Printed in U.S.A.. Inhibition of Inositol Trisphosphate-stimulated Calcium Mobilization ... coupling receptor occupation with Ca2+ channel open- ing has not been ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 263,No. 31, Issue of November 5,pp, 16479-16464,1988 Printed in U.S.A.
Inhibition of Inositol Trisphosphate-stimulated Calcium Mobilization by Calmodulin Antagonists in Rat Liver Epithelial Cells* (Received for publication, February 22, 1988)
Timothy D. Hill, Roberto Campos-Gonzalez, Henrik Kindmark$, andAlton L. BoyntonQ From the Basic Science Program, Cancer Research Center of Hawaii, University of Hawaii, Honolulu, Hawaii 96813
Inositol 1,4,5-trisphosphate (Ins(l,4,5)Ps), an intra- tablished intracellular second messengers (for review seeRef. cellular second messenger produced from the hydrol- 1).Ins(1,4,5)P3mediates surface-elicited signals through moysis of phosphatidylinositol 4,5-bisphosphate, inter- bilization of free Ca2+from storage sites located within the acts withcytoplasmic membrane structures to elicit the endoplasmic reticulum (2-4). Subsequent elevation in cytorelease of stored Ca2+. Ins(l,l,Fi)PS-induced Ca2+ mo- plasmic Ca2+levels is thought to initiate a wide diversity of bilization is mediated through high affinity receptor cellular processes (5-8). bindingsites;however,the biochemical mechanism Studies byWorley et al. (9, 10) have characterized the coupling receptor occupation with Ca2+channel open- binding properties of Ins(1,4,5)P3 to membrane preparations ing has not been identified. In studies presented here, of rat cerebellum. These sites display high affinity and selecwe examined the effects of naphthalenesulfonamide calmodulin antagonists, W 7 and W 13, and a new selec- tivity for Ins(1,4,5)P3 compared with other phosphorylated tive antagonist,CGS 9343B, on Ca2+mobilization stim- inositol compounds, suggesting that Ca2+ mobilization inulated by Ins( 1,4,5)Ps in neoplastic rat liver epithelial duced by Ins(1,4,5)P3 is membrane receptor-coupled. Our (261B) cells. Intact fura-2 loaded cells stimulated by recent findings demonstrate that these high affinity binding thrombin, a physiological agent that causes phospha- sites represent physiological Ins( 1,4,5)P3 receptors involved in triggering Ca2+release in permeabilized rat liver cells (11). tidylinositol 4,5-bisphosphate hydrolysis and Ins (1,4,5)Ps release,responded with a rise incytoplasmic However, the biochemical nature of the mechanism by which free Ca2+levels that wasdose dependently inhibited by Ins(1,4,5)P3opens Ca2+channels has not been described. In W 7 (Ki = 25 MM), W 1 3 (Ki = 45 p ~ ) and , CGS 9343B this study, based on the selective inhibitory effects of calmod( K i= 110 PM). Intracellular Ca2+release stimulatedby ulin antagonists (W7, W13, and CGS 9343B), we present the additionof Ins(1,4,5)Ps directly to electropermea-evidence that calmodulin is an integral component of the bilized 261B cells was similarly inhibitedby pretreat- Ins( 1,4,5)P3 receptor-activated Ca2+ release mechanism in ment with anti-calmodulin agents. W7 and CGS neoplastic rat liver epithelial (261B) cells. 9343B, which potently blocked Ca2+/calmodulin-dependent protein kinase, had no significant effect on EXPERIMENTALPROCEDURES protein kinase A or C in the dose range required for Materials"W5, W7, W13, CAMP, thrombin, ATP, phosphatidylcomplete inhibition of Ca2+mobilization. Ca2+release serine, histone, creatine phosphokinase, and creatine phosphatewere channels and Ca2+-ATPasepump activity were also obtained from Sigma. Ins(1,4,5)P3 was bought from Calbiochem. unaffected by calmodulin antagonist treatment. These Fura-2 acetoxymethyl ester, and potassium salt forms were purchased results indicate that calmodulin is tightly associated from Molecular Probes (Eugene, OR). CGS9343Bwas a generous with the intracellular membranemechanism coupling gift from the CIBA-GEIGY Corp. [Y-~'P]ATPwas obtained from Du Ins( 1,4,5)Ps receptors Ca2+ to release channels. Pont-New England Nuclear. Electrophoresis reagents were acquired from Bio-Rad. All other reagents used were analytical grade. Cell Culture-261B neoplastic cells were plated at a density of 7 X lo3 cells/cm2 on 100-mm round tissue culture dishes (Falcon) for One consequence of plasma membrane receptor interaction permeabilization and in vitro enzyme assay studies, or plated on 12by various physiological agents including hormones, neuro- mm round glass coverslips (Fisher) placed in multiwell sterile culture transmitters, andgrowth factors is the hydrolysis of phospha- plates (Miles Laboratories) for whole cell Caz+ measurements, and tidylinositol 4,5-bisphosphate that liberates inositol 1,4,5- grown to confluency (3 X 10' cells/cm2) in Eagle's Basal Medium trisphosphate (Ins(1,4,5)P3)l and diacylglycerol, two well es- (GIBCO) supplemented with 10% bovine calf serum (Colorado Serum Co., Denver) and 25 pg/ml gentamicin (37 "C, 95% air/5% COZ,water* This work was supported by National Cancer Institute Grants saturated). Permeabilizatwn of 261B Cells-Cells were removed from culture CA39745 and CA42942 (to A. L. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This dishes by mild trypsin (0.005%) treatment. After washing twice in article must therefore be hereby marked "advertisement" in accord- cold phosphate-buffered saline (pH 7.4), cell samples (15 X lo6 cells in 300 pl of K+ buffer: 110 mM KC1, 10 mM NaCl, 25 mM HEPES, 2 ance with 18 U.S.C. Section 1734 solelyto indicate this fact. 3 On leave from the Department of Medical Cell Biology, Univer- mMMgC12, pH 7.4) were electroporated with 20 discharges at 2 kV. Cells were washed twice with cold K+ buffer and placed at 4 "C. sity of Uppsala, Biomedicum, Uppsala, Sweden. Ca2+Release Measurement-Monolayers of confluent intact 261B 5 To whom correspondence should be addressed: Basic Science cells on glass coverslips were loaded with 5 p~ fura-2am for 45 min Program, Cancer Research Center of Hawaii, 1236 LauhalaSt., in 90% Eagle's basal medium/lO% bovine calf serum, washed for 5 Honolulu, HI 96813. The abbreviations used are: Ins(1,4,5)P3, inositol 1,4,5-trisphos- min in 1.8 mM Ca2+/HEPESbuffer (25 mM HEPES, 125 mM NaCl, W7, N - ( 6 - 6 mM KCl, 1 mMMgC12, and 0.55 mM glucose, pH 7.4) and placed in phate; W5, N-(6-aminohexyl)-l-naphthalenesulfonamide; aminohexyl)-5-chloro-l-naphthalenesulfonamide; W13, A"(4-amino- cuvettes (1cm) filledwith2 ml of 1.8mM CaZ+/HEPESbuffer (37 OC). butyl)-5-chloro-2-naphthalenesulfonamide; [Ca2+],,intracellular free Samples of permeabilized cells (5 X lo6 cells in 0.5 ml K+ buffer) Ca2+ concentration; HEPES, 4-(2-hydroxyethyl)-l-piperazineeth- were incubated for 15 min at 30"C following addition of an ATP anesulfonic acid; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; regenerating system: 4 mM ATP, 10 mM creatine phosphate, and 25 EGTA, [ethylenebis (oxyethylenenitrilo)]tetraaceticacid. mg/ml creatine phosphokinase. Fura-2 (4 p ~ was ) then added, and
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Calmoddin Antagonists Inhibit the Action of Im(l,4,5)P3
changes in fluorescence were monitored at 340 nm excitationand 500 nm emission at 30 "C using a fluorescence spectrophotometer (Perkin-Elmer). Calcium concentration was calculated according to the formula: [Ca2+]= K d X ( F - Fmin)/(Fmin F ) as previously described (12), where F is the fluorescence of released Ca2+,Fm..is the fluorescence following saturation of fura-2 with Ca2+,and Fmi,isthe fluoreswas determined by the calculation Fmi, cence of unbound fura-2. Fmin = FM"+ 0.243 (Fmax - FM"),where Fhlnis the fluorescence following MnZ+displacement of Ca2+bound to fura-2. Cell Fractiomtion-Following removal of the growth media, confluent 261B cells were washed with cold phosphate-buffered saline, pH 7.4, and scraped from the culture dishes in 3 ml of fractionation buffer (20 mM Tris, pH 7.5,250 mM sucrose, 2 mM EGTA, 2 mM DTT). Cells were homogenized with 10 strokes of a Potter-Elvehjem homogenizer, and thesoluble fraction was obtained after centrifugation a t 131,000 X g for 20 min (4 "C) in a Beckman TL-100 ultracentrifuge. Protein determinations were made according to the method of Bradford (13). In Vitro Phosphorylation-Protein kinase A was assayed by a modification of the method of Corbin and Reimann (14). The 80-pl reaction mixture contained 20 mM Tris, pH 7.5, 12.5 p~ CAMP, 5 pM MgC12, 37.5 mM D T T 34.3pgof histone, 10 p~ ATP, soluble fraction enzyme (15 pg of protein), and 1pCi of [y3'P]GTP. Protein kinase C was measured according to the method of Zwiller et al. (15). The 80-pl reaction mixture contained 20 mM Tris, pH 7.5, 10 mM MgC12,0.2 mM DTT, 1 gM ATP, 20 pg of histone, 1.6 mM CaCl,, 10 pg of phosphatidylserine, soluble fraction enzyme (30 pg of protein), and 1 pCi of [Y-~'P]ATP.Ca2+/calmodulin-activatedprotein kinase was measured by the method of protein kinase C, except that phosphatidylserine was deleted from the reaction mixture. Reactions were initiated by the addition of histone/[y3'P]ATP or [y3'P]ATP alone and incubated for 5 min at 30 "C, with shaking. Quantitation of reaction samples by liquid scintillation was made as follows: samples were dispensed onto 3-cm2pieces of Whatman No. 3MM paper, fixed with ice cold 10% trichloroacetic acid for 15 min, washed for 30 min with 5% trichloroacetic acid (22 "C), extracted with ethanol for 10 min, and extracted with petroleum ether for 10 min. After drying, the paper pieces were placed in vials containing liquid scintillation mixture, and the radioactivity was measured using a Beckman LS3801 scintillation counter. Polyacrylamide gel electrophoresis separation of phosphorylated endogenous substrates was made on reaction samples treated with 20 pl of SDS-electrophoresis sample buffer (312.5 mM Tris, pH 7.5,50% glycerol,10% SDS, 10% mercaptoethanol, 0.04% bromphenol blue) and heated at 80 "C for 10 min. Electrophoresis protein separation was performed according to the method of Laemmli (16) in 12% acrylamide-acrylaide gel cross-linked to GelBond. Gels were exposed to Kodak SB-5 photographic film for 3 days at -80 "C, and thefilm wasdeveloped for visualization of 32P-labeled protein bands.
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-
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W13
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t
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THROMBIN
FIG. 1. Influence of calmodulin antagonists on Ca2+release induced by thrombin in whole 261B cells. Fura-2 loaded confluent 261B cells on glass coverslips were monitored for changes in [CaZ+li stimulated by0.05 unit/ml of thrombin following a 5-min pretreatment with: A, 100 pM W5; B, 50 p~ W7; C, 85 p M W13; D, 130 p~ CGS 9343B. Tracings are of the change in fluorescence at 340 nm. Left of the tracings are scales indicating the change in Ca2' levels before and after thrombin addition. The individual experiments presented were repeated 20 times. It should be noted that these calmodulin antagonists did not alter the amount of Ca2+ stored within intracellular pools, since ionomycin-releasable Caz+ in treated cells was identical with controls (i.e. approximately 750 nM).
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0
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30
60
90
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CALMOOULIN ANTAGONIST fyM)
RESULTS
Influence of Calmoddin Antagonists on Thrombin-stimuluted Ca" Mobilization in Whole261B Cells-Previous studies demonstrated a disruptive effect of calmodulin antagonists upon cell physiology and proliferative activity (17-19). These effects were interpreted to be caused by inhibition ofCa"/ calmodulin-dependent processes within cells and not associated with alterations in the agonist-induced Ca2+signal generation mechanism. Experiments were performed with fura-2 loaded intact 261B cells to determine if calmodulin antagonists alteredthe Ca2+signal generation mechanism stimulated by thrombin, a physiological agent shown to raise [Ca2+]iin several cell types (20,21). Resultsshown in Fig. 1 demonstrate that thrombin-induced changes in [Ca'+]i are completely inhibited after a 5-min incubation with 50 p M W7,85 p M W13, or 130 p~ CGS 9343B, but not by 100 ~ L MW5, a w 7 analog and weak calmodulin inhibitor (22). Dose-response studies (Fig. 2) of the effect of calmodulin antagonism on thrombininduced Ca2+ release indicate that these agents are potent inhibitors of Ca" release. Inhibition by CGS 9343B required a higher dose than the naphthalenesulfonamide compounds presumably due to the low aqueous solubility of CGS 9343B. We noticed that small grains of CGS 9343B adhered to the
FIG. 2. Dose-response curve of the influence of calmodulin antagonists upon thrombin-induced Caa+release. The amount of Ca2+released from intracellular pools of intact 261B cells following 0.05 unit/ml of thrombin addition was determined from an experiment similar to that shown in Fig. 1. Pretreatment for 5 min with W7, W13, or CGS 9343B inhibited the cell response to thrombin dose dependently, while W5 was without effect. Values are mean f. S.E. of four determinations using different cell preparations.
surface of cells which reduced the amount of available drug for antagonism of cell calmodulin. Influence of CalmodulinAntagonists on Ins(l,4,5)P3-induced Ca2+ Releasefrom P e r m d l e 261B Cells-In 261B cells, stimulation with thrombin mobilized Ca2+ solely from storage pools within cells as determined by comparing the rise in [Ca2+Iiin thepresence and absence of extracellular Ca2+(data not shown). Since ample evidence exists indicating that the thrombin-induced [Ca2+Iiincrease is mediated by the second messenger Ins(1,4,5)P3 (23, 24), we hypothesized that the effect of calmodulin antagonists on Ca'+ mobilization was caused by inhibition of the intracellular membrane mechanism activated by Ins(1,4,5)PS. To test this theory, we electroporated 261B cells to allow access of Ins(1,4,5)P3to intracellular Ca2+ storage sitesand examined the influence of
Calmodulin Antagonists Inhlibit the Action of Im(1,4,5)P3 calmodulin antagonism on Ins( 1,4,5)P&imulated Ca2+ release. Fig. 3 depicts tho results after treating permeable cells with 50 p~ W7, 100 pM W13, or 120 pM CGS 9343B. Ca2+ release was completely inhibited by these agents suggesting that calmodulin is tightly coupled to the mechanism linking Ins(1,4,5)P3 receptor binding to Ca2+channel opening. The dose-dependent inhibition of these agentswas nearly identical with results obtained with intact 261B cells (Fig. 4).
16481
ICa 9 "Y
(COS 93438)
(W7)
FIG.5. Influence of calmodulin antagonists on passive Ca2+ flux and ATP-dependent Ca2+uptake. To induce passive movement of Ca2+out of storage pools, permeable 261B cells were rapidly chilled to 4 "C. Cold cell suspensions were placed in the spectrophotometer (37 "C) andallowed to warm. Tracings of fura-2 fluorescence changes were recorded for: no addition ( A ) ,120 p~ CGS 9343B ( B ) , and 50 p~ W7 (C). Shown are individual experiments representative of eight replications.
Effect of Calmodulin Antagonists on Passive Ca2+Flux and ATP-dependent Ca2+Uptake-To rule out possible inhibitory ws I P 3 effects of calmodulin antagonists on the ability of Ca2+ to traverse membranes, we treated 261B cells with either 50 p~ W7 or 120 p~ CGS 9343B and observed the changes in [Ca2+] by alternately cooling to 4 "C and rewarming to 37 "C (Fig. 5). Passive Ca2+flux, induced by rapidly chilling the cells, and the subsequent ATP-dependent Ca2+sequestration mediated by Ca2+-ATPasesupon slowly warming to 37 "C were unaffected by W7 or CGS 9343B, indicating that Ca2+movement across intracellular membranes is not impeded by calmodulin antagonism. IP3 IP3 IP3 Selectivity of the Inhibitory Action of W7 and CGS 9343B (CGS93438) ( W 13) toward Calmodulin-Because high doses of W7 and other U calmodulin antagonists have been shown to alter cellular 0 2 MINUTES processes apart from their influence on calmodulin, studies FIG.3. Effect of calmodulin antagonists on Ins(1,4,6)Ps- were performed to determine if secondary effects occurred induced Ca" release from permeable 261B cells. Suspensions within the dose range required for inhibition of Ca2+release of permeable 261B cells containing 4p~ fura-2 were treated with 0.5 induced by Ins(1,4,5)P3. To demonstrate the influence of W7 p M Ins(1,4,5)P3 to release intracellular Ca2+.In A , 100 p M W5 and 50 and CGS 9343B on an endogenous calmodulin-dependent p~ W7 were added to the same cells after an Ins(l,4,5)P3-induced enzyme, we first phosphorylated endogenous substrates of the Ca2+release/sequestration cycle, and the cells were allowed to incubate for 5 min. W7 inhibition of the cell response to Ins(1,4,5)P3 was 131,000 X g supernatant fraction of 261B cells in an in vitro reversed by washing with buffer. B, shows individual experiments reaction mixture containing excess Ca2+ (Fig. 6) and then representative of the effects of 100 p~ W13 and 120 p~ CGS 9343B separated the phosphoproteins by SDS electrophoresis. Auon Ins(l,4,5)P3-induced Ca2+release. Antagonists were incubated 5 toradiographs of these phosphoproteins revealed a bandof M , min prior to the addition of 0.5 p~ Ins(1,4,5)P3. The tracings shown = 100,000corresponding to thesubstrate previously identified here represent findings repeated 20 times. by calmodulin-dependent protein kinase I11 (25). Treatment ) the phoswith W7 (50 p ~ or) CGS 9343B (50 p ~ blocked phorylation of this M , = 100,000 substrate. Two major phosphorylating enzyme activities, protein kinase A and protein kinase C, which are not dependent on Ca2+/calmodulin for metabolic activity, were monitored for possible secondary inhibition by calmodulin antagonists. The autoradiograph of separated 32P-labeledproteins in Figs. 7 and 8 indicates that W7 and CGS 9343B have no significant effect on the phosphorylation reactions catalyzed by protein kinase A and protein kinase C. When the effects of W7 and CGS 9343B on protein kinase A and protein kinase C were quantitated in an in vitro assay using histone as a substrate, it was apparent that neither agent inhibited protein kinase A or protein kinase C at a dose that completely blocks Ins(l,4,5)P3-induced Ca2+ mobilization (Fig. 9). A dose of CGS 9343B as high as 200 p~ was without effect on both enzyme activities; however, partial inhibition of both protein kinase A and protein kinase C 0 30 60 90 I20 I6 0 resulted from treatment with W7 in a concentration-dependCALYODULIW AWTA00NIST ( p Y ) ent manner.
. .
FIG.4. Dose-response effect of calmodulin antagonists on Ins(l,4,S)Ps-stimulatedCaa+release. Changes in [Ca2+]induced by 0.5 PM Ins(1,4,5)P3 were determined following pretreatment (5 min) with increasing doses of W5, W7, W13,and CGS 9343B. Values arethe mean & S.E. of five determinations using different cell preparations.
DISCUSSION
The results presented here demonstrate that anti-calmodulin agents (W7, W13, CGS 9343B) inhibit both thrombininduced Ca2+ mobilization in whole cells and Ins(1,4,5)P3-
CalmodulinAntagonists Inhibit the Action of In~(1,4,5)P3
16482 ”
A
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4 e 38 3
1 2 3 I 24 3 4 5 6 7 I 28 3 4 5 6 7 8
I2 I 2 3 4 5 I 2 3 4 5 I 2 3 4 w5 w7 CGS NO
FIG.7. Investigation of the effect of W5 and W 7 on 261B
endogenous protein kinase A and protein kinase C activities. Dose-dependent studies were made with the 131,000 X g supernatant A D D 93438 fraction of 261B cells treated with either W5 or W7. Electrophoretic separation of in vitro 32P-labeledendogenous substrates was visualized by autoradiography (shown) revealing a 40-kDa substrate stimulated A D B C and two substrates stimulated by cAMP (protein kinase A (PKA)), FIG.6. Effect of calmodulin antagonists on Ca*+/calmodu- by Ca2+/phosphatidylserineof M,= 35,000 and 38,000 (protein kinase lin-dependent phosphorylation of a 261B cytosol substrate. In C (PKC)).Protein kinase A was assayed in the 131,000 X g supervitro assay of the 131,000 X g supernatant fraction stimulated by the natant fraction in an 80-pl reaction mixture which contained 20 mM addition of Ca2+alone revealed increased 32P-labelingof a 100-kDa Tris, pH 7.5, 12.5 p~ CAMP, 5 p M MgCI2, 37.5 mM DTT, 34.3 pg of endogenous substrate. Shown is an autoradiograph of 32P-labeled histone, 10 p~ ATP, 1pCi of [y3’P]ATP, andsoluble enzyme fraction proteins separatedby gelelectrophoresis after treatment with increas- (15 pg of protein). Protein kinase C was assayed in the 131,000 X g ing doses of CGS 9343B, W7, or W5. The in vitro reaction mixture supernatant fraction in an 80-pl reaction mixture which contained of 80 ml contained 20 p M Tris, pH 7.5,lO p M MgC12,0.2 p M DTT, 1 20 mM Tris, pH 7.5, 10 mMMgC12, 0.2 mM DTT, 1 p~ ATP, 1 pCi p~ ATP, 1 pCi of [y-3ZP]ATP,1.6 p M CaC12, 20 pg of histone, and of [y3’P]ATP, 20 pg of histone, 10 pg of phosphatidylserine, and 1.6 soluble enzyme (30 pg of protein). Reactions were initiated by the mM CaCI2. Assays for protein kinase A and protein kinase C were addition of [y3’P]ATP andincubated for 5 min a t 30 “Cwith shaking. initiated by the addition of [ Y - ~ ~ P I A and T P incubated for 5 min a t Reactions were terminated by the addition of 20 pl of SDS-electro- 30 “C, with shaking. Reactionswere terminated by the addition of 20 phoresis sample buffer and prepared for SDS electrophoresis sepa- pl of SDS electrophoresis buffer and prepared for SDS electrophoresis ration of labeled proteins as described under “Experimental Proce- separation of labeled proteinsas described under“Experimental dures.” A, controls: enzyme alone (lane I ) , enzyme + Ca2+( l a n e 2). Procedures.” Treatment of supernatant fractions with increasing B, enzyme + Ca2++ CGS 9343B: 1, 10,50, 100, 200 p~ (lanes 1-5, doses of W5 and W7 are shown as follows: A, controls: enzyme alone, respectively). c, enzyme + Ca2++ w? 1, 5, 10, 50, 100 p M (lanes 1- enzyme + Ca2+, enzyme + Caz+ + phosphatidylserine, enzyme + 5,respectively). D,enzyme + Ca2++ W 5 5, 10, 50, 100 p M (lanes 1- cAMP (lanes 1-4, respectively). B, protein kinase A activity(enzyme 4, respectively). + CAMP): 10, 50, 100, 150 p M W5 (lanes 1 4 , respectively); 10, 50, 100,150 p M W7 (lanes 5-8, respectively). c, protein kinase c activity (enzyme + Ca2++ phosphatidylserine): 10,50, 100,150 p~ W5 (lanes induced Ca2+ mobilization fromintracellular storage sites of 1 4 , respectively); 10,50, 100, 150 p~ W7 (lanes 5-8, respectively).
permeabilized 261B cells.The inhibitory effect of these agents appears to be caused by a direct interaction with calmodulin for several reasons. First, although naphthalenesulfonamide compounds are known to have secondary effects upon cellular enzymatic activities apart from their calmodulin antagonism (26), our studies here indicate that theeffects of W7 had only a minimal influence on two major activities in 261B cells, namely protein kinase A and protein kinase C, at a dose required to blockCa2+release stimulated byIns(1,4,5)P3. Second, a new selective calmodulin antagonist, CGS 9343B, which has been previously shown to lack inhibitory action toward protein kinase C and only weakly displaces [3H]spiperone from postsynaptic dopamine receptors (27), had no detectable influence on protein kinase C or protein kinase A activities of261B cells and dose dependently blocked Ca2+ mobilization. Third, calmodulin antagonism did not alter the amount of Ca2+stored within intracellular pools, since ionomycin-releasable Ca2+in treated cells was identical with the control (data not shown). Fourth, membrane Ca2+channels appear to remain functional since passive leakage of Ca2+ occurred in treated cells upon cooling to 4 “C. Fifth, Ca2+ATPase mediated Ca2+ sequestration of passively released Ca2+remained unaffected by W7 or CGS 9343B treatment.
Sixth, W7 and CGS 9343Bdid not block Ins(1,4,5)P3binding to high affinity receptors in an assay using rat brain cerebellum membranes as a receptor source.’ Calmodulin, a low molecular weight protein, binds Ca2+ with high affinity (28) and interacts with a wide variety of enzymes in eukaryotic cells (29). Since calmodulin modifies cellular processes in a Ca2+-dependentmanner, it is reasonable to accept a role for calmodulin in a receptor-mediated mechanism controlling intracellular membrane Ca2+channel opening. Several investigations have indirectly provided evidence suggesting a functional role for calmodulin in Ca2+ release. Smooth muscle cells of isolated rabbit aorta strips exposed to W7 were shown to relax as a result of inhibition of actin and myosin filament interaction, a Ca2+-dependent event (30). Studies by Douglas and Nemeth (32) on mast cell secretion induced by antigens, which elicit a rapid rise in [Ca”]i upon binding to surface receptors (31), revealed inhibitory effects of phenothiazine and naphthalenesulfonamide S. Nakamoto, J. Zwiller, and A. L. Boynton, unpublished observations.
Calmodulin Antagonists Inhibit the Action of Ins(1,4,5)P3 A
B PKA
C PKC
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the divalent ionophore A23187. Ca2+transport across membranes by the action of A23187 occurs through a mechanism separate from the Ca2+release channel opening (33); therefore, the authors were unable to detect possible alterations in an Ins( 1,4,5)P3receptor-coupledCa2+response. Gig1 et al. (34) extended these observations in mast cells by examining the -100 effects of a broader range of antagonistic agents on various 100 stimuli which raise [Ca2+Ii.Although they noted that several calmodulin antagonists inhibited exocytosis regardless of the source from which Ca2+was derived following agonist stimulation, two agents (prenylamine and thioridazine) had a more potent effect on the secretory response when the Ca2+rise was mediated through Ins(l,4,5)P3-induced mobilization of 40A0 intracellular Ca2+pools.Moreover, phosphate transport in 58 LLC-PKI cells, which is stimulated by calcitonin receptor 1 binding and Ins(l,4,5)P3-induced Ca2+release, was inhibited 35' 35 by W7 treatment (35). Calcium ions are considered to play an important role in regulating non-neoplastic cell proliferation, being essential for Go to GI transition in some cell types, and GI to S cell cycle transition in most cell types (36). Investigations by Boynton et al. (37) into the biochemical nature of the Ca2+ dependency for non-neoplastic cell proliferation found cal1 2 3142 3142 3 4 modulin antagonist sensitivity during the late GIphase of the FIG. 8. Examination of the influence ofCGS 9343B on pro- cellcycle aftertreatment with trifluoperazine or W7. In tein kinase A and protein kinase C activities of 261B cell- another study, Hidaka et al. (38) observed inChinese hamster soluble fraction. Shown is the autoradiograph of in vitro "P-labeled ovary cells that W7 arrested cell proliferation at the GI/S proteins separated by electrophoresis after stimulation with Ca2+/ boundary. The effects on cell cycle progression were interphosphatidylserine to activate protein kinase Cor cAMP to activate preted based on the assumption that calmodulin antagonists protein kinase A. Details of protein kinase A and protein kinase C stimulation of endogenous proteins are as described in the legend to exert their inhibitory action distal to Ca2+signal generation. Fig. 7. A, controls: enzyme alone, enzyme + Ca", enzyme + Ca2+/ Experiments presented here suggest that defective intracelphosphatidylserine, enzyme + cAMP (lanes 1-4, respectively). B, lular Ca2+signal generation accompanies modified enzymatic protein kinase A activity(enzyme + CAMP):10,50,100,200p~ CGS activity in cells as a consequence of calmodulin antagonism 9343B (lanes 1-4, respectively). C,protein kinase C activity (enzyme and may beresponsible for arresting cell proliferative activity. + Caz++ phosphatidylserine): 10, 50, 100, ~LM CGS 2009343B (lanes Calmodulin modulation of Ca2+release channels has been 1-4, respectively). proposed from studies by Meissner (39) on skeletal muscle sarcoplasmic reticulum vesicle Ca2+efflux. Exogenous addiA' 0. tion of calmodulin partially inhibited 45Ca2+release, while 4.0 4 0.4 inhibition of endogenous vesicle membrane calmodulin had no effect, leading the author to conclude that calmodulin played a regulatory but not essential role in Ca2+channel inactivation. Wolf et al. (40) reported that W7 treatment potentiated Ins(1,4,5)PS-induced4sCa2+efflux from digitoninpermeabilized pancreatic islet cells and from isolated endoplasmic reticulum membrane fractions, and addition of exogenous calmodulin inhibited by >50% the Ca2+efflux stimulated by Ins(1,4,5)P3. These results cannot bereconciled with our studies of calmodulin antagonist in 261B cells. Differences in cell type as a possible reason for the opposing observations is highly unlikely, since we have repeated our results using cultured C3H/lOT1/2 mouse fibroblasts? We OL aO "have found that commercially available calmodulin always contains Ca2+,which might alter cell responsiveness depend0 100 200 0 100 200 ing on the dose used. High [Ca2+]ihas been shown to inhibit CALMODULIN ANTAGONIST (yM) Ca2+release (39);therefore, it is probable that high Ca2+levels FIG. 9. Quantitative study of the action of CGS 9343B and caused bythe exogenous addition of calmodulin were responW7 on 261B cell endogenous soluble protein kinase C and sible for the inhibitory effects observed in previous studies. protein kinase A activities. Dose-response effects of calmodulin antagonists on enzymatic activity was determined by in vitro assay In addition, Wolf et al. administered W7 and Ins(1,4,5)P3 at and liquid scintillation counting methods described under "Experi- the same time point-we found that while Ins(1,4,5)P3 momental Procedures." CGS 9343B had no significant inhibitory effect bilized Ca2+within a few seconds, the inhibitory action of on protein kinase A ( A )or protein kinase C( B ) ;however, high doses W7, W13, and CGS 9343B was realizedonly following 5 min of W7 partially blocked the phosphorylating activityof these enzymes. of incubation, ample time for maximal Ca2+release to occur. Values are mean f S.D. of three determinations. The presence of calmodulin in the biochemical apparatus linking Ins(1,4,5)P3receptors to Ca2+release channels implies calmodulin antagonists upon the exocytotic process. They that a membrane-bound protein regulated by calmodulin is dismissed the possibility that calmodulin antagonists affect T. Hill and A. Boynton, unpublished observations. the Ca2+release mechanism based on results acquired using
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Calmodulin Antagonists Inhibit the Action of Im(1,4,5)P3
an integral component of the Ca2+mobilization mechanism as well. This proposed proteinconstituent may require calmoddin interaction to change its conformation, Or for enzymatic activity. We have shown that physiological Ins(1,4,5)P~binding receptors are sensitively and selectively inhibited by the phosphataseinhibitor heparin ( l l ) , suggesting that these receptors might have enzymatic function. However, isolation and characterization of Ins(1,4,5)P3receptors and ca2+ from necessary for establishing the nature of these structures and the mechanism by which Ca2+channels are regulated. Acknowledgments-We wish to thank Nathan Yanagi and Sherilyn Boynton for their excellent technical assistance. REFERENCES 1. Berridge, M. J. (1987) Annu. Reu. Biochem. 56,159-193 2. Streb, H., Irvine, R. F., Berridge, M., and Schulz, J. (1983) Nature 306,6749 3. Streb, H., Bayerdorffer, E., Haase, W., Irvine, R. F., and Schulz, I. (1984) J. Membr. Biol. 81,241-253 4. O’Rourke, F. A., Halenda, S. P., Zavoico, G. B., and Feinstein, M. D. (1985) J. Biol. Chem. 2 6 0 , 956-962 5. Volpe, P., Salviati, G.,DiVirgilio, F., and Pozzan, T. (1985) Nature 316,347-349 6. Authi, K. S., Evenden, B. J., and Crawford, N. (1986) Biochem. J. 233, 707-718 7. Slack, B. E., Bell, J. E., and Benos, D. J. (1986) Am. J. Physiol. 2 5 0 , C340-C344 8. Fein, A., Payne, R., Corson, D. W., Berridge, M. J., and Irvine, R. F. (1984) Nature 3 1 1 , 157-160 9. Worley, P. F., Baraban, J. M., Colvin, J. S., and Snyder, S. H. (1987) Nature 325, 159-161 10. Worley, P. F., Baraban, J. M., Supattapone, S., Wilson, V. S., and Snyder, S. H. (1987) J. Biol. Chem. 2 6 2 , 12132-12136 11. Hill, T. D., Berggren, P.-O., and Boynton, A. L.(1987) Biochem. Biophys. Res. Commun. 149,897-901 12. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1984) J. Biol. Chem. 260,3440-3450 13. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 14. Corbin, J. D., and Reiman, E. M. (1974) Methods Enzymol. 3 8 , 287-290 15. Zwiller, J., Revel, M.-O., and Malviya, A. N. (1985) J.Biol. Chem. 260,1350-1353 16. Laemmli, U. K. (1970) Nature 2 2 7 , 680-685
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