CHANGES IN OUABAIN AFFINITY OF NA-ATPASE DURING FOCAL CEREBRAL-ISCHEMIA IN THE MOUSE(,K+)

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 fol lowing ischaemia. In the sham group, the dose-response curves for ouab ain disclosed three inhibitory states which contribute, respectively, 24.9 +/- 6.7%, 39.1 +/- 7.5% and 36.0% of the total activity (low affi nity, 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 (IC 50 = 2.0 X 10(-4) M) but its relative contribution to the total activi ty (46.1 +/- 5.2%) is twice that noted for the LA site in non-ischaemi c tissues. The second site presents an affinity intermediate between t hose 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 activit y of the second site explains that of the total activity. The most lik ely explanation in the presence of only two ouabain sites of Na+,K+-AT Pase following ischaemia may be a change in ouabain affinity of alpha( 2) and/or alpha(3) isoforms, as the presence of all three alpha isofor ms has been observed by Western blotting. These results suggest that i schaemia induces intrinsic modifications in Na+,K+-ATPase which result from perturbations in membrane integrity and/or association of the al pha isoforms of this enzyme. (C) 1997 Elsevier Science B.V.