(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]
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.
The importance of the inositol phosphate (InsP) ()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.
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) 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.
Figure 1:
Ca 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.
L cells also responded to UTP and ADP
with a release of stored Ca, 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) .
Further aspects of the
[Ca]
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.
Figure 2:
ATP-induced
[Ca]
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.
Figure 3:
[Ca]
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.
The addition of
U-73122 (2-5 µM) also produced a drop in resting
[Ca]
(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.
Similar phenomena could be observed in single attached
cells in a static bath, i.e. one without continuous perfusion.
Both single and repeated [Ca]
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).
Figure 4:
[Ca]
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.
Figure 5:
Accumulation of labeled inositol
1-phosphate and inositol trisphosphate in L cell populations. Cells
were loaded with [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.).
Also examined was the
profile of InsP 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).
In this investigation we show that mouse L cells respond to
applied ATP with increased PPI turnover and Ca 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 appeared to be a
relatively weak stimulator of phospholipase C, because when compared to
the InsP-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
similarity of the effect of apyrase addition to that of putative
phospholipase C inhibitor, U-73122, in reducing
[Ca]
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.
The oscillations in membrane potential previously
observed in multinucleate L cell giants (27, 28) appear unrelated to
[Ca]
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) .
To our knowledge, endothelial
cells do not show Ca 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.
To speculate on a possible role that tonic ATP stimulation
and induction of [Ca]
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.