Calcium waves in colonic myocytes produced by mechanical and
receptor-mediated stimulation
S. H.
Young,
H. S.
Ennes,
J. A.
McRoberts,
V. V.
Chaban,
S. K.
Dea, and
E. A.
Mayer
CURE: Digestive Diseases Research Center/Neuroenteric Disease
Program, Departments of Medicine and Physiology, University of
California at Los Angeles, Los Angeles, California 90024
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ABSTRACT |
The mechanisms underlying intracellular
Ca2+ waves induced by either
mechanical or receptor-mediated stimulation of myocytes isolated from
the longitudinal muscle layer of the rabbit distal colon were compared
using fura 2 and fluorescence videomicroscopy. Light focal mechanical
deformation of the plasma membrane or focal application of substance P
resulted in localized intracellular Ca2+ concentration
([Ca2+]i)
transients that propagated throughout the cell. In both cases, the
Ca2+ response consisted of a
transient peak response followed by a delayed-phase response. Substance
P-mediated
[Ca2+]i
responses involved generation of inositol 1,4,5-trisphosphate and
release of Ca2+ from
thapsigargin-sensitive stores, whereas mechanically induced responses
were partially (29%) dependent on
La3+-sensitive influx of
extracellular Ca2+ and partially
on release of intracellular Ca2+
from thapsigargin-insensitive stores gated by ryanodine receptors. The
delayed-phase response in both cases was dependent on extracellular Ca2+. However, although the
response to substance P was sensitive to
La3+, that after mechanical
stimulation was not. In the later case, the underlying mechanism may
involve capacitative Ca2+ entry
channels that are activated after mechanical stimulation but not by
substance P.
smooth muscle; tissue culture; substance P; inositol
1,4,5-trisphosphate; ryanodine receptor; stretch-activated cation
channels
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INTRODUCTION |
IN SEVERAL CELL TYPES, intracellular
Ca2+ concentration
([Ca2+]i)
transients, stimulated either mechanically or chemically (via plasma
membrane receptors), show intracellular propagation in the form of
[Ca2+]i
waves (26, 30). Regenerative Ca2+
release by a diffusible messenger such as inositol 1,4,5-trisphosphate [Ins(1,4,5)P3; see Refs. 1,
4, 18, 30] or Ca2+ itself
(2) have been demonstrated to play a role in different cell types (4,
10, 18, 30). Using brief mechanical stimulation, we have recently
provided evidence in myocytes from the circular muscle layer of the
rabbit colon that
[Ca2+]i
wave propagation is fully dependent on
Ca2+ release from
thapsigargin-sensitive intracellular stores (35). On the other hand,
the generation and propagation of
Ca2+ waves within the stimulated
myocyte are partially dependent on Ca2+ influx via
La3+-insensitive
Ca2+ pathways in addition to
release of Ca2+ from
thapsigargin-sensitive intracellular stores (36). In smooth muscle from
the circular muscle layer of the guinea pig intestine, agonists have
been shown to stimulate phosphoinositol hydrolysis, resulting in
Ins(1,4,5)P3 generation and
release of Ca2+ from
thapsigargin-sensitive stores (25). In contrast, in freshly isolated
myocytes from the longitudinal layer of the guinea pig intestine,
Ins(1,4,5)P3 does not appear to
play a role in receptor-mediated cell activation (17). Furthermore,
Ca2+-induced
Ca2+ release initiated by
receptor-mediated influx of Ca2+
appears to be the major mechanism underlying intracellular
Ca2+ release in intestinal
longitudinal smooth muscle cells (24).
In the current study, we wanted to address the following questions.
1) What are the mechanisms
underlying mechanically induced [Ca2+]i
transients and their propagation, and how do they differ from mechanisms previously identified in myocytes from the circular muscle
layer of the colon? 2) Do the
mechanisms underlying mechanically induced and receptor-mediated
[Ca2+]i
transients and their propagation differ? We report that mechanically induced
[Ca2+]i
waves differ from those previously characterized in cells from the
circular muscle layer. They are partially dependent on influx of
extracellular Ca2+ through a
La3+-sensitive pathway in the
plasma membrane and partially on release from a
thapsigargin-insensitive, ryanodine- and caffeine-sensitive store. In
contrast,
[Ca2+]i
transients induced by substance P in this cell type are fully dependent
on release of Ca2+ from
thapsigargin-sensitive stores.
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METHODS |
Solutions and chemicals. The normal
external solution was Hanks' buffered saline solution (GIBCO BRL)
containing (in mM) 1.3 CaCl2, 5 KCl, 0.5 MgCl2 · 6H20,
0.4 MgSO47 · H20,
138 NaCl, 4 NaHCO3, 0.3 KH2PO4,
0.3 NaHPO4, and 5.6 D-glucose with 20 mM HEPES added, and the pH was adjusted
to 7.4 with addition of NaOH. The Ca2+-free solutions were prepared
without any CaCl2 and with
addition of EGTA (0.1 or 1 mM). A stock solution of the phospholipase C (PLC)-dependent process inhibitor U-73122 (Calbiochem) was prepared in
DMSO (5 mM). The stock solution was then diluted in external saline at
a final concentration of 10 µM immediately before use. This compound
was present in the cell culture for <120 s (3).
Cell culture. Cell cultures were
prepared following the methods of Ennes et al. (11). In brief, a
segment of the distal colon of the rabbit is removed and placed in
oxygenated incubation buffer at 4°C consisting of (in mM) 115 NaCl,
5.8 KCl, 0.6 MgCl2, 1.2 CaCl2, 25 KH2PO4, 2 glutamine, and 12 glucose, 2.6% (vol/vol) basal Eagle's medium essential amino acids,
and 25 mM HEPES, pH 7.4. The segment was then slit longitudinally along
the antimesenteric border, the luminal contents were removed, and the
segment was initially washed in cold buffer containing 100 µg/ml
gentamicin, 100 U/ml penicillin, 10 µg/ml streptomycin, and 10 µg/ml Fungizone. After this washing procedure, the antibiotic
composition in the buffer was changed to 0.004 µg/ml gentamicin, 120 µg/ml penicillin, 270 µg/ml streptomycin, and 1 µg/ml Fungizone.
Under a microscope, the mesentery, serosa, longitudinal muscle, and
mucosa were removed. Tissue containing primarily longitudinal muscle
was slit several times along the muscular axis. Dispersion of smooth
muscle cells was accomplished by three incubations at 31°C with
agitation in incubation buffer containing 0.1 mg/ml mg BSA, 0.1%
(wt/vol) collagenase, and 0.1% (wt/vol) soybean trypsin inhibitor.
Isolated cells were collected by low-speed centrifugation, and cultures
were initiated by resuspension in DMEM containing high glucose, sodium
pyruvate (1 mM), nonessential amino acids (1% vol/vol, 100×),
heat-inactivated fetal calf serum (10% vol/vol),
L-glutamine (4 mM), gentamicin (0.004 mg/ml), and
amphotericin B-penicillin-streptomycin (2% vol/vol). Myocytes were
plated on sterile collagen-coated glass coverslips (type 1 rat tail
collagen; Collaborative Biomedical Products) and incubated at 37°C
in a humidified 5% CO2 atmosphere.
Measurement of
[Ca2+]i.
Cells were labeled in external saline containing fura 2-AM (5 µM;
Molecular Probes) for 1 h at 37°C, followed by rinse in external
saline. The cells were mounted in a coverslip chamber (volume 1 ml) on
the stage of a Zeiss 100TV inverted microscope equipped with an oil
immersion objective (40× Fluar; Zeiss). All experiments were
performed at room temperature (20-23°C).
Mechanical stimulation. Light, focal
mechanical stimulation was provided by a 1- to 3-mm tip diameter
fire-polished glass pipette that depressed the muscle cell membrane for
0.5 s and then retracted. The pipette was connected to an
electronically controlled micropositioner (Eppendorf) that controlled
the depression depth and duration. At the start of an experimental
series, the tip of the pipette was placed above the surface of the
cell, and image acquisition was initiated. The tip was then programmed
to travel in a downward direction for a distance of 2 µm for a
duration of 0.5 s, followed by a return to the start position. When no response was observed, the transient tip deflection was increased by
2-µm increments until a cellular
[Ca2+]i
response was observed. If only a very small mechanoresponse was
measured initially, the deflection increment was decreased to 1 µm,
and the cell was restimulated. At the end of the series, the tip
deflection was observed under transmitted light to verify that the
membrane depression was slight (no more than 3 µm) and had not
visibly damaged the cell.
Chemical stimulation. Chemicals were
administered with both global saline perfusion and local microejection.
Perfusion was accomplished with a peristaltic pump (Rainin) with source
and drain lines to perfuse the experimental chamber (volume 1 ml) at 1 ml/min. Microejection was performed by filling a fine glass pipette
with ejection solution, placing the tip of the pipette near the muscle
cell surface perpendicular to the bath flow, and ejecting a small
amount of fluid under controlled pressure and timing with a controlling
unit (Eppendorf).
Data analysis.
[Ca2+]i
was measured with a commercially available videomicroscopy system
(RatioVision; Atto Instruments) that includes processing software with
options for background subtraction, shading correction, and
calibration. Calibration of the fura 2 fluorescence was performed in
vitro by the use of a series of buffered
Ca2+ standards containing
magnesium (Molecular Probes) and fura 2 potassium salt.
[Ca2+]i
data were collected at a rate of ~1/s (0.8-1.3 s/sample).
Ca2+ concentrations
([Ca2+]) at points
within the cell were measured within regions of interest (ROI)
consisting of 100-point pixel arrays (8 × 6 µm). Because of
limitations in the temporal resolution of our system, ROIs were
selected within the cytoplasm. We did not attempt to measure differential changes in
[Ca
2]i
near the plasma membrane. To maximize temporal resolution, individual
elongated cells (30- to 50-mm-long axis) were stimulated near one end
of the cell where the first ROI was located and were measured at
another ROI near the opposite end of the cell. Averaged concentration
and time information from each ROI were stored on computer disk.
Velocity of wave propagation was calculated from the difference in
arrival times of the start or half-maximal
[Ca2+] of the
propagated wave at different ROI divided by the distance between the ROI.
Data analysis and statistics. Data are
reported as mean values ± SE. Statistical analysis was performed on
data using paired Student's t-test.
Significance was expressed at the P < 0.05 level throughout. Paired experiments were performed for most
conditions, where pretreatment values from a single cell in normal
saline were compared with posttreatment values from the same cell
obtained after exposure to a given experimental solution. In
circumstances where paired experiments could not be performed,
[Ca2+]i
responses to stimulation after treatment were compared with responses
obtained on the same day on cells from untreated parallel or
"sister" cultures. These situations are indicated in the text.
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RESULTS |
[Ca2+]i
responses to focal mechanical and chemical stimulation were studied in
110 myocytes in primary cultures obtained from the longitudinal layer
of the distal rabbit colon.
General characteristics of mechanically induced
[Ca2+]i
transients.
Ninety percent of smooth muscle cells (63 out of 70 cells) showed a
[Ca2+]i
increase in response to focal mechanical stimulation, followed by
intracellular propagation of the
[Ca2+]i
transient. No
[Ca2+]i
oscillations were observed. The response of a representative cell to
focal mechanical stimulation is shown in Fig.
1. The
[Ca2+]i
response showed a rapidly rising initial peak, occurring within 1 s of
the stimulus, and a slow, biphasic recovery to resting levels of
[Ca2+]i.
The duration of this latter component, which we refer to as the
"delayed phase," ranged from 100 to 1,000 s and was seen in ~80% of the cells stimulated. Mean resting
[Ca2+]i
in cells cultured for 6-9 days was 115.9 ± 9.2 nM
(n = 53), and the mean amplitude of
the mechanically induced
[Ca2+]i
transient under control conditions was 668.0 ± 28.1 nM (range 147-948 nM; n = 53). In
contrast to previously reported age-dependent differences in
[Ca2+]i
transient amplitude for myocytes from the circular muscle layer (36),
the amplitude of the
[Ca2+]i
transient in myocytes that had been cultured for 11-14 days ("old" cells) were not statistically different from those
cultured for 6-9 days (Table 1). The
amplitude of
[Ca2+]i
transients varied significantly between cells; however, repeated mechanical stimulation of a given cell produced relatively stable [Ca2+]i
responses when applied after recovery of
[Ca2+]i
to resting levels. For example, when cells were mechanically stimulated
a second time in normal saline (containing 1.3 mM
Ca2+), the amplitude and
velocity of the second
[Ca2+]i
wave were not different from that of the first
[Ca2+]i
wave (Fig. 1A and Table
2).

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Fig. 1.
General characteristics of intracellular
Ca2+ concentration
([Ca2+]i)
in response to focal mechanical and substance P (Sub P)-mediated
stimulation. A: light focal
mechanostimulation of the plasma membrane produces
[Ca2+]i
increase. At time marked by the arrow, a single myocyte was
mechanically stimulated with a small fire-polished glass rod. Within 1 s,
[Ca2+]i
increases to a peak value of ~950 nM. Return to resting
[Ca2+]i
occurs with both a rapid and slow (delayed phase, marked by * ) time
course. Inset: second
mechanostimulation, given 400 s after the first mechanostimulation,
produces a similar response. B: focal
application of substance P results in a
[Ca2+]i
transient. Substance P (10 6
M) was pressure ejected (duration 0.5 s) from a micropipette (time
marked by arrow). Myocyte responded with a rapid increase in
[Ca2+]i
of amplitude 400 nM, followed by a slower recovery (delayed phase,
marked by *) to baseline
[Ca2+]i.
Inset: ~800 s after the first pulse
of substance P, a second ejection produced a similar
[Ca2+]i
transient, followed by a similar recovery phase.
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Table 1.
Comparison of [Ca2+]i wave parameters
induced by either focal mechanical stimulation or by focal
substance P application in young (6-9 days) and old
(11-14 days) SMC cultures
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The mechanically induced
[Ca2+]i
response was unlikely to be the result of nonspecific leakage of
Ca2+ secondary to injury to the
plasma membrane, since we monitored the fluorescence intensity changes
to mechanostimulation at both 334 and 360 nm. The response at 334 nm
was a rapid increase in intensity followed by a slower recovery toward
baseline, whereas the response at 360 nm remained unchanged, indicating
that there was no significant loss of dye from the cell (Fig.
2).

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Fig. 2.
Mechanoresponse is not the result of damage to the plasma membrane.
Response to mechanical stimulation was monitored at 334 and 360 nm at a
single region of interest at the point of stimulation. At the 360-nm
wavelength, fura 2 fluorescence is independent of
[Ca2+]i
and indicates only the concentration of dye.
Top: ratio of 334/360 nm shows
features of a typical mechanoresponse, with both a rapid rise after
stimulation (marked by arrow) followed by a slower recovery.
Bottom: individual 334- and 360-nm
signals. Whereas the 334-nm signal shows both the rapid rise and slower
recovery phases, the signal at 360 nm is unchanged after
mechanostimulation, showing only a slight constant rate of decline
reflecting photobleaching of fura 2 over time.
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Two lines of evidence suggest that the
[Ca2+]i
response is an actively propagated wave and not the result of
Ca2+ diffusion from a single
point. As shown in Fig.
3A, the
speed of propagation showed no significant correlation with the
amplitude of the
[Ca2+]i
peak as might be expected from simple diffusion. In addition, the
amplitude of the
[Ca2+]i
wave remained relatively undiminished over distances as great as 40 µm from the point of stimulation (Fig.
3B), another indication that the
[Ca2+]i
is the result of regenerative propagation and not simple diffusion. There were also no age-dependent differences in the velocity of propagation (Table 1), nor was there any difference with repeat stimulation (Table 2).

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Fig. 3.
Characteristics of the
[Ca2+]i
waves produced by mechanical stimulation and focal substance P
application. A: velocity of the
mechanically induced
[Ca2+]i
wave is not correlated with the amplitude of the wave. Pooled data are
from 66 cells. B: amplitude of the
mechanically induced
[Ca2+]i
wave does not decline with distance from the point of stimulation.
Pooled data are from 54 cells. C:
velocity of substance P-induced
[Ca2+]i
wave is not correlated with the amplitude of the wave. Pooled
data are from 42 cells. D: amplitude
of the substance P-induced
[Ca2+]i
wave does not decline with distance from the point of
stimulation. Pooled data are from 32 cells.
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General characteristics of
[Ca2+]i
transients induced by substance P.
When cells were perfused with substance P
(10
9 to
10
7 M), 56% of cells (99 out of 176) showed an increase in
[Ca2+]i.
The mean amplitude of the
[Ca2+]i
increase was 402 ± 47 nM above an average resting level of 176 ± 6 nM. A similar proportion (75%) of the smooth muscle cells responded to focal application of substance P
(10
6 M) with an increase in
[Ca2+]i.
With focal application, the initial response occurred immediately adjacent to the point of stimulation and was followed by intracellular propagation of the
[Ca2+]i
transient throughout the muscle cell. We never observed a "locus" or preferential initiation point for the
[Ca2+]i
waves induced by focal substance P application.
[Ca2+]i
oscillations in response to focal substance P application were observed
in 2 out of 42 cells. The response of a representative cell to focal
substance P stimulation is shown in Fig.
1B. Like the mechanically induced
response, the
[Ca2+]i
response typically showed an initial peak, occurring within 1 s of the
stimulus, and a biphasic recovery to resting levels of
[Ca2+]i
(delayed phase). The duration of the delayed phase ranged between 100 and 1,000 s and was observed in ~40% of the cells stimulated. Even
though the amplitude of
[Ca2+]i
transients varied significantly between cells,
[Ca2+]i
responses to repeated substance P applications in a single cell were
relatively stable when applied after recovery of
[Ca2+]i
to resting levels (Fig. 1B,
inset, and Table 1), suggesting that
significant desensitization did not occur in the majority of cells at
the concentrations and recovery times used in our protocols. Mean
resting
[Ca2+]i
in "young" cells was 124.1 ± 12.9 nM, and the mean amplitude of the substance P-induced
[Ca2+]i
transients under control conditions was 483.0 ± 33.5 nM (range 84-915 nM, Table 1). This value was significantly lower than that
produced by mechanical stimulation of similar cells
(P < 0.001). As with mechanical
stimulation, there was no significant difference between young and old
cells (Table 1).
Mean propagation velocity of
[Ca2+]i
transients after chemical stimulation was 14.4 ± 2.1 mm/s
(n = 41), a value not significantly different from that observed after mechanical stimulation (Table 1).
There was no significant correlation between the amplitude of the
substance P-induced
[Ca2+]i
transient and the velocity of propagation (Fig.
3C). In addition, the amplitude of
the
[Ca2+]i
wave remained relatively undiminished over distances as great as 35 mm
from the point of stimulation (Fig.
3D), suggesting that the
[Ca2+]i
response was the result of regenerative propagation.
Contribution of
Ca2+ influx
through the plasma membrane.
A portion of the transient peak response to mechanical stimulation was
dependent on extracellular Ca2+.
When measured within 5 min, changing the external solution to a
Ca2+-free solution did not affect
the resting
[Ca2+]i
levels but reduced the average peak amplitude of the
[Ca2+]i
response by 29% from 628.5 ± 140.4 to 456.0 ± 112.0 nM
(n = 6;
P = 0.02; Table 1). In addition, the
delayed phase was completely abolished. In the presence of normal
extracellular [Ca2+],
addition of 100 mM La3+ caused a
mean reduction of the
[Ca2+]i
response of 18% (n = 7;
P = 0.04) but did not result in
complete block of the
[Ca2+]i
response in any of the cells tested. Furthermore, addition of
La3+ had no significant effect on
the delayed response. These results indicate that a portion of the
initial
[Ca2+]i
peak is dependent on external
[Ca2+], whereas the
delayed phase is entirely dependent on the influx of external
Ca2+ through
La3+-insensitive pathways. In
contrast, neither inhibition of voltage-sensitive Ca2+ channels with
Gd3+ (100 µM) or nifedipine
(10
6 M), nor inhibition of
certain stretch-activated channels with Gd3+ (100 µM) or amiloride (100 µM; see Ref. 13), had a significant effect on mechanically induced
peak
[Ca2+]i
responses (Table 2). Similarly, none of these compounds had a
significant effect on the delayed phase of the mechanically induced transient.
In contrast to the situation with mechanical stimulation, the transient
peak response to focal application to substance P was not changed by
removing Ca2+ from the bathing
solution in seven out of nine cells tested (Table 2). In two out of
nine cells, the
[Ca2+]i
response was completely abolished. In all seven responding cells, the
delayed phase was completely abolished, similar to the results from
mechanical stimulation in
Ca2+-free external solution. In
the presence of normal extracellular [Ca2+], addition of
La3+ (100 µM) blocked both
initial
[Ca2+]i
transient and delayed response in two of seven cells. In the remaining
five cells, peak transient amplitude was not different from control,
but the delayed phase was completely blocked. Addition of
Gd3+ (100 µM) had no effect on
the substance P-induced transient or delayed phase of the
[Ca2+]i
response (Table 2). These results indicate that in most cells the
initial
[Ca2+]i
peak response to substance P is independent of external
[Ca2+], but the
delayed phase is entirely dependent on the influx of external
Ca2+ through
La3+-sensitive pathways.
Contribution of release from intracellular
Ca2+ stores.
Based on mean responses obtained from populations of freshly isolated
myocytes from the longitudinal muscle layer of the guinea pig
intestine, the
[Ca2+]i
response to plasma membrane receptor-mediated stimulation is thought to
be mediated largely by influx of extracellular
Ca2+, and to a smaller degree by
release from
Ins(1,4,5)P3-insensitive stores
(25). Addition of thapsigargin or caffeine to the external solution
produced a small transient increase in baseline
[Ca2+]i
that then returned to resting levels. The response to thapsigargin was
seen in 33 out of 39 cells (85% responding, mean amplitude 91.2 nM),
whereas a response to caffeine was seen in 21 out of 41 cells (51%
responding, mean amplitude 20 nM).
Depletion of intracellular stores by pretreatment of cells with
thapsigargin had no effect on the mechanically induced
[Ca2+]i
transient, a result similar to that reported in circular smooth muscle
cells (35). Pretreatment of cells with either caffeine (10 mM) or
ryanodine (50 µM) had no significant effect on resting [Ca2+]i
or on
[Ca2+]i
transients. However, treatment with a combination of ryanodine (100 µM) and caffeine (10 mM) reduced the amplitude of the transient peak
response (average ratio 0.73 ± 0.06, n = 7, P = 0.005) but did not significantly
affect propagation velocity (average ratio 0.88 ± 0.17, n = 7, P = 0.52; Table
3). Addition of thapsigargin (1 µM) to the ryanodine-caffeine mixture further reduced but did not
eliminate the transient amplitude (average ratio 0.50 ± 0.10, n = 5, P = 0.008) and did not affect the
propagation velocity. Delayed-phase responses to mechanical
stimulation were significantly reduced by caffeine pretreatment or
pretreatment with the combination of ryanodine, caffeine, and
thapsigargin. Inhibition of PLC with U-73122 (10 µM; see Ref. 3) had
no significant effect on the mechanically induced
Ca2+ transients (Table 3).
In marked contrast, depletion of intracellular
Ca2+ stores by thapsigargin (1 µM) abolished both the peak and delayed response to substance P in
eight out of nine cells and reduced the response in the remaining cell
by 86% (Table 3). Pretreatment with the PLC inhibitor U-73122 (1 µM)
also completely abolished both peak and delayed
[Ca2+]i
responses in seven out of seven cells. Neither pretreatment with
caffeine (10 mM), ryanodine (50 µM), nor the combination of ryanodine
and caffeine produced a significant effect on peak responses.
Characterization of the interaction between substance P and
mechanical stimulation on
[Ca2+]i
transients.
To test whether activation of receptor-mediated signal transduction
pathways influences the mechanically induced response, cells were
mechanically stimulated before and after stimulation with substance P. After an initial mechanical stimulation and recovery of
[Ca2+]i
to resting levels, substance P
(10
6 M) was perfused in the
bath. The same cell was then mechanically stimulated a second time in
the continued presence of substance P. As shown in Table
4, stimulation with substance P did not change either the amplitude of the mechanically induced
[Ca2+]i
transient or the velocity of propagation of the transient. Addition of
La3+ (100 µM) together with
substance P (10
6 M) also
had no effect on the amplitude or velocity of propagation of the
mechanically induced
[Ca2+]i
transient.
Refilling of intracellular
Ca2+ stores.
In many cell types, depletion of intracellular
Ca2+ stores by agonists or by
thapsigargin results in Ca2+
influx through the plasma membrane (29). Excitable cells such as smooth
muscle have been thought to rely on voltage-operated or
receptor-operated Ca2+ channels
for refilling of Ca2+ stores. Only
recently has a role for capacitative or store-operated Ca2+ channels (SOCC) been
recognized in this cell type (12). In the current experiments, the
delayed response to mechanical stimulation was abolished in the absence
of extracellular Ca2+, but not by
La3+ or nifedipine, suggesting the
existence of this type of Ca2+
influx is activated by intracellular store depletion. To demonstrate the presence of capacitative Ca2+
entry in these cells, we first depleted intracellular stores with
thapsigargin in the absence of extracellular
Ca2+, followed by reintroduction
of extracelluar Ca2+. As shown in
a representative experiment (Fig.
4A),
reintroduction of Ca2+ caused a
large increase in
[Ca2+]i,
confirming that longitudinal smooth muscle cells express functional SOCC. To test whether mechanical stimulation of the myocytes activates this SOCC channel, we performed the analogous experiment after mechanically stimulating the cells in the absence of extracellular Ca2+ (Fig.
4B). Elevation of
[Ca2+]i
after reintroduction of extracellular
Ca2+ occurred in five out of a
total of seven cells responding to mechanical stimulation. In contrast,
no
[Ca2+]i
increase was seen upon reintroduction of extracellular
Ca2+ in 36 out of 37 cells
stimulated by perfusion with substance P in the absence of
Ca2+, despite a substantial
[Ca2+]i
response due to release of Ca2+
from intracellular stores (Fig.
4C).

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Fig. 4.
Myocytes exhibit capacitative Ca2+
entry. A: 21 cells were exposed to 1 µM thapsigargin in Ca2+-free
saline. All 21 cells showed a transient increase in
[Ca2+]i
of average amplitude 150 nM, followed by a return to baseline
[Ca2+]i.
After the return to baseline, the external solution was changed to
normal saline. Reintroduction of normal saline containing 1.3 mM
Ca2+ produced another transient
increase in
[Ca2+]i.
B: myocytes were mechanically
stimulated in Ca2+-free saline,
and, after return of
[Ca2+]i
to baseline, normal Ca2+ was
perfused back to the cells. Five out of 7 cells displayed an increase
in
[Ca2+]i
after reintroduction of Ca2+.
C: myocytes were stimulated by
perfusion with substance P (1 µM) in
Ca2+-free saline. Reintroduction
of normal saline failed to cause an increase in
[Ca2+]i
in 36 out of 37 cells. In A-C, the
first heavy arrow marked by "0" indicates the removal of
extracellular Ca2+ by addition of
either 0.5 µM EGTA (A and
C) or 10 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (B); narrow arrow indicates the
time of stimulation; and the second heavy arrow marked by "+"
indicates start of perfusion with normal saline (1.3 mM
Ca2+).
A-C show a representative
experiment.
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DISCUSSION |
Comparison of mechanically and chemically induced
[Ca2+]
transients.
Cultured myocytes from the longitudinal muscle layer of the rabbit
colon respond to focal plasma membrane deformation or focal application
of substance P with a transient biphasic increase in
[Ca2+]i
that propagates across the cell. The initial
[Ca2+]i
transient is largely (mechanical stimulation) or solely (substance P
application) dependent on release of
Ca2+ from intracellular stores. In
both cases, the delayed-phase response is dependent on extracellular
Ca2+, implying a role for
Ca2+ influx. The propagation of
the Ca2+ transient across the cell
occurs at a similar velocity and appears to be regenerative after both
types of stimuli.
Apart from these similarities, there were a number of differences found
between these two methods of stimulation. First, as noted above, a
portion of the mechanically induced initial
Ca2+ transient was dependent on
extracellular Ca2+ and could be
blocked by La3+. The
La3+-sensitive contribution to the
initial
[Ca2+]i
peak in the current study ranged from 2 to 27%. The fact that the
influx was blocked by La3+ and was
not associated with a detectable decrease in intracellular dye
concentration makes cell wounding as a cause for the
[Ca2+]i
transient unlikely. However, very small changes in dye concentration (<1%) that might occur from mechanical damage would not be detected, and it remains possible that the
La3+-insensitive portion of the
[Ca2+]i
transient could be due to nonspecific leakage. One possible mechanism
underlying the La3+-sensitive
influx could be stretch activation of voltage-sensitive Ca2+ channels, as recently
reported for myocytes from the circular muscle layer of the guinea pig
antrum (34). However, L-type Ca2+
channels are unlikely to be involved, since neither
Gd3+ nor nifedipine had a
significant effect on the mechanically induced [Ca2+]i
peak. Several pharmacologically distinct stretch-activated cation
channels (SACC) have been reported in a variety of cell types (13),
including epithelial cells (5), as well as vascular and nonvascular
smooth muscle cells (22, 23). Some of these SACC are inhibited by
Gd3+ or amiloride; however, in the
current study, neither of these agents had an inhibitory effect.
Similar to our findings, the mechanically induced
Ca2+ influx in epithelial cells
was also sensitive to La3+ (5).
Our data suggest that longitudinal smooth muscle cells have SACC that
are sensitive to La3+ but
insensitive to Gd3+ or amiloride.
Mechanical and substance P focal stimulation also appeared to mobilize
different intracellular Ca2+
stores. Our findings clearly implicate substance P-mediated activation of PLC, Ins(1,4,5)P3 generation,
and release of Ca2+ from
thapsigargin-sensitive stores as the principal mechanism by which focal
application of substance P results in the initial [Ca2+] transient. The
occurrence of Ca2+ oscillations in
some cells indicates the existence of positive and negative feedback
mechanisms on Ca2+ release.
Furthermore, the fact that caffeine and ryanodine were unable to
significantly attenuate the amplitude of the
[Ca2+]i
transient suggests that the
Ins(1,4,5)P3 store is functionally independent from ryanodine-sensitive stores.
On the other hand, mechanically induced
[Ca2+]i
transients were entirely independent of
Ins(1,4,5)P3 and did not involve
thapsigargin-sensitive Ca2+
stores. Neither addition of the PLC inhibitor U-73122 nor depletion of
Ca2+ stores with thapsigargin had
any effect on the mechanically induced peak response. To rule out the
possibility that basal intracellular concentrations of
Ins(1,4,5)P3 (which may not be
affected by inhibition of PLC) may be sufficient to mediate activation
of Ins(1,4,5)P3 release channels
when
[Ca2+]i
levels in vicinity of the channel are increased by
Ca2+ influx, we repeated the
experiments with thapsigargin in the presence of
La3+. No significant reduction of
the mechanical response was observed under these circumstances, making
such an explanation unlikely. Our observations are most consistent with
Ca2+ release from a
thapsigargin-insensitive store, which is most likely gated by ryanodine
receptor Ca2+ release channels.
Even though caffeine and ryanodine added individually failed to inhibit
the mechanically induced peak response, the combination of ryanodine
and caffeine did inhibit a portion of the response. This observation is
consistent with the known properties of ryanodine receptor
Ca2+ channels, which are blocked
by high-dose ryanodine in a use-dependent manner (9). The observation
that thapsigargin in combination with ryanodine and caffeine further
inhibited the response is also consistent with this explanation, since
elevated
[Ca2+]i
could activate additional ryanodine receptor channels, allowing ryanodine to bind and inactivate them. In analogy with our previous findings in circular muscle, this compartment may be directly mechanically coupled to the plasma membrane via actin filaments. Indeed, ryanodine receptors have been shown to interact with ankyrin in
T lymphocytes (6).
Even though the delayed phase of both the mechanical and substance
P-stimulated responses required the influx of extracellular Ca2+, the two responses differed
in La3+ sensitivity, implying a
role for different Ca2+ channels.
The delayed-phase response to mechanical stimulation occurs through a
La3+-insensitive
Ca2+ influx, whereas the
delayed-phase response to substance P appears to be mediated through
La3+-sensitive channels. In the
case of the mechanical response,
Ca2+ uptake could be mediated
through a SOCC (8). An alternate possibility is that mechanical
stimulation causes the release of ATP and elevation of
[Ca2+]i
through autocrine stimulation of P2 receptors. This mechanism plays an
important role in intercellular communication between hepatocytes and
other cells (32). However, it seems unlikely to account for the
delayed-phase response reported here, since the cells in our system
were continuously perfused, and
[Ca2+]i
transients in noncontacting adjacent cells we never observed. Furthermore, as demonstrated in Fig. 4, longitudinal smooth muscle cells functionally express SOCCs that are activated by depletion of
intracellular Ca2+ stores with
thapsigargin in Ca2+-free saline.
We found that mechanical stimulation in
Ca2+-free saline could also
activate SOCCs in the majority of the cells tested, as evidenced by the
influx of Ca2+ after
reintroduction of normal saline. In contrast, substance P did not
appear to open SOCC that could be measured in this manner. One
possibility is that the delayed-phase response to substance P may be
mediated by La3+-sensitive
voltage-activated Ca2+ channels.
We have previously demonstrated (20) that
La3+ decreased the amplitude of
spontaneous
[Ca2+]i
increases in freshly isolated intestinal longitudinal smooth muscle
cells. Whether or not this
La3+-sensitive, delayed-phase
response to substance P is due to activation of L-type
Ca2+ channels remains to be investigated.
Mechanism underlying
Ca2+ transient
propagation after mechanical and chemical stimulation.
Both mechanical and substance P-induced transients propagated through
the cell. The velocity of propagation for the
[Ca2+]i
wave induced by the two stimuli was 10.4 ± 0.8 and 14.4 ± 2.1 µm, respectively, and was not statistically different. Furthermore, the velocity of propagation was also similar to the value of 13.2 ± 2.5 µm/s found previously in myocytes from the circular muscle layer
(35). Reported propagation velocities in a variety of cell types range
from 4 to 100 µm/s (2, 26, 30). Both
Ins(1,4,5)P3 and
Ca2+ have been identified as the
diffusible messenger underlying the propagation of intracellular
Ca2+ waves after the initial
signal transduction. Although
Ins(1,4,5)P3-mediated Ca2+ release has been implicated
in endothelial, epithelial, and glial cells (7, 10, 14),
Ca2+-induced
Ca2+ release has been identified
as the underlying mechanism in cardiac (33), pancreatic (28), hepatic
(27), and vascular smooth muscle cells. Based on the severalfold
greater diffusion length of
Ins(1,4,5)P3 compared with
Ca2+ (16), simple
Ins(1,4,5)P3 diffusion from its
original site of generation could be responsible for a wave propagated
over 70 µm (average length of colonic smooth muscle cell). Reported propagation velocities of mechanically induced
Ca2+ waves mediated by
Ins(1,4,5)P3 diffusion range from
10 to 30 µm/s (30). For the substance P-induced
Ca2+ wave, our data suggest that
the increase in Ins(1,4,5)P3 that is responsible for the Ca2+
transient is followed by a diffusion of
Ins(1,4,5)P3 to other release
sites within the cell. However, in the vascular smooth muscle cell line
A7r5, despite the role of
Ins(1,4,5)P3 in the initial
Ca2+ transient to focal
application of vasopressin, the propagation of the
Ca2+ transient (average 16 µm/s)
involved regenerative Ca2+-induced
Ca2+ release (2). Because our data
on the effects of ryanodine and caffeine on the substance P-induced
Ca2+ wave are limited, we cannot
exclude this possibility.
For the mechanically induced Ca2+
wave, both mechanotransduction and propagation were independent of
Ins(1,4,5)P3 production and
thapsigargin-sensitive stores. These results suggest the presence of a
diffusible messenger, which is induced mechanically and which diffuses
throughout the cell. Given the role of ryanodine receptor Ca2+ release channels in
initiating the mechanical response and the documented role of these
channels in Ca2+-induced
Ca2+ release (9), this messenger
is likely to be Ca2+ itself.
Alternatively, cADP-ribose or another chemical second messenger that
activates ryanodine receptor channels (9) could be involved.
Comparison of mechanically induced
[Ca2+]i
responses between myocytes from circular and longitudinal muscle layer.
In response to the same mechanical stimulus as used in the current
study (36), we have previously demonstrated that myocytes from the
circular muscle layer of the rabbit colon produced a propagated
[Ca2+]i
transient. In view of the reported differences in receptor-mediated cell activation between myocytes from longitudinal and circular muscle
layers of the mammalian gastrointestinal tract (19), we expected to see
differences in the response to a mechanical stimulus. Indeed, in cells
from the circular muscle layer, the average contribution of
Ca2+ influx to the initial peak
was >50%, and, in contrast to the current findings, this
Ca2+ influx was insensitive to
La3+ (36). These findings suggest
that mechanical deformation of the plasma membrane activates
Ca2+ pathways that are
qualitatively and quantitatively different between the two cell types.
In addition, in the current study, we found no evidence for the
contribution of Ca2+ release from
thapsigargin-sensitive stores, in contrast to our previous finding that
25% of the response to mechanical stimulation of circular myocytes was
thapsigargin sensitive. Finally, longitudinal and circular smooth
muscle cells appeared to respond to culture differently. In the current
study, we did not find any evidence for a decrease in the transient
amplitude with increasing age of the cells in culture while previously
with circular muscle we had described this effect. On the other hand,
we found that smooth muscle cells from the two layers share the
following mechanisms. 1)
Ins(1,4,5)P3 does not play a
significant role in mechanically induced intracellular
Ca2+ release.
2) The delayed phase of the
[Ca2+]i
response is related to Ca2+ influx
via a store-operated,
La3+-insensitive
Ca2+ influx pathway, possibly a SOCC.
In summary, we have shown that mechanical stimulation of myocytes from
the longitudinal muscle layer of the colon produces [Ca2+]i
responses that appear similar in their pattern (transients with
intracellular propagation) to those produced by activation of plasma
membrane receptors and to those previously demonstrated by mechanical
stimulation of myocytes from the circular muscle layer. Yet, despite
the similarities in pattern, the intracellular mechanisms underlying
the generation of the transient, its propagation, and the refilling of
involved Ca2+ stores differ
significantly between the three situations.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-40919.
 |
FOOTNOTES |
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.
Address for reprint requests and other correspondence: E. A. Mayer,
UCLA/CURE Neuroenteric Disease Program, West Los Angeles VA Medical
Center, Bldg. 115, Los Angeles, CA 90073 (E-mail:
emayer{at}ucla.edu).
Received 29 September 1998; accepted in final form 3 February
1999.
 |
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