Mu opioid receptor-mediated release of endolysosome iron increases levels of mitochondrial iron, reactive oxygen species, and cell death

Abstract

Objectives: Opioids including morphine and DAMGO activate mu-opioid receptors (MOR), increase intracellular reactive oxygen species (ROS) levels, and induce cell death. Ferrous iron (Fe²⁺) through Fenton-like chemistry increases ROS levels and endolysosomes are "master regulators of iron metabolism" and contain readily-releasable Fe²⁺ stores. However, mechanisms underlying opioid-induced changes in endolysosome iron homeostasis and downstream-signaling events remain unclear.

Methods: We used SH-SY5Y neuroblastoma cells, flow cytometry, and confocal microscopy to measure Fe²⁺ and ROS levels and cell death.

Results: Morphine and DAMGO de-acidified endolysosomes, decreased endolysosome Fe²⁺ levels, increased cytosol and mitochondria Fe²⁺ and ROS levels, depolarized mitochondrial membrane potential, and induced cell death; effects blocked by the nonselective MOR antagonist naloxone and the selective MOR antagonist β-funaltrexamine (β-FNA). Deferoxamine, an endolysosome-iron chelator, inhibited opioid agonist-induced increases in cytosolic and mitochondrial Fe²⁺ and ROS. Opioid-induced efflux of endolysosome Fe²⁺ and subsequent Fe²⁺ accumulation in mitochondria were blocked by the endolysosome-resident two-pore channel inhibitor NED-19 and the mitochondrial permeability transition pore inhibitor TRO.

Conclusions: Opioid agonist-induced increases in cytosolic and mitochondrial Fe²⁺ and ROS as well as cell death appear downstream of endolysosome de-acidification and Fe²⁺ efflux from the endolysosome iron pool that is sufficient to affect other organelles.

Keywords: cell death; deferoxamine; endolysosomes; iron; mitochondria; opioids.