Abstract
OBJECTIVE: The presence of both alpha1- and alpha2-isoforms of the
Na$^+$/K$^+$-ATPase (NKA) in cardiomyocytes indicates different
functions. We hypothesized that preferential localization of the
alpha2-isoform to the t-tubules, locally controlling the Na$^+$/Ca$^2+$-exchanger
(NCX), underlies a specific role in Ca$^2+$ handling. METHODS:
We studied NKA isoform distribution in isolated cardiomyocytes from
Wistar rats using immunocytochemistry. NKA pump and NCX currents
(I(pump) and I(NCX)) were measured in control and detubulated cardiomyocytes.
Intracellular Na$^+$ concentration Na$^+$i was assessed
with the fluorescent dye SBFI. RESULTS: The alpha2-isoform abundance
was higher in the t-tubules than in the surface sarcolemma. We established
that 0.3 microM ouabain specifically blocked the alpha2-isoform in
isolated rat cardiomyocytes. This low concentration blocked 10.7+/-0.6\%
of I(pump) in control, but only 6.0+/-0.5\% in detubulated cardiomyocytes.
Moreover, measured and calculated alpha1-specific and alpha2-specific
I(pump) in control (547+/-29 pA and 66 pA, respectively) and in detubulated
cells (495+/-30 pA and 31 pA, respectively) showed that 53\% of the
alpha2-isoform, but only 9.5\% of the alpha1-isoform, were localized
to the t-tubules. Despite the small abundance of the alpha2-isoform
(approximately 11\% of total NKA), selective inhibition of this isoform
induced a 40\% increase in contractility in field stimulated cardiomyocytes,
but no increase in global Na$^+$i. However, inhibition of the
alpha2-isoform increased I(NCX) indicating local subsarcolemmal accumulation
of Na$^+$ near NCX. CONCLUSIONS: The alpha2-isoform of the NKA
is functionally coupled to the NCX and can regulate Ca$^2+$ handling
without changing global Na$^+$i.
- /&/
- analysis/antagonists
- analysis/metabolism;
- animals;
- atpase,
- blotting,
- calcium,
- cardiac,
- cell
- confocal;
- contraction,
- electric
- enzymology/physiology;
- exchanger,
- immunohistochemistry;
- inhibitors/metabolism
- male;
- metabolism;
- methods;
- microscopy,
- myocardial
- myocytes,
- ouabain,
- patch-clamp
- pharmacology;
- physiology;
- rats,
- rats;
- size;
- sodium,
- sodium-calcium
- sodium-potassium-exchanging
- stimulation;
- techniques;
- western,
- wistar;
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