 · 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.