We investigated the effect of focal cerebral ischaemia on the activity and the affinity of the ouabain sites of Na+,K+-ATPase in the mouse. The Na+,K+-ATPase activity was decreased by 38% as early as 30 min following ischaemia. In the sham group, the dose-response curves for ouabain disclosed three inhibitory states which contribute, respectively, 24.9 +/- 6.7%, 39.1 +/- 7.5% and 36.0% of the total activity (low affinity, LA; high affinity, HA and very high affinity, VHA, respectively). Their computed IC50 values are, respectively: 1.3 X 10(-3) M, 4.5 X 10(-6) M and 2.9 X 10(-9) M. Surprisingly, in ischaemic cortices, only two sites for ouabain were detected. The first site exhibits a LA (IC50 = 2.0 X 10(-4) M) but its relative contribution to the total activity (46.1 +/- 5.2%) is twice that noted for the LA site in non-ischaemic tissues. The second site presents an affinity intermediate between those of KA and VHA sites of the sham group (IC50 = 1.7 X 10(-7) M) and contributes 53.9% to the total activity. Loss in the specific activity of the second site explains that of the total activity. The most likely explanation in the presence of only two ouabain sites of Na+,K+-ATPase following ischaemia may be a change in ouabain affinity of alpha(2) and/or alpha(3) isoforms, as the presence of all three alpha isoforms has been observed by Western blotting. These results suggest that ischaemia induces intrinsic modifications in Na+,K+-ATPase which result from perturbations in membrane integrity and/or association of the alpha isoforms of this enzyme. (C) 1997 Elsevier Science B.V.
