Down-regulation of Na+/K+-ATPase alpha(2) isoform in denervated rat vas deferens

In the rat vas deferens, an organ richly innervated by peripheral sympathet ic neurons, we have demonstrated recently the expression of alpha(1) and al pha(2), but not alpha(3) isoforms of the a subunit of Na+/K+-ATPase (EC 3.6 .1.37), a membrane-bound enzyme of vital function for living cells (Noel et al., Biochem Pharmacol 55: 1531-1535, 1998). In the present work, we chara cterized, qualitatively and quantitatively, Na+/K+-ATPase a isoforms in den ervated rat vasa deferentia. [H-3]Ouabain binding at concentrations defined for high-affinity isoforms (alpha(2) and/or alpha(3)) detected only one cl ass of specific binding sites in control (C) and denervated (D) vas deferen s. Although the dissociation constant was similar for both groups [K-d = 13 8 +/- 14 nM (C) and 125 +/- 8 nM (D)], a marked decrease in density was obs erved after denervation [716 +/- 81 fmol(.)mg protein(-1) (C) and 445 +/- 3 4 fmol(.)mg protein(-1) (D), P < 0.05]. In addition, western blotting revea led that denervated vasa deferentia produce the alpha(1) and alpha(2) isofo rms but not alpha(3), just as we reported for the controls previously (Noel et al., Biochem Pharmacol 55: 1531-1535, 1998). Densitometric analysis sho wed a decrease of the alpha(2) isoform by about 4094 in denervated organs, in very good agreement with what was shown with the [H-3]ouabain binding te chnique, but no significant change in oil isoform density. Truncated alpha( 1) (alpha(1)T), an isoform suggested to exist in the guinea pig vas deferen s, was not detected. Altogether, our results demonstrated that Na+/K+-ATPas e alpha(2) is down-regulated after sympathetic denervation of the rat vas d eferens. (C) 2000 Elsevier Science Inc.