The plasma membrane Na+/Ca2+ exchange inhibitor KB-R7943 is also a potent inhibitor of the mitochondrial Ca2+ uniporter.
OBJECTIVE
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.


METHODS
[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.


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
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.
Santo-Domingo
J
J
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.
Vay
L
L
Hernández-Sanmiguel
E
E
Lobatón
C D
CD
Moreno
A
A
Montero
M
M
Alvarez
J
J
D000331:Aequorin / Q000096:biosynthesis; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002478:Cells, Cultured; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D006367:HeLa Cells; D006632:Histamine / Q000494:pharmacology; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors*; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology
C101670:2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate; D015220:Calcium Channels; D005456:Fluorescent Dyes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D019831:Sodium-Calcium Exchanger; C034703:mitochondrial calcium uniporter; D000331:Aequorin; D006632:Histamine; D015544:Inositol 1,4,5-Trisphosphate; D013890:Thiourea; D002118:Calcium; D016257:Fura-2