Intracellular binding of spin‐labeled amiloride: An alternative explanation for amiloride's effects at high concentration

Abstract

Amiloride, an important inhibitor of Na+ transport and Na+/H+ exchange, has been used in nontransporting tissues to investigate the relationship between ionic fluxes or intracellular pH change and proliferative or synthetic events. Reports that amiloride is permeant and had direct effects on intracellular processes have led us to investigate the possibility that amiloride binds intracellularly to nuclei, mitochondria, and to purified nucleic acids. Using a nitroxide spin-labeled derivative of amiloride (ASp) and electron paramagnetic resonance (EPR) spectroscopy, we have demonstrated that nuclei and mitochondria isolated from trout liver bind significant amounts of ASp especially at the high amiloride concentrations (approximately mM) commonly used to inhibit proliferative events. While the chemical component responsible for ASp binding in these organelles was not identified, native DNA binds significant amounts of ASp whereas single stranded DNA and RNA bind much less. When these observations are taken together with reports of amiloride's direct action on cellular processes, they support the possibility that some of the effects attributed to inhibition of a transport event are caused by amiloride directly.

Hexamethylene amiloride engages a novel reactive oxygen species- and lysosome-dependent programmed necrotic mechanism to selectively target breast cancer cells

“…While our data support mitochondrial dysfunction in mediating HMA associated cell death, evidenced by increased mitochondrial swelling, aggregation and ROS production, it is probable that endosomal trafficking may only be a consequence of amiloride derivative action and not required for cell death. It is likely that amiloride derivatives such as HMA directly interact with mitochondrial transporters or channels, a notion supported by previous work investigating the association of particular amiloride derivatives with intracellular organelles [42]. …”

Cross‐reactivity of acid‐sensing ion channel and Na+–H+ exchanger antagonists with nicotinic acetylcholine receptors

“…Nevertheless, zoniporide has been reported to damage sensory nerve fibres, causing axonal degeneration in the spinal cord and dorsal root ganglia (Pettersen et al 2008). Furthermore, long-term use of inhibitors can lead to their excessive accumulation due to reduced clearance (Knauf et al 1985) and, in the case of amiloride, due to its uptake by intracellular organelles such as nuclei and mitochondria, subsequently affecting various cellular processes (Costa et al 1987).…”