Bradykinin-induced Ca2+ Entry, Release, and Refilling of Intracellular ...

THEllOURNAL 'L

Val . 267, No. 1, Issue af'.January 5 , pp. 108-118, 1992 Prrnted in 11 S A.

OF BIOLOC.ICA1. CHEMISTRY

1992 hy T h e American Society for Biochemistry and Molecular B i o l o g y . Inc.

Bradykinin-induced Ca2+Entry, Release, and Refillingof Intracellular Ca2+Stores RELATIONSHIPSREVEALED

OF INDIVIDUAL HUMANFIBROBLASTS*

BY IMAGEANALYSIS

(Received for publication, September 4, 1991)

Kenneth L. Byron, Gyorgy Babnigg,and Mitchel L. VillerealS From the Department of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, Illinois 60637

Lys-Bradykinin (BK), a mitogen for human foreskin stimuli evoke dramatic changes in[Ca'+], in their targetcells, fibroblasts (HSWP cells) (Owen, N. E.,and Villereal, the complex regulation of the mechanisms involved in these M. L. (1983)Cell 32, 979-985), elicits a rapid, tran- changesremains poorly defined. Extracellular signals can sient elevationof intracellular free Ca2+ concentrationtrigger an elevation of [Ca'+], by increasing entryof Ca" from ([Ca"+Ii)in these cells. We have used image analysisof the extracellular space and/or by releasing Ca'+ from intrafura-%loaded HSWP cells to examine theBK-induced cellular stores. The release of intracellular Ca2+ is generally [Ca2+li changes in individual cells. BK-stimulated Ca"+ attributedtothebinding of inositol 1,4,5-trisphosphate entry and release of intracellular Ca'+ stores can be (Ins(1,4,5)P:J1 to a receptor located on an intracellular Ca2+distinguished by stimulating cells in the presence or sequestering organelle which results in the activation of a absence of extracellular Ca"', or by inhibiting Ca'+ of that organelle and the entry with 5 mM NiCla. BK-sensitive intracellular Ca'+ Ca2+ channel in the membrane consequent release of Ca2+ into the cytosol (2). stores canbe depletedby exposure of the cells to BK in A great deal of recent research has been devoted to examCa"+-free medium; refilling of the stores requires extracellular Ca2+. A component of BK-stimulated Ca'+ ining the relationship between release of intracellular Ca'+ entry persists after removalof agonist, but inactivates stores and increased entry of Ca2+ across the plasma memwith a tliz of approximately 5 min. Although previous brane. A model developed by J. W. Putney, Jr. (3) in 1986 studies have attributed the Ca2+ entry which persists suggests that agonist-induced Ca2+ entry in parotid acinar cells is regulated by the Ca'+ content of the intracellular Ca'+ after agonist removal to a "capacitative Ca2+ entry" pathway activatedby the depletion of the intracellular stores. This model arose largely from some earlier work from Ca'+ stores, we find that a large component of this BK- Putney's lab which examined the emptying and refilling of stimulated Ca2+entry is not due to capacitative Ca"+ the intracellular Ca2+ stores in parotid acinar cells (4, 5). entry since (1) ionomycin can deplete the BK-sensitive Based on those findings, Putney proposed that the intracellular Ca"' stores without appreciably stimulat- Ins(l,4,5)P,-releasable intracellular Ca'+ stores arelocated in ing Ca"+ entry and without inhibiting the BK-stimu- close apposition to the plasma membrane. In the event of lated Ca2+ entry and (2) Ca"+ this entry pathway inac- agonist-mediatedphosphatidyl inositol turnover, elevated tivates at a time when the Ca'+ pools are still empty Ins(1,4,5)Pc3 levels trigger the release of Ca2+from these stores and a capacitance entry pathway should still be open. into the cytosol. This lowers the Caz+ content within the On the other hand, refilling of the intracellular Ca" stores and relieves inhibition of a Ca2+ entry pathway in the stores can occur after the noncapacitative Ca2+entry adjacent plasma membrane. Ca2+ then enters through this component has inactivated or when it is inhibited by pathway across the plasma membrane and into the intracelNi"+;in these cases refilling occurs without a detectable lular stores,by-passing the bulk of the cytosol. Putney coined elevation of [Ca'+Ii suggesting that refilling of internal the phrase"capacitative Ca'+ entry" todescribe this phenomCa"+pools might occur by a capacitative route. enon. According to this model, while Ins(1,4,5)P, levels remain elevated, the Ca2+which enters the intracellular stores will leak into the cytosol, accounting for the sustained elevaThe signal transduction pathway for mitogen-stimulated tion of [Ca'+], observed during agonist stimulation in Ca2+cell proliferation in cultured fibroblasts involves a sequence containing medium. When Ins(1,4,5)Pa levels fall (as on terof biochemical events which begin at the cell surface and mination of agonist binding to receptor), the intracellular progress temporally and spatially to thecell nucleus. Among release channel closes, buttheentry pathwayacross the the earliest of these events are dramatic changes in [Ca'+],. plasma membrane remains open, and Ca2+ can enter to refill These changes are believed to be an importantsignal for later the intracellular stores. As the Ca2+content of the storesrises, events in a sequence which includes changes in gene expres- the entry pathwayis inhibited until, when the stores arefull, sion and initiationof DNA synthesis. no further Ca2+entry occurs. Although itis well establishedthatmany physiological An earlier report by Poggioli et al. (6) had suggested that a reduction of [Ca'+] on the inner surface of the plasma mem* This work was supported by National Institutes of Health Grants brane could increase the permeability of that membrane to

GM-28359 and S10-RR-03322 and Training Grant 5T32-GM-07151. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ T o whom correspondence should be addressed Dept. of Pharmacological and Physiological Sciences, The University of Chicago, 947 E. 58th St., Chicago, IL 60637.

The abbreviations used are: Ins(1,4,5)P:), inositol 1,4,5-trisphosphate;Ins(1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate;BK, Lysbradykinin; [Ca'+],, intracellular free Ca2+concentration;EGTA, [ethylenebis(oxyethylenenitrilo)]tetraaceticacid; fura-2-AM, acetoxymethyl ester form of fura-2; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid HBSS, Hanks' balanced salt solution.

108

Bradykinin-induced [Ca2+IiChanges in Human Fibroblasts

109

Ca'+. Putney's model elaborated on that finding to speculate of [Ca'+]i after inactivation or inhibition of the noncapacitathat, if the intracellular Cay+ storeswere located adjacent to tive Cay+ entry pathway.Heterogeneity of responses of indithe plasma membrane, then depleting these stores ofCa'+ vidual cells to BK and to removal of BK supports a role for might reduce [Ca'+] on the innersurface of the plasma mem- cell-to-cellvariability in receptor numberindetermining these responses in individual cells. The implications of these brane and increase the permeability of that membrane as described by Poggioli et al. (6). Evidence has been presented findings with regard to the possibility of multiple Ca" entry of refillfor localization of agonist-sensitive intracellular Ca2+ stores pathways in human fibroblasts and the mechanisms discussed. adjacent to the plasma membrane in pancreatic (7) and pa- ing the intracellular Caz+ stores are rotid acinar cells (8). EXPERIMENTALPROCEDURES Since Putney's model was first published, many reports relating to Ca'+ entry and refilling of the intracellular Ca2+ Cell Culture-HSWP human foreskin fibroblasts were obtained stores have appeared in the literature (9-21, 23, 35, 36). An and cultured as previously described (25). Cells between the 10th and early report by Merritt and Rink (9) used fura-%loaded pa- 20th passages were subcultured onto 25-mm round glass coverslips days prior to the startof the experiment. rotid acinar cells to arrive at very similar conclusions to 1-5Loading Cells withfura-2-Essentially as described previously (24), Putney. These authors suggested that a gap junction-like cells were incubated in a HEPES-buffered Hanks' balanced salt channel might exist between the plasma membrane and the solution (HEPES-HBSS), with 1mg/ml bovine serum albumin, 5 ~ I M fura-2-AM, and 0.025% Pluronic F127 detergent (26), for 90 min at intracellular Ca2+ stores, and that Ca'+ entry through this channel would be regulated by the Ca2+ content of the stores. room temperature, then washed free of this solution and incubated More recent studies(10-18) have continued to supporta role for 30 min more in HEPES-HBSS. Thecoverslip was then mounted into a perfusion chamber, and HEPES-HBSSwas added to cover the for the Ca'+ content of the intracellular stores in regulating cells. Ca'+ entry, but with the qualification that Ca2+ enters the Capturing Cell Images-The perfusion chamber was mounted onto cytosolbefore it is takenupintotheintracellularstores. the stage of a Nikon inverted epifluorescence microscope. The cells These findings have led to the speculation that the intracel- were excited alternately with light of wavelengths 340 and 380 nm by lular Ca2+ stores might be affecting Ca'+ entry while acting at computer-controlled switching of narrow band interference filters in some distance from the siteof entry (11)and that these effects front of a 200-watt mercury arc lamp. The emittedfluorescence passed through a dichroic mirror (400 nm) anda high pass barrier filter (420 may be mediated by some diffusible messenger (12, 19). nm) and was directedtoa Videoscope KS1380 image intensifier Depending on the cell type, depletion of the intracellular coupled in series to a Dage Model 70 vidicon camera. The video signal Ca'+ stores may not besufficient to stimulate Ca" entry. was processed (16frames averaged) by an Avionics Image Sigma Kass et al. (20) have shown that depletionof the Ins(1,4,5)PZ3- image processor. Averaged image pairs (340 and 380 nm) were capsensitive Ca'+ stores in rat hepatocytes canbe accomplished tured at 5-10-s intervals and stored in the memory of a Joyce-Loebl image analyzer. Background images for each wavelength by inhibiting Ca2+ uptake into these stores with 2,5-di-(tert- Magiscan (captured from a portion of the coverslip which contained no cells) butyl)-1,4-benzohydroquinone,a potent inhibitor of liver mi- were subtracted from the cell images. The resulting images were crosomal ATP-dependent Ca2+ sequestration. Depleting the ratioed (340380) pixel by pixel to produce ratio images. At the image stores with this compound blocked further release of intracel- intensifier gain settings used, no autofluorescence was detected in lular Ca'+ by vasopressin, but did not stimulate Ca'+ entry or cells not loaded with fura-2. Estimation of Intracellular-free Ca" Concentration-Quantitation inhibit vasopressin-stimulated Ca2+ entry. Stauderman and of [Ca"], was performed as described previously (24). Briefly, soluPruss (21) found that angiotensin I1 stimulates Ca'+ entry tions of known ca2+ concentration (0-2000 nM) with 5 ~ I Mfura-2 into bovine adrenalchromaffin cells bya noncapacitative free acid were imaged in the same manner as the cells. 340380 ratio mechanism. These findings do not rule out the possibility values were stored in a look-up table for analysis of the cell images. Drug Additions-All experiments were conducted a t room temperthat agonistsmay activate more than oneCa" entry pathway. In support of this notion, Schilling et ai. (22) suggested that ature. The chamber containing the cells was perfused by gravityBK may activate two Ca2+ entry pathways in bovine aortic driven syringes mounted above the microscope. A 4-way valve controlled flow from four different syringes. Switching solutionsproduced endothelial cells. And Sage et al. (23) have presented evidence a lag time of approximately 2 s while the solution passed through fortwo Ca2+ entry pathways activated by ADP in human tubing to thechamber. The entirevolume of the chamber turned over platelets, one receptor-operated and another which is regu- in less than 20 s. Nominally Ca'+-free medium was prepared by treating Ca'+-free, lated by the Ca2+ content of the intracellular stores. T o date, no studies have been publishedon the relationships Mg'+-free, bicarbonate-free HBSS (+20 mM HEPES) with Chelex100 then adding MgC1, to a final concentration of 1 mM. between Ca2+ entry and the release and refilling of the intraImage Analysis-Images of as many as 50 cells/field were analyzed cellular Ca2+ stores in mitogen-stimulated fibroblasts.Disusing Joyce-Loebl Tardis software. This program allows the user to cerning the relationships between the transient and sustained select, usingalightpen, areas of the image for analysis ofCa'+ concentration. The [Ca"], value reported at each time point is the phases of mitogen-stimulated [Ca2+], elevations and subsequent events in the sequenceleading to DNA synthesis is mean [Ca'+], value for all pixels within the selected area, Each cell in image was independently analyzed for each time point in the fundamental to our understanding role the of Ca" in regulat- the captured sequence. All individual cell [Ca"], traces shown are repreing cell proliferation. T o further our insight into these rela- sentative responses for a given field of cells, which were observed in tionships, we have used imageanalysis techniques to monitor at least three seperate experiments. Measurement of Inositol Phosphates-For assays of inositol phosmitogen-induced [Ca'+], changes in individual human fibroblasts. We have shown previously that individual cells exhibit phate release in intact cells, labeling with 1 pCi/ml 3H-myo-inositol done for 18 h in sterile HBSS containing 2.5 mM NaHC03, 25 asynchronous andheterogeneous responses to BKwhich may was pg/ml gentamicin, 20 mM HEPES (pH 7.45), and 0.1% dialyzed fetal be attributed to cell-to-cell variabilityin receptor number bovine serum. Cells were washed twice and then incubated in assay (24). The present study expands on that work to demonstrate medium (labeling medium lacking gentamicin, serum, and isotope) that BK stimulates both the release of intracellularCa2+ for 1 h at room temperature. The medium was then aspirated and stores and Ca2+ entry from the extracellular space. BK-stim- replaced with 2.7 ml of fresh medium. After a5-minincubation, ulated Ca2+ entry occurs, a t least in part, through a nonca- assays were started by adding 0.3 ml of 100 ng/ml BK into the medium. After a I-, 5-, or 10-minincubation, the medium was pacitativepathway which inactivates slowly afterBKreaspirated, and the assay was quenched with 2 ml of ice-cold 10% moval. Refilling of the intracellular Cay+ stores requires ex- trichloroacetic acid. Following extraction of the trichloroacetic acid tracellular Ca'+, but mayoccur without a detectable elevation with diethyl ether and neutralization with 50 mM borax, the sample

Bradykinin-induced (Ca2+Ii Changesin Human Fibroblasts

110

t co2'

BK 5 ng/ml

BK 5 nq/ml

EK 5 ng/ml

FIG. 1. BK-induced [Ca2+Iichanges in the presence or absence of extracellular Ca2+.Truces are from an individual cell in a field of cells which was stimulated three times consecutively with 5 ng/ml BK. Between the left and center traces, the cells were washed for 8 min with HEPES-HBSS, and then2 min with nominally CaZ+free HEPES-HBSS. Between the center and right hand truces, the cells were washed for 10 min with HEPESHBSS. BK was added as indicated by the arrows. During the generation of center trace, BK wasadded in nominally Ca2+-freemedium. was applied to an anion-exchange column, from which the inositol phosphates were eluted by using ammonium formate buffers as detailed previously (27). The mono-, bis-, and trisphosphate forms of inositol were eluted with 9 ml of buffer directly into scintillation vials, 12 ml of scintillation fluid was added, and the samples were counted. Materials-fura-2 free acid, fura-2 AM, and Pluronic F127 were purchased from Molecular Probes, Inc. EGTA was purchased from Fluka Chemical Co. Lys-bradykinin was purchased from Peninsula Laboratories, Inc. Bovine serum albumin was purchased from Sigma. Chelex-100 was purchased from Bio-Rad. HBSS without phenol red or NaHCO,, and 10 X HBSS without phenol red, NaHCO,, CaC12, MgC12, or MgS04 were purchased from GIBCO. NPC-415 was provided by Nova Pharmaceutical Corporation. RESULTS

BK Stimulates Both the Release of Intracellular ea2+Stores and ea2+Entry from the Extracellular Space-[Ca2+], changes in human foreskin fibroblasts (HSWP cells) treated with BK in thepresence or absence of extracellular Ca2+are shown in Fig. 1. The traces shown are from an individual cell which was stimulated repetitively with BK (with 10-min washes between each recording). In Ca2+-containing medium, BK typically produces a biphasic response: after an initial lag period, [Ca2+Iirises abruptly to a peak, then declines to a plateau of elevated [Ca2+];which remains elevated above basal levels for many minutes. In Ca2+-freemedium (center trace, Fig. l),the plateau phase is absent, and themagnitude of the peak is lower than that of the same cell stimulated with BK in Ca2+-containing medium. It appears, therefore, that the plateau of elevated [Ca"], results from entry of Ca2+from the extracellular space into thecytosol. This is further evidenced by the observation that removal of extracellular Ca2+during the plateau phase results in a rapid return of [Caz+]ito basal levels (not shown). Also, the presence of Ni", a nonspecific Ca2+-channelblocker (28), prevents the plateau phase when added before (Fig. 2 A ) or at the same time (not shown) as BK, without inhibitingthe component of the response which occurs independently of extracellular Ca2+. In addition, Ni2+terminates theplateau phase when added after thepeak [Ca"], response has occurred (Fig. 2B). The BK-induced [Ca2+],changes which occur in the absence of extracellular Ca2+ (Fig. 1, center) or in the presence of extracellular Ni2+(Fig. 2 A ) can be attributed to therelease of Ca2+ bound or sequestered within the cell. Since previous studies from our laboratory have indicated that BK stimulates release of Ins(1,4,5)P3 in HSWP cells (27), it is likely that Ca2+is being mobilized via Ins( 1,4,5)P3release of intracellular Ca" stores. A timecourse of BK-stimulated inositol trisphos-

phate production at room temperature reveals that InsP3 levels peak at about 1 min and then decline, but remain elevated for at least 10 min in the continued presence of BK (Table I). The data in Table I also show that removal of BK leads to a rapid decline in the levels of inositol trisphosphates with values rapidly returning to near basal levels (within 4 min of BK removal). When averaged over three experiments the Ins(1,4,5)P3 levels decline to 10 f 3% of the maximal stimulated level within 4 min following BK removal, while the Ins(1,4,5)P3 levels for cells in the continued presence of BK for 5 min was 83 f 10% of the maximal stimulated levels. Emptying the Intracellular ea2+Stores-Since we were interested ininvestigating the emptying and refilling of the BKsensitive intracellular Ca2+ stores, we first determined the conditions under which these stores could be emptied. The BK-sensitive Ca2+ stores can apparently be depleted by a single exposure to BK in Ca2+-free medium; subsequent exposure of the same cells to BK in Ca*+-freemedium produces no elevation of [Ca2+];(Fig. 3B). The inability of a second exposure to BK to elicit an elevation of [Ca'+], in Ca2+-free medium does not reflect a refractory state of the cells following the first BK treatment since a second BK response can be elicited within 2 min after terminating the first BK exposure when the cells are maintained in Ca2+-containing medium; in Ca2+-freemedium the response is not restored even as long as 30 min after terminating the first BK response (data not shown). Addition of Extracellular Ca2+ Produces an Elevation of [Ca2+];-When extracellular Ca2+ is added to unstimulated cells previously incubated in Ca2+-freemedium, little or no elevation of[Ca"], is observed (Fig. 3A). However, Ca2+ addition following exposure of the cells to BK frequently elicits a substantial elevation of [Ca'+], (Fig. 3B). This Ca2+induced elevation of [Ca"]; occurs even though BK has been removed by perfusing with BK-free medium* (see "Experimental Procedures"). The magnitude of the [Ca'+], elevation varies greatly from cell to cell, with some cells exhibiting only small elevations (200 nM) as the interval between BK membranes, we measured the effect of adding Ca2+back to removal and Ca2+addition is increa~ed.~ In order to assess Ca2+-depletedcells closer to thetime of depletion. When Ca2+ addition 4min after ionomycin removal was compared to Ca2+ what effect varying this intervalhason refilling the BKsensitive intracellular Ca2+ stores,we examined the response addition4 min after BK removal, a pattern of responses to BK after the refilling period (that is, after the transient almost identical to that shown in Fig. 5B was observed. That exposure to extracellular Ca2+).For each cell, the peak [Ca2+], is, the peak Ca2+ observed with Ca2+ addition 4 min after value of this response to BK (BK,) was normalized by dividing ionomycin removal was approximately 100 nM above basal by the peak of the initial response of that same cell to BK levels, whereas Ca2+addition 4 min after BK removal produced a peak Ca2+ of 800 nM above the basal level. This indicates that there is not a decline in Ca2+influx with time In two similar experiments, only five out of 116 cells exhibited peak [Ca2+Iielevations > 200 nM when Ca2+was added more than 10 after ionomycin depletion of ca2+stores as was observed in min after BK removal. In contrast, 23 out of 45 cells displayed peak [Ca'+], elevations > 200 nM when Ca" was added 1 min after BK removal.

That theratio BK,/BK, is consistently < 1indicates some desensitization to BK, as was found previously (24).

112

Bradykinin-induced [Ca2"li Changes in Human Fibroblasts

FIG. 3. Emptying and refilling of intracellular Ca2+stores. A, the trace shown is from an individualcell in a field of cells. Medium changes are indicated in the boxes at the bottom of the figure ("- Ca2+" indicates nominally Ca2+-free HEPES-HBSS, "+ Cas+" indicates HEPES-HBSS). Thistrace is representative of several similar experiments. B , the truce shown is from an individual cell in a field of cells. Medium changes are indicated in the boxes at the bottom of the figure.

- ca2t

A + co2+

- c02+

" -

iB

I

the case where the stores were depleted by addition of BK (Fig. 4B).These results indicate that depleting the intracellular Ca2+ stores is not sufficient to produce the large elevation of [Ca"], which is observed on Ca2+ addition afterBK treatment. Heterogeneity of Individual Cell Responses to B K Removal: Ca2' Entry but Not Elevated [Ca2+IiIs Necessary for Refilling the Intracellular Ca2+Stores-When BK is removed from cells during the plateau phase of elevated [Caz+]l, amarked heterogeneity in the [Ca2+Iiresponse is frequently observed among the cells in the field (Fig. 6). A fraction of the cells exhibits no plateau phase and [Ca2+Iiin thesecells has usually declined to near basal levels by the time that BK is removed (Fig. 6 A ) . Of the cells that do exhibit a plateau, some return rapidly to near basal [Ca2+Iiwhen BK is removed (Fig. 6 B ) ,while others maintain aplateau even after BK is removed (Fig. 6 C ) . Subsequent removal of extracellular Ca2+ resultsin a drop in [Ca2+I1 to basal levels in all of the cells in the field. It appears that, in some cells (Fig. 6C), BK-activated Ca2+entry inactivates very slowly when BK is removed, while in other cells (Fig. 6 B ) , the predominant BK-induced Ca2+entry component requires the continued presence of BK for activation. When the cells of Fig. 6 were re-exposed to BK in Ca2+-free medium, it was revealed that those cells which initially exhibit no plateau phase rarely respond to a second BK challenge (Fig. 6 A ) . On the other hand, cells which exhibit a plateau

I

I

I

after the initial exposure to BK do respond to the second challenge with a substantial elevation of [Ca2+Ii(Fig. 6 , B and C). This suggests that, in the cells exhibiting a second response to BK, the intracellular stores were either notdepleted or had refilled after the initial BK exposure. In a parallel experiment, BK and Ca2+were simultaneously removed during the plateau phase of the BK-induced [Ca'+], elevation (Fig. 7). Readdition of medium produced no elevation of [Ca2+Ii (in any of the 25 cells in thefield), though subsequent addition of BK with extracellular Ca2+produced a large elevation of [Ca2+Iiin many cells, indicating that these cells are still responsive to BK (Fig. 7). Theseresults suggest that the intracellular Ca2+stores had emptied and thatrefilling of the intracellular storesdoes not occur while BK is present. If this interpretation is extended to the responses illustrated in Fig. 6 , B and C, refilling of the Ca2+ stores must have occurred during the period when BK was absent but extracellular Ca2+ present. It is thus apparent that refilling occurred to thesame extent in cells which had returned to near basal [Ca2+]ilevels (Fig. 6 B ) as incells which maintained a very elevated [Ca"], level followingBK removal (Fig. 6 C ) . Nz2+ Blocks [Ca"], Elevation but Not Refilling of Intracellular Stores-We were interested in determiningwhether Putney's capacitative Ca2+ entry model might be applicable to fibroblasts. From the data of Fig. 4, it was apparent that the refilling of the stores was not related to themagnitude of

Bradykinin-induced [Ca2+IiChanges in Human Fibroblasts

113

A 5 m,n __ I

1

200 nM

1

-

d

+

2 6 K 10 ng/rnl (- Co2+)

II

1

2

3

-C2+

4

+C2+

5

-CJ+

Time Betwean BK Ramoval and Ca2+ Addition (min)

6 EK 10 ng/ml (- Ca2+)

3

4

5

6

.. . 1

2

3

1 2

3

I

5

6

FIG. 4. Elevation of [Ca2'Ii, but not refilling of intracellular Ca2' stores, varieswith the interval between BK removal and Ca2+addition. A , each trace shown is from an individual cell in a field of cells which was treated as indicated by the arrows labeled 16. Each trace represents adifferentcoverslip(see "Experimental Procedures"). The time interval between BK removal (arrow 3 ) and Ca" addition (arrow4 ) was different for each coverslip. B , mean data from thesameexperiment.Foreach cell inthe field, the basal [Ca'+], (average [Ca'+], value measured in Ca"-containing medium before the first BK exposure), was subtracted from the peak [Ca'+], value attained during theperiod when extracellular Ca" was present after BK treatment. The resulting values were averaged and plotted as the open circles. The peak [Ca'+], values attained during the 1st and 2ndexposures to BK (BK, and BK2, respectively) were also determined for each cell. The mean values of BK,/BK, are plotted as the open triangles. Error bars for both plots represent the standard error.

125

114

Bradykinin-induced[Ca2+IiChanges in H u m a n Fibroblasts

TABLE 111

BK but not depletwn of Ca2+pools increases Ca2+ entry:averages for

all cells studied Basal [Ca2+];values were determined by averaging 10 successive time points for each cell (measured in Ca2+-containingmedium before ionomycin treatment). Peak [Ca2+Iivalues were determined for each cell in the field during exposure to ionomycin, Ca2+addition after ionomycin (Ca",), BK, and Ca2+addition after BK (Ca2+J.Individual cell traces from this experiment are shown in Fig. 5B. [Ca2+],values in parentheses are not significantly different from each other, but each is significantly different from the underlined value (Student's t test. D < 0.0001). Cells responding

BK"

to

Cells not responding to B K ~ *I1

n 14 6 20 48.6 f 5.7 47.4 f 4.3 44.7 f 5.8 Basal [Ca2+], Peak ionomycin 1321 f 42 1345 f 35 1398 f 62 [Ca2+l, Peak Ca2+1 (163f 16) f 13 (151 f 20) 159 [Ca'+l, Peak BK [Ca2+Ii 1167 f 67d114 f 17d851 f 120 Peak Ca2+2 872 f 2 (142f 22)653 f 103 [Ca2+]; a [Ca2+],values (nM) are mean f S.E. for cells which responded to BK with a peak [Caz+lielevation > 600 nM. * [Ca2+Iivalues (nM) are mean f S.E. for cells which responded to BK with a peak [Ca2+Iielevation