Na+/Ca2+ exchange is inhibited in both guinea pig cardiac membrane vesicles and papillary muscles in a concentration-dependent fashion by several analogs of the pyrazine diuretic amiloride. Structure/activity studies based on transport measurements in vesicles prepared from guinea pig left ventricle indicate that hydrophobic substitutions at the terminal nitrogen atom of the guanidinium moiety of amiloride improved the inhibitory potency almost 100-fold over that of the parent compound. 3', and that this transport system may be an important source of Ca2+ supplying the sarcoplasmic reticulum in guinea pig heart. Moreover, these amiloride analogs function as potent inhibitors of the positive inotropic effect caused by increased intracellular Na+ concentration.Both extracellular Na+ and Ca2+ affect the contractile force generated by isolated cardiac muscle (1). Using 45Ca2+ flux measurements in guinea pig atria, Reuter and co-workers found that both Ca"+ influx (2) and Ca>2 efflux (3) were dependent on external Na+ concentration. From these data, a Na+/Ca2+ countertransport system in the cardiac sarcolemmal membrane was conceptualized. Studies with cardiac sarcolemmal membrane vesicles exhibiting Na+/Ca2+ exchange (4) provided confirmation of the existence of a Na+ gradient-dependent Ca2+ antiport mechanism and defined the interaction between Na+ and Ca2+ at the level of the transporter (5). The above findings implicate Na+/Ca2+ exchange in the regulation of Ca2+ homeostasis in the cardiac cell and suggest that this antiport system may be an important determinant of the contractile state.In the cardiac sarcolemmal membrane a number of processes are involved in controlling intracellular Ca2+ concentration on a beat-to-beat basis. Ca2+ influx occurs during the action potential as a consequence of a depolarization-induced opening of the slow calcium channels (6). A high-affinity ATP-driven Ca2+ transport system has been described (7) that is responsible for Ca2+ efflux from cardiac cells. The contribution of Na+/Ca2" exchange to transsarcolemmalCa2+ fluxes relative to Ca2+ influx via the "slow" channel and Ca2' efflux via Ca2+-ATPase is unknown. Since Ca2+ fluxes are important determinants of both force development (Ca2+ influx) and relaxation (Ca2+ efflux) in cardiac muscle, it is important to determine the relative contribution of Na+/Ca"+ exchange.To investigate this area, specific inhibitors of Na+/Ca2+ exchange must be developed. In this communication, relatively potent inhibitors of Na+/Ca2+ exchange in sarcolemmal membrane vesicles are described. The primary purpose of this study was to examine the effects of one such inhibitor, 3',4'-dichlorobenzamil (DCB), on mechanical responses of isolated papillary muscles from guinea pig heart and to assess the role of Na+/Ca2+ exchange in regulating contractility. METHODSVesicles were prepared from freshly isolated guinea pig left ventricle by the procedure of Reeves and Sutko (4). For these studies sarcolemmal membranes were not routinely purified further beca...

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.

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