Pharmacological and physiological stimuli do not promote Ca(2+)-sensitive K+ channel activity in isolated heart mitochondria.
OBJECTIVE
Mitochondrial calcium-activated K(+) (mitoK(Ca)) channels have been described as channels that are activated by Ca(2+), inner mitochondrial membrane depolarization and drugs such as NS-1619. NS-1619 is cardioprotective, leading to the assumption that this effect is related to the opening of mitoK(Ca) channels. Here, we show several weaknesses in this hypothesis.


METHODS
Isolated mitochondria from rat hearts were tested for evidence of mitoK(Ca) activity by analyzing functional parameters in K(+)-rich and K(+)-free media.


RESULTS
NS-1619 promoted mitochondrial depolarization both in K(+)-rich and K(+)-free media. Respiratory rate increments were also seen in the presence of NS-1619 for both media. In parallel, NS-1619 promoted respiratory inhibition, as evidenced by respiratory measurements in state 3. Mitochondrial volume measurements conducted using light scattering showed that NS-1619 led to swelling, in a manner unaltered by inhibitors of mitoK(Ca) channels, antagonists of adenosine triphosphate-sensitive potassium channels or inhibitors of the permeability transition. Swelling was also maintained when K(+) in the media was substituted with tetraethylammonium (TEA(+)), which is not transported by any known K(+) carrier. Electron microscopy experiments gave support to the idea that NS-1619-induced mitochondrial swelling took place in the absence of K(+). In addition to testing the pharmacological effects of NS-1619, we attempted, unsuccessfully, to promote mitoK(Ca) activity by altering Ca(2+) concentrations in the medium and inducing mitochondrial uncoupling.


CONCLUSIONS
Our data indicate that NS-1619 promotes non-selective permeabilization of the inner mitochondrial membrane to ions, in addition to partial respiratory inhibition. Furthermore, we found no specific K(+) transport in isolated heart mitochondria compatible with mitoK(Ca) opening, whether by pharmacological or physiological stimuli. Our results indicate that NS-1619 has extensive mitochondrial effects unrelated to mitoK(Ca) and suggest that tissue protection mediated by NS-1619 may occur through mechanisms other than activation of these channels.
Cancherini
Douglas V
DV
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.
Queliconi
Bruno B
BB
Kowaltowski
Alicia J
AJ
D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D003470:Culture Media; D018698:Glutamic Acid / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects*; D008293:Malates / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000378:metabolism; D008933:Mitochondrial Swelling / Q000187:drug effects; D009249:NADP / Q000378:metabolism; D009840:Oligomycins / Q000494:pharmacology; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D010619:Phenazines / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D024681:Potassium Channels, Calcium-Activated / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012402:Rotenone / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D019802:Succinic Acid / Q000494:pharmacology; D019789:Tetraethylammonium / Q000378:metabolism / Q000494:pharmacology; D046509:Tissue Culture Techniques; D014475:Uncoupling Agents / Q000494:pharmacology
D001562:Benzimidazoles; D003470:Culture Media; D008293:Malates; D009840:Oligomycins; D010619:Phenazines; D024681:Potassium Channels, Calcium-Activated; D014475:Uncoupling Agents; D012402:Rotenone; C086491:NS 1619; D018698:Glutamic Acid; D009249:NADP; D019789:Tetraethylammonium; C030298:malic acid; D012964:Sodium; D019802:Succinic Acid; D011188:Potassium; D002118:Calcium; C009195:safranine T