Relationship between Secretagogue-induced Ca2' Release and ...

Vol. 260, No. 12, h u e of June 25. pp. 7309-7315,1985 Printed in U S A .

THEJOURNAL OF &OLOGICAL CHEMIsTRV 0 1985by The Ametican Society of Biological Chemists. Inc.

Relationship between Secretagogue-induced Ca2' Release and Inositol Polyphosphate Production inPermeabilized Pancreatic Acinar Cells* (Received for publication, November 15,1984)

Hanspeter Strebl, JohnP. Hesloprj, Robin F. Irvinell, Irene Schulzl, and Michael J. Berridgerj From the $Max-Planck-lnstitut fiir Biophysik, Kennedyallee 70,6000 Frankfurt (Main) 70, Federal Republic of Germany, the nDepartment of Biochemistry, AFRC Institute of Animal Physiology, Babraham, Cambridge CB2 4AT,United Kingdom, and the SAFRC Unit of Insect Neurophysiology and Pharmacology, Department of Zoology, University of Cambridge, Cambridge C32 3EJ, United Kingdom

We have previously shown that inositol trisphosphate (IPS)releases Cas+ from a nonmitochondrial pool of permeabilized rat pancreatic acinar cells (Streb, H., Irvine, R. F., Berridge, M. J., and Schulz, I. (1984) Nature 306,67-69). This pool was later identified as endoplasmic reticulum (Streb, H., Bayerdorffer, E., Haase, W., Irvine, R. F., and Schulz, I. (1984) J. Membr. Bioi. 81, 24 1-253). As IPS is produced by hydrolysis of phosphatidylinositol bisphosphate on activation of many "Ca2+-mobilizingreceptors," our observation supported the proposal that IPSfunctions as a second messenger to release Caa+ from the endoplasmic reticulum. We have hereused the same preparationof permeabilked acinar cells to study the relationship of secretagogue-induced Ca" release and IPSproduction. We show that: 1) secretagogue-induced Ca" release in permeabilized cells is accompanied by a parallel production of inositol trisphosphate. 2) When the secretagogue-induced increase in intracellular free Ca2+ concentration was abolished by ethylene glycol bis(l8aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering, secretagogue-induced IPS production was unimpaired. 3) When secretagogue-induced IPSproduction was reduced by inhibiting phospholipase C with neomycin, secretagogue-induced ea2' release was also abolished. 4) When the IPS breakdown was reduced either by lowering thefree M e + concentration of the incubation medium or by adding 2.3-diphosphoglyceric acid, the rise in IPS and the release o f Ca" induced by secretagogues were both increased. These results further support the role of I P S as a second messenger to induce Ca2+mobilization.

Release of digestive enzymes from exocrine pancreas in response to secretagogues isstimulated by an increase in intracellular free Ca2+concentration (33). This calcium signai is initially derived from intracellular stores andis maintained by increased Ca2+influx into the cell during sustained secretion (33).Activation of receptors that induce their physiological effects via an increase in intracellular free Ca" concentration isusually accompanied by hydrolysis of inositol phospholipids. Michell (24) has proposed that this "phospholipid effect," which was first described by Hokin and Hokin (17), might play some rolein calcium signalling. Almost all possible *This workwas supported by Grant Schu 429/2-1 from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

products of this hydrolysis have been suggestedto play a role in Ca2+ mobilization (5, 241, but the proposal that has attracted most attention recently is the suggestion that inositol trisphosphate (IPa'), which is formed by receptor-coupled hydrolysis of phosphatidylinositol bisphosphate, is the second messenger responsible for mobilizing intracellular calcium (6, 7). Experimental support for this proposal was obtained using permeabilized pancreatic acinar cells where IP3 was shown to stimulate the release of Ca2+from a nonmitochondrial calcium pool (35). This observation has now been confirmed in a variety of tissues such as liver, insulinoma celis, and smooth muscle(10, 20,29, 37). By studying the effect of IP3 on isolated subcellular fractions from exocrine pancreas, the nonmitochondrial store was identified as endoplasmic reticulum (34). These observations therefore suggested that IP, functions as a second messengerof Ca2+-mobilizingreceptors to release Ca2+ fromthe endoplasmic reticulum. In order to further test this hypothesis, we have used the same preparation of permeabilizedcellswhich retainthe ability to respond to secretagogues by releasing Ca2+ from intracellular stores (36). If our hypothesis is correct, they should still be able to produce IPS on receptor activation, which would then be released to the incubation medium. If so, permeabilized cells would provide a powerful system to further analyze the re~ationship of secretagogue-inducedCa2+ release and IPS production, as the cell interior is accessible and Ca2+ and phospholipid metabolism can be influenced specifically. In the datadescribed below we showthat thesecretagogueinduced Ca" release from permeabilized cells is indeed accompanied by formation of IPS. When the amount of IP3 formed is decreased or increased by manipulation of the incubation conditions, Ca2+release is changed in parallel. In contrast, when the increase in intracellular Ca2+concentration is abolished, IP3 production is unimpaired. Our results thus provide further evidence that secretagogue-induced Ca2+ release is mediated by inositol trisphosphate. EXPERIMENTAL PRO~EDURES

Materials-Reagents were obtained from the following sources: KsATP, phosphocreatine (sodium salt), 2,3-diphosphoglyceric acid (sodium salt), andneomycin sulfate from Sigma; creatine kinase and soybean trypsin inhibitor from Boehringer Mannheim; and bovine serum albumin (lyophilized) from Serva (Heidelberg, Germany). ColThe abbreviations used are: IP3,inositol trisphosphate; Hepes, 4(2-hydroxyethyl)-l-piperazineethanesulfonic acid; IP2,inositol bisphosphate; CCK-OP, cholecystokinin octapeptide; IP,,inositol monophosphate; EGTA, ethylene glycol bis(P-aminoethyl ether)diphosphoglyceric acid; PIPz, N,N,N',N'-tetraacetic acid; 2,3-PGA, phosphatidylinositol 4,5-bisphosphate.

7309

Ca2+Release and IP3 Production

7310

lagenase (from clostridium histolyticum) type I11 was purchased from Worthington and the synthetic octapeptide of cholecystokinin from Paesel (Frankfurt, Germany). The Phadebas test kit for amylase assay was obtained from Pharmacia (Freiburg, Germany) and myo[2-3H]inositolfrom New England Nuclear. All other reagents were of analytical grade. Ca’+-selective electrode membranes containing the neutral carrier N,N’-di((ll-ethoxycarbonyl)undecyl)-N,N”4,5-tetramethyl-3,6dioxaoctane amide were purchased from Glasbliiserei W. Moller, Zurich, Switzerland. “P-labeled inositol-1,4,5-trisphosphatewas prepared from human erythrocytes essentially by the method of Downes et al. (14) as modified by Irvine et d. (18)and Burgess et aL (10). Preparation of Isolated Acinar Cells and Prelabeling of L@&Isolated acinar cells from rat pancreas (approximately 200 g of male Wistar rats) were prepared by a collagenase digestion method accord/”c-* ing to Amsterdam and Jamieson (4) with slight modifications (36). The plasma membranes of isolated cells were permeabilized by wash8 . ; ing the cells twice with a nominally Caz+-freesolution containing (in millimolars): KCI, 135;Hepes, 10;MgClp, 1; 0.1 m g / d trypsin inhibitor, pH 7.4. The contaminating Ca2+concentration of this solution . treatment in Ca2+-free solution was approximately 2-3 p ~ This resulted in an increase in trypan blue uptake, as an indicator of cell o-o-e-” leakiness, from below 10% before washing to 70-90% within 30 min CPm lP’ 1500 I I I I 1 I I I after washing (36).Cells were stored as a concentrated suspension on 0 10 20 30 ice in the same medium until measurement (up to 4 h). Time [min] Inositol phospholipids were prelabeled by addition of 100 pCi/ml my0-[2-~H]inositol(approximately 6 p M ) during the last hour of FIG.1. Cholecystokininoctapeptide-inducedCa4+release collagenase digestion. Cells were then washed three times with the and formation of inositol phosphates in permeabilized pansolution described above supplemented with 1 mM cold myo-inositol. creatic acinar cells. Permeabilized cells (4.4mg ofprotein/ml) were Incubation Procedure and Determination of Inositol Phosphatesincubated under standardconditions as described under “ExperimenIsolated cells were incubated a t 25 “C in 3 ml of a solution containing tal Procedures.” Where indicated, calcium chloride (20 nmol) or (in millimolars): KCI, 110; MgClp, 6; KpATP, 5; creatine phosphate cholecystokinin octapeptide to a final concentration of 3 p~ were (sodium salt), 10; K+-succinate, 5;K+-pyruvate, 5;creatine kinase, 8 added. Upper curve: medium free-Ca2+concentration measured with units/ml, Hepes, 25, pH 7.4. The free M e concentration of this the Ca’+-specific electrode. Lower three curves: radioactivity resolution was calculated as 1.3 mM (36).In some experiments the total covered as inositol trisphosphate,).( inositol bisphosphate (A),and M e concentration was reduced to 3.9 mM (free M$+ concentration inositol monophosphate (0)from 2004 medium samples after sep= 0.3 mM) which will be referred to as“low M P . ” T h esolution was aration on Dowex anion exchange columns. Typical for five esperistirred and gassed continuously with 100% oxygen. The medium-free ments. Ca2+concentration was measured continuously with a Caz+ specific macroelectrode (neutral carrier ETH 1001). Electrodes were ma& tration was rapidly re-established following addition of a small and calibrated as described previously (1, 36). For analysis of inositol phosphates, 200-4aliquots were removed pulse of calcium (Fig. 1, upper curve). Stimulation with the a t given time points during incubation. The samples were rapidly secretagogue cholecystokinin resulted in Ca” release from the mixed with the same volume of ice-cold 20% (w/v) trichloroacetic cells to themedium followed byreuptake (Fig. 1,upper curve). acid and stored on ice. At the end of the experiment, the samples Parallel measurements of the inositol phosphates showed that were centrifuged for 4 min a t 12,000 rpm in an Eppendorf 3200 this release of Ca2+was associated with a rapid production of centrifuge and the supernatant removed. Trichloroacetic acid was removed from the supernatantsby extracting eachsample three times ,H-labeled inositol trisphosphate (Fig. 1).A parallel increase with approximately 1 ml of water-saturated diethyl ether. Inositol in thelevel of inositol bisphosphate (IP,), possibly formedby phosphates were separated on Dowex anion exchange columns as hydrolysis of IPS, wasalso observed (Fig. 1). In five out of six described previously (8). separate experiments performed under the conditions of Fig. In some experiments we analyzed the lipids which remained in the 1, IP3 increased by 106 k 35% S.E.(range 62-245%) above pellet after removing the water-soluble metabolites. The pellet was basal levels and IP, by 64 k 15% (range 23-110%). In one extracted three times with 200 pl of chloroform/methanol/concentrated HCI (200/100/1).After drying, the lipids were deacylated to experiment there was no Ca2+ release and no measurable the corresponding glycero derivatives which werethen separated from increase in IP3production (see below). Both CCK-OP-induced Ca2+release and formation of inositol phosphates was aboleach other as described previously (6). Other Analytical Procedures-For determination of protein, sam- ished by the CCK-OP antagonist dbc GMP(3 mM, one experples were precipitated in 10% (w/v) trichloroacetic acid and dissolved iment). in 1M NaOH. Protein content was then measured according to Lowry The time course of inositol phosphate formation was vari(22)using bovine serum albumin as standard. able between different cell preparations. In the five experiAmylase was determined by measuring the hydrolysis of a watersoluble cross-linked starch polymer carrying a blue dye (Phadebas ments performed under the conditions of Fig. 1, IP3 decreased at later time points in four experiments (e.g. controls in Figs. test kit).

25001

RESULTS

Simultaneous Secretagogue-induced Inositol Polyphosphate Production and ea2+Release As shown previously (36), when permeabilized cells were added to theincubation medium, the free CaZ+concentration decreased due to calcium uptake into mitochondria and endoplasmic reticulum. A steady state was reached at about 0.4 FM free Ca2+(Fig. 1, upper curue). This steady-state concen-

6 and 7) while a steady state was reached in one experiment (Fig. 1). Similarly, IP, decreased in two of five experiments (Fig. 1) and approached a steady state in three (e.g. control in Fig. 6). The secretagogue-induced increase in free Ca2+ concentration was always reversed before the inositol phosphates had returned to basal levels. This might be explained by the fact that the action of IP, in permeabilized cells will not only be terminated by IP3hydrolysis, but also by loss of IP, to themedium. This will result in a dilution of about 100fold, thus terminating theeffect.

Ca2' Release and IP3 Production

7311

There was verylittle evidence of any secretagogue-induced CCK-OP-induced Ca2+release decreased during storage on change in the level of inositol monophosphate (IP,) (Fig. 1) ice, there was a parallel decrease of the amount of inositol which only in a few experiments could be discriminated from phosphates produced on stimulation. There was a good corscatter (e.g. Fig. 2). In all experiments performed, no signifi- relation between CCK-OP-induced Ca2+release and produccant changes in the concentration of glycerophosphoinositol tion of either IP, or IP2 in three different cell preparations (Fig. 3). occurred. In subsequent experiments we tried specifically to alter Similar results were obtained when cells were stimulated with carbamylcholine (carbachol) (Fig. 2). In general, both either the increase in Ca2+concentration or the amount of Ca2+release and inositol polyphosphate production tended to inositol phosphates produced,in order to establish more be smaller with carbachol (50 p ~ than ) with a comparably f i i l y the way in which these two processes are related to high dose of CCK-OP (3 PM) (compare also control curve in each other. Fig. 7). In the experiment showninFig. 2, therefore, IP3 Inhibition of Intracellular Ca2+Increase formation was enhanced by reducingthe free Me concentration and by adding 2,3-diphosphoglyceric acid.This resulted In order to examine whether or not secretagogue-induced in increased accumulation of inositol phosphates andinIP3 production was due to or influenced by the parallel increased Ca2+release (see below). crease in free Caz+concentration of the incubation medium, The amount of CCK-OP-induced Ca2+ releaseand change the latterwas bufferedwith EGTA. As the plasma membrane in inositol phosphate production showed considerable scatter of the cells is permeable, EGTA will enter into the cells and between different cell preparations. In some experiments, reduce any changes in Ca2+concentration by about 100-fold. secretagogue-induced Ca2+ release also tended to decrease The free Ca2+concentration of the EGTA-buffered solution progressively during storage on ice. When comparing different was adjusted to approximately 0.2 pM, a concentration equivconcentratreatments, therefore, it was necessary alwaysto use the same alent, or at least very similar, to the cytosolic Ca2+ preparation with control responses carried out either before tion in intact cells (26, 27, 36). Thus IP3formation could be or after each experimental treatment. Despite this gradual investigated not only at the physiological intracellular free deterioration of the cell preparations, there was always a good Ca2+concentration, but also in the absence of any secretacorrelation of secretagogue-induced Ca2+release and produc- gogue-inducedchanges in Ca2+concentration. The usual CCK-OP-induced increase in the medium-free tion of inositol polyphosphates. In one out of six preparations that were analyzed under the conditions of Fig. 1, there was Ca2+concentration was abolished in the presence of EGTA no CCK-OP-induced Ca2+ release. In parallel, there was no measurable formation of inositol phosphates. When cells from LO-) this preparation were incubated under conditions which promote the formation of IP3 (low M e ,see below), there was a 0.8measurable release of Ca2+indicating that thelack of responsiveness was not due to receptor damage. Similarly, when 0.6-

P

,.I?

o.4: 0.2

0

I

0

I

r a 0.99

1

I

I

0.2 0.4 0.6 0.8 1.0 IP3 production

1.0

-t

1

1

0

1

1

10

1

1

1

I

I

-

20 30 Time [min]

FIG.2. Carbamylcholine-induced Cas* release and formation of inositol phosphates in permeabilized pancreatic cells. Permeabilized cells (3.2 mg of protein/ml) were incubated at low M$+ concentration as described under "Experimental Procedures." In addition, the incubation medium contained 1 mM 2,3diphosphoglyceric acid. Where indicated, calcium chloride (20 nmol) or carbamylcholine (carbachol)to a final concentration of 50 p~ were added (IP3,0, IPz,A; IPI,0).One experiment.

I

I

1

-

0.2 0.4 0.6 0.8 1.0 production FIG. 3. Correlation of cholecystokinin octapeptide-induced Cas+release and formation of inositol phosphates during cell aging. Cholecystokinin octapeptide-induced Ca2* release and formation of inositol phosphates was determined as shown in Fig. 1. For each cell preparation, data from the first incubation obtained directly acinar after the cells had been prepared (open symbds) was compared with that of the last incubation after cells had been stored on ice for 2-3 h (closed symbols). Different symbols refer to different cell preparations. Ftesults are expressed as fraction of the total Cas+ release or increase in inositol trisphosphate or inositol bisphosphate measured in both incubations from the same cell preparation. r = correlation coefficient.

0

ea2+Release and IP3 Production

7312

control

(upper curue), whereas the formation of inositol phosphates was unaltered (Fig. 4). This result indicates that the CCKOP-induced production of inositol phosphates in the permeabilized cells is not due to a change in free Ca2+concentration.

neomycine,lmM CCK-OP

CCK-OP

Inhibition of Phospholipase C Neomycin has been reported to inhibit the polyphosphoinositide phosphodiesterase of erythrocyte membranes and thus prevent the formation of IP, from phosphatidylinositol 4,5-bisphosphate (13). Neomycin (1 mM) completely blocked CCK-OP-induced IP3 production and calcium-release in permeabilized pancreatic acinar cells (Fig. 5). The above result is probably not due to some deleterious effect on the cholecystokinin receptor because neomycin did not alter the ability of CCK-OP to stimulate amylase release in intact cells (one experiment, data not shown). Inhibition of IPSHydrolysis

1

-6.44

500

'PZ

cprn 400 -:4.j"

300

;zrn

200

3000 20003 1000

o-#?-+%

,

, 10 min

.

-

oo.~+"o~ 10 min

If CCK-OP-induced Ca2+release is mediated by IP3, inhiFIG. 5. Comparison of cholecystokinin octapeptide-induced bition of IP, hydrolysis should result in increased Ca" release. Ca" release and formation of inositol phosphates in the presLithium-Li+ has been known for some time to inhibit the ence or absence of neomycin. Permeabilized cells were incubated as described under "Experimental Procedures" (low Me concentrahydrolysis of IP, to form free inositol and Pi (16). Recent tion) with and without 1 mM neomycin. Where indicated, cholecysreports indicate, that athigher concentrations Li+ also inhib- tokinin octapeptide was added to a final concentration of 3 pM. Bars its the hydrolysis of IPS and IPz(32, 38). We therefore tested in upper curues: change in free Ca2+concentration due to addition of Li' (10 mM) on CCK-OP-induced Ca" release and formation 20 nmol of Ca2+determined as shown in Figs. 1 and 2. (IPB,0 IP,, of inositol phosphates in the permeabilized cells. There were A , IP1, 0).Typical for two experiments. no significant effects of Li' either on the release of calcium or on the formation of IP3 (datanot shown). The basal levels Ca2+ release and production of inositol polyphosphates in of IP1 were increased, but as in the absence of Li' there was permeabilized cells. 2,3-DiphosphoglycericAcid-In erythrocyte membranes, no significant accumulation of IP,. Li' therefore did not 2,3-diphosphoglyceric acid (2,3-PGA) inhibits the inositol appear a very useful tool to correlate secretagogue-induced trisphosphate phosphomonoesterase competitively, with a Ki of approximately 0.35 mM (14). When applied to permeabilcontrol EGTA, 1mM ized pancreatic cells at 1mM, 2,3-PGA had little effect on the amount of calcium released at early time periods but it did CCK-OP CCK-OP slow down the subsequent reuptake process (Fig. 6). Inkeeping with these observations on calcium release, 2,3-PGA had little effect on the initial rate and extentof IP3 formation but it did appear to stabilize the IPS level. While the control response to CCK-OP showed a distinct maximum, the increase in IP, in the presence of 2,3-PGA went on increasing. Thus, at later time points,IP3 concentration was about double - 6.3 the control in the presence of 2,3-PGA. IP2 concentration tended to rise slower in thepresence of 2,3-PGA compared to the control, as would be expected from an inhibition of IP3 hydrolysis. At 2.5 mM 2,3-PGA there was a clear increase in the amount of Ca2+released (Fig. 7). This became most obvious whenthe secretagogue-induced Ca2+release was small in the control (Fig. 7). Note that the reuptake of calcium was considerably 200' delayed which would be consistent with an inhibitory action of 2,3-diglyceric acid on IP3 hydrolysis. Magnesium-The inositol-1,4,5-trisphosphate phosphommin %z? monoesterase of erythrocyte membranes shows a strong deFIG. 4. Comparison of cholecystokinin octapeptide-induced pendence on the free Mg2+ concentration: enzyme activity at formation of inositol phosphates in the presence or absence of 0.1 p M free Mg' was only about 20% that at 1 mM (14). EGTA.Permeabilized cells (2.6 mg of protein/ml) were incubated as described under "Experimental Procedures" (low M e concentration) Reducing the free Mg2' concentration of the medium bathing with and without 1 mM EGTA. The free Ca2+concentration in the permeabilized cells from 1.3 to 0.3 mM resulted in a doubling presence of EGTA was adjusted to about 0.2 p~ by addition of CaClz of the amount of IP, formed in response to CCK-OP (Fig. 9). before cells were added. Where indicated, cholecystokinin octapeptide At the low Mg?+ concentration, the level of IPSremained high was added to a final concentrationof 3 pM. Bar in upper trace on the throughout the experiment while it decreased at later time left: change in medium free-Ca2+concentration due to addition of 20 points at the higher Mg?+ concentration. In parallel to the nmol of CaZ+determined as shown in Figs. 1 and 2. Addition of 10 times this amount (200 nmol) did not result in any measurable change pronounced increase of IP, forpation, CCK-OP-induced Ca2+ release was about doubled. To assess the amount ofCa" in free Ca2+concentration in the presence of 1 mM EGTA (IPB,0 released under the two conditions it is important to note that IP,, A; IP,, 0).Typical for two experiments.

-7

'

'

Ca2+Release and IF3Production control CCK-OP

2,3-phospho-glycsrate 1mM

73 13 Mg2* , 1.3mM CCK-OP

CCK-OP

Mg"

, 0.3mM

CCK-OP

1

-6.2'

100 J

loo01

96x2

w

FIG. 6. Comparison of cholecystokinin octapeptide-induced CaZ+release and formationof inositol phosphates in the presence or absence of 2.3-diphosphoglyceric acid. Permeabilized cells (3.6 mg of protein/ml) were incubated under standard conditions as described under "Experimental Procedures" with and without 1 mM 2,3-diphosphoglyceric acid. Where indicated, cholecystokinin octapeptide was added to a final concentrationof 3 pM. Bars in upper curves: change in free Ca2+concentration due to addition of 20 nmol of Ca2+ determined as shown in Figs. 1 and 2. Typical for two experiments. control

2,3-phospho-glycerote.

CO~'

Carbachol

1 1

co2*

1

25mM

Corbochol

'

lornin

'

'

tornin

'

FIG. 8. Comparison of cholecystokinin octapeptide-induced Caz+release andformation of inositol phosphates at different free M 8 + concentrations. Permeabilized cells (2.6 mg of protein/ ml) were incubated as described under "Experimental Procedures" in the presence of 1.3 mM or 0.3 mM free M e . Where indicated, cholecystokinin octapeptide was added to a final concentration of 3 pM. Bars in Ca" traces: change in free Ca2+concentration due to addition of 10 nmol of Ca2+determined as shown in Figs. 1 and 2. Note that the reduction of the medium-free MgZ' concentration results in increased Ca2+binding to ATP, so that the change in free Ca2+concentration for the same amount of Ca2+added is about halved (IP3, 0 , IP2,A;IPI, 0).Typical for five experiments.

1

- 6.L

FIG. 7. Comparison of carbamylcholine-induced CaZ+ release in thepresence or absence of 2,3-diphosphoglyceric acid. Permeabilized cells were incubated as described under "Experimental Procedures" (low MgZ' concentration) with and without 2.5 mM 2,3diphosphoglyceric acid. Where indicated, carbamylcholine (carbachol) to a final concentration of 50 pM was added. Typical for three incubations from one cell preparation.

to permeabilized cells at a concentration of 5 PM and therate of hydrolysis measured over the first 5 min. The half-time of [32P]IP3hydrolysis was reduced both in the presence of 2,3diphosphoglyceric acid (1 mM:7.5 min, 2.5mM:20 min) or 0.3 mM free M$+ (5.8 min) compared to the control without 2,3-diphosphoglycericacid and 1.3 mM free M e (4.4 min). It should be emphasized that comparison of these hydrolysis with those for trirates for pure inositol-1,4,5-trisphosphate tiated IP3 produced by the permeabilized cells is complicated by the fact that the latter is a mixture oftwo isomers of inositol trisphosphate (Ref. 19 and see below).

Effects of Li+ and 2,3-PGA on Intact Cells

One of the problems of studying leaky cells is that the permeabilization procedure is not totally effective. It is necessary, therefore, to rule out the possibility that theeffects of secretagogues on IP3formation in permeabilized cells was due decreasing the free Mg2' concentration results in increased to increases occurring in a small number of intact cells. The Ca2+binding to ATP, i.e. Ca2+ buffering of the incubation responsiveness of such intact cells to Li', which inhibits IP, medium is increased. Therefore, the two calcium traces inFig. breakdown, and 2,3-PGA,which inhibitsIP3 breakdown, 9 are not directly comparable because the increase in free should be qualitatively different to that of the leaky cells Ca2+concentration recorded at thelow MgZ+ concentration is described earlier. First, 2,3-PGA is a highly polar molecule considerably larger (compare bars for 10 nmol of calcium in that should not enter intact cells. Indeed, when cells were Ca2+ traces). As might be expected from a reduction in the incubated in thepresence of 1.5 mM Ca2+so that their plasma hydrolysis of IP3, the rate of Ca2+reuptake was also reduced membranes remained tight, 2,3-PGA had no effect whatsoever at thelower magnesium concentration (Fig. 8). on CCK OP-induced formation of inositol phosphates (Fig. In Fig. 9, CCK-OP-induced Ca2+release and changes in IP3 10). Li' produced a pronounced increase in IP3 and IP, and IP, production at 0.3 mM and 1.3 mM free M 2 + concen- production in intact cells and a continuous accumulation of tration were correlated from five separate experiments. An IPl (Fig. lo), whereas in permeabilized cells it augments the excellent correlation (correlation coefficient, r = 0.99 for IPS IPl level only. These results together effectively rule out the possibility that the changes in IP3 thatwe are monitoring in and r = 0.95 for IP,) was obtained. In one experiment the rate of hydrolysis of 32P-labeled the permeabilized cells, are occurring in a small population of inositol-1,4,5-trisphosphate was measured. [32P]IP3was added intact cells.

Ca2+Release and IP3 Production

7314

0

1

0

,'

a/ I

I

0.2

I

I

I

0.4 0.6 0.8 1.0

-

"

1 P3 production

1.0

1 FIG. 10. Effect ofLi+ or 2.3-diphosphoglyceric acidon cholecystokinin-induced productionof inositol phosphates in intact cells. Isolated acinar cells were prepared, prelabeled, and incubated as described under "Experimental Procedures"with the follow-

ing modifications. The nominally Caz*-free solution used for washing was replaced by a medium containing (in milliiolars): NaCl, 120; KCI, 4.7; KH2P0,, 1.2; MgCIZ,1.2; CaCI2, 1.5;Hepes, 10; myo-inositol, 01 I I I I 1 1; glucose, 15; 0.1 mg/ml trypsin inhibitor, 0.2% albumin; pH 7.4. 0 0.2 0.4 0.6 0.8 1.0 Trypan blue uptake was below 10%.Cells were incubated in the same medium without inositol with either 10 mM LiCl (A) or 1 mM 2,3IP2 production diphosphoglyceric acid (0)or no further addition (a).CholecystokiFIG. 9. Correlation of cholecystokinin octapeptide-induced nin octapeptide to a final concentration of 3 p M was added where CaB+release and formationof inositol phosphates at different indicated. Protein concentration = 5.0 mg/ml. One experiment. free M$+ concentrations. Cholecystokinin octapeptide-induced Ca2+release and production of inositol phosphates was determined In order to provide further evidence forthis hypothesis we at 0.3 mM and 1.3 mM free M$+ concentration as shown in Fig. 8. Results were standardized by setting the sum of CCK-OP-induced have used permeabilized pancreatic cells to explore the proCa" release and increase in IPS and IPz production measured for posed relationship between receptor-mediated IP3 formation each pair of incubation at 1.3mM and 0.3 mM free M e concentration and calcium mobilization. A unique aspect of these permeato 1. Open symbols, 0.3 mM free M$+ concentration. Closed symbols, biliied cells is that they have retained the ability to release 1.3 mM free M P concentration. Different symbols refer to different calcium in response to secretagogues(35,36). The most cell preparations. In A, A and 0, the incubation medium contained also 1 mM 2,3-diphosphoglycericacid at both M$+ concentrations. r significant observation to emerge from the current study is that this secretagogue-induced releaseof calcium in permea= correlation coefficient.

bilized cells is accompanied by a parallel production of IP3 and IP,. There was generally a good correlation between Caz+ DISCUSSION release and IPSproduction between different cell preparations During recent years increasing evidence has accumulated, and under different conditions (Figs. 3 and 9).Obviously it is suggesting that inositol trisphosphate might function as a a difficult task to study the effect of a second messenger in second messenger of Ca2+-mobiliigreceptors to release in- an open system, as a large part of it will be lost t o the medium tracellular Ca2+(7).It was shown in a variety of tissues that instead of fulfilling its role as anintracellular signal. However, the polyphosphoinositides phosphatidylinositol 4-phosphate the disadvantages of this approach are more than counterbaland phosphatidylinositol 4,5-bisphosphate (PIP,) ratherthan anced by the advantage of being able to control the intracelphosphatidylinositol itself form the primary target of receptor lular environment. The qualitatively different effects of Li+ coupled hydrolysis (2,6, 12, 15, 21, 23, 28,30, 31, 39, 40, 41). and 2,3-PGA indicates that IPSwas produced by the permeaWhere inositol phosphates were investigated, cell stimulation bilized rather than the small proportion of intact cells. Incucaused the formation of inositol trisphosphate and inositol bation of intact cells with CCK-OP in the presence of Li+ bisphosphate, i.e. there was evidence for an activation of a resulted in a continuous accumulation of IP, indicating an PIP, phosphodiesterase (2, 6, 8, 15, 23, 38). Hydrolysis was enhanced turnover of the inositol phosphates throughout the sufficientlyrapid to precede or at least not follow Ca" release period of stimulation. In contrast, there was no such rise in (38). Furthermore, IP3 in micromolar concentrations was IP, in the permeabilized cellsnor was there any amplification shown to release Ca2+ from intracellular stores in a variety of of the IP3and IP, responses to CCK-OP suggesting that the tissues (10,20,29,35,37). For exocrine pancreas these stores rapid initial formation of IP3 was not maintained. Neverthewere identified as endoplasmic reticulum (34).These obser- less, it seems an important observation that cells which have vations taken as a whole strongly support the hypothesis that lost 80% of their lactate dehydrogenase (36)retain theability IP3 functions as a second messengerto mobilize intracellular to convert PIP, to IP3. Thus permeabilized cells, where the calcium. intracellular environment can be easily controlled, present an

Ca2’ Release and IPSProduction

7315

3. Akhtar, R.A., and Abdel-Latif, A. A. (1980) Biochem. J. 192, ideal model system to investigate some of the unsolved prob783-791 lems concerning the transduction mechanisms responsible for 4. Amsterdam, A., and Jamieson, J. D. (1972) P m . Natl. A d . Sci generating IP, and to provide further evidence for its role in U.S. A. 69,3028-3032 calcium mobilization. 5. Bemdge, M. J. (1981) MOL CeU. Endocr. 24,115-140 One of the long-standing controversies concerning the pro6. Berridge, M. J. (1983) Biochem. J. 212,849-858 posed link between inositol lipids and calcium mobilization 7. Bemdge, M. J. (1984) Biochem. J. 220,345-360 concerns the calcium dependencyof the PIP,phosphodiester- 8. Berridge, M. J., Dawson, M. C., Downes, C . P., Heslop, J. P., and Irvine, R. F. (1983) Biochem. J. 212,473-482 ase responsible for forming IPS(525). In the case of pancreas, 9. Billah, M. M., and Lapetina, E. G. (1982) J. Biol. Chem. 2 6 7 , as in many other cells, the agonist-dependent hydrolysis of 12705-12708 PIP, is not affected by removing extracellular calcium (9,21, 10. Burgess, G. M.,Godfrey, P. P., McKinney, J. S., Berridge, M. J., 30, 41). It might be argued, however, that PIP, hydrolysis Irvine, R. F.,and Putney, J. W.(1984) Nature 309,63-66 could be activated by a small amount of calcium released from 11. Cockcroft, S., Bennett, J. P., and Gomperts, B. D. (1981) Biochem. J. 200,501-508 intracellular stores. A similar uncertainty surrounds the studies using calciumionophores (A 23187 and ionomycin) which 12. Creba, J. A., Downes, C.P., Hawkins, P. T., Brewster, G., Michell, R. H., and Kirk, C . J. Biochem J. 212,733-747 have no effect oninositol lipid hydrolysisin some tissues (30, 13. Downes, C. P., and Michell, R. H. (1981) Biochem. J. 198, 13341) but did in others (3, 11).One way of resolving this issue 140 is to study the activity of the enzyme in situunder conditions 14. Downes, C. P., Mussat, M. C., and Michell, R. H. (1982) Biochem. J. 203,169-177 where the intracellular level is clamped at a value close to that found in cells at rest. Stimulation of inositol polyphos- 15. Downes, C. P., and Wusteman, M. M. (1983) Biochem. J. 2 1 6 , 633-640 phate formation by CCK-OP was unchanged when the intraL. M., and Sherman, W. R. (1980) J. BioL Chem. 2 6 6 , cellular free Ca2+concentration of the cells was buffered with 16. Hallcher, 10896-10901 EGTA (Fig. 4). Conditions were chosen, so that a free Ca2+ 17. Hokin, M. R., and Hokin, L. E. (1953) J. B i d Chem. 2 0 3 , 967concentration equivalent to the physiological intracellular 977 Ca2+level was present (26, 27, 36), but any changes in free 18. Irvine, R. F.,Letcher, A. J., and Dawson, R. M. C. (1984) Biochem. J. 2 1 8 , 177-185 Ca2+concentration were reduced by about 100-fold. It was thus established that the enzyme that converts phosphatid- 19. Irvine, R. F., Letcher, A. J., Lander, D. J., and Downes, C. P. (1984) Biochem. J. 223,237-243 ylinositol bisphosphate to IP, was not sensitive to changes in 20. Joseph, S. K., Thomas, A. P., Williams, R. J., Irvine, R. F., and Ca2+concentration over the normal physiological range.The Williamson, J. R. (1984) J. Bwl. Chem. 2 6 9 , 3077-3081 result proves that the production of inositol polyphosphates 21. Kirk, C. J., Creba, J. A., Downes, P., and Michell, R. A. (1981) in pancreas is notsecondary to the change in Ca2+concentraBiochem. Soc. Tram. 9,377-379 tion. These studies on permeabilized cells are entirely con- 22. Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275 sistent with observations on the PIP,phosphodiesterasestud23. Martin, T. F. J. (1983) J. Bwl. Chem. 2 6 8 , 14816-14822 ied in vitrowhere enzymeactivity was unchanged over a range 24. Michell, R. H. (1975) Biochim Biophys. Acta 416,81-147 of calcium concentrations from to lo-‘ M (18). 25. Michell, R.H., Kirk, C. J., Jones, L. M., Downes, C. P., and In contrast, changes in phospholipid metabolismled to Creba, J. A. (1981) Philos. Trans.R. Soc. Land. B BioL Sci. iarallel changes in Ca2+ release. When the amount of IP3was 296,123-137 diminished by inhibiting the phospholipase C with neomycin, 26. Ochs, D. L., Korenbrot, J. I., and Williams, J. A. (1983) Bioehem. Bwphys. Res. Commun. 1 1 7 , 122-128 Ca2+release was abolished (Fig. 5), when it was increased by J., and Stark,R. J. (1982) Am. J. Physiol. 2 4 2 , G513inhibiting IPShydrolysis with 2,3-diphosphoglyceric acid (Fig. 27. ODoherty, G521 7) or low M g + concentration (Fig. 8), Caz+ release was in- 28. Orchard, J. L., Davis, J. S., Larson, R. E., and Farese, R. V. creased. The latter result would be equivalent to the stimu(1984) Biochem. J. 217,281-287 latory action of theophylline on processes triggered by CAMP. 29. Prentki, M., Biden, T. J., Janjic, D., Irvine, R. F., Berridge, M. J., and Wollheim, C. B. (1984) Nature 309,562-564 It should finally be noted, that no differentiation of the inositol trisphosphates formed was done in our study. It is 30. Putney, J. W., Burgess, G. M., Halenda, S. P., McKinney, J. S., and Rubin, R. P. (1983) Biochem. J. 212,483-488 therefore possible, that some of the “IP3 counts” observed 31. Rhodes, D., Prpik, V., Exton, J. H., and Blackmore, P. F. (1983) were due to ineffective isomers as suggested by Irvine et al. J. Biol. Chem. 268,2770-2773 (19). Indeed, preliminary examination of the [‘H]IP3 formed 32. Rubin, R. P., Godfrey, P. P., Chapman, D. A., and Putney, J. W. in these experiments showed that both isomers 1,4,5-IP3and (1984) Bioehem. J. 219,655-659 1,3,4-IP3 are present.’ Nevertheless, the correlation of IP, 33. Schulz, I. (1980) Am. J. Physwl. 2 3 9 , G335-G347 formation and CaZ+release observed in this study under a 34. Streb, H., Bayerdorffer, E., Haase, W., Irvine, R. F.,and Schulz, I. (1984) J. Membr. Bwl. 81,241-253 variety of conditions provides further confirmation for a sec35. Streb, H., Irvine, R.F., Berridge, M. J., and Schulz, I. (1983) ond messenger function of IP, to release intracellular Ca2+. Nature 3 0 6 , 6 7 4 9 36. Streb, H., and Schulz, I. (1983) Am. J. Physwl. 246, G347-G357 Acknowledgment-H. S. and J. S. thank Prof. Dr. K. J. Ullrich for 37. Suematsu, E., Hirata, M., Hashimoto, T., and Kuriyama, H.

valuable discussions.

REFERENCES 1. Affolter, H., and Sigel, E. (1979). A d . Biochem. 97,315-319 2. Agranoff, B.W., Murthy, P., and Seguin, E. B. (1983) J. Bwl. Chem. 268,2076-2078

*R. F. Irvine, F. Thbvenod, and I. Schulz, unpublished observations.

(1984) Bwchem. Bwphys. Res. Commun. 120,481-485 38. Thomas, A. P., Alexander, J., and Williamson, J. R. (1984) J. Biol. Chem. 269,5574-5584 39. Thomas, A. P., Marks, J. S., Coll, K. E., and Williamson, J. R. (1983) J. Bwl. Chem. 268,5716-5725 40. Volip, M., Yassin, R., Naccache, P. H., and Sha’afi, R. I. (1983) Biochem. Bwphys. Res. Commun. 112,957-964 41. Weiss, S. J., McKinney, J. S., and Putney, J. W. (1982) Biochem. J. 206,555-560