(+)-Doxapram

The mode of action by which doxapram acts as a respiratory stimulant in humans is controversial. Studies in rodent models, have shown that doxapram is a more potent and selective inhibitor of TASK‐1 and TASK‐1/TASK‐3 heterodimer channels, than TASK‐3. Here we investigate the direct effect of doxapram and chirally separated, individual positive and negative enantiomers of the compound, on both human and mouse, homodimeric and heterodimeric variants of TASK‐1 and TASK‐3.

Whole‐cell patch clamp electrophysiology on tsA201 cells was used to assess the potency of doxapram on cloned human or mouse TASK‐1, TASK‐3 and TASK‐2 channels. Mutations of amino acids in the pore‐lining region of TASK‐3 channels were introduced using site‐directed mutagenesis.

Doxapram was an equipotent inhibitor of human TASK‐1 and TASK‐3 channels, compared with mouse channel variants, where it was more selective for TASK‐1 and heterodimers of TASK‐1 and TASK‐3. The effect of doxapram could be attenuated by either the removal of the C‐terminus of human TASK‐3 channels or mutations of particular hydrophobic residues in the pore‐lining region. These mutations, however, did not alter the effect of a known extracellular inhibitor of TASK‐3, zinc. The positive enantiomer of doxapram, GAL‐054, was a more potent antagonist of TASK channels, than doxapram, whereas the negative enantiomer, GAL‐053, had little inhibitory effect.

These data show that in contrast to rodent channels, doxapram is a potent inhibitor of both TASK‐1 and TASK‐3 human channels, providing further understanding of the pharmacological profile of doxapram in humans and informing the development of new therapeutic agents.

The augmentation by doxapram (DOP) of the reduction in viability and of the apoptosis of cells induced by acetaminophen (AA) was examined in mouse primary cultured hepatocytes. Loss of viability on exposure to AA and/or DOP in cultured hepatocytes was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and the apoptosis of cultured hepatocytes was detected by nuclear morphologic observation and from a ladder-like DNA fragmentation pattern. The combination of AA (5 mM) and DOP (10, 20, 50 or 100 microM) potentiated the reduction in cell viability and increased the oxidative stress. Hepatocytes exposed for 24 h to AA (5 mM) plus DOP (100 microM), showed atrophy of nuclei, including chromatin condensation and a ladder-like DNA fragmentation pattern, characteristic of apoptosis. Benzyl-oxycarbonyl-Asp-CH2OC (O)-2,6-dichlorobenzene (Z-Asp-CH2-DCB, 50 microM), an inhibitor for caspases, improved the viability and ladder-like DNA fragmentation in cells exposed to DOP (200 or 500 microM) alone or AA ( 5 mM) plus DOP (100 microM). However, loss of viability on exposure to a high concentration of AA (10 mM) and ladder-like DNA fragmentation were not affected by Z-Asp-CH2-DCB. These results indicated that the synergistic increase in oxidative stress, activation of caspases and DNA fragmentation induced by DOP potentiated the hepatotoxicity of AA.