The Agonist-sensitive Calcium Pool in the Pancreatic Acinar Cell

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 262,No. 35, Issue of December 15,pp. 16963-16968,1987 Printed in U.S.A.

The Agonist-sensitive Calcium Poolin the PancreaticAcinar Cell ACTIVATION OF PLASMA MEMBRANE Ca2+ INFLUX MECHANISM* (Received for publication, December 29, 1986)

Stephen J. PandolSP, Mari S.SchoeffieldS,Claus J. Fimmelll, and Shmuel Muallemn From the $Department of Medicine, Veterans Administration Medical Center, and University of California, San Diego, California 92161 and the VResearchInstitute, Cedars-Sinai Medical Center, and University of California, School of Medicine, Los Angeles, California 90048

an intracellular agonist-sensitive pool (1-4), that is probably The purposes of the present study were to investigate the characteristics and regulation of Ca" influx across the rough endoplasmic reticulum (6-8). The Ca2+release is the plasma membrane in pancreatic acini andto dem- due to anagonist-induced increase in thepermeability of the onstrate therole of this Ca" influx in the mechanism pool to Caz+(4-7) that is thought to be mediated by inositol of reloading of the agonist-sensitive Ca2+pool. In pan- 1,4,5-trisphosphate (5-11), the agonist-stimulated hydrolysis creatic acini, depleted of intracellar Ca2+ by stimula- product of phosphatidylinositol 4,5-bisphosphate (8-16). This tion with carbachol in the absence of extracellular Ca2+ release results in an increase in freecytosolic Caz+ Caz+,25 NM LaCl, inhibited the increase in free cyto- ([Ca"]J and subsequent efflux of the Ca2+from the cell (1solic Ca'+ ([ca"]~) and reloading of the agonist-sensi- 4, 17-20). In the continued presence of the agonist, [Caz+Ii tive pool that occurred with the addition of extracel- returns to theresting or near resting level (4,20), but thecell lular CaCl, to the medium. LaCl, also inhibited the does not respond to a second agonist with an increase in increase in cellular 4aCa2+ uptake that occurred during agonist stimulation andits termination butnot cellular [Ca2+],(4). At the termination of agonist stimulation, the ''Ca2+ uptake intounstimulated acini. In acini depleted ability of the agonist-sensitive pool to release Ca2+and inreturns (4). The return in responsiveness is of intracellular Cap+,increased cellularCa2+influx and crease [Ca2++Ii reloading of the agonist-sensitive pool occurred even dependent on extracellular Ca2+.Such findings suggest the if extracellular CaCI, was added 10 min after the ter- presence of a Ca2+entry mechanism in theplasma membrane that allows Ca2+influx during the reloading of the agonistmination of agonist action. Maximal reloading was independent of the extracellular Ca2+ concentration sensitive pool. In a variety of tissues, agonists increase the rate of cellular between 0.5 and 2.0 mM CaCl,. However, the time to maximal reloading was longer at lower extracellular Ca2+influx (2, 11, 21-28). However, neither the mechanism Caz+ concentrations. These resultsdemonstratea of the increased Ca2+influx nor its role in reloading of the plasma membrane Ca2+ influx mechanism in the pan- intracellular agonist-sensitive Caz+ pool are established (29, creatic acinar cell that is activated during cell stimu- 30). Thus, thepurpose of the present study was to investigate lation. This transportremains activated as long as the the characteristics and regulation of Ca2+influx across the agonist-sensitive pool is not completely loaded with plasma membrane and itsrole in the reloading of the agonistCa2+suggesting that theCa" influx mechanism is reg- sensitive pool with Ca2+in the pancreatic acinar cell. ulated by the quantity of Ca2+in the agonist-sensitive pool. The activation of this Ca" transport mechanism EXPERIMENTALPROCEDURES functions to allow Caz+influx across theplasma membrane andCaz+reloading of the agonist-sensitive pool. Materials Furthermore, these results suggest that duringreloadGuinea pigs(175-225 g) were obtained from Murphy Breeding ing Caa+crosses the plasma membrane into thecytosol Laboratories, Plainfield, IN. Hepes and bovine serum albumin (fracbefore entering the agonist-sensitive pool. tion V) were from Boehringer Mannheim. Soybean trypsin inhibitor,

Previous studies indicate that the stimulation of the pancreatic acinar cell with secretagogues such as carbachol or cholecystokinin (CCK)' results in arapid release of Ca2+from

* This work was supported by funds from the Research Service of the Veterans Administration and National Institutes of Health Grants DK-33010 (to S. U. P.) and DK 38938 (to S. M.). 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 18U.S.C. Section 1734 solelyto indicate this fact. I TOwhom correspondence should be addressed VA Medical Center, Gastroenterology Section (111D), 3350 La Jolla Village Dr., San Diego, CA 92161. The abbreviations used are: CCK, cholecystokinin; CCK-OP, synthetic COOH-terminal octapeptide of cholecystokinin; Hepes, 4(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid.

EGTA, carbachol, and atropine were fromSigma. Purified collagenase (type CLSPA) was from Cooper Biomedical, Inc., Malvern, PA. Ionomycin was from Behring Diagnostics. &CaC12 (4-50Ci/g calcium) was from Du Pont-New England Nuclear. Fura-2/AM wasfrom Molecular Probes, Junction City, OR. Synthetic COOH-terminal octapeptide of cholecystokinin (CCK-OP) was a gift from M.A. Ondetti, Squibb Institute for Medical Research, Princeton, NJ. The incubation solution contained 20 mM Hepes (pH 7.4), 120 mM NaCI, 5 mM KCl, 1 mM MgCl,, 10 mM sodium pyruvate, 10 mM ascorbate, 0.1% (w/v) bovine serum albumin, and 0.01% (w/v) soybean trypsin inhibitor.

Methods Tissue Preparation-Dispersed pancreatic acini were prepared from guinea pigs using the procedure previously published (31, 32). Measurement of Cellular *Ca2+-For these experiments acini from the pancreas of one animal were suspended in 30-50mlof the indicated solution and incubated with &CaC12at a specific activity of approximately 5 X los cpm/nmol Ca2+. Allincubations were a t 37 'C. Cellular '%a2+ was determined in 0.5-ml aliquots of cell suspension by addition of the aliquot to 10 mlof iced incubation solution

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Membrane Plasma

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containing 1.0 mM Lac&. Theacini were separated by centrifugation and then washed once with another 10 ml of the iced incubation solution containing 1 mM Lac& followed by 10 ml of iced incubation solution containing 1 mM EGTA by alternate centrifugation and resuspension. The acini were then dissolved by heating a t 60 'C in 0.5 ml of 1 M NaOH for 30 min and &CaZ+in the acini was measured using standard liquid scintillation counting techniques. Fura-2 L o a d i n g and Measurement of [Ca2+l~-Dispersed acini from one animal were suspended in 10 ml of incubation solution containing 0.5 mM CaClz and 2 PM fura-B/AM. The acini were incubated at 37 "C for 15 to 30 min. These fura-2-loaded acini were then washed and suspended in 60-80 ml of the indicated solution. Fluorescent measurements for [Caz+]idetermination were performed using a PerkinElmer spectrofluorometer. Settings were 340 nm excitation and 510 nm emission. The measurements were done in 2.0-ml aliquots of cell suspension that were continually stirred and maintained at 37 "C. [Ca2+];was calculated using the following formula (4,33-35).

All fluorescence values were measured relative tothe EGTAquenched signal ( i e . autofluorescence was subtracted from all values) determined as described below. F was the relative fluorescence measurement of the sample. To determine F- with fura-2-loaded acini, cells were perrneabilized with digitonin (100 pglml), 2.5 mM CaC12 was added, and relative fluorescence was measured. After determination of F-, 20.0 mM EGTA was added and the pH increased to 8.5 with NaOH to totally quench the fura-2 signal. The totally quenched signal was F-. RESULTS

Ca2+Influx bachol-stimulated acini did not result in an additional increase in [Ca2+]i,indicating that thepool was either depleted of Ca2+or desensitized to thesecond agonist (4).In the present study when the action of carbachol was terminated with atropine in Caz+-containingmedia, the ability of CCK-OP to cause a transient increase in [Ca2+Iireturned (Fig. 1, upper tracing). This "reloading" of the intracellular agonist-sensitive Ca2+pool was absolutely dependent on extracellular Ca2+in that itdid not occur in the absence of extracellular Ca2+(Fig. 1,lower tracing). In our first attempt to determine if the Ca2+reloading of the agonist-sensitive pool is dependent on activation of a plasma membrane Ca2+transport mechanism which allows Ca2+influx, we performed the experiments illustrated inFigs. 2-3. In the tracings illustrated inFig. 2 we demonstrated that the nonspecific Caz+ channel blocker, La3+ (36-38), had no effect on the CCK-OP-induced transient increase in [Caz+Ii, indicating that La3+had no effect on the ability of CCK-OP to release Ca2+from the agonist-sensitive pool into the cytosol. La3+did increase the rate at which the pancreatic acinar cell decreased [Ca2+Iito the resting level during continued agonist stimulation. For the upper tracing in Fig. 3, the agonist-sensitive Ca" pool was completely depleted by incubating acini with carbachol in the absence of extracellular Ca2+. Carbachol caused its typical transient increase in [Ca"], and then thecells decreased [Ca"], to values less than that observed at rest. Then atropine was added to terminate the action of carbachol. The addition of CaC1, to the extra-

In the experiment illustrated in Fig. 1, we determined the effect of extracellular Ca2+on theagonist-induced increase in [Ca2+Ii and the reloading of the agonist-sensitive Ca2+pool at the termination of agonist stimulation. Both in the presence or absence of extracellular Ca2+,carbachol induced a similar increase in [Ca2+Iifrom about 150 nM to 1 p ~ In. both conditions, the carbachol-induced increase in [Ca2+]iwas transient, but the rate of reduction of [Ca2+Ii afterthe carbacholinduced peak was morerapid in acini incubated in the absence of extracellular Ca2+.Because the carbachol-induced increase FIG. 2. Effect of LaCls on CCK-OP-induced changes in in peak [Ca2+Iiwas independent of medium Ca2+, these results [Cap+],.Fura-%loaded pancreatic acini were suspended in incubation indicated that the initial increase in [Ca2+Iiwas mainly due solution containing 2.0rnM CaCl2. Fluorescence was measured and to Ca2+release from intracellular stores. Our previous studies [Caz+liwas determined as described under "Experimental Proce(4)indicated that CCK-OP causes a similar transient extra- dures." CCK-OP and LaC1, were added where indicated. These tracings are of a single experiment representative of eight others. cellular Ca2+ independent increase in [Ca2+Ii.Furthermore, these studies also showed that addition of CCK-OP to car-

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lmin.

FIG. 1. Extracellular calcium dependence of reloading of the agonist-sensitive Cap+ poolin dispersed pancreatic acini. Fura-2-loaded pancreatic acini were suspended in either incubation solution containing 2.0 mM CaClz (upper tracing) or incubation solution containing 0.2 mM EGTA (lower tracing). Carbachol (Curb), atropine (Atro), and CCK-OP were added where indicated and [Caz+]iwas determined as described under "Experimental Procedures." These tracings arefrom a single experiment representative of at least ten others.

FIG. 3. The effect of La" on [Cap+],and reloading of the agonist-sensitive Ca2+pool. Fura-%loaded pancreatic acini were suspended in incubation solution containing 0.2 mM EGTA with no added CaClZ.Fluorescence was measured and [Ca2+]iwas determined as described under "Experimental Procedures." Carbachol (Curb), atropine (Atro),CaCl,, CCK-OP, and LaC13 were added where indicated. These tracings are of a single experiment representative of six others.

Plasma Membrane Ca2+Influx cellular medium after terminationof carbachol action resulted both in an increase in [Ca2+Iiand complete reloading of the agonist-sensitive pool because the subsequent addition of CCK-OP caused a maximal transient increase in [Ca*+],. As illustrated in the lower tracing in Fig. 3, we next determined the effect of low concentrations of La3+on [Ca2+Iiand reloading of the agonist-sensitive pool. In contrast to the experiment in the upper tracing, when 25 p~ excess LaC1, (225 PM LaC13 with 200 p~ EGTA in the extracellular medium) was added before the addition of atropine and CaC12, both the increase in [Ca2+Ji andthe CCK-OP-induced transient increase in [CaZ+liwere prevented. Because LaC13 prevented a rise in [Ca2+Ji in the unstimulated state with the addition of extracellular CaC12,these findings suggested that Lac& prevented Ca2+flux across the plasma membrane into the cytosol. The observation that LaCb prevented the CCKOP-induced transient increase in [Ca2+Jj suggested that it prevented reloading of the agonist-sensitive pool with Ca2+. Thus, the experiment suggested that the path of Ca2+ for reloading of the agonist-sensitive pool is from the extracellular space, across the plasma membrane into the cytosol, and then into theagonist-sensitive pool. The effect ofLaC13 on Ca2+ influx both during agonist stimulation and at its termination was next determined by measuring Y.!a2+ uptake into the acini (Figs. 4-5). In the experiment illustrated in Fig. 4, the agonist-sensitive pool of Caz+was depleted by a preincubation with carbachol in incubation solution containing no CaC12. With the addition of 2 mM "CaC12 to these carbachol-stimulated acini, &Ca2+uptake was greater than thatobserved in unstimulated acini. 30 pM Lac& had no effect on &Ca2' uptake intothe unstimulated acini butblocked T a 2 +uptake intothe carbachol-stimulated acini. In similar experiments with 2 mM CaC12present in the incubation solution during both the preincubation and the incubation with &CaZ+,30 p~ LaCl, also specifically blocked carbachol-induced increase in 46Ca2+ uptake (data notshown). As illustrated in Fig. 5, LaC1, also prevented the increase in "Ca" uptake that occurred when 2.0 mM %aC12 and atropine were added to carbachol-stimulated acini preincubated in Caz+-freeincubation solution. The finding was sim-

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y'

CONTROL

0

CARE-ATRO

0

5

10

n M - min. FIG. 5. The effect of La3+on cellular '%aa+uptake at the termination of carbachol action with atropine. Aliquota of dispersed pancreatic acini were suspended in incubation solution containing no CaC12and preincubated for 10 min with no agonist (0,O) or with 1mM carbachol (0,m). At time 0 of the illustrated experiment, 2.0 mM '6CaC12 was added to each aliquot with either no other addition (0).with 0.1 mM atropine (0,m) or with 30 p M Lac4 (a,m). Cellular %aZ+ was then measured at the indicated times as described under "Experimental Procedures" and expressed as a percent of that observed in control acini at 10 min of incubation. Results are the mean of four separate experiments. Vertical bars represent 1 f SE.

bb

264 155

I

CCK OP 25nM

-4-

I

Alro

am

I

CCK OP 2 . M

FIG. 6. The effect of the duration of incubation with La3+ on reloading of the agonist-sensitive pool with eaa+. Fura-2loaded pancreatic acini were suspended in incubation solution containing 2.0 mM CaC12. Fluorescence was measured and [Ca2+Iiwas determined as described under "Experimental Procedures." Carbachol (Curb),atropine (Atro),LaCb, and CCK-OP were added where indicated. These tracings are of one experiment representative of two others.

~

0

5

10

n w - min. FIG. 4. The effect of La3+on carbachol-stimulated cellular %aa+ uptake. Aliquota of dispersed pancreatic aciniwere preincubated for 10 min in incubation solution without added CaClz and either alone (0,a) or with 1 mM carbachol (Curb) (A, A). At time 0 of the illustrated experiment, 2.0 mM "CaClzwas added to each aliquot with either no further addition (0,A) or with the addition of 30 p~ LaCb (a,A). Cellular %az+ was then measured at theindicated times as described under "Experimental Procedures" and expressed as a percent of that observed in control aciniat 10 min of incubation. Results are the mean of four separate experiments. Vertical burs represent 1 f SE.

ilar in acini incubating in 2.0 mM CaC1, during the entire experiment (data not shown). Because LaCb blocked "Ca2+ uptake, only into acini during carbachol stimulation and at the termination of carbachol stimulation and because LaCb also prevented reloading of the agonist-sensitive pool during these periods (Fig. 3), these experiments indicated that the agonist-induced Ca2' influx and reloading of the agonistsensitive Ca2+pool depend on the activation of a La3+-sensitive plasma membrane Ca2+influx mechanism. To further demonstratethe effect of La3+on Ca2+reloading of the agonist-sensitive pool, we performed the experiment illustrated in Fig. 6. For these experiments, acini were stimulated with carbachol in the presence of extracellular CaC12. For the upper and middle tracings,atropine was added after the carbachol-stimulated transient increase in [Ca2+Iiwas complete. The extent of reloading of the agonist-sensitive

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Plasma Membrane Ca2+Influx

pool with Ca" was then determined by measuring the effect of a subsequent addition of CCK-OP on [Ca2+Ii.Addition of LaCL, 15 s after atropine addition, partially decreased the ability of CCK-OP to increase [Ca2+Ji(Fig. 6, upper tracing). In contrast, when Lac& was added before carbachol (Fig. 6, middle tracing), the ability of CCK-OP to increase [Ca"], after atropine addition was further inhibited. The inhibition was a function of the time of incubation with La3+ prior to the addition of atropine (Fig. 6, lower tracing). In the next series of experiments we determined whether activation of the plasma membrane Ca2+influx mechanism that occurred during agonist stimulation required the continuous presence of agonist. As demonstrated in Fig. 7, in acini treated with carbachol in the absence of extracellular Ca2+, the addition of CaC1, to the medium, 10 min after carbachol stimulation was terminated with atropine, resulted in an increase in resting [Ca2+Iiand complete reloading of the agonist-sensitive Ca" pool as indicated by the ability of CCKOPto cause a maximal transient [Ca2+Iiresponse (upper tracing). La3+blocked both the increase in [Ca2+Iiand reloading of the agonist-sensitive Ca" pool (lower tracing). These findings suggested that removal of agonist was not sufficient to inactivate the plasma membrane Ca2+influx mechanism. Despite the removal of agonist 10 min before the addition of

I

0 2 k

LaCl

225&

FIG. 7. Latereloadingoftheagonist-sensitivepoolwith Ca". Fura-2-loaded pancreatic acini were suspended in incubation solution containing 0.2 mM EGTA and no CaCIZ.Fluorescence was measured and [Caz+]i was determined as described under "Experimental Procedures." Carbachol (Curb),atropine, (Atro),CaCI2,Lac&, and CCK-OP were added where indicated. These tracings are of one experiment representative of five others.

5

10

15

5

10

15

Ca", normal reloading through a La3+-sensitiveCa2+transport occurred. To further establish that theplasma membrane Ca2+influx mechanism remains active despite the removal of agonist and as long as theagonist-sensitive Ca2+pool is depleted of Ca2+, 45CaZ+ uptake was measured.In theexperiment in Fig. 8, acini were pretreated with carbachol either in the presence of 2.0 mM extracellular Ca" ( A ) or absence of extracellular Ca2+ ( B ) . For condition A , 45Ca2+was added to the incubation media at thesame time carbachol stimulation was terminated with atropine. In condition B, 2.0 mM "CaCl2 was added to the incubation media 10 min after carbachol stimulation was terminated with atropine. In both cases 45Ca2+uptake into the carbachol/atropine-treated acini was greater than that observed in unstimulated acini. Because increased %a2+ uptake occurred despite the addition of atropine 10 min prior to the Ca2+addition and because the kinetics of 45Ca2+uptake were similar in Fig. 8, A and B, these results suggested that the plasma membrane Ca2+influx mechanism remained active despite the removal of the agonist. The rate of '5Ca2+uptake into carbachol- and atropine-treated acini returned to the control level within 5 min of incubation, which is the time required for maximal reloading of the pool with Ca". Thus, the plasma membrane Ca2+influx mechanism remained active until the intracellular pool reloaded with Ca2+.This suggested that the level ofCa" in the pool may somehow affect the activity of the plasma membrane Caz+influx mechanism. This phenomenon has also been described in the perfused liver preparation after stimulation with epinephrine (21) and in parotid gland slices after stimulation with carbachol (39). In another demonstration of the relationship between reloading of the agonist-sensitive pool with Ca2+and the plasma membrane Ca2+influx mechanism, we performed the experiment illustrated in Fig.9. This experiment demonstrated that, in incubation solution containing 2.0 mM CaC12,reloading of the agonist-sensitive pool with Ca2+was complete within 2.5 min as measured by the ability of CCK-OP to cause a maximal transient increase in [CaZ+li after termination of carbachol stimulation. When the CaC12 concentration in the extracellular medium was 0.5mM, maximal reloading could be obtained but required about 25 min. These findings indicated that the rate, but not the final extent of reloading of the

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TlME - min

FIG. 8. Cellular '%aa+ uptake several minutes after termination of carbachol stimulation. Aliquots of dispersed pancreatic acini were suspended in incubation solution containing either 2.0 mM CaClZ(A) or 0.2 mM EGTA (B) and preincubated for 10 min with either no agonist (0)or 1.0 mM carbachol (B).For A , either 0.1 mM atropine (B) or no agent (0)plus '%a2+ were added at time 0 of the illustrated experiment. For B, 0.1 mM atropine was added to the carbachol-stimulated acini (m) and the preincubation continued another 10 min. Then 2.0 mM %aC12 was added and T a n +uptake was measured as described under "Experimental Procedures." For both A and B, values for cellular &Can+were expressed as a percent of that observed in control acini at 15 min of incubation. Results are the mean f 1 S.E. of four experiments.

CAR

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4TRO

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C"0P

cu(-W

FIG. 9. Effect of extracellular Caa+ concentration on the pool. Furaduration ofCa" reloading of the agonist-sensitive 2-loaded pancreatic acini were suspended in incubation solution containing either 2.0 mM CaCh or 0.5 mM CaClZ. Fluorescence was measured and [Ca2+Iiwas determined as described under "Experimental Procedures." Carbachol (Curb),atropine (Atro),and CCK-OP were added where indicated. In the lower tracing the times indicate the duration between the addition of atropine and CCK-OP. These tracings are of one experiment representative of three others.

Plasma Membrane Ca2+Influx agonist-sensitive pool, wasdependent on the concentration of extracellular Ca2+. DISCUSSION

The combined findings in the present study indicate that there is a plasma membrane Ca2+influx mechanism in pancreatic acinar cells that is regulated by the quantity of Ca2+ in the agonist-sensitive pool. The activation of this Ca2+ transport mechanism results in increased cellular Ca2+influx, during and at the termination of agonist stimulation, and functions to reload the agonist-sensitive pool with Ca2+. Observations that demonstrate the existence of this Ca2+ influx mechanism are as follows. Reloading of the agonistsensitive pool with Ca2+is dependent on extracellular Ca2+. When the pool is depleted of Ca2+by carbachol stimulation in the absence of extracellular Ca2+,the complete reloading of the pool takes place with the subsequent addition of atropine and CaClz to the incubation medium. This reloading is prevented by relatively low concentrations of the nonselective Ca2+channel blocker, La3+.In acini depleted of intracellular Ca2+,La3+also inhibits the increase in cellular 45Ca2+uptake that occurs with the addition of 45Ca2+to the extracellular media at the termination of carbachol action with atropine. However, this concentration of La3+has no effect on 45Ca2+ uptake into controlacini. Other experiments in the present study indicate that this Ca2+transport mechanism is active during agonist stimulation as well as at the termination of agonist action. That is, La3+ blocks the increased cellular 45Ca2+uptake that occurs both during agonist stimulation and its termination. Furthermore, in experiments with extracellular Ca2+ present during carbachol stimulation, La3+ inhibits reloading of the agonistsensitive pool as measured by the ability of CCK-OP to increase [Ca2+Iiafter the carbachol action is terminated. The La3+inhibition depends on the incubation time of acini with La3+prior to the addition of atropine. Thus, it appears that the plasma membrane Ca2+ influx mechanism is activated during agonist stimulation andthat Ca2+is continually incorporated into the cell. Inhibition of the uptake mechanism by La3+ causes depletion of the intracellular agonist-sensitive pool during stimulationin Ca2+-containingmedia because the agonist continues to cause Ca2+ release while uptakeis blocked. Our study suggests that the agonist-sensitive pool reloads by Ca2+uptake from the cytoplasm and thatcytoplasmic Ca2+ is replenished with Ca2+by the plasma membrane Ca2+influx mechanism. This suggestion comes from the findings in the present study that La3+ prevents the increase in [Ca2+Ji as well as reloading of the agonist-sensitive pool that occurs with the addition of extracellular CaC12 to Ca2+-depleted acini. Furthermore, we have recently demonstrated that theagonistsensitive pool can take up Ca2+from the cytosol in gastric glands (40).That is, in the absence of extracellular Ca2+,the addition of atropine duringthe carbachol-induced increase in [Caz+liresults in an accelerated decrease in [Ca2+Ii.That decrease in [Ca2+Iiresults from Ca2+moving into theagonistsensitive pool because a subsequent addition of CCK-OP causes a transient increase in [Ca2+li.We have observed the same phenomenon in pancreatic acinar cells.2 These observations and those of the present study suggest that the cytoplasm is in the pathway of Ca2+movement from the extracellular space to theagonist-sensitive pool. Our study does not address the mechanism of activation or the nature of the plasma membrane Ca2+influx mechanism

* S. J. Pandol, M.S. Schoeffield, C. J. Fimmel, and S. Muallem, unpublished observations.

16967

in the pancreatic acinar cell. However, the findings indicate that theactivity of this mechanism is sensitive to the amount of Ca2+in the agonist-sensitive pool. Specifically, the influx mechanism remains activated when the pool is not replete with Ca2+,even when the agonist is removed. For example, in acini depleted of intracellular Ca2+by carbachol stimulation in the absence of extracellular Ca2+,the addition of CaC12 to the incubation media several minutes after carbachol stimulation is terminated with atropine resulted in accelerated Ca2+ influx across the plasma membrane intothe cytosol and reloading of the agonist-sensitive pool. Furthermore, decreasing extracellular Ca2+concentration results in prolongation but maximal reloading. The same phenomenon has been described in liver (21). This Ca2+ influx and reloading is inhibited by La3+.Thus, these results indicate that theplasma membrane Ca2+transport is probably activated by depletion of the agonist-sensitive pool of Ca2+and notby direct agonist activation. A recent study (41) in hepatocytes also suggests that changes in intracellular Ca2+ regulate a plasma membrane Ca2+influx mechanism. In this study, a decrease in [Ca2+Ii resulted in cellular influx of Ca2+. Such results led to the suggestion that a Ca2+channel could be regulated by a local decrease in the concentration of Ca2+on the inner surface of the plasma membrane (11,30, 41). These findings also led to the speculation (11, 30) that the reloading of the agonistsensitive pool could cause the local decrease in Ca2+at the inner surface of the plasma membrane, resulting in activation of a plasma membrane Ca2+influx mechanism. This model required a close anatomical relationship between the agonistsensitive pool and the plasma membrane and that agonistsensitive pool Ca2+ release and reuptake occur at separate sites. However, as discussed above, our findings suggest that the whole cytoplasmic pool of Ca2+ could be the source of Ca2+for reloading of the agonist-sensitive pool and that Ca2+ release and uptake into the pool can occur from the same cytosolic site of the pool. The mechanism of activation of the plasma membrane Ca2+ influx mechanism is presently not known. It is possible that a second messenger such asinositol 1,3,4,5-tetrakisphosphate (42)activates this Ca2+influx mechanism. Acknowledgments-We thank Diane Foster for preparation of the manuscript. S.M. thanks F. Bygrave for illuminating discussions. REFERENCES 1. Williams, J. A. (1980) Am. J. Physiol. 2 3 8 , G269-G279 2. Schulz, I. (1980) Am. J. Physiol. 2 3 9 , G335-G347 3. Gardner, J. D., and Jensen, R. T. (1981) inPhysiology of the Gastrointestinal Tract (Johnson, L. R., ed) pp. 831-871, Raven Press, New York 4. Pandol, S. J., Schoeffield, M. S., Sachs, G., and Muallem, S. (1985) J. Bwl. Chem. 260, 10081-10086 5. Streb, H., Irvine,R. F., Berridge, M. J., and Schulz, I. (1983) Nature 306,67-69 6. Streb, H., Bayerdorffer, E., Haase, W., Irvine, R. F., and Schulz, I. (1984) J. Membr. BioE. 8 1 , 241-253 7. Muallem, S., Schoeffield, M., Pandol, S., and Sachs, G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,4433-4437 8. Streb, H., Heslop, J. P., Irvine, R. F., Schulz, I., and Berridge, M. J. (1985) J. Biol. Chem. 260, 7309-7315 9. Berridge, M.J., and Irvine, R. F. (1984) Nature 3 1 2 , 315-321 10. Williamson, J. R., Cooper, R. H., Joseph, S. K., and Thomas, A. P. (1985) Am. J. Physiol. 248, C203-C216 11. Putney, J. W. (1986) Annu. Reu. Physiol. 48, 75-88 12. Rubin, R. P., Godfrey, P. P.,Chapman, D. A., and Putney, J. W. (1984) Biochem. J. 219,655-659 13. Powers, R. E., Saluja, A. K., Houlihan, M. J., and Steer, M. L. (1985) Biochem. Biophys. Res. Commun. 131, 284-288 14. Putney, J. W., Jr., Burgess, G. M., Halenda, S. P., McKinney, J.

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