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• W1969498405 abstract "Positively charged cyclic hexapeptides have been synthesized and tested for their effects on the cardiac sarcolemma Na+-Ca2+ exchange activities 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 hydrophobicity), and two cysteines (to form an intramolecular S-S bond). The effect of cyclic hexapeptides were tested on Na+-Ca2+ exchange and its partial reaction, the Ca2+-Ca2+ exchange, by measuring the 45Ca fluxes in the semi-rapid mixer or monitoring the calcium-sensitive dye Arsenazo III and voltage-sensitive dyes (Oxanol-V or Merocyanine-540). Seven cyclic hexapeptides inhibit Na+-Ca2+ exchange with a different potency (IC50 = 2-300 μM). Phe-Arg-Cys-Arg-Cys-Phe-CONH2 (FRCRCFa) inhibits the Na+o-dependent 45Ca uptake (Na+-Ca2+ exchange) and Ca2+i-dependent 45Ca uptake (Ca2+-Ca2+ exchange) in the isolated cardiac sarcolemma vesicles with IC50 = 10 ± 2 μM and IC50 = 7 ± 3 μM, respectively. Interaction of FRCRCFa with a putative inhibitory site does not involve a “slow” binding (a maximal inhibitory effect is already observed after t = 1 s of mixing). The inside positive potential, generated by Na+o-dependent Ca2+ efflux, was monitored by Oxanol-V (A635-A612) or Merocyanine-540 (A570-A500). In both assay systems, FRCRCFa inhibits the Na+-Ca2+ exchange with IC50 = 2-3 μM, while a complete inhibition occurs at 20 μM FRCRCFa. The forward (Na+i-dependent Ca2+ influx) and reverse (Na+o-dependent Ca2+ efflux) modes of Na+-Ca2+ exchange, monitored by Arsenazo III (A600-A785), are also inhibited by FRCRCFa. The L-Arg4→D-Arg4 substitution in FRCRCFa does not alter the IC50, meaning that this structural change may increase a proteolytic resistance without a loss of inhibitory potency. At fixed [Na50]50 (160 mM) or [Ca50]50 (250 μM) and varying 50Ca50(2-200 μM), FRCRCFa decreases Vmax without altering the K50. Therefore, FRCRCFa is a noncompetitive inhibitor in regard to extravesicular Ca50 either for Na+-Ca2+ or Ca2+-Ca2+ exchange. It is suggested that FRCRCFa prevents the ion movements through the exchanger rather than the ion binding. Positively charged cyclic hexapeptides have been synthesized and tested for their effects on the cardiac sarcolemma Na+-Ca2+ exchange activities 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 hydrophobicity), and two cysteines (to form an intramolecular S-S bond). The effect of cyclic hexapeptides were tested on Na+-Ca2+ exchange and its partial reaction, the Ca2+-Ca2+ exchange, by measuring the 45Ca fluxes in the semi-rapid mixer or monitoring the calcium-sensitive dye Arsenazo III and voltage-sensitive dyes (Oxanol-V or Merocyanine-540). Seven cyclic hexapeptides inhibit Na+-Ca2+ exchange with a different potency (IC50 = 2-300 μM). Phe-Arg-Cys-Arg-Cys-Phe-CONH2 (FRCRCFa) inhibits the Na+o-dependent 45Ca uptake (Na+-Ca2+ exchange) and Ca2+i-dependent 45Ca uptake (Ca2+-Ca2+ exchange) in the isolated cardiac sarcolemma vesicles with IC50 = 10 ± 2 μM and IC50 = 7 ± 3 μM, respectively. Interaction of FRCRCFa with a putative inhibitory site does not involve a “slow” binding (a maximal inhibitory effect is already observed after t = 1 s of mixing). The inside positive potential, generated by Na+o-dependent Ca2+ efflux, was monitored by Oxanol-V (A635-A612) or Merocyanine-540 (A570-A500). In both assay systems, FRCRCFa inhibits the Na+-Ca2+ exchange with IC50 = 2-3 μM, while a complete inhibition occurs at 20 μM FRCRCFa. The forward (Na+i-dependent Ca2+ influx) and reverse (Na+o-dependent Ca2+ efflux) modes of Na+-Ca2+ exchange, monitored by Arsenazo III (A600-A785), are also inhibited by FRCRCFa. The L-Arg4→D-Arg4 substitution in FRCRCFa does not alter the IC50, meaning that this structural change may increase a proteolytic resistance without a loss of inhibitory potency. At fixed [Na50]50 (160 mM) or [Ca50]50 (250 μM) and varying 50Ca50(2-200 μM), FRCRCFa decreases Vmax without altering the K50. Therefore, FRCRCFa is a noncompetitive inhibitor in regard to extravesicular Ca50 either for Na+-Ca2+ or Ca2+-Ca2+ exchange. It is suggested that FRCRCFa prevents the ion movements through the exchanger rather than the ion binding. The cell membrane Na+-Ca2+ exchanger is a major regulator of intracellular calcium in cardiac and neuronal cells during the resting and action potentials(1Hilgemann D.W. Collins A. Cash D.P. Nagel G.A. Ann. N. Y. Acad. Sci. 1991; 639: 126-139Crossref PubMed Scopus (37) Google Scholar, 2Langer G.A. Trends Cardiovasc. Med. 1994; 4: 103-109Crossref PubMed Scopus (10) Google Scholar). The cardiac sarcolemma Na+-Ca2+ exchange is the only electrogenic system (3Na50:Ca50) that provides a voltage-sensitive extrusion of intracellular calcium that has entered the cell via the Ca50 channels(3Bridge J.H.B. Smolley J.R. Spitzer K.W. Science. 1990; 248: 376-378Crossref PubMed Scopus (228) Google Scholar, 4Noble D. Noble S.J. Bett G.C.L. Earm Y.E. Ho W.K. So I.K. Ann. N. Y. Acad. Sci. 1991; 639: 334-353Crossref PubMed Scopus (124) Google Scholar). The cardiac Na+-Ca2+ exchanger is a typical carrier-type system(5Stein W.D. Transport and Diffusion across Cell Membranes. Academic Press, Orlando, FL1986: 326-337Google Scholar, 6Khananshvili D. Curr. Opin. Cell Biol. 1990; 2: 731-734Crossref PubMed Scopus (4) Google Scholar), which can also catalyze the Ca2+-Ca2+ and Na50-Na50 exchanges. The Na+-Ca2+ exchange cycle and its partial reactions can be described as separate movements of Na50 and Ca50 (so called consecutive or ping-pong mechanism) through the exchanger(7Khananshvili D. Biochemistry. 1990; 29: 2437-2442Crossref PubMed Scopus (75) Google Scholar, 8Khananshvili D. J. Biol. Chem. 1991; 266: 13764-13769Abstract Full Text PDF PubMed Google Scholar, 9Niggli E. Lederer W.J. Nature. 1991; 349: 621-624Crossref PubMed Scopus (117) Google Scholar, 10Hilgemann D.W. Nicoll D.A. Philipson K.D. Nature. 1991; 352: 715-718Crossref PubMed Scopus (178) Google Scholar, 11Li J. Kimura J. Ann. N. Y. Acad. Sci. 1991; 639: 48-60Crossref PubMed Scopus (16) Google Scholar). A contribution of exchange modes to cellular activities as well as their catalytic and regulatory mechanisms are poorly understood(12Khananshvili D. Ann. N. Y. Acad. Sci. 1991; 639: 85-95Crossref PubMed Scopus (14) Google Scholar, 13Matsuoka S. Hilgemann D.W. J. Gen. Physiol. 1992; 100: 963-1001Crossref PubMed Scopus (121) Google Scholar, 14Philipson K.D. Nicoll D.A. Int. Rev. Cytol. 1993; 137: 199-227Google Scholar). Amiloride and its derivatives have been identified as relatively effective inhibitors of Na+-Ca2+ exchange(15Slaughter R.S. Garcia M.L. Cragoe E.J. Reeves J.P. Kaczorowski G.I. Biochemistry. 1988; 27: 2403-2409Crossref PubMed Scopus (52) Google Scholar, 16Kaczorowski G.I. Slaughter R.S. King V.F. Garcia M.L. Biochim. Biophys. Acta. 1989; 988: 287-302Crossref PubMed Scopus (121) Google Scholar), but their application is strictly limited for most biomedical experiments. For example, most popular amiloride analogs (e.g. benzamil, dichlorobenzamil, or benzobenzamil) inhibit Na+-Ca2+ exchange with a relatively low potency, exhibiting IC50 = 1050-1050M(15Slaughter R.S. Garcia M.L. Cragoe E.J. Reeves J.P. Kaczorowski G.I. Biochemistry. 1988; 27: 2403-2409Crossref PubMed Scopus (52) Google Scholar, 16Kaczorowski G.I. Slaughter R.S. King V.F. Garcia M.L. Biochim. Biophys. Acta. 1989; 988: 287-302Crossref PubMed Scopus (121) Google Scholar). However, the main problem is that amiloride derivatives inhibit a number of other Na50-transport systems (e.g. Na50,K50-ATPase, Na50-H50 exchanger), displaying IC50 in a micromolar range(15Slaughter R.S. Garcia M.L. Cragoe E.J. Reeves J.P. Kaczorowski G.I. Biochemistry. 1988; 27: 2403-2409Crossref PubMed Scopus (52) Google Scholar, 16Kaczorowski G.I. Slaughter R.S. King V.F. Garcia M.L. Biochim. Biophys. Acta. 1989; 988: 287-302Crossref PubMed Scopus (121) Google Scholar, 17Garty H. FASEB J. 1994; 8: 522-528Crossref PubMed Scopus (113) Google Scholar). Likewise, some ligand-gaited Na50-channel(s) can be inhibited with nanomolar concentrations of amiloride derivatives(17Garty H. FASEB J. 1994; 8: 522-528Crossref PubMed Scopus (113) Google Scholar). Therefore, more selective, potent and bioavailable ligands are badly needed for biomedical research and for a development of effective drugs. The cardiac Na+-Ca2+ exchanger (NCX1) contains a large regulatory intracellular loop(18Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-564Crossref PubMed Scopus (628) Google Scholar, 19Philipson K.D. Nicoll D.A. Curr. Opin. Cell Biol. 1992; 4: 678-683Crossref PubMed Scopus (45) Google Scholar). A 20-amino acid sequence was identified on the intracellular loop as a possible calmodulin-binding domain with an auto-inhibitory potency(20Li Z. Nicoll D.A. Collins A. Hilgemann D.W. Filoteo A.G. Penniston J.T. Weiss J.N. Tomich J.M. Philipson K.D. J. Biol. Chem. 1990; 266: 1014-1020Abstract Full Text PDF Google Scholar). Similar sequences were found before in a number of calmodulin-binding proteins (for review, see Ref. 21). On the basis of this information, the XIP peptide has been synthesized and tested for inhibition of Na+-Ca2+ exchange activities. The XIP peptide inhibits most of the exchanger activity with IC50 = 0.1-1.5 μM(21Carafoli E. FASEB J. 1994; 8: 993-1002Crossref PubMed Scopus (363) Google Scholar), but the inhibitory effect does not tend to completion(22Kleiboeker S.B. Milanick M.A. Hale C.C. J. Biol. Chem. 1992; 267: 17836-17841Abstract Full Text PDF PubMed Google Scholar). Although the XIP peptide is more potent and specific than dichlorobenzamil (a most potent amiloride derivative), this peptide inhibitor may also interact with other calmodulin binding proteins. Likewise, the XIP-binding site is situated at the intracellular surface and thus is inaccessible for most physiological experiments(20Li Z. Nicoll D.A. Collins A. Hilgemann D.W. Filoteo A.G. Penniston J.T. Weiss J.N. Tomich J.M. Philipson K.D. J. Biol. Chem. 1990; 266: 1014-1020Abstract Full Text PDF Google Scholar). We recently found that in the cardiac sarcolemma vesicles the Phe-Met-Arg-Phe-CONH2 (FMRFa)1( 1The abbreviations used are: FMRFaPhe-Met-Arg-Phe-CONH2Mops3-(N-morpholino)propanesulfonic acidArsenazo III2,7-bis(arsenophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acidOxanol-Vbis-(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxanolMerocyanine-5403(2H)-benzoxazolepropanesulfonic acid, 2-(4-(1,3-dibytyltetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-2-butenylideneFRCRCFaPhe-Arg-Cys-Arg-Cys-Phe-CONH2. )tetrapeptide and its analogs yield a complete inhibition of Na+-Ca2+ and Ca2+-Ca2+ exchanges, exhibiting IC50 = 1050-1050M(23Khananshvili D. Price D. Greenberg M. Sarne Y. J. Biol. Chem. 1993; 268: 200-205Abstract Full Text PDF PubMed Google Scholar). The FMRFa-like peptides and opiate agonists and antagonists are mutually exclusive inhibitors of Na+-Ca2+ exchange, suggesting that they may bind to the same site(23Khananshvili D. Price D. Greenberg M. Sarne Y. J. Biol. Chem. 1993; 268: 200-205Abstract Full Text PDF PubMed Google Scholar). But this putative “opiate-like” site lacks the pharmacological properties of known opiate receptors and may be located on the exchanger or at its vicinity (23Khananshvili D. Price D. Greenberg M. Sarne Y. J. Biol. Chem. 1993; 268: 200-205Abstract Full Text PDF PubMed Google Scholar). The inhibitory FMRFa peptides behave as noncompetitive inhibitors in regard to extravesicular calcium and, like the XIP peptide, may interact with the intracellular surface(23Khananshvili D. Price D. Greenberg M. Sarne Y. J. Biol. Chem. 1993; 268: 200-205Abstract Full Text PDF PubMed Google Scholar, 24Khananshvili D. Price D. Greenberg M. 8th International Symposium on Calcium-binding Proteins In Health and Disease. 1992; (Poster Presentation (Abstr. Py 17), Davos/Switzerland)Google Scholar). It was found recently that the XIP and FMRFa peptides can also inhibit the Na+-Ca2+ exchange in squid axons, suggesting that the putative XIP and FMRFa sites are also present in neuronal tissue(25Di Polo R. Beauge L. Am. J. Physiol. 1994; 267: C307-C311Crossref PubMed Google Scholar). Linear peptide inhibitors attribute common structural disadvantages, which seem to be difficult to overcome without application of alternative approaches. For example, both the XIP and FMRFa peptides contain positively charged amino acids Arg and/or Lys, which make them attractive for proteolytic enzymes. It is widely recognized that the short linear peptides undergo numerous conformational transitions, which may decrease the specificity and affinity of peptide-receptor interaction(26Hruby V.J. Al-Obeidi F. Kazmierski W. Biochem. J. 1990; 268: 249-262Crossref PubMed Scopus (552) Google Scholar, 27Toniolo C. Int. J. Pept. Protein Res. 1990; 35: 287-300Crossref PubMed Scopus (268) Google Scholar). The chemical model studies show that the intramolecular cyclization may restrict a conformational flexibility of a peptide structure, resulting improved affinity, selectivity, and stability (26, 28-30). Phe-Met-Arg-Phe-CONH2 3-(N-morpholino)propanesulfonic acid 2,7-bis(arsenophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid bis-(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxanol 3(2H)-benzoxazolepropanesulfonic acid, 2-(4-(1,3-dibytyltetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-2-butenylidene Phe-Arg-Cys-Arg-Cys-Phe-CONH2. In this work, a number of positively charged cyclic hexapeptides (with intramolecular disulfide bond) have been designed and tested for their inhibitory activity. All of the cyclic hexapeptides have the same amino acid composition (Arg2, Cys2, Phe2), differing only in sequence, and all have a C-terminal amide (CONH2). The inhibitory potency of synthetic cyclic hexapeptides was tested on Na+-Ca2+ and Ca2+-Ca2+ exchanges by using the preparation of isolated cardiac sarcolemma vesicles. The present findings may be an attractive starting point for design of even better inhibitors of the Na+-Ca2+ exchanger. The calf sarcolemmal vesicles were isolated at 4°C(7Khananshvili D. Biochemistry. 1990; 29: 2437-2442Crossref PubMed Scopus (75) Google Scholar, 8Khananshvili D. J. Biol. Chem. 1991; 266: 13764-13769Abstract Full Text PDF PubMed Google Scholar, 31Jones L.R. Methods Enzymol. 1988; 157: 85-91Crossref PubMed Scopus (72) Google Scholar, 32Khananshvili D. Weil-Maslansky E. Biochemistry. 1994; 33: 312-319Crossref PubMed Scopus (25) Google Scholar). Ventriculus and intraventrical septa (700-900 g) were minced in meat grinder (Braun, meatmincer/600, Germany) and resuspended in medium I (20 mM Mops/Tris, pH 7.4, 160 mM NaCl, 1 mM EGTA). The pH of suspension was adjusted to 7.4 by 2 M Tris and centrifuged (12,000 50 g for 20 min) to remove blood. The pellets were resuspended in medium I, briefly homogenized at 11,000 rpm (3 50 5 s) in PT-3000 (Kinematica AG/Polytron, Luzern, Switzerland), equipped with PT-DA3030/2M knives, and centrifuged (12,000 50 g for 20 min). The pellets were suspended (1:2) in medium II (20 mM Mops/Tris, pH 7.4, 0.25 M mannitol) containing DNase (10-25 μg/ml) and protease inhibitors (0.2 mM phenylmethanesulfonyl fluoride, 1 μg/ml pepstatin, leupeptin, and aprotenin). The suspension was homogenized 3 50 30 s in PT-3000 (11,000 rpm), followed by centrifugation (12,000 50 g for 20 min). The supernatant was saved, and the pellet was homogenized once again as described above. Combined supernatants were centrifuged at 190,000 50 g (rotor Ti-45) for 30 min, and the pellets were resuspended in 20 mM Mops/Tris, pH 7.4, 0.2 M sucrose. Equal amounts of 1.8 M sucrose was added to the membrane suspension and divided in Ti-45 tubes. On top of this suspension, 0.77 M sucrose/Mops/Tris buffer was next layered, and 0.25 M sucrose/Mops/Tris was then layered on top of 0.77 M sucrose. The samples were centrifuged (190,000 50 g for 2 h), and the membranes at the 0.25M/0.77 M interface were collected. The membrane suspension was diluted 3-fold with water and centrifuged at 190,000 50 g for 1 h. The sarcolemma vesicles (5-14 mg of protein/ml) were stored at −70°C in 20 mM Mops/Tris, pH 7.4, and 0.25 M sucrose. The Na+o-dependent 45Ca uptake of various preparations were 1-5 nmol of Ca50 mg50 s50. The Na50- or Ca50-loaded vesicles were obtained by their incubation either with sodium ([Na50]50 = 160 mM) or calcium ([Ca50]50= 250 μM) at 4°C for 12-18 h or at 37°C for 1 h. The 45Ca uptake in cardiac sarcolemma vesicles was measured by filtration on glass microfiber filters (GF/C Whatman)(32Khananshvili D. Weil-Maslansky E. Biochemistry. 1994; 33: 312-319Crossref PubMed Scopus (25) Google Scholar, 33Reeves J.P. Methods Enzymol. 1988; 157: 505-510Crossref PubMed Scopus (16) Google Scholar). The filters were presoaked in 0.3% polyethylenimine at 4°C for 4-12 h and washed with cold filtration buffer (20 mM Mops/Tris, pH 7.4, 160 mM KCl, 0.5 mM EGTA) before the experiment. The initial rates (t = 1 or 2 s) of Na+o- or Ca2+i-dependent 45Ca uptake were measured at 37°C. The 45Ca uptake was initiated by 20-50-fold dilution of Na+o- or Ca2+i-loaded vesicles (50-120 μg of total protein) in assay medium by using the semi-rapid mixing device(7Khananshvili D. Biochemistry. 1990; 29: 2437-2442Crossref PubMed Scopus (75) Google Scholar, 8Khananshvili D. J. Biol. Chem. 1991; 266: 13764-13769Abstract Full Text PDF PubMed Google Scholar, 24Khananshvili D. Price D. Greenberg M. 8th International Symposium on Calcium-binding Proteins In Health and Disease. 1992; (Poster Presentation (Abstr. Py 17), Davos/Switzerland)Google Scholar, 32Khananshvili D. Weil-Maslansky E. Biochemistry. 1994; 33: 312-319Crossref PubMed Scopus (25) Google Scholar). The assay medium (0.25-0.5 ml) contained 20 mM Mops/Tris, pH 7.4, 0.25 M sucrose, 2-200 μM50CaCl2 (105-106 cpm/nmol) plus various concentrations of tested cyclic hexapeptide. The “blanks” contained 160 mM NaCl in the assay medium. The cyclic hexapeptides were added to the assay medium 1-5 min before the initiation of 45Ca uptake. The 45Ca uptake reaction was quenched by automatic injection of cold 20 mM Mops/Tris, pH 7.4, 5 mM EGTA, and 160 mM KCl(8Khananshvili D. J. Biol. Chem. 1991; 266: 13764-13769Abstract Full Text PDF PubMed Google Scholar, 24Khananshvili D. Price D. Greenberg M. 8th International Symposium on Calcium-binding Proteins In Health and Disease. 1992; (Poster Presentation (Abstr. Py 17), Davos/Switzerland)Google Scholar, 32Khananshvili D. Weil-Maslansky E. Biochemistry. 1994; 33: 312-319Crossref PubMed Scopus (25) Google Scholar). Quenched solutions were filtered on GF/C filters (the filtration rate was controlled by a Gilford-3021 pressure regulator), and collected vesicles were washed (5 50 5 ml) with cold buffer (Tris/Mops/KCl plus 0.5 mM EGTA) by using Eppendorf Multipette 4780. The reaction timing was controlled by RTB-MP-2N timer (IDEC, Japan) connected to a computerized high performance peristaltic pump (Perifill IQ 200, Zinsser-Analytic, UK/Germany). The kinetic parameters (IC50, K50 and Vmax) and their standard errors (± S.E.) were calculated by GraFit v3.0 (written by R. J. Leatherbarrow, Erithacus Software Ltd). When varying concentrations of 45Ca were added to the assay medium, the calcium concentrations plotted as [Ca50]50 = [50Ca]50+ [Ca50]50+ [Ca50]50, and the specific radioactivity was corrected as [50Ca]50/[Ca50]50 for each concentration of added [50Ca]50. [Ca50]50 represents the endogenous (ambient) calcium in the assay medium and [Ca50]50 is the final concentration of calcium obtained by dilution of vesicles (in the case of Na50-loaded vesicles, the [Ca50]50 = 0). Free calcium concentrations were measured by Arsenazo III(34Scarpa A. Methods Enzymol. 1979; 56: 301-338Crossref PubMed Scopus (229) Google Scholar, 35Bauer P.J. Anal. Biochem. 1981; 110: 61-72Crossref PubMed Scopus (102) Google Scholar). Protein was determined as outlined before(36Markwell M.A.K. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5331) Google Scholar). The voltage-sensitive dyes Oxanol-V and Merocyanine-540 (37Bashford C.L. Smith J.C. Methods Enzymol. 1979; 55: 569-586Crossref PubMed Scopus (95) Google Scholar) were used for measuring the inside positive potential, generated by Na+-Ca2+ exchange. The Na+o-dependent Ca2+i efflux (25°C) was done in 2 ml of assay medium (20 mM Mops/Tris, pH 7.4, and 0.25 M sucrose) with 3 μM Oxanol-V or Merocyanine-540. The vesicles were preloaded with 1 mM CaCl2 at 4°C for 12-18 h. The Ca50-loaded vesicles (60 μg of protein) were diluted in the assay medium, and the reaction of Na+o-dependent [Ca50]50 efflux was initiated by addition of 4 M NaCl to give a final concentration of 100 mM. The spectral changes of Oxanol-V (from 550 to 650 nm) or Merocyanine-540 (from 450 to 600 nm) were measured in computerized Hewlett Packard 8452A diode array spectrophotometer with 0.5-s intervals. For kinetic studies, the double wavelength differences, A50-A50 (Merocyanine-540) or A50-A50 (Oxanol-V), were measured with 0.1-s intervals, and the data were automatically plotted versus time. Stock solutions (1 mM) of Oxanol-V and Merocyanine-540 were prepared in absolute ethanol and stored in the dark at −20°C. The cyclic hexapeptides were designed by Dr. Khananshvili and synthesized by Chiron Co. (Drs. Angela DiPasquale and Joe Maeji). Intramolecular disulfide bond has been formed by oxidation of cysteine in the parent peptide. Since the efficiency of cyclization reaction is sequence dependent, after the oxidation step the synthetic cyclic peptides were purified on high pressure liquid chromatography to 75-95% purity, and the formation of intramolecular S-S bond has been confirmed for each peptide by ion spray mass spectrometry. Different batches of cyclic hexapeptides show very similar inhibitory potency. Stock solutions of cyclic peptides were prepared in deionized water to give final concentrations of 1050-1050M (pH 6.3-7.0) and stored at −20°C. No loss of inhibitory potency has been detected within at least 3 months. Deoxyribonuclease I (type DN-25, obtained from bovine pancreas), protease inhibitors (phenylmethanesulfonyl fluoride, pepstatin, leupeptin, aprotenin), and EGTA were purchased from Sigma. Chelex-100 (100-200 mesh) was from Bio-Rad. Arsenazo III was from ICN Pharmaceuticals (Plainview, NY). The glass microfiber filters (GF/C Whatman) were from Tamar (Jerusalem, Israel) or Whatman Int. Ltd. (Maidstone, UK). 50CaCl2 (10-30 mCi/mg) was purchased from DuPont NEN. The scintillation mixture Opti-Fluor for radioactivity counting was from Packard (Groningen, Netherlands). Oxanol-V and Merocyanine-540 were from Molecular Probes, Inc. (Eugene, Oregon). All other reagents used in this work were of analytical or reagent (>99.9%) grade purity. The solutions were prepared with deionized water (17-18 megaohms/cm). The time course of Na+-Ca2+ exchange (Fig. 1A) and its partial reaction the Ca2+-Ca2+ exchange (Fig. 1B) were measured in the absence or presence of extravesicular FRCRCFa. The Na+o-dependent or Ca2+i-dependent 45Ca uptake was measured by mixing the vesicles with the reaction mixture in the semi-rapid mixer. The Na50- or Ca50-loaded vesicles were rapidly diluted (50-fold) in the assay medium (20 mM Mops/Tris, pH 7.4, 0.25 M sucrose, 14 μM50CaCl2) without or with 70 μM FRCRCFa. The exchange reactions were stopped at various times (t = 1-10 s) by injecting the quenching solution (Mops/Tris/KCl buffer with 5 mM EGTA) in the reaction mixture. As can be seen from Fig. 1, the FRCRCFa peptide inhibits both the Na+-Ca2+ exchange (Fig. 1A) and Ca2+-Ca2+ exchange (Fig. 1B). Likewise, the peptide-induced inhibition for both exchange reactions is already maximal at t = 1 s (shortest time available for mixing). These data suggest that the binding of FRCRCFa to a putative inhibitory site does not involve a “slow” process. The inhibitory effect of seven cyclic hexapeptides were examined on Na+-Ca2+ exchange with a goal to identify a most potent peptide inhibitor. The initial rates (t = 2 s) of Na+o-dependent 45Ca uptake were measured with unsaturating [Ca50]50 = 12-15 μM50CaCl2 and saturating [Na50]50 = 160 mM and varying concentrations of cyclic hexapeptides (). Among the tested peptides, the FRCRCFa is a most potent inhibitor of Na+-Ca2+ exchange, showing IC50 = 10 ± 3 μM. Similar results were obtained with five batches of synthetic FRCRCFa peptide (75-95% purity) and with five different preparations of sarcolemma vesicles (n = 25). Shorter cyclic peptides (4-5 amino acids) that contain only one Arg exhibit IC50 > 250 μM (not shown). Since the Na+-Ca2+ exchange can operate in forward (Na5050-dependent Ca50 influx) and reverse (Na5050-dependent Ca50 efflux) modes, the effect of 20 μM FRCRCFa was tested on both exchange modes. In these experiments, the extravesicular calcium concentrations were measured, by monitoring OD (0.1-s intervals) of Arsenazo III (A50-A50) (Fig. 2). The Na+o-dependent Ca50 influx was initiated by addition of Na50-loaded vesicles to the assay medium with 9.5 μM CaCl2 and 20 μM Arsenazo III, and then the exchange mode was reversed to the Na+o-dependent Ca50 efflux by addition of 100 mM NaCl (Fig. 2, curvea). This protocol has been used to examine the effect of FRCRCFa on both modes of Na+-Ca2+ exchange (Na5050-dependent Ca50 influx and Na+o-dependent Ca50 efflux). As can be seen from Fig. 2(curvesb and c), 20 μM FRCRCFa is able to block both the forward and reverse modes of Na+-Ca2+ exchange. Although the reverse mode of Na+-Ca2+ exchange can be observed by following a time course of Na+o-dependent 45Ca efflux from 50Ca-loaded vesicles(23Khananshvili D. Price D. Greenberg M. Sarne Y. J. Biol. Chem. 1993; 268: 200-205Abstract Full Text PDF PubMed Google Scholar), a quantitative estimation of exchange rates is not an easy task. For example, less than 5-10% release of loaded 45Ca has to be measured for estimating the initial rates of exchange (expected signal might be very close to the experimental error). Here, we used an alternative approach. Since the cardiac sarcolemma Na+-Ca2+ exchanger is able to generate a membrane potential (3Na50:Ca50), we measured a positive-inside potential by using the voltage-sensitive probes Oxanol-V (Fig. 3A) and Merocyanine-540 (Fig. 3B). The reverse mode of Na+-Ca2+ exchange (Na5050-dependent Ca50 efflux) was measured in the absence (curvea) or presence of 1-20 μM FRCRCFa (curvesb-f). The Ca50-loaded vesicles ([Ca50]50 = 1 mM) were added to the assay medium containing the optical probe (3 μM) plus various concentrations of FRCRCFa. The Na+o-dependent Ca50 efflux was initiated by injection of 100 mM NaCl. The optical signals were measured at two different wavelengths, and OD differences were plotted as A50-A50 for Oxanol-V (Fig. 3A) or A50-A50 for Merocyanine-540 (Fig. 3B). In both dye-assay systems, FRCRCFa is a potent inhibitor, showing IC50 = 2-3 μM, while a complete inhibition of optical signal is achieved at 20 μM FRCRCFa. Similar inhibitory potency was observed for VRCRCFa (not shown). By using the same method of assay, FCRRCFa shows IC50≈ 10-20 μM (not shown). A dose response of FRCRCFa was tested on the initial rates (t = 2 s) of Na+-Ca2+ exchange and its partial reaction the Ca2+-Ca2+ exchange. The Na+o- and Ca2+i-dependent 45Ca uptake was measured with unsaturating [50Ca]50 = 13 μM50CaCl2 and saturating concentrations of intravesicular calcium (250 μM CaCl2) or sodium (160 mM NaCl). As can be seen from Fig. 4, FRCRCFa inhibits both the Ca2+-Ca2+ exchange (IC50 = 6.8 ± 3.2 μM) and the Na+-Ca2+ exchange (IC50 = 10.0 ± 1.6 μM) with a similar potency. Although the rate of Na+-Ca2+ exchange is 503-fold higher than the rate of Ca2+-Ca2+ exchange, the fraction of inhibition at each concentration of FRCRCFa is similar for both exchange modes (Fig. 4B). Other cyclic hexapeptides inhibit the Na+-Ca2+ and Ca2+-Ca2+ exchanges in a similar way, although the IC50 values are higher (not shown). The [L-Arg4]- and [D-Arg4]FRCRCFa peptides show a similar dose response (not shown), suggesting that the L-Arg450D-Arg4 substitution does not significantly effect the inhibitory potency of the cyclic hexapeptide. Therefore, this substitution may increase the proteolytic resistance of FRCRCFa without decreasing the inhibitory potency. As in the case of FLRFa(23Khananshvili D. Price D. Greenberg M. Sarne Y. J. Biol. Chem. 1993; 268: 200-205Abstract Full Text PDF PubMed Google Scholar), the FRCRCFa hexapeptide inhibits the Na+-Ca2+ and Ca2+-Ca2+ exchanges in trypsin-treated vesicles (not shown). The inhibitory potency of FRCRCFa was tested in the sucrose medium at various pH levels (6.4, 7.4, 8.4) or at fixed pH 7.4 in the sucrose, choline-Cl, and KCl media. At fixed pH 7.4, the observed IC50 values were not significantly different for sucrose, choline-Cl, or KCl medium (). By increasing pH from 7.4 to 8.4, the inhibitory potency of FRCRCFa is decreased (IC50 is increased 501.5-2-fold). Similar effect was observed before for linear FMRFa tetrapeptides(23Khananshvili D. Price D. Greenberg M. Sarne Y. J. Biol. Chem. 1993; 268: 200-205Abstract Full Text PDF PubMed Google Scholar, 24Khananshvili D. Price D. Greenberg M. 8th International Symposium on Calcium-binding Proteins In Health and Disease. 1992; (Poster Presentation (Abstr. Py 17), Davos/Switzerland)Google Scholar). To characterize the FRCRCFa-induced inhibition, the initial rates (t = 1 s) of Na+-Ca2+ exchange were measured with varying concentrations of extravesicular 45Ca (2-200 μM) and fixed [Na50]50 = 160 mM in the absence or presence of 40 μM FRCRCFa. Eadie-Hofstee analysis of the data shows that FRCRCFa affects Vmax rather than K50, suggesting that this peptide is a noncompetitive inhibitor for extravesicular calcium (Fig. 5). Similar inhibitory type was observed with [D-Arg4]FRCRCFa and FCRRCFa (not shown). Likewise, for Ca2+-Ca2+ exchange FRCRCFa is also a noncompetitive inhibitor in regard to extravesicular calcium under condition in which [50Ca]50 = 2-200 μM and [Ca50]50 = 250 μM (not shown). These data indicate that similar inhibitory mechanisms may involve both the Na+-Ca2+ and Ca2+-Ca2+ exchanges, while the cyclic hexapeptide may prevent the ion movements through the exchanger rather than the ion binding. The present work is a first attempt for identifying the short cyclic peptides with a potency to inhibit the Na+-Ca2+ exchange. The basic idea was to restrict (at least partially) a conformational flexibility of positively charged hexapeptides by cyclization of the peptide with intramolecular S-S bond. The structure of cyclic hexapeptides was different in their amino acid sequence, although the amino acid content was the same for all cyclic hexapeptides. Namely, each cyclic hexapeptide consists of two arginines (to maintain a positive charge), two phenylalanines (to control hydrophobicity), and two cysteines (to form an intramolecular S-S bond). It was found that the seven cyclic hexapeptides show a quite distinct inhibitory potency for Na+-Ca2+ exchange and its partial reaction Ca2+-Ca2+ exchange, displaying the characteristic IC50 values in the range of 2-300 μM (, Fig. 1, 3, and 4). The effect of FRCRCFa on the time course (t = 1-10 s) of Na+-Ca2+ and Ca2+-Ca2+ exchanges shows that after 1 s of the peptide exposure to the vesicles, a maximal inhibitory effect is observed (Fig. 1). This means that the interaction of FRCRCFa with a putative inhibitory site does not involve a “slow” binding process. The forward (Na5050-dependent Ca50 influx) and reverse (Na5050-dependent Ca50 efflux) modes of Na+-Ca2+ exchange were measured in the absence or presence of 20 μM FRCRCFa, when the calcium concentration in the assay medium was assayed by optical probe Arsenazo III (Fig. 2). These data demonstrate that both the forward and reverse modes of Na+-Ca2+ exchange are effectively blocked by 20 μM cyclic hexapeptide. FRCRCFa inhibits both the Na+o- or Ca2+i-dependent 45Ca uptake with IC50 = 10 ± 2 μM and IC50 = 7 ± 3 μM, respectively (Fig. 4), suggesting that the cyclic hexapeptide inhibits not only the forward and reverse modes of Na+-Ca2+ exchange but also the partial reaction of the main mode, the Ca2+-Ca2+ exchange. The reverse mode of Na+-Ca2+ exchange (Na5050-dependent Ca50 efflux) was monitored by voltage-sensitive optical probes Oxanol-V (Fig. 3A) or Merocyanine-540 (Fig. 3B). In both dye-assay systems, FRCRCFa inhibits Na+-Ca2+ exchange with IC50 = 2-3 μM, reaching a complete inhibition at 20 μM FRCRCFa (Fig. 3). Complete inhibition of exchange reactions was also observed before for FMRFa tetrapeptides(23Khananshvili D. Price D. Greenberg M. Sarne Y. J. Biol. Chem. 1993; 268: 200-205Abstract Full Text PDF PubMed Google Scholar, 24Khananshvili D. Price D. Greenberg M. 8th International Symposium on Calcium-binding Proteins In Health and Disease. 1992; (Poster Presentation (Abstr. Py 17), Davos/Switzerland)Google Scholar). It was recently found that the addition of FMRFa to the axoplasmic side inhibits the Na+-Ca2+ exchange in squid axons, suggesting that the relevant site is conserved in neuronal tissue (25Di Polo R. Beauge L. Am. J. Physiol. 1994; 267: C307-C311Crossref PubMed Google Scholar). It is worthwhile to note that the inhibitory potency of FRCRCFa, observed for Na+o-dependent Ca50 efflux (IC50 = 2-3 μM) is at least 3-5-fold higher than the inhibitory potency observed for Na+o-dependent 45Ca uptake (IC50 = 8-12 μM). These quantitative differences were observed on a regular basis (more than 20 independent experiments, in which five different preparations of sarcolemma vesicles were used) and cannot be explained by experimental error. It is not clear at this moment whether these differences reveal distinct properties of the forward (Na5050-dependent Ca50 influx) and reverse (Na5050-dependent Ca50 efflux) modes of Na+-Ca2+ exchange or whether they reflect the methodological disparities of applied procedures. In contrast to FRCRCFa, XIP is a more potent inhibitor for 50-dependent Ca50 influx (IC50 = 0.5-1 μM) as compared with the Na+o-dependent Ca50 efflux (IC50 > 5 μM)(22Kleiboeker S.B. Milanick M.A. Hale C.C. J. Biol. Chem. 1992; 267: 17836-17841Abstract Full Text PDF PubMed Google Scholar). These diverse properties suggest that FRCRCFa and XIP may bind to distinct inhibitory sites. A substitution of L-Arg4 by D-Arg4 in the FRCRCFa peptide does not alter significantly the inhibitory potency of the cyclic hexapeptide (not shown). Therefore, this type of substitution may increase the stability of cyclic hexapeptides against proteases. Since it is expected that the peptide cyclization already increases the proteolytic resistance(39Zukermann R.N. Curr. Opin. Struct. Biol. 1994; 3: 580-584Crossref Scopus (38) Google Scholar), the L-Arg450D-Arg4 substitution may produce an even more stable peptide structure. Likewise, a potency of FCRCRFa-induced inhibition is relatively insensitive to pH and potassium (Table II) and to extravesicular calcium concentrations (not shown).TABLE II Open table in a new tab At fixed [Na50]50 = 160 mM and varying [50Ca]50 = 2-200 μM, FRCRCFa decreases Vmax with a little change of K50 (Fig. 5), suggesting that FRCRCFa is a noncompetitive inhibitor for extravesicular calcium during the Na+-Ca2+ exchange. Similar results were obtained for Ca2+-Ca2+ exchange with [50Ca]50 = 2-200 μM and [Ca50]50 = 200 μM (not shown). In the frame of consecutive mechanism(7Khananshvili D. Biochemistry. 1990; 29: 2437-2442Crossref PubMed Scopus (75) Google Scholar, 8Khananshvili D. J. Biol. Chem. 1991; 266: 13764-13769Abstract Full Text PDF PubMed Google Scholar, 32Khananshvili D. Weil-Maslansky E. Biochemistry. 1994; 33: 312-319Crossref PubMed Scopus (25) Google Scholar), the inhibition of the calcium transport step (Fig. SI) can be interpreted as follows. The binding of FRCRCFa to the inhibitory site (located either on the exchanger or at its vicinity) prevents the calcium and/or sodium translocation through the exchanger (β50 0) rather than affecting the ion binding to the exchanger (α50 1). The inhibitory potency of FRCRCFa is significantly higher than the most popular amiloride derivatives (benzamil, dichlorobenzamil, or benzobenzamil). Although the inhibitory potency of FRCRCFa (or VRCRCFa) and XIP peptides seem to be not so different, in contrast to XIP, the FRCRCFa-induced inhibition attends to completion (Figure 2:, Figure 3:). FCRCRFa has a number of advantages as compared with the active linear tetrapeptides (e.g. HMRFa and VMRFa): (a) the cyclic hexapeptide represents a conformationally more stable peptide structure; (b) it does not contain chemically unstable amino acids (e.g. Met, the oxidation of which may decrease the inhibitory potency of the tetrapeptide); and (c) the cyclic peptides are expected to be more resistant to proteolytic enzymes than the linear peptides. A systematic application of more sophisticated molecular approaches may produce new “peptido-mimetic” blockers with improved pharmacokinetics and therapeutic potency. The cardiac sarcolemma vesicles (11-14 mg of protein/ml) were preloaded with 160 mM NaCl at 4°C for 14-18 h (see “Materials and Methods”). The initial rates (t = 2 s) of Na+o-dependent 45Ca uptake were measured at 37°C by using a semi-rapid mixer, as described under “Materials and Methods.” The standard assay medium contained 20 mM Mops/Tris, pH 7.4, 0.25 M sucrose, 12-15 μM50CaCl2, and various concentrations of cyclic hexapeptides. The IC50 values and their standard errors (±S.E.) were estimated by fitting the calculated lines to the experimental lines (GraFit program) as outlined under “Materials and Methods.” The Na50-loaded vesicles were obtained by incubation of cardiac sarcolemma vesicles (14 mg of protein/ml) with 160 mM NaCl at 37°C for 1 h (see “Materials and Methods”). The Na+-Ca2+ exchange was assayed by measuring the initial rates (t = 2 s) of Na+o-dependent 45Ca uptake in the semi-rapid mixer (see also Fig. 1 and Table I). The standard assay medium contained 20 mM Bis-Tris propane (fixed at pH 6.4, 7.4, or 8.4) plus indicated concentrations of either sucrose, choline-Cl, or KCl. Other components of the assay medium were 15-18 μM ▨ ▨ 50CaCl2 and varying concentrations of FRCRCFa (0.1-150 μM). The IC50 values were estimated by computing and fitting the curves to the experimental points (see “Materials and Methods” and Table I).TABLE I Open table in a new tab" @default.
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