Effects of WIN 55,212‐2 and AM281 in mitochondrial respiration of skeletal muscle

Abstract

The endocannabinoid system is emerging as a critical agent of cellular metabolism regulation, through its effects on the cellular respiration and energy production. Most of the cannabinoid and endocannabinoid effects have been mediated predominantly via the activation of the cannabinoid type 1 (CB1) receptor, which is widely distributed in the brain and peripheral organs as the skeletal muscle and recently were confirmed to be localized on the outer mitochondrial membrane of these tissues. It is known that their activation by synthetic cannabinoids is able to cause changes in mitochondrial respiration of brain mitochondria, however, little is known about its effect in skeletal muscle. Thus, the aim of this work was to explore the effects of the cannabinoid agonist WIN 55,212‐2 and the CB1 antagonist receptor AM281 on oxygen consumption in the state 3 from mitochondrial respiration in isolated mitochondria derived from rat skeletal muscle.MethodsMitochondria from hindlimb muscles of male Wistar rats weighing between 300 and 350 were isolated by differential centrifugation. The rate of oxygen consumption was determined in a sealed glass chamber with constant stirring at room temperature, using a Clark‐type oxygen electrode coupled to an oxygen monitor YSI 5300 and a computer for data acquisition. The determinations started after adding 10 mM glutamate/malate as respiratory substrate for complex I (state 4) and 1 mM ADP was added to determine oxygen consumption in the phosphorylating state (state 3). Finally, the inhibitor of complex IV (1mM KCN) were added to inhibit mitochondrial respiration. To explore the effects of the experimental drugs on mitochondrial respiration, a concentration‐response curve was performed for each of them: WIN 55,212‐2 (0.1, 1, 5 and 10 μM) and AM281(1, 5, 10 and 20 μM).Our results show that both drugs have similar effects by decrease mitochondrial respiration, this inhibitory effects on oxygen consumption is dose‐dependent by WIN 55,212‐2 being more prominent with 10 μM (40%) and to a lesser extent with 0.1 μM (≈ 20%), while with AM281 decreased 50% with 1 μM. These results suggest that the effect by this drugs on mitochondrial respiration exert a similar effect as has been previously reported in brain mitochondria and that could be acting on other respiratory chain components.Support or Funding InformationWe thank to Universidad de Colima, Universidad Michoacana de San Nicolás de Hidalgo (UMSNH) and Consejo Nacional de de Ciencia y Tecnología (CONACyT) the partial financial of this research. ESD was a receipient of a CONACyT PhD fellow. We acknowledge the partial financing to Coordinación de la Investigación Científica of UMSNH: ASM‐CIC‐2.16; RMP‐CIC 182171.