Abstract. The present study reports on the location of the Na+-Ca2+ exchanger in cardiac sarcolemma with immunofluorescence and immunoelectron microscopy. Both polyclonal and monoclonal antibodies to the Na+-Ca2+ exchanger were used. The mAb was produced from a hybridoma cell line generated by the fusion of mouse myeloma NS-1 cells with spleen cells from a mouse repeatedly immunized with isolated reconstituted canine cardiac Na+-Ca2+ exchanger (Philip-

We used a panel of monoclonal antibodies raised against the canine cardiac Na(+)-Ca2+ exchanger expressed in Sf9 insect cells to analyze the immunoreactive domains and the topological organization of this membrane protein. Antibodies, which reacted strongly on Western blots of the recombinant protein, were used to screen an expression sublibrary composed of exchanger cDNA fragments. Positive clones thus indicated the expression of antibody binding sites. Linear epitopes, 16-155 amino acids in length, could be identified for four antibodies. One antibody recognized two neighboring, but nonoverlapping, sequences. All epitopes were localized to the large hydrophilic region of the exchanger connecting the putative transmembrane segments 5 and 6. The immunodominant region of the protein is a highly charged domain in the carboxy-terminal half of the hydrophilic region. Binding studies with the 3H-labeled high-affinity antibody R3F1 establish that the immunodominant region is located on the intracellular surface of the membrane. The same antibody was used to directly determine the membrane concentration of the exchanger in different cell types. Newborn rat heart cells contain approximately 6 x 10(5) exchanger molecules per cell. Exchanger densities in different cells seem to correlate with the Na(+)-dependent Ca2+ transport activity in the corresponding membrane vesicles.

Striking correlations are found between cardiac contractility and Ca2+ binding to isolated cardiac sarcolemma in rabbit, neonatal rat, and frog ventricular tissue. Deviations from this correlation are seen in the adult rat ventricle and rabbit atrium. The observation of this correlation in the three former tissues and under various ionic conditions suggests that this correlation is not coincidental and that Ca2+ bound to the cardiac sarcolemma is of major importance in the control of myocardial contractility. The data are consistent with a functional Ca2+-induced Ca2+ release system in the sarcoplasmic reticulum (SR) of all the tissues (which is controlled by Ca2+ entry from sarcolemmal sites), with the adult rat ventricular and rabbit atrial SR Ca2+ release being much more sensitive to CA2+. It is suggested that the frog, neonatal rat, and rabbit ventricles depend more directly on the entry of Ca2+ from sarcolemmal sites for the control of tension development, whereas the adult rat ventricle and rabbit atrium depend to a greater extent on CA2+ released from the SR.

L-lactate is taken up by cardiac sarcolemmal vesicles in a process that is saturable with respect to L-lactate, stereospecific, associated specifically with the sarcolemmal membrane, and inhibited by other monocarboxylic acids and by the protein modifiers p-chloromercuriphenyl-sulfonate and N-ethylmaleimide. 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of the inorganic anion transporter, is without effect. The L-lactate transport is very sensitive to pH. Uptake is stimulated by a proton gradient directed inward and decreased when internal pH is lower than external pH. Passive diffusion of nonionized lactic acid into the vesicles is negligible at physiological pH and appears to remain minor even when external pH is lowered by more than one unit. Also, the mechanism does not require specific Na+-L-lactate contransport. The properties of the L-lactate transporting system in cardiac sarcolemmal vesicles appear similar to those of the monocarboxylate transporter in erythrocytes, hepatocytes, and Ehrlich ascites cells. The present results do not allow a distinction to be made between stepwise interaction of lactate- and H+ or association of nonionized lactic acid with the carrier.

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