1Department of Zoology, Stockholm University, SE-106 91 Stockholm, Sweden; and 2Institute of General Zoology and Animal Physiology, Friedrich-Schiller-University, D-07743 Jena, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Wegener, Christian and
Dick R. Nässel.
Peptide-Induced Ca2+ Movements in a Tonic Insect
Muscle: Effects of Proctolin and Periviscerokinin-2.
J. Neurophysiol. 84: 3056-3066, 2000.
Although
most of the characterized insect neuropeptides have been detected by
their actions on muscle contractions, not much is known about the
mechanisms underlying excitation-contraction coupling. Thus we
initiated a pharmacological study on the myotropic action of the
peptides periviscerokinin-2 (PVK-2) and proctolin on the hyperneural
muscle of the cockroach Periplaneta americana. Both peptides
required extracellular Ca2+ to induce muscle
contraction, and a blockage of sarcolemmal Ca2+
channels by Mn2+ or La3+
inhibited myotropic effects. The peptides were able to induce contractions in dependence on the extracellular
Ca2+ concentration in muscles depolarized with
high K+ saline. A reduction of extracellular
Na+, K+, or
Cl did not effect peptide action. Nifedipine,
an L-type Ca2+-channel blocker, partially blocked
the response to both peptides but to a much lesser extent than
contractions evoked by elevated K+. Using calcium
imaging with fluo-3, we show that proctolin induces an increase of the
intracellular Ca2+ concentration. In calcium-free
saline, no increase of the intracellular Ca2+
concentration could be detected. The inhibiting effect of ryanodine, thapsigargin, and TMB-8 on peptide-induced contractions suggests that
Ca2+ release from the sarcoplasmic reticulum
plays a major role during peptide-induced contractions. Preliminary
experiments suggest that the peptides do not employ cyclic nucleotides
as second messengers, but may activate protein kinase C. Our results
indicate that the peptides induce Ca2+ influx by
an activation or modulation of dihydropyridine-sensitive and
voltage-independent sarcolemmal Ca2+ channels.
Ca2+-induced Ca2+ release
from intracellular stores, but not inositol trisphosphate-induced Ca2+ release, seems to account for most of the
observed increase in intracellular Ca2+.
Additionally, both peptides were able to potentiate glutamate-induced contractions at threshold concentrations.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Most of the numerous
insect neuropeptides known to date can induce stimulatory or inhibitory
responses in different types of muscle (see Gäde
1997; Holman et al. 1991
; Schoofs et al. 1997
). Nevertheless our knowledge about the mechanisms of
peptide-induced excitation-contraction coupling in insect muscles is
very limited. Most studies have been made on the actions of proctolin.
Proctolin (RYLPT) is found in different arthropods and has
myostimulatory actions or potentiates neurally evoked contractions in a
variety of arthropod muscles (see Orchard et al. 1989
).
There is compelling evidence that proctolin in insect muscles
activates the phospholipase C (PLC) pathway leading to
production of the second messengers 1,4,5-inositoltrisphosphate
(InsP3) and diacylglycerol (DAG) (Baines et al. 1990
; Hinton and Osborne 1995
,
1996
; Lange 1988
; Mazzocco-Manneval et
al. 1998
). Proctolin also increases muscle membrane resistance through a reduction of the resting K+ conductance
(Baines et al. 1996
; Erxleben et al.
1995
; Hertel and Penzlin 1986
; Hertel et
al. 1997
; Walther et al. 1998
). The action of
proctolin is dependent on the presence of extracellular Ca2+ (Cook and Holman 1985
;
Hertel and Penzlin 1986
; Hinton et al. 1998
; Lange et al. 1987
; Penzlin
1994
; Washio and Koga 1990
) and comprises an
influx of Ca2+ into the muscle (Baines and
Downer 1991
; Dunbar and Huddart 1982
; Wilcox and Lange 1995
). A proctolin-induced increase of
the intracellular Ca2+ concentration
([Ca2+]i) has been
demonstrated for barnacle muscle fibers (Bittar and Nwoga
1989
). An involvement of both voltage-dependent and
non-voltage-dependent Ca2+ channels during
proctolin action has been proposed for the oviduct of Locusta
migratoria (Lange et al. 1987
) and the hindgut of
the cockroach Leucophaea maderae (Cook and Holman
1985
). It is, however, not clear to what extent
proctolin-induced muscle contractions in insects depend on
intracellular Ca2+ mobilization by
InsP3 or by Ca2+-induced
Ca2+ release (CICR). There is also little data
available on the myotropic mode of action of other insect
neuropeptides. Thus we initiated the present pharmacological study on
the action of proctolin on muscle contractions and calcium movements
and made a comparison to the actions of another myotropic cockroach
peptide, periviscerokinin-2.
Periviscerokinins (PVKs) are abundant myotropic neuropeptides in
the abdominal neurosecretory system of the cockroach Periplaneta americana and are stored in the abdominal perisympathetic organs (Eckert et al. 1999; Predel et al. 1995
,
1998
; Wegener et al. 1999
). The two known PVKs
(PVK-1: GASGLIPVMRNamide, and PVK-2: GSSSGLISMPRVamide) both have
myotropic effects on the heart, segmental vessels, and the hyperneural
muscle (HNM) of P. americana (Eckert et al.
1999
; Predel et al. 1995
, 1998
).
We chose the HNM of P. americana to study peptide
action because the HNM is sensitive to both PVKs and proctolin
(Hertel and Penzlin 1986; Penzlin 1994
;
Predel et al. 1995
, 1998
) and has ideal properties for
pharmacological studies (see Moss and Miller 1988
).
Also, as we will show, the HNM is suitable for
Ca2+ monitoring using
Ca2+-sensitive dyes. This property enabled us to
investigate for the first time the effect of proctolin on
[Ca2+]i in an intact
insect muscle. The close proximity of the PVK-2-containing abdominal
perisympathetic organs to the HNM (see Klass 1999
)
suggests that, on release from these neurohemal organs, PVK-2 activates the muscle in vivo. Evidence for a physiological role of proctolin on
the HNM of P. americana is that proctolin-immunoreactive
nerve fibers innervate the muscle (Witten and O'Shea
1985
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Insects
American cockroaches (P. americana) were reared under a 16-h-light, 8-h-dark photoperiod at a constant temperature of 28°C. Adult males were used throughout the experiments. They had free access to water and were fed dog food pellets and occasionally fruits.
Chemicals
Methoxy-verapamil, tetraethylammonium (TEA), MDL-12,330-A, ryanodine, and TMB-8 were from RBI (Natick, MA). The acetoxymethyl ester form of fluo-3 (fluo-3 AM) was from TEF Labs (Austin, TX). All other chemicals were purchased from Sigma (Stockholm). PVK-2 was a kind gift of Reinhard Predel (Jena).
Preparation
For both bioassay and Ca2+ imaging, the
HNM was prepared according to Predel et al. (1994). The
abdominal sternites 3 to 8 were taken away and the HNM was removed
along with the 2nd and 9th sternite, to which the muscle is attached.
The ventral nerve cord was removed and the preparation was cleaned from
attached fat body and tracheae.
Bioassay
For measurement of isotonic contractions, the HNM was mounted in
a glass chamber filled with 2.5 ml continuously aerated cockroach saline [containing (in mM) 140 NaCl, 5 KCl, 5 CaCl2, 1 MgCl2, 5 glucose,
and 9.2 HEPES; pH 7.25]. During the application of 3-isobutyl-1-methylxaanthine (IBMX), H-7, Rp-cAMPS, MDL-12,330, phorbol
12-myristate 13-acetate (TPA), and cyclic nucleotide analogues, the
saline additionally contained 1% DMSO, which by itself had no effect
on peptide-induced muscle contraction (n = 6). A small amount of NaOH was used to solubilize EGTA prior to the preparation of
an appropriate stock solution in saline. Application of stock solution
aliquots did not change the pH in the bathing saline. One end of the
muscle was attached to the lever of a photoelectric transducer, the
other was fixed at the bottom of the chamber. The jacketed saline
reservoirs and glass chamber were adjusted to 28°C using a heating
circulator (MP-5A; Julabo, Seelbach, Germany). A detailed description
of the bioassay is given by Penzlin (1994).
After mounting in the glass chamber, the muscle was allowed to relax for 20 min under a tension of approximately 0.5 mN. Further elongation was prevented by a stopping lever. During the pharmacological experiments, peptide and drug applications, usually in aliquots of 25 µl, followed the scheme shown in Fig. 1. Contractions were simultaneously recorded using a flatbed recorder (Goerz Servogor 124, Wr. Neudorf, Austria) and the software LabView 5.0 (National Instruments, Austin, TX) on a Macintosh G3 computer with built-in PCI-MIO-16E-4 card (National Instruments, Austin, TX).
|
In Na+-, Ca2+- or
K+-free solutions, the ions were substituted by
an osmotically equivalent amount of choline. For high
K+ or Li+ solutions, an
osmotically equivalent amount of Na+ was omitted.
For reduced Cl solution, NaCl was substituted
by an osmotically equivalent of the sodium salt of isethionic acid.
Ca2+ imaging
Loading of the HNM with the fluorescent
Ca2+ indicator fluo-3 (Minta et al.
1989) was performed as follows: the HNM was incubated for
1 h at room temperature in darkness in 1 ml continuously aerated saline containing 20 µM fluo-3. To reduce dye leakage out of the muscle, 2 mM probenecid was included in the saline. Before adding fluo-3 to the saline, an appropriate amount of the dye was taken from a
stock solution in dry DMSO and mixed with 1 µl 15% pluronic F-127 in
dry DMSO to prevent dye precipitation. After loading, the muscle was
washed three times with 1 ml saline containing 2 mM probenecid and
mounted in an open Plexiglas chamber with a capacity of 2 ml by fixing
the attached sternites to the silicone elastomer (Sylgard, Dow Corning,
Midland, MI) lining of the chamber. Solutions were continuously aerated
and gravity-fed to the chamber at a flow-rate of approximately 3 ml/min. The muscle was viewed with an Zeiss Axioplan 2 microscope
equipped with a Zeiss water immersion objective (Achroplan 20x/NA 0.5).
The light source was a HBO 100 W lamp (Zeiss, Jena, Germany). A normal
filter combination for fluorescein (FITC) fluorescence was used. Images
were captured with a chilled CCD camera (C4742-95, Hamamatsu, Japan)
at a frequency of 0.33 or 1 picture/s and stored and analyzed on a
Macintosh PowerPC 8600 using Openlab 2.06 (Improvision, Coventry, UK).
Regions of interest were selected and relative fluo-3 fluorescence was measured. If necessary, regions of interest were adjusted on a frame-to-frame basis to keep track of the muscle movements.
Fluorescence intensity is presented as relative gray scale intensity
after subtraction of the acellular background. Individual muscles were used for a single proctolin application only.
Statistics
Data were evaluated with StatView 5.0 (SAS Institute, Cary, NC) using a Wilcoxon signed-rank test. Differences in contraction before and after drug application (compare Fig. 1) were defined to be significant when P(control 1/control 2) > 0.1, P(control 2/drug) < 0.05. Data are presented in percentages as means ± SE of control 1.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Proctolin and PVK-2 induce long-lasting tonic contractions
Proctolin and PVK-2 induced long-lasting tonic contractions of the HNM when applied at 10 nM (Fig. 2, B and C). These contractions were reproducible even after several applications of the same peptide concentration, which allowed a quantification of the contractile effect of the peptides. Usually it took around 10-20 s after peptide application until the muscle started to contract. Likewise, relaxation after peptide wash-out was not immediate, and some preparations needed several minutes to return to the resting state (see Fig. 2C). Peptide-induced contractions were different from contractions evoked by depolarization with 100 mM K+. Application of high-K+ saline resulted in an immediate strong contraction of the HNM without any plateau-phase (Fig. 2). On saline wash, the muscle returned immediately to the resting length.
|
Peptide-induced contractions are dependent on extracellular
Ca2+ but not on extracellular Na+,
K+, or Cl
Ca2+-free saline containing 2 mM of the
Ca2+-chelator EGTA completely suppressed the
myotropic action of both peptides (n = 6). This effect
was not reversible, even after several washes with normal saline.
Incubating the muscle in Na+-free saline
(n = 6) or Cl-reduced saline
(50 mM Cl
, n = 4) neither
altered the basal tonus of the HNM nor significantly changed
peptide-induced contractions. Incubation of the muscle in
K+-free saline led to a higher basal tonus, but
PVK-2 was still able to induce contractions similar to the controls in
normal saline, i.e., peptide-induced contractions were additive to the effect of omitting K+ (n = 8).
Proctolin-induced contractions were slightly but significantly decreased in K+-free saline and were not additive
to the effect of omitting K+ (Penzlin
1994
).
Sarcolemmal Na+/Ca2+ exchanger is active during peptide-induced contractions
The HNM relaxed normally after peptide-induced contractions when washed with Na+-free saline. However, after a subsequent 20 min in Na+-free saline, peptide-induced contractions were significantly increased (Fig. 3A). After washing with normal saline, the peptide response returned to the level of the first control. This indicated the presence of a Na+/Ca2+ exchange mechanism that counterbalances the peptide-induced increase in [Ca2+]i. Therefore we tested the effect of benzamil, a blocker of the Na+/Ca2+ exchanger in the sarcolemmal membrane. At 50 µM, benzamil significantly increased proctolin-induced contractions to 166% (n = 6) and PVK-2-induced contractions to 173% (n = 5) of the control (Fig. 3B). This reversible effect of benzamil suggests the presence of a Na+/Ca2+ exchanger in the sarcolemmal membrane of the HNM.
|
Effect of K+ channel blockers on peptide-induced contractions
The resting potential of insect muscle fibers is primarily
determined by K+ (Pichon and Ashcroft
1985; Walther et al. 1998
). In many cells, inward rectifying K+ channels regulate the
resting membrane potential and can be blocked by extracellular
application of Cs+. At 2 mM,
Cs+ attenuated proctolin-induced contractions
significantly to 81% (n = 6) and PVK-2-induced
contractions not significantly to 86% (n = 6) of the
controls (Fig. 3D). The unspecific K+
blocker 4-amino pyridine (4-AP) produced a transient twitch-like contraction of the muscle when directly applied at 1 mM but did not
reduce PVK-2-induced contractions (n = 6). The same was
found for proctolin (Hertel and Penzlin 1986
). Also TEA,
another unspecific K+ blocker, had no significant
effect on contractions evoked by proctolin (n = 7) or
PVK-2 (n = 9) when applied at 1 mM.
Effect of Ca2+ channel blockers on peptide-induced contractions
The dependence of peptide-induced contractions on extracellular Ca2+ indicated that the contractile effect is due to an influx of Ca2+ into the HNM. This was substantiated by the complete block of peptide-induced contractions by 2 mM Mn2+ or 100 µM La3+. Both ions are unspecific Ca2+ channel blockers. Their effect was not reversible after several washes with saline without these ions (n = 6).
In body-wall muscles of larval Drosophila melanogaster, the
main Ca2+ channel current consists of a
dihydropyridine (DHP)-sensitive ("L-type") and an
amiloride-sensitive ("T-type") component (Gielow et al.
1995). Voltage-dependent L-type Ca2+
channels are also present in crustacean muscles (Araque et al. 1998
; Erxleben and Rathmayer 1997
). Therefore we
used the DHP nifedipine and the phenylalkylamine (PAA)
methoxy-verapamil, another antagonist of vertebrate L-type channels,
and amiloride to investigate if similar channels are involved during
peptide-induced Ca2+ influx of the HNM.
Even at 100 µM, methoxy-verapamil had no inhibitory effect on
PVK-2-induced contractions (n = 6). At the same
concentration, verapamil also did not prevent proctolin-induced
contractions (Penzlin 1994). Nifedipine at 5 µM
reduced peptide-evoked contractions significantly to 72% of the
controls for PVK-2 (n = 7) and to 56% for proctolin
(n = 8, Fig. 3C). The effect of nifedipine
was, however, more pronounced on high-K+ (100 mM)-induced contractions, which were significantly reduced to an
average of 20% of the control with some contractions being completely
blocked (n = 6). At 60 µM nifedipine, PVK-2-induced contractions were significantly reduced to 65% (n = 6), which is comparable to the effect at 5 µM. Both proctolin and
PVK-2 were also able to further contract the muscle during a
depolarization with 100 mM K+ and in presence of
5 µM nifedipine. The effect of nifedipine was partially reversible
(Fig. 3C).
Amiloride had no significant effect at a concentration of 100 µM on either PVK-2- or proctolin-induced contractions (n = 6).
Proctolin and PVK-2 can induce muscle contractions in depolarized muscles
The much more pronounced effect of nifedipine on high-K+-induced contractions than on peptide-induced contractions suggested that the peptides use mechanisms other than or additional to membrane depolarization to induce Ca2+ influx. Since depolarization of the HNM by an incubation in saline with high (100 mM) K+ resulted in a near-to-maximum contraction (Fig. 2) of the muscle, it was not possible to test in normal saline whether peptide-induced contractions are additive to high-K+-induced contractions. To circumvent this problem, we first depolarized the muscle with 25 or 100 mM K+ in nominal Ca2+-free saline (without EGTA) and then increased the extracellular Ca2+ concentration stepwise. Following a wash with 10 ml high-K+, Ca2+-free saline, the same procedure was repeated but in presence of 10 nM of the peptides. Most preparations reacted with a short twitch-like contraction and an immediate relaxation to the depolarization with high K+ in nominally Ca2+-free saline. With introduction of extracellular Ca2+, muscle contractions reappeared. In general, the contractile response to high K+ under this experimental regime was strongly reduced compared with that in normal Ca2+-containing saline. A possible explanation for this effect could be that the voltage-dependent Ca2+ channels became activated by the introduction of high-K+, nominally Ca2+-free saline and that their activity decreased with time and was already diminished when extracellular Ca2+ was stepwise added after 3 min. This would result in a smaller Ca2+ influx and hence a smaller contraction and is also consistent with only transient contractions being evoked by high K+ in normal saline. Proctolin or PVK-2, however, induced normal or even increased tonic contractions in high K+-containing saline. These contractions first increased with increasing extracellular Ca2+ concentration before becoming smaller or unchanged at higher extracellular Ca2+ concentrations (Fig. 4).
|
Intracellular Ca2+ is mobilized during peptide action
To test whether intracellular Ca2+ from the
sarcoplasmic reticulum is released during peptide action, we used
TMB-8, an unspecific blocker of intracellular
Ca2+ release. TMB-8 had a strong inhibitory
effect on the myotropic action of both peptides (Fig.
5A). Proctolin-induced
contractions were significantly reduced to about 20%
(n = 5) and PVK-2-induced contractions to 40% of the
controls (n = 5). Thapsigargin, a blocker of the
sarcoplasmic Ca2+-ATPase (see Treiman et
al. 1998), also reduced peptide-induced contractions
significantly (Fig. 5B). These results indicated that both
peptides mobilize Ca2+ from the sarcoplasmic
reticulum. Therefore we tested the effect of the ryanodine receptor
(RyR) blocker ryanodine on peptide-induced contractions.
|
Ryanodine at 10 µM suppressed proctolin-induced contractions significantly to 50% (n = 6) and PVK-2-induced contractions to 60% of the control (Fig. 5C, n = 6). Increasing the ryanodine concentration to 100 µM did not result in a further suppression. Proctolin-induced contractions were significantly reduced to 51% (n = 5), those of PVK-2 to 63% (n = 5).
Functional ryanodine receptors are present in the HNM
The preceding results suggest that intracellular Ca2+ release via RyR is functionally important for peptide-induced contractions. To show that functional RyR are present in the HNM, we used caffeine, a RyR agonist. When applied at 20 mM, caffeine produced a short twitch-like contraction (Fig. 6). In most preparations, this twitch-like contraction was followed by a long-lasting tonic or phasic contraction (Fig. 6). The twitch-like contraction could be completely blocked by 10 µM ryanodine in most preparations (n = 6). Application of 20 mM caffeine in Ca2+-free Ringer containing 2 mM EGTA caused only twitch-like contractions, a tonic phase was never observed (n = 6). Therefore the tonic component appears to be dependent on extracellular Ca2+ influx.
|
Preliminary evidence for an activation of the PLC signaling pathway by proctolin and PVK-2
Activation of PLC leads to production of the second
messengers InsP3 and diacylglycerol (DAG).
Whereas InsP3 induces Ca2+
release from the sarcoplasmic reticulum, DAG activates protein kinase C
(PKC). Since proctolin activates the PLC pathway in several insect
muscles, we tested the effect of Li+, an
inhibitor of several enzymes in the phosphoinositol cycle, and the
unspecific PKC blocker H-7 (Hidaka et al. 1984) on
peptide-induced contractions. It has been proposed that
application of Li+ leads either to an
accumulation or a reduction of InsP3 and DAG (Nahorski et al. 1991
). When applied at 50 mM 20 min
before peptide application, Li+ caused a slight
but significant increase of proctolin-induced contractions to 109% of
the control (n = 9, Fig.
7A). PVK-2-induced contractions were not significantly increased to 105%
(n = 8, Fig. 7A).
|
At 10 µM, H-7 significantly reduced proctolin-induced contractions to 87% (n = 7) and PVK-2-induced contractions to 86% (n = 6) of the control (Fig. 7B), suggesting that a protein kinase is involved in the action of both peptides. The phorbol ester TPA can activate PKC and thus mimics the action of DAG. When TPA was applied at 1-5 µM, it caused a tonic contraction of the muscle that was slower but very similar to peptide-induced contractions (Fig. 6B). Like peptide-induced contractions, the myotropic action of TPA could be completely blocked by 100 µM La3+ (n = 4). The effect of TPA was not reversible: the muscle remained in its tonic state even after longer periods of washing.
Cyclic nucleotides seem not to be involved in the action of proctolin and PVK-2
Since H-7 also blocks the protein kinases A and G (Hidaka
et al. 1984), it seemed possible that also cyclic nucleotides
could mediate the myotropic effect of the peptides. Therefore we tested the effect of several chemicals known to interfere with cyclic nucleotide signaling pathways.
The phosphodiesterase blocker IBMX at 100 µM had no effect on contractions evoked by PVK-2 (n = 9) or proctolin (n = 6). The specific protein kinase A blocker Rp-cAMPS at 100 µM did not reduce PVK-2-induced contractions (n = 4). The adenylate cyclase blocker MDL-12,330 A always evoked tonic contractions when applied at 10-50 µM (n = 7) and did not alter proctolin- or PVK-2-induced contractions at lower drug concentrations (MDL-12,330 A alone at lower concentrations did not induce HNM contractions). The membrane permeable cAMP analogue 8-bromo-cAMP, which is resistant to phosphodiesterase degradation, had no contractile effect when applied at 0.2-1 mM for up to 40 min (n = 5). Also the membrane permeable cGMP analogue db-cGMP had no contractile effect on the HNM when applied at 100-200 µM for 40 min together with 100 µM IBMX (n = 4).
Threshold levels of proctolin and PVK-2 potentiate glutamate-induced contractions
The HNM is antagonistically controlled by excitatory and
inhibitory neurons running in the transverse nerves 1-7 and the ramus hyperneuromuscularis of the nerve VIII A/1 of the terminal ganglion (Penzlin 1994). Glutamate is probably the main
excitatory neurotransmitter at the HNM (Moss and Miller
1988
). We tested the effect of proctolin and PVK-2 on
glutamate-induced contractions of the HNM. Both peptides, applied at
threshold level of 0.5 nM together with 100 µM glutamate, significantly potentiated contractions compared with the effect of 100 µM L-glutamate alone to 202 ± 38% for proctolin
and 257 ± 61% for PVK-2 (n = 5 for each
combination). Control 3 of the glutamate-induced contractions remained
increased even in the absence of the peptides (168 ± 52 and
242 ± 59% following proctolin and PVK-2 application,
respectively), although this effect was only significant for PVK-2.
Ca2+ imaging
The Ca2+-sensitive dye fluo-3 has often been
used for Ca2+ imaging in muscles (e.g.,
Pagala and Taylor 1998; Somlyo et al.
1992
; Vergara et al. 1991
) and was applied to
investigate proctolin-induced changes of
[Ca2+]i in the HNM. One
advantage of fluo-3 is its large rise in fluorescence on
Ca2+ binding, which makes the fluo-3 signal
relatively insensitive to movement artifacts and relatively free of
noise (Caputo et al. 1994
).
After loading with fluo-3, the fluorescence intensity was not evenly
distributed throughout the HNM due to the varying muscle thickness. We
therefore selected homogenous regions of interest within which
fluorescence intensity was measured. At higher proctolin concentrations, muscle movements could not be completely eliminated due
to elastic elements within the HNM. We thus had to determine whether an
increase in fluorescence, caused by an increasing dye concentration due
to muscle shortening, interfered with the fluorescence signal caused by
increasing [Ca2+]i during
peptide action. For this, we monitored the effect of 100 nM proctolin
on the fluo-3 signal in muscles immobilized with hypertonic saline
containing 300 mM sucrose. Hypertonic sucrose salines do not
significantly change membrane properties and intracellular Ca2+ release (Gallagher and Huang
1997; Lamb et al. 1993
) and did not affect the
signal of Ca2+-sensitive dyes in frog muscle
fibers at up to two times normal tonicity (Parker and Zhu
1987
; Taylor et al. 1975
). Incubation of the HNM
in hypertonic saline resulted in a transient decrease of the fluo-3
signal (Fig. 8D). Proctolin was able to induce an increase
of the fluo-3 signal that was similar in shape and intensity to that
induced in normal saline and that was reversible by washing with
hypertonic saline (Fig. 8D,
n = 2). This showed that movement artifacts are
negligible and that fluo-3 can be used to monitor peptide-induced
relative changes in
[Ca2+]i in the HNM.
However, the onset and time course of the proctolin-induced increase of
[Ca2+]i were considerably
delayed in hypertonic saline.
|
Typical traces of the fluo-3 signal during stimulation with 1, 10, or 100 nM proctolin in normal saline are shown in Fig. 8, A-C (n = 5 for each concentration). After application of 1 nM proctolin, the increase in fluorescence intensity was sometimes only transient even in the presence of the peptide. The increase of fluorescence caused by 10 nM proctolin (Fig. 10) lasted until the peptide was washed out. Following an initial peak, the fluorescence intensity invariantly declined and stabilized on a somewhat lower level that returned to the basic level after peptide wash-out. Proctolin at 100 mM consistently caused an increase in fluorescence intensity that did not decline but slightly increased until the peptide was washed out. As with peptide-induced contractions, there was a delay between the application of proctolin and the onset of the proctolin-induced increase of [Ca2+]i. This increase was fast at 10 and 100 nM proctolin, with a slope independent of the applied proctolin concentration. The increase seems therefore to be caused by intracellular Ca2+ release rather than Ca2+ influx.
In Ca2+-free saline containing 2 mM EGTA, 100 nM proctolin was unable to produce a detectable increase in fluorescence intensity, whereas 2 mM caffeine produced a strong signal (Fig. 9A, n = 5). The time course of high-K+-induced changes in fluorescence intensity differed from that induced by proctolin (Fig. 9B). A steep initial increase was followed by a steady decline, no plateau phase could be detected (n = 4).
|
Figure 10 shows a typical example of a Ca2+ imaging experiment using fluo-3.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Action potentials in arthropod muscles are in general
Ca2+ dependent but Na+
independent (Ashcroft 1981; Deitmer and Rathmayer
1976
; Fatt and Ginsborg 1958
; Hagiwara
and Naka 1964
; Hencek and Zachar 1977
; Washio 1972
), whereas action potentials in nerves are
Na+ dependent. The ability of proctolin and PVK-2
to induce contractions in Na+-free saline thus
suggests that both peptides act postsynaptically at the muscle
membrane. Neurally evoked contractions of the HNM disappeared
completely in Na+-free saline, whereas proctolin
was still able to induce contractions in the absence of
Na+ (Hertel and Penzlin 1986
;
Penzlin 1994
).
The myotropic effects of proctolin and PVK-2 on the HNM are completely
dependent on an influx of extracellular Ca2+.
This is consistent with the complete dependence of proctolin-induced contractions on extracellular Ca2+ influx in
other insect muscles (Cook and Holman 1985;
Hertel and Penzlin 1986
; Hinton et al.
1998
; Lange et al. 1987
; Penzlin 1994
; Washio and Koga 1990
). We showed that
peptide-induced Ca2+ entry is not through
amiloride-sensitive voltage-dependent (T-type-like) channels, but at
least partially through nifedipine-sensitive/verapamil-insensitive Ca2+ channels that most likely are voltage
dependent since also high-K+-induced contractions
were inhibited by nifedipine. Similarly it has been shown that the
DHP-sensitive voltage-dependent Ca2+ channels of
larval Drosophila melanogaster body-wall muscles are
relatively insensitive to verapamil (Gielow et al.
1995
). For P. americana muscles, only PAA binding
sites that were DHP sensitive have so far been characterized
(Skeer and Sattelle 1993
; Skeer et al.
1992
). Our findings suggest that also a DHP-sensitive but
(relatively) PAA-insensitive Ca2+ channel exists
in sarcolemmal membranes of P. americana. Also in the
Schistocerca gregaria foregut, up to 1 mM verapamil had no
effect on proctolin-induced contractions but dose-dependently reduced
acetylcholine-induced contractions (Hinton et al. 1998
). Verapamil was also much less effective than nifedipine in reducing proctolin-induced contractions of the oviduct of L. migratoria (Lange et al. 1987
). Taken together with
the findings in D. melanogaster, it seems thus likely that a
DHP-sensitive, PAA-insensitive voltage-dependent Ca2+ channel is a general feature of insect
muscle membranes.
An involvement of DHP-sensitive voltage-dependent
Ca2+ channels in the HNM seems to be in conflict
with the finding that the HNM is not electrically excitable
(Hertel and Penzlin 1986; Miller and Adams
1974
). Recently, however, Monterrubio and co-workers (2000)
described "silent" nifedipine-sensitive
Ca2+ channels that occur in low density in the
tonic abdominal flexor muscles of the crustacean Atya
lanipes. These muscles, like the HNM, are not electrically
excitable. Under voltage-clamp conditions, Ca2+
inward currents were too small to measure in response to depolarizing currents but seem to be sufficient to induce CICR. This is probably due
to the close proximity of the silent Ca2+
channels to the sarcoplasmic reticulum in the dyad (Monterrubio et al. 1999
).
The ability of PVK-2 and proctolin to induce contractions in
depolarized muscles suggests that the mode of action of both peptides
comprises additional mechanisms besides activation of voltage-dependent
Ca2+ channels. This is also suggested by the
differences between peptide-induced and
high-K+-induced contractions and increases of
[Ca2+]i and the more
pronounced inhibiting effect of nifedipine on high-K+-induced contractions than on
peptide-induced contractions. An activation of non-voltage-dependent
Ca2+ channels by proctolin was suggested by the
fact that proctolin was able to induce contractions of high
K+-depolarized cockroach hindgut (Cook and
Holman 1980, 1985
), and locust oviduct (Lange et al.
1987
) which could not be blocked by nifedipine (Lange et
al. 1987
). Due to a lack of specific antagonists of
receptor-operated channels, we could not obtain direct pharmacological evidence for an activation of receptor-operated channels during peptide
action. An alternative explanation for the myotropic effect of the
peptides on depolarized muscles is that the peptides up-modulate Ca2+ channels that were activated by
high-K+ depolarization. In crayfish, proctolin
markedly increased the opening probability of
Ca2+ channels in depolarized patches of the tonic
flexor muscle (Bishop et al. 1991
); several
neuropeptides enhance the DHP-sensitive L-type current of the accessory
radula closer muscle of the mollusc Aplysia californica
(Brezina et al. 1994
).
Although influx of extracellular Ca2+ is a
prerequisite for the myotropic effect of the peptides on the HNM, the
peptide-induced [Ca2+]i
increase appears largely to be caused by intracellular
Ca2+ release via RyR as suggested by the
inhibiting effect of TMB-8, thapsigargin, and ryanodine and the slope
of the proctolin-induced [Ca2+]i increase. This
resembles the situation in crab muscle fibers, where extracellular
Ca2+ is essential for mechanical activity, but
Ca2+ release from the sarcoplasmic reticulum is
required to induce contractions (Mounier and Goblet
1987). Ryanodine receptors have been characterized for P. americana muscle membranes (Lehmberg and Casida
1994
; Schmitt et al. 1996
, 1997
), and the
effects of caffeine on contraction and
[Ca2+]i in
Ca2+-free saline show that they are also
functional in the HNM. A peptide-induced depolarization (by
non-voltage-dependent Ca2+ influx?) could
activate DHP-sensitive Ca2+ channels and CICR as
described for A. lanipes by Monterrubio and
co-workers (2000)
. This is suggested by the presence of dyad structures associated with the transverse tubular system of the HNM
(Miller and Adams 1974
), the demonstrated involvement of
DHP-sensitive Ca2+ channels, the small
proctolin-induced depolarization (Hertel and Penzlin 1986
), and
the proctolin-induced increase of the input resistance of the HNM
membrane, which increases membrane excitability (Hertel and
Penzlin 1986
). The observation of proctolin-induced Ca2+ waves in some preparations suggests that
peptide-induced Ca2+ influx may trigger local
CICR that then spreads over the whole muscle fiber.
Agonists that increase InsP3 can induce smooth
muscle contractions and raise
[Ca2+]i also in
Ca2+-free extracellular medium containing 2 mM
EGTA (e.g., Itoh et al. 1992; Kasuya et al.
1989
). In the absence of extracellular Ca2+ or after a block of sarcolemmal
Ca2+ channels, proctolin and PVK-2 were not able
to contract the HNM. Proctolin at 100 mM was also not able to elevate
[Ca2+]i in the absence of
extracellular Ca2+, although 100 nM proctolin
elicits a near maximal contraction of the HNM and a strong increase of
the fluo-3 signal when applied in the presence of extracellular
Ca2+. The increase of
[Ca2+]i and resulting
contractions induced by caffeine in Ca2+-free
saline showed that the lack of effect of proctolin in
Ca2+-free saline was not caused by a prior
depletion of stored Ca2+ in the sarcoplasmic
reticulum. Although we cannot rule out that InsP3-induced Ca2+ release
occurs at higher [Ca2+]i
than was present during our experiments, our findings still suggest
that InsP3-induced Ca2+
mobilization is not a key mechanism during peptide action on the HNM.
This is in contrast to the models of proctolin action on the oviduct of
L. migratoria (Lange and Nykamp 1996
) and the foregut of S. gregaria (Hinton et al. 1998
),
which suggest a role of InsP3-induced
Ca2+ mobilization. Our findings are, however, in
agreement with experiments on the locust hindleg extensor tibia muscle
with the proctolin analog [Afb
(p-NO2)2]-proctolin
(Baines et al. 1996
). [Afb
(p-NO2)2]-proctolin was
equipotent to proctolin in the ability to raise InsP3 but was about 1,000 times less efficacious
than proctolin in evoking contractions and to reduce the resting
potassium conductance. Thus the contractile effect of proctolin is
obviously not linked to InsP3 production in the
hindleg extensor tibia muscle. Perhaps there are differences in the
importance of InsP3-induced
Ca2+ mobilization during proctolin action between
"visceral" insect muscles such as the oviduct or foregut and
"skeletal" insect muscles such as the HNM and the hindleg extensor
tibiae muscle.
|
Although we used a range of drugs influencing cyclic nucleotide
signaling pathways, no evidence was found for an involvement of cyclic
nucleotides in proctolin- or PVK-2-induced contractions. Thus the
myoinhibitory effect of the unspecific protein kinase blocker H-7
appears to be due to an inhibition of PKC. This assumption is supported
by the tonic contractions of the HNM evoked by the PKC activator TPA
that could be blocked by La3+ similar to
peptide-induced contractions. A role of PKC in proctolin-induced contractions was also proposed for the oviduct of L. migratoria (Lange and Nykamp 1996) and the foregut
of S. gregaria (Hinton et al. 1998
) but
rejected for the mandibular closer muscle of L. migratoria
(Baines and Downer 1992
). Clearly further
research is needed to clarify the role of PKC during proctolin- and
PVK-2-induced contractions.
The most likely primary excitatory transmitter at the HNM is
L-glutamate (Moss and Miller 1988). The
potentiating effect of proctolin and PVK-2 on glutamate-induced
contractions of the HNM suggests a modulatory role of the peptides when
present in sub-nanomolar concentrations. This assumption is supported
by the potentiating effect of proctolin on neurally elicited
contractions of the HNM (Hertel and Penzlin 1986
) and
the increase in spontaneous HNM activity after application of proctolin
and PVK-1 on in situ preparations of the HNM (Predel et al.
1995
; S. Marsch, unpublished data). Proctolin is also
known to both induce contractions and modulate neurally evoked
contractions of the locust oviduct (Noronha and Lange
1997
).
In conclusion, we have presented evidence that proctolin and PVK-2
induce contractions of the HNM by an activation of
Ca2+ influx through DHP-sensitive and
voltage-independent channels in the sarcoplasmic membrane. The main
increase of [Ca2+]i is
due to CICR triggered by the Ca2+ influx. This
mechanism for excitation-contraction coupling in the HNM is thus the
same as proposed for crustacean skeletal muscles (Györke
and Palade 1993a,b
, 1994
).
InsP3-induced Ca2+
mobilization from the sarcoplasmic reticulum seems not to contribute to
the peptide-induced increase of
[Ca2+]i. The peptide
actions on the muscle at lower concentrations may serve to potentiate
neurally evoked contractions.
![]() |
ACKNOWLEDGMENTS |
---|
We thank M. Axelsson (Göteborg) for help with the LabView software, K. Åkermann (Uppsala) for the kind gift of fluo-3 and helpful suggestions concerning Ca2+ imaging, E. Krause (Jena) for teaching us the bioassay, R. Predel (Jena) for the kind gift of PVK-2, and S. Kreissl (Konstanz), D. Wicher (Jena), and A. Lange (Toronto) for valuable comments on an earlier draft of the manuscript.
This investigation was supported by the Swedish Natural Science Research Council (NFR) (D. R. Nässel). C. Wegener was supported by a predoctoral grant of the German Academic Exchange Service (DAAD; Hochschulsonderprogramm III von Bund und Ländern).
![]() |
FOOTNOTES |
---|
Address for reprint requests: D. R. Nässel, Dept. of Zoology, Stockholm University, Svante Arrhenius väg 14, SE-106 91 Stockholm, Sweden (E-mail: dnassel{at}zoologi.su.se).
Received 23 February 2000; accepted in final form 22 August 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|