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.

Effects of Different Opioid Drugs on Oxidative Status and Proteasome Activity in SH-SY5Y Cells

“…Indeed, the ROS-increasing effect of morphine is not shared by the other opioid analgesics here investigated, thus suggesting that the specific drug pharmacological profile can influence this parameter. In accordance with previous studies that indicated morphine’s ability to induce an oxidative stress in SH-SY5Y cells [ 33 , 34 ], our findings support the hypothesis that agonist stimulation of MOR could contribute to the increase in intracellular ROS. Indeed, the MOR-agonist-induced reactive oxygen species generation is completely blocked by the non-selective MOR antagonist naloxone and the selective MOR antagonist β-funaltrexamine (β-FNA).…”

Weak base drug-induced endolysosome iron dyshomeostasis controls the generation of reactive oxygen species, mitochondrial depolarization, and cytotoxicity

“…Thus, others and we use PhenGreen SK with confidence to measure effects of various treatments on levels of cytosolic Fe 2+ . Previously, we reported that endolysosome stores of Fe 2+ when released were sufficiently large enough to account for insult-induced increases in cytosolic Fe 2+ levels ([Fe 2+ ] cyt ) and in reactive oxygen species (ROS) levels [ 14 , 15 ]. Accordingly, we next investigated the extent to which atropine, azithromycin, fluoxetine, metoprolol, and tamoxifen increased levels of [Fe 2+ ] cyt ( Figure 4A ) and cytosolic ROS ( Figure 4B ).…”

“…Drug-induced cytotoxicity and the pathogenesis of diverse disease states are linked to increased iron levels and iron dysregulation [ 10 , 11 ]; Fe 2+ via Fenton-like chemistry generates reactive oxygen species (ROS) and induces cytotoxicity [ 12 ]. Furthermore, endolysosome Fe 2+ release induced by endolysosome de-acidification causes iron dysregulation [ 13 ], [ 14 ], [ 15 ]. We reported recently in numerous cell types that de-acidification of endolysosomes decreased Fe 2+ levels in endolysosomes, increased Fe 2+ and ROS levels in the cytosol and mitochondria, as well as induced cell death [ 14 ], [ 15 ], [ 16 ].…”

“…Furthermore, endolysosome Fe 2+ release induced by endolysosome de-acidification causes iron dysregulation [ 13 ], [ 14 ], [ 15 ]. We reported recently in numerous cell types that de-acidification of endolysosomes decreased Fe 2+ levels in endolysosomes, increased Fe 2+ and ROS levels in the cytosol and mitochondria, as well as induced cell death [ 14 ], [ 15 ], [ 16 ]. Because endolysosome sequestration of weak base drugs can result in endolysosome de-acidification [ 17 ], we tested the hypothesis that ion trapping of weak base drugs in endolysosomes might be a mechanism common to changes in inter-organellar signaling and increases in cytotoxicity caused by weak base drugs.…”

Sodium Sulfite-Triggered Hepatocyte Ferroptosis via mtROS/Lysosomal Membrane Permeabilization-Mediated Lysosome Iron Efflux

“…Therefore, when the lysosomal membrane permeabilization increases, the Fe 2+ contained in lysosomes may flow out of the lysosome and become an important reason for the increase of iron ion concentration in the cytoplasm. 13 Lysosomal membrane permeabilization can be triggered by appropriate stimuli such as ROS, lipid mediators, and lysosomal detergents. 14 ROSmediated lysosomal membrane permeabilization has received increasing attention in recent years.…”