Sites and modes of action of proctolin and the FLP F2 on lobster cardiac muscle
1 Department of Biological Sciences, University of Calgary, Calgary,
Canada
2 Department of Cardiovascular Medicine, Tohoku Graduate School of Medical
Sciences, Sendai, Japan
3 Department of Biology, Tokyo Metropolitan University, Minamiohsawa 1-1,
Hachioji, Japan
4 Faculty of Medicine, University of Calgary, Calgary, Canada
* Author for correspondence (e-mail: wilkens{at}ucalgary.ca)
Accepted 6 December 2004
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Summary |
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Key words: F2, proctolin, calcium, cardiac muscle, lobster
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Introduction |
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The basic heart rhythm is modified by extrinsic nervous and hormonal inputs
(Wilkens, 1995,
1999
). A variety of aminergic
and peptidergic cardioactive hormones directly influence heart function and
may also affect heart function indirectly via actions in the central
nervous system. For those hormones with chronotropic effects the site of
action is the cardiac ganglion. Those that produce inotropic effects can act
at the level of the cardiac ganglion to change burst characteristics that will
alter the level of myocardial depolarization and they may act directly on the
myocardium to change its contractile response to a burst. The neuropeptide
hormones proctolin (PR) (RLYPT; Sullivan,
1979
) and the FRLFamide-like peptides (FLP) F1
(TNRNFLRFamide) and F2 (SDRNFLRFamide)
(Trimmer et al., 1987
) have
chronotropic and inotropic effects on decapod hearts
(Wilkens and Mercier, 1993
;
Saver and Wilkens, 1998
). It
is assumed that each class of peptide binds to separate myocardial receptors
since there is no homology between PR and the FLPs.
PR and F2 act directly on a variety of crustacean, chelicerate
and insect skeletal, cardiac and visceral muscles to increase tonus, the
amplitude of contractions and in some cases induce or increase the frequency
of myogenic contractions (Lange et al.,
1987; Lange, 1988
;
Griffiths, 1990
; Mercier et
al., 1991
,
1997
,
2003
;
Wilkens and Mercier, 1993
;
Groome et al., 1994
;
Skerrett et al., 1995
;
Worden et al., 1995
;
Fuse and Orchard, 1998
;
Saver and Wilkens, 1998
;
Saver et al., 1998
). These
peptides appear to exert their actions by binding to sarcolemmal receptors
and, in the case of PR, a variety of different signal transduction pathways
have been identified in different animals and tissues. PR has been shown to
increase Na+ efflux by activating Ca2+ channels in
barnacle muscle (Nowaga and Bittar, 1985; Bittar and Nowaga, 1989). PR
increases Na+ conductance in lobster cardiac ganglion neurons
(Freschi, 1989
). PR decreases
K+ conductance in isopod skeletal muscle and in lobster cardiac
ganglion neurons (Erxleben et al.,
1995
; Sullivan and Miller,
1984
). In locust skeletal muscle PR decreases K+
conductance via a G protein mediated pathway;
(Walther et al., 1998
). The
mode of action of F2 is much less well understood
(Mercier et al., 2003
).
Furthermore, PR and FLP effects on a variety of arthropod neuromuscular
synapses and muscles requires external Ca2+ (cockroach,
Wegener and Nassel, 2000
;
crab, Rathmayer et al., 2002
;
limulus, Watson and Hoshi,
1985
; crayfish, Wilcox and
Lange, 1995
; Bishop et al.,
1991
). This diversity of hormone-induced effects may represent
true differences in response from animal to animal or it may merely reflect
the focus of the different investigations.
It is known that PR increases contractile force in semi-isolated and intact
crab and lobster heart (Wilkens and
Mercier, 1993; Saver and
Wilkens, 1998
; Wilkens and
Kuramoto, 1998
). This could arise from actions of the peptide on
the cardiac ganglion or directly on the myocardium. In the present study, we
have used the valve muscles of the six ostia of the lobster heart (the
orbicularis ostii muscle or OOM) as a model system
(Yazawa et al., 1999
) to study
of the mode(s) of action of PR and F2 on the myocardium itself. We
have already reported on the role of sarcolemmal and sarcoplasmic reticular
(SR) Ca2+ fluxes in excitation-contraction coupling in this ostial
muscle preparation (Shinozaki et al.,
2002
). We now report on hormonal effects on Ca2+
dynamics at the level of the sarcolemma and SR. The time course of the
peptide-induced responses point to the possible role of second messengers, but
this will be the subject of a future study.
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Materials and methods |
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Membrane potential was measured by hanging or dog-leg shaped
(Fedida et al., 1990)
microelectrodes filled with 3 mol l-1 KCl, tip resistance of 10-30
M
. Hyperpolarizing 5-15 nA current pulses of 100 ms were used to
estimated the input resistance of the fibers. Force was measured isometrically
by means of a Pixie (Endevco Corp., San Juan Capistrano, CA) or SensoNor AE801
(AME, Sensonor, Horten, Norway) force transducer mounted on a micromanipulator
(Yazawa et al., 1999
). The
transducer was moved to pull slack out of the valve leaflets. The force
recorded by this initial stretch is referred to as basal tonus. Data were
displayed on an oscilloscope and chart recorder during initial experiments and
on an analog-to-digital data acquisition system (Chart4; ADInstruments,
Toronto, Ontario, Canada) for later experiments.
The cytosolic [Ca2+]i was measured by measurement of
fura-2 fluorescence (Shinozaki et al.,
2002). Briefly, the acid-free form of fura-2 was loaded into the
tip of a microelectrode. Then, after observing stable membrane potential, dye
was electrophoretically microinjected into the ostial fibers by applying 8-15
nA for 20-30 min. After completion of one or two injection cycles,
fluorescence, excited by 360 nm UV light, at the injected site and at the site
farthest from the injected site increased to 3-4-fold and 0.5-1.5-fold above
background level, respectively. Electrical stimulation for 15-20 min
facilitated the diffusion of fura-2 over the muscle, resulting in homogeneous
distribution of fluorescence (2-3-fold above background). This fluorescence
level allowed for stable recording during more than 2 h with intermittent
exposure to UV light. Muscles showing a membrane potential of less than -45 mV
or showing a decrease in force of more than 10% of control force were
discarded. After subtraction of autofluorescence of the OOM measured before
fura-2 loading, [Ca2+]i was calculated according to the
equation (Grynkiewicz et al.,
1985
):
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Several drugs which are known to modify components of
excitation-contraction coupling were tested both for their intrinsic effects
on resting tension and active force development, and for their ability to
modify the effects of PR (Sigma, St Louis, MO, USA) and F2 (gift
from Ian Orchard, Toronto). We used drugs that act at the sarcolemma L-type
Ca2+ channels (nifedipine and verapamil; Sigma) and Cd2+
(added to normal saline) and a T-channel blocker mibefradil dihydrochloride
(Hoffmann-LaRoche Ltd, Basel, Switzerland). Two drugs that affect the SR were
also tested (ryanodine and caffeine, Sigma). Ryanodine stock (5 mmol
l-1 in water) was diluted to 10 µmol l-1, a
concentration known to lock the ryanodine receptor (RYR, SR
Ca2+-release channel) in a state of sub-maximal conductance and
blocks Ca2+-induced Ca2+ release (CICR). It took 50 min
exposure to ryanodine to reach a steady state where force was reduced by 82%
from control. Caffeine (10 µmol l-1) was applied to cause
Ca2+ release from the SR (Lea,
1996). Caffeine (dissolved in saline) directly gates the ryanodine
sensitive SR Ca2+ release channels. Joro spider toxin (JSTX; Wako
Pure Chemical Industries, Osaka, Japan) was used to block glutamate
postsynaptic receptors. PR and F2, when applied during the
perfusion of one of the modified salines, were diluted in that modified
saline. Hormones and other drugs were added by switching the perfusion pump
source from control saline to one containing the desired concentration of
hormone.
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Results |
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Glutamic acid (10 mmol l-1) also induced strong contractures, but, in contrast to responses to peptides, tetani following the contracture were depressed for up to 15 min (Figs 1F and 8D) and the rate of onset and recovery of the effect of glutamate was always rapid.
|
Occasionally an OOM showed spontaneous oscillations in membrane potential and force following PR or F2 (Figs 1D and 2). These oscillations could disappear in a few minutes, as in the traces shown, or occasionally persist for hours and lead to a small contracture. OOM which displayed continuous large amplitude oscillations were discarded.
|
Effects of PR and F2 on membrane potential and input resistance
The resting membrane potential of fibers varied from -30 to -80 mV
(Table 2). Threshold
concentrations of PR and F2 that augmented tetani but did not
produce contracture (1 pmol l-1 and 10 pmol l-1,
respectively) did not change the resting membrane potential. Fibers were
depolarized by up to 45 mV during exposure to peptide concentrations that
produced contracture (Fig. 2,
Table 2). The duration of the
depolarization corresponded to that of the contracture. The resting membrane
potential, but not amplitude of the tetanus, returned to near control level by
the end of the contracture period.
|
The input resistance (Rinput) of fibers exposed to PR, F2 or glutamic acid did not change significantly from the control values at the time of peak contracture or during the period of augmented tetani that followed. (Fig. 2, Table 2).
Effects mediated by the cardiac ganglion
Since the distal portions of the motoneurons of the cardiac ganglion and
their presynaptic terminals on the OOM could have been depolarized by the
electrical stimuli, it was necessary to determine whether the evoked tetani or
the hormone responses were due, at least in part, to activation of the nerve
terminals. Perfusing OOM with JSTX (5 µmol l-1, 10 min minimum)
blocked glutamic acid-induced (10 mmol l-1) contractures, but did
not alter the amplitude of stimulated tetani in control saline nor did it
prevent PR- and F2-induced contracture and enhanced tetani (data
not shown).
Dopamine (N=4) and 5-hyroxytryptamine (5-HT or serotonin,
N=8) (0.1 µm to 1.0 mmol l-1) exert strong chronotropic
effects on intact crab and lobster hearts
(Wilkens and Mercier, 1993;
Wilkens and McMahon, 1994
;
Wilkens and Kuramoto, 1998
),
presumably by their actions on the cardiac ganglion. Neither amine affected
basal tonus or tetani of OOM (data not shown).
Effects of PR and F2 on Ca2+ dynamics
The amplitude of tetani in saline was reduced at
[Ca2+]o 2 mmol l-1, but not at
[Ca2+]o >6 mmol l-1 (data not shown). The
absolute magnitude of the peptide-enhanced tetani, where amplitude was
normalized relative to control conditions, also decreased with reduced
[Ca2+]o. PR had no effect at
[Ca2+]o 2 mmol l-1 while F2
augmented the tetani. At higher [Ca2+]o both peptides
increased tetani (Fig. 3).
|
Short exposure to PR at 1 nmol l-1 and higher caused an increase in [Ca2+]i and force following each depolarization compared with control conditions (Fig. 4), while PR at 10 nmol l-1 and 1 µmol l-1 caused a sustained increase in resting [Ca2+]i and a dose-dependent contracture. Unfortunately, F2 was not tested at the time of the fura-2-based [Ca]i measurements.
|
The force/pCa2+ relationship during relaxation of tetani is
assumed to reflect the Ca2+-sensitivity of the contractile
apparatus (Ashley et al.,
1993). The force/pCa curves for drug-free conditions and after
addition of PR are illustrated in Fig.
5. The recovery portion of these curves is shifted to the right
following exposure to PR at both 0.01 (data not shown) and at 1 µmol
l-1.
|
The increased [Ca2+]i following hormone treatment could come from either or both an increased influx across the sarcolemma and an increased storage and release from the SR. We tried to distinguish between these two possibilities by applying PR or F2 in the presence of the sarcolemmal L-type Ca2+ channel blockers Cd2+, nifedipine, and verapamil and the T-type Ca2+ blocker mibefradil, as well as in Na+-free saline, which blocks the Na+/Ca2+ exchanger. The efficacy of the peptides was also tested during treatment with ryanodine, which blocks SR Ca2+ release, and caffeine, which stimulates the release of SR Ca2+.
Sarcolemmal influx
Verapamil (N=3) and nifedipine (N=2) alone (each at 0.1
mmol l-1) caused a 10% attenuation of tetani (P<0.05,
N=5). These L-type Ca2+ channel blockers reduced but did
not prevent the peptide-induced contracture followed by increased tetani (10
nmol l-1 to 1 µmol l-1;
Fig. 6A,B). Contracture
amplitude in the presence of verapamil and nifedipine was reduced in three
experiments and slightly increased in three other trials compared with that in
normal saline. Cd2+ (1 mmol l-1) substantially
attenuated tetani (70-95%), but did not block peptide-induced contracture
(Fig. 6C). These blockers did
not prevent caffeine-induced (10 mmol l-1) contracture (data not
shown). When the peptides were applied a threshold concentration, verapamil
(0.1 mmol l-1) and Cd2+ (10 µmol l-1)
attenuated the tetani by 10% for PR and 26% for F2 (three trials,
N=2). Mibefradil had no effect on OOM or on peptide-induced
responses. Na+-free saline caused contracture. Na+-free
saline reduced the amplitude of, but did not block, peptide contractures
(Fig. 7). Peptide induced
contractures were also observed in unstimulated OOM
(Fig. 8B,C).
|
|
Role of the SR
SR Ca2+ sequestration could account for both the slow onset and
recovery of the peptide-induced potentiation of tetani. The increase in
[Ca2+]i during a contracture could, by up-regulating the
SR Ca2+-ATPase pumps (SERCA, Timmerman, in
Ashley et al., 1993), increase
the amount of releasable Ca2+ from the SR and facilitate subsequent
tetani. As expected, prolonged electrical stimulation potentiated the brief
tetani after the period of prolonged stimulation
(Fig. 8A). This potentiation
decayed in less than a minute. To study the effects of Ca2+
sequestration by the SR, we applied pulses of caffeine (10 mmol
l-1, 30 s) before and immediately after PR- and
F2-induced contractures (Fig.
8B,C). The caffeine-contractures following the peptide-induced
contracture were indeed augmented by 32% by PR and 54% by F2. The
caffeine-induced contracture following the glutamic acid-induced contracture
(Fig. 8D) was 11% smaller than
the one before.
Continuous perfusion of caffeine (10 mmol l-1), which opens the RYR of the SR continuously so that sequestered Ca2+ immediately returns to the cytosol, depleted the SR Ca2+ store in about 8 min and this dramatically reduced or abolished tetani (Fig. 9). In the presence of caffeine PR and F2 induced faster and stronger contractures (Fig. 9). OOM recovered completely after their return to normal saline, and tetani recovered rapidly following a peptide contracture when caffeine had been withdrawn prior to the end of the peptide contracture (Fig. 9B).
|
We have previously shown that sarcolemmal Ca2+ entry induces
Ca2+-induced Ca2+ release (CICR,
Shinozaki et al., 2002).
Ryanodine (10 µmol l-1, a concentration that locks SR
Ca2+ release channels in the open state) by itself caused an
elevation in [Ca2+]i and an increase in basal tonus (0
time levels in Fig. 10C,E).
Continuous treatment with ryanodine eliminated most of the PR-augmented
increase in [Ca2+]i and force during stimulation, but it
did not prevent the dose-dependent PR-induced increase in
[Ca2+]i and contracture. Ryanodine treatment abolished
caffeine-induced contracture (data not shown).
|
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Discussion |
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Force generation, membrane potential and Rinput
At threshold concentration each neuropeptide increases tetani without a
change, or at most a small increase, in basal tonus. We argue below that these
effects arise from peptide modulation of the voltage-gated Ca2+
channels. The threshold concentration found here was as low as or lower than
the values reported in the literature
(Mercier et al., 2003;
Kobierski et al., 1987
;
Erxleben et al., 1995
;
Lange, 2002
). The response to
threshold concentrations of the peptides combined was less than the sum of the
responses to the individual peptides. The lack of full additive effects would
be expected if each peptide activated the same or a similar pathway. The
relatively slow onset and recovery, requiring several minutes, of the
heightened tetani may suggest involvement of second messenger pathways.
Conversely, SR sequestration of Ca2+ that enters the cells owing to
the peptides effects must play an important buffering role in the time course
of the response because caffeine exposure dramatically accelerated the
contractures (Fig. 9).
At higher concentration each peptide produces a prominent contracture
possibly by activating a lower affinity receptor-mediated Ca2+
entry mechanism. During contractures, the tetani are reduced. Since
[Ca2+]i is elevated during the contracture period, the
smaller tetani probably indicate that the fibers are near the upper limit of
their [Ca2+]i and of their force generating ability.
Since the responses last a long time after a brief exposure to peptide there
may be strong receptor binding and a slow off rate. Similar response durations
are seen in Aniculus (hermit crab) hearts (T.Y., unpublished). These
effects on tonus and tetani may be widespread in arthropod muscles (force in
Limulus myocardium, Grome et al., 1994; force and Ca2+ in
barnacle, Griffiths, 1990;
lobster heart, Wilkens and Mercier,
1993
; crab skeletal muscle,
Mercier and Wilkens, 1985
;
FLPs reviewed by Mercier et al.,
2003
). Receptor desensitization to prolonged exposure to PR is
minimal.
PR and F2 at contracture-inducing concentrations depolarize the muscle cells by up to 45 mV from resting membrane potential as low as -80 mV. The magnitude and duration of this depolarization is proportional to the contracture and may be responsible for it. Similar results to PR have been found in Aniculus (hermit crab) and Bathynomus (isopod) heart (T.Y., unpublished).
PR increases the [Ca2+]i following each depolarizing
stimulus train. The potentiated tetani following PR exposure could arise
directly from increased Ca2+ entry or indirectly by enhancing
Ca2+ entry as a consequence of decreasing K+ currents.
We did not attempt to measure or modify potassium currents, but PR can reduce
K+ currents in neurons of the cardiac ganglion
(Sullivan and Miller, 1984)
and in other muscles (locust, Walther et
al., 1998
; isopod, Erxleben et
al., 1995
).
The rate of rise and fall of force of the tetani is increased in the
presence of PR and F2. PR, and presumably F2, increases
the rate of Ca2+ accumulation in and removal from the cytoplasm.
Increased SR Ca2+ loading would, in turn, increase the rate of
Ca2+ release as a result of an increased diffusion gradient from
the SR into the sarcoplasm. Increased [Ca2+]i may
increase the activity of the SERCA, an action that would increase the rate of
removal (Shinozaki et al.,
2004).
Some OOM exhibited spontaneous or myogenic membrane potential and force
oscillations. The peptides and glutamic acid often induced periods of myogenic
oscillation, similar to such peptide-induced myogenic activity observed in
other arthropod muscles (shrimp, Meyrand
and Marder, 1991; cockroach,
Fuse and Orchard, 1998
;
Limulus, Watson and Hoshi,
1985
; locust, Evans,
1984
; Steele et al.,
1997
). Peptide-induced membrane potential oscillations were always
associated with these mechanical oscillations. This observation allows for a
plethora of mechanisms that we have not pursued during this study.
We felt it necessary to eliminate the possibility that the peptides were
augmenting tetani by increasing neurotransmitter release from nerve terminals.
PR should increase presynaptic transmitter release since it increases
Na+ and decreases K+ conductances
(Sullivan and Miller, 1984;
Walther et al., 1998
;
Freschi, 1989
;
Golowasch and Marder, 1992
;
Erxleben et al., 1995
) and FLPs
increase transmitter release from lobster and crayfish motoneuron terminals
(Worden et al., 1995
;
Mercier et al., 2003
). The
cardiac ganglion motoneurons appear to be glutamatergic in lobsters
(Anderson, 1973
) and a variety
of other crustaceans (see Sakurai and
Yamagishi, 1996
; Yazawa et
al., 1998
). Field stimulation near the threshold for muscle
activation by electrodes parallel to the muscle is unlikely to stimulate nerve
terminals. Furthermore, JSTX blocks responses to bath applied glutamic acid
but does not reduce electrically evoked tetani or the responses to PR or
F2. This indicates that the evoked tetani arise from direct
stimulation of the muscle fibers rather than from activation of the nerve
terminals and that PR and F2 are acting directly on the myocytes
rather than modulating transmitter release (also in crayfish,
Quigley and Mercier,
1997
).
The input resistance of fibers was little changed from control both during
the contracture (glutamic acid, PR and F2) and during the
subsequent period of augmented tetani by PR or F2. That glutamic
acid fails to alter Rinput is puzzling since it is known
to depolarize insect and other crustacean muscle fibers and decrease input
resistance (Usherwood, 1967;
Aonuma et al., 1998). During prolonged exposure of insect muscle to glutamic
acid the initial fall in Rinput recovers toward the
control level during the first minute of exposure, presumably due to
desensitization of the glutamate receptors
(Usherwood, 1967
). Some FLPs
modestly increase the input resistance in deep abdominal extensor muscles of
crayfish while others do not, and they do not alter input resistance in
lobster opener muscle or Cancer borealis muscles associated with the
stomach (reviewed by Mercier et al.,
2003
). Presumably, the effects of the peptides on intracellular
calcium could be produced either by increasing Ca2+ conductance
and/or decreasing K+ conductance. If both occur simultaneously, it
is possible that the net effect would be little change in input resistance.
Although this may not be a parsimonious explanation, there is no reason to
exclude it. Activation or inactivation of membrane carriers can lead to
substantial changes in input resistance (e.g.
Spanswick, 1972
). The main
carrier-mediated transport systems that may be affected by the peptides,
Ca2+-ATPase and the Na+/Ca2+ exchanger,
transport Ca2+ out of the cell. Inhibition of these carriers by the
peptides would increase input resistance in the absence of any other
conductance changes; however, it does not appear that the peptides block the
Na+/Ca2+ exchanger since they still cause contracture in
Na+-free saline. We have not ruled out that the peptides may
enhance Na/Ca2+ exchange and thereby induce Ca2+ entry
into the cell. This puzzle is the subject of ongoing study.
Hormone influences on Ca2+ dynamics
External Ca2+ is required for PR-induced effects on arthropod
muscles (Wilcox and Lange,
1995; Wegener and Nassel,
2000
; Rathmayer et al.,
2002
). Reducing [Ca2+]o reduces tetani in
OOM (Shinozaki et al., 2002
)
and reduces the responses to PR and F2. At the lowest
[Ca2+]o, tested here, tetani were reduced by 50% and the
contraction-enhancing effect of PR, but not F2, was almost totally
eliminated.
At threshold concentration, PR and F2 appear to increase only
voltage-sensitive Ca2+ entry into the cell since there is no
increase in tonus. This is consistent with previous studies on insect muscle
(Wegener and Nassel, 2000)
This observation would impute the L-type Ca2+ channel as the high
affinity receptor. This increased Ca2+ current will augment CICR
from the SR and increase Ca2+ uptake by the SR by increasing the
activity of SERCA. We have shown previously
(Yazawa et al., 1999
) that
tetani of the OOM are initiated by action potentials of up to
60 mV,
which is sufficient to activate crustacean voltage-Ca2+ current
dependent (Tazaki and Cooke,
1986
) and hence induce Ca2+ release from the SR
(Shinozaki et al., 2002
). The
peptides would increase the rate of rise, the rate of recovery and the
amplitude of tetani by increasing CICR and SERCA.
At higher concentration PR causes an increase in the Ca2+
transient and the resting [Ca2+]i, membrane
depolarization and a contracture. These contractures are not significantly
reduced by the ICa(v) antagonists Cd2+, nifedipine and
verapamil (Tazaki and Cooke,
1986; Maunier and Goblet,
1987
; Shinozaki et al.,
2002
). The shift of the force pCa curves suggests a decreased
sensitivity to Ca2+i after exposure to PR; however, it
is recognized that shifts during hormone exposure may also indicate that the
hormone is having effects in addition to those controlling
[Ca2+]i alone
(Brustle et al., 2001
). The
responses of tetani to higher concentrations of F2 are similar to
those to PR and, although we did not measure intracellular [Ca2+]
in the presence of F2, we assume that there are parallel changes in
[Ca2+]i. A sustained Ca2+ influx may account
for the depolarization that accompanies the contracture. However, we have no
data that would help identify the system that carries this Ca2+
entry. These peptides do not seem to cause contracture by blocking the
sarcolemmal Na+/Ca2+ exchanger (see above). It is also
unlikely that the peptides induce contracture by enhancing depolarization
dependent Na+ entry and subsequent Na/Ca2+ exchange,
because their effect is still present in the absence of stimulation.
The peptide-induced contracture and increase in
[Ca2+]i presumably is caused by a sarcolemmal
Ca2+ influx because it was not blocked by blocking CICR with
ryanodine (Rousseau et al.,
1987; Sitsapesan et al.,
1991
; Hwang et al.,
1987
) or depleting SR Ca2+ stores by caffeine. Both
caffeine and ryanodine dramatically attenuates tetani as has also been
observed in insect muscle (Wegener and
Nassel, 2000
), but do not prevent peptide induced contracture
indicating that the peptide induced induced sarcolemmal Ca2+ influx
is enough to directly activate contraction in the absence of SR
Ca2+ release.
Caffeine-induced contractures are greater following a PR or F2
contracture than before. Since caffeine directly stimulates SR Ca2+
release (Bianchi, 1962;
Lea, 1996
), the larger
caffeine contractures following peptide pulses indicate that the releasable SR
Ca2+ pool has been increased, similar to the potentiation of tetani
following a prolonged tetanus. The increased influx of Ca2+ during
peptide contractures is likely to lead to increased uptake of Ca2+
by the SR via SERCA, thereby increasing the releasable pool of SR
Ca2+. This would also explain the augmented tetani after the
contracture; however, glutamic acid-induced contracture, which should increase
Ca2+ influx, does not augment the tetani. The difference between
the effect of the peptides and the effect of glutamate may reside in
differences of the degree of filling of the SR and the degree of
desensitization of the contractile filaments. [Ca2+]i
rises quickly and falls rapidly in a stepwise fashion with glutamic acid. Such
a [Ca2+]i increase is known to increase the
EC50 of the F-pCa relationship
(Shinozaki et al., 2004
). If
the EC50 decreases slowly after a stepwise decrease in
[Ca2+]i without a decline of SR Ca2+ release,
one may expect an undershoot of force, as is seen following glutamic acid
contracture. Other neuropeptide-induced processes may be involved; for
example, PR may modulate other intracellular components such as the
phosphorylation of a 30 kDa protein associated with thin filaments in an
isopod (Brustle et al.,
2001
).
Continuous perfusion with caffeine saline depletes the SR of Ca2+ and eliminates tetani; however, this accelerates and enhances peptide-induced contractures. This is consistent with the proposal that the low affinity receptor effect of each peptide is to enhance net Ca2+ entry into the cells and that normal SR Ca2+ sequestration blunts the response to peptide-induced Ca2+ entry.
The force-pCa relationships show that PR augments force by increasing the
[Ca2+]i. However, the sensitivity of the contractile
components to Ca2+ is actually reduced at this time. This decrease
in myofibrillar responsiveness to Ca2+ following a period of
elevated [Ca2+]i, induced here by PR treatment, has also
been observed following prolonged tetanic stimulation of OOM
(Shinozaki et al., 2004). In
mammalian cardiac muscle such [Ca2+]i increase is known
to cause decreases in pH and protein phosphorylation, both of which decrease
Ca2+ sensitivity of the contractile apparatus
(Hoerter et al., 1986
).
In conclusion, our data are consistent with the hypothesis that the high
affinity inotropic effects of proctolin and F2 arise from
modulation of voltage-gated Ca2+ channels and the low affinity
effects are mediated by activation of ligand-gated Ca2+
transporters in the sarcolemma. Even though the responses may be reduced in
amplitude by some sarcolemmal Ca2+ channel blockers, the
contracture and augmentation of tetani is still present and qualitatively
similar to the responses in normal saline. Continuing studies are
investigating the possibility that these peptide hormones may modulate
intracellular signal transduction second messenger pathways. Overall, in vivo
the dual effects of PR and F2 to increase heart rate by acting on
the cardiac ganglion (Wilkens and Mercier,
1993; Wilkens and Kuramoto,
1998
) and to increase contractile force will have a dramatic
effect on cardiac output.
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Acknowledgments |
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References |
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