J Santo-Domingo1, L Vay, E Hernández-Sanmiguel, C D Lobatón, A Moreno, M Montero, J Alvarez. 1. Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Instituto de Biología y Genética Molecular, Universidad de Valladolid and Consejo Superior de Investigaciones Científicas, Valladolid, Spain.
Abstract
BACKGROUND AND PURPOSE: The thiourea derivative KB-R7943, originally developed as inhibitor of the plasma membrane Na(+)/Ca(2+) exchanger, has been shown to protect against myocardial ischemia-reperfusion injury. We have studied here its effects on mitochondrial Ca(2+) fluxes. EXPERIMENTAL APPROACH: [Ca(2+)] in cytosol, mitochondria and endoplasmic reticulum (ER), and mitochondrial membrane potential were monitored using both luminescent (targeted aequorins) and fluorescent (fura-2, tetramethylrhodamine ethyl ester) probes in HeLa cells. KEY RESULTS: KB-R7943 was also a potent inhibitor of the mitochondrial Ca(2+) uniporter (MCU). In permeabilized HeLa cells, KB-R7943 inhibited mitochondrial Ca(2+) uptake with a Ki of 5.5+/-1.3 microM (mean+/-S.D.). In intact cells, 10 microM KB-R7943 reduced by 80% the mitochondrial [Ca(2+)] peak induced by histamine. KB-R7943 did not modify the mitochondrial membrane potential and had no effect on the mitochondrial Na(+)/Ca(2+) exchanger. KB-R7943 inhibited histamine-induced ER-Ca(2+) release in intact cells, but not in cells loaded with a Ca(2+)-chelator to damp cytosolic [Ca(2+)] changes. Therefore, inhibition of ER-Ca(2+)-release by KB-R7943 was probably due to the increased feedback Ca(2+)-inhibition of inositol 1,4,5-trisphosphate receptors after MCU block. This mechanism also explains why KB-R7943 reversibly blocked histamine-induced cytosolic [Ca(2+)] oscillations in the same range of concentrations required to inhibit MCU. CONCLUSIONS AND IMPLICATIONS: Inhibition of MCU by KB-R7943 may contribute to its cardioprotective activity by preventing mitochondrial Ca(2+)-overload during ischemia-reperfusion. In addition, the effects of KB-R7943 on Ca(2+) homeostasis provide new evidence for the role of mitochondria modulating Ca(2+)-release and regenerative Ca(2+)-oscillations. Search for permeable and selective MCU inhibitors may yield useful pharmacological tools in the future.
BACKGROUND AND PURPOSE: The thiourea derivative KB-R7943, originally developed as inhibitor of the plasma membrane Na(+)/Ca(2+) exchanger, has been shown to protect against myocardial ischemia-reperfusion injury. We have studied here its effects on mitochondrial Ca(2+) fluxes. EXPERIMENTAL APPROACH: [Ca(2+)] in cytosol, mitochondria and endoplasmic reticulum (ER), and mitochondrial membrane potential were monitored using both luminescent (targeted aequorins) and fluorescent (fura-2, tetramethylrhodamine ethyl ester) probes in HeLa cells. KEY RESULTS:KB-R7943 was also a potent inhibitor of the mitochondrial Ca(2+) uniporter (MCU). In permeabilized HeLa cells, KB-R7943 inhibited mitochondrial Ca(2+) uptake with a Ki of 5.5+/-1.3 microM (mean+/-S.D.). In intact cells, 10 microM KB-R7943 reduced by 80% the mitochondrial [Ca(2+)] peak induced by histamine. KB-R7943 did not modify the mitochondrial membrane potential and had no effect on the mitochondrial Na(+)/Ca(2+) exchanger. KB-R7943 inhibited histamine-induced ER-Ca(2+) release in intact cells, but not in cells loaded with a Ca(2+)-chelator to damp cytosolic [Ca(2+)] changes. Therefore, inhibition of ER-Ca(2+)-release by KB-R7943 was probably due to the increased feedback Ca(2+)-inhibition of inositol 1,4,5-trisphosphate receptors after MCU block. This mechanism also explains why KB-R7943 reversibly blocked histamine-induced cytosolic [Ca(2+)] oscillations in the same range of concentrations required to inhibit MCU. CONCLUSIONS AND IMPLICATIONS: Inhibition of MCU by KB-R7943 may contribute to its cardioprotective activity by preventing mitochondrial Ca(2+)-overload during ischemia-reperfusion. In addition, the effects of KB-R7943 on Ca(2+) homeostasis provide new evidence for the role of mitochondria modulating Ca(2+)-release and regenerative Ca(2+)-oscillations. Search for permeable and selective MCU inhibitors may yield useful pharmacological tools in the future.
Authors: Amit K Chouhan; Maxim V Ivannikov; Zhongmin Lu; Mutsuyuki Sugimori; Rodolfo R Llinas; Gregory T Macleod Journal: J Neurosci Date: 2012-01-25 Impact factor: 6.167