©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Shear Stress-induced Ca Transients and Oscillations in Mouse Fibroblasts Are Mediated by Endogenously Released ATP (*)

(Received for publication, September 13, 1994; and in revised form, November 28, 1994)

Jeremy P. Grierson (§) Jacopo Meldolesi

From the Department of Pharmacology, University of Milano, CNR and B. Ceccarelli Centers and DIBIT-S. Raffaele Scientific Institute, via Olgettina 58, 20132 Milan, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The importance of the inositol phosphate (InsP) (^1)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(3)) 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.


EXPERIMENTAL PROCEDURES

Materials

Fura-2 and thapsigargin were obtained from Calbiochem and dissolved in dimethyl sulfoxide. U-73122 (1-[6-[[17beta-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(2) atmosphere.

[Ca](i) Measurements

Cell 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 KH(2)PO(4), 1.2 MgSO(4), 2 CaCl(2), 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 times 10^6/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 2times concentration. For additional details, see Grohovaz et al.(13) .

Inositol Phosphate Determination

Monolayer cultures were labeled for 24 h with myo-[^3H]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 times 10^6 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(3) 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(3) fraction also contained some fraction of the inositol tetrakisphosphate content, although this was not determined.


RESULTS

Nucleotide-induced [Ca](i) Mobilization in L Cells

Populations of fura-2-loaded L cells, in a fluorimeter with constant stirring, exhibited an average resting [Ca](i) 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](i) was consistently higher (200-250 nM, data not shown). This change in resting [Ca](i) 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](i) (peak 320 ± 23 nM S.E. of mean), which then subsided gradually to prestimulus values (Fig. 1A). Unlike the InsP(3)-mediated [Ca](i) 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](i) 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).


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 (bullet), 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](o) (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](i) 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(3)(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](i) 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](i) 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](i) 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.



Unstimulated Ca Activity in L Cells

A characteristic behavior of these cells was a transient increase in [Ca](i) 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.


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](i) (Fig. 3C) of the same magnitude as that of apyrase. When U-73122 and apyrase were administered sequentially, the induced drop in [Ca](i) 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](i) 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](i), 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](i) 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](i) 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](i) 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.



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).


Figure 5: Accumulation of labeled inositol 1-phosphate and inositol trisphosphate in L cell populations. Cells were loaded with [^3H]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 times 10^6 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 [^3H]InsP(3) 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(3) 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(3)] to rise, although moderately and slowly, and that this could be suppressed with apyrase. When stimulated with 100 µM ATP, InsP(3) 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(3) within the first 30 s and following that subsided to prestimulus levels (Fig. 5B).


DISCUSSION

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](i) 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.

The ATP receptor of L cells appeared to be a relatively weak stimulator of phospholipase C, because when compared to the InsP(3)-liberating action of LPA, the release of InsP(3) 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](i), and the peak amplitude is proportional to the applied shear(4, 10) . Intracellular [Ca](i) 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](i), 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(3) 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.

The similarity of the effect of apyrase addition to that of putative phospholipase C inhibitor, U-73122, in reducing [Ca](i) and PPI metabolism implicated the involvement of extracellular ATP in setting the resting [Ca](i). The existence of tangible levels of ATP in the extracellular milieu was also suggested by the induction of small [Ca](i) 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](i) oscillations. Apyrase was found to eliminate this EGTA-induced hump and yet had no effect on [Ca](i) mobilization provoked by LPA. The ATPase also inhibited repetitive [Ca](i) 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](i) release.

The oscillations in membrane potential previously observed in multinucleate L cell giants (27, 28) appear unrelated to [Ca](i) 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](i) 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](i) 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](i) 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](i) 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](i) 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](i) 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.


FOOTNOTES

*
This work was supported by a grant from the CNR Target Project on Biotechnology and Bioinstruments. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
EMBO Fellow.

(^1)
The abbreviations used are: InsP, inositol phosphate; InsP(3), 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.


ACKNOWLEDGEMENTS

We thank Ed Westhead and Francesco Di Virgilio for helpful suggestions and Fabio Grohovaz and Daniele Zacchetti for help with the video imaging equipment.


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