Synchronized oscillation of Ca2+ entry and Ca2+ release in agonist ...

THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 266, No. 3, Issue of January 25. pp. 1363-1366,1991 Printed in U.S. A.

Communication Synchronized Oscillation of Ca2+ Entry and Ca2+Release in Agonist-stimulated AR42 J Cells* (Received for publication, July 9, 1990)

Peggy A. Loessberg, Hong Zhao, and Shmuel Muallem$ From the Departmentof Physiology, The University of Teras Southwestern Medical Center, Dallas, Texas 75235

Oscillation in [Ca2+Iiinduced by agonists has been described in many cell types and is thought to reflect Ca2+release from and uptake into internal stores. We measured [Ca2+Iiand Mn2+entry in single cells of the pancreatic acinar cell line AR42J loaded with Fura 2 to examine the behavior of Ca2+ influx across the plasma membrane (Ca2+entry) during agonist-evoked [Ca2+Ii oscillation. Addition of extracellular Ca2+ (C4:J to agonist-stimulated cells bathed in Ca2+-free medium resulted in a marked [Ca2+Iiincrease blocked by La3+.The use of Mn2+as a congener of Ca2+ to follow unidirectional Ca2+ movement reveals an oscillatory activation of Ca2+ entry by Ca2+-mobilizingagonists. The frequency at which Ca” entry oscillated matched the frequency of Ca” release from intracellular stores. Ca2+entry is activated after completion of Ca2+release and is inactivated within the time span of each [Ca2+Ii spike. These studies reveal a new aspect of [Ca2+]i oscillation in agonist-stimulated cells, that is the oscillatory activation of [Ca2+]ientry during [Ca2+]ioscillation.

the oscillation persists for some time in the absence of extracellular Ca2+ (Cdit), albeit at reduced frequency (13) and amplitude (7-12). The behavior of Ca2+influx across the plasma membrane (Ca2+entry) during [Ca2+];oscillation is not known. Studies in cell suspension show that activation of this pathway coincides (14-16) or lags behind (17, 18) Ca2+release from intracellular stores(IS).Furthermore, the pathway appears to remain continuously activated as long as the cells are stimulated and the IS are depleted of Ca2+(19-22). To understand the role and behavior of Ca2+ entryduring [Ca2+]ioscillation, we measured [Caz+li insingle AR42J cells and Fura2 fluorescence quench by Mn2+. We report here the synchronized oscillation of both Ca2+release from IS and Ca2+entry across the plasma membrane during agonist-triggered [Ca2+];oscillation. Ca2+entry is activated after completion of Ca2+release and is inactivated within the time span of each [Ca2+],spike. MATERIALSANDMETHODS

Cell Culture and Loading of Fura 2-AR42J cells were plated on glass cover slips and cultured at 37 “C in a humidified 95% air, 5% C02 atmosphere in Ham’s F-l2/Dulbecco’s modified Eagle’s medium supplemented with 14.3 mM NaHC03, 1.2 mM L-glutamine, 7.5% fetal calf serum, 0.1 mg/ml streptomycin, and 100 units/ml penicillin. On the day of the experiments, thecells were washed twice with serumfree tissue culture medium and then incubated with the same medium containing 2 p M Fura 2/acetoxymethyl ester for 45-60 min at 37 “ c . Finally, the cells were washed once and maintained in serum-free medium at room temperature until use. Fluorescence Recording-Cover slips with Fura 2-loaded cells attached to them were mounted in an open perfusion chamber. The perfusate volume in the chamber was adjusted to 0.3 ml and thecells were perfused at a flow rate of 10-12 ml/min and maintained at 37 “C. The standardperfusion medium contained (in mM): NaCll40; KC1 5; MgC12 1; HEPES 10 (pH 7.4 with NaOH); glucose 10; and bovine serum albumin 0.1%. This medium was supplemented with 1 Agonist-evoked changes in intracellular free Ca2+([Ca”]J’ mM CaC12 unless otherwise specified. The recording system included initiates and regulates a variety of cellular functions (1).In a Nikon Diaphot inverted microscope equipped with a CL Fluor 40X pancreatic acini, an increase in [Ca2+Iiis the primary activator oil immersion objective attached to a Photon Technology Internaof digestive enzymes and fluid secretion (2, 3).[Ca2+Ii in tional-Delta Scan l spectrofluorometer which provided dual waveresting and stimulatedcells is regulated by Ca2+-transporting length excitation light. The excitation light (340 and 380 nm for [CaZ+li measurementsor 360 and 380 nm for Mn2+ entry measurepathways located in the plasma and intracellular membranes ments), was selected by a spinning mirror anddirected to thesample (4-6). Sequential activation of these pathways results in a by a dichroic mirror. The emitted light of 510 nm wavelength was transient increase in [Ca2+Ii(1, 5, 6). Monitoring [Ca2+Ii in monitored by a photomultiplier tube type R955. The light monitored different single cells (7-9), including pancreatic acinar cells a t excitation wavelengths of 340 and 380 nm was used to calculate using a K d ratio of 340/380 and the ratiowas converted to [CaZ+li (10-12), shows that the agonist-evoked transient change in the O f 224 nM (26).

[Ca2+],is periodically repeated to give [Ca2+];oscillation. [Ca’+]; oscillation is thought to involve the repetitive Ca2+ release from and reuptake into intracellular stores (7-9), since

RESULTSANDDISCUSSION

Continuous stimulation of AR42J cells with carbachol resulted in a long lasting [Ca2+Iioscillation. In a first attempt DK 38938 and AR 39245 and by an Established Investigatorship to determine the role and properties of Ca2+ entry during Award from the American Heart Association. The costs of publication on [Ca2+Iiin cells of this article were defrayed in part by the payment of page charges. oscillation we tested the effect ofCd:, whose IS were depleted of Ca2+.Fig. 1 (upper panel) shows This article must therefore be hereby marked “aduertisement”in that in the absence of carbachol caused a rapid [Ca2+]i accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ T o whom correspondence and reprint requests should be ad- rise from a resting level of 105 (96 f 19) to 960 nM (915 f dressed Dept. of Physiology, University of Texas Southwestern Med- 103). In 12 out of 14 experiments, no oscillation was observed ical Ctr., 5323 Harry Hines Blvd., Dallas, T X 75235. Tel.: 214-688in the absence ofCd:,. In two experiments, oscillation with 2593. decreased amplitude occurred during the first 6 min of stim’ The abbreviationsused are: [Ca”],, free cytosolic Ca2+concentration; Ca%,, extracellular Caz+;IS, intracellular stores; HEPES, 4-(2- ulation. To demonstrate that theagonist-mobilizable IS were hydroxyethy1)-1-piperacineethanseulfonic acid; EGTA, [ethylene depleted of Ca2+by the first stimulation,the cell wasperfused bis(oxyethylinenitrilo)]tetraacetic acid. with Ca2+-freesolution in the absence of carbachol for 7.5 * This work was supported by National Institutesof Health Grants

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Oscillation of ea2+Entry 10 min

EGTA 0.2mM Ca2'lmM Carbachol 50 uM

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La3'5@M

10 min

llOOr

EGTA+ca2+ +Carbachol

entry is required to maintain the oscillation. It is therefore possible that Ca2+entry, and not only Ca2+release from IS, oscillates. To obtain direct evidence for this we followed Fura 2 fluorescence quench by Mn2+.Mn2+was shown to act as a congener of Ca2+ inpassive Ca2+pathways, thereby entering cells through an agonist-activated Ca2+entry pathways and quench Fura 2 fluorescence when excited at 380 nm (Ca2+sensitive) and 360 nm (isosbestic point, Ca2+-insensitive)(17, 25). Hence, measurements of Fura 2 fluorescence quenching in single cells which report the unidirectional Mn2+ influx across the plasma membrane, should reveal the behavior of Ca2+ entry during [Ca2+Iioscillation. Fig. 2, a and c, shows that Mn2+ entered relatively slowly and at a constant rate into an unstimulated cell. When a cell was stimulated with 50 ~ L Mcarbachol to initiate [Ca2+];oscillation, Mn2+ entry alternated between fastand slow rates with a frequency matching that of the [Ca2+];oscillation (Fig. 2, b and d ) . Thus, recording at 380 nm shows that [Ca"]; oscillated also in the presence of Mn2+at a frequency of 1.43 -+ 0.21 min" ( n= 4). Recording at 360 nm (to follow Mn2+ entry independent of [Ca2+];changes) showed the time course of change between the slow and fast rates of Mn2+entry, which occurred at a frequency of 1.40 f 0.34 min" ( n = 4). To obtain further evidence for the intimate relationship

"

FIG. 1. Effect of Ca% on carbachol-evoked [Ca2+Iiincrease. Single AR42J cells loaded with Fura 2 were continuously perfused (37 "C). The cells in the upper and lower panels were perfused with standard solution containing either 1 mM Ca2+or 0.2 mM EGTA and as indicated, 50 p~ carbachol, and 50 p~ La3+.

min. Under these conditions restimulation with carbachol had no effect on [Ca2+Ii.Similar results were obtained when the second stimulation was with other Ca2+-mobilizingagonists such as cholecystokinin octapeptide, substance P, and bombesin (not shown). Raising C&it to 1 mM now resulted in a rapid [Ca2+];increase to 635 nM (622 f 48) which was completed within 2.5 f 0.4 s ( n = 12), and wasfollowedby continuous [Ca2+Iioscillation. None of the voltage-operated Ca2+ channel blockers tested (nitrendipine and nisoldipine) prevented the marked initial [Ca2+];increase. La3+is the most effective blocker of agonist-activated Ca2+ influx (19).Accordingly, when the stimulated cell was perfused with Ca2+-free Ca:, in the presence of medium and then exposed to 1 mM 50 p~ La3+,the rapid increase in [Ca2+];was inhibited. Removal of La3+ wasfollowedby rapid [Ca2+];increase and sustained oscillation as expected from the resumed Ca2+ entry. Therefore, the Ca2+ responsible for the marked and rapid increase in [Ca2+Iiupon Cd;, addition must have been due to Ca2+influx across the plasma membrane. Fig. 1 (lower panel) also shows that thesurge of Ca2+across the plasma membrane required the presence of agonist. Thus, when the agonist wasremoved prior to addition of Cdi,, 100 to 240 nM (262 12, n = 5), [Ca2+];roseonlyfrom although the IS were depleted of Ca2+.During the incubation with C&:, the IS were reloaded with Ca2+(19-25), as evident from the [Ca2+Iiincrease and the oscillation observed on restimulation with carbachol. The marked increase in plasma membrane permeability to Ca2+observed during agonist-stimulated [Ca2+];oscillation (Fig. 1, upper panel) is unlikely to persist continuously since the cytosol willrapidly become saturated with Ca2+.Yet, Ca2+

'tr FIG. 2. Time course of Fura 2 fluorescence quench by Mn2+. Traces a, c and b, d were recorded from the same cell at excitation wavelengths of 380 (a,b)and 360 nm ( c , d ) ,respectively. Traces a and care from unstimulated cell. For traces 6 and d the cell was stimulated with 50 p~ carbachol and when [Caz+],oscillation became uniform (Fig. 1) the cell was exposed to standard medium containing 50 /LM carbachol, 1 m M CaC12,and 100 p~ Mn2+.Similar behavior was seen in four control and four stimulated cells. Additional experiments ( n = 6) showed similar oscillation of Caz+entry in cells stimulated with another Caz+-mobilizingagonist, JMV-180. Oscillation of Caz+entry always correlated with oscillation of [Ca2+Ii.

Oscillation of Ca2+Entry between oscillation of [Ca2+]iand Ca2+ entry, we measured the rateof Mn2+ entry into stimulated cells in the absenceof [Ca2+], oscillation (Fig. 3). This was achieved by perfusing AR42J cells with standardmedium t o which no Ca" or EGTA was added. Fura 2 fluorescence quench in control and stimulated cells wasmeasured in the presence of 25 p~ Mn2+.Mn2+ concentration was reduced to 25 p~ to better resolve the rate of fluorescence quench in the absence of Ca:., Stimulation with carbachol resulted in a single transient [Ca2+Ii increase with no furtheroscillation (Fig. 3b). Fluorescence quench due to Mn" monitored a t 380 and 360 nm followed smooth curves with no apparent alterationbetween fast andslow rates. Cell stimulation increased the rate of Mn2+ influxby 6.1 k 0.73fold (n = 4). As expected, the stimulatedMn'+ entry was not inhibited by the voltage-operatedCa2+channel blockers, but was inhibited by La3+(not shown)as was foundfor Caz+ entry (Fig. l a ) . Hence, when [Ca2+Iioscillation was prevented by preventing reloading of the IS, the Ca2+ entry pathway remained continuously activated. This experiment also demonstrates the abilityof the Mn2+ quench technique resolve to oscillations in Ca2+ entry (compareFig. 3 with Figs. 2 and 4) when Fura 2 fluorescence is recorded from single cells. While this work was in progress, a similar approach was used to study Ca2+ influx in single human umbilicalvein endothelial cells stimulated with histamine (22). In contrast to what is reported here, it was suggested that Ca'' entry remained continually activated even when [CaZ+lioscillated, although at least one endothelial cell showed oscillation of Ca2+ entry during [Caz+li oscillation (22). One possible explanation for the difficulty in observing oscillation of Ca'+ entry in endothelialas compared to AR42J cells (10 out of 10 cells) 2

Ca

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is that the experiments reported for endothelial cells were performed in the absence of Cd:, (22), while in our experiments Ca:, was present in the perfusion medium. In the absence ofCd:t, the amplitude of the oscillation decreased with time (22). The results in Fig. 3 suggest that theincrease in [Ca2+Ii during oscillation is probably responsible for the periodic inhibition of Ca2+ entry. Hence, it is possible that the reduced amplitude observed in the absence of Ca% precluded the periodical inhibition of Ca2+ entry(22). Additional effect of removing Cd:, is that theIS were never completely reloaded with Ca2+between the [Ca2+Iispikes. As was shown previously for several cell types (20, 21), including pancreatic acinar (19, 23, 24) andendothelial cells (22), Ca2+entry IS are not completely reremains activated as long as the loaded with Ca'+. This probably also hampered the ability to observe oscillation of Ca2+ entry in endothelial cells. The experiments with Mn2+clearly demonstrate the oscillation of CaZcentry during oscillation of [CaZ+li.Fig. 4 shows the relationshipbetween the time course of Ca" release from IS and Ca2+ entry. In this experiment the signals recorded at 380 and 360 nm excitation from the same stimulated cell were superimposed. It can be seen thatexposing the cell to MnZ+ when [Ca2++li was a t resting level was followed by slow quench ( i e . Mn2+ entry). The fast rate of Mn2+ entry commenced only when [Ca2+]; reached maximal level during each spike, and lasted for approximately 8.8 s. Hence, it appears that Ca2+ release from IS preceded the activation ofCa'+ entry and mostof the rapid Ca2+ entry occurred during theelevated phase of [Ca2+Ii.This accounts for the stair-shape signal at 380 nm excitation in the presenceof Mn'+ (Figs. 2 and 4). In this case Mn'+ entry is reflected in reduced levels of fluorescence between the [Ca2+Iispikes. It is also clear from the recording at 380 nm that during the time interval between spikes the rate of Mn2+ entrywas slow and corresponded to that measured in unstimulated cells. AR42J cells, agonist-evoked Ourresults show thatin [Ca2++Ii oscillation is due to the synchronizedoscillatory activation ofCa'+ release and Ca2+ entry. The oscillating Ca" entry contributes to the magnitude and in particular to the duration of [Ca2+], increase, and probably serves to reload the IS with Ca2+ during oscillation. The nature of the agonistactivated Ca'" entry pathway is notwell understood (1,4-6). MnZ+lOO pM 380nrn

I

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a

Carbachol 50 pM

1 fin

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Excitation 360 nm

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FIG. 3. Fura 2 fluorescence quench by Mn2+ inthe absence of [Ca2+Iioscillation. Traces a, c and b, d were recorded from the same cell a t excitation wavelength of 360 (a,b) and 380 nm (c,d), respectively. The cells were perfused with standard solution towhich no CaY+or EGTA were added. Traces a and c are from a resting cells. For traces b and d the cell was stimulated with50 p~ carbachol prior t o perfusion with Ca*+-free standard solution containing 50 pM carbachol and 25 p~ Mn2+.

1 min

I

FIG. 4. Time course of Ca2+release and Ca2+entry. The cell was stimulated with 50 p~ carbachol. Where indicated,100 p~ Mn2+ The signals recorded a t was includedinthe perfusionmedium. excitation wavelengths of 360 and 380 nm from the same cell are superimposed. Solid lines indicate initiation of [Ca"], increase and dashed lines indicate initiation ofCa2' entry. Note that entry lags behindCa2+ release. Similarrelationship between [Ca"Ii increase and Ca2+ entrywas observed in 10 experiments (four with carbachol and six with JMV-180).

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Oscillation of Ca2+Entry

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(1988) FASEB J. 2,3074-3082 mechanism by which [Ca2+Iican regulate Ca2+entryis 9. Berridge, M. J., and Irvine, R. F. (1989) Nature 3 4 1 , 197-205 through cyclical changes in membrane potential, thereby in- 10. Stuenkel, E. L.,Tsunoda, Y., and Williams, J. A. (1989) Biochem. Biophys. Res. Commun. 158,863-869 ducing a sequence of hyperpolarization during [CaZ+li increase11. Yule, D. I., and Gallacher, D.V. (1988) FEBS Lett 239,358-362 and repolarization after [Ca2+Ii reduction back to basallevels. 12. Parlong, W. J., Wollheim, C. B., and Bruzzone, R. (1988) FEBS This is considered unlikely since Caz+ entry remained actiLett. 2 4 2 , 79-84 vated even when [Ca2+];was kept a t or below resting levels 13. Rooney, T. A., Sass, E. J., and Thomas, A. P. (1989) J. Biol. Chem. 2 6 4 , 17131-17141 during continuous stimulation (Figs. 1 and 3) and, as judged 14. Sage, S. 0..and Rink. T. J. (1987) . . J. B i d . Chem. 262. 16364fromtheMn2+ signal, Ca2+entry was inactivatedwhen 16369 [Ca2+],was still elevated during each spike(Fig. 4).Hence, it 15. Merritt, J. E., and Rink, T. J. (1987) J. Biol. Chern. 2 6 2 , 4958seems that Ca2+-mediated changes in membrane potential are 4960 16. Merritt, J. E., and Rink, T. J. (1987) J. Biol. Chem. 262, 14912not the primary mechanism controlling activation and inac14916 tivation of Ca2+ entry during [Ca2+li oscillation. 17. Merritt, J. E.,Jacob, R., and Hallam, T.J. (1989) J. Biol. Chern. 2 6 4 , 1522-1527 A possible alternative mechanismwhich we favor is a direct 18. Sam. S. 0.. Merritt. J. E.. Hallam. T. J.. and Rink. T. J. (1989) . . regulation of Ca2+ entryby [Ca2+Ii. It was shown previously &chem.'J. 258,'923-926 that Ca2+releasefrom IS is required (19-25) and may be 19. Pandol. S. J.. Schoeffield. M. S.. Fimmel, C. J.. and Muallem, S. (1987) J. Biol. Chem. 2 6 2 , 16963-16968 sufficient (32) for activation of Ca2+ entry. The results in Figs. 3 and 4 are in agreement with this suggestion. Hence, 20. Merritt, J. E., and Rink, T. J. (1987) J. Biol. Chem. 262, 1736217368 Ca2+release from IS mediated by inositol 1,4,5-trisphosphate 21. Hughes, A. K., Takemura, H., and Putney, J. W., Jr. (1988) J. will result in activation of Ca2+ entry. When [Ca2+], reaches Biol. Chem. 2 6 3 , 10314-10319 a critical level due to the combined entry and release, Ca2+ 22. Jacob, R. (1990) J. Physiol. (Lond.) 421,55-77 S., Schoeffield, M. S., Fimmel, C. J., and Pandol, S. J. entry is inhibited ain time-dependent manner. The possibility 23. Muallem, (1988) Am. J.Physwl. 255, G221-C228 that high [Ca2+IiinhibitsCa2+entryissupported by the 24. Muallem, S., Schoeffield, M. S., Fimmel, C. J., and Pandol, S. J. (1988) Am. J. Physiol. 255, G2294235 findings that in the absence ofCz:t (and therefore [CaZ+li oscillation) Mn2+ entry into stimulatedcells was continuous 25. Muallem, S., Khademazad, M., and Sachs, G. (1989) J. Bid. Chem. 265.2011-2016 and did notoscillate (Fig. 3). The proposed inhibition of Ca2+ 26. Grynkiewicz, ~G.,Poenie, M., andTsien, R. Y. (1985) J. Biol. entry by [Ca2+Iiis reminiscent of the inhibition of voltageChem. 260,3440-3450 operated Ca2+ channels by [Caz+li(33). Feedback inhibition 27. Irvine. R. F.. and Moor. R. M. (1986) Biochern J. 240.917-920 of Ca2+release as demonstrated in several cells (34-37) in- 28. Morris, A. P., Gallacher, D. V., Irvine, R. F., and Petersen, 0. H. (1987) Nature 330, 653-655 cluding AR42J cells,' and inhibition of Ca2+ entryby [Ca'+]i 29. Changya, L.,Gallacher, D. V., Irvine, R. F., Potter, B. V. L., and will allow Ca2+ uptakeback into the IS. Periodical repeats of Petersen, 0. H.(1989) J. Membr. Biol. 109,85-93 this sequence of events will result in [Ca"]i oscillation. Re- 30. Penner, R., Matthews, G., and Neher, E. (1988) Nature 3 3 4 , 499-504 gardless of the regulatory mechanism, the present studies 31. Snyder, P. M., Krause, K.-H., and Welsh, M. J. (1988) J . Bid. reveal a new, unsuspected,aspect of [Ca2+];oscillation in Chem. 2 6 3 , 11048-11051 agonist-stimulated AR42J cells. This is the oscillatory acti- 32. Takemura, H., Hughes, A. R., Thastrup, O., and Putney, J. W., Jr. (1989) J. Biol. Chem. 264,12266-12271 vation of Ca2+ entry during[Ca2+Ii oscillation. '

Acknowledgment-We thank Constance Johnson for excellent administrative assistance.

Zhao, H., and Muallem, S. (1990) J . Biol. Chem. 2 6 5 , 2141921422.

33. Tsien, R. W. (1983) Annu. Reu. Physiol. 45,341-358 34. Cheuch, S.-H., and Gill, D.L. (1986) J. Biol. Chem. 261, 1388313886 35. Jean, T., and Klee, C. B. (1986) J. Biol. Chem. 261,16414-16420 36. Suppatopone, S., Worley, P. F., Baraban, J. M., and Snyder, S. H.(1988) J. Biol. Chem. 2 6 3 , 1530-1534 37, Parker, I., and Ivorra, I. (1990) Proc. Natl. Acad. Sci. U.S. A. 87,260-264