Inhibition of Na+/Ca2+ exchange in pituitary plasma membrane vesicles by analogues of amiloride.

Amiloride is a weak inhibitor of Na+/Ca2+ exchange in isolated plasma membrane vesicles prepared from GH3 rat anterior pituitary cells. However, substitution on either a terminal guanidino nitrogen atom or the 5-amino nitrogen atom can increase inhibitory potency ca. 100-fold (I50 approximately 10 microM). A structure-activity study indicates that defined structural modifications of guanidino substituents are associated with increases in inhibitory activity. In contrast, analogues bearing 5-amino substituents generally increase in potency with increasing hydrophobicity of the substitution. Specificity in action of either class is indicated by several criteria. These inhibitors do not disrupt the osmotic integrity of the membrane, nor do they significantly interfere with plasmalemmal Ca2+-ATPase-driven Ca2+ uptake, Na+,K+-ATPase enzymatic activity, or the function of Ca2+ or K+ channels. Inhibition is freely reversible, further indicating a lack of nonspecific membrane effects. The mechanism by which each inhibitor class blocks exchange was found to be identical. Protonation of the guanidino moiety (i.e., cationic charge) is essential for activity. Analysis of transport inhibition as a function of Ca2+ concentration indicates noncompetitive kinetics. However, inhibition was reversed by elevating intravesicular Na+, indicating a competitive interaction with this ion. These results suggest that the inhibitors function as Na+ analogues, interact at a Na+ binding site on the carrier (presumably the site at which the third Na+ binds), and reversibly tie up the transporter in an inactive complex. In addition to blocking pituitary exchange, these analogues are effective inhibitors of the bovine brain and porcine cardiac transport systems.