POSITIVELY CHARGED CYCLIC HEXAPEPTIDES, NOVEL BLOCKERS FOR THE CARDIAC SARCOLEMMA NA-CA2+ EXCHANGER()

Positively charged cyclic hexapeptides have been synthesized and teste d for their effects on the cardiac sarcolemma Na+-Ca2+ exchange activi ties with a goal to identify a potent blocker, The cyclic hexapeptides , having the different amino acid sequence, contain two arginines (to retain a positive charge), two phenylalanines (to control hydrophobici ty), and two cysteines (to form an intramolecular S-S bond). The effec t of cyclic hexapeptides were tested on Na+-Ca2+ exchange and its part ial reaction, the Ca2+-Ca2+ exchange, by measuring the Ca-45 fluxes in the semi-rapid mixer or monitoring the calcium-sensitive dye Arsenazo III and voltage-sensitive dyes (Oxanol-V or Merocyanine-540). Seven c yclic hexapeptides inhibit Na+-Ca2+ exchange with a different potency (IC50 = 2-300 mu M). Phe-Arg-Cys-Arg-Cys-Phe-CONH2 (FRCRCFa) inhibits the Na-i(+)-dependent Ca-45 uptake (Na+-Ca2+ exchange) and Ca-i(2+)-de pendent Ca-45 uptake (Ca2+-Ca2+ exchange) in the isolated cardiac sarc olemma vesicles with IC50 = 10 +/- 2 mu M and IC50 = 7 +/- 3 mu M, res pectively. Interaction of FRCRCFa with a putative inhibitory site does not involve a ''slow'' binding (a maximal inhibitory effect is alread y observed after t = 1 s of mixing). The inside positive potential, ge nerated by Na-o(+)-dependent Ca2+ efflux, was monitored by Oxanol-V (A (635)-A(612)) or Merocyanine-540 (A(570)-A(500)). In both assay system s, FRCRCFa inhibits the Na+-Ca2+ exchange with IC50 = 2-3 mu M, while a complete inhibition occurs at 20 mu M FRCRCFa. The forward (Na-i(+)- dependent Ca2+ influx) and reverse (Na-o(+)-dependent Ca2+ efflux) mod es of Na+-Ca2+ exchange, monitored by Arsenazo III (A(600)-A(785)), ar e also inhibited by FRCRCFa. The L-Arg(4) --> D-Arg(4) substitution in FRCRCFa does not alter the IC50, meaning that this structural change may increase a proteolytic resistance without a loss of inhibitory pot ency. At fixed [Na+](i) (160 mM) or [Ca2+](i) (250 mu M) and varying C a-45(o) (2-200 mu M), FRCRCFa decreases V-max without altering the K-m . Therefore, FRCRCFa is a noncompetitive inhibitor in regard to extrav esicular Ca2+ either for Na+-Ca2+ or Ca2+-Ca2+ exchange. It is suggest ed that FRCRCFa prevents the ion movements through the exchanger rathe r than the ion binding.