©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Positively Charged Cyclic Hexapeptides, Novel Blockers for the Cardiac Sarcolemma Na-Ca Exchanger (*)

Daniel Khananshvili (§) , Gilat Shaulov , Evelyne Weil-Maslansky , David Baazov

From the (1)Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Positively charged cyclic hexapeptides have been synthesized and tested for their effects on the cardiac sarcolemma Na-Ca 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-Ca exchange and its partial reaction, the Ca-Ca exchange, by measuring the Ca 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-Ca exchange with a different potency (IC = 2-300 µM). Phe-Arg-Cys-Arg-Cys-Phe-CONH (FRCRCFa) inhibits the Na-dependent Ca uptake (Na-Ca exchange) and Ca-dependent Ca uptake (Ca-Ca exchange) in the isolated cardiac sarcolemma vesicles with IC = 10 ± 2 µM and IC = 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-dependent Ca efflux, was monitored by Oxanol-V (A-A) or Merocyanine-540 (A-A). In both assay systems, FRCRCFa inhibits the Na-Ca exchange with IC = 2-3 µM, while a complete inhibition occurs at 20 µM FRCRCFa. The forward (Na-dependent Ca influx) and reverse (Na-dependent Ca efflux) modes of Na-Ca exchange, monitored by Arsenazo III (A-A), are also inhibited by FRCRCFa. The L-Arg D-Arg substitution in FRCRCFa does not alter the IC, meaning that this structural change may increase a proteolytic resistance without a loss of inhibitory potency. At fixed [Na] (160 mM) or [Ca] (250 µM) and varying Ca(2-200 µM), FRCRCFa decreases V without altering the K. Therefore, FRCRCFa is a noncompetitive inhibitor in regard to extravesicular Ca either for Na-Ca or Ca-Ca exchange. It is suggested that FRCRCFa prevents the ion movements through the exchanger rather than the ion binding.


INTRODUCTION

The cell membrane Na-Ca exchanger is a major regulator of intracellular calcium in cardiac and neuronal cells during the resting and action potentials(1, 2) . The cardiac sarcolemma Na-Ca exchange is the only electrogenic system (3Na:Ca) that provides a voltage-sensitive extrusion of intracellular calcium that has entered the cell via the Ca channels(3, 4) . The cardiac Na-Ca exchanger is a typical carrier-type system(5, 6) , which can also catalyze the Ca-Ca and Na-Na exchanges. The Na-Ca exchange cycle and its partial reactions can be described as separate movements of Na and Ca (so called consecutive or ping-pong mechanism) through the exchanger(7, 8, 9, 10, 11) . A contribution of exchange modes to cellular activities as well as their catalytic and regulatory mechanisms are poorly understood(12, 13, 14) .

Amiloride and its derivatives have been identified as relatively effective inhibitors of Na-Ca exchange(15, 16) , but their application is strictly limited for most biomedical experiments. For example, most popular amiloride analogs (e.g. benzamil, dichlorobenzamil, or benzobenzamil) inhibit Na-Ca exchange with a relatively low potency, exhibiting IC = 10-10M(15, 16) . However, the main problem is that amiloride derivatives inhibit a number of other Na-transport systems (e.g. Na,K-ATPase, Na-H exchanger), displaying IC in a micromolar range(15, 16, 17) . Likewise, some ligand-gaited Na-channel(s) can be inhibited with nanomolar concentrations of amiloride derivatives(17) . Therefore, more selective, potent and bioavailable ligands are badly needed for biomedical research and for a development of effective drugs.

The cardiac Na-Ca exchanger (NCX1) contains a large regulatory intracellular loop(18, 19) . A 20-amino acid sequence was identified on the intracellular loop as a possible calmodulin-binding domain with an auto-inhibitory potency(20) . 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-Ca exchange activities. The XIP peptide inhibits most of the exchanger activity with IC = 0.1-1.5 µM(21) , but the inhibitory effect does not tend to completion(22) . 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(20) .

We recently found that in the cardiac sarcolemma vesicles the Phe-Met-Arg-Phe-CONH (FMRFa)()tetrapeptide and its analogs yield a complete inhibition of Na-Ca and Ca-Ca exchanges, exhibiting IC = 10-10M(23) . The FMRFa-like peptides and opiate agonists and antagonists are mutually exclusive inhibitors of Na-Ca exchange, suggesting that they may bind to the same site(23) . 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 (23). The inhibitory FMRFa peptides behave as noncompetitive inhibitors in regard to extravesicular calcium and, like the XIP peptide, may interact with the intracellular surface(23, 24) . It was found recently that the XIP and FMRFa peptides can also inhibit the Na-Ca exchange in squid axons, suggesting that the putative XIP and FMRFa sites are also present in neuronal tissue(25) . 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(26, 27) . 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).

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 (Arg, Cys, Phe), differing only in sequence, and all have a C-terminal amide (CONH). The inhibitory potency of synthetic cyclic hexapeptides was tested on Na-Ca and Ca-Ca 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-Ca exchanger.


MATERIALS AND METHODS

The calf sarcolemmal vesicles were isolated at 4 °C(7, 8, 31, 32) . 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 g for 20 min) to remove blood. The pellets were resuspended in medium I, briefly homogenized at 11,000 rpm (3 5 s) in PT-3000 (Kinematica AG/Polytron, Luzern, Switzerland), equipped with PT-DA3030/2M knives, and centrifuged (12,000 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 30 s in PT-3000 (11,000 rpm), followed by centrifugation (12,000 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 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 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 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-dependent Ca uptake of various preparations were 1-5 nmol of Ca mg s.

The Na- or Ca-loaded vesicles were obtained by their incubation either with sodium ([Na] = 160 mM) or calcium ([Ca]= 250 µM) at 4 °C for 12-18 h or at 37 °C for 1 h. The Ca uptake in cardiac sarcolemma vesicles was measured by filtration on glass microfiber filters (GF/C Whatman)(32, 33) . 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- or Ca-dependent Ca uptake were measured at 37 °C. The Ca uptake was initiated by 20-50-fold dilution of Na- or Ca-loaded vesicles (50-120 µg of total protein) in assay medium by using the semi-rapid mixing device(7, 8, 24, 32) . The assay medium (0.25-0.5 ml) contained 20 mM Mops/Tris, pH 7.4, 0.25 M sucrose, 2-200 µMCaCl (10-10 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 Ca uptake. The Ca uptake reaction was quenched by automatic injection of cold 20 mM Mops/Tris, pH 7.4, 5 mM EGTA, and 160 mM KCl(8, 24, 32) . 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 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 (IC, K and V) and their standard errors (± S.E.) were calculated by GraFit v3.0 (written by R. J. Leatherbarrow, Erithacus Software Ltd). When varying concentrations of Ca were added to the assay medium, the calcium concentrations plotted as [Ca] = [Ca] + [Ca] + [Ca], and the specific radioactivity was corrected as [Ca]/[Ca] for each concentration of added [Ca]. [Ca] represents the endogenous (ambient) calcium in the assay medium and [Ca] is the final concentration of calcium obtained by dilution of vesicles (in the case of Na-loaded vesicles, the [Ca] = 0). Free calcium concentrations were measured by Arsenazo III(34, 35) . Protein was determined as outlined before(36) .

The voltage-sensitive dyes Oxanol-V and Merocyanine-540 (37) were used for measuring the inside positive potential, generated by Na-Ca exchange. The Na-dependent Ca 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 CaCl at 4 °C for 12-18 h. The Ca-loaded vesicles (60 µg of protein) were diluted in the assay medium, and the reaction of Na-dependent [Ca] 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, A-A (Merocyanine-540) or A-A (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 10-10M (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). CaCl (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).


RESULTS

Effect of FRCRCFa on the Time Course of Na- and Ca-dependent Ca Uptake

The time course of Na-Ca exchange (Fig. 1A) and its partial reaction the Ca-Ca exchange (Fig. 1B) were measured in the absence or presence of extravesicular FRCRCFa. The Na-dependent or Ca-dependent Ca uptake was measured by mixing the vesicles with the reaction mixture in the semi-rapid mixer. The Na- or Ca-loaded vesicles were rapidly diluted (50-fold) in the assay medium (20 mM Mops/Tris, pH 7.4, 0.25 M sucrose, 14 µMCaCl) 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-Ca exchange (Fig. 1A) and Ca-Ca 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.


Figure 1: Effect of extravesicular FRCRCFa on the time course of Na- or Ca-dependent Ca uptake. The time course (t = 1-10 s) of Na-dependent Ca uptake (A) or Ca-dependent Ca uptake (B) were measured in the absence (, ) or presence (, ) of 70 µM FRCRCFa in the assay medium. Before the experiment, the sarcolemma vesicles (13.8 mg of protein/ml) were preloaded with 160 mM NaCl or 250 µM CaCl at 4 °C for 14-18 h as described under ``Materials and Methods.'' The Na-loaded (, ) or Ca-loaded (, ) vesicles (138 µg) were diluted 50-fold in 0.5 ml of assay medium (20 mM Mops/Tris, pH 7.4, 0.25 M sucrose, and 13 µMCaCl) at 37 °C. At the indicated times, the Ca uptake was stopped by injecting the quenching buffer in the semi-rapid mixer. Intravesicular Ca was measured by filtration of quenched solutions on GF/C filters (see ``Materials and Methods''). The blanks were taken for each time point and subtracted.



Inhibitory Potency of Different Cyclic Hexapeptides

The inhibitory effect of seven cyclic hexapeptides were examined on Na-Ca exchange with a goal to identify a most potent peptide inhibitor. The initial rates (t = 2 s) of Na-dependent Ca uptake were measured with unsaturating [Ca] = 12-15 µMCaCl and saturating [Na] = 160 mM and varying concentrations of cyclic hexapeptides (). Among the tested peptides, the FRCRCFa is a most potent inhibitor of Na-Ca exchange, showing IC = 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 IC > 250 µM (not shown).

FRCRCFa-induced Inhibition of Na-dependent CaInflux and Na-dependent CaEfflux, Monitored by Arsenazo III

Since the Na-Ca exchange can operate in forward (Na-dependent Ca influx) and reverse (Na-dependent Ca 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 (A-A) (Fig. 2). The Na-dependent Ca influx was initiated by addition of Na-loaded vesicles to the assay medium with 9.5 µM CaCl and 20 µM Arsenazo III, and then the exchange mode was reversed to the Na-dependent Ca 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-Ca exchange (Na-dependent Ca influx and Na-dependent Ca 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-Ca exchange.


Figure 2: Effect of FRCRCFa on the Na-dependent Ca influx and Na-dependent Ca efflux, measured with a calcium probe Arsenazo III. The Na-dependent Ca influx and Na-dependent Ca efflux modes of Na-Ca exchange were measured in the absence (curvea) or presence (curvesb and c) of 20 µM FRCRCFa. The assay medium contained 20 mM Bis/Tris propane, pH 7.4, 0.25 M sucrose, 9.5 µM CaCl (2.5 µM ambient calcium plus 7 µM of added calcium), and 20 µM Arsenazo III. The sarcolemma vesicles (11 mg of protein/ml) were preloaded with 160 mM NaCl at 4 °C for 12-18 h. The Na-dependent Ca influx was initiated by addition of 77-µg vesicles, and the exchange was reversed to the Na-dependent Ca efflux by addition of 4 M NaCl to give a final concentration of 100 mM (curvea). For inhibition, the Na-dependent Ca influx or Na-dependent Ca efflux FRCRCFa was added either before (curveb) or after (curvec) addition of vesicles. The differences in A-A were measured with 0.1-s intervals in a computerized Hewlett Packard 8452A diode array spectrophotometer, equipped with a controlled stirring device.



Effect of FRCRCFa on the Positive Inside Potential, Generated by Na-dependent CaEfflux

Although the reverse mode of Na-Ca exchange can be observed by following a time course of Na-dependent Ca efflux from Ca-loaded vesicles(23) , a quantitative estimation of exchange rates is not an easy task. For example, less than 5-10% release of loaded Ca 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-Ca exchanger is able to generate a membrane potential (3Na:Ca), 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-Ca exchange (Na-dependent Ca efflux) was measured in the absence (curvea) or presence of 1-20 µM FRCRCFa (curvesb-f). The Ca-loaded vesicles ([Ca] = 1 mM) were added to the assay medium containing the optical probe (3 µM) plus various concentrations of FRCRCFa. The Na-dependent Ca efflux was initiated by injection of 100 mM NaCl. The optical signals were measured at two different wavelengths, and OD differences were plotted as A-A for Oxanol-V (Fig. 3A) or A-A for Merocyanine-540 (Fig. 3B). In both dye-assay systems, FRCRCFa is a potent inhibitor, showing IC = 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 IC 10-20 µM (not shown).


Figure 3: Effect of FRCRCFa on membrane potential, generated by Na-dependent Ca efflux. The positive inside membrane potential, generated by Na-Ca exchange, was measured by using the voltage-sensitive dyes Oxanol-V (A) or Merocyanine-450 (B) in the absence or presence of FRCRCFa. The assay medium contained 20 mM Mops/Tris, pH 7.4, 0.25 M sucrose). A, 3 µM of Oxanol-V was added to the assay medium in the absence (curvea) or presence of 2, 5, 10, or 20 µM FRCRCFa (curvesb-e). B, the assay medium contained 3 µM Merocyanine-450 in the absence (curvea) or presence of 1, 2, 5, 10, or 20 µM FRCRCFa (curvesb-f). The sarcolemma vesicles were preloaded with 1 mM CaCl for 12-18 h at 4 °C. 60 µg of Ca-loaded vesicles were added to the assay medium containing the optical probe and various concentrations of FRCRCFa. The Na-dependent Ca efflux was initiated by addition of 4 M NaCl to give a final concentration of 100 mM as indicated by an arrow. The differences in A-A (for Oxanol-V) or A-A (for Merocyanine-540) were measured with 0.1-s intervals by using a computerized Hewlett Packard 8452A diode array spectrophotometer.



Comparison of FRCRCFa-induced Inhibition of Na-Ca and Ca-Ca Exchanges

A dose response of FRCRCFa was tested on the initial rates (t = 2 s) of Na-Ca exchange and its partial reaction the Ca-Ca exchange. The Na- and Ca-dependent Ca uptake was measured with unsaturating [Ca] = 13 µMCaCl and saturating concentrations of intravesicular calcium (250 µM CaCl) or sodium (160 mM NaCl). As can be seen from Fig. 4, FRCRCFa inhibits both the Ca-Ca exchange (IC = 6.8 ± 3.2 µM) and the Na-Ca exchange (IC = 10.0 ± 1.6 µM) with a similar potency. Although the rate of Na-Ca exchange is 3-fold higher than the rate of Ca-Ca exchange, the fraction of inhibition at each concentration of FRCRCFa is similar for both exchange modes (Fig. 4B). Other cyclic hexapeptides inhibit the Na-Ca and Ca-Ca exchanges in a similar way, although the IC values are higher (not shown). The [L-Arg]- and [D-Arg]FRCRCFa peptides show a similar dose response (not shown), suggesting that the L-Arg D-Arg 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(23) , the FRCRCFa hexapeptide inhibits the Na-Ca and Ca-Ca exchanges in trypsin-treated vesicles (not shown).


Figure 4: Inhibition of Na-Ca and Ca-Ca exchanges by various concentrations of FRCRCFa. Before the experiment, the sarcolemma vesicles were preloaded with 160 mM NaCl or 250 µM CaCl as described in Fig. 1. A, the Na-loaded () or Ca-loaded () vesicles were mixed with the assay medium containing 20 mM Mops/Tris, pH 7.4, 0.25 M sucrose, 13 µMCaCl, and 0-100 µM FRCRCFa. The Ca uptake reaction was quenched (t = 2 s) with Mops/Tris/EGTA buffer by using the semi-rapid mixer as described under ``Materials and Methods.'' Intravesicular Ca was measured by filtration as outlined under ``Materials and Methods.'' The lines were calculated to give an optimal fit to the experimental points by using the GraFit program (see ``Materials and Methods''). The IC values were estimated as 6.8 ± 3.2 µM and 10.0 ± 1.6 µM for Ca-Ca () and Na-Ca () exchanges, respectively. B, the data obtained in A were plotted as % of control in the absence of FRCRCFa. 100% corresponds to 0.44 nmol of Ca mg 2 s for Na-Ca exchange and 0.17 nmol of Ca mg 2 s for Ca-Ca exchange.



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 IC 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 (IC is increased 1.5-2-fold). Similar effect was observed before for linear FMRFa tetrapeptides(23, 24) .

Type of FRCRCFa-induced Inhibition

To characterize the FRCRCFa-induced inhibition, the initial rates (t = 1 s) of Na-Ca exchange were measured with varying concentrations of extravesicular Ca (2-200 µM) and fixed [Na] = 160 mM in the absence or presence of 40 µM FRCRCFa. Eadie-Hofstee analysis of the data shows that FRCRCFa affects V rather than K, suggesting that this peptide is a noncompetitive inhibitor for extravesicular calcium (Fig. 5). Similar inhibitory type was observed with [D-Arg]FRCRCFa and FCRRCFa (not shown). Likewise, for Ca-Ca exchange FRCRCFa is also a noncompetitive inhibitor in regard to extravesicular calcium under condition in which [Ca] = 2-200 µM and [Ca] = 250 µM (not shown). These data indicate that similar inhibitory mechanisms may involve both the Na-Ca and Ca-Ca exchanges, while the cyclic hexapeptide may prevent the ion movements through the exchanger rather than the ion binding.


Figure 5: Effect of extravesicular FRCRCFa on K and V of Na-Ca exchange, measured with varying [Ca] and fixed [Na]. The Na-dependent Ca uptake was measured in the semi-rapid mixer as described in Figs. 1 and 4 (see ``Materials and Methods''). The Na (160 mM)-loaded vesicles were mixed with 20 mM Mops/Tris, pH 7.4, 0.25 M sucrose, containing 2-200 µMCaCl in the absence () or presence () of 40 µM FRCRCFa. The initial rates (t = 1 s) of Na-Ca exchange were measured as described under ``Materials and Methods.'' Blanks were taken for each concentration of CaCl and subtracted. The specific radioactivity was corrected according to the [Ca]/[Ca] ratio as outlined under ``Materials and Methods.'' The experimental points were fitted (GraFit program) to the calculated lines, and the data were presented as the Eadie-Hofstee plot. In the absence of FRCRCFa, the parameters were: K = 63.4 ± 8.4 µM and V = 3.4 ± 0.2 nmol Ca mg s; in the presence of FRCRCFa, K = 47.4 ± 5.8 µM and V = 1.6 ± 0.1 nmol Ca mg s.




DISCUSSION

The present work is a first attempt for identifying the short cyclic peptides with a potency to inhibit the Na-Ca 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-Ca exchange and its partial reaction Ca-Ca exchange, displaying the characteristic IC 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-Ca and Ca-Ca 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 (Na-dependent Ca influx) and reverse (Na-dependent Ca efflux) modes of Na-Ca 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-Ca exchange are effectively blocked by 20 µM cyclic hexapeptide. FRCRCFa inhibits both the Na- or Ca-dependent Ca uptake with IC = 10 ± 2 µM and IC = 7 ± 3 µM, respectively (Fig. 4), suggesting that the cyclic hexapeptide inhibits not only the forward and reverse modes of Na-Ca exchange but also the partial reaction of the main mode, the Ca-Ca exchange.

The reverse mode of Na-Ca exchange (Na-dependent Ca 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-Ca exchange with IC = 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(23, 24) . It was recently found that the addition of FMRFa to the axoplasmic side inhibits the Na-Ca exchange in squid axons, suggesting that the relevant site is conserved in neuronal tissue (25).

It is worthwhile to note that the inhibitory potency of FRCRCFa, observed for Na-dependent Ca efflux (IC = 2-3 µM) is at least 3-5-fold higher than the inhibitory potency observed for Na-dependent Ca uptake (IC = 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 (Na-dependent Ca influx) and reverse (Na-dependent Ca efflux) modes of Na-Ca exchange or whether they reflect the methodological disparities of applied procedures. In contrast to FRCRCFa, XIP is a more potent inhibitor for Na-dependent Ca influx (IC = 0.5-1 µM) as compared with the Na-dependent Ca efflux (IC > 5 µM)(22) . These diverse properties suggest that FRCRCFa and XIP may bind to distinct inhibitory sites. A substitution of L-Arg by D-Arg 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(39) , the L-Arg D-Arg substitution may produce an even more stable peptide structure. Likewise, a potency of FCRCRFa-induced inhibition is relatively insensitive to pH and potassium () and to extravesicular calcium concentrations (not shown).

At fixed [Na] = 160 mM and varying [Ca] = 2-200 µM, FRCRCFa decreases V with a little change of K (Fig. 5), suggesting that FRCRCFa is a noncompetitive inhibitor for extravesicular calcium during the Na-Ca exchange. Similar results were obtained for Ca-Ca exchange with [Ca] = 2-200 µM and [Ca] = 200 µM (not shown). In the frame of consecutive mechanism(7, 8, 32) , 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 ( 0) rather than affecting the ion binding to the exchanger ( 1).


Figure SI: Scheme I.



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 ( Fig. 2and Fig. 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.

  
Table: Effect of different cyclic hexapeptides on Na-Ca exchange

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-dependent Ca 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 µMCaCl, and various concentrations of cyclic hexapeptides. The IC 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.''


  
Table: &cjs0822; &cjs0822;FRCRCFa-induced inhibition of Na-Ca exchange at various extravesicular pH levels and assay medium

The Na-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-Ca exchange was assayed by measuring the initial rates (t = 2 s) of Na-dependent Ca 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 &cjs0822; &cjs0822; CaCl and varying concentrations of FRCRCFa (0.1-150 µM). The IC values were estimated by computing and fitting the curves to the experimental points (see ``Materials and Methods'' and Table I).



FOOTNOTES

*
This work is supported by the Israeli Science Foundation (administrated by the Israel Academy of Sciences and Humanities Grant 196/93-1) and the Ministry of Science and the Arts. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Holds the Igal Alon Career Development Fellowship. To whom correspondence should be addressed.

The abbreviations used are: FMRFa, Phe-Met-Arg-Phe-CONH; Mops, 3-(N-morpholino)propanesulfonic acid; Arsenazo III, 2,7-bis(arsenophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid; Oxanol-V, bis-(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxanol; Merocyanine-540, 3(2H)-benzoxazolepropanesulfonic acid, 2-(4-(1,3-dibytyltetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-2-butenylidene; FRCRCFa, Phe-Arg-Cys-Arg-Cys-Phe-CONH.


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