From the
Positively charged cyclic hexapeptides have been synthesized and
tested for their effects on the cardiac sarcolemma
Na
The cell membrane Na
Amiloride and its
derivatives have been identified as relatively effective inhibitors of
Na
The cardiac
Na
We recently found that
in the cardiac sarcolemma vesicles the Phe-Met-Arg-Phe-CONH
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
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
The
Na
The voltage-sensitive dyes Oxanol-V and
Merocyanine-540 (37) were used for measuring the inside positive
potential, generated by Na
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
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).
The present work is a first attempt for identifying the short
cyclic peptides with a potency to inhibit the
Na
The forward
(Na
The reverse mode of
Na
It is worthwhile to note that the inhibitory potency of FRCRCFa,
observed for Na
At fixed
[Na
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
The
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.
-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) .
-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
-10
M(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.
-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) .
(FMRFa)
(
)tetrapeptide and its analogs
yield a complete inhibition of Na
-Ca
and Ca
-Ca
exchanges,
exhibiting IC
=
10
-10
M(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).
, 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.
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
.
- 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 µM
CaCl
(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) .
-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.
-10
M (pH 6.3-7.0) and stored at -20 °C. No
loss of inhibitory potency has been detected within at least 3 months.
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).
Effect of FRCRCFa on the Time Course of
Na
The time course of
Na- and
Ca
-dependent
Ca
Uptake
-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 µM
CaCl
) 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 µM
CaCl
) 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 µM
CaCl
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
Since the Na-dependent Ca
Influx and Na
-dependent
Ca
Efflux, Monitored by Arsenazo
III
-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
Although the reverse mode of
Na-dependent Ca
Efflux
-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
A dose
response of FRCRCFa was tested on the initial rates (t = 2 s) of Na-Ca
and
Ca
-Ca
Exchanges
-Ca
exchange
and its partial reaction the Ca
-Ca
exchange. The Na
- and
Ca
-dependent
Ca
uptake was measured with unsaturating
[
Ca]
= 13 µM
CaCl
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 µM
CaCl
, 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 µM
CaCl
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
.
-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.
-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.
-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).
-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).
]
= 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.
Table: Effect of different cyclic hexapeptides
on Na-Ca
exchange
-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 µM
CaCl
, 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
-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).
; 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
.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.