Imidazoline-guanidinium receptive site in renal proximal tubule: Asymetric distribution, regulation by cations and interaction with an endogenous clonidine displacing substance

In the present report we have used [³H] idazoxan to charaterize the rabbit renal imidazoline preferring site by defining its plasmalemma distribution, its regulation by cations and the type of interaction with the clonidine displacing substance (CDS), a putative endotenous ligand for the imidazoline receptor. The density of [³H]idazoxan binding sites was 12-fold higher in purified basolateral membranes than in brush-border membranes (maximal binding activity, 566 ± 118 vs. 46 ± 2 fmol/mg of protein). In basolateral membranes, [³H]idazoxan binding was inhibited not only by imidazoline compounds but also by guanidinium analogs such as guanabenz, amiloride, 5-(M-ethyl-N-isopropyl)amiloride and phenamylamiloride. Amiloride had no effect on the dissociation rate of [³H]idazoxan, suggesting a direct interaction of this molecule with the ligand binding site. [³H]Idazoxan binding was 80% inhibited by 150 mM K⁺ or Rb⁺. The effect of K⁺ appeared to occur through the interaction with an allosteric site in as much as both the apparent dissociation constant and the dissociation rate of [³H]idazoxan were increased in the presence of 75 mM K⁺. CDS inhibited [³H]idazoxan binding with a half-maximal effective concentration of 2 U/250 μl. The competitive nature of CDS effect was indicated by the increase in the apparent dissociation constant of [³H]idazoxan (K(d) from 3 ± 0.3 to 8.5 ± 0.2 nM, P < .01) in the presence of CDS. In conclusion, our findings showed that the imidazoline-guanidinium receptive site is located mainly in the basolateral side of the tubular cell, recognizes CDS and is regulated by K⁺. These specific characteristics of imidazoline-guanidinium receptive site suggest that this membrane protein could have a physiological relevance in the renal proximal tubule.