|
|
|
|||||||
|
|||||||||
(Received for publication, September 13, 1994; and in revised form, November 28,
1994) From the
The effects of ATP, U-73122, apyrase, and saline shear stress on
[Ca The importance of the inositol phosphate (InsP) ( The L cell line has been widely
employed for the expression of recombinant cDNA vectors and also in
studies of wound contraction in skin (see (7) ). It is perhaps
in the wound response that the similarity between fibroblasts and
endothelial cells is most clearly seen, since both cell types can
migrate and generate force via the action of actin and myosin
fibrils(8) . This contractile response can produce important
adverse effects in both tissues: scarring of the skin and permeability
of the vascular lining(9) . Thus, it is important to understand
the signaling mechanisms at work in these cells. From in vitro experiments on endothelial cells, it is becoming clear that an
increase in fluid shear stress has several direct effects, including a
rapid release of ATP and other transmitters, a transient stimulation of
inositol 1,4,5-trisphosphate (InsP
Figure 1:
Ca
L cells also responded to UTP and ADP
with a release of stored Ca Further aspects of the
[Ca
Figure 2:
ATP-induced
[Ca
Figure 3:
[Ca
The addition of
U-73122 (2-5 µM) also produced a drop in resting
[Ca Similar phenomena could be observed in single attached
cells in a static bath, i.e. one without continuous perfusion.
Both single and repeated [Ca
Figure 4:
[Ca
Figure 5:
Accumulation of labeled inositol
1-phosphate and inositol trisphosphate in L cell populations. Cells
were loaded with [
Also examined was the
profile of InsP In this investigation we show that mouse L cells respond to
applied ATP with increased PPI turnover and Ca
The ATP receptor of L cells appeared to be a
relatively weak stimulator of phospholipase C, because when compared to
the InsP
The
similarity of the effect of apyrase addition to that of putative
phospholipase C inhibitor, U-73122, in reducing
[Ca The oscillations in membrane potential previously
observed in multinucleate L cell giants (27, 28) appear unrelated to
[Ca To our knowledge, endothelial
cells do not show Ca To speculate on a possible role that tonic ATP stimulation
and induction of [Ca
Volume 270,
Number 9,
Issue of March 3, 1995 pp. 4451-4456
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ca
Transients and Oscillations in Mouse Fibroblasts Are
Mediated by Endogenously Released ATP (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
] homeostasis were
studied in fura-2 loaded, mouse fibroblast cells (L929), both in
suspension and plated on glass. Release of internal Ca was induced by ATP, via a receptor identified pharmacologically
as a P
type. In single cells, low concentrations of ATP
evoked [Ca] oscillations. These events were blocked by the putative
phospholipase C inhibitor, U-73122 (but not by the inactive analog
U-73343) and by the ATP/ADPase, apyrase. In addition, both these agents
reduced the [Ca] of
unstimulated cells, especially after stirring, and blocked
spontaneously occurring [Ca] oscillations, which suggested an already activated state of
the ATP receptor, independent from exogenous stimulations. Moreover, it
was found that stirring of the cells was correlated with a steady
accumulation of inositol phosphates, also blockable by apyrase, and
that [Ca] mobilization
could be induced by puffs of saline in single cells. The transition to
a Ca-free environment also provoked
[Ca] oscillations,
most likely via the increase in ATP
concentration.
This evidence suggests that endogenous ATP is released from L
fibroblasts in response to fluid shear stress, and this results in an
autocrine, tonic up-regulation of the phosphoinositide signaling system
and an ensuing alteration in Ca homeostasis. Up until
now, such a response to shear stress was believed to be unique to
endothelial cells.
)signaling pathway is well recognized in controlling a wide
variety of physiological functions, and it is clear that the level of
activation of this pathway acutely regulates the release of free
Ca from intracellular storage pools and, perhaps
indirectly, the influx of Ca across the plasma
membrane(1) . What is less clear, however, is how such systems
can be tonically modulated by a constant low level of receptor
activation, and the possibility should be considered that a subtle
regulation of this pathway might exist via some sort of autocrine
mechanism. One cell type which seems to have evolved this kind of
mechanism is the endothelial cells of the vasculature. The fluid shear
stress of blood flow appears to dynamically regulate the
polyphosphoinositide (PPI) metabolism in these cells, and changes in
flow rate can alter this balance(2) . This, in turn, leads to
changes in nitric oxide synthesis and
[Ca](3, 4, 5) ,
which produce the physiological response of dilation, or contraction,
of the surrounding muscle wall(6) . To our knowledge, a
response analogous to this has not been previously reported in cell
types other than endothelia. However, from our studies of
Ca homeostasis in the mouse fibroblast cell line (L,
clone 929), we find striking parallels with the fluid shear stress
response of endothelial cells.
) and rise in
[Ca] and cytoskelatl
rearrangement(2, 10, 11) . Similarly, we have
found that increases in vortical stirring of fibroblast cell
suspensions correlated with increases in phospholipid metabolism and in
resting [Ca]. The
involvement of extracellular ATP in mediating these changes was
inferred from the observation that the responses were blocked by the
addition of a purified ATP/ADPase, or by the phospholipase C inhibitor,
U-73122(12) . Responses to fluid shear stress and endogenous,
extracellular ATP were also found in individual, adherent cells using
fura-2 [Ca] imaging.
In these experiments, [Ca] oscillations were induced by short bursts of saline
perfusion, which were dependent on extracellular ATP. Thus, we provide
evidence for a tonic stimulation of PPI metabolism in L cells which is
sustained by extracellular ATP, apparently acting via a P type receptor. We propose that, in a fashion similar to that of
vascular endothelial cells, ATP is released from the fibroblasts in
response to fluid shear stress, and this results in a tonic
up-regulation of the InsP signaling system and an alteration in
Ca homeostasis.
Materials
Fura-2 and thapsigargin were obtained from Calbiochem and
dissolved in dimethyl sulfoxide. U-73122
(1-[6-[[17
-3-methoxyestra-1-3-5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione)
from Biomol Research Laboratories, was also prepared as a 2 mM stock in dimethyl sulfoxide. Apyrase (EC 3.6.1.5) grade III and
all additional chemicals were purchased from Sigma.Cells
Mouse L cells (ATCC CCL 1, clone 929, a kind gift from Dr A.
Mantovani, Mario Negri Inst. of Pharmacology, Milan) were plated on
either 10-cm plastic dishes or 24-mm glass coverslips and maintained in
Dulbecco's modified Eagle's medium (Bio-Whittaker),
supplemented with 10% fetal bovine serum, 2 mM glutamine and
antibiotics, in an incubator at 37 °C with a humidified, 5%
CO
atmosphere.[Ca
]
MeasurementsCell Populations
Cells were detached from Petri
dishes using a trypsin/EDTA solution, then mixed with a
Ca/Mg-free Hank's buffered
salt solution, 0.1% albumin, centrifuged and resuspended in a
Hepes-buffered, Krebs-Ringer saline (KRH) (mmol/liter): 125 NaCl, 5
KCl, 1.2 KHPO
, 1.2 MgSO, 2
CaCl, 6 glucose, 25 Hepes (pH 7.4). They were then loaded
with 2 µM fura-2/AM (acetoxymethylester form) and pluronic
acid (0.025%) for 45 min at 24 °C, centrifuged, and resuspended in
KRH containing 250 µM sulfinpyrazone (to block dye
extrusion). Cells were stored at room temperature, at a density of 2
10
/ml until use. For each experiment, cells were
diluted 1:2 with KRH and transferred to a quartz cuvette of either 1.5-
or 3-ml volume (volume of cell suspension 0.75 or 1.5 ml,
respectively). Fluorescence measurements were made, at 37 °C, using
LS-50 fluorimeters (Perkin-Elmer), mostly in single-wavelength mode for
increased speed. Traces were calibrated at the end of the experiment
with membrane permeabilization and EGTA and Ca. Data
was transferred to a spreadsheet program for off-line analysis and
plotting.Single Cells
Cells attached to glass coverslips
were fura-2-loaded as above, rinsed, and then mounted on a
thermostatted microscope stage (37 °C, PDMI-2, Medical Systems
Corp.) in 1 ml of KRH. The digital fluorescence imaging system used a
Zeiss Axiovert 135 TV microscope connected to a dual monochromator
light source (Jasco CAM-230, emitting at 340 and 380 nm) in
epifluorescent mode. Fluorescent images (510 nm, 1 pair at each
excitation wavelength every 2 s) were collected by an intensified CCD
camera (Photonic Science), digitized and integrated in real time
(Imaging Technology), and stored on magnetic media. Ratio images and
mean ratio values in discrete areas were computed off-line. Stimulation
of perfused cultures was achieved by changing the reservoir connected
to the peristaltic pump (delay time, 30 s). For experiments which used
a static bath, additions were made by removing 0.5 ml of KRH from the
cell chamber and replacing it with prewarmed KRH containing the test
solution at 2 concentration. For additional details, see
Grohovaz et al.(13) .Inositol Phosphate Determination
Monolayer cultures were labeled for 24 h with myo-[H]inositol (1 µCi/ml) in a
medium consisting of 28% Dulbecco's modified Eagle's
medium, 61.5% Hank's buffered salt solution, 0.5% fetal bovine
serum. They were then rinsed twice with Hank's buffered salt
solution, detached with trypsin/EDTA, and resuspended in KRH (1
10 cells/ml). Ten min before agonist addition, LiCl (10
mM) was added to inhibit the hydrolysis of inositol
1-phosphate. After stimulation, aliquots were removed at the
appropriate times and added to trichloroacetic acid (10% w/v) to stop
the reaction. Following extraction with ether (5 times), the samples
were neutralized and the inositol phosphates were separated on Dowex
1-X8 (Fluka) columns. Inositol 1-phosphate (InsP), inositol
1,4-phosphate, and InsP were eluted with 10 ml of 0.2,
0.45, and 1.0 M ammonium formate, all in 0.1 M formic
acid, respectively. It is likely that the InsP fraction
also contained some fraction of the inositol tetrakisphosphate content,
although this was not determined.
Nucleotide-induced
[Ca
Populations of fura-2-loaded L cells, in a fluorimeter
with constant stirring, exhibited an average resting
[Ca] Mobilization in L
Cells] of 110 ± 28.7
nM (± S.E. of mean). This value, however, was only
attained after 1-2 min in the cuvette since immediately following
transferral of the cells [Ca] was consistently higher (200-250 nM, data not
shown). This change in resting [Ca] appeared not to be due to a change in temperature, but it could
be avoided with an extremely gentle transferral procedure. The putative
inhibitor of phospholipase C, U-73122 (2-5
µM)(12) , was also found to block the initial rise
(not shown). Once stabilized, the populations responded to 100
µM ATP with a swift elevation of
[Ca] (peak 320 ± 23
nM S.E. of mean), which then subsided gradually to prestimulus
values (Fig. 1A). Unlike the InsP-mediated
[Ca] transients in other cell
types, there was no plateau following the initial spike, although in
some cases there was a small and gradual increase in
[Ca] 2-3 min after
stimulation. The response to ATP was dose-dependent from 0.1 to 100
µM (Fig. 1A). Above 100 µM,
the peak of Ca release was progressively reduced.
There appeared to be no contribution from external Ca to the ATP-induced rise, since the response looked unchanged in
Ca-free (1 mM Ca, 2 mM EGTA) KRH (not shown).
mobilization
induced by ATP, UTP, and ADP in L cell suspensions. A-C show
traces of agonist-induced [Ca] rises in fura-2-loaded, fibroblast suspensions. A,
ATP: 100, 5, 1, and 0.1 µM. B, UTP: 100, 5, 1,
and 0.1 µM. C, ADP: 500, 200, 100, and 50
µM. The agonists were added to the cuvette at the break in
the traces, indicated by 
. D, dose dependence curves
for increasing concentrations of ATP (
), UTP (), and ADP
(
) on intracellular Ca release. Data are
expressed as the percent increase in peak
[Ca] above the
initial, resting level. The curves were drawn by hand, and the values
are means ± S.E. of three separate
experiments.
, but we observed no
response to adenosine. The Ca transient evoked by UTP
was almost identical with that of ATP, and the two nucleotides were
equipotent (Fig. 1, B and D). ADP, on the
other hand, was a substantially weaker agonist (Fig. 1, C and D). The response to UTP or ADP was prevented by prior
stimulation of the cells with the maximally effective concentration of
ATP (100 µM). This was true both in the presence and
absence of [Ca]
(results not
shown), but only in experiments where the first stimulus was not rinsed
out. A similar result was obtained if a maximally effective
concentration of UTP (100 µM) was applied before ATP or
ADP. This was not due to depletion of Ca stores
because subsequent application of either thapsigargin (1
µM, a blocker of the sarcoplasmic-endoplasmic reticulum
Ca ATPases that pump Ca into the
store lumen) or of lysophosphatidic acid (LPA, 1 nM, an
agonist which is known to induce GTP-dependent PPI hydrolysis and
intracellular Ca release in a variety of cells (14) including fibroblasts(15) ) induced an appreciable
[Ca] spike (not shown).
Submaximal concentrations of the primary nucleotides, however, did
permit a second stimulation, a feature common to the quantal release
properties of stored Ca by agonists which liberate
InsP(16) . These data suggest that a single
receptor might mediate the Ca release by the
nucleotides and that it could be of the P subtype(17) .] responses were revealed
when adherent, fura-2-loaded, L cells were examined with the video
imaging system. Under resting conditions, the majority of cells were
apparently silent, but a few cells showed occasional
[Ca] spikes. When the coverslip
was perfused with 1 µM ATP (Fig. 2), about 50% of
the population responded with a Ca transient and, in
about half of those, repetitive spikes or sinusoidal oscillations in
[Ca] were observed. Higher
concentrations of ATP elicited a response from more cells, but
oscillatory behavior was less common. The
[Ca]
transients did not appear to spread from one cell to the next, and
responsive cells did not look morphologically different from
unresponsive ones. Oscillatory activity was gradually blocked by the
addition of 0.5 mM excess EGTA; cells showed two or three
spikes before stopping. The dihydropyridine blocker of
voltage-activated Ca channels, nitrendipine, had no
effect on the oscillations, but, in contrast, an immediate cessation
was produced by the addition of U-73122 (2 µM) to the
perfusate. When U-73122 was rinsed out, the oscillations would
recommence.
] responses in
single L cells. The [Ca] responses of five representative, fura-2-loaded, fibroblasts
in a videoimaged field. The bars at the bottom indicate perfusion of the coverslips with 1 µM ATP, 2
mM Ca, followed by 1 µM ATP,
0.5 mM EGTA, 0 Ca; 1 µM ATP, 2
mM Ca; then 10 µM ATP, 2 mM Ca and finally wash out. A range of
Ca responses to the various conditions can be seen,
ranging from single transients to repetitive spikes and sinusoidal
oscillations.
Unstimulated Ca
A characteristic behavior of these cells was a transient
increase in [Ca Activity in L
Cells] in the form of
a ``hump,'' which occurred after the addition of EGTA to L
cell suspensions. Fig. 3A shows an example of this hump,
immediately after the EGTA-induced fluorescence drop (due to
extracellular fura-2). We found that this slow transient could not be
reproduced when EGTA was administered to cells suspended in
Ca
-free KRH or when the cells in complete KRH were
subjected to a small drop in pH, as might occur upon EGTA addition
(data not shown). We then considered that it might arise from the
conversion of endogenous CaATP to the ATP
form,
known to be more efficacious at the P
receptors(17) . This was tested by using a naturally
occurring ATPase, apyrase, which rapidly hydrolyzes both ATP and ADP.
At concentrations between 0.2 and 1.0 unit per ml (2-10 µg of
protein), this purified protein eliminated the EGTA-induced hump (Fig. 3B), but did not significantly affect the
response to LPA (Fig. 3D). It can also be seen from Fig. 3B that a drop in
[Ca] of around 20-30
nM occurred following apyrase addition. These results suggest
that there is some occupancy of ATP receptors in the presence of
``normal'' [Ca]
(2
mM), and this produces some tonic stimulation of InsP
production and Ca release.
]
mobilization by EGTA in L cell suspensions. Trace A shows the
addition of EGTA (E) to L cell suspensions produced a sharp
downward deflection in the fluorescence trace (due to extracellular
fura-2) followed by a transient rise (the hump). 10 µM ATP, then added at the arrowhead, induced a swift
[Ca] elevation. In B, apyrase (apyr, 0.5 unit/ml) produced a slow decline in
[Ca], and subsequent
addition of EGTA produced the sharp drop, but no hump. Thapsigargin (TG, 100 nM) elicited a slow
[Ca]increase. In C, U-73122 (U, 2 nM) produced a decrease in
resting [Ca], but then
the subsequent addition on apyrase had no effect. The EGTA-induced hump
was eliminated as in B, and 100 nM ionomycin (IONO), added at the arrowhead, induced a rapid
[Ca] transient. In D, apyrase (0.5 unit/ml), added after the EGTA-induced hump,
showed no effect on the immediate, large
[Ca] transient induced
by the subsequent addition of 10 nM LPA.
] (Fig. 3C)
of the same magnitude as that of apyrase. When U-73122 and apyrase were
administered sequentially, the induced drop in
[Ca] was found not to be
additive (Fig. 3C), suggesting that a common pathway
was affected by the two agents. U-73122 also eliminated the
EGTA-induced hump and prevented the initial elevation in
[Ca] which occurred during
transferral of the cells to the cuvette (not shown), indicating that
these three phenomena were causally related. In contrast, the inactive
analog of U-73122, U-73343(12) , used at 2-5
µM, had no effect on resting
[Ca], the EGTA-induced hump, or
the initial rise. None of the compounds tested, apyrase, U-73122, or
U-73343, was found to have any intrinsic fluorescence at the settings
used, nor did they show any quenching of fura-2 fluorescence. These
data therefore suggest that ``resting'' L cells are
maintained at a plateau level of Ca mobilization and
that this is caused by endogenous ATP activating the cell surface
receptors.] transients could be initiated in individual cells simply by the
addition of EGTA (Fig. 4). In 75% of cells of such experiments,
the response to EGTA was a brief spike and, for half of those, the
spikes were repetitive. The remaining 25% of cells exhibited longer
lasting transients or complex spikes. These ``spontaneous''
spikes were of a peak height similar to an ATP-induced transient, as
can be seen when 100 µM ATP was added at the end of a
series of spontaneous spikes (Fig. 4). When apyrase (0.5
unit/ml) was included in the extracellular saline (Fig. 4), we
found that some random spikes remained, but the EGTA-induced spikes and
oscillations were absent. In some cells, bathed in
Ca-containing KRH, a single
[Ca] spike could apparently be
provoked by a puff of saline alone, following a lag time of about 3 s.
To produce these saline puffs, saline was drawn up and re-expelled from
a tube at a short distance from the imaged cells; thus, the cells
within the microscope field received saline which had washed over
neighboring cells. The [Ca] spikes induced in this manner were usually not repetitive, but
were otherwise similar in form to the EGTA-induced spikes. They were
blocked by U-73122 (2 µM, not shown).
]
responses in single L cells in response to EGTA addition and saline
changes. The responses of two cells, in a static bath, are shown to the
addition of EGTA (1 mM excess), followed by the readdition of
2 mM Ca, as indicated by the bottom
bars. 0.5 unit/ml apyrase was then added for the duration
indicated by the bar, and this was rinsed out with fresh
KRH/EGTA. Finally, 100 µM ATP was added at the arrow.
ATP-induced Inositol Hydrolysis
Further
confirmation that ATP exerted a tonic activation of receptors was
obtained by examination of InsP accumulation under conditions where
inositol-1-phosphatase was blocked by Li
. Fig. 5A shows that, for L cells analyzed in a stirred
suspension, addition of exogenous ATP (100 µM) induced a
steady accumulation of InsPs (+100%) for the 10 min of the
experiment. A more moderate but clear steady increase was observed also
in the unstimulated control cells. Only when 0.5 unit/ml apyrase was
included in the KRH was this rise prevented, indicating that PPI
hydrolysis had virtually stopped (Fig. 5A). A similar
result was obtained if U-73122 (2 µM), but not U-73343,
was used instead of apyrase (data not shown).
H]inositol (1 µCi/ml) for
24 h, rinsed, detached, and resuspended in KRH. 10 mM LiCl was
added 10 min before beginning sample collection (1 10 cells/sample). The accumulation of labeled InsP is shown in A. ATP stimulation (100 µM, open
circles) was made immediately after the first sample was taken,
and, in controls (filled circles), the vehicle alone was added
(KRH, 7.5 µl). The third curve (open squares)
shows the effect of apyrase addition (0.5 unit/ml) immediately after
the first control sample was taken. In B, the levels of
[H]InsP are shown for the same
samples as A. The treatment conditions are therefore the same,
with the inclusion here of stimulation by 1 µM LPA (open diamonds). Values are averages of three experiments
(± S.E.).
formation under the same conditions (in the
presence and absence of Li). Fig. 5B shows that the stirring of the unstimulated cell suspension caused
[InsP] to rise, although moderately and slowly,
and that this could be suppressed with apyrase. When stimulated with
100 µM ATP, InsP formation was stimulated over
a period of 2 min and then was maintained at an elevated plateau. For
comparison, we also tested the effect of LPA; stimulation by LPA
dramatically increased levels of InsP within the first 30 s
and following that subsided to prestimulus levels (Fig. 5B).
release. As a plated monolayer, the cells frequently responded to
exogenous ATP with a series of [Ca] spikes. Our evidence suggests that the experimental manipulation
of these cells also leads to their stimulation in an autocrine fashion.
This, we suggest, is due to the release of ATP in response to fluid
shear stress.The ATP Receptor of L Cells
The ATP receptor expressed
by mouse L cells, as evidenced by pharmacological results, appeared to
be of the P subtype (recently redefined as
P
, (18) ). This receptor displays equal
sensitivity to ATP and UTP and a low sensitivity for ADP(14) .
It also shows a relatively greater sensitivity for ATP ions(17) , confirmed in the present study. A receptor
with a similar agonist specificity appears to be expressed by variety
of cells, including fibroblasts (19, 20) and neural
cells(21, 22, 23) . The P
receptor, on the other hand, which is expressed by endothelial
cells(24) , has a sensitivity ATP = ADP > UTP, and so
is clearly different.-liberating action of LPA, the release of
InsP by ATP was small and slow. This could be due to
partial, homologous desensitization of a type which seems frequent in
phospholipase C-coupled receptors(25) . We also noted that ATP
was never able to deplete Ca stores (in single cells
or suspension), whereas LPA was able to, thus precluding any further
release by the sarcoplasmic-endoplasmic reticulum Ca ATPase blocker, thapsigargin.The Effect of Fluid Shear Stress
The fact that
fluid shear stress can induce the release of ATP, prostacyclin, and
other vasoactive substances from endothelial cells is relatively
known(5, 10) , and, in vivo, this process is
thought to mediate the vasodilation which occurs when blood flow
increases(3, 6) . The signaling pathway involved and
the mechanism of transmitter release, however, is less. In single
cells, shear stress produces a transient rise in
[Ca], and the peak amplitude is
proportional to the applied shear(4, 10) .
Intracellular [Ca] oscillations
can occur, and PPI metabolism, as well as several other lipid pathways,
is also stimulated (2, 4) .Shear Stress Responses in L Cells
The responses of
mouse L cells, observed in the present study to the physical stress of
saline movement, appeared similar to the shear stress-response of
endothelial cells, although differences were also apparent. Agitation
of the cells by either transferral or stirring was associated with an
increase in [Ca], but, since
this could be rapidly reduced with U-73122, or apyrase, it suggests
that it was an elevated plateau. Vortical stirring also resulted in an
increase in InsP production which was similarly and
dramatically reduced by the addition of apyrase. These data point
toward a shear-induced stimulation of PPI metabolism leading to an
enhanced discharge of intracellular Ca pools.] and PPI metabolism
implicated the involvement of extracellular ATP in setting the resting
[Ca]. The existence of tangible
levels of ATP in the extracellular milieu was also suggested by the
induction of small [Ca] transients by EGTA addition. The chelation of Ca by EGTA could lead to a 2-fold increase in the effective
ATP concentration(26) , presumably enough to
elicit a burst of Ca release or
[Ca] oscillations. Apyrase was
found to eliminate this EGTA-induced hump and yet had no effect on
[Ca] mobilization provoked by
LPA. The ATPase also inhibited repetitive
[Ca] spikes induced by EGTA in
plated cells. Thus, we believe that ATP, released into the medium by L
cells experiencing fluid shear stress, is responsible for the
activation of PPI metabolism and [Ca] release.] oscillations, since only
the first were inhibitable by dehydropyridine antagonists of
voltage-activated Ca channels. Furthermore,
spontaneously occurring cell giants (n = 2) were found
to have very small [Ca] oscillations. This, and the finding that oscillations could
continue for a short time in a Ca-free saline,
suggests that it is the kinetics of discharging and recharging the
internal Ca pools which most likely create the
oscillatory behavior(29) . oscillations when exposed to low
concentrations of ATP, but do in response to histamine(30) . It
is not clear whether the lack of effect of ATP is due to the high level
of ectonucleotidase activity associated with these cells (24, 31) or that the response is intrinsic to the ATP
receptor subtype. It is perhaps significant that, in endothelial cells,
1-10 µM ATP produced a
[Ca] transient 3-5-fold
higher than that of stress-induced response(10) , whereas in L
cells exogenous ATP (10-100 µM) produced a
Ca transient of a similar height, but broader than a
spontaneous spike. We found that L cells exhibited a variety of
oscillatory forms; from repetitive Ca spikes with no
elevated baseline to cells which showed sinusoidal
[Ca] oscillations on an
elevated baseline. The continuum of forms of oscillations exhibited by
these cell indicates that the mechanism for each must be fundamentally
the same.] oscillation might play in the living animal, we might suggest
that these events are important during fibroblast migration. It is well
known that, after cutaneous wounding, quiescent fibroblasts become
activated and migrate to the fibronectin-fibrin wound
interface(32) . It has been suggested that
[Ca] oscillations occur during
the migration of both fibroblasts and endothelial cells(33) .
The oscillations and thus, perhaps, cell movement, could be modulated
by autocrine release of low concentrations of ATP. It has been reported
recently that mast cells also migrate into the wound area and establish
a special relationship with the activated fibroblasts(34) .
Mast cells release ATP upon antigen binding, and this spreads the
activation response from cell to cell. It is thus likely that
fibroblasts in the wound site would also be party to this flow of
information.Conclusions
We have demonstrated that L
fibroblasts express a P-subtype ATP receptor, a relatively
weak stimulator of PPI metabolism, which, however, can elicit
[Ca] oscillations. In addition,
this receptor appears to play a key role in the change of
Ca homeostasis induced in fibroblast cells by fluid
shear stress, a response that appears to be sustained by the autocrine
release of ATP. We advise, therefore, that particular care be employed
when using mouse L cells in models of signaling in view of their
sensitivity to handling. Clearly, many aspects of the above processes
remain to be investigated, including the nature of the shear stress
receptor and the mechanism(s) of ATP secretion. Further studies should
also address fibroblasts from various origins, since it is clear from
the endothelial cells (2) that origin has a profound effect on
physiological response.
),
inositol 1,4,5-trisphosphate;
[Ca] and
[Ca], free
Ca concentration in the cytosol and in the
extracellular medium, respectively; KRH, Krebs-Ringer HEPES-buffered
saline; PPI, polyphosphoinositide; LPA, lysophosphatidic acid.
We thank Ed Westhead and Francesco Di Virgilio for
helpful suggestions and Fabio Grohovaz and Daniele Zacchetti for help
with the video imaging equipment.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Kawai, M. Kaidoh, and T. Ohhashi MDA-MB-231 produces ATP-mediated ICAM-1-dependent facilitation of the attachment of carcinoma cells to human lymphatic endothelial cells Am J Physiol Cell Physiol, November 1, 2008; 295(5): C1123 - C1132. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Martinon Detection of immune danger signals by NALP3 J. Leukoc. Biol., March 1, 2008; 83(3): 507 - 511. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Yamamoto, N. Shimizu, S. Obi, S. Kumagaya, Y. Taketani, A. Kamiya, and J. Ando Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1646 - H1653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. D. Douillet, W. P. Robinson III, P. M. Milano, R. C. Boucher, and P. B. Rich Nucleotides induce IL-6 release from human airway epithelia via P2Y2 and p38 MAPK-dependent pathways Am J Physiol Lung Cell Mol Physiol, October 1, 2006; 291(4): L734 - L746. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Li, D. Liu, H. Z. Ke, R. L. Duncan, and C. H. Turner The P2X7 Nucleotide Receptor Mediates Skeletal Mechanotransduction J. Biol. Chem., December 30, 2005; 280(52): 42952 - 42959. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Chivasa, B. K. Ndimba, W. J. Simon, K. Lindsey, and A. R. Slabas Extracellular ATP Functions as an Endogenous External Metabolite Regulating Plant Cell Viability PLANT CELL, November 1, 2005; 17(11): 3019 - 3034. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Lappas, J. M. Rieger, and J. Linden A2A Adenosine Receptor Induction Inhibits IFN-{gamma} Production in Murine CD4+ T Cells J. Immunol., January 15, 2005; 174(2): 1073 - 1080. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Feng, A. G. Mery, E. M. Beller, C. Favot, and J. A. Boyce Adenine Nucleotides Inhibit Cytokine Generation by Human Mast Cells through a Gs-Coupled Receptor J. Immunol., December 15, 2004; 173(12): 7539 - 7547. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Boudreault and R. Grygorczyk Cell swelling-induced ATP release is tightly dependent on intracellular calcium elevations J. Physiol., December 1, 2004; 561(2): 499 - 513. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. E. Pafundo, P. Mut, M. Perez Recalde, R. M. Gonzalez-Lebrero, V. Fachino, G. Krumschnabel, and P. J. Schwarzbaum Effects of extracellular nucleotides and their hydrolysis products on regulatory volume decrease of trout hepatocytes Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2004; 287(4): R833 - R843. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Lu, A. Pierron, and K. Ravid An Adenosine Analogue, IB-MECA, Down-Regulates Estrogen Receptor {alpha} and Suppresses Human Breast Cancer Cell Proliferation Cancer Res., October 1, 2003; 63(19): 6413 - 6423. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. R. Lazarowski, R. C. Boucher, and T. K. Harden Mechanisms of Release of Nucleotides and Integration of Their Action as P2X- and P2Y-Receptor Activating Molecules Mol. Pharmacol., October 1, 2003; 64(4): 785 - 795. [Full Text] [PDF]
|
|
|
|
|
|
C. I. Seye, N. Yu, R. Jain, Q. Kong, T. Minor, J. Newton, L. Erb, F. A. Gonzalez, and G. A. Weisman The P2Y2 Nucleotide Receptor Mediates UTP-induced Vascular Cell Adhesion Molecule-1 Expression in Coronary Artery Endothelial Cells J. Biol. Chem., June 27, 2003; 278(27): 24960 - 24965. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. T. Neary, Y. Kang, K. A. Willoughby, and E. F. Ellis Activation of Extracellular Signal-Regulated Kinase by Stretch-Induced Injury in Astrocytes Involves Extracellular ATP and P2 Purinergic Receptors J. Neurosci., March 15, 2003; 23(6): 2348 - 2356. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. V. Gerasimovskaya, S. Ahmad, C. W. White, P. L. Jones, T. C. Carpenter, and K. R. Stenmark Extracellular ATP Is an Autocrine/Paracrine Regulator of Hypoxia-induced Adventitial Fibroblast Growth. SIGNALING THROUGH EXTRACELLULAR SIGNAL-REGULATED KINASE-1/2 AND THE Egr-1 TRANSCRIPTION FACTOR J. Biol. Chem., November 15, 2002; 277(47): 44638 - 44650. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. F. Weidema, S. J. Dixon, and S. M. Sims Activation of P2Y but not P2X4 nucleotide receptors causes elevation of [Ca2+]i in mammalian osteoclasts Am J Physiol Cell Physiol, June 1, 2001; 280(6): C1531 - C1539. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Yamamoto, R. Korenaga, A. Kamiya, and J. Ando Fluid Shear Stress Activates Ca2+ Influx Into Human Endothelial Cells via P2X4 Purinoceptors Circ. Res., September 1, 2000; 87(5): 385 - 391. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Roman, A. P. Feranchak, K. D. Salter, Y. Wang, and J. G. Fitz Endogenous ATP release regulates Cl- secretion in cultured human and rat biliary epithelial cells Am J Physiol Gastrointest Liver Physiol, June 1, 1999; 276(6): G1391 - G1400. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Kittel Lipopolysaccharide Treatment Modifies pH- and Cation-dependent Ecto-ATPase Activity of Endothelial Cells J. Histochem. Cytochem., March 1, 1999; 47(3): 393 - 400. [Abstract] [Full Text]
|
|
|
|
|
|
R. K. Palmer, J. L. Boyer, J. B. Schachter, R. A. Nicholas, and T. K. Harden Agonist Action of Adenosine Triphosphates at the Human P2Y1 Receptor Mol. Pharmacol., December 1, 1998; 54(6): 1118 - 1123. [Abstract] [Full Text]
|
|
|
|
|
|
L. Tenneti, S. J. Gibbons, and B. R. Talamo Expression and Trans-synaptic Regulation of P2x4 and P2z Receptors for Extracellular ATP in Parotid Acinar Cells. EFFECTS OF PARASYMPATHETIC DENERVATION J. Biol. Chem., October 9, 1998; 273(41): 26799 - 26808. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Ralevic and G. Burnstock Receptors for Purines and Pyrimidines Pharmacol. Rev., September 1, 1998; 50(3): 413 - 492. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. Mohaupt, T. Fischer, J. Schwobel, R. B. Sterzel, and E. Schulze-Lohoff Activation of purinergic P2Y2 receptors inhibits inducible NO synthase in cultured rat mesangial cells Am J Physiol Renal Physiol, July 1, 1998; 275(1): F103 - F110. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. C. Watt, E. R. Lazarowski, and R. C. Boucher Cystic Fibrosis Transmembrane Regulator-independent Release of ATP. ITS IMPLICATIONS FOR THE REGULATION OF P2Y2 RECEPTORS IN AIRWAY EPITHELIA J. Biol. Chem., May 29, 1998; 273(22): 14053 - 14058. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Lazarowski, L. Homolya, R. C. Boucher, and T. K. Harden Direct Demonstration of Mechanically Induced Release of Cellular UTP and Its Implication for Uridine Nucleotide Receptor Activation J. Biol. Chem., September 26, 1997; 272(39): 24348 - 24354. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |