Intracellular calcium events activated by ATP in murine
colonic myocytes
Orline
Bayguinov1,
Brian
Hagen1,
Adrian D.
Bonev2,
Mark T.
Nelson2, and
Kenton M.
Sanders1
1 Department of Physiology and Cell Biology, University of
Nevada School of Medicine, Reno, Nevada 89557; and
2 Department of Pharmacology, University of Vermont College
of Medicine, Burlington, Vermont 05405
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ABSTRACT |
ATP is a candidate enteric inhibitory neurotransmitter
in visceral smooth muscles. ATP hyperpolarizes visceral muscles via activation of small-conductance, Ca2+-activated
K+ (SK) channels. Coupling between ATP stimulation and SK
channels may be mediated by localized Ca2+ release.
Isolated myocytes of the murine colon produced spontaneous, localized
Ca2+ release events. These events corresponded to
spontaneous transient outward currents (STOCs) consisting of
charybdotoxin (ChTX)-sensitive and -insensitive events.
ChTX-insensitive STOCs were inhibited by apamin. Localized
Ca2+ transients were not blocked by ryanodine, but these
events were reduced in magnitude and frequency by xestospongin C
(Xe-C), a blocker of inositol 1,4,5-trisphosphate receptors. Thus we
have termed the localized Ca2+ events in colonic myocytes
"Ca2+ puffs." The P2Y receptor agonist
2-methylthio-ATP (2-MeS-ATP) increased the intensity and frequency of
Ca2+ puffs. 2-MeS-ATP also increased STOCs in association
with the increase in Ca2+ puffs.
Pyridoxal-phospate-6-azophenyl-2',4'-disculfonic acid tetrasodium, a
P2 receptor inhibitor, blocked responses to 2-MeS-ATP. Spontaneous Ca2+ transients and the effects of 2-MeS-ATP on
Ca2+ puffs and STOCs were blocked by U-73122, an inhibitor
of phospholipase C. Xe-C and ryanodine also blocked responses to
2-MeS-ATP, suggesting that, in addition to release from IP3
receptor-operated stores, ryanodine receptors may be recruited during
agonist stimulation to amplify release of Ca2+. These data
suggest that localized Ca2+ release modulates
Ca2+-dependent ionic conductances in the plasma membrane.
Localized Ca2+ release may contribute to the electrical
responses resulting from purinergic stimulation.
calcium puffs; local calcium transients; P2Y receptors; enteric neurotransmission
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INTRODUCTION |
ENTERIC INHIBITORY
NEURONS express and utilize multiple neurotransmitters to
regulate relaxation of gastrointestinal muscles. Numerous studies have
demonstrated that nitric oxide (NO) is a key enteric inhibitory
neurotransmitter (see Ref. 35 for review); however, in many
gastrointestinal muscles, there are multiple components to the
inhibitory response (see Refs. 15, 23, 36, 39). A number of
reports suggest that, in addition to NO, ATP serves as a primary
inhibitory neurotransmitter released from enteric motor neurons, and it
is thought that ATP works by activation of an apamin-sensitive ionic
conductance (13, 20). Recent studies have
identified small-conductance, Ca2+-activated K+
(SK) channels in gastrointestinal muscles that are activated by
purinergic stimulation (24, 41), but the
mechanism of coupling receptor activation to channel opening is not understood.
Colonic muscles express P2Y receptors that are thought to
mediate relaxation responses to ATP (12, 42).
Several isoforms of P2Y receptors, but not all, have been
shown to couple to activation of phospholipase C (PLC) and to stimulate
production of inositol 1,4,5-trisphosphate (IP3; see Refs.
3, 11, 31). Ahn and co-workers (1) proposed that some of
the effects mediated by activation of P2Y receptors in
gastrointestinal smooth muscles may be mediated by release of
Ca2+ from intracellular stores. Release of Ca2+
from IP3 receptor-operated stores may be a common mechanism
by which ATP initiates intracellular signaling via P2Y
receptors. For example, others have demonstrated ATP-dependent
Ca2+ release from stores mediated by P2Y
receptors in striatal and neurohypophysial astrocytes (14,
40).
Upon first consideration, it is unclear how inhibitory responses in
gastrointestinal muscles could be mediated by Ca2+ release,
but it is now apparent that localized Ca2+ release can
occur in smooth muscles without significant effects on global
cytoplasmic Ca2+ concentration. Localized Ca2+
release events (sparks) were first observed in vascular smooth muscle
cells (30), and these events were associated with
spontaneous transient outward currents (STOCs) that result from
activation of large-conductance, Ca2+-activated
K+ channels (BK channels; see Refs. 4 and 43). Periodic
Ca2+ sparks in multicellular tissues yield a
hyperpolarizing influence on vascular muscles (22). Recent
studies have shown that a variety of smooth muscles manifest
Ca2+ sparks or periodic Ca2+ waves, and these
events regulate the open probabilities of Ca2+-dependent
conductances in the plasma membrane (17, 33,
37, 43). Ca2+ release mediated by
IP3 receptor-operated channels has also been reported in a
variety of cell types (5, 8,
25). These events, termed Ca2+ blips (i.e.,
elementary Ca2+ release events) or Ca2+ puffs
(i.e., release from clusters of IP3 receptors), could also be involved in regulating membrane ionic conductances
(26), but coupling of IP3-dependent
Ca2+ release to regulation of membrane conductances has not
been demonstrated in smooth muscles. If localized Ca2+
transients have a net effect of activating K+ conductances,
then these events could couple inhibitory responses in gastrointestinal
smooth muscles to G protein-coupled receptors.
We investigated the nature of Ca2+ release events in
colonic muscles. We also investigated the hypothesis that
P2Y receptors are coupled to localized Ca2+
transients via activation of PLC and release of Ca2+ from
IP3 receptor-operated stores. Local Ca2+
transients might activate Ca2+-dependent conductances in
the plasma membrane without significant changes in global cytoplasmic
Ca2+ concentration. Local Ca2+-dependent
regulation of ionic conductances might mediate the responses of
gastrointestinal muscles to inhibitory purinergic neurotransmission.
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METHODS |
Cell preparation.
BALB/C mice (15-30 days old) of either sex were anesthetized with
chloroform and were killed by decapitation. The large intestine was
removed and opened along the mesenteric border, and the luminal contents were washed with Krebs-Ringer bicarbonate buffer (see Solutions and drugs). Tissues were pinned to the base of a
Sylgard-coated dish, and the mucosa and submucosa were removed by peeling.
Colonic muscles were equilibrated in Ca2+-free solution for
60 min, and then the buffer was replaced with an enzyme solution containing collagenase F (Sigma, St. Louis, MO) to disperse single smooth muscle cells. The tissues were incubated with the enzyme 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 through a series of three blunt pipettes of
decreasing tip diameter. Isolated smooth muscle cells were freed from
the tissue matrix by trituration.
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 and to complete the
deesterification. All measurements were made within 15-45 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 (numeric aperture = 1.2) was used to image
the cells. The cells were scanned using INTERVISION software (Noran
Instruments) 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
30 or
40 mV using pCLAMP software (version 7.0; Axon
Instruments). Currents were filtered at 1 kHz and were digitized at 2 kHz. 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 Krebs solution used in this study contained (in mM) 137.4 Na+, 5.9 K+, 2.5 Ca2+, 1.2 Mg2+, 134 Cl
, 15.5 HCO3
,
1.2 H2PO4
, 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 (in mM) 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES,
and 0.05 EGTA (pH 7.2). The pipette solution also contained 250 µg/ml
amphotericin B.
Drugs used.
Nicardipine, cyclopiazonic acid (CPA), ryanodine apamin, charybdotoxin
(ChTX), and 2-methylthio-ATP (2-MeS-ATP) were obtained from Sigma.
Pyridoxal-phospate-6-azophenyl-2',4'-disculphonic acid tetrasodium
(PPADS), U-73122, and U-73343 were obtained from RBI (Natick, MA).
Xestospongin C (Xe-C) was obtained from Calbiochem. Concentrations of
drugs used were determined from the literature or by empirical testing.
Analysis of data.
Image analysis was performed using custom-written analysis programs
using Interactive Data Language software (Research Systems, Boulder,
CO), as previously described (33). Baseline fluorescence (F0) was determined by averaging 10 images (out of 600)
with no activity. Ratio images were then constructed and replayed for careful examination to detect active areas where sudden increases in
F/F0 occurred (33). F/F0 vs. time
traces were further analyzed in Microcal Origin (Microcal Software,
Northampton, MA) and AcqKnoledge Software (Biopac Systems, Santa
Barbara, CA) and represent the averaged F/F0 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 (33). Rise time of
puffs was calculated as the time required to reach peak fluorescence from the baseline. The rate of spread of Ca2+ waves was
calculated as the time required for the peak fluorescence to move 10 µm.
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),
as described in RESULTS. Some drug treatments changed the
characteristics of the Ca2+ transients from brief
Ca2+ puffs to events of more extended duration. As a
result, it was not accurate to analyze the data as simple changes in
frequency. Therefore, we analyzed the data as the area of the
Ca2+ transients above baseline during 20-s scans.
To determine the amplitude of the STOCs, analysis was performed
off-line, using a Mini Analysis Program (Synaptosoft Software, Leonia,
NJ). The threshold of STOCs was set at three times the single
Ca2+-activated K+ (KCa) channel
amplitude at
40 mV or at 6 pA. The activity of KCa
channels in the absence of Ca2+ release events is very low
at
40 mV (number of channels × open probability ~ 10
3; see Ref. 9), with the probability of three
simultaneous openings being exceedingly low.
Statistical analysis.
Results are expressed as means ± SE where applicable. All
statistical analysis was made with SigmaStat 2.03 software (Jandel Scientific Software, San Rafael, CA). The Spearman rank order correlation test was used for correlation analysis.
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RESULTS |
Characterization of spontaneous Ca2+ transients and
STOCs.
Colonic myocytes loaded with fluo 3 produced spontaneous, transient
elevations in intracellular calcium concentration
([Ca2+]i). These events occurred as either
localized events or more widely dispersed, spreading events
(Ca2+ waves; Fig. 1).
Localized Ca2+ events were characterized by a rapid focal
rise in [Ca2+]i (mean rise time was 160 ± 34 ms) and slower decay (mean time to half amplitude was 742 ± 87 ms; n = 60). Frequently, the Ca2+
transients were clustered into groups consisting of multiple events
that did not fully relax to the resting level between events (see Fig.
1B, inset). Clusters of transients were highly
variable in duration but often lasted for more than a second.

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Fig. 1.
Spontaneous Ca2+ transients in colonic
myocytes. A: localized Ca2+ transients that
occurred at 2 different sites during a single 20-s scan. Frames shown
are representative images taken at the maximum of the Ca2+
transients. B: traces 1 and 2 show the
temporal relationship between the 2 sites displaying spontaneous
Ca2+ transients shown in A. Inset in
B shows an expanded time scale of a Ca2+
transient at site 1. Ca2+ waves could be
recorded from the same cells that generated Ca2+ localized
transients. C: additional images of the same cell shown in
A during the same 20-s scan. A Ca2+ wave was
detected in the lower one-half of the cell. Colored circles represent
sites at which fluorescence was monitored during the Ca2+
wave. The wave was first detected at the red site and spread through a
significant part of the bottom one-half of the cell. D:
superimposed traces from the points marked in C. Traces show
the progression of the fluorescence maxima, indicating a spreading
Ca2+ wave. F/F0, ratio of recorded fluorescene
to baseline fluorescence.
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Ca2+ transients, beginning at discrete sites within cells,
often spread through part or all of the cell. From 40 analyzed records, 60% of the cells displayed spontaneous Ca2+ waves.
Ca2+ spread with an average propagation velocity of 32 ± 3 µm/s (n = 9; Fig. 1, C and
D). Localized Ca2+ transients and
Ca2+ waves could be recorded from the same cell and were
observed to originate from the same or from different sites (see Fig.
1, A and C).
In 11 experiments, fluo 3 fluorescence and whole cell membrane currents
were recorded simultaneously. Cells were held at
30 mV while membrane
currents and spontaneous Ca2+ transients were recorded. The
current records showed STOCs in association with the Ca2+
transients (Fig. 2, A-E). A
correlation between STOCs and localized Ca2+ transients was
demonstrated by plots of STOC amplitude vs. the amplitude of the
corresponding Ca2+ transients (Fig. 2F;
correlation coefficient was 0.931; n = 56; P < 0.005). STOCs were reduced in amplitude and
frequency by ChTX (200 nM; treatment for 15 min), as previously
reported (30, 33); however, ChTX did not
fully block STOC activity (Fig. 3). STOCs
remaining after ChTX were further reduced in amplitude and frequency by
apamin (n = 7; Fig. 3), suggesting that SK channels also contribute to STOCs in colonic muscles as previously reported (26).

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Fig. 2.
Ca2+ transients in colonic myocytes were
associated with spontaneous transient outward currents (STOCs).
A: cell with a Ca2+ transient that developed
into a local wave. Frames represent images taken during the black bar
in B. B-D: Ca2+ transients that were
recorded from 3 areas of interest during a 20-s scan of the cell in
A. E: STOCs recorded simultaneously from the same
cell. There was a high degree of correlation between Ca2+
transients and STOCs. However, a few STOCs (denoted by asterisks) were
not apparently associated with resolvable Ca2+ transients
in the 3 areas of interest. These STOCs may have resulted from
Ca2+ transients at nonimaged sites. F: plot of
STOC amplitudes as a function of the amplitude of the Ca2+
transients. Data were fit to a straight line via linear regression
analysis (r = 0.854; n = 56;
P < 0.001).
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Fig. 3.
STOCs persist after charybdotoxin (ChTX). A:
STOC activity under control conditions at a holding potential of 30
mV. B: STOCs after application of ChTX (200 nM).
C: apamin (1 µM) reduced the amplitude and frequency of
ChTX-resistant STOCs. D: histograms showing counts of STOCs
under control conditions (filled bars) and after ChTX (open bars) and
apamin (shaded bars).
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The occurrence of spontaneous Ca2+ transients and the
propagation of Ca2+ waves was not significantly affected by
nicardipine (1 µM). The average area of Ca2+ transients
was 138 ± 31% of control after nicardipine (n = 5; P > 0.5). Ca2+ transients were also not
significantly affected by Ni2+ or Cd2+ (200 µM each); average areas of Ca2+ transients were 100 ± 16% for Ni2+ and 96 ±17% of control area for
Cd2+, respectively (each n = 10 and
P > 0.5). Incubation of cells in nominally
Ca2+-free buffer for at least 30 min, however, resulted in
complete blockade of spontaneous Ca2+ transients.
Spontaneous Ca2+ transients recovered in cells that had
been incubated in Ca2+-free buffer by the readdition of 2 mM Ca2+ to the bath solution.
Spontaneous Ca2+ transients were reduced by CPA (10 µM).
After CPA, the area of Ca2+ transients was 24 ± 4% of the control area (n = 5; P < 0.01). STOCs were also blocked by CPA (n = 5).
Ryanodine (10 µM; 15 min) had little effect on spontaneous
Ca2+ transients; the average area of Ca2+
transients was 97 ± 8% of the control area (n = 10;
P > 0.5), and ryanodine was shown to have no
resolvable effect on STOCs. Figure 4
shows the effects of CPA and ryanodine on spontaneous Ca2+
transients and STOCs.

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Fig. 4.
Effects of ryanodine and cyclopiazonic acid (CPA) on
Ca2+ puffs. A: spontaneous Ca2+
transients before (top) and after (middle)
addition of ryanodine (10 µM; 15 min exposure to ryanodine before
scan). There was no significant change in the area of the
Ca2+ transients after ryanodine. Trace on bottom
shows the same cell after exposure to 2-methylthio-ATP (2-MeS-ATP).
Ryanodine inhibited the increase in Ca2+ transients
typically observed in response to 2-MeS-ATP. B: spontaneous
Ca2+ transients before (top) and after
(middle) addition of CPA (10 µM; 5 min exposure to CPA
before scan). CPA inhibited spontaneous Ca2+ and blocked
the increase in the area of Ca2+ transients observed in
response to 2-MeS-ATP (bottom). C: STOCs recorded
continuously during exposure to ryanodine (10 µM) and CPA (10 µM).
Ryanodine did not affect STOCs, but these events were greatly reduced
by CPA.
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Cells pretreated with ryanodine (10 µM; 15 min), which had no effect
on spontaneous activity (i.e., 97 ± 8% of control
Ca2+ transient area), were exposed to Xe-C (5 µM), a
membrane-permeable antagonist of IP3 receptors
(16). After addition of Xe-C, spontaneous Ca2+
transients were reduced to 33 ± 4% (n = 5;
P < 0.01; Fig.
5A) of the control area. In
additional experiments, we found that Xe-C (5 µM), added in the
absence of ryanodine, also blocked spontaneous Ca2+
transients (n = 6; discussed below). In patch-clamped
cells held at
30 mV, ryanodine did not affect STOCs, but Xe-C added
after ryanodine reduced STOCs to an unresolvable level (Fig.
5B). Thus the localized Ca2+ transients in
colonic muscle cells appeared to be due to release from IP3
receptor-operated stores and are referred to below as "Ca2+ puffs."

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Fig. 5.
Block of Ca2+ transients and STOCs by
xestospongin C (Xe-C). A: lack of effect of ryanodine on
spontaneous Ca2+ transients. After ryanodine, addition of
Xe-C (5 µM) completely blocked spontaneous Ca2+
transients. B: another cell studied with the patch-clamp
technique. Similar to the findings with fluorescence, ryanodine did not
affect STOCs, but these events were greatly reduced in amplitude and
frequency by Xe-C. The effects of Xe-C did not depend on ryanodine
pretreatment, but this sequence was shown to demonstrate the effects of
both drugs on the same cells.
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With all of the spontaneous Ca2+ transients measured, we
did not observe cell shortening, suggesting that, even in cells in which Ca2+ waves were observed, these events did not raise
global Ca2+ to the threshold for contraction. Addition of
caffeine (1 mM), however, caused significantly larger Ca2+
transients in cells and produced cell shortening (see Fig. 10).
Effects of 2-MeS-ATP on intracellular Ca2+ transients.
In 98% of the cells that produced spontaneous Ca2+
transients, 2-MeS-ATP (200 µM) increased the frequency and amplitude
of Ca2+ puffs (average increase to 200 ± 19% of
control area; n = 15; P < 0.05; Fig.
6) and STOCs. In eight cells pretreated
with ChTX (200 nM), we also found that 2-MeS-ATP enhanced the
occurrence of ChTX-insensitive STOCs (Fig. 7,
B-D). In 50% of the cells
that produced only Ca2+ puffs under control conditions, the
addition of 2-MeS-ATP generated propagating Ca2+ waves. In
the other one-half of these cells, 2-MeS-ATP either increased the
frequency of puffs from the same site or introduced new sites of puffs
(Fig. 6, C and D). In cells that displayed propagating waves before 2-MeS-ATP, addition of this drug increased the
area of propagation (i.e., spontaneous Ca2+ waves spread
over an area of 112.6 ± 11.7 µm2 in control cells
and 318.8 ± 84.6 µm2 after 2-MeS-ATP;
n = 7, P < 0.005). Although
stimulation of cells with 2-MeS-ATP increased Ca2+
transients and the spread of Ca2+, the level of
Ca2+ reached was apparently below the threshold for
contraction, and shortening of cells was not observed. As a positive
control, subsequent addition of caffeine (1 mM) caused a relatively
massive increase in global Ca2+ and cell shortening
(n = 8; see Fig. 10). To control for nonspecific activation of P2X receptors, which may also be expressed by
colonic myocytes, we tested
,
-methylene-ATP (200 µM), and this
compound was without resolvable effects on Ca2+ transients
(n = 5, P > 0.5).

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Fig. 6.
Ca2+ puffs in colonic myocytes were
associated with STOCs. A: cell with a single spontaneous
Ca2+ puff site during control conditions. Frame shown
represents the maximum intensity and spatial spread of the puff.
B: simultaneous recordings of whole cell current and
fluorescence. Note that the 3 puff events recorded during the 20-s scan
were associated with STOCs. C and D: addition of
2-MeS-ATP increased the number of puff sites and increased the
frequency and intensity of Ca2+ puffs from the original
site. The frequency of STOCs was increased in association with the
increase in Ca2+ puffs after 2-MeS-ATP. Note that two
fluorescence traces are shown in D to describe the temporal
changes in fluorescence at the 2 puff sites (areas of interest).
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Fig. 7.
2-MeS-ATP increased ChTX-insensitive STOCs. A:
control STOCs. B: reduction in the number and amplitude of
STOCs after ChTX (200 nM). C: in the presence of ChTX,
2-MeS-ATP (200 µM) increases ChTX-insensitive STOC activity.
D: histogram describing the number of STOCs under control
conditions (filled bars), after ChTX (open bars), and after 2-MeS-ATP
(shaded bars).
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The nonspecific Ca2+ entry blockers Ni2+ and
Cd2+ (200 µM) had no significant effects on the increases
in Ca2+ transients elicited by 2-MeS-ATP [i.e., 225 ± 38% (n = 5; P < 0.01) of control area
for cells pretreated with Ni2+ and 213 ± 25%
(n = 5; P < 0.01) for cells pretreated
with Cd2+]. Pretreatment of cells with CPA blocked the
effects of 2-MeS-ATP (i.e., no significant increase over the effect of
CPA alone; 100.2 ± 12%; n = 5;
P > 0.5). Ryanodine (10 µM) inhibited the
amplification of spontaneous Ca2+ transients caused by
2-MeS-ATP (i.e., after ryanodine, 2-MeS-ATP caused no increase over the
effects of ryanodine alone; 101 ± 8%; n = 5;
P > 0.5). The effects of CPA and ryanodine on
responses to 2-MeS-ATP are shown in Fig. 4.
Pretreatment of cells with PPADS (10 µM), a P2 receptor
antagonist (12), for 15 min did not significantly affect
Ca2+ transients (76 ± 21% of control area;
n = 7; P > 0.1), but this compound
blocked the increase caused by 2-MeS-ATP (95 ± 14%;
n = 7; P > 0.5 of pre-2-MeS-ATP level
in the presence of PPADS; Fig.
8A). An inhibitor of PLC
(U-73122, 2.5 µM; see Ref. 38), which prevents IP3
production, reduced spontaneous calcium transients (36 ± 7% of
control; n = 9; P < 0.01; Fig.
8B) and ChTX-insensitive STOCs (Fig.
9). After addition of U-73122, 2-MeS-ATP
did not significantly affect spontaneous Ca2+ transients
(i.e., 83 ± 9% of pre-2-MeS-ATP level in the presence of U-73122;
n = 9; P > 0.5). The inactive analog
U-73343 had no effect on control Ca2+ transients or the
response to 2-MeS-ATP (not shown). After pretreatment of cells with
Xe-C (5 µM; 10 min), 2-MeS-ATP had no effect on Ca2+
transients (108 ± 7%; n = 6; P > 0.5 of area for cells pretreated with Xe-C; Fig.
10). As a positive control, caffeine
was added at the end of experiments. This caused a massive increase in
Ca2+ and cell shortening (Fig. 10F).

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Fig. 8.
Effects of a P2Y receptor blocker and an
inhibitor of phospholipase C on spontaneous and stimulated
Ca2+ transients. A-C: spontaneous
Ca2+ transients before (A) and after
(B) addition of
pyridoxal-phosphate-6-azophenyl-2',4'-disculfonic acid
tetrasodium (PPADS; 10 µM). PPADS had no significant effect on
spontaneous Ca2+ transients, but it blocked the increase in
Ca2+ transients produced by 2-MeS-ATP (C).
D-F: Ca2+ transients (D) were
reduced by addition of U-73122 (2.5 µM; E). U-73122
blocked spontaneous Ca2+ puffs, suggesting that ongoing
production of IP3 is required for these events. There was
no response to 2-MeS-ATP in the presence of U-73122 (F).
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Fig. 9.
STOCs were blocked by U-73122. A: control STOC
activity in a cell held at 30 mV. B: U-73122, an inhibitor
of phospholipase C, blocked STOCs. C: histogram describing
the number of STOCs in control conditions (filled bars) and after
U-73122 (open bars).
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Fig. 10.
A: image of a cell with spontaneous
Ca2+ puff activity. C: fluorescence recorded
from the puff site during a 20-s scan. D: significant
reduction in the puff activity after exposure to Xe-C (5 µM).
E: Xe-C also inhibited the response to 2-MeS-ATP.
B and F: cells treated with Xe-C retained
significant internal stores of Ca2+, however, and this was
demonstrated by a large Ca2+ transient in response to
caffeine (1 mM; B and F) and contraction of the
cell (B).
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|
 |
DISCUSSION |
This study has demonstrated Ca2+ puffs and waves in
isolated murine colonic myocytes. In contrast to the localized
Ca2+ transients previously described in vascular, tracheal,
and small intestinal smooth muscles (17, 30,
33, 37, 43), spontaneous Ca2+ transients in colonic muscle cells were not primarily
mediated by ryanodine receptors. IP3 receptor-operated
stores appear to be the main source of spontaneous Ca2+
transients in these cells. Enhanced Ca2+ release by this
mechanism may be a major mechanism for coupling between G
protein-regulated receptors and activation of
Ca2+-dependent conductances in the plasma membrane. Showing
that stimulation with the P2Y agonist, 2-MeS-ATP, increased
the occurrence of Ca2+ puffs and waves supports this
hypothesis. The data also suggest that stimulation with 2-MeS-ATP also
"recruits" additional Ca2+ release from ryanodine
receptors. A similar phenomenon was recently observed in rat portal
vein myocytes in response to stimulation with norepinephrine
(8). Stimulation with 2-MeS-ATP also increased the
tendency of Ca2+ puffs to become regenerative and develop
into Ca2+ waves. Despite the dynamic mechanisms to mobilize
Ca2+ in response to P2Y receptor occupation,
the Ca2+ transients did not raise global Ca2+
sufficiently to activate the contractile apparatus. Thus the Ca2+ puffs and waves in colonic myocytes appear to be
compartmentalized in microdomains near the plasma membrane where
Ca2+-dependent ionic conductances can be regulated.
Localized Ca2+ transients are an important mechanism for
regulating ionic conductances in the plasma membrane in smooth muscles (30, 37, 43). We found that the
Ca2+ puffs and waves in murine colonic myocytes were of
sufficient magnitude to activate Ca2+-dependent
conductances. STOCs (4), which have been related to the
activation of BK channels, were correlated with spontaneous Ca2+ transients. We have previously suggested that
additional Ca2+-dependent conductances may also be
regulated by localized Ca2+ transients in colonic myocytes
(26). This concept has also been demonstrated by studies
of guinea pig tracheal myocytes in which individual Ca2+
sparks initiated both inward currents (via a Ca2+-activated
Cl
conductance) and outward currents via activation of BK
channels, depending on the holding potential (43). In the
present study, Ca2+ puffs were associated with activation
of ChTX-insensitive STOCs. These events were reduced by apamin, a
blocker of SK channels. Because ATP-sensitive hyperpolarization
responses are also reduced by apamin in gastrointestinal muscles
(2, 13), it is possible that coupling between
Ca2+ puffs and SK channel activation is the mechanism
coupling ATP to postjunctional hyperpolarization in situ.
Regulating the frequency and amplitude of localized Ca2+
transients is an important means of coupling receptor activation to electrical responses. Several second messenger mechanisms have been
shown to regulate Ca2+ release events in smooth muscles.
Ca2+ sparks recorded from rat coronary and cerebral
arteriole myocytes were increased in frequency by cAMP-dependent
mechanisms (34) and were reduced by protein kinase
C-dependent mechanisms (9). Spark frequency in these
studies may have been modulated by affecting Ca2+ uptake in
the sarcoplasmic reticulum (SR; i.e., modifying luminal Ca2+ content) or by changing the properties of ryanodine
receptors, such as altering the sensitivity to Ca2+.
In porcine tracheal muscles, discrete Ca2+ sparks
developed into Ca2+ oscillations when cells were stimulated
with ACh (37). In these studies, the Ca2+
oscillations were attributed to release from ryanodine receptors; however, it is also possible that amplification via IP3
receptors may have participated in the cholinergic responses, as
described in studies of duodenal myocytes (8). The present
study suggests that localized Ca2+ release is mediated by
IP3-dependent mechanisms in murine colonic myocytes, and,
as part of the response, regenerative responses involving
Ca2+ release from IP3 receptors and ryanodine
receptors may be important. Both Ca2+ release mechanisms
are facilitated by cytoplasmic Ca2+ and thus are capable of
regenerative responses (see Ref. 10). Factors such as luminal
concentration of Ca2+ in the SR (29,
32), basal IP3 levels (27), basal
Ca2+ levels in the microdomain near ryanodine and
IP3 receptors (6, 18,
21, 28), receptor isoform and density, and
the spatial relationship between receptors could all be important in
determining the mechanism of local Ca2+ transients and the
responses to agonist stimulation in specific types of smooth muscle. A
recent study in which caged Ca2+ was released in portal
vein smooth muscle cells also supports the concept of cooperativity
between IP3 receptors and ryanodine receptors
(7). These authors provided evidence that
IP3-dependent Ca2+ release is amplified by
ryanodine receptors, and this facilitates the development of
Ca2+ waves.
The results of this study offer new insights into the mechanisms of
enteric inhibitory regulation of gastrointestinal muscles and suggest
that localized Ca2+ transients are a means of
coupling receptors with inhibitory effectors such as plasma membrane
K+ channels (Fig. 9). There is significant evidence that at
least a portion of the inhibitory neural response in many species is due to release of ATP from enteric inhibitory neurons (13,
15, 20). Previous studies showed that ATP
activates SK channels in murine colonic (24) and small
intestinal (41) myocytes, and activation of these channels
is likely to explain the apamin-sensitive hyperpolarization response to
enteric inhibitory neurotransmission in intact gastrointestinal
muscles. The actions of ATP appeared to be mediated via P2Y
receptors because they were blocked by PPADS and mimicked by 2-MeS-ATP
(24). The mechanism for coupling between P2Y
receptors and SK channels, however, has not been previously described.
Our studies suggest the following model: 2-MeS-ATP increases localized
Ca2+ release via a mechanism involving P2Y
receptors, PLC, and IP3 receptors. Because SK channels are
highly sensitive to Ca2+, the increase in Ca2+
near the plasma membrane provides a plausible mechanism for increasing the open probability of SK channels. In the present study,
Ca2+ puffs and waves were increased by 2-MeS-ATP. In
association with the increase in Ca2+
transients, STOCs were increased. Ca2+ transients were
correlated with ChTX-sensitive large-amplitude STOCs and
ChTX-insensitive STOCs that were reduced by apamin. ChTX-insensitive
STOCs increased in response to 2-MeS-ATP, and this occurred in parallel
with increases in Ca2+ transients. Thus activation of
apamin-sensitive K+ channels by localized Ca2+
release provides a mechanism for hyperpolarization responses (inhibitory junction potentials) caused by enteric inhibitory nerve
stimulation in gastrointestinal muscles.
 |
ACKNOWLEDGEMENTS |
We thank Dr. C. W. R. Shuttleworth and Julia Bayguinov
for technical assistance with the confocal microscope and preparation of smooth muscle cells.
 |
FOOTNOTES |
This study was supported by National Institutes of Health Grants
DK-41315 (to K. M. Sanders, O. Bayguinov, and B. Hagen) and HL-44455 (to M. T. Nelson and A. D. Bonev). The Noran
Confocal microscope was purchased by shared equipment Grant HL-44455
Address for reprint requests and other correspondence:
K. M. Sanders, Dept. of Physiology and Cell Biology, Univ. of
Nevada School of Medicine, Anderson Medical Bldg., Reno, NV 89557 (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. §1734 solely to indicate this fact.
Received 19 November 1999; accepted in final form 19 January 2000.
 |
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