Mechanical strain-induced Ca2+
waves are propagated via ATP release and purinergic
receptor activation
H.
Sauer,
J.
Hescheler, and
M.
Wartenberg
Department of Neurophysiology, University of Cologne, D-50931
Cologne, Germany
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ABSTRACT |
Mechanical strain
applied to prostate cancer cells induced an intracellular
Ca2+ (Cai2+) wave spreading with a
velocity of 15 µm/s. Cai2+ waves were not
dependent on extracellular Ca2+ and membrane potential
because propagation was unaffected in high-K+ and
Ca2+-free solution. Waves did not depend on the
cytoskeleton or gap junctions because cytochalasin B and nocodazole,
which disrupt microfilaments and microtubules, respectively, and
1-heptanol, which uncouples gap junctions, were without effects.
Fluorescence recovery after photobleaching experiments revealed an
absence of gap junctional coupling. Cai2+ waves
were inhibited by the purinergic receptor antagonists basilen blue and
suramin; by pretreatment with ATP, UTP, ADP, UDP, 2-methylthio-ATP, and
benzoylbenzoyl-ATP; after depletion of ATP by 2-deoxyglucose; and after
ATP scavenging by apyrase. Waves were abolished by the anion channel
inhibitors 5-nitro-2-(3-phenylpropylamino)benzoic acid, tamoxifen,
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, niflumic acid, and
gadolinium. ATP release following strain was significantly inhibited by
anion channel blockers. Hence, ATP is secreted via mechanosensitive
anion channels and activates purinergic receptors on the same cell or
neighboring cells in an autocrine and paracrine manner, thus leading to
Cai2+ wave propagation.
calcium wave; adenosine 5'-triphosphate release; purinergic
receptor; anion channel
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INTRODUCTION |
CALCIUM WAVES HAVE BEEN
OBSERVED in various excitable and nonexcitable cells where they
coordinate physiological responses within the respective tissues. In
nonexcitable cells the most intensively investigated mechanism for wave
propagation is based on strain-induced inositol trisphosphate
[Ins(1,4,5)P3]
formation and diffusion though gap junctional pores.
Ins(1,4,5)P3
then releases intracellular Ca2+ from
Ins(1,4,5)P3-sensitive
Ca2+ stores in the neighboring cells (2,
36, 37). Recent studies on basophil
leukemic cells, hepatocytes, and osteoblastic cell lines
(21) have shown that intracellular Ca2+
(Cai2+) waves may be alternatively propagated
via activation of purinergic receptors of the G protein-coupled P2Y
class that activate phospholipase C (PLC), resulting in the generation
of
Ins(1,4,5)P3
and intracellular Ca2+ release from
Ins(1,4,5)P3-sensitive
Ca2+ stores. Although it has been speculated that
purinergic receptor activation and Cai2+ wave
propagation may be mediated by ATP release from the mechanically stimulated cells, the molecular mechanism of purinergic receptor stimulation following mechanical strain remains poorly defined. ATP
release triggered by mechanical strain has been recently reported and
apparently did not involve the cystic fibrosis transmembrane regulator
(CFTR) (47). Furthermore, it has been demonstrated that
connexins regulate, via a still unraveled mechanism, strain-mediated Ca2+ signaling by controlling ATP release. In this study
ATP release was inhibited by anion channel blockers (12).
The present study reports on mechanical strain-elicited
Cai2+ waves in confluent prostate cancer cells
of the DU-145 cell line, which were independent of intercellular
communication because they persisted after uncoupling of gap junctions.
Cai2+ wave propagation could be inhibited by
antagonists of purinergic receptors and by preincubation with several
nucleotides, indicating that activation of multiple purinergic
receptors, including P2Y2 receptors, which have been
previously shown to be present in DU-145 prostate cancer cells
(46), may underlie wave propagation. In hypotonic solution
these cells secreted ATP. Because ATP release could be inhibited by
blockers of anion channels, we concluded that purinergic receptor
activation during Cai2+ wave propagation is
mediated via ATP release through volume-activated Cl
channels. ATP released by mechanical strain will diffuse radially in
the extracellular space and will propagate a
Cai2+ wave spreading up to a distance of ~300
µm from the stretched cell area.
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MATERIALS AND METHODS |
Chemicals.
Fluo 3-acetoxymethyl ester (AM) and 5-carboxyfluorescein diacetate
(5-CFDA) were purchased from Molecular Probes (Eugene, OR). ATP, ADP,
UTP, UDP, 2-methylthio-ATP (2-MeS-ATP), benzoylbenzoyl-ATP (Bz-ATP),
apyrase, 2-deoxyglucose, 1-heptanol, tamoxifen, niflumic acid, suramin,
basilen blue, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid
(DIDS), U-73122, histamine, and GdCl3 were from Sigma
(Deisenhofen, Germany). 5-Nitro-2-(3-phenylpropylamino)benzoic acid
(NPPB) and glibenclamide were from RBI (Natick, MA).
Cell culture.
The human prostate cancer cell line DU-145 was kindly provided by Dr.
J. Carlsson (Uppsala, Sweden). Cells were cultivated in
25-cm2 tissue culture flasks (Costar, Fernwald, Germany) in
5% CO2 and humidified air at 37°C with Ham's F-10
medium (GIBCO, Life Technologies, Gaithersburg, MD) supplemented with
10% fetal calf serum (Boehringer Mannheim, Mannheim, Germany), 2 mM
glutamine, 0.1 mM
-mercaptoethanol, 2 mM minimal essential
medium, 100 IU/ml penicillin, and 100 µg/ml streptomycin (ICN
Flow, Meckenheim, Germany). For the experiments, cells grown to
confluency in tissue culture flasks were enzymatically dissociated in
Ca2+-free phosphate-buffered saline supplemented with
0.05% EGTA and 0.1% trypsin. Single cells were plated to coverslips
and cultivated to confluency.
Bioluminescence experiments.
ATP release from confluent DU-145 cells was determined using a
luciferin-luciferase assay (Sigma) in a chemiluminescence apparatus (Bioluminiscence Analyzer XP2000, SKAN, Basel, Switzerland) under dim
light. For data sampling, the output of the photomultiplier tube of the
setup was connected to a multimeter (Voltcraft M-3610D, Conrad
Electronics, Hirschau, Germany) and a Tandon 286/N personal computer
(Tandon, Moorpark, CA). Cells grown to confluency on 20 × 20-mm
coverslips were washed five times in F-10 cell culture medium, which
resulted in a background luminescence signal that was not significantly
different from the signal obtained with cell culture medium in the
absence of cells. Cells were subsequently immersed in 1 ml of F-10 cell
culture medium that was diluted 1:1 with distilled water, resulting in
an osmolality of 150 mosmol/kgH2O. In control experiments,
cells grown to confluency on coverslips were immersed in an equal
volume of isotonic medium. For the experiments with anion channel
inhibitors, cells were preincubated for 5 min in isotonic F-10 cell
culture medium that was supplemented with the respective inhibitor.
Subsequently, cells were immersed in 1 ml of hypotonic F-10 medium
supplemented with inhibitors. After different times, during which the
cells were gently shaken, a 200-µl aliquot was removed and pressure
injected via a light-tight access into a 3-ml glass cuvette containing
50 µl of the ATP assay mix and 1.5 ml of ATP assay mix dilution
buffer (Sigma). Calibration measurements with ATP were performed in a
concentration range of 0-100 nM. The lowest concentration of ATP
that could be detected under the applied experimental conditions was
0.5 nM. From these calibration curves the total picomoles of ATP
released per 105 cells were calculated. Each of the applied
anion channel inhibitors was tested for its effects on the activity of
luciferase enzyme activity. No significant effects of the compounds on
luciferase enzyme activity were observed. Furthermore, dilution of F-10
cell culture medium to yield an osmolality of 150 mosmol/kgH2O did not affect luciferase activity
(n = 3 for each experimental condition) (see Fig.
1). The chemiluminescence output curve
was integrated, and the resulting values were set in relation to the
calibration curve. To correlate ATP release to the cell number, the
cells from which ATP release had been determined were enzymatically dissociated with a 0.2% trypsin-0.05% EDTA solution, and the
cells were counted using an automated cell counter (Cell Analyzer Casy 1, Schärfe System, Reutlingen, Germany).

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Fig. 1.
Effects of either hypotonic solution or anion channel
inhibitors on the activity of the luciferin-luciferase assay. A
200-µl aliquot of either isotonic F-10 cell culture medium containing
10 nM ATP or hypotonic F-10 medium (150 mosmol/kgH2O)
containing 10 nM ATP and supplemented with either niflumic acid (100 µM), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 100 µM),
tamoxifen (50 µM), gadolinium (100 µM), or DIDS (500 µM) was
pressure injected into a glass cuvette containing 1.55 ml of the
luciferin-luciferase solution. Note that neither hypotonic solution nor
addition of any of the compounds significantly impaired the
luminescence signal.
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Fluorescence recovery after photobleaching.
Cell cultures were incubated with 20 µM 5-CFDA for 20 min and
postincubated in the absence of 5-CFDA for a further 10 min. Excitation
of 5-CFDA was provided by the 488-nm line of the argon ion laser of the
confocal setup. Emission was recorded using a long-pass LP 515 filter
set. Single cells were selected using an overlay mask. 5-CFDA
fluorescence was photobleached in the selected cells using the "point
scan" mode of the confocal setup. By switching the attenuation filter
wheel of the confocal setup, we elevated the laser power from 0.125 to
12.5 mW for 3 s, which resulted in photobleaching of the dye.
After photobleaching, the microscope settings were returned to the
recording configuration, and fluorescence recovery in the photobleached
cell was monitored every 10 s.
Ca2+ imaging and confocal laser scanning microscopy.
Measurement of Cai2+ was performed using the
fluorescent Ca2+ indicator fluo 3-AM. Cells
adherent to 20 × 20-mm-diameter glass coverslips were incubated
for 60 min at 37°C in cell culture medium containing 10 µM fluo
3-AM dissolved in dimethylsulfoxide (final concentration 0.1%) and
Pluronic F-127 (final concentration <0.025%; Molecular Probes). After
loading, the coverslips were rinsed in E1 buffer containing (in mM) 135 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 10 glucose,
and 10 HEPES (pH 7.4 at 37°C) and mounted to the bottom of an
incubation chamber that was fixed to the table of an inverted confocal
laser scanning microscope (LSM 410, Zeiss, Jena, Germany). Fluorescence
was excited by the 488-nm line of an argon-ion laser. Emission was
recorded using a long-pass LP 515 filter set. The experiments were
performed with a ×25 objective (NA 0.85; Neofluar, Zeiss). Processing
of images was carried out with the Time software facilities of the
confocal setup. The minimum, maximum, mean, standard deviation, and
integrated sum of the pixel values in a region of interest (selected
using an overlay mask) were written to a data file and routinely
exported for further analysis to the commercially available SigmaPlot
(Jandel Scientific, Erkrath, Germany) graphics software. Data are
presented in arbitrary units as percentages of fluorescence variation
(F/F0) with respect to the resting level fluorescence
(F0).
For the quantification of Cai2+ concentrations,
calibration experiments were performed as described previously
(22), assuming a dissociation constant of 1,100 nM at
vertebrate ionic strength. Cai2+ waves were
induced by stimulating a group of 8-10 cells with a blunt-end (tip
diameter 50 µm) borosilicate glass pipette affixed to a Narishige
micromanipulator (Narishige International, Tokyo, Japan). Images were
recorded in 1-s intervals. Conduction velocities of
Cai2+ waves were measured by determining the
distance and the amount of time required for the wave to spread from
the mechanically stretched cells (0 µm) to cells within a distance of
100 and 200 µm, respectively.
Statistical analysis.
Data are given as means ± SE, with n denoting the
number of experiments. Student's t-test for unpaired data
was applied as appropriate. A value of P < 0.05 was
considered significant.
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RESULTS |
DU-145 prostate cancer cells propagate
Cai2+ waves upon mechanical
strain.
When confluent cells of the prostate cancer cell line DU-145 were
mechanically stimulated with the tip of a glass pipette, a transient
rise of Cai2+ from 109 ± 38 nM
(n = 16 cells in 5 independent experiments) under
resting conditions to 440 ± 61 nM (n = 17 cells
in 5 independent experiments) upon mechanical strain occurred with a
delay of 1-3 s after the cell membrane was stretched
(n = 8) (Fig.
2A). The Cai2+ signal spread radially from the stretched
area with a velocity of ~15 µm/s and declined within a distance of
200-300 µm. Cai2+ waves could be elicited
from the same stretched area up to three times (n = 3)
(Fig. 2B), indicating that cell membranes were not ruptured
during mechanical stimulation. However, the distance covered by the
Cai2+ wave declined with repetitive mechanical
strain. After two periods of mechanical strain of the same cell area, a
third application of mechanical strain elicited a transient
Cai2+ response that was restricted to the
stretched area but did not spread to more distant parts of the
coverslip. The absence of Cai2+ wave propagation
was not caused by the desensitization of purinergic receptors upon
mechanical strain because exogenous addition of 10 µM ATP elicited a
pronounced transient Cai2+ response
(n = 3) (Fig. 2C). Furthermore,
Ins(1,4,5)P3
consumption following repetitive mechanical strain could be excluded
because 50 µM histamine, which uses the
Ins(1,4,5)P3
signal transduction pathway, transiently raised
Cai2+ in the cell area that had been stretched
three times (n = 4) (Fig. 2D).

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Fig. 2.
Mechanical strain-induced intracellular Ca2+
(Cai2+) wave in confluent prostate cancer cells.
A: false color images of a Cai2+ wave
spreading radially from the cell area touched with the tip of a
blunt-end glass pipette (indicated by circle). The images were recorded
2, 4, 6, 8, and 12 s after strain application (from upper
left to lower right panel). Bar, 100 µm.
B: representative tracing of a strain-induced
Cai2+ wave. Fluo 3 fluorescence, presented as
relative fluorescence increase (F/F0), was evaluated in
single cells in the center of the stretched cell area (0 µm) and at
distances of 100 and 200 µm. Note that mechanical strain could be
elicited several times, indicating that the glass pipette did not
disrupt cell membranes. C: representative tracing of the
Cai2+ response in the cell area that was touched
by the blunt-end glass pipette. After the third mechanical stimulus,
cells were superfused with 10 µM ATP, which resulted in a transient
Cai2+ response in all cells, including the cells
that had been mechanically distorted, indicating that the P2 receptors
were not desensitized following mechanical strain. D:
representative tracing of the Cai2+ response in
the cell area that was touched by the blunt-end glass pipette. After
the third mechanical stimulus, cells were superfused with 50 µM
histamine, which resulted in a transient Cai2+
response in all cells, including the cells that had been mechanically
distorted, indicating that inositol trisphosphate was not depleted
following mechanical strain. The times at which strain was applied are
indicated by arrows. The times of agonist administration are indicated
by horizontal lines. The dotted line (D) represents the
Cai2+ signal in a control cell that was not
mechanically distorted.
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Wave propagation is not dependent on membrane potential,
extracellular Ca2+, an intact
microfilament/microtubular network, or functional gap junctions.
To evaluate whether the mechanism for wave propagation was a
strain-induced membrane depolarization, cells were superfused with a
solution containing 140 mM K+ to depolarize the membrane
potential. Figure 3A shows
that under these conditions wave propagation and the amplitude of the
Cai2+ response in the mechanically stretched
cell area (see Fig. 3G) were not impaired (n = 3). Because superfusion with high-K+ solution in the
absence of mechanical strain did not raise
Cai2+, we concluded that no voltage-dependent
Ca2+ channels were present in DU-145 cancer cells (data not
shown). The source of the Cai2+ response
following mechanical strain was further evaluated by superfusion with
nominally Ca2+-free solution. Figure 3B
demonstrates that under this experimental condition neither wave
propagation nor the amplitude (see Fig. 3G) of the
Cai2+ response in the mechanically stretched
cell area was impaired, suggesting an involvement of intracellular
Ca2+ stores in the Cai2+ response
elicited by mechanical stimulation (n = 3). After
preincubation with thapsigargin, which depletes intracellular
Ca2+ stores and inhibits the Ca2+-ATPase of the
sarcoplasmic reticulum (42), Cai2+
wave propagation upon mechanical strain was abolished
(n = 3) (Fig. 3C) and the amplitude (see
Fig. 3G) of the Cai2+ response in the
mechanically stretched cell area was significantly reduced to 169 ± 60 nM (n = 3).

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Fig. 3.
Dependence of Cai2+ wave
propagation on Ca2+ release from intracellular stores but
not on extracellular Ca2+, membrane potential, intact
microfilaments and microtubules, and intercellular communication via
gap junctions. A: Cai2+ wave
propagation was not impaired after membrane potential depolarization in
140 mM extracellular K+. B:
Cai2+ wave propagation was not impaired in
nominally Ca2+-free solution. C: mechanical
strain-induced Cai2+ responses were abolished
after store depletion with 1 µM thapsigargin.
Cai2+ wave propagation was not impaired after
disruption of the microfilament cytoskeleton with 50 µM cytochalasin
B (D), disruption of microtubules with 20 µM nocodazole
(E), or after uncoupling of gap junctions with 2.5 mM
1-heptanol (F). Data are presented as representative
tracings. The times at which strain was applied are indicated by an
arrow. G: amplitudes of the Cai2+
responses in the cell area stretched by the glass pipette under the
experimental conditions presented in A-F. *P < 0.05, significantly different from the untreated control.
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Mechanical strain may be transduced to intracellular Ca2+
stores, i.e., the endoplasmic reticulum, via cytoskeletal elements, i.e., the microfilament or microtubular network. To evaluate whether this mechanism underlies the observed phenomenon, cells were
preincubated for 30 min with either 50 µM cytochalasin B (Fig.
3D) or 20 µM nocodazole (Fig. 3E), which
disrupts actin filaments or microtubules, respectively. Under these
conditions neither the amplitude (see Fig. 3G) of the
transient Cai2+ rise in the mechanically
stretched cells nor the propagation of the Cai2+
wave was significantly impaired (n = 3 for each
experimental condition), indicating that an intact cytoskeleton is not
necessary for signal transduction.
The most common mechanism for the propagation of
Cai2+ waves in nonexcitable cells relies on
Ins(1,4,5)P3
diffusion through gap junctions from the mechanically stretched cells
to more distant cell areas. To evaluate whether this mechanism holds
true for DU-145 prostate cancer cells, cells were preincubated for 30 min with 2.5 mM 1-heptanol, which is known to uncouple gap junctions (10). We observed that this agent did not impair
Cai2+ wave propagation (Fig. 3F) and
the amplitude of the Cai2+ response in the
stretched cell area (see Fig. 3G) (n = 4),
which indicates that signals leading to Cai2+
transients in cells neighboring the stretched cell area were not
transduced via gap junctions. To get a closer view of gap junctional
communication in DU-145 cells and to exclude misinterpretation of our
results due to nonspecific effects of 1-heptanol, fluorescence recovery
after photobleaching (FRAP) experiments were performed by
photobleaching single 5-CFDA-labeled cells and monitoring fluorescence recovery in the bleached cell. Figure 4
clearly shows that fluorescence recovery of carboxyfluorescein after
photobleaching was low, which demonstrates that DU-145 cells grown to
confluency possess only marginal gap junctional communication. Further
evidence that gap junctional communication is not required
for Cai2+ waves in DU-145 cells came from
experiments in which subconfluent cells were mechanically
stretched. These islands of cells were not in physical contact. We
observed that the Cai2+ response was not
restricted to the cell island that was mechanically stretched but
spread to nearby cell islands that were not in physical contact with
the stretched cells, indicating that a diffusible mediator may be
involved in Cai2+ wave propagation (data not
shown).

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Fig. 4.
Absence of intercellular communication in confluent
DU-145 cells as revealed by fluorescence recovery after photobleaching
experiments. Single 5-carboxyfluorescein diacetate (CFDA)-loaded cells
were photobleached, and the fluorescence recovery was monitored. Note
that fluorescence recovery after bleaching is marginal, indicating the
absence of functional gap junctions. Data are presented as a
representative tracing.
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Involvement of purinergic receptor activation in the propagation of
mechanical strain-induced
Cai2+ waves.
Cai2+ wave propagation has been recently shown
to be mediated by purinergic receptor activation (21). The
presence of P2Y2 receptors in the DU-145 prostate cancer
cell line used in the present study has been previously demonstrated
(46). To investigate the types of purinergic
receptors present in DU-145 cells in more detail,
Cai2+ transients following treatment with 10 µM ATP (Fig. 5A), UTP (Fig.
5B), ADP (Fig. 5C), UDP (Fig. 5D),
2-MeS-ATP (Fig. 5E), or Bz-ATP (Fig. 5F) were
recorded. All applied reagents elicited a transient
Cai2+ response in DU-145 cells, suggesting the
presence of multiple P2 receptors in DU-145 prostate cancer cells
(n = 3 for each experimental condition).

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Fig. 5.
Cai2+ responses in DU-145 cells after
treatment with 10 µM ATP (A), UTP (B), ADP
(C), UDP (D), 2-methylthio-ATP (2-MeS-ATP)
(E), and benzoylbenzoyl-ATP (Bz-ATP) (F). Data
are presented as representative tracings. The times of agonist
administration are indicated by horizontal lines.
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Further evidence for an involvement of purinergic receptors in
strain-induced Cai2+ waves was obtained from
experiments in which cells were preincubated with 300 µM suramin
(n = 3) (Fig.
6A) or 300 µM basilen blue
(Fig. 6B) (n = 3), both of which are known
as antagonists of purinergic receptors (3). Preincubation
with suramin and basilen blue abolished Cai2+
wave propagation. However, a transient Cai2+
response not significantly different in amplitude from the control was
observed in the cell area that was directly stretched by the glass
pipette (see Fig. 6F). Because activation of purinergic receptors of the P2Y subfamily involves the
Ins(1,4,5)P3
signaling pathway, experiments were performed in which PLC was
inhibited by U-73122 (Fig. 6C) (n = 4). This
treatment resulted in inhibition of wave propagation. However,
mechanical strain elicited a transient Cai2+
response in the stretched cell area that was not significantly different in amplitude compared with the control (see Fig.
6F).

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Fig. 6.
Involvement of purinergic receptor activation in the
mechanical strain-induced Cai2+ waves.
Cai2+ wave propagation was inhibited in the
presence of the purinergic receptor antagonists suramin (300 µM)
(A) and basilen blue (300 µM) (B) and in the
presence of the phospholipase C (PLC) antagonist U-73122 (10 µM)
(C). Wave propagation was furthermore inhibited after
depletion of intracellular ATP by inhibition of glycolysis with 5 mM
2-deoxyglucose (D) and after scavenging of extracellular ATP
by 2 U/ml apyrase (E). Data are presented as representative
tracings. The times at which strain was applied are indicated by an
arrow. F: amplitudes of the Cai2+
responses in the cell area stretched by the glass pipette under the
experimental conditions presented in A-E.
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Purinergic receptor stimulation may be mediated via ATP released from
the mechanically stretched cells. Hence, the
Cai2+ wave propagation should be absent under
conditions in which either intracellular ATP was depleted or
extracellular ATP was scavenged. Downregulation of intracellular ATP
was achieved by incubation of cells for 72 h with 5 mM
2-deoxyglucose, which is known to deplete ATP by the inhibition of
glycolysis (44) (Fig. 6D). Enzymatic
degradation of extracellular ATP was achieved by addition of 2 U/ml
apyrase (Fig. 6E) to the incubation medium. Under these experimental conditions the strain-induced Cai2+
response remained restricted to the stretched area but was not significantly different in amplitude compared with the control (see
Fig. 6F) (n = 3 for each experimental condition).
Receptor desensitization by extracellular nucleotides may impair
the strain-induced Cai2+ response. To evaluate
this issue, 10 µM ATP, UTP, ADP, UDP, MeS-ATP, or Bz-ATP was added to
the incubation medium, and the mechanical strain was applied after the
nucleotide-induced Cai2+ response had declined
to baseline Ca2+ levels. Treatment of cells with either ATP
(Fig. 7A) or UTP (Fig. 7B) totally abolished Cai2+ wave
propagation and significantly reduced the amplitude of the Cai2+ response in the stretched area to 217 ± 26 or 205 ± 18 nM, respectively (Fig. 7G)
(n = 3 for each experimental condition). As observed with ATP and UTP, treatment with ADP (Fig. 7C), UDP (Fig.
7D), MeS-ATP (Fig. 7E), and Bz-ATP (Fig.
7F) inhibited wave propagation (n = 3 for
each experimental condition). However, the amplitude of the
Cai2+ response in the mechanically stretched
cell area was not significantly different from that of the control
under the latter experimental conditions (see Fig. 6G).

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Fig. 7.
Effects of receptor desensitization with 10 µM ATP
(A), UTP (B), (ADP) (C), UDP
(D), 2-MeS-ATP (E), and Bz-ATP (F) on
the propagation of mechanical strain-induced
Cai2+ waves. Cells were mechanically stretched
after the transient Cai2+ responses elicited by
the agonists had declined to resting Cai2+
levels. Data are presented as representative tracings. The times at
which strain was applied are indicated by an arrow. G:
amplitudes of the Cai2+ responses in the cell
area stretched by the glass pipette under the experimental conditions
presented in A-F. Note that preincubation with ATP and UTP
significantly (*P < 0.05) reduced the amplitude of the
Cai2+ response in the stretched cell area
compared with the untreated control.
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Inhibition of Cai2+ waves
by anion channel inhibitors.
It has been previously demonstrated that ATP is secreted via anion
channels. CFTR or P-glycoprotein has been described as a possible
candidate for ATP release (1, 32). It is,
however, also likely that other, not-yet-characterized volume-sensitive anion channels are able to conduct ATP. To evaluate a possible role of
anion channels for the mediation of ATP release, DU-145 cells were
incubated for 10 min with NPPB (100 µM; n = 8) (Fig. 8A), DIDS
(500 µM; n = 10) (Fig. 8B), niflumic acid
(100 µM; n = 5) (Fig. 8C), or tamoxifen
(50 µM; n = 8) (Fig. 8D). The latter compound is known to inhibit anion channels but, in addition, exerts
inhibitory effects of protein kinase C (34). Additionally, cells were treated with gadolinium (n = 5) (Fig.
8E), which is known to be an antagonist of stretch-activated
cation channels but has also been described to inhibit
Ca2+-activated (43) and stretch-activated
Cl
channels (33, 48). All
applied anion channel blockers inhibited Cai2+
wave propagation upon mechanical strain. However, a
Cai2+ transient not significantly different in
amplitude compared with the control was observed in the cell area
mechanically distorted by the patch pipette (Fig. 8I). To
evaluate a possible role of CFTR and P-glycoprotein in ATP release,
cells were preincubated for 10 min with verapamil (90 µM) (Fig.
8F) and quinidine (50 µM) (Fig. 8G), which
inhibit P-glycoprotein-associated Cl
channels, as well as
with glibenclamide (100 µM) (Fig. 8H), which inhibits
CFTR. However, none of these agents impaired
Cai2+ waves elicited upon mechanical strain
(n = 3 for each experimental condition).

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Fig. 8.
Effects of anion channel inhibitors on the mechanical
strain-induced Cai2+ waves. Wave propagation was
inhibited in the presence of NPPB (100 µM) (A), DIDS (500 µM) (B), niflumic acid (100 µM) (C),
tamoxifen (50 µM) (D), and gadolinium (100 µM)
(E). Wave propagation was unaffected in the presence of the
P-glycoprotein-associated anion channel blockers verapamil (90 µM)
(F) and quinidine (50 µM) (G) and the CFTR
antagonist glibenclamide (100 µM) (H). Data are presented
as representative tracings. The times at which strain was applied are
indicated by an arrow. I: amplitudes of the
Cai2+ responses in the cell area stretched by
the glass pipette under the experimental conditions presented in
A-H .
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ATP release via Cl
channels following hypotonic
incubation.
To obtain stronger evidence of a mechanism of ATP release,
bioluminescence experiments were performed to directly access the amount of released ATP in the supernatant. In this series of
experiments, mechanical strain was applied by incubating cells in
hypotonic medium, and aliquots of the supernatant were analyzed.
Incubation of DU-145 cells in hypotonic medium increased the medium
concentration of ATP within 10 s. Maximum ATP was yielded within 2 min, whereas longer incubation times resulted in a gradual decay of the
ATP concentration, presumably because of ATP consumption or degradation (Fig. 9A). The effects of
anion channel inhibitors were evaluated after 2 min of hypotonic
incubation (Fig. 9B). The absolute amount of ATP released
within 2 min after swelling was 1.57 ± 0.5 pmol/105
cells (n = 8). Preincubation of cells for 10 min with
niflumic acid (100 µM; n = 3), NPPB (100 µM;
n = 3), tamoxifen (50 µM; n = 4), and
gadolinium (100 µM; n = 3) reduced ATP release by 51 ± 7%, 87 ± 4%, 84 ± 5%, and 55 ± 12%,
respectively. It has been previously estimated that the local ATP
concentration at the plasma membrane upon hypotonic stimulation should
be in the micromolar range (25). Concentrations of
externally added ATP exceeding 1 µM elicit transient
Cai2+ responses in DU-145 cancer cells (M. Wartenberg, unpublished results). Interestingly, DIDS (500 µM;
n = 3) did not significantly impair ATP release
following hypotonic incubation.

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|
Fig. 9.
ATP release upon hypotonic incubation of confluent DU-145
cells. A: time course of ATP release in a 150 mosmol/kgH2O hypotonic solution. B: effects of
the anion channel inhibitors niflumic acid (100 µM), NPPB (100 µM),
tamoxifen (50 µM), gadolinium (100 µM), and DIDS (500 µM) on ATP
release following hypotonic incubation. Confluent cells were pretreated
for 5 min with the agents in isotonic (300 mosmol/kgH2O).
Subsequently, cells were immersed for 2 min in hypotonic solution
containing the respective anion channel inhibitors. The same
experimental protocol was applied for the untreated isotonic control.
ATP released from the cells was determined in the supernatant with the
luciferin-luciferase assay and was quantified with the use of
calibration curves obtained at different concentrations of ATP. Note
that DIDS did not impair hypotonic ATP release. *P < 0.05, significantly different from the sample treated with hypotonic
solution in the absence of anion channel inhibitors.
|
|
 |
DISCUSSION |
The present study reports on Cai2+ waves
elicited by mechanical strain in confluent prostate DU-145 cancer
cells. The mechanism of wave propagation did not operate via diffusion
of
Ins(1,4,5)P3 or Ca2+ through gap junctional pores because chemical
uncoupling of cells did not impair wave propagation and because FRAP
experiments revealed that gap junctional coupling in DU-145 cells was
marginal. This observation is in line with recent results of Carruba et
al. (6a), who investigated cell-cell communication in different
cultured human prostate tumor cell lines and demonstrated only minor
intercellular communication in these preparations. The transient
Cai2+ response was apparently mediated by
Ca2+ release from intracellular stores because it persisted
in nominally Ca2+-free solution but was abolished after
store depletion with the Ca2+-ATPase inhibitor
thapsigargin. The signal transduction pathway connecting the primary
stretch site and the intracellular Ca2+ store, furthermore,
did not require an intact cytoskeleton because pretreatment with
cytochalasin B, which destroys the microfilament network, and
nocodazole, which depolymerizes microtubules, were without any effect
on wave propagation.
The data of the present study strongly support the notion that the
mechanism underlying wave propagation operated via ATP release, because
receptor desensitization with exogenous nucleotides, incubation with
suramin and basilen blue (which are antagonists of purinergic
receptors), intracellular ATP depletion by preincubation with
2-deoxyglucose, and the presence of apyrase in the incubation medium
abolished the observed effects.
The secreted ATP may subsequently stimulate multiple purinergic P2
receptors, which were demonstrated to be present in DU-145 cells.
Interestingly, a synergism of different purinergic receptors seems to
be necessary for the initiation of wave propagation, because receptor
desensitization with agonists of P2Y subtypes and P2X receptors
abolished wave propagation upon mechanical strain. This finding
indicates that the activation of multiple receptors is required for
Ca2+ wave propagation and explains why the inhibition of
PLC, which is involved in the
Ins(1,4,5)P3
signaling pathway following binding of nucleotides to subtypes of P2Y
receptors, abolished wave propagation despite the possible presence of
P2X receptors in the investigated cell line. It should, however, be
mentioned that the nucleotides used in the present study are not
conclusively selective and that a contamination of the commercial
preparations with either ATP or UTP could not be excluded.
Interestingly, under most conditions in which Ca2+ wave
propagation was inhibited, a transient Cai2+
response persisted in the cell area that was mechanically distorted by
the glass pipette. This points toward the notion that Ca2+
wave initiation in the stretched cell area may operate via a direct
Ins(1,4,5)P3
release from the plasma membrane upon mechanical strain. Strain-induced
increases of
Ins(1,4,5)P3
have been previously reported for several preparations
(13, 15), and the possibility that PLC may
act as a mechanotransducer mediating
Ins(1,4,5)P3 generation following mechanical perturbation has been discussed (4, 18, 41). Because in the
DU-145 cell line used in the present study,
Ins(1,4,5)P3,
due to the absence of gap junctional communication, cannot diffuse to
neighboring cells. The Cai2+ response remained
restricted to the stretched cell area. It was furthermore observed that
the Cai2+ transient in the stretched cell area
was significantly reduced in amplitude after preincubation with ATP and
UTP, which involves the
Ins(1,4,5)P3,
signal transduction pathway, whereas it remained unchanged when cells
were pretreated with ADP, UDP, 2-MeS-ATP, or Bz-ATP. This points toward
the notion that ATP and UTP desensitized purinergic receptors in the
stretched cell area more efficiently than the latter nucleotides and
argues against the notion that Ins(1,4,5)P3
had been consumed by the externally added purinergic receptor
agonists. Whereas externally added nucleotides apparently inhibited wave propagation via receptor desensitization,
inactivation of receptors did not occur after repetitive
mechanical strain, because addition of ATP to the incubation
medium elicited a transient Cai2+
response. Furthermore, depletion of intracellular
Ins(1,4,5)P3 by repetitive stimulation could be obscured because histamine transiently raised Cai2+ in all cells under
investigation, including the cells that had been stretched by the glass
pipette. We therefore concluded that the lack of wave propagation
following repetitive mechanical strain was caused by depletion of
intracellular ATP in the stretched cell area.
There is increasing evidence for extracellular pathways of
Ca2+ wave propagation and intercellular communication based
on ATP release. Ca2+ wave propagation via ATP release has
been previously reported for rat mast cells and basophil leukemic cells
(27), hepatocytes (38), osteoblastic cell
lines (21), C6 glioma cells, HeLa cells, and U373
glioblastoma cells (12). Furthermore, ATP release following mechanical strain or hypotonic swelling has been demonstrated for several preparations, including urinary bladder epithelial cells
(14), guinea pig ileal smooth muscle (24),
hepatoma cells (45), tracheal epithelial cells
(26), and red blood cells (40). The
physiological significance of ATP release has not yet conclusively been
unraveled. However, the possibility that ATP release mediated by
hypotonic stimulation of ciliary epithelial cells may modulate aqueous
humor flow by paracrine and/or autocrine mechanisms within the two cell
layers of this epithelium (25) has been discussed. In
liver cells (45) and bilary epithelial cells
(35), recovery from swelling is mediated by an autocrine
pathway involving conductive release of ATP. In endometrial,
intestinal, and epididymal epithelial cells, regulation of
Cl
release is mediated by extracellular ATP
(8, 18). The ATP release observed in urinary
bladder epithelial cells by changes in hydrostatic pressure has been
suggested as a sensory mechanism for the degree of distension of the
urinary bladder (14). A similar sensor mechanism may be
true in prostatic epithelial tissues.
In the present study ATP release was significantly inhibited by the
Cl
channel blockers tamoxifen, NPPB, niflumic acid, and
gadolinium. Consequently, propagation of Ca2+ waves was
inhibited in the presence of anion channel inhibitors. This points
toward the notion that ATP released through Cl
conductive
pathways by mechanical strain activates P2 receptors in the plasma
membrane. This results in transient Cai2+
responses in cells that are distant to the area directly touched by the
glass pipette. Interestingly, DIDS inhibited
Cai2+ wave propagation but did not impair ATP
release upon hypotonic stimulation. An increase of ATP release in the
presence of DIDS following hypotonic incubation has been recently
reported (25) and has been interpreted as inhibition of
ecto-ATPases, which rapidly degrade extracellular ATP. The inhibitory
effects of DIDS on Cai2+ wave propagation
therefore may be explained by its previously demonstrated effect as an
antagonist of purinergic receptors (5, 11,
29).
The molecular mechanisms of ATP release are still a matter of debate.
Some evidence suggests that ATP release from mammalian epithelial cells
can proceed through members of the ATP-binding cassette family of
proteins such as the cAMP-activated CFTR or P-glycoprotein, which plays
a pivotal role in multidrug resistance (1, 6,
28, 30, 32, 38).
These observations have been challenged by others (16,
23, 31, 47). In the present study these pathways were excluded because the P-glycoprotein antagonist cyclosporin A (data not shown) and the 4E3 antibody, which
is directed against an extracellular domain of the transporter (data
not shown), as well as verapamil and quinidine, which inhibit the
P-glycoprotein-associated Cl
conductance, failed to
inhibit wave propagation. Likewise, glibenclamide, which interferes
with CFTR Cl
channels, was without any affect on wave
propagation. However, it has been recently shown that the permeation
pathway associated with CFTR-modulated ATP release is independent of
the Cl
conductance pathway in the channel pore
(20). A comparable mechanism may likewise hold true for
P-glycoprotein. Because it has been recently demonstrated that CFTR may
regulate other epithelial ion channels such as the epithelial
Na+ channel and the outward rectifying Cl
channel (ORCC), an indirect effect of CFTR and P-glycoprotein on ATP
release through other swell-activated anion channels cannot be
excluded. A recent publication (38) demonstrating that
CFTR regulates the ORCC through pathways that involve P2Y receptors points in this direction.
The data of the present study support a model by which mechanical
strain raises Cai2+ via
Ins(1,4,5)P3
generation in the cell area touched by the glass pipette. The
mechanical strain opens volume-sensitive Cl
channels,
which either directly release ATP or activate associated, not-yet-described release mechanisms for ATP. The released ATP then
activates P2 receptors in neighboring cells, which results in a
Cai2+ wave that spreads radially from the
stretched cell area. A strain-induced release mechanism for ATP may
provide a sensor for the distension of the prostate tissue.
Furthermore, an autocrine/paracrine model of humor formation, as has
been recently proposed for ciliary epithelial cells (25),
likewise may hold true for prostate epithelial cells. In this model
released ATP is hydrolyzed by membrane ectoenzymes to adenosine, which
stimulates aqueous humor formation by activating Cl
channels in the nonpigmented epithelial cell layer. In the pigmented cell layer, extracellular ATP stimulates aqueous humor formation by
directly activating anion conductances. The purinergic regulation of
anion secretion may involve Cai2+ mobilization
including Cai2+ waves, as has been recently
demonstrated for pancreatic duct cells and retinal pigment epithelium
(7, 28). Cai2+ waves
may provide a means to transduce a localized strain event into an
extended signaling cascade in distant cell layers that are not affected
by the mechanical strain. By this signal transduction cascade,
Cai2+ waves may trigger fluid secretion in the
tissue of the prostate gland.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: M. Wartenberg, Dept. of Neurophysiology, Robert-Koch-Str. 39, D-50931 Cologne, Germany (E-mail:
hs{at}physiologie.uni-koeln.de).
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 9 August 1999; accepted in final form 16 March 2000.
 |
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