Muscarinic stimulation increases basal Ca2+ and
inhibits spontaneous Ca2+ transients in murine colonic
myocytes
Orline
Bayguinov,
Brian
Hagen, and
Kenton M.
Sanders
Department of Physiology and Cell Biology, University of Nevada
School of Medicine, Reno, Nevada 89557-0046
 |
ABSTRACT |
Localized Ca2+ transients in
isolated murine colonic myocytes depend on Ca2+ release
from inositol 1,4,5-trisphosphate (IP3) receptors.
Localized Ca2+ transients couple to spontaneous transient
outward currents (STOCs) and mediate hyperpolarization responses in
these cells. We used confocal microscopy and whole cell patch-clamp
recording to investigate how muscarinic stimulation, which causes
formation of IP3, can suppress Ca2+ transients
and STOCs that might override the excitatory nature of cholinergic
responses. ACh (10 µM) reduced localized Ca2+ transients
and STOCs, and these effects were associated with a rise in basal
cytosolic Ca2+. These effects of ACh were mimicked by
generalized rises in basal Ca2+ caused by ionomycin
(250-500 nM) or elevated external Ca2+ (6 mM).
Atropine (10 µM) abolished the effects of ACh. Pretreatment of cells
with nicardipine (1 µM), or Cd2+ (200 µM) had no effect
on responses to ACh. An inhibitor of phospholipase C, U-73122, blocked
Ca2+ transients and STOCs but did not affect the increase
in basal Ca2+ after ACh stimulation. Xestospongin C (Xe-C;
5 µM), a membrane-permeable antagonist of IP3 receptors,
blocked spontaneous Ca2+ transients but did not prevent the
increase of basal Ca2+ in response to ACh. Gd3+
(10 µM), a nonselective cation channel inhibitor, prevented the increase in basal Ca2+ after ACh and increased the
frequency and amplitude of Ca2+ transients and waves.
Another inhibitor of receptor-mediated Ca2+ influx
channels, SKF-96365, also prevented the rise in basal Ca2+
after ACh and increased Ca2+ transients and development of
Ca2+ waves. FK-506, an inhibitor of
FKBP12/IP3 receptor interactions, had no effect on
the rise in basal Ca2+ but blocked the inhibitory effects
of increased basal Ca2+ and ACh on Ca2+
transients. These results suggest that the rise in basal
Ca2+ that accompanies muscarinic stimulation of colonic
muscles inhibits localized Ca2+ transients that could
couple to activation of Ca2+-activated K+
channels and reduce the excitatory effects of ACh.
calcium puffs; nonselective cation current; muscarinic receptors; enteric neurotransmission
 |
INTRODUCTION |
COORDINATED CONTRACTIONS
OF visceral smooth muscle organs depend on a complicated array of
regulatory processes. Key to normal motor behavior is the regulation
provided by autonomic and/or enteric motor neurons. The efferent motor
outflow in most visceral organs is organized into both excitatory and
inhibitory neurons. In the gastrointestinal (GI) tract neural
excitatory regulation comes from the release of acetylcholine (ACh) and
neuropeptides such as substance P or neurokinin A (cf. Ref.
20). ACh impinges on muscarinic receptors (M2
and M3 isoforms; see Refs. 35 and 36) in
postjunctional cells, and activation of these receptors initiates a
complex series of events that lead to enhanced contractile force.
Prominent among the many responses attributable to postjunctional muscarinic stimulation of GI muscles are: 1) activation of
nonselective cation channels (3, 14-16, 27, 32) and
2) G protein-dependent stimulation of phopholipase C and
generation of inositol 1,4,5-trisphosphate (IP3; e.g., Ref.
4). The former is thought to depolarize GI muscles and
increase the entry of Ca2+ through L-type Ca2+
channels. IP3 production initiates Ca2+
release from IP3 receptor-operated stores, a common feature
of most smooth muscles. Ca2+ release from stores is thought
to summate with Ca2+ entering cells to augment the strength
of contraction. Recent studies, however, have provided an expanded view
of the role of Ca2+ release from internal stores. When
localized to specific membrane areas, Ca2+ release can
activate Ca2+-dependent conductances in the plasma membrane
(24, 26, 29, 38), and activation of these channels can
transduce the release of Ca2+ (which would normally be an
excitatory signal) to inhibitory events leading to hyperpolarization
and relaxation (see Ref. 17). Both ryanodine and
IP3 receptors have been linked to localized Ca2+ release and regulation of Ca2+-dependent
conductances in the plasma membrane (1, 2, 26).
In GI smooth muscles G protein-coupled receptors mediate excitatory
(e.g., muscarinic or neurokinin receptors) and inhibitory responses
(e.g., P2Y receptors) that couple to responses through IP3-dependent pathways and Ca2+ release. How
coupling through similar second messenger pathways can elicit opposite
responses is not understood. We have reported that stimulation of
P2Y receptors of colonic smooth muscles cells is coupled to
inhibitory responses via IP3 production and localized Ca2+ release (1, 19). Stimulation of
P2Y receptors increased the occurrence and amplitude of
spontaneous Ca2+ transients and spontaneous transient
outward currents (STOCs). The STOCs were due to activation of
large-conductance Ca+-activated K+ channels (BK
channels) and small-conductance Ca2+-activated
K+ channels (SK channels) (1, 19). Thus
localized Ca2+ release from stores in these cells led to
membrane hyperpolarization. Stimulation of muscarinic receptors also
increases production of IP3. Since enhanced IP3
production greatly increases the occurrence of spontaneous
Ca2+ transients and STOCs, we hypothesize that another
mechanism must be present and additively coupled to muscarinic
stimulation to suppress the increase in Ca2+ transients
and/or coupling between Ca2+ transients and activation of
Ca2+-activated K+ channels. Previous studies
have demonstrated that muscarinic stimulation can reduce the open
probability of BK channels (11), but there is no known
inhibitory regulation of SK channels mediated by muscarinic receptor
stimulation. Thus it would seem that, to suppress the K+
conductances activated by IP3 receptor-operated
Ca2+ release, a mechanism to suppress localized
Ca2+ release from stores may exist.
In the present study we have characterized localized Ca2+
transients in response to muscarinic stimulation and investigated the
coupling of these responses to activation of outward currents. We have
observed that an increase in basal Ca2+ that occurs via
activation of receptor-operated conductances in the plasma membrane
suppresses the natural tendency for muscarinic stimulation to increase
localized Ca2+ transients in colonic myocytes. In
addition to depolarization, suppression of spontaneous Ca2+
transients is a new mechanism attributed to the receptor-operated channels activated by muscarinic stimulation.
 |
METHODS |
Cell preparation.
BALB/C mice (15-30 days old) of either sex were anesthetized with
chloroform and killed by decapitation. After removal, colons were
opened along the mesenteric border, and the luminal contents were
washed away with Krebs-Ringer bicarbonate buffer (KRB; see Solutions and drugs). Tissues were pinned to the base of a
Sylgard-coated dish, and the mucosa and submucosa were removed by peeling.
Strips of colonic muscle were equilibrated in Ca2+-free
solution for 60 min, and then the tissues were digested to free single cells with an enzyme solution containing Collagenase F (Sigma, St.
Louis, MO). During digestion the tissues were incubated at 37°C for
16 min without agitation. After three to four washes with
Ca2+-free Hanks' solution to remove the enzyme, the
tissues were triturated with blunt pipettes of decreasing tip diameter
to mechanically free smooth muscle cells.
Confocal microscopy.
Cell suspensions were placed in a specially designed 0.5-ml chamber
with a glass bottom. The cells were incubated for 35 min at room
temperature in Ca2+-free buffer containing fluo 3-AM (10 µg/ml; Molecular probes, Eugene, OR) and pluronic acid (2.5 µg/ml;
Teflabs, Austin, TX). Cell loading was followed by a 25-min incubation
in a solution containing 2 mM Ca2+ to restore normal
extracellular Ca2+ concentration, allow the cells to
tightly adhere to the bottom of the chambers, and to complete the
deesterefication of fluo 3. All measurements were made within 45 min
after restoring extracellular Ca2+.
An Odyssey XL confocal laser scanning head (Noran Instruments,
Middleton WI) connected to a Nikon Diaphot 300 microscope with a ×60
water immersion lens (numerical aperture = 1.2) was used to image the cells. The cells were scanned using INTERVISION software (Noran Instruments, Middleton WI) running on an Indy workstation (Silicon Graphics, Mountain View, CA). Changes in the fluo 3 fluorescence (indicating fluctuations in cytosolic Ca2+)
were recorded for 20-s test periods using T-series acquisition and a
laser wavelength of 488 nm (excitation for FITC). Six hundred frames
were acquired per test period (one frame every 33 ms), creating 20-s
movie files.
Single cell measurements of ionic currents.
Ionic currents were measured in isolated muscle cells using the whole
cell, perforated-patch (amphotericin B) configuration of the
patch-clamp technique. Average cell capacitance was 56.1 ± 4.2 pF. An Axopatch 200B amplifier with a CV 203BU headstage (Axon
Instruments, Foster City, CA) was used to measure ionic currents and
membrane potential. Membrane currents were recorded while holding cells
at
40 or
70 mV (after corrections of junction potentials) using
pCLAMP software (version 7.0, Axon Instruments). Currents were
digitized at 1 kHz in dual recordings and were digitized at 500 Hz in
experiments that were stepped repetitively between
40 and
70 mV. In
some experiments patch-clamped cells were simultaneously scanned for
fluorescence changes in cells preloaded with fluo 3 as described above.
All experiments were performed at room temperature (22-25°C).
Solutions and drugs.
The standard KRB used in this study contained (in mM) 120.35 NaCl, 5.9 KCl, 2.5 CaCl, 1.2 MgCl2, 15.5 NaHCO3, 1.2 NaH2PO4, and 11.5 dextrose. This solution had a
final pH of 7.3-7.4 after equilibration with 97%
O2-3% CO2. The bathing solution used in confocal microscopy studies and patch-clamp studies contained (in mM)
134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose,
and 10 HEPES (pH 7.4). The enzyme solution used to disperse cells
contained 1.3 mg/ml collagenase F, 2 mg/ml papain, 1 mg/ml BSA, 0.154 mg/ml L-dithiothreitol, 134 mM NaCl, 6 mM KCl, 1 mM
MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4). The pipette
solution used in patch-clamp experiments contained 110 mM potassium
aspartate, 30 mM KCl, 10 mM NaCl, 1 mM MgCl2, 10 mM HEPES,
0.05 mM EGTA (pH 7.2), and 250 µg/ml amphotericin B.
ACh, atropine, cadmium chloride, gadolinium (III) chloride,
and nicardipine were obtained from Sigma.
(1-{6-[(17
)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)- 1H-pyrrole-2,5-dione
(U-73122) was obtained from RBI. Xestospongin C (Xe-C) was obtained
from Calbiochem.
1-{2-(4-Methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl}-1H- imidazole
(SKF-96365 hydrocloride) was obtained from Tocris Coocson. FK-506 was a
generous gift from Fiji Sawa. Concentrations of drugs used were
determined from previous studies in the literature or by empirical
testing of effective concentrations on murine colonic myocytes.
Analysis of data.
Image analysis was performed using custom analysis programs using
Interactive Data Language software (Research Systems, Boulder, CO), as
previously described (1). Baseline fluorescence
(F0) was determined by averaging 10 images (of 600) with no
activity. F0 from control experiments was used to determine
the ratio of the records of following drug treatments. Ratio images
were then constructed and replayed for careful examination to detect
active areas where sudden increases in F/F0 occurred. To
detect changes in basal Ca2+ concentration, areas free of
Ca2+ transients were selected. F/F0 vs. time
traces were further analyzed in Microcal Origin (Microcal Software,
Northampton, MA) and AcqKnowledge Software (Biopac Systems, Santa
Barbara, CA) and represent the averaged F/Fo from a box
region of 2.2 × 2.2 µm centered in the active area of interest
to achieve the fastest and sharpest changes. This box size (4.8 µm2) was determined empirically to be the best compromise
between temporal and spatial precision of Ca2+ release
events and the signal-to-noise ratio (1, 29). Fluorescence records from single colonic myocytes were composed of Ca2+
transients of multiple characteristics (i.e., single Ca2+
puffs, clusters of puffs, and Ca2+ waves) (1).
As a result, the data could not be accurately analyzed simply as
changes in transient amplitude or frequency. Therefore, we analyzed the
data as the area of the Ca2+ transients above a baseline
drawn between the end points of 20-s scans. Areas are expressed in
terms of amplitude units (F/F0) times horizontal units
(time). Changes of basal Ca2+ concentration were calculated
as the increase in average ratio in areas of interest. The amplitudes
of STOCs were determined off-line using the Mini Analysis Program
(Synaptosoft software, Leonia, NJ). The threshold of STOCs was set at
three times the single Ca2+-activated-K+
channel amplitude at
40 mV or at 6 pA. The open probability of
Ca2+-activated K+ channels in the absence of
Ca2+ release events is very low at
40 mV (number of
channels times open probability
10
3; see Ref.
5), and the probability of three simultaneous openings is
far less.
Statistical analysis.
Results are expressed as means ± SE where applicable. All the
statistical analysis was made with SigmaStat 2.03 software (Jandel Scientific Software, San Rafael, CA). ANOVA on Ranks test was used to
compare the results from different treatments.
 |
RESULTS |
Effect of ACh on spontaneous Ca2+
transients in murine colonic myocytes.
Spontaneous, transient elevations in intracellular
Ca2+ concentration
([Ca2+]i) were observed in freshly dispersed
murine colonic myocytes, as previously reported (1). These
events occurred either as localized events (Ca2+ puffs) or
as more widely dispersed, propagating events (Ca2+ waves).
Previous studies have indicated that the spontaneous Ca2+
transients in colonic myocytes are mediated by Ca2+ release
from IP3-receptor operated channels (1).
We tested the effects of ACh (10 µM) on spontaneous Ca2+
transients in murine colonic myocytes. ACh reduced the area of
Ca2+ transients to 46 ± 6% of control
(P < 0.005; n = 11; Fig.
1, A-D). The
reduction in Ca2+ transients was accompanied by a
significant increase in the basal Ca2+ level (i.e., to
120 ± 5%) of control. Both of the observed effects (inhibition
of the spontaneous activity and increase in basal Ca2+)
began within 1 min after application of ACh, and the effects were
stable for at least 10 min of exposure. Figure 1E summarizes the results of 11 experiments. It is unlikely that the inhibition of
Ca2+ transients in response to ACh was due to simple
unloading of stores because addition of caffeine (1 mM) caused a
massive release of Ca2+ and contraction of the cells (data
not shown).

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 1.
Effects of muscarinic stimulation on basal Ca2+ and
localized Ca2+ transients. A: a cell loaded with
fluo 3. A spontaneous localized Ca2+ event is pictured at
the bottom, middle region of the cell. Areas of interest (AOI) are
noted by arrows. B: same cell after exposure to ACh (10 µM
for 5 min). A generalized increase in basal Ca2+ was
observed. C: Ca2+ transients (fluorescence ratio
F/F0) that occurred within AOI-SA (spontaneously active)
before (left trace) and after (right trace) ACh
(10 µM) during 20-s scans (600 images/20 s). D:
F/F0 from AOI-NSA (not spontaneously active) before
(left trace) and after (right trace) ACh during
the same 20-s scans as in C (600 images/20 s). AOIs were the
same before and after ACh. Note the increase in baseline
Ca2+ in both AOIs. E: time-dependent changes in
Ca2+ transients (solid bars) and basal Ca2+
(open bars) after muscarinic stimulation. The changes in ratio are
relative to a point at time = 0 where ratio = 100%
(control). Data like those shown in A-D were tabulated at 4 time points after addition of ACh (10 µM; n = 11 cells for each time point). There were significant reductions in
Ca2+ transients within 1 min of addition of ACh, and these
persisted through 10 min of constant exposure. Basal Ca2+
increased significantly within 1 min of exposure, and this effect also
persisted throughout the exposure. All points are significant to at
least P < 0.05.
|
|
Pretreatment with atropine (10 µM) eliminated both effects of ACh. In
the presence of atropine 1) the areas of the
Ca2+ transients after the 1st and 3rd min of exposure to
ACh (10 µM) were 101 ± 10% and 102 ± 16% of the control
Ca2+ transients (10 µM; n = 7;
P > 0.1) and 2) basal Ca2+
levels were 102 ± 4% and 101 ± 3% of control at the same
time points (Fig. 2).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
Responses to ACh were inhibited by atropine.
A: spontaneous Ca2+ transients under control
conditions in an AOI-SA (left trace) and an AOI-NSA
(right trace). B: response to ACh of the cell
after pretreatment for 10 min with atropine (10 µM). ACh did not
reduce spontaneous transients or increase the level of basal
Ca2+. Data from 7 cells (P > 0.1)
pretreated with atropine and stimulated with ACh for 1 and 3 min are
tabulated in C (solid bars, Ca2+ transients;
open bars, basal Ca2+).
|
|
Effects of ACh on spontaneous transient outward currents.
We investigated the effects of ACh on STOCs in 11 experiments. In the
first series of experiments six cells were stepped repetitively between
40 and
70 mV for 15-s intervals. As reported previously (1,
19), colonic myocytes held at
40 mV displayed STOCs. STOCs
were not observed at
70 mV. Application of ACh (10 µM) reduced the
amplitude and occurrence of STOCs (Fig.
3).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
Effects of ACh on spontaneous transient outward currents (STOCs).
A: a continuous record before and during a response to ACh
(10 µM). After ACh there was a decrease in the amplitude and
frequency of STOCs. B and C: STOC amplitude
histograms before (B) and after (C) 5-min
exposure to ACh (n = 8; P < 0.001).
|
|
Five cells were held at
40 mV, and membrane currents and
spontaneous Ca2+ transients were recorded simultaneously.
The current records demonstrated that the STOCs were associated with
the Ca2+ transients (Fig. 4).
The reduction in Ca2+ transients and rise in basal
Ca2+ caused by ACh were accompanied by significant
reduction in STOCs.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 4.
Spontaneous Ca2+ transients were coincident
with STOCs. A: a cell with 2 active AOI during control
scans. After ACh, a more generalized increase in basal Ca2+
was observed (pairs of images at 1 and 3 min after addition of ACh).
B: Ca2+ transients from the 2 AOI depicted in
A. Note the occurrence of multiple Ca2+
transients at each site during the 20-s scan. Traces to the right show
Ca2+ transients after exposure to ACh for 1 and 3 min. Note
the rise in basal Ca2+ at each AOI and decrease in the
Ca2+ transients above baseline. C: simultaneous
recordings of whole cell currents during the fluorescence measurements.
STOCs are coincident with Ca2+ transients, but the
relationship between Ca2+ transients at different AOI and
STOC amplitude differs, suggesting nonhomogenous coupling between
Ca2+ transients and activation of
Ca2+-activated K+ channels.
|
|
Participation of IP3-producing mechanism on effects of
ACh.
As reported previously (1) an inhibitor of phospholipase C
(PLC), U-73122 (2.5 µM), which inhibits IP3 production,
significantly reduced spontaneous Ca2+ transients
(P < 0.05). In six experiments, we investigated the effects of U-73122 on the effects of ACh. Figure
5A shows that inhibition of
PLC did not prevent the increase of basal Ca2+, suggesting
that the rise in basal Ca2+ may be largely due to
Ca2+ influx (P < 0.05).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of various agents on spontaneous Ca2+
transients and responses to ACh. A: spontaneous
Ca2+ transients were inhibited by U-73122 as previously
reported [see Bayguinov et al. (1)]. After U-73122, ACh
(3- and 5-min time points tabulated) caused further reduction in
spontaneous Ca2+. The increase in basal Ca2+
was not significantly reduced by pretreatment with U-73122. Data are
summarized from 6 cells. B: xestospongin C (Xe-C) inhibited
spontaneous Ca2+ transients as previously reported (see
Ref. 1) but had no significant effect on baseline
Ca2+. In the presence of Xe-C, ACh had no effect on
Ca2+ transients. The increase in basal Ca2+ was
not significantly reduced by pretreatment with Xe-C. Data are
summarized from 6 cells. C: nicardipine (Nicard) had no
significant effect on Ca2+ transients and basal
Ca2+. After nicardipine ACh continued to cause an increase
in basal Ca2+ and a reduction in Ca2+
transients. Data are summarized from 6 cells. D: effects of
Cd2+ (200 µM). Cd2+ did not reduce
Ca2+ transients or basal Ca2+ and did not
significantly affect responses to ACh. *Significance of at least
P < 0.05. Solid bars, Ca2+ transients;
open bars, basal Ca2+.
|
|
As reported previously (1), Xe-C (5 µM), a
membrane-permeable antagonist of IP3 receptors, reduces the
magnitude and frequency of Ca2+ puffs and STOCs. In
experiments on six cells we found that Xe-C inhibited spontaneous
Ca2+ transients (P < 0.05) but did not
prevent the increase in basal Ca2+ after ACh
(P < 0.05; Fig. 5B). ACh had no
significant effect on Ca2+ transients in the presence of
Xe-C (P > 0.05; Fig. 5B).
Influence of Ca2+ channel blockers on
the action of ACh.
We have previously reported that dihidropyridines (e.g., nicardipine, 1 µM) or inorganic Ca2+ channels blockers [e.g.,
Ni2+ (200 µM) or Cd2+ (200 µM)]
did not affect spontaneous Ca2+ transients in
murine colonic myocytes (1). Pretreatment of cells with
nicardipine (1 µM) or Cd2+ (200 µM) in the present
study did not affect responses to ACh (10 µM). Figure 5, C
and D, shows summaries of results from experiments on 12 cells in which the effects of nicardipine (n = 6, P < 0.05) and Cd2+ (n = 6, P < 0.05) were tested on responses to ACh.
ACh induces inward currents in colonic myocytes.
Many previous reports have shown that cholinergic (muscarinic)
stimulation of GI smooth muscle cells induces a nonselective cation
conductance (cf. Refs. 3, 14-16,
27, 32). We found that ACh (10 µM) induced
a persistent net inward current that averaged
26.3 ± 6.5 pA
(holding potential
70 mV) in murine colonic myocytes (Fig.
6A).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 6.
Gd3+ and SKF-96365 inhibit inward current
activated by ACh. A: voltage-clamp recordings from a cell
stepped from 40 to 70 mV before (control) and after addition of ACh
(10 µM). There was a persistent increase in inward current noted in
the presence of ACh. B: effects of Gd3+.
Gd3+ reduced the basal inward conductance of cells
(middle trace; cell stepped from 40 to 70 mV) and
blocked the increase in inward current after ACh (right
trace). C: effects of SKF-96365. SKF-96365 also
decreased basal inward current (middle trace; cell stepped
from 40 to 70 mV) and inhibited inward current stimulated by ACh
(right trace). Summary data are reported in the text. In
some of the traces shown spontaneous outward currents (which were
observed at 40 mV but not at 70 mV) can be seen just before the
step to 70 mV. Dotted line denotes zero current in each
panel.
|
|
Gadolinium (Gd3+), a blocker of nonselective cation
currents including the nonselective cation conductance activated by
muscarinic stimulation (H. K. Lee, O. Bayguinov, and K. M. Sanders, unpublished observations), reduced basal inward
current at
70 mV (i.e., from
22.6 ± 2.2 to
14.0 ± 1.5 pA; n = 4; P < 0.05) and inhibited the
increase in basal current on exposure to ACh (10 µM; i.e., from
14.0 ± 1.5 pA with Gd3+ to
13.1 ± 1.4 pA
with Gd3+ and ACh; n = 4; P > 0.5; e.g., Fig. 6B). Another inhibitor of receptor-mediated Ca2+ influx channels, SKF-96365
(22), also inhibited basal inward current at
70 mV
(i.e., from
12.2 ± 5.4 to
6.0 ± 2.7 pA;
n = 4; P < 0.01) and inhibited the
inward current induced by ACh (i.e., from
6.0 ± 2.7 pA with
SKF-96365 to
6.0 ± 1.3 pA with SKF-96365 and ACh;
n = 4; P > 0.5; e.g., Fig.
6C).
Gd3+ and SKF-96365 reduce the rise in
basal Ca2+ and block the inhibition of
Ca2+ transients caused by ACh.
Pretreatment of cells with Gd3+ (10 µM) caused no
significant effect in Ca2+ transients (112 ± 13% of
control; n = 6, P > 0.05), and
application of ACh (10 µM) significantly increased the area of
Ca2+ transients (to 129 ± 15% of control;
P < 0.05). Basal Ca2+ remained unchanged
in response to ACh in the presence of Gd3+ (i.e.,
97 ± 2% of control; n = 6, P > 0.05; Fig. 7).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 7.
Gd3+ blocks the effects of ACh on spontaneous
Ca2+ transients. A: Ca2+ transients
during 20 s from a spontaneously active AOI (AOI-SA). Middle and
bottom traces show Ca2+ transients from the same AOI-SA
after addition of Gd3+ (10 µM) and ACh (10 µM).
B: data during the same scans as in A from an AOI
that was not spontaneously active (AOI-NSA). Note that ACh did not
increase basal Ca2+ or inhibit Ca2+ transients
in the presence of Gd3+. C: summary of 6 experiments in which ACh was added after Gd3+. There was no
statistical change in basal Ca2+ (open bars) after ACh in
the presence of Gd3+, and there was a significant increase
in Ca2+ transients (solid bars). *P < 0.05 after ACh in the presence of Gd3+.
|
|
Pretreatment with SKF-96365 (10 µM) had no significant effect on
Ca2+ transients, and application of ACh (1 µM) caused a
significant increase in Ca2+ transients over control
activity (e.g., to 146 ± 7% of control in the 10th min after
exposure; n = 6, P < 0.05; Fig.
8). Basal Ca2+ levels were
unchanged by ACh in the presence of SKF-96365 (e.g., 100 ± 7% of
control after 10 min; n = 6, P > 0.05).

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 8.
SKF-96365 blocks the inhibitory effects of ACh on spontaneous
Ca2+ transients. A: a cell with 3 active AOI
(numbered 1-3; arrows). One nonactive AOI is also noted. Addition
of SKF-96365 did not significantly affect the occurrence of
Ca2+ transients or basal Ca2+. Addition of ACh
after pretreatment with SKF-96365 caused a significant increase in
spontaneous Ca2+ transients in some cells, and in others
localized Ca2+ transients developed into spreading waves
(e.g., cell depicted in A; sequence of Ca2+ wave
as numbered). B: fluorescence traces from the AOIs noted in
A. Note the occurrence of localized Ca2+
transients in AOIs 1-3 and lack of activity in AOI 4. After ACh a
large Ca2+ transient was recorded at each AOI as a wave
spread through the cell. C: tabulations of Ca2+
transients (solid bars) and basal Ca2+ (open bars) after
SKF- 96365 and after ACh (5- and 10-min exposures). *Significance of
at least P < 0.05.
|
|
Ca2+ influx blocks spontaneous
Ca2+ transients.
The experiments with Gd3+ and SKF-96365 reveal a
dual effect of ACh in murine colonic myocytes. It appears from our
study that activation of inward current in colonic myocytes by ACh is
responsible for the rise in basal Ca2+ after ACh.
Additionally, G protein-dependent increases in IP3 leads to
release of Ca2+ from internal stores (4). Our
experiments have shown that the latter couples to activation of
K+ channels (i.e., STOCs). It is unclear how muscarinic
stimulation overrides the activation of K+ channels to
yield a dominantly excitatory response. We hypothesized that the rise
in basal Ca2+ caused by ACh inhibits spontaneous
Ca2+ transients and thus deactivates the coupling between
spontaneous Ca2+ release and K+ channel
activation. We tested this hypothesis by adding ionomycin, a compound
that would tend to raise cytoplasmic Ca2+ levels without
the concomitant stimulation of Ca2+ release by
PLC-dependent increases in IP3 production. In experiments on six cells we compared the effects of ACh with ionomycin (250 and 500 nM) and found that both compounds increased basal Ca2+ and
inhibited spontaneous Ca2+ transients (Fig.
9, A and C).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 9.
Ionomycin and elevated external Ca2+
increased basal Ca2+ and inhibited spontaneous
Ca2+ transients. A: spontaneous Ca2+
transients from an active AOI during control recording and after
addition of ionomycin (500 nM). Note the increase in basal
Ca2+ and inhibition of Ca2+ transients. Other
spontaneously active AOI were similarly affected. B: similar
effects when external Ca2+ was elevated to 6 mM. Data are
shown after a 3-min exposure to 6 mM Ca2+. Elevated
Ca2+ caused an increase in basal Ca2+ and
inhibition of Ca2+ transients. C: summary of the
average decreases in spontaneous transients (solid bars) and increases
in basal Ca2+ (open bars) caused by ionomycin and elevated
external Ca2+. All points were significant to at least
P < 0.05.
|
|
In addition to increasing Ca2+ influx, it is possible that
ionomycin could also have unloaded internal Ca2+ stores and
thereby affected Ca2+ transients. Therefore, we also tested
the effects of increasing external Ca2+ on basal
cytoplasmic Ca2+ levels and spontaneous Ca2+
transients. Exposure of cells to 6 mM (n = 6) caused a
progressive substantial increase in basal Ca2+ (to 205 ± 33%; P < 0.001) and reduced Ca2+
transients (to 47 ± 8.6; P < 0.005). Figure 9,
B and C, shows an example and a summary of the
effects of increasing extracellular Ca2+ on basal
Ca2+ and Ca2+ transients.
Effects of ACh on Ca2+ transients
were inhibited by FK-506.
Our data suggest that the rise in basal Ca2+ may inhibit
spontaneous Ca2+ transients in murine colonic myocytes.
Part of the regulation of IP3-dependent Ca2+
release may be due to FKBP12 tethering of calcineurin to
IP3 receptors (8, 9). We tested the effects of
FK-506, which disassociates FKBP12 from its targets, on the effects of
ACh in colonic myocytes. Pretreatment of six cells with FK-506 (1 µM) had no effect on spontaneous Ca2+ transients or basal
Ca2+ (P > 0.5 for both parameters).
Addition of ACh (10 µM) in the continued presence of FK-506 caused a
normal increase in basal Ca2+ (e.g., 139 ± 11% after
5 min exposure, n = 6, P < 0.05);
however, spontaneous Ca2+ transients were not inhibited
(P > 0.05; Fig. 10).
In the presence of FK-506, Ca2+ waves were generated after
addition of ACh in two of six cells.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 10.
FK-506 prevents the inhibition of Ca2+ transients by
ACh. A: Ca2+ transients from an active AOI.
FK-506 had no significant effect on the occurrence of spontaneous
Ca2+ transients (2nd trace). Addition of ACh did not
inhibit spontaneous Ca2+ transients (3rd and 4th traces),
and in some cells Ca2+ waves developed (5th trace).
B: fluorescence traces from an inactive AOI. Note that
FK-506 did not inhibit the increase in basal Ca2+ after
addition of ACh. C: summary of data from 6 cells pretreated
with FK-506 and then exposed to ACh (1-, 3-, and 5-min exposures).
Solid bars, spontaneous transients; open bars, basal Ca2+.
*Significance of at least P < 0.05.
|
|
We also tested the effects of FK-506 and cyclosporin A on the rise in
basal Ca2+ and inhibition of Ca2+ transients
observed when cells were exposed to elevated external Ca2+.
Exposure of cells to 6 mM caused an increase in basal Ca2+
and reduced Ca2+ transients (see above). After pretreatment
of cells with FK-506 (1 µM), increased external Ca2+
increased basal Ca2+ to 221 ± 24% of control
(P < 0.005), but Ca2+ transients were not
significantly affected (i.e., 97 ± 20% of control;
n = 6; P = 0.64). Pretreatment of
cells with cyclosporin A had similar effects. In the presence of
cyclosporin A (1 µM) elevated external Ca2+ increased
basal Ca2+ to 225 ± 20% of control
(P < 0.005), but the elevation in basal Ca2+ was not accompanied by effects on Ca2+
transients (i.e., 139 ± 31% of control; P = 0.89).
 |
DISCUSSION |
Localized Ca2+ release couples to
activation of K+ channels in colonic
myocytes.
Localized Ca2+ transients have been reported in vascular,
tracheal, and small intestinal smooth muscles (12, 26, 29, 31, 38), and, in general, these events have been attributed to
Ca2+ release from ryanodine receptors. In colonic smooth
muscle cells ryanodine was found to have no effect on spontaneous
Ca2+ transients, and it was recently reported that
localized Ca2+ transients predominantly resulted from
Ca2+ release from IP3 receptor-operated stores
(1, 19). Studies of localized Ca2+ transients
in smooth muscle cells have supported the unorthodox concept that
release of stored Ca2+ in smooth muscle cells may not
always couple to contraction, but, in fact, Ca2+ release
may be an inhibitory signal in many smooth muscles due to activation of
Ca2+-dependent K+ currents in the plasma
membrane (26, 29). For example, localized Ca2+
transients in colonic myocytes activate Ca2+-dependent ion
channels, including BK and SK channels (1, 19), and
activation of these channels results in membrane hyperpolarization and
reduced excitability. Stimulation of colonic myocytes cells with ATP,
via P2Y receptors and activation of PLC, increased
Ca2+ transients, increased the tendency for localized
Ca2+ release to develop into Ca2+ waves, and
increased coupling between Ca2+ transients and activation
of BK and SK channels. Thus inhibitory stimulation by ATP (and the
P2Y agonist 2-methylthio-ATP) increased K+
channel open probability via a mechanism involving localized Ca2+ release.
Muscarinic stimulation of colonic myocytes inhibits spontaneous
Ca2+ release from IP3
receptor-operated stores.
In the present study we investigated how muscarinic stimulation, which
is also coupled by G proteins to activation of PLC and IP3
formation, can overcome the tendency for activation of IP3
receptor-operated Ca2+ release to drive activation of
K+ channels, which is predominantly an inhibitory response.
The main implications of the present study are that muscarinic
stimulation of colonic muscles includes at least two important phases:
1) IP3 receptor-operated Ca2+
release generates Ca2+ transients, and 2) global
Ca2+ rises, due at least in part to Ca2+ entry,
and this suppresses Ca2+ transients and reduces coupling
between localized Ca2+ release and
Ca2+-activated K+ currents. We suggest that
reducing the activation of Ca2+-activated K+
channels by this mechanism facilitates the development of excitatory responses to muscarinic stimulation. Deactivation of localized Ca2+ transients is a previously unreported aspect of
muscarinic stimulation in GI smooth muscles.
Increased basal Ca2+ due to
Ca2+ entry inhibits
Ca2+ release from IP3
receptor-operated stores.
Muscarinic stimulation of colonic myocytes resulted in a conversion in
the pattern of Ca2+ transients from spontaneous localized
Ca2+ transients to a sustained elevation in global
Ca2+. Conversion of the Ca2+ response pattern
from localized Ca2+ transients to a global increase in
Ca2+ in response to agonist stimulation has been observed
in nonexcitable cells (e.g., Ref. 7). In these experiments
conversion from local responses to global increases in Ca2+
were characterized by increased frequency, amplitude, and/or distribution of localized transients, and spatial and/or temporal summation of transients led to massive Ca2+ waves. The
responses of colonic myocytes differed in that global Ca2+
increased tonically in response to ACh, not as repetitive waves. Second, there were two processes that contributed to the
Ca2+ response. Ca2+ entry through
receptor-operated cation channels was necessary for the global
increase, and the global increase appeared to inhibit the
IP3-dependent Ca2+ transients. When
Ca2+ entry was prohibited, ACh stimulation increased
Ca2+ transients (much the same way that ATP increased
Ca2+ transients via an IP3-dependent mechanism;
see Ref. 1). In some cells in which Ca2+ entry
was blocked, Ca2+ waves occurred in response to ACh, and
this response may have been equivalent to the changes observed in
nonexcitable cells during agonist stimulation (7).
There are many reports in the literature about the nonselective cation
conductance activated in visceral smooth muscles by muscarinic
stimulation (e.g., Refs. 6, 15,
16, 27, 33). The cation
conductance activated by muscarinic stimulation requires the
simultaneous activation of pertussis toxin-sensitive (M2) receptors and PLC (M3)-linked receptors (6, 15, 16,
27, 33). Although this conductance is permeable to
Ca2+, it is unclear whether much Ca2+ entry
occurs via this pathway in physiological Ca2+ gradients
(14). Pacaud and Bolton (29)
reported that the global Ca2+ transients in guinea pig
jejunal muscle cells in response to muscarinic stimulation included an
initial transient phase that was dependent on release of
Ca2+ from IP3 receptor-operated stores and a
second, more sustained phase that depended on Ca2+ entry by
a non-L-type Ca2+ conductance. We confirmed that
the rise in basal Ca2+ in response to ACh was not blocked
by nicardipine. Ca2+ entry that was responsible for the
increase in basal Ca2+ occurred via a conductance blocked
by Gd3+ and SKF-96365; however, further studies will be
necessary to fully determine the nature of this conductance and whether
it is related to the nonselective cation conductance activated in other
GI muscles.
In previous studies ACh transiently increased STOCS in tracheal [Wade
and Sims (37), Saunders and Farley (34)],
esophageal [Hurley et al. (14)], jejunal [Benham and
Bolton (2)], and vascular smooth muscle cells.
Reductions in or cessation of STOCs often followed the initial
stimulation. These authors concluded that the initial burst of STOCs
following ACh application was due to release of Ca2+ from
intracellular stores, and the inhibitory phase was due to unloading of
stores because a similar pattern of STOC activity was observed when
high concentrations of caffeine were applied. Application of ACh to
murine myocytes did not result in a significant initial burst of STOC
activity, but, within a short period, we observed decreased localized
Ca2+ transients and reduction in STOCs. The reasons for the
lack of an initial increase in STOCs in murine myocytes are not
entirely clear, but this may be due to a relatively lower store content in phasic colonic muscles vs. tonic muscles such as tracheal and esophageal muscles.
Our experiments suggest that the inhibitory effects of ACh on STOCs
were directed at inhibition of the localized Ca2+
transients that underlie STOCs. We concluded that this effect was not
due to store unloading because: 1) the inhibitory effects of
ACh on Ca2+ transients were absent (and in fact we
observed a sustained increase in Ca2+ transients)
when the rise in global Ca2+ in response to ACh was blocked
by Gd3+ and SKF-96365; and 2) addition of
caffeine after inhibition of Ca2+ transients by ACh caused
a massive increase in Ca2+ and contraction of cells.
Blockade of the rise in basal Ca2+ converted muscarinic
responses into effects that were similar to those observed previously
via stimulation of P2Y receptors (i.e., enhancement in
localized Ca2+ transients; and a tendency for localized
Ca2+ transients to organize into Ca2+ waves;
see Ref. 1). Furthermore, the rise in basal
Ca2+ and inhibition of spontaneous Ca2+
transients and STOCs in response to ACh were mimicked by treatment of
cells with ionomycin and elevated external Ca2+, more
generic stimulants of Ca2+ entry that were unlikely to
involve signaling via metabotrophic receptors. These observations led
to the conclusion that the rise in basal Ca2+ promotes the
inhibition of Ca2+ transients (and STOCs). These findings
highlight some interesting similarities between two stimuli (i.e., ACh
and ATP) that have not previously been explained: both agonists couple
to responses through IP3 formation, but ACh and ATP elicit
opposite responses in colonic smooth muscles. In the case of muscarinic
stimulation, the superposition of a mechanism to enhance basal
Ca2+ suppresses the underlying IP3-driven
increase in localized Ca2+ transients that naturally elicit
activation of outward currents.
How does increased basal Ca2+ inhibit
Ca2+ release from IP3
receptor-operated stores?
Our results suggest that the rise in basal Ca2+ that
accompanies muscarinic stimulation inhibits IP3
receptor-operated Ca2+ release events. The complex effects
of cytoplasmic Ca2+ on the sensitivity of IP3
receptors might explain the inhibition of
Ca2+ release caused by a rise in
basal Ca2+. Studies on skinned smooth muscle fibers have
shown that release of Ca2+ from IP3
receptor-operated stores is regulated by
[Ca2+]i in a biphasic manner with positive
feedback operating below 300 nM [Ca2+]i and
negative feedback acting above 300 nM [Ca2+]i
(13). According to studies of single
IP3-receptor channels, the bell-shaped relationship between
open probability (Po) and [Ca2+]i has a sharp peak when
[Ca2+]i is <1 µM and levels of
IP3 are low (i.e., <20 nM) (21). These studies suggested that the effects of [Ca2+]i
on Po of IP3 receptors would be to
increase IP3 sensitivity as
[Ca2+]i increases from low basal levels, but,
with further increases in [Ca2+]i,
IP3 sensitivity would decrease. Thus, at low levels of
[Ca2+]i, IP3 levels may be
sufficient to produce spontaneous Ca2+ transients, but, as
basal Ca2+ rises, even though IP3 levels may
also rise in response to muscarinic stimulation, the spontaneous
openings of IP3 receptor-operated channels could be reduced
by the negative feedback exerted by rising
[Ca2+]i.
The expression and distribution of the different isoforms of
IP3 receptors in visceral smooth muscles may influence the
pattern of responses to changes in [Ca2+]i
and IP3. Studies have shown that the three isoforms of
IP3 receptors respond differently to cytoplasmic agonists
such as IP3, Ca2+, and ATP (25),
and expression of multiple isoforms can result in complex
spatiotemporal patterns. Future studies will investigate relative
isoform expression in GI smooth muscle that manifest spontaneous
IP3-dependent Ca2+ transients to determine the
unique combination of receptors responsible for the patterns of
Ca2+ transients in these cells. IP3 receptor
expression becomes an important issue because studies have now shown
that Ca2+-release events via IP3
receptor-operated channels contribute to resting membrane potential,
the inhibitory response to purinergic neurotransmission (1,
18), and the development of excitatory responses to muscarinic stimulation.
There are several mechanisms by which elevated Ca2+ or
Ca2+ binding proteins might decrease the tendency of
IP3 to release Ca2+. IP3 receptors
have intrinsic Ca2+ binding sites (23), and it
is possible that direct binding of Ca2+ could decrease the
sensitivity of IP3 receptors for IP3. Two of
the sites for Ca2+ binding lie within the IP3
binding domain, and thus Ca2+ could exert antagonism on
IP3 binding to its receptor (30). It is also
possible that interactions with calmodulin could be involved in
regulation or IP3 sensitivity. IP3 receptor
subunits bind one calmodulin molecule in the absence of
Ca2+ and two when Ca2+ is present
(10). The effectiveness of IP3 in releasing
Ca2+ from IP3 receptor-operated stores is
reduced by calmodulin binding (28).
Ca2+-dependent enzymes may also regulate the interactions
of IP3 and its receptor. IP3 receptors are
tightly associated with the immunophilin, FKBP12 (8), and
calcineurin (which is expressed by colonic smooth muscle cells; G. Amberg, B. Perrino, and K. M. Sanders, unpublished observations)
is physiologically associated with the IP3 receptor-FKBP12
complex. The immunosuppressant drug FK-506 binds to FKBP12 and
inhibits its association with IP3 receptors (8). It is thought that anchoring calcineurin to
IP3 receptors with FKBP12 controls the level of
phosphorylation of the receptor and contributes to the
Ca2+-dependent regulation of Ca2+ release from
IP3 receptor-operated stores (9, 34). Our data showing that FK-506 decreased the inhibition of spontaneous
Ca2+ transients associated with the rise in basal
Ca2+ due to ACh or elevated external Ca2+
suggest that IP3 receptor-FKBP12 association, possibly via
tethering of calcineurin, might be important in regulating
Ca2+ sensitivity of IP3 receptors.
Additionally, we found that cyclosporin A, which disrupts calcineurin
activity by interacting with cyclophilin to form a
calcineurin-inhibitory complex [see Perrino and Soderling (32)], also inhibited the tendency of elevated external
Ca2+ to inhibit Ca2+ transients. Together,
these data suggest that cytoplasmic Ca2+ regulation of
IP3 receptors, possibly via the actions of calcineurin, might be an important aspect of muscarinic responses in colonic myocytes.
In summary, the response of colonic myocytes to muscarinic stimulation
includes enhanced IP3 receptor-operated Ca2+
release that is potentially coupled to activation of
Ca2+-dependent K+ channels. This tendency is
suppressed in colonic cells by Ca2+ entry and a rise in
basal Ca2+ that inhibits spontaneous Ca2+
transients. Ca2+-dependent negative feedback of
Ca2+ release from IP3 receptor-operated
channels may be an important step in defining whether muscarinic
stimulation is an excitatory or inhibitory signal in GI muscles.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Program Project Grant DK-41315. The Noran
confocal microscope was purchased by National Heart, Lung, and Blood
Institute shared equipment Grant HL-44455.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: K. M. Sanders, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Reno, NV 89557-0046 (E-mail:
kent{at}physio.unr.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 June 2000; accepted in final form 4 October 2000.
 |
REFERENCES |
1.
Bayguinov, O,
Hagen B,
Bonev AD,
Nelson MT,
and
Sanders KM.
Intracellular calcium events activated by ATP in murine colonic myocytes.
Am J Physiol Cell Physiol
279:
C126-C135,
2000[Abstract/Free Full Text].
2.
Benham, CD,
and
Bolton TB.
Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of the rabbit.
J Physiol (Lond)
381:
385-406,
1986[Abstract].
3.
Benham, CD,
Bolton TB,
and
Lang RJ.
Acetylcholine activates an inward current in single mammalian smooth muscle cells.
Nature
316:
345-347,
1985[ISI][Medline].
4.
Boittin, FX,
Coussin F,
Macrez N,
Mironneau C,
and
Mironneau J.
Inositol 1,4,5-trisphosphate- and ryanodine-sensitive Ca2+ release channel-dependent Ca2+ signalling in rat portal vein myocytes.
Cell Calcium
23:
303-311,
1998[ISI][Medline].
5.
Bolton, TB,
Prestwich SA,
Zholos AV,
and
Gordienko DV.
Excitation-contraction coupling in gastrointestinal and other smooth muscles.
Annu Rev Physiol
61:
85-115,
1999[ISI][Medline].
6.
Bolton, TB,
and
Zholos AV.
Activation of M2 muscarinic receptors in guinea-pig ileum opens cationic channels modulated by M3 muscarinic receptors.
Life Sci
60:
1121-1128,
1997[ISI][Medline].
7.
Bonev, AD,
Jaggar JH,
Rubart M,
and
Nelson MT.
Activators of protein kinase C decrease Ca2+ spark frequency in smooth muscle cells from cerebral arteries.
Am J Physiol Cell Physiol
273:
C2090-C2095,
1997[Abstract/Free Full Text].
8.
Bootman, MD,
Berridge MJ,
and
Lipp P.
Cooking with calcium: the recipes for composing global signals from elementary events.
Cell
91:
367-373,
1997[ISI][Medline].
9.
Cameron, AM,
Steiner JP,
Roskams AJ,
Ali SM,
Ronnett GV,
and
Snyder SH.
Calcineurin associated with the inositol 1,4,5-trisphosphate receptor-FKBP12 complex modulates Ca2+ flux.
Cell
83:
463-472,
1995[ISI][Medline].
10.
Cameron, AM,
Steiner JP,
Sabatini DM,
Kaplin AI,
Walensky LD,
and
Snyder SH.
Immunophilin FK506 binding protein associated with inositol 1,4,5-trisphosphate receptor modulates calcium flux.
Proc Natl Acad Sci USA
92:
1784-1788,
1995[Abstract].
11.
Cardy, TJ,
and
Taylor CW.
A novel role for calmodulin: Ca2+-independent inhibition of type-1 inositol trisphosphate receptors.
Biochem J
334:
447-455,
1998[ISI][Medline].
12.
Cole, WC,
Carl A,
and
Sanders KM.
Muscarinic suppression of Ca2+-dependent K+ current in colonic smooth muscle.
Am J Physiol Cell Physiol
257:
C481-C487,
1989[Abstract/Free Full Text].
13.
Gordienko, DV,
Bolton TB,
and
Cannell MB.
Variability in spontaneous subcellular calcium release in guinea-pig ileum smooth muscle cells.
J Physiol (Lond)
507:
707-720,
1998[Abstract/Free Full Text].
14.
Hurley, BR,
Preiksaitis HG,
and
Sims SM.
Characterization and regulation of Ca2+-dependent K+ channels in human esophageal smooth muscle.
Am J Physiol Gastrointest Liver Physiol
276:
G843-G852,
1999[Abstract/Free Full Text].
15.
Iino, M.
Biphasic Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca release in smooth muscle cells of the guinea pig taenia caeci.
J Gen Physiol
95:
1103-1122,
1990[Abstract].
16.
Inoue, R,
and
Isenberg G.
Acetylcholine activates nonselective cation channels in guinea pig ileum through a G protein.
Am J Physiol Cell Physiol
258:
C1173-C1178,
1990[Abstract/Free Full Text].
17.
Inoue, R,
and
Isenberg G.
Effect of membrane potential on acetylcholine-induced inward current in guinea-pig ileum.
J Physiol (Lond)
424:
57-71,
1990[Abstract].
18.
Inoue, R,
and
Isenberg G.
Intracellular calcium ions modulate acetylcholine-induced inward current in guinea-pig ileum.
J Physiol (Lond)
424:
73-92,
1990[Abstract].
19.
Jaggar, JH,
Porter VA,
Lederer WJ,
and
Nelson MT.
Calcium sparks in smooth muscle.
Am J Physiol Cell Physiol
278:
C235-C256,
2000[Abstract/Free Full Text].
20.
Koh, SD,
Dick GM,
and
Sanders KM.
Small-conductance Ca2+-dependent K+ channels activated by ATP in murine colonic smooth muscle.
Am J Physiol Cell Physiol
273:
C2010-C2021,
1997[Abstract/Free Full Text].
21.
Kong, ID,
Koh SD,
and
Sanders KM.
Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes.
Am J Physiol Cell Physiol
278:
C352-C362,
2000[Abstract/Free Full Text].
22.
Kunze, WA,
and
Furness JB.
The enteric nervous system and regulation of intestinal motility.
Annu Rev Physiol
61:
117-142,
1999[ISI][Medline].
23.
Mak, DO,
McBride S,
and
Foskett JK.
Inositol 1,4,5-trisphosphate [correction of tris-phosphate] activation of inositol trisphosphate [correction of tris-phosphate] receptor Ca2+ channel by ligand tuning of Ca2+ inhibition.
Proc Natl Acad Sci USA
95:
15821-15825,
1998[Abstract/Free Full Text].
24.
Merritt, JE,
Armstrong WP,
Benham CD,
Hallam TJ,
Jacob R,
Jaxa-Chamiec A,
Leigh BK,
McCarthy SA,
Moores KE,
and
Rink TJ.
SK&F 96365, a novel inhibitor of receptor-mediated calcium entry.
Biochem J
271:
515-522,
1990[ISI][Medline].
25.
Mignery, GA,
Johnston PA,
and
Sudhof TC.
Mechanism of Ca2+ inhibition of inositol 1,4,5-trisphosphate (InsP3) binding to the cerebellar InsP3 receptor.
J Biol Chem
267:
7450-7455,
1992[Abstract/Free Full Text].
26.
Mironneau, J,
Arnaudeau S,
Macrez-Lepretre N,
and
Boittin FX.
Ca2+ sparks and Ca2+ waves activate different Ca2+-dependent ion channels in single myocytes from rat portal vein.
Cell Calcium
20:
153-160,
1996[ISI][Medline].
27.
Miyakawa, T,
Maeda A,
Yamazawa T,
Hirose K,
Kurosaki T,
and
Iino M.
Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes.
EMBO J
18:
1303-1308,
1999[Abstract/Free Full Text].
28.
Nelson, MT,
Cheng H,
Rubart M,
Santana LF,
Bonev AD,
Knot HJ,
and
Lederer WJ.
Relaxation of arterial smooth muscle by calcium sparks.
Science
270:
633-637,
1995[Abstract].
29.
Pacaud, P,
and
Bolton TB.
Relation between muscarinic receptor cationic current and internal calcium in guinea-pig jejunal smooth muscle cells.
J Physiol (Lond)
441:
477-499,
1991[Abstract].
30.
Patel, S,
Morris SA,
Adkins CE,
O'Beirne G,
and
Taylor CW.
Ca2+ -independent inhibition of inositol trisphosphate receptors by calmodulin: redistribution of calmodulin as a possible means of regulating Ca2+ mobilization.
Proc Natl Acad Sci USA
94:
11627-11632,
1997[Abstract/Free Full Text].
31.
Perez, GJ,
Bonev AD,
Patlak JB,
and
Nelson MT.
Functional coupling of ryanodine receptors to KCa channels in smooth muscle cells from rat cerebral arteries.
J Gen Physiol
113:
229-238,
1999[Abstract/Free Full Text].
32.
Perrino, BA,
and
Soderling TR.
Biochemistry and pharmacology of calmodulin regulated phosphatase calcineurin.
In: Calmodulin and Signal Transduction. New York: Academic, 1998, p. 170-236.
33.
Pietri, F,
Hilly M,
and
Mauger JP.
Calcium mediates the interconversion between two states of the liver inositol 1,4,5-trisphosphate receptor.
J Biol Chem
265:
17478-17485,
1990[Abstract/Free Full Text].
34.
Saunders, HH,
and
Farley JM.
Pharmacological properties of potassium currents in swine tracheal smooth muscle.
J Pharmacol Exp Ther
260:
1038-1044,
1992[Abstract].
35.
Sieck, GC,
Kannan MS,
and
Prakash YS.
Heterogeneity in dynamic regulation of intracellular calcium in airway smooth muscle cells.
Can J Physiol Pharmacol
75:
878-888,
1997[ISI][Medline].
36.
Vogalis, F,
and
Sanders KM.
Cholinergic stimulation activates a non-selective cation current in canine pyloric circular muscle cells.
J Physiol (Lond)
429:
223-236,
1990[Abstract].
37.
Wade, SM,
and
Sims GR.
Muscarinic stimulation of tracheal smooth muscle cells activates large-conductance Ca2+-dependent K+ channel.
Am J Physiol Cell Physiol
265:
C658-C665,
1993[Abstract/Free Full Text].
38.
Wang, YX,
and
Kotlikoff MI.
Signalling pathway for histamine activation of non-selective cation channels in equine tracheal myocytes.
J Physiol (Lond)
523:
131-138,
2000[Abstract/Free Full Text].
39.
Zhang, BX,
Zhao H,
and
Muallem S.
Ca2+-dependent kinase and phosphatase control inositol 1,4,5-trisphosphate-mediated Ca2+ release. Modification by agonist stimulation.
J Biol Chem
268:
10997-11001,
1993[Abstract/Free Full Text].
40.
Zhang, LB,
and
Buxton IL.
Muscarinic receptors in canine colonic circular smooth muscle. II. Signal transduction pathways.
Mol Pharmacol
40:
952-959,
1991[Abstract].
41.
Zhang, LB,
Horowitz B,
and
Buxton IL.
Muscarinic receptors in canine colonic circular smooth muscle. I. Coexistence of M2 and M3 subtypes.
Mol Pharmacol
40:
943-951,
1991[Abstract].
42.
Zholos, AV,
and
Bolton TB.
Muscarinic receptor subtypes controlling the cationic current in guinea-pig ileal smooth muscle.
Br J Pharmacol
122:
885-893,
1997[Abstract].
43.
ZhuGe, R,
Sims SM,
Tuft RA,
Fogarty KE,
and
Walsh JV, Jr.
Ca2+ sparks activate K+ and Cl
channels, resulting in spontaneous transient currents in guinea-pig tracheal myocytes.
J Physiol (Lond).
513:
711-718,
1998[Abstract/Free Full Text].
Am J Physiol Cell Physiol 280(3):C689-C700
0363-6143/01 $5.00
Copyright © 2001 the American Physiological Society