Ouabain stimulates unidirectional and net potassium efflux in resting mammalian skeletal muscle

Abstract

The present study compared ouabain-sensitive unidirectional K+ flux into (JinK) and out of (JoutK) perfused rat hindlimb skeletal muscle in situ and mouse flexor digitorum brevis (FDB) in vitro. In situ, 5 mM ouabain inhibited 54 +/- 4% of the total JinK in 28 +/- 1 min, and increased the net and unidirectional efflux of K+ within 4 min. In contrast, 1.8 mM ouabain inhibited 40 +/- 8% of the total JinK in 38 +/- 2 min, but did not significantly affect JoutK. In vitro, 1.8 and 0.2 mM ouabain decreased JinK to a greater extent (83 +/- 5%) than in situ, but did not significantly affect 42K loss rate compared with controls. The increase in unidirectional K+ efflux (JoutK) with 5 mM ouabain in situ was attributed to increased K+ efflux through cation channels, since addition of barium (1 mM) to ouabain-perfused muscles returned JoutK to baseline values within 12 min. Perfusion with 5 mM ouabain plus 2 mM tetracaine for 30 min decreased JinK 46 +/- 9% (0.30 +/- 0.03 to 0.16 +/- 0.02 micromol x min(-1) x g(-1)), however tetracaine was unable to abolish the ouabain-induced increase in unidirectional K+ efflux. In both rat hindlimb and mouse FDB, tetracaine had no effect on JoutK. Perfusion of hindlimb muscle with 0.1 mM tetrodotoxin (TTX, a Na+ channel blocker) decreased JinK by 15 +/- 1%, but had no effect on JoutK; subsequent addition of ouabain (5 mM) decreased JinK a further 32 +/- 2%. The ouabain-induced increase in unidirectional K+ efflux did not occur when TTX was perfused prior to and during perfusion with 5 mM ouabain. We conclude that 5 mM ouabain increases the unidirectional efflux of K+ from skeletal muscle through a barium and TTX-sensitive pathway, suggestive of voltage sensitive Na+ channels, in addition to inhibiting Na+/K+-ATPase activity.

K⁺ Transport and Volume Regulatory Response by NKCC in Resting Rat Hindlimb Skeletal Muscle

“…For experiments with ouabain, an appropriate volume of the ouabain stock solution was added to perfusate plasma to give 5 mM ouabain. While concentrations of 1 mM have typically been used in studies using intact skeletal muscle [22], we have found that 5 mM ouabain provides a more rapid and pronounced inhibition of JinK than 1 mM ouabain, with no known non-specific effects [23]. The final arterial perfusate DMSO concentration from all DMSO-containing solutions did not exceed 0.8%.…”

“…Cellular shrinkage or membrane depolarization may have resulted in the transient opening of mechanosensitive or voltage-gated K + channels, allowing K + to flow down its chemical gradient. Clearly, these channels would have to be relatively insensitive to barium, and the use of barium also rules out the likelihood of K + efflux through voltage-gated Na + channels noted previously in ouabain-treated muscles [23].…”

“…In this experiment, bumetanide had a tendency (p=0.16) to .min -1 and the addition of bumetanide had no effect (0.38±0.03 µmol.g Barium, followed by barium with bumetanide It remained to be confirmed that the bumetanidesensitive J in K (NKCC activity) was not a component of J in K attributed to K + channels. We have previously shown that the barium-sensitive J in K is insensitive to inhibition by ouabain, and that the ouabain-sensitive J in K is insensitive to inhibition by barium [5,23]. Perfusion of …”