Anion Dependence of Ca2+ Transport and (Ca2' + K+)-stimulated ...

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1987 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 262, N o . 28, Issue of October

Anion Dependenceof Ca2+Transport and (Ca2’+ K+)-stimulated Mg2+-dependent Transport ATPase in Rat Pancreatic Endoplasmic Reticulum* (Received for publication, December 30, 1986)

Thomas P. Kemmer, Eckehard Bayerdorffer,Heike Will, and IreneSchulz From the Max-Planck-Institut fur Biophysik, Kennedyallee70, 0-6000Frankfurt 70, Federal Republic of Germany

Anion dependence of (Ca” + K+)-stimulated M 8 + - port ATPase (7, 8). Further characterization of this enzyme dependent transport ATPase and its phosphorylated in different steps of its turnover cycle showed formation of intermediate have been characterized in both “intact” an acid-stable 100-kDa phosphoproteinin the presence of and ”broken” vesicles from endoplasmic reticulum of Ca2+in the micromolar concentration range. For dephospho] rat pancreatic acinar cells using adenosine ~ ’ - [ Y - ~ ’ Prylation Mg2+ and monovalent cations such as K+ or Na+ triphosphate ([y3’P]ATP). In intact vesicles (Ca2++ were necessary, the Na+being less effective than K+ (8).We K+)-Mg2+-ATPase activity was higher in thepresence have therefore termed this enzyme (Ca2+ + K+)-stimulated of C1- or Br- as compared to NO;, SCN-, cyclamate-, M$+-dependent ATPase (8). SO?- or SO;-. Incorporation of 32Pfrom [-p3’P]ATP Since Ca2+ uptake into vesicles from rough endoplasmic into the 100-kDa intermediate of this Ca2+ATPasewas reticulum was also anion-dependent (7), it was the aim of the also higher in the presence of C1-, Br-, NO; or SCNas compared to cyclamate-, SO:- or SO;-. When the present study to further characterize the properties of Ca2+ membrane permeability barrier to anions was abol- transport by differentiating between the effect of anions on ished by breaking vesicle membrane with the detergent both Ca2+ATPaseand Ca2+-dependent phosphorylationof its activ- intermediate in “intact” and “broken” vesicles. The present Triton X-100 (0.015%)(Ca” + K+)-Mg2+ATPase ity in thepresence of weakly permeant anions, such as study shows that (Ca2++ K+)-MgZ+ATPase-promotedCa2+ SOf- and cyclamate-, increased to the level obtained transport into intactER vesicles was stimulated in the preswith C1-. However, 32Pincorporation into 100-kDa ence of permeant anions, whereas in broken vesicles anion protein was still higher in thepresence of C1- as com- stimulation sequence was abolished. An electrical membrane pared to cyclamate-, indicating a direct effect ofC1potential (vesicle inside-negative) created by a K+ gradient on the Ca2+ATPase molecule. The anion transport over the membrane in thepresence of valinomycin stimulated 4,4-diisothiocyanostilbene-2,2-disulfonate Ca2+ATPaseactivity, whereas a vesicle inside-positive memblocker (DIDS) inhibited (Ca2++ K+)-Mg2+ATPase activity to brane potential inhibited it. We therefore conclude that Ca2+ about 10%of the C1- stimulation level, irrespective of transport intopancreatic endoplasmic reticulum is coupled to the sort of anions present in both intact and broken ion movements which must occur to maintain electroneutralvesicles. This indicates a direct effect of DIDS on(Ca’+ ity. Ca2+transport is either electrogenic and permeant anions + K+)-Mg2+ATPase.K+ ionophore valinomycin influ- are necessary for charge compensation of (Ca2+ + K+)enced (Ca2+ + K+)-Mg2+ATPaseactivity according to the actual K+ gradient: KZ > Kt caused inhibition, K: Mg2‘ATPase-promoted Ca2+transport or charge compensac K t caused stimulation. From these results we con- tion is necessary for electrogenic transport of another cation clude that Ca2+ transport into endoplasmic reticulum into ER that then serves as counterion for a neutral Ca2+ is coupled to ion movements which must occur to main- cation countertransport. tain electroneutrality. EXPERIMENTALPROCEDURES

Materials-All reagents were of analytical grade. EGTA, EDTA, ATP (as Tris, Mg, or K, salt), the protease inhibitor benzamidine, Changes of intracellular free Ca2+ concentration play an pyronin Y, carbonic anhydrase, egg albumin, bovine albumin, phosimportant role in receptor-mediated activation of enzyme, phorylase b, P-galactosidase, myosin, FeS04, citric acid, and lithium NaCl, and fluid secretion from the exocrine pancreas (1-4). dodecyl sulfate were bought from Sigma. Collagenase Worthington was from Worthington. Soybean trypsin inhibitor was purchased We have demonstrated the presence of ATP-dependent mi- I11 from Boehringer Mannheim. Hepes, acrylamide, Coomassie Brilliant tochondrial and non-mitochondrial calcium pools in isolated Blue R-250, Serva blue G, Triton X-100, bovine serum albumin, permeabilized acinar cells (5-7). Steady state free Ca2+con- oligomycin, and antimycin A were bought from Serva (Heidelberg, centration is regulated at 4 X IOT7mol/l’ by the rough endo- Federal Republic of Germany) and Tris, 2-mercaptoethanol, N,N‘plasmic reticulum (6). Caz+uptake intothisstructure is methylenediacrylamide, active charcoal, ascorbic acid, and sodium cation- and anion-dependent and promoted by a Ca2+trans- azide were bought from Merck (Darmstadt, F. R. G.). 45CaC12(4-50 Ci/g) and adenosine 5’-[y-32P]triphosphate (tetra/triethylammonium * This work was supported by Deutsche Forschungsgemeinschaft salt, 1000-3000 Ci/mmol) were obtained from New England Nuclear Grant Ke 354/1-1 and Schu 42912-2.The costs of publication of this Chemicals (Dreieich, F. R. G.), and scintillator Rotiszint 22X from article were defrayed in part by the payment of page charges. This Roth (Karlsruhe, F. R. G.). For autoradiography of 32P-labeledproarticle must therefore be hereby marked “nduertisement” in accord- teins, Kodak X-Omat AR-5 films from Siemens (Frankfurt/Main, F. R. G.) were used. 4,4-Diisothiocyanostilbene-2,2-disulfonate(DIDS) ance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: 1, liter; ER, endoplasmic reticulum; was synthesized by Prof. H. Fasold, Johann Wolfgang Goethe-UnivLDS, lithium dodecyl sulfate; EGTA, [ethylenebis(oxyethylene- ersitat (Frankfurt/Main, F. R. G.). Preparation of Acinar Cells and Rough Endoplasmic Reticulum nitri1o)ltetraacetic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; DIDS, 4,4-diisothiocyanostilbene-2,2-disulfonate. Vesicles-Rat pancreaticacinar cells were prepared as described

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Anion-dependent Ca2+Transport inEndoplasmic Reticulum previously (9). Briefly, pancreatic tissue from six male Wistar rats (200-250 g), which had fasted overnight was digested for 15 min at 37 "C in a collagenase (150 units/ml) containing Krebs-Ringer-Hepes solution, followed by a washing step with a 2 mmol/l EDTA containing Krebs-Ringer-Hepes solution for 10 min. Single cells were then obtained by a second collagenase digestion (225 units/ml) for 60 min. Rough endoplasmic reticulum vesicles were prepared as described recently (8). Briefly, cells were washed twice after isolation ina mannitol buffer containing (in millimoles/liter) 280 mannitol, 10 Hepes, 1 benzamidine, pH 7.0, adjusted with Tris. Cells were then homogenized in 18 ml of mannitol buffer using a tight-fitting Teflonglass Potter-Elvehjem homogenizer by 50 strokes at 900 rpm. The homogenate was centrifuged for 15 min a t 11,000 X g and theresulting supernatant for 15 min a t 27,000 X g. The resulting pellet was resuspended in sucrose buffer containing (in millimoles/liter) 280 sucrose, 18 Hepes, 1 benzamidine, pH 7.0, adjusted with Tris. The protein concentration of 5 mg/ml was adjusted using the protein measurement of Bradford (10). Vesicles werestored in liquid nitrogen for a maximum of 14 days. Measurement of 45Ca2+Uptake-Calcium uptake was measured using "Ca2+. Fifty to 100 pg of rough endoplasmic reticulum membrane protein were preincubated for 20 min at 25 "C in 500 pl of an incubation medium containing basically (in millimoles/liter) 130 KCl, 30 Hepes, 0.01 antimycin A, 0.05 oligomycin, pH 7.0, adjusted with Tris. Theamount of radioactivity varied from 4 to 12 pCi/ml according to the desired total Ca2+concentration. Uptake was initiated by adding Tris-ATP toa final concentration of 1 mmol/l. At given time points triplicate samples were filtered rapidly through cellulose nitrate filters with a pore size of 0.65 p m (Satorius, Gottingen, F. R. G.), which had been presoaked in isotonic KC1 solution. Filters were washed with 4 ml of an ice-cold solution containing (in millimoles/ liter) 140 KCI, 10 Hepes, 1 MgC12, pH 7.0, adjusted with KOH. The radioactivity was quantitated using Rotiszint 22X scintillator (Roth, Karlsruhe, F. R. G.) in a MarkI11 Liquid Scintillation System, Model 6880 Searle Analytic Inc., Des Plaines, IL. The values for ATP-driven Ca2+transport into vesicles were calculated as thedifference between Ca2+content in the presence and absence of ATP in all experiments. Free Ca2+and M e concentrations were calculated with a computer program using the trueproton, Ca2+,and M e dissociation constants for ATP, EDTA, and EGTA as described previously (8, 11). Phosphorylation Procedure-The phosphorylation reaction was carried out as described previously (8) for 20 s a t 4 "C or at room temperature (22-25 "C) and was started by addition of ATP solution containing 0.1 mmol/l Tris-ATP and [y-32P]ATP(10 pCi, 5 pmol/l final concentration) to the incubation medium. The reaction mixture contained 100 pg of 27,000 X g pellet protein in 200 pl of incubation medium with 130 mmol/l potassium salt of Br-, C1-, NO;, SCN-, or cyclamate-. When divalent anions were tested, incubation medium contained 65 mmol/l potassium2 salt of SO:- or SO:- and 65 mmol/l mannitol to adjust osmolarity. Further additions were 0.01 mmol/l antimycin A, 0.01 mmol/l oligomycin, 5 mmol/l sodium azide, 1 mmol/l benzamidine, 28 mmol/l sucrose, 18 mmol/l Hepes/Tris (pH 7.0), 3 mmol/l EDTA, 3.7 mmol/l total magnesium, and 0.3 mmol/l total calcium corresponding to 1 mmol/l free Mg2+ and 1 pmol/l free Ca", respectively. In "Ca2+-free" media, 3 rnmol/l EGTA without added calcium was used. Total magnesium concentration was 1.1 mmol/l (corresponding to 1 mmol/l free M$+) under theseconditions. The reaction was terminated by addition of ice-cold stop solution containing 10%trichloroacetic acid, 10 mmol/l KH2P04, and 1 mmol/ 1 ATP. The samples were kept on ice for 10 min and were then centrifuged for 5 min a t 2,250 X g a t 4 "C. After aspiration of the supernatant, the pellets were washed once with 1 mlof 50 mmol/l KH2POl/H3PO, (pH 2.0) solution and centrifuged again. The final pellets were dissolved in 50 pl of a solution containing (in millimoles/ liter) I sucrose, 9.2 citric acid, 1.2 phosphoric acid, 1.2 Tris, 18 lithium dodecyl sulfate (LDS), 2.4% (v/v) mercaptoethanol, and 40 pg/ml pyronin Y (pH 4.0) for LDS-polyacrylamide gel electrophoresis (12). LDS-Polyacrylamide Gel Electrophoresis at Acidic pH-Following solubilization of proteins with the anionic detergent LDS, 25-p1 aliquots of the solubilized samples were immediately subjected to LDS-polyacrylamide slab gel electrophoresis a t acidic pH according to themethod of Lichtner and Wolf (12) with small modifications as described previously (8). The stacking gel (1.5 X 18 X 0.7 cm) consisted of 5% acrylamide/bisacrylamide, 36 mrnol/l LDS, 93.8 mmol/l citric acid, 12.4 mmol/l phosphoric acid, and 12 mmol/l Tris (pH 2.4). The running gel (12 X 18 X 0.7 cm) consisted of 8% acrylamide/bisacrylamide.Otherwise it was the same as thestacking gel. Polymerization of acrylamide was catalyzed by freshly prepared

13759

0.00025% FeS04, 0.083% ascorbic acid, and 0.025% HzO,. The electrode buffer contained 93.8 mmol/l citric acid, 12.4 mmol/l phosphoric acid, 12 mmol/l Tris, and 36 mmol/l LDS (pH 2.4). Electrophoresis was performed at 4 "C at 40 mA/gel for 3-4 h. When electrophoresis was finished, the gel was soaked in 1%glycerol for 5 min and dried on filter paper (LKB-Producter AB, Bromma, Sweden) using a slab gel dryer unit (LKB). Molecular weights of phosphoproteins were determined by calibration with marker proteins. Standard proteins for molecular weight determination were carbonic anhydrase (29,0001, egg albumin (45,000), bovine albumin (66,000), phosphorylase b (97,400), 8-galactosidase (116,000), and myosin (205,000). Proteins in the gel were stained by 0.25% Coomassie Brilliant Blue R-250 in a methanol/ acetic acid/water solution (5:1:5) and destained in the same solution, following fixation in 12.5%trichloroacetic acid for 30 min. Autoradiography and Determinution of 32P Incorporation into 100kDa Phosphoprotein-For autoradiography of 32P-labeledproteins, Kodak X-Omat AR-5 films were exposed to dried gels at -20 "C for 3 h. Quantitative measurement of 32Pincorporation was carried out with an Automatic TLC LinearAnalyzer System (Berthold, Wildbad, F. R. G.). Additionally, phosphorylated protein bands on the dried gel were excised according to the superimposed autoradiogram, and radioactivity of 32Pincorporated into protein was counted with 4 ml of scintillator in a liquid scintillation counter (Mark 111). Assay for Determination of ATPase Activity-ATPase activity was determined in parallel with the assay for phosphorylation by measuring 32Piliberated from [T-~'P]ATPduring the reaction according to the method of Bais (13). After termination of the phosphorylation reaction and subsequent centrifugation, a 20-4 aliquot of the supernatant was mixed with 500 p1 of active charcoal solution (125 mg/ml 1N HCI). The sample was centrifuged at 2500 X g for 10 min at 4 "C. 100-pl aliquots of the resulting supernatant were mixed with 4 ml of scintillator Rotiszint 22X, and 32Piliberated was counted in a liquid scintillation counter (Mark111). The radioactivity of a control sample obtained in the absence of membrane protein was subtracted from each sample. The Ca2+ATPaseactivity was estimated by subtracting the ATPase activity in the absence of Ca2+from that in the presence of Ca2+.Free Ca2+and M 2 + concentrations were adjusted with EDTA or EGTA and calculated as indicated above under "Measurement of "Ca2+ Uptake." When oxalate was used, its true proton, Ca2+,and M$+ dissociation constants were included for calculation of free Ca2+ and M$+ concentrations. RESULTS

+

Effect of Anions on(Ca2+ K+)-stimulated M$+-dependent ATPase Activity and 32PIncorporation into 100-kDa Protein in Intact Vesicles-When t h e 27,000 X g pellet protein with purified endoplasmic reticulum from rat pancreatic acini was incubatedinthepresence of differentanions, an aniondependent 32Pincorporation into a 100-kDa phosphoprotein could be demonstrated using LDS-polyacrylamide gel electrophoresis at acidic pH and autoradiography. The autoradiogram in Fig. 1 illustrates the effect of anions o n 32Pincorporation into a 100-kDa protein in the presence and absence of Ca2+ i n the incubation medium. It c a n be seen that Ca2+dependent 32Pincorporation was increased in the presence of C1-, NO,, and SCN- as compared to cyclamate- andSO:-. A second band of about 115 kDA showing Ca2+-independent phosphoprotein formation could be a Mg2+ATPase,whose origin, however, is not known. Fig. 2a shows the dependence of 45Ca2+uptake into rough and endoplasmicreticulumvesiclesondifferentpermeant impermeant anions. Membranes are permeable for C1-, Br-, NO;, and SCN-, whereas SO:- and cyclamate- hardly penetrate through membranes. Maximal 45Ca2+uptake was found i n the presence of C1- and decreased 45Ca2+uptake in the presence of Br-, NO,, SCN-, cyclamate-, or SO:- (Fig. 2a). As shown in Fig. 2b and Table I, at 22 "C Ca2+-dependent Mg'ATPase activity was significantly higher the in presence of C1- as compared to NO, ( p c 0.011, t o SCN- ( p < 0.001), to cyclamate- ( p < 0.001), to SO:- ( p < 0.001), a n d to SO$

Anion-dependent Ca2+Transport Endoplasmic in

13760

Molecular

KC1 +Co -Ca

KNO,

KSCN

+Ca -Ca

+Co -Ca

Kcycl. +Ca -Ca "

100000+

.21

K,SO, +Ca -Ca

weight

-205000 -116000 - 97000

- 66000 - 45000 - 29000 FIG.1. Autoradiogram of LDS-polyacrylamide gel illustrating anion dependence of Caz+-dependentphosphoprotein formation in rough endoplasmicreticulum. Phosphorylation and gel electrophoresis were performed as described under "Experimental Procedures." Standard proteins for molecular weight determination were carbonic anhydrase (29,000), egg albumin (45,000), bovine albumin (66,000), phosphorylase b (97,400), 8-galactosidase (116,000), and myosin (205,000).Arrow indicates the 100-kDa phosphoprotein. K cycl., potassium cyclamate.

FIG.2. Anion dependence of "Ca2+ uptake (a),Ca2+ATPase activity ( b ) , and "P incorporation ( c ) into 100-kDa protein in intact membrane vesicles from pancreatic endoplasmic reticulum at room temperature in the presence of different anions. Phosphorylation and gel electrophoresis were performed as described under "Experimental Procedures." Asterisks indicate significance against C1- control, as calculated by Student's t test for unpairedsamples(**,p Br- > SO:- 7 NO, > I- > cyclamate- > SCN- (7) suggestedelectrogenicity of eaz+ uptakefacilitated by permeation of accompanying anions through a hypothetical Cl- conductancepathway.Onthe 0 other hand,a direct effect of these anions on the Ca*+ATPase Time [s] intermediate could be possible. In order to decide between both possibilities, we have inFIG. 3. Time course of Ca"(M$+)-ATPase activity in the of bothATPase presence of Cl- (a)or SO:- (A) in intact membrane vesicles vestigated inthisstudythedependence from pancreatic endoplasmic reticulum at roam temperature. activity and phosphoprotein formation on different anions At indicated time points Ca2+ATPase activity was measured by 32Pi and theeffect of the anion transportblocker DIDS. Since we liberation from [y32pjATPas described under "Experimental Pro- wanted to differentiate between direct effects of anions on cedures." Lines represent resultsof linear regression analysesfor C1(-, y = 0.0442 - 0.090, r = 0.966, p < 0.001, n = 15) and SO!- Ca2'ATPase and indirect effects due to different membrane permeabilities for these anions by which electrogenic cation (- - -, y = 0.022~- 0.043, r = 0.896, P < 0.001, n = 15).

+

i

/

Anion-dependent

0'

KC1

W

ea2+Transport in Endoplasmic Reticulum

L

FIG. 4. Effect of electrical membrane potential on Ca2+(Mg')-ATPase activityin membrane vesicles from pancreatic endoplasmic reticulum at room temperature. Upper panel: Vesicles were preincubated for 15 min at room temperature (22 "C) without or with valinomycin (lod6M) in the absence of K' salts in sucrose buffer containing (in millimoles/liter)280 sucrose, 18 Hepes, 1 benzamidine, pH 7.0, adjusted with Tris. Twenty pl of vesicleswere then transferred to 180 pl of the regular incubation medium containingindicatedpotassium salts (130mmol/l) or potassiumzsalts (65 mmol/l) (K: > KT) without or with valinomycin M). Ca2+ATPase activity was measuredby determination of liberated 32Pias described under "Experimental Procedures." Lower panel: Vesicles were preincubated for 15 min at room temperature (22 "C) in the presence of valinomycin M) with KC1 (130 mmol/ 1) or (65 mmol/l) and Kz oxalate (10 mmol/l). After the preincubation period, phosphorylation reaction was started by adding 20 pl of preincubated vesicles to 180 p1 of the incubation medium M) with containing KZ oxalate (10 mmol/l) and valinomycin KC1 (115 mmol/l) or &SO4 (57.5 mmol/l) (K: = KT) or without the K' salts ( I C < KT), respectively. CaZ+ATPase activity was measured as described under "Experimental Procedures." Columns represent of protein X 20 s) which was Ca2+(Mg2+)-ATPase activity (nmol/mg calculated by subtraction of 32Pliberation inthe absence of Ca2+from the values obtained in the presence of Ca2+.Asterisks indicate signifas calculated by Student's t test icant changes against control values, for paired samples (*, p < 0.05; **, p < 0.01). Vertical bars represent S.E. of 4-6 experiments.

transport intoclosed roughendoplasmic reticulum membrane vesicles could be influenced, we compared effects of different anions for Ca2'ATPase activity and 32Pincorporation into 100-kDa protein in both intact and broken vesicles. Evidence for AnionMovements for Maintenanceof Electroneutralityin Ca2+ Transport-Halogenes, suchas C1- and Br-, facilitate 45Ca2' uptakeintomembrane vesicles from rough endoplasmic reticulum (7) and, as shown inFigs. 1and 2, enhance Ca2+ATPase activity aswell as 32Pincorporation into the 100-kDa intermediate of intact membrane vesicles. Furthermore, higher Ca2+ATPase activity in the presenceof C1- or Br- as compared to cyclamate- and SO:- ions which do not easily move through membranes could be observed. In order to examine whether this preferenceof C1- and Br- for Ca2+ATPase stimulation mightbe due to anion conductance pathways in the membrane, which can beeasily passed by C1and Br- but less by cyclamate- and SO:-, we have opened vesicles by treatment with the detergent Triton X-100. When

13763

Ca2'ATPase activity was compared with and without Triton X-100 treatment, preference of C1- and Br- for stimulation of Ca2'ATPase disappeared, and weakly permeant anions were no longer rate-limiting. (Table I). These results are in agreement with the hypothesis that CaZ+ATPaseactivity is tightly coupled with passive ion movements which must occur to maintain electroneutrality. Valinomycin increases K' conductance in artificial lipid bilayers and naturally occurring biological membranes (1416) and has been shown to act aasmobile carrier (14,15).In biological membranes this property has been used to create membranepotentialsfrompreformed K' gradientsorto collapse membrane potentials in the presence of K+ salts (16). Using valinomycin, Zimniak and Racker (17) gave evidence forelectrogenic Ca2+ uptake in reconstituted sarcoplasmic reticulum systems. In contrast, Chiu and Haynes (18) have concluded that Ca2+ transport into sarcoplasmic vesicles is electroneutral due t o countertransport of cations, K+ being the major countertransported ion. A direct effect of valinomycin on Ca2+ATPase activity has also to be considered (16). Thus Davidson and Berman (16) described that valinomycin (200 nmol/mg), in the absence of monovalent cations, decreased sarcoplasmic ATPase activity by 30% and abolished the stimulatory effects of 150 mM KC1 or NaCl on Ca2+ATPase turnover. The authorsconcluded that the ionophore interactsdirectly with the Ca2'ATPase, independent of its K'-conductive effects on the lipid bilayer, and that this modifies the affinity andspecificity of the monovalent cation site, either by direct interaction or by the formation of a valinomycin-monovalent cation-enzyme complex. In our study a direct effect of valinomycin (10"j M) on Ca2'ATPase could not be observed. This had been verified by measuring Ca2+ATPase activity in the presence andabsence of valinomycin without monovalent cations, osmolarity kept a t 300 mosm/l with sucrose (data not shown). Oxalate itself also did not influence Ca2+ATPase activity. When vesicles were preincubated in a high K' medium in the presence of oxalate but without valinomycin and were then transferred toa medium without K' but with valinomycin, the stimulatory effect on Ca"ATPase activity as shown in Fig. 4, lower panel, could not be observed. We therefore conclude that pancreatic ER membrane is tightfor K' and that, in the absence of valinomycin, vesicles were not preloaded with K' sufficiently as to generate a KT > K,' gradient. The reasonwhy oxalate had to be present also in the preincubationmedium to see the effect of vesicle inside to outside directedK' gradient could be due to non-ionicdiffusion of the lipophilic oxalic acid into vesicles and accumulation of K' oxalate yielding higher K+ concentrations in the vesicle than in the absence of oxalate. It should be also considered that thevalinomycin-K' complex can ion(19, 20). This pairwithanionsandtranslocateionpairs reaction will lead to increase in cation and anionfluxes to a greater extent than can be accounted for by the membrane conductance for these ions. These ion-pairing reactions (19, 20) are quite avid of lipophilic anions, but are also expected C1- (18). Since valinomycin influences to a smaller extent with our Ca2+ATPase activity in the presence of preformed K' gradients only, showing inhibition at a vesicle inside-positive electrical membrane potential and stimulation at an insidenegative potential, we conclude that either transport of Ca2+ is electrogenic or transport of another cation is electrogenic to which Ca2+is coupled in a countertransport. Passivemovement of permeant anions serves for charge compensation of electrogenically transported cation (Fig. 4). Effect of DIDS on Ca2+ATPasein Endoplasmic ReticulumIn sarcoplasmic reticulum, DIDS has been shown to inhibit

13764

Anion-dependent Ca2' Transport in Endoplasmic Reticulum

anionpathways (21). If a C1- pathway separate from the Ca2+ATPase was present in the pancreatic ER membrane, one should expect that DIDS inhibited Ca2+ATPase activity in intact but not in broken vesicles. However, Table I shows that Ca2+ATPaseactivity was abolished by DIDS in the presence of either anion,irrespective of whether or not Triton X-100 was present. 32Pincorporation into the100-kDa protein was also reduced in the presence of DIDS and was even lower than that obtained with cyclamate- without DIDS treatment (Table 11). At 4 "C, at which temperature theeffect on phosphorylationwithout dephosphorylation could be studied, DIDS also inhibited 32Pincorporation (Table 11).Since DIDS reacts with NH, and SH side groups, the observation that the anion transport blocker DIDS (22, 23) inhibited Ca2+ATPase activity and 32Pincorporation into 100-kDa protein in the presence of all anionstested could indicate that, in the Ca2+ATPasemolecule, such side groups are necessary for the process of phosphorylation. Similarly, directinhibition of purified and reconstituted Ca2+ATPase by DIDS was observed inerythrocytes (24). The inhibitory effect of DIDSon Ca2+ATPase activityis also reflected in 45Ca2+accumulation. As shown in Table 111, the same concentration of DIDS that inhibited Ca2+ATPasenearly completely, reduced Mg2'ATPdependent 45Ca2' accumulation by -50% (Table 111). Are There "Chaotropic" Effects of Anions on Endoplasmic Reticulum Calcium Pump?-Ca2+ATPase of pancreatic endoplasmic reticulum has many similarities to that of sarcoplasmic reticulum (25-35). Both Ca2+ATPases are dependent on cations and on anions in a similar way (36-43). Since the membrane of isolated sarcoplasmic reticulum vesicles is permeable to anions and small cations (44-47), the study of electrical properties of the Ca2+ATPaseis difficult (17). IfC1- and Br- would stimulate electrogenic Caz+ uptake and consequently alsoCa2+ATPaseactivity inintact ER vesicles by charge compensation, it should be expected that a lipophilic anion such as SCN-, which easily penetrates membranes, should stimulate even better than C1-. However, as shown inTableI and Fig. 2b, the effect of SCN- on Ca2+ATPase activity in intact vesicles compares with relatively impermeantanions, such as cyclamate- and SO:-, rather than with C1- or Br-. This could be explained by a direct effect of SCN- on the Ca2+ATPasemolecule, due to a chaotropic action of this anion. In sarcoplasmic reticulum Ca2+transport is dependent on anions in a sequence (Cl- L CH3S0, L Br- > NO: > I- > SCN- (48)) which is similar to Ca2+transport in pancreatic endoplasmic reticulum in thepresent study. The andHasselbach (49) have tested large anions denoted as chaotropic on sarcoplasmic calcium pump. They found that chaotropic anions inhibited Ca2+transport, Ca2+-dependent ATPase activity, phosphoprotein formation, and ATP binding, and that the effectiveness of this inhibiting effect increased in the order urea < C1- < NO: < SCN- < C10; < CCl3COO-. The authors came to theconclusion that theeffects of anions such as SCN-, C10; and CC1,COO- could not be explained by chaotropic action as a result of interfering with hydrophobic interactions or by disruption of hydrogen bonds, and they explained the effects of anions by interference with ATPbinding sites (49). Similarly, in our studies, a direct effect on the Ca2+ATPasemolecule byanions might be possible, as also indicated by the inhibitory effect of cyclamate- as compared to C1- and Br- on phosphoprotein formation in both intact and broken vesicles. Mainly, however, the effects of anions

are explained by different membrane permeabilities to these anions in intactvesicles. Our data, together with the effect of electrical membrane potentials on (Ca2++ K+)-M$+ATPase activity in intact vesicles, suggest that Ca2+ transport into endoplasmic reticulum from pancreatic acinar cells is coupled to passive movements of ions which must occur to maintain electroneutrality. Whether the Ca2+pump is electrogenic or coupled to other ions in an electroneutral way remains for further investigation. Acknowledgments-We wish to thank Prof. Dr. K. J. Ullrich for valuable discussions during the course of the study. REFERENCES 1. Schulz, I. (1980) Am. J. Physiol. 2 3 9 , G335-G347

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