Neuroscience and Biomedical Systems, Institute of Biomedical and Life Sciences, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK
* Author for correspondence (e-mail: j.mccarron{at}bio.gla.ac.uk )
Accepted 11 March 2002
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Summary |
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Introduction |
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Ca2+ release from the SR store is controlled by two
receptor-channel complexes: the ryanodine receptor (RyR), which mediates
Ca2+-induced Ca2+ release (CICR); and the
InsP3 receptor, which is involved in transmitter/ligand
activity at the sarcolemma. Stores are classified, on the basis of the
receptors they express, into ryanodine-sensitive and
InsP3-sensitive stores. Whether or not a single store or
multiple stores containing different arrays of receptors exist in smooth
muscle is controversial (e.g. Bolton and
Lim, 1989; Yamazawa et al.,
1992
; Golovina and Blaustein,
1997
; Sims et al.,
1997
; Janiak et al.,
2001
). We have recently proposed the existence of two stores in
smooth muscle, one with exclusively RyR, the other with both RyR and
InsP3 receptors (Flynn
et al., 2001
) (see also
Baró and Eisner,
1995
).
SR store receptor activation releases Ca2+ to generate
intracellular signals, the best known of which are the Ca2+
`sparks' spontaneous transient releases of Ca2+ from the
RyR (reviewed by Bootman and Lipp
1999; Niggli,
1999
; Jaggar et al.,
2000
; Bootman et al.,
2001
; Sanders,
2001
). Ca2+ sparks in turn activate a number of
intracellular effectors including large conductance Ca2+-activated
K+ channels [BKCa
(Nelson et al., 1995
)
(reviewed by Berridge, 1997
)],
which regulate [Ca2+]i, and a variety of smooth muscle
contractile responses (e.g. Nelson et al.,
1995
; Ganitkevich and
Isenberg, 1996
; Kume et al.,
1995
; Khan et al.,
1998
; Porter et al.,
1998
). The activation of up to 100 BKCa by a
Ca2+ spark from RyR, gives rise to spontaneous transient outward
currents [STOCs (Benham and Bolton,
1986
)] at the sarcolemma, although the precise relationship
between them is controversial (Perez et
al., 1999
; ZhuGe et al.,
2000
; Kirber et al.,
2001
). STOCs are sarcolemma-based hyperpolarizing currents that
stabilise the membrane potential and oppose contraction. Which of the two SR
Ca2+ stores is responsible for the generation of STOCs is unclear
but cholinergic agonists, which generate InsP3, suppress
STOCs (Bolton and Lim, 1989
;
Komori and Bolton, 1991
;
Kitamura et al., 1992
). Thus,
whereas activation of RyR generates STOCs, sarcolemma agonist that generate
InsP3 and so evoke InsP3 receptor
activity suppress them. Whether or not suppression of STOCs by sarcolemma
agonists is due to the generation of other second messengers besides
InsP3, such as protein kinase C, acting on either the RyR
or BKCa, is uncertain (Kitamura
et al., 1992
; Schubert et al.,
1999
; Jaggar and Nelson,
2000
).
Hence modulation of SR Ca2+ release, as indicated by STOC activity, may be an important regulator of smooth muscle contraction and account for phasic and tonic phenomena. This possibility was explored in the present study and the relationship between STOCs and muscarinic agonists that generate InsP3 was examined using whole cell patch-clamp and fluorescence techniques in single dissociated smooth muscle cells. In particular, the relationship between InsP3 receptor activity and the ability to suppress STOCs and regulate contraction was examined. The results show that InsP3 receptor activity can account for the suppression of STOCs by cholinergic agonists and raise the possibility that generation of InsP3 accounts not only for the phasic component (by releasing Ca2+ from the SR Ca2+ store) but also for the tonic component of contraction since InsP3, by depleting the SR store, suppresses STOCs and so depolarises the sarcolemma and facilitates entry via voltage-dependent Ca2+ channels.
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Materials and Methods |
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Methods
From male guinea-pigs (500-700 g), killed by cervical
dislocation then immediately bled following the guidelines of the Animal
(Scientific Procedures) Act 1986, a segment of the intact distal colon (5
cm) was removed and transferred to a Sylgard-coated (Dow Corning) Petri dish
containing an oxygenated (95% O2, 5% CO2) physiological
saline solution (PSS; 118.4 mM NaCl, 25 mM NaHCO3, 4.7 mM KCl, 1.13
mM NaH2PO4, 1.3 mM MgCl2, 2.7 mM
CaCl2 and 11 mM glucose, pH 7.4).
Pieces of intact colon were cleaned, by perfusing with PSS, then mounted in a vertical, heated organ bath (10 ml, 37°C) filled with oxygenated PSS (95% O2, 5% CO2). One end of each piece was fixed to a hook on the bottom of the bath and the other attached to a force displacement transducer (GrassFT03C). Contractions were recorded (Grass polygraph, Model 79E) in response to drugs added to the bath (5-300 µl). Drugs were washed out by emptying and refilling the bath. Ca2+-free solutions were prepared without compensation. Signals were digitized (10 Hz; Data translation board 2801-A) using a software program (kindly provided by F. L. Burton, University of Glasgow). To compare contractile responses to agonists, before and after 2-APB, the phasic component was taken as the force developed at the time required to reach 80% of the initial (control) peak contraction minus baseline. The tonic response was measured as the force developed from an average of 50 seconds of recording beginning 3.3 minutes (2000 data points) after agonist was added, minus baseline.
Single smooth muscle cells were enzymatically dissociated from guinea-pig
colonic muscle (McCarron and Muir,
1999). Membrane currents were measured using conventional tight
seal whole-cell recording. The composition of the extracellular solution was:
80 mM Na glutamate, 60 mM NaCl, 4.7 mM KCl, 1.1 mM MgCl2, 3 mM
CaCl2, 10 mM Hepes and 10 mM glucose, pH 7.4 with 1 M NaOH.
Tetraethylammonium chloride (20 mM), where used, replaced equimolar amounts of
NaCl. Ca2+-free solutions contained MgCl2 (3 mM) and
ethylene glycol-bis(ß-aminoethyl ether)N,N,N',N'-tetra-acetic
acid (EGTA, 1 mM). Unless otherwise stated, the pipette solution contained:
105 mM KCl, 1 mM MgCl2, 3 mM MgATP, 2.5 mM Pyruvic acid, 2.5 mM
Malic acid, 1 mM NaH2PO4, 5 mM creatine phosphate, 0.5
mM guanosine triphosphate, 30 mM Hepes, 0.1 mM fluo-3 penta-ammonium salt and
25 µM caged Ins(1,4,5)P3 trisodium salt, pH 7.2 with 1
M KOH. Whole-cell currents were amplified by an Axopatch 1D (Axon Instruments,
Union City, CA), low-pass filtered at 500 Hz (8-pole bessel filter, Frequency
Devices, Haverhill, MA), digitally sampled at 1.5 kHz using a digidata
interface and Axotape (Axon Instruments) and stored for analysis.
[Ca2+]i was measured using the membrane-impermeable dye fluo-3 (penta-ammonium salt) introduced into the cell from the patch pipette. Fluorescence was measured using a microfluorimeter that comprised an inverted fluorescence microscope (Nikon diaphot) and a photomultiplier tube with a bi-alkali photocathode. Fluo-3 was excited at 488 nm (bandpass 9 nm) from a PTI Delta Scan (Photon Technology International, East Sheen, London, UK) through the epi-illumination port of the microscope (using one arm of a bifurcated quartz fiber optic bundle). Excitation light was passed through a field stop diaphragm to reduce background fluorescence and reflected off a 505 nm long-pass dichroic mirror; emitted light was guided through a 535 nm barrier filter (bandpass 35 nm) to a photomultiplier in photon counting mode. Longer wavelengths, from bright field illumination with a 610 nm Shott glass filter, were reflected onto a CCD camera (Sony model XC-75) mounted on to the viewing port of the Delta Scan allowing the cell to be monitored during experiments. Interference filters and dichroic mirrors were obtained from Glen Spectra (London, UK). To photolyse caged-Ins(1,4,5)P3 (referred to in the text as InsP3) the output of a xenon flashlamp (Rapp Optoelecktronic, Hamburg, Germany) was passed though a UG-5 filter to select ultraviolet light and merged into the excitation light path of the microfluorimeter using the second arm of the quartz bifurcated fiber optic bundle. The nominal flash lamp energy was 57 mJ, measured at the output of the fiber optic bundle and the flash duration was about 1 millisecond. Single cell experiments were conducted at room temperature (18-22°C).
STOCs were activated by slowly depolarising the membrane potential from
-70 mV to avoid activation of a large Ca2+ current. STOCs
varied widely in amplitude, duration and frequency; therefore, to summarize
STOC activity, they were integrated for 5 or 20 second periods as described in
the text. A Student's t-test was applied to the raw data; results are
expressed as means±s.e.m. of n cells (except where otherwise
stated) with a value of P<0.05 being considered significant.
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Results |
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The nature of the periodic outward currents
The results suggested that InsP3 receptors could be
involved in both phasic and tonic components and prompted an investigation of
the basis of this involvement. Ca2+ release from stores reportedly
regulates contraction via spontaneous transient outward currents [STOCs
(Nelson et al., 1995)].
Accordingly, to explore the possibility that modulation of STOCs may form the
basis of InsP3 effects on contraction, single smooth
muscle cells were isolated and the effects of
InsP3-inducing agonists on STOCs examined using patch
clamp techniques. Depolarisation to between -20 mV and 0 mV, from a holding
potential of -70 mV, increased [Ca2+]i and activated periodic
outward currents (Fig. 2Ai,ii)
which increased in frequency and amplitude over several seconds even as
[Ca2+]i declined (Fig.
2Aiv). Currents varied widely in amplitude, frequency and duration
(expanded time base Fig. 2Ai,
and Fig. 2Aiii, which is an
`all-points histogram' of the membrane current recording at -20 mV). The mean
outward current amplitude (±s.e.m.) was 116±30 pA, the rise time
19±13 milliseconds, the t0.5 of decay 26±7
milliseconds and the t0.9 of decay 36±1 milliseconds
(n=758 from three cells). The periodic outward currents were indeed
STOCs (Benham and Bolton,
1986
); they were inhibited, in separate experiments, each by the
potassium channel blocker TEA (20 mM, Fig.
2Bi) and by ryanodine (50 µM,
Fig. 2Ci). Thus, before TEA,
STOCs produced a charge entry of 123±32 pC whereas, after TEA, this had
declined to -1±5 pC (n=3, P<0.05, 5 second
integral). Before ryanodine (50 µM), the 5 second integral was 47±8
pC; after the drug, this was 3±5 pC (n=3,
P<0.05).
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Effects of InsP3, caffeine and CCh on STOCs
Depolarisation from -70 mV to -10 mV elevated [Ca2+]i and
activated STOCs (Fig. 3i-iv).
InsP3, caffeine (10 mM) and CCh (50 µM) each
transiently increased [Ca2+]i, and reversibly inhibited STOCs. In
six identical experiments, a 5 second integral of the membrane current
decreased from 143±33 pC to 9±14 pC (P<0.05) after
InsP3, from 105±24 pC to -20±15 pC
(P<0.01) after caffeine, and from 86±15 pC to -33±17
pC (P<0.05) after CCh. CCh suppression of STOCs was reproducible,
being seen in each of 42 cells examined and up to five times in the same cell.
The first CCh application increased [Ca2+]i (by 1.0±0.2
F/F0 units above baseline, n=5) and suppressed
STOCs to 4±2% of their pre CCh value (89±21 pC vs 3±2 pC,
n=5, P<0.05). The second application of CCh also
increased [Ca2+]i (by 0.9±0.2
F/F0,
n=5) and suppressed STOCs to -2±3% of their pre CCh value
(56±7 pC to -1±9 pC, n=5, P<0.05). Both the
CCh-evoked increase in [Ca2+]i and the suppression of STOCs were
blocked by atropine (10 µM); the [Ca2+]i increase evoked by CCh
was reduced by 97% to 3±3% of controls in the same cells
(1.4±0.6
F/F0 vs 0.1±0.1
F/F0, n=3, P<0.05) and CCh suppression
of STOCs was reduced. In controls, STOCs were reduced to 12±12% of
their value by CCh (99±35 pC vs 15±14 pC, n=3,
P<0.05). After atropine, in these same cells, the extent of the
suppression of STOCs by CCh had been reduced and they remained at
77±17% of their control value (85±21 pC vs 73±28 pC,
n=3, P>0.05).
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Caffeine suppressed STOCs presumably by activating RyR, thus depleting the
RyR-sensitive store of Ca2+ (e.g.
Bolton and Lim, 1989;
Ganitkevich and Isenberg,
1995
); InsP3 may also deplete the
RyR-sensitive store of Ca2+, but via a different route. To explore
this possibility, the effects of ryanodine on InsP3-evoked
Ca2+ release were examined. Ryanodine, significantly reduced the
InsP3-evoked Ca2+ transient to 68±15% of
control values (P<0.05, n=8;
Fig. 4).
InsP3 increased [Ca2+]i by 1.9±0.3
F/F0 units above baseline under control conditions (the
sixth control InsP3 release; n=8); the sixth
InsP3-mediated Ca2+ release in the presence of
ryanodine (50 µM; 1.3±0.4
F/F0 units above
baseline, n=8, P<0.05) was significantly less than in the
absence of the drug. In the same experiment, activation of RyR by caffeine
both increased [Ca2+]i and apparently depleted the store of
Ca2+ so that a second caffeine application failed to increase
[Ca2+]i (by 0.07±0.02
F/F0 units above
baseline, n=8, P>0.05;
Fig. 4). Significantly, after
the second caffeine application, InsP3 no longer evoked
Ca2+ release. This latter finding suggests that the
InsP3 receptor and RyR have access to a common
Ca2+ store (Flynn et al.,
2001
), thus InsP3 receptor activity could
inhibit STOCs by reducing the Ca2+ available for release via the
RyR.
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The extent to which the store's Ca2+ content had to be reduced
to inhibit STOCs was next examined. The store's Ca2+ content was
assessed from the amplitude of InsP3-evoked transients and
compared with the probability of STOC occurrence (PSTOC) as the
store's content decreased. The store was depleted of Ca2+ by
incubating cells in a Ca2+-free solution [containing 3 mM
MgCl2 and 1 mM EGTA (McCarron
et al., 2000)]. Ca2+-entry induced Ca2+
release, from the RyR, plays a minor role in the generation of STOCs since the
currents persist for some time in the presence of Ca2+ channel
blockers such as cadmium (Benham and
Bolton, 1986
; Nelson et al.,
1995
). The time course of disappearance of STOCs (at -10 mV) in
the Ca2+-free solution was first examined, then the time course of
reduction of InsP3 store content (as indicated by the
amplitude of Ca2+ transients) at various times (30 seconds to 8
minutes) was investigated. Both STOCs and InsP3-evoked
Ca2+ transients were abolished in the Ca2+ free solution
(Fig. 5). STOCs were the more
sensitive. The probability of STOC occurrence was reduced by 70% from
0.3±0.05 to 0.09±0.025 after 30 seconds in Ca2+-free
solution. At the same time (30 seconds), the
InsP3-sensitive Ca2+ store content was reduced
by only 16±7% of control values as assessed by the magnitude of the
InsP3-evoked Ca2+ transient
(Fig. 5iv). The store therefore
seems to require a substantial Ca2+ load to generate STOCs.
Together (Figs 4,
5), these results suggest that
a relatively modest reduction in SR Ca2+ content is required to
inhibit STOCs.
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The contribution of CICR, at the RyR, to InsP3-evoked
increases in [Ca2+]i
The finding that ryanodine, by itself, decreased the
InsP3-evoked Ca2+ transient
(Fig. 4) may be explained in
two ways. First, InsP3-evoked Ca2+ release
could have triggered CICR at the RyR; in the presence of ryanodine this would
be prevented so that the Ca2+ transient evoked by
InsP3 would appear reduced. Secondly, by locking the RyR
into a subconductance level (Smith et al.,
1988; Anderson et al.,
1989
; Xu et al.,
1994
), ryanodine may have rendered the store leaky, reducing both
the total SR Ca2+ content and that available to
InsP3. To distinguish between these possibilities,
tetracaine (100 µM), a local anaesthetic with RyR blocking activity, was
used to inhibit RyR (Pizarro et al.,
1992
; Gyorke et al.,
1997
). Tetracaine did not reduce the
InsP3-evoked Ca2+ transient (n=8) but
inhibited STOCs (300±61 pC vs 72±23 pC, 20 second integrals,
n=5, P<0.05), a finding consistent with its inhibitory
action on the RyR (Fig. 6A,B).
The latter result could not be explained by an inhibitory action of tetracaine
on the Ca2+-activated K+ channel itself since the peak
Ca2+-activated K+ current activated by
InsP3-evoked Ca2+ release was unaltered by the
drug (740±91 pA vs 679±91 pA in the presence of tetracaine,
n=6, P>0.05, data not shown). The reduction in the
InsP3-evoked Ca2+ transient by ryanodine alone
(Fig. 4), probably occurred as
a result of the drug's rendering the SR leaky to Ca2+ so reducing
its Ca2+ content. Collectively, these experiments
(Fig. 6A,B) suggest that: (1)
InsP3 did not activate CICR at the RyR (had it done so,
the amplitude of the InsP3-evoked Ca2+
transient would have been decreased by tetracaine); and (2) STOCs arise from
the RyR and not the InsP3 receptor (since tetracaine
blocked STOCs while leaving the InsP3-evoked
Ca2+ transient unaffected).
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The contribution of InsP3 and protein kinase C to
CCh-evoked suppression of STOCs
CCh suppression of STOCs could have arisen from its ability to produce
InsP3 and so deplete the SR Ca2+ store. If so,
blocking the InsP3 receptor with, for example, 2-APB,
should prevent the suppression. This proved to be the case. The increase in
[Ca2+]i evoked by CCh was significantly
(P<0.05) reduced by 2-APB to 23±14% of control values (by
0.9±0.14 F/F0 vs 0.2±0.14
F/F0, n=6; Fig.
7). CCh by itself reduced STOCs to 9±14% of their pre-CCh
value (145±31 pC vs 13±12 pC, n=6, P<0.05).
After 2-APB (50 µM), in these same cells, CCh-suppression of STOCs was
reduced and the currents remained at 85±19% of their pre-CCh value
(110±29 pC vs 94±33 pC, n=6, P<0.05;
Fig. 7). Similar results were
obtained with the impermeable InsP3 receptor inhibitor
heparin. After heparin (2.5 mg/ml,
15 minutes), the
[Ca2+]i increase in response to
InsP3 was insignificant: 0.03±0.02
F/F0 units above baseline (resting 1.3±0.1
F/F0 vs 1.4±0.1 F/F0 after
InsP3, n=5, P>0.05); nor were STOCs
significantly different in either frequency or amplitude (126±53 pC vs
126±48 pC after InsP3, n=5,
P>0.05) from controls. In these same cells, the CCh (50
µM)-evoked [Ca2+]i increase in the presence of
heparin was, like the InsP3 response, attenuated to
0.2±0.04
F/F0 units above baseline so that the rise in
[Ca2+]i was insignificant (from a resting value of
1.3±0.1 F/F0 in the absence of CCh to 1.6±0.2
F/F0 after CCh, n=5, P>0.05) as was the
suppression of STOCs (106±40 pC before and 88±35 pC after CCh,
n=5, P>0.05). Together these results indicate that the
InsP3 receptor is essential for the CCh-evoked suppression
of STOCs and that this mechanism underlies the depletion of the SR
Ca2+ store by the agonist.
|
Other second messengers, such as protein kinase C (PKC) may also have
contributed to the inhibition of STOCs by mechanisms other than by changes in
either InsP3 or the SR Ca2+ store content. To
examine this possibility, the effect of the broad spectrum protein kinase
inhibitor H-7 (10 µM) was examined. H-7 did not reduce the ability of CCh
to inhibit STOCs (Fig. 8A).
Under control conditions, at depolarised membrane potentials ( -20 mV),
STOCs produced an integrated current of 103±44 pC, which was reduced to
-3±2 pC after CCh (n=7, P<0.05). In the presence
of H-7, perfused some 10 minutes beforehand, STOCs produced an integrated
current of 98±30 pC whereas, after CCh, the charge entry was reduced to
2±3 pC (n=7, P<0.05;
Fig. 8A). Other protein kinase
C inhibitors were no more effective. After the protein kinase C inhibitory
peptide (PKC19-36, 3 mM,
10 minutes;
Fig. 8B),
InsP3 and CCh each inhibited STOCs. Before
InsP3, STOCs evoked a 5 second integrated current of
107±27 pC whereas, after InsP3, the charge entry
was reduced to -6±1.2 pC (n=3, P<0.05;
Fig. 8B). Charge entry before
CCh was 64±14 pC and, after CCh, was -32±18 pC (n=3,
P<0.05). These results indicate that CCh suppression of STOCs is
independent of protein kinase C.
|
The latter finding may occur because protein kinase C cannot suppress STOCs
or, alternatively, because the effect of CCh on STOCs is not mediated via the
kinase. To distinguish between these possibilities the protein kinase C
activator indolactam was used. Indolactam (10 µM) significantly inhibited
STOCs (Fig. 8C). Before
indolactam, [Ca2+]i was 1.6±0.2
F/F0 units and STOCs was 386±62 pC (20 second
integrated current) whereas, after indolactam, [Ca2+]i
was 1.4±0.1
F/F0 units (P>0.05) and STOCs
was 161±51 pC (20 second integral, P<0.05, n=9 in
all cases). The inhibitory action of indolactam (10 µM) on STOCs was fully
blocked by the inhibitory peptide PKC19-36 (3 mM) introduced into
the cell via the patch pipette (304±85 pC before and 395±152 pC
after indolactam, n=6, 20 second integrals). Together, these results
indicate that while protein kinase C activation can suppress STOCs, muscarinic
receptor activation with CCh does not suppress STOCs by activating the
kinase.
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Discussion |
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STOCs arise from the Ca2+ store, access to which is shared by
RyR and the InsP3 receptors. Suppression of STOCs is a
direct consequence of depletion of Ca2+ in this store by
InsP3. InsP3 did not, for example,
deplete a separate store by releasing Ca2+ and activating CICR at
the RyR; tetracaine, which blocks RyR, had no effect on the
InsP3-evoked Ca2+ transient. Furthermore, at
negative sarolemma potentials (-70 mV), where RyR activity is reduced
(Jaggar et al., 1998),
ryanodine had no effect on the InsP3-evoked
Ca2+ transient (Flynn et al.,
2001
). We have previously proposed the existence of two SR
Ca2+ stores in these cells
(Flynn et al., 2001
), one
containing both InsP3 receptors and RyR, the other
containing RyR alone. Since block of the RyR did not affect the
InsP3-evoked Ca2+ transient, only the store
with both receptors was responsible for the generation of STOCs
(Bolton and Lim, 1989
).
The finding that both InsP3 receptors and RyR have
access to a common Ca2+ store is based partly on the conclusion
that InsP3-evoked Ca2+ release did not activate
CICR at the RyR. Notwithstanding, ryanodine reduced the
InsP3-evoked Ca2+ transient in the present
study at depolarised membrane potentials ( -20 mV), appearing to indicate
that InsP3-evoked release activated CICR at the RyR. An
alternative explanation to CICR involvement is that ryanodine, by maintaining
the RyR in an open configuration could have attenuated the
InsP3-evoked Ca2+ transient by reducing the SR
Ca2+ content. In support, tetracaine, which does not open but
blocks the RyR, did not reduce the InsP3-evoked
Ca2+ transient. At membrane potentials of
-20 mV (used in
this study) the RyR are active, as shown by their electrical manifestation at
the sarcolemma (i.e. STOCs). Ryanodine binds to the open state of the RyR
(Meissner and El-hashem, 1992
;
McPherson and Campbell, 1993
;
Ogawa, 1994
) and, in the
concentration range used in the present study, may prolong its open time of
the receptor albeit at a lower conductance
(Xu et al., 1994
). {Higher
concentrations of ryanodine than those presently used [e.g. 300 µM
(Janiak et al., 2001
)] may
stabilise the channel in the closed state.} The persistent opening of the RyR
would increase Ca2+ leak from the SR, lower its Ca2+
content and thus reduce the InsP3-evoked Ca2+
transient. Consistent with this scheme, at a membrance potential of -70 mV,
ryanodine did not alter the InsP3-evoked Ca2+
transient (Flynn et al.,
2001
). RyR, in smooth muscle, is less active at negative membrane
potentials (e.g. -70 mV) and increases with depolarisation presumably
reflecting increasing [Ca2+]i and/or voltage-dependent
Ca2+ channel activity (Jaggar
et al., 1998
). Because of the reduced opening of the RyR,
ryanodine will be less effective and would not be expected to reduce the
InsP3-evoked Ca2+ transient. Together these
results provide the evidence that Ca2+ release from the
InsP3 receptor does not activate CICR at the RyR but that
ryanodine reduces the InsP3-evoked Ca2+
transient by increasing Ca2+ leak from the store.
The conclusion that InsP3-evoked Ca2+
release does not trigger CICR at the RyR disagrees with that of others in
which the reduction of the Ca2+ transient evoked by
InsP3-generating agents, by ryanodine, was interpreted as
evidence that InsP3-evoked Ca2+ activates CICR
at the RyR (Boittin et al.,
1999; Jaggar and Nelson,
2000
). The sarcolemma agonists used in these latter studies to
generate InsP3 (as opposed to caged
InsP3 used in the present study) may also have activated
other second messengers that sensitized the RyR to Ca2+.
Alternatively, since the ability of Ca2+ release to activate CICR
at neighbouring RyR increases with SR Ca2+ content, release may
activate further release under conditions of `store overload'
(Cheng et al., 1996
;
Trafford et al., 1995
). Some
smooth muscle types may maintain a higher SR Ca2+ content
facilitating CICR at the RyR.
The mechanism by which muscarinic receptor activation suppresses STOCs was
deduced from studies on the effects of the muscarinic agonist CCh. CCh
suppressed STOCs; this inhibition was prevented by each of the
InsP3 receptor blockers 2-APB and heparin but not by the
protein kinase C inhibitors H-7 or PKC19-36. Photolysed caged
InsP3 also suppressed STOCs, confirming the view that the
ability of CCh to suppress STOCs is solely dependent on the production of
InsP3 (see also Komori
and Bolton, 1990). These present results do not preclude the
involvement, in other smooth muscles, of other second messenger systems where
other neurotransmitters may be operative [e.g. in rabbit portal vein
(Kitamura et al., 1992
) and
rat cerebral artery (Jaggar and Nelson,
2000
)]. In murine colonic myocytes, muscarinic receptor activation
was reported to inhibit STOCs by increasing the bulk average
[Ca2+]i (Bayguinov et
al., 2001
). However, no evidence was presented that the store's
Ca2+ content had not been decreased by the treatments used to
elevate [Ca2+]i (e.g. ionomycin or ACh)
(Bayguinov et al., 2001
). Such
a decrease would seem the most likely explanation for the results obtained. By
contrast, in the present study, an increase in [Ca2+]i
is unlikely to account for the suppression of STOCs; STOC inhibition by CCh or
InsP3 persisted even when [Ca2+]i
had been restored to levels existing before InsP3 or CCh
had been applied. The persistent inhibition of STOCs in the present study
presumably reflected the time course of store refilling. Indeed, the
Ca2+ content of the stores needs to fall by only a relatively small
amount to suppress STOCs. Estimates from the present study suggest that a
decrease in the Ca2+ content by
16% resulted in a 70%
inhibition of STOCs. Consistent with this observation, after depletion, the
store [Ca2+] must exceed 80% of normal capacity before there is
steep relationship between Ca2+ content and STOC occurrence
(ZhuGe et al., 1999
).
If both agonist-induced phasic and tonic components each depend on
InsP3 production, this substance must be available
throughout the presence of the agonist. There is evidence that this is the
case. InsP3 formation, as deduced both from the
disappearance of its precursor phosphatidylinositol 4,5,bisphosphate and by
direct measurement of InsP3 itself, is indeed sustained
throughout the period of agonist stimulation up to 1 hour
(Akhtar and Abdel-Latif, 1984;
Baron et al., 1984
;
Takuwa et al., 1986
;
Marc et al., 1988
). For
example, in guineapig intestinal smooth muscle stimulated by CCh for 1 minute,
elevated InsP3 levels were detected for more than 5
minutes (Salmon and Bolton,
1988
), consistent with present observations where STOCs remained
inhibited for periods of minutes after CCh washout. In other tissues, the
levels of InsP3 oscillate after receptor activation as a
result of receptor desensitisation, metabolism of the neurotransmitter or
feedback regulation of production. Significantly, InsP3
concentration is maintained above resting levels during these oscillations
(Duncan et al., 1987
;
Hirose et al., 1999
).
The present results demonstrate that activation of muscarinic receptors on
smooth muscle evokes a tonic contraction by the generation of
InsP3. However, such a result raises the controversial
issue of whether or not the neurotransmitter itself directly contacts the
smooth muscle cell. Recent evidence has proposed that interstitial cells of
Cajal (ICCs) serve as intermediate transducers of the nerve response in
certain smooth muscles. Reduction or elimination of these cells abolished both
inhibitory (nitrergic) and excitatory (cholinergic) transmission
(Ward et al., 1998;
Ward et al., 2000
). Others
have failed to repeat these findings and found neurotransmission unimpaired in
preparations from mice lacking ICCs
(Sivarao et al., 2001
). Since
some extrinsic nerves make close synaptic contact (20 nm) with smooth muscle
as well as innervating ICCs, neural transmission may persist in the absence of
ICCs (FaussonePellegrini et al.,
1989
). A difficulty, with the use of mice lacking ICCs, is that
differences in smooth muscle contractility exist that are unrelated to the
innervation (Sivarao et al.,
2001
). Thus stomachs of the ICC-deficient mice lack basal tone and
are more compliant than the corresponding atropine-treated controls
(Ward et al., 2000
). The
origin of such differences must await further investigation.
Agonist activation evokes smooth muscle contraction via the activation of
several signalling systems which include activation of non-capacitative
Ca2+ entry pathways (Broad et
al., 1999) such as cationic channels
(Pacaud and Bolton, 1991
;
Zholos and Bolton, 1997
),
alterations in the myofilament Ca2+ sensitivity
(Somlyo and Somlyo, 2000
) and
Ca2+ release from the internal stores
(Sims et al., 1997
). The
present results reveal that the traditionally recognised phasic and tonic
components of agonist-induced smooth muscle contraction may be mediated, at
least in part, by InsP3. The mechanism proposed helps to
explain why excitatory G-protein-coupled agonists, such as ACh, triggered
biphasic changes in both [Ca2+]i and the contractile
state (Himpens and Somlyo,
1985
; Williams and Fay,
1986
). The first component is transient, reflecting
Ca2+ release from the internal store. In the second (tonic
component), depletion of the store leads to Ca2+ entry via
voltage-dependent Ca2+ channels because of the depolarisation that
arises from the suppression of STOCs, which generates a tonic contractile
phase; each component requires InsP3.
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