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The expression of the canine alpha 2 and 3 subunit isoenzymes of NA,K-ATPase has been investigated in plasma membranes isolated from dog heart, brain and kidney by immunoblotting, employing polyclonal anti rat fusion protein, and enzymological techniques. Western blot analysis revealed with purified dog membrane Na,K-ATPase preparations, one immunoreactive signal with rat specific alpha 3 antisera in cardiac tissues, and two immunoreactive signals with rat alpha 2 and alpha 3 antisera in cerebral tissues. These findings suggested the specific expression of alpha 3 polypeptide in dog heart (99 kDa), whereas dog brain expressed the alpha 2 and 3 polypeptides. The stained bands were superimposed. The antibody to rat brain alpha 1 fusion protein did not cross-react with dog antigens whatever the three tissues tested. Expression of the alpha 3-subunit isoform in dog heart membranes was consistent with a high affinity digitoxigenin-sensitive class of Na,K-ATPase (IC50 = 7 +/- 2 nM). A single component with low affinity to digitoxigenin (IC50 = 110 +/- 10 nM) characterized the alpha 1 kidney form. The mixture of alpha 2 and alpha 3 isoforms in dog brain exhibited an apparent affinity for digitoxigenin (IC50 = 17 +/- 5 nM) lower than the heart. The sodium dependences of the high affinity digitoxigenin sites were for the cardiac alpha 3 form (K0.5 = 10 +/- 1.9 mM) and for the cerebral alpha 2 and alpha 3 mixture (K0.5 19.6 +/- 4.9 mM). The sensitivities for Na+ of the low affinity sites (alpha 1) were: 6.7 +/- 1.4 mM, 6.3 +/- 1.2 mM and 11.6 +/- 2.9 mM in heart, brain and kidney respectively. This is the first report of the catalytic characteristics of the alpha 3 subunit isoenzyme in canine cardiac plasma membranes.

Serine-specific reagents, anticholinesterase organophosphorus compounds like Vx provoke, in the micromolar range, digitalis-like ventricular arrhythmias of non-cholinergic origin in rodent hearts. The sensitivities of the two rat cardiac Na+,K(+)-ATPase isoforms (alpha 1 and alpha 2) to Vx (0.1-100 microM) were measured in sarcolemma vesicles. At 1 microM Vx, the inhibition of the total activity averaged 18% but never exceeded 75% with 100 microM. When the alpha 2 isoform activity was inhibited by 0.1 microM ouabain, alpha 1 was 35% inhibited by 1 microM Vx, i.e. a 16 +/- 4% inhibition of the total activity. The cardiac alpha 1 being related to the digitalis-induced toxicity, its selective inhibition by a micromolar dose of Vx fully accounts for the cardiotoxicity of Vx. Inasmuch as Vx had no effect on the rat kidney alpha 1, differentially inactivated the cardiac isozymes and specifically reacted with serine residues, the putative binding-site(s) of the organophosphorus compound on the Na+-K(+)-ATPase molecules has been considered.