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Purified red blood cell Ca2+-pump ATPase: Evidence for direct inhibition by presumed anti-calmodulin drugs in the absence of calmodulin
Cell
Calcium
3: 545-559,
1982
PURIFIED RED BLOOD CELL CaZ+-PUMP ATPase: EVIDENCE FOR DIRECT INHIBITION BY PRESUMED ANTI-CALMODULIN DRUGS IN THE ABSENCE OF CALMODULIN Frank F. Vincenzi, Evans S. Adunyah, Verena Niggli and Ernest0 Carafoli Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland and Department of Pharmacology, University of Washington, Seattle, WA 98195, USA. (reprint requests to FFV) SUMMARY A variety of presumed anti-calmodulin (anti-CaM) drugs was tested for their potential inhibit0 effects on the isolated, purified and reconstituted CaZY-pump ATPase of human d blood cell membranes. Anti-CaM drugs inhibited the Caz8-pump ATPase both in the absence and presence of added CaM. Qualitatively similar inhibition was observed in two different ATPase assay systems. In asolectin vesicles in the absence of added CaM trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-l-naphthalene- sulfonamide (W-7), vinblastine, dibucaine, imipramine, propranolol and dimethylpropranolol (UM-272) were all inhibitory. Potency of anti-CaM drugs was generally greater on the enzyme reconstituted in asolectin vesicles than on the enzyme reconstituted in phosphatidylcholine vesicles, either in the presence or absence of CaM. The results emphasize that anti-CaM drugs have actions other than to bind to CaM. Possible direct i teraction of amphipathic cationic anti-CaM drugs with the Ca'2+ -pump ATPase and/or its lipid environment is suggested. IN!CRODUCTION Calmodulin (CaM) is involved in the regulation of a wide variety of enzymes (1,2). Among those enzymes which are activa ed above a characteris ic "basal" activity by the complex is the Ca*+-pump ATPase of human red CaM(Ca$+) blood celf (RBC) membranes (3-6). From early studies it was implied, but not demonstr$jed, that ther5+was a direct interaction between CaM(Ca )n and the Ca -pump ATPase. More recently, studies using a photoaffinity derivative of radiolabeled CaM have pf+oviFnddt"h",ipa3$efor a direct interaction between CaM(Ca ) -pump ATPase (7). Niggli & al. (8-10f succeeded in isolating the gw -pump ATPase by using CaM affinity chromatography. This approa not only demonstrated direct interaction between the Ca% -pump ATPase and the important regulatory protein, but al?? allowed reconstitution and functional studies on the Ca pump and measurement of the stoichiometry of the 545
purified plasma membrane Ca2+-pump (10) as well as the preparation of specific antibodies (11). Thus, there are severas+different ways in whic.j+direct interaction between complex and been CaM(Ca ) Ca -pump ATPase have demonstrated. An important chapter in the study of CaM-regulated processes was begun when Levin and Weiss reported that anti-psychotic phenothiazin2+ drugs such as trifluoperazine (TFP) bind to CaM in Ca -dependent manner (12). This could account for selective antagonism of CaM-dependent processes such as reported for many enzymes. Correlation was found between the clinical anti-psychotic potency of various drugs and their potency for in yitro inhibition of cyclic nucleotide phosphodiesterase (13). Thus, a model was presented that anti-psychoy+ 'c drugs antagonize CaM by virtue of binding to the CaM(Ca )n complex. In short, it was that inferred antipsychotics are CaM-binding drugs (CaM-BDs). However, it appears that anti-CaM drugs do more than only bind to CaM. Drugs which selectively antagonize the effects of CaM have been va iously called "CaM antagonists" (14), "intracellular Ca2+ antagonists (15), etc. We will use the term "anti-Can drugs" because this describes the observed effects without implying any particular mechanism. It appears that many investigators now accept that selective inhibition of some process by a presumed anti-CaM drug is good evidence for implicating CaM in the regulation of that process (16). However, a growing body of evidence suggests that great caution should be exercised in interpreting such data (16,17). The present . . work emphasizes this and reconstituted Ca2P$ikp A~~~s~~'~~~~'t~~?~ $~e"~::f:~~ of presumed anti-CaM drugs inhibit the enzyme in the absence of CaM. Two preliminary reports on the problem have been presented from our laboratories (18,19). Methods
BC membranes were isolated as previously described. Ca2$-pump ATPase activity was determined in membranes or the ATPase was purified from the membranes by the methods described by Niggli & nl. (8-10). In short, HBC membranes were solubilized in 0.4% Triton X-100 (w/v) for 10 min. Non- solubilized material was removed by centrifugation at 100,000 x g at 2O C for 30 min. The soluble material was passed over a calmodulin affinity chromatography column in 0.4% Triton X-100, 130 mM KCl, 20 mM HEPES pH 7.4, 1 mM MgCl2, 2 mM dithiothreitol, 50 PM CaCl with 0.5 mg/ml of asolectin or phosphatidyl choline (PCP. The ATPase was eluted with the'same buffer containing 2 mM EDTA instead of CaC12. Phospholipid vesicles incorporating the ATPase were formed by cholate dialysis as described previously (9).
546
Ca2+-pump ATPase activity was determined in two different First, the isolated and reconstituted enzyme was ways. measured at 37' C using a the spectrophotometric assay described by Niggli & al (9). The spectrophotometric procedure involves a coupled enzyme assay system. The production of ADP is coupled to the oxidation of NADH. This is detected as a change in absorbance at 366 nm compared to 550 nm. Thus, the spectrophotometric assay is a rapidly performed assay which allows continuous measurement of ADP production. It lends itself to rapid assay of a small number of samples, and to detection of rapidly occurring events. On the other hand, the spectrophotometric assay is limited in the number of samples which can be measured in, say a few hours, and it is usually not performed with any preincubation of drugs, etc. Second, the ATPase activity was determined at 37' C in an assay system described by Raess and Vincenzi (21, 22). This assay system was originally used for measurement of ATPase activities of isolated RBC membranes. It routinely employs preincubation It is set up with a of enzyme, CaM, drugs, etc. semi-automated inorganic phosphate (Pi) assay and can thus accomodate up to 200 samples in a day. A minor modification of the system was that the ATPase reactions were terminated with 0.33% rather than 3.3% sodium dodecyl sulfate (SDS). Appropriate controls showed that in the concentration ranges studied, the drugs did not interfere with the coupled enzyme assay. In addition, the effect of solvents alone was found to be egligible. All ATPase assays of the reconstituted Ca9+ -pump were performed in the presence of 0.4 HIM A23187, a divalent cation ionophore, in order to dissipate Ca2+ ion gradients which could inhibit ATPase activity. N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W-7) was a gift of Professor H. Hidaka, Mie University, TSU, Mie, Japan. Vinblastine was a gift of Eli Lilly & Co., Indianapolis, IN. Trifluoperazine was a gift of Smith, Kline & French Laboratories, Philadelphia, PA. Dimethylpropranolol (UM-272) was a gift of Professor B. Lucchesi, University of Michigan, Ann Arbor, MI. Other drugs were purchased from Sigma, St. Louis, MO. Drug potencies and IC50 values (+ 95% confidence intervals) were estimated using programs presented by Tallarida and Murray (20) adapted to run on an Apple II Plus Microcomputer. Results
As described previously (91, the Ca2+-pump ATPase enzyme was isolated by CaM affinity chromatography and was reconstituted into vesicles of phosphatidylcholine (PC) or in asolectin by cholate dialysis. A series of drug 547
inhibition experiments performed was using the spectrophotometric enzyme assay system. The addition of CaM to the enzyme in PC vesicles increased its activity. In asolectin vesicles the enzyme had high activity in the absence of CaM because of the presence of acidic phospholipids (9). For initial drug experiments enzyme activity was determined over the course of several minutes. Then, drugs were added in increasing concentrations. After each drug addition the enzymatic activity was determined over the course of several minutes. Fig. 1 shows the cumulative concentr tion-effect curves for TFP-induced inhibition of the Ca9+ -pump ATPase. TFP was a more potent inhibitor (IC50 = 69 +, 1 $JJanon the yhr,ifie\;,,e,nzy;ns reconstituted in asolectin when reconstituted in PC and activated with 2.5 pg/ml of purified CaM (IC 9 = 229 f 1 CM). Thus, when activated by acidic phosphoIipids in the absence of CaM the enzyme was even more sensitive to inhibition by the classical anti-CaM drug, TFP. It could be argued that the decreased sensitivity of the enzyme to TFP in the presence of CaM (2.5 rg/ml) was due to the fact that CaM acted as sink for the drug. This seemed unlikely considering the relative molar ratios of TFP and CaM (approximately 1OO:l). Nevertheless, to test this idea a similar experiment was performed with 0.25 rg/ml of CaM. This was a concentration of CaM that gave approximately half-maximal activation in the spectrophotometric assay system. The results were qualitatively and quantitatively similar to those in Fig. 1. The respective IC values for TFP inhibition of asolectin-dependent and PC/s!M-dependent ATPase activities were 60 + 1 PM and 168 + 1 pM. For comparison the Ca2+-pump ATPase activity of isolated RBC membranes was determined by the spectrophotometric technique. The resultant curve for antagonism of 0.25 pg/ml CaM gave an IC50 value for TFP of 132 PM. A wide range of amphipathic cations has been reported to antagonize CaM-induced activation of various enzymes (18) Fig. 2 shows that a vari+ety of these cationic compounds inhibit the purified Ca -pump ATPase reconstituted in asolectin vesicles withou Data were plotted as % CaM. inhibition of the total Ca5+ -dependent ATPase activity. If anything, this underestimates inhibition of CaM-like activation produced by acidic phospholipids in asoJpc;&;;, All of the putative anti-CaM drugs inhibited the Ca ATPase. As shown above for TFP (Fig.l), the potency of the various anti-CaM drugs was somewhat greater against the enzyme in asolectin vesicles than against the enzyme in PC vesicles with 0.25 pg/ml CaM (data not shown). l
It was initially surprising to find that the drugs were more potent against the asolectin-activated enzyme than against the CaM-activated enzyme. Also, TFP appeared to be CaM-activation of the potent in antagonizing less reconstituted ATPase than was previously reported for 548
100,
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,* l
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o_/ , 20
Fig.
1.
ip //
/
4’ I
Ia
//” 1
100 TRIFLUOPERAZINE
1
I
1000 (uM)
inhibition of Trifluoperazin (TFP) induced -pump ATPase. The activity of re onstituted Ca !S+ Ca S+ -pump ATPase reconstituted in asolectin (solid line) or phosphatidyl choline (PC) (dashed line) the was determined at 37' C using vesicles spectrophotometric assay (10 IIMCa2+, 500 PM ATP, 1 After determination of the mM MgC12, PH 7). initial enzyme rate TFP was added cumulatively. The incubation mixture for PC vesicles contained Values 2.5 pg/ml of purified calmodulin (CaM). TFP was a represent the mean of two experiments. less potent inhibitor of the CaM-activated enzyme in PC vesicles than of the asolectin-activated TFP was also apparently less potent when enzyme. added cumulatively than when preincubated (see Fig. 3.).
antagonizing CaM-activation of the ATPase in RBC membranes When measured using the spectrophotometric assay (21). system, TFP was less potent in antagonizing CaM activation of the isolated enzyme (IC5 = 168 FM) PI;.RBC membranes 132 PM) than in an eagonism of CaM activation of (I%opu;p membranes tested in a more Ca ATPase in RBC traditional calorimetric phosphate assay system (IC50 = 18 PM) (23). The difference in apparent potency appears to be due mainly to the assay conditions and procedures and not the state of the enzyme. In the ATPase assay method of Raess and Vincenzi (22) drugs are preincubated with the RBC membranes. By contrast, the data presented in Fig. 1 were obtained without preincubation of drugs with the enzyme.
549
20
100 DRUG
Fig.
2.
(JIM)
Drug-induced inhibition of Ca'+-pump ATPase reconstituted in asolectin vesicles. TFP (O-O), vinblastine . -.) dibucaine w-7 (X-X), imipramine (a-m), ~ropranblol (A.-& (+-a), and UM-272 (V-V) were added cumulatively as in Fig. 1 and enzyme activity was determined as in Fig. 1. Results in this figure are representative of several similar experiments. All the presumed anti-CaM drugs inhibited the asolectin-activated enzyme.
It was considered that if the dissociation rate of CaM from the enzyme were slow (23) and if these drugs only inhibit the enzyme by binding to CaM not already bound to the enzyme (or to the enzyme without CaM) then the cumulative concentration-effect procedure as performed in Figs. 1 and 2 would underestimate potencies of drugs for antagonism of CaM. To examine this idea several drugs were cumulative procedure and by tested both by the usual preincubating drug and enzyme together for at least 15 min before addition of substrate. In the former case a complete concentration-effect curve was obtained from a single cuvette. In the latter case each concentration was For each drug the determined in a separate cuvette. apparent potency against the CaM-activated enzyme in PC when were vesicles was considerably greater drugs preincubated with the enzyme than when they were added cumulatively. For example, vinblastine was over 5 times as potent, and TFP about 10 times, in this comparison.
550
DRUG
Fig.
3.
(104)
Drug-induced inhibition of ca2+-pump ATPase reconstituted in PC. The reconstituted enzyme was preincubated in a 2 ml assay system for 15 min at 37O C in the absence (solid lines) or presence (dashed lines) of 0.16 rg/ml of CaM and TFP (0-O) or imipramine (n-0) or dibucaine (0-O). Assay method was as described by Raess and Vincenzi (17). ATPase activity was initiated by the addition of ATP and carried out for 120 min at 37' C. TFP was apparently more potent against the CaM-activated ATPase in PC under these conditions than without preincubation (Fig. 1). All drugs inhibited basal activity at high concentrations. None inhibited the basal activity at concentrations less than those which were antagonistic to CaM. Values represent the mean of two experiments
Similar results were obtained using the Ca'+-pump ATPase in isolated REX membranes as the test system (not shown). In contrast to the above results, the potency of drugs against the asolectin-activated enzyme was not very dependent on whether they were preincubated or added cumulatively. In most cases tested, the drugs were even more potent when added cumulatively. For example, the respective preincubation and cumulative IC50 values for TFP were 113 x 2 and 91 ?: 1 pM. For vinblastine the respective values were 489 f 1 and 257 f 10 FM.
551
DRUG
Fig.
4.
(uM)
Drug-induced inhibition of ca2+-pump ATPase reconstituted in asolectin vesicles. Assay was carried out as in Fig. 3. in the absence (solid lines) and presence (dashed lines) of 0.16 rg/ml of CaM and TFP (0-O) or imipramine (m-m) or Addition of CaM resulted in a dibucaine (+-+I. modest increase in enzyme activity. TFP was the most potent inhibitor, both in the absence and presence of CaM. In contrast to the results in inhibited the basal Fig. 3., all three dF_LU+4s (asolectin-activated) Ca -pump ATPase activity at than which were concentrations less those antagonistic to CaM. Values represent the mean of two experiments.
Purified and reconstituted enzyme was assayed under conditions previously reported for REX membrane ATPase assays (21, 22). All the drugs tested were inhibitory under these conditions. In this assay procedure enzyme and buffer with or without CaM or drugs was incubated together at 37' C for 15 min before addition of ATP. The enzyme reaction was also carried out at 37' C. Aliquots were removed at various times and the reaction was terminated by addition.of SDS. The reaction was linear for at least 120 min. Fig. 3 shows the effects of three drugs, Tg+P, imipramine and dibucaine on the activity of purified Ca -pump ATPase reconstituted in PC vesicles. As expected, activity was very low in the absence of added CaM. Addition of 0.16 rg/ml CaM resulted in an approximately eight-fold increase
552
in activity. All three drugs inhibited the CaM-activated portion of the activity, with TFP being the most potent and imipramine and dibucaine being less potent, respectively. All these drugs also inhibited the basal activity of the enzyme but there was some selectivity against the CaM-dependent fraction of the activity. This was not very evident in the reconstituted enzyme which had very low basal activity at high concentrations. When reconstituted in asolectin, the enzyme had higher activity (Fig. 4) but the addition of 0.16 pg/ml of CaM resulted in an approximately 25% increase in activity. Evidently, incorporation into asolectin was less than optimal in this case. In any event, each of the three drugs tested inhibited the enzyme either in the presence or absence of CaM. TFP was the most potent inhibitor, but all the drugs inhibited both the asolectin-dependent and CaM-dependent activities. In particular it should be noted that all of the drugs significantly inhibited the asolectin-dependent enzyme activity at concentrations considerably lower than those needed to inhibit either the basal PC-dependent activity or the CaM-dependent activity. This curious result is not explained by these data but corresponds to the results shown in Figs. 1 and 2. Discussion
The present results show that a wide variety of rmacological agents antagonized the CaM-dependent ;;9+-pump ATPase of human RBC membranes both in the absence and in the presence of CaM. This was true for the enzyme activity measured in membranes and for the purified, reconstituted enzyme. Similar results were obtained using both a spectrophotometric coupled-enzyme ATPase assay and a more traditional calorimetric phosphate assay. The itative nature of the results (i.e., apparent drug potencies) were somewhat dependent on the ATPase assay system. As might be expected, drug potency depends on the lipid content of the system (19). On the other hand, the tative nature of the results (i.e., the finding that anti-CaM drugs inhibit the enzyme even in the absence of CaM) was not dependent on the ATPase assay system. The purified enzyme has been shown to consist of essentially a single polypeptide of approximately 138,000 M, (8). From the present result there seems to be little question that presumed anti-CaM drugs inhibit this enzyme in the absence of endogenous CaM. The specificity of both the "calmodulinomimetic" (17) effect and the anti-CaM effect questioned. Thus, for example, t;~ed;a2ZJ?pur$$zzst; human RBC membranes is activated by CaM (3,4), by acidic phospholipids (10, 24) and by simple amphipathic anions such as oleate (18). Similar activation by such agents has been 553
demonstrated for another CaM-dependent enzyme, cyclic nucleotide phosphodiesterase (25). It has been reported that putative anti-CaM drugs such as trifluoperazine antagonize the activation of these enzymes not only by CaM but by the calmodulinomimetic anions such as acidic phospholipids and fatty acids (16-18). One possible interpretation is that calmodulinomimetic anions bind at the same site as CaM to activate these enzymes. Another possible interpretation is that calmodulinomimetic anions interact with CaM-dependent enzymes, probably at different site(s), but in a manner which produces a state which is very much like that produced when CaM binds. This interpretation fits the observed data in many ways and seems a fair assumption. If this is so, then the effects of anti-CaM drugs can probably be clarified. One need only discard the notion that anti-CaM drugs simply act only as CaM-binding drugs. While this is a useful and perhaps applicable interpretation in certain systems under some conditions, it is, quite obviously, not generally applicable. If one assumes instead, or in addition, that anti-CaM drugs have the capacity to interact directly with CaM-dependent enzymes even (or perhaps only) in the absence of bound CaM then the present data fit into and contribute to a comprehensive pattern. The finding that higher concentrations of anti-CaM drugs are Cas$:;s,a,ry,,,;;E inhibition of the basal activity of the reconstituted in PC than for comparable inhibition of the enzyme activated by CaM or acidic phospholipids may indicate that the drugs interact preferentially with the "activated" A similar differentiation might be form of the enzyme. observed with other CaM-dependent enzymes. The present results would not be expected if TFP acted only by binding to the CaM(Ca2+) complex and preventing its binding to the enzyme, but the? might be expected if TFP exerted a direct inhibitory effect on the enzyme. This could occur by the binding of TFP directly to the enzyme and/or by insertion into the phospholipid vesicle. The not distinguish between these present results do possibilities. The fact that TFP and other anti-CaM drugs are amphipathic and that the concentrations employed were very high makes disruption of the phospholipid environment a very likely possibility, in any event. It has been shown elsewhere that a wide variety of amphipathic cationic dr s are non-competitive antagonists of the activation of Ca" -pump ATPase by CaM (16,18). This finding led to the idea of a direct interaction of drugs with the ATPase and to several related predictions. Among these was the prediction that such drugs would antagonize CaM-activated enzymes, no matter whether activated by CaM z &, by-acidic phospholipids, or by proteolysis (26, 27), Preliminary results in our laboratorp+s indicate that a f;agment of the proteolyzed, purified Ca -pump ATPase is 554
retained by a phenothiazine affinity chromatography column. In addition, some of the presumed anti-CaM drugs described here prevent activation by acidic phospholipids even when the enzyme is in the micellar form (19). Also, these drugs inhibit the enzyme reconstituted in PC after activation by limited proteolysis in the absence of CaM or acidic phospholipids.
This work was supported in part by a grant from the Swiss Nationalfonds.
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Cheung, W.Y. (1980). Calmodulin plays a pivotal role in cellular regulation. Science 207, 19-27.
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Niggli, V., Penniston, J.T. and Carafoli, E. (1979). Purification of the (Ca2+-Mg2+) ATPase from human erythrocyte membranes using a calmodulin affinity column. Journal of Biological Chemistry 254, 9955-9958.
9.
Niggli, V., Adunyah, E.S. and Carafoli, E. (1981). Acidic phospholipids, unsaturated fatty acids, and limited proteolysis mimic the effect of calmoduulin on the purified erythrocyte Ca2+-ATPase. Journal of Biological Chemistry 2356, 8588-8592.
10.
Niggli, V., Adunyah, E.S., Penniston, J.T. and Carafoli, E. (1981). Purified (Ca2+-Mg2+) ATPase of the erythrocyte membrane: Reconstitution and effect of calmodulin and phospholipids. Journal of Biological Chemistry 256, 395-401.
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Verma, A.K., Gorski, J.P. and Penniston, J.T. (1982). Antibodies directed toward human erythrocyte Ca2+-ATPase: Effect on enzyme function and immunoreactivity of Ca2+-ATPases from other sources. Archives of Biochemistry and Biophysics 215, 345-354.
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Levin, R.M. and Weiss, B. (1977). Binding of trifluoperazine to the calcium-dependent activator of cyclic nucleotide phosphodiesterase. Molecular Pharmacology 13, 690-697.
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Lynch, J.J., Rahwan, R.G. and Witiak, D.T. (1981). Effects of 2-substituted 3-dimethylamino-5, 6-methylenedioxyindenes on calcium-induced arrhythmias. Journal of Cardiovascular Pharmacology 3, 49-60.
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Adunyah, E.S., Niggli, V. and Carafoli, E. (1982). The anticalmodulin drugs trifluoperazine and R 24 571 remove the activation of purified erythrocyte Ca2+ ATPase by acidic phospholipids and by controlled proteolysis. FEBS Lett. 143, 65-68.
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Received Revised
4:8:82 version
received
and accepted
559
4:lO:82
Calcium
3: 545-559,
1982
PURIFIED RED BLOOD CELL CaZ+-PUMP ATPase: EVIDENCE FOR DIRECT INHIBITION BY PRESUMED ANTI-CALMODULIN DRUGS IN THE ABSENCE OF CALMODULIN Frank F. Vincenzi, Evans S. Adunyah, Verena Niggli and Ernest0 Carafoli Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland and Department of Pharmacology, University of Washington, Seattle, WA 98195, USA. (reprint requests to FFV) SUMMARY A variety of presumed anti-calmodulin (anti-CaM) drugs was tested for their potential inhibit0 effects on the isolated, purified and reconstituted CaZY-pump ATPase of human d blood cell membranes. Anti-CaM drugs inhibited the Caz8-pump ATPase both in the absence and presence of added CaM. Qualitatively similar inhibition was observed in two different ATPase assay systems. In asolectin vesicles in the absence of added CaM trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-l-naphthalene- sulfonamide (W-7), vinblastine, dibucaine, imipramine, propranolol and dimethylpropranolol (UM-272) were all inhibitory. Potency of anti-CaM drugs was generally greater on the enzyme reconstituted in asolectin vesicles than on the enzyme reconstituted in phosphatidylcholine vesicles, either in the presence or absence of CaM. The results emphasize that anti-CaM drugs have actions other than to bind to CaM. Possible direct i teraction of amphipathic cationic anti-CaM drugs with the Ca'2+ -pump ATPase and/or its lipid environment is suggested. IN!CRODUCTION Calmodulin (CaM) is involved in the regulation of a wide variety of enzymes (1,2). Among those enzymes which are activa ed above a characteris ic "basal" activity by the complex is the Ca*+-pump ATPase of human red CaM(Ca$+) blood celf (RBC) membranes (3-6). From early studies it was implied, but not demonstr$jed, that ther5+was a direct interaction between CaM(Ca )n and the Ca -pump ATPase. More recently, studies using a photoaffinity derivative of radiolabeled CaM have pf+oviFnddt"h",ipa3$efor a direct interaction between CaM(Ca ) -pump ATPase (7). Niggli & al. (8-10f succeeded in isolating the gw -pump ATPase by using CaM affinity chromatography. This approa not only demonstrated direct interaction between the Ca% -pump ATPase and the important regulatory protein, but al?? allowed reconstitution and functional studies on the Ca pump and measurement of the stoichiometry of the 545
purified plasma membrane Ca2+-pump (10) as well as the preparation of specific antibodies (11). Thus, there are severas+different ways in whic.j+direct interaction between complex and been CaM(Ca ) Ca -pump ATPase have demonstrated. An important chapter in the study of CaM-regulated processes was begun when Levin and Weiss reported that anti-psychotic phenothiazin2+ drugs such as trifluoperazine (TFP) bind to CaM in Ca -dependent manner (12). This could account for selective antagonism of CaM-dependent processes such as reported for many enzymes. Correlation was found between the clinical anti-psychotic potency of various drugs and their potency for in yitro inhibition of cyclic nucleotide phosphodiesterase (13). Thus, a model was presented that anti-psychoy+ 'c drugs antagonize CaM by virtue of binding to the CaM(Ca )n complex. In short, it was that inferred antipsychotics are CaM-binding drugs (CaM-BDs). However, it appears that anti-CaM drugs do more than only bind to CaM. Drugs which selectively antagonize the effects of CaM have been va iously called "CaM antagonists" (14), "intracellular Ca2+ antagonists (15), etc. We will use the term "anti-Can drugs" because this describes the observed effects without implying any particular mechanism. It appears that many investigators now accept that selective inhibition of some process by a presumed anti-CaM drug is good evidence for implicating CaM in the regulation of that process (16). However, a growing body of evidence suggests that great caution should be exercised in interpreting such data (16,17). The present . . work emphasizes this and reconstituted Ca2P$ikp A~~~s~~'~~~~'t~~?~ $~e"~::f:~~ of presumed anti-CaM drugs inhibit the enzyme in the absence of CaM. Two preliminary reports on the problem have been presented from our laboratories (18,19). Methods
BC membranes were isolated as previously described. Ca2$-pump ATPase activity was determined in membranes or the ATPase was purified from the membranes by the methods described by Niggli & nl. (8-10). In short, HBC membranes were solubilized in 0.4% Triton X-100 (w/v) for 10 min. Non- solubilized material was removed by centrifugation at 100,000 x g at 2O C for 30 min. The soluble material was passed over a calmodulin affinity chromatography column in 0.4% Triton X-100, 130 mM KCl, 20 mM HEPES pH 7.4, 1 mM MgCl2, 2 mM dithiothreitol, 50 PM CaCl with 0.5 mg/ml of asolectin or phosphatidyl choline (PCP. The ATPase was eluted with the'same buffer containing 2 mM EDTA instead of CaC12. Phospholipid vesicles incorporating the ATPase were formed by cholate dialysis as described previously (9).
546
Ca2+-pump ATPase activity was determined in two different First, the isolated and reconstituted enzyme was ways. measured at 37' C using a the spectrophotometric assay described by Niggli & al (9). The spectrophotometric procedure involves a coupled enzyme assay system. The production of ADP is coupled to the oxidation of NADH. This is detected as a change in absorbance at 366 nm compared to 550 nm. Thus, the spectrophotometric assay is a rapidly performed assay which allows continuous measurement of ADP production. It lends itself to rapid assay of a small number of samples, and to detection of rapidly occurring events. On the other hand, the spectrophotometric assay is limited in the number of samples which can be measured in, say a few hours, and it is usually not performed with any preincubation of drugs, etc. Second, the ATPase activity was determined at 37' C in an assay system described by Raess and Vincenzi (21, 22). This assay system was originally used for measurement of ATPase activities of isolated RBC membranes. It routinely employs preincubation It is set up with a of enzyme, CaM, drugs, etc. semi-automated inorganic phosphate (Pi) assay and can thus accomodate up to 200 samples in a day. A minor modification of the system was that the ATPase reactions were terminated with 0.33% rather than 3.3% sodium dodecyl sulfate (SDS). Appropriate controls showed that in the concentration ranges studied, the drugs did not interfere with the coupled enzyme assay. In addition, the effect of solvents alone was found to be egligible. All ATPase assays of the reconstituted Ca9+ -pump were performed in the presence of 0.4 HIM A23187, a divalent cation ionophore, in order to dissipate Ca2+ ion gradients which could inhibit ATPase activity. N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W-7) was a gift of Professor H. Hidaka, Mie University, TSU, Mie, Japan. Vinblastine was a gift of Eli Lilly & Co., Indianapolis, IN. Trifluoperazine was a gift of Smith, Kline & French Laboratories, Philadelphia, PA. Dimethylpropranolol (UM-272) was a gift of Professor B. Lucchesi, University of Michigan, Ann Arbor, MI. Other drugs were purchased from Sigma, St. Louis, MO. Drug potencies and IC50 values (+ 95% confidence intervals) were estimated using programs presented by Tallarida and Murray (20) adapted to run on an Apple II Plus Microcomputer. Results
As described previously (91, the Ca2+-pump ATPase enzyme was isolated by CaM affinity chromatography and was reconstituted into vesicles of phosphatidylcholine (PC) or in asolectin by cholate dialysis. A series of drug 547
inhibition experiments performed was using the spectrophotometric enzyme assay system. The addition of CaM to the enzyme in PC vesicles increased its activity. In asolectin vesicles the enzyme had high activity in the absence of CaM because of the presence of acidic phospholipids (9). For initial drug experiments enzyme activity was determined over the course of several minutes. Then, drugs were added in increasing concentrations. After each drug addition the enzymatic activity was determined over the course of several minutes. Fig. 1 shows the cumulative concentr tion-effect curves for TFP-induced inhibition of the Ca9+ -pump ATPase. TFP was a more potent inhibitor (IC50 = 69 +, 1 $JJanon the yhr,ifie\;,,e,nzy;ns reconstituted in asolectin when reconstituted in PC and activated with 2.5 pg/ml of purified CaM (IC 9 = 229 f 1 CM). Thus, when activated by acidic phosphoIipids in the absence of CaM the enzyme was even more sensitive to inhibition by the classical anti-CaM drug, TFP. It could be argued that the decreased sensitivity of the enzyme to TFP in the presence of CaM (2.5 rg/ml) was due to the fact that CaM acted as sink for the drug. This seemed unlikely considering the relative molar ratios of TFP and CaM (approximately 1OO:l). Nevertheless, to test this idea a similar experiment was performed with 0.25 rg/ml of CaM. This was a concentration of CaM that gave approximately half-maximal activation in the spectrophotometric assay system. The results were qualitatively and quantitatively similar to those in Fig. 1. The respective IC values for TFP inhibition of asolectin-dependent and PC/s!M-dependent ATPase activities were 60 + 1 PM and 168 + 1 pM. For comparison the Ca2+-pump ATPase activity of isolated RBC membranes was determined by the spectrophotometric technique. The resultant curve for antagonism of 0.25 pg/ml CaM gave an IC50 value for TFP of 132 PM. A wide range of amphipathic cations has been reported to antagonize CaM-induced activation of various enzymes (18) Fig. 2 shows that a vari+ety of these cationic compounds inhibit the purified Ca -pump ATPase reconstituted in asolectin vesicles withou Data were plotted as % CaM. inhibition of the total Ca5+ -dependent ATPase activity. If anything, this underestimates inhibition of CaM-like activation produced by acidic phospholipids in asoJpc;&;;, All of the putative anti-CaM drugs inhibited the Ca ATPase. As shown above for TFP (Fig.l), the potency of the various anti-CaM drugs was somewhat greater against the enzyme in asolectin vesicles than against the enzyme in PC vesicles with 0.25 pg/ml CaM (data not shown). l
It was initially surprising to find that the drugs were more potent against the asolectin-activated enzyme than against the CaM-activated enzyme. Also, TFP appeared to be CaM-activation of the potent in antagonizing less reconstituted ATPase than was previously reported for 548
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100 TRIFLUOPERAZINE
1
I
1000 (uM)
inhibition of Trifluoperazin (TFP) induced -pump ATPase. The activity of re onstituted Ca !S+ Ca S+ -pump ATPase reconstituted in asolectin (solid line) or phosphatidyl choline (PC) (dashed line) the was determined at 37' C using vesicles spectrophotometric assay (10 IIMCa2+, 500 PM ATP, 1 After determination of the mM MgC12, PH 7). initial enzyme rate TFP was added cumulatively. The incubation mixture for PC vesicles contained Values 2.5 pg/ml of purified calmodulin (CaM). TFP was a represent the mean of two experiments. less potent inhibitor of the CaM-activated enzyme in PC vesicles than of the asolectin-activated TFP was also apparently less potent when enzyme. added cumulatively than when preincubated (see Fig. 3.).
antagonizing CaM-activation of the ATPase in RBC membranes When measured using the spectrophotometric assay (21). system, TFP was less potent in antagonizing CaM activation of the isolated enzyme (IC5 = 168 FM) PI;.RBC membranes 132 PM) than in an eagonism of CaM activation of (I%opu;p membranes tested in a more Ca ATPase in RBC traditional calorimetric phosphate assay system (IC50 = 18 PM) (23). The difference in apparent potency appears to be due mainly to the assay conditions and procedures and not the state of the enzyme. In the ATPase assay method of Raess and Vincenzi (22) drugs are preincubated with the RBC membranes. By contrast, the data presented in Fig. 1 were obtained without preincubation of drugs with the enzyme.
549
20
100 DRUG
Fig.
2.
(JIM)
Drug-induced inhibition of Ca'+-pump ATPase reconstituted in asolectin vesicles. TFP (O-O), vinblastine . -.) dibucaine w-7 (X-X), imipramine (a-m), ~ropranblol (A.-& (+-a), and UM-272 (V-V) were added cumulatively as in Fig. 1 and enzyme activity was determined as in Fig. 1. Results in this figure are representative of several similar experiments. All the presumed anti-CaM drugs inhibited the asolectin-activated enzyme.
It was considered that if the dissociation rate of CaM from the enzyme were slow (23) and if these drugs only inhibit the enzyme by binding to CaM not already bound to the enzyme (or to the enzyme without CaM) then the cumulative concentration-effect procedure as performed in Figs. 1 and 2 would underestimate potencies of drugs for antagonism of CaM. To examine this idea several drugs were cumulative procedure and by tested both by the usual preincubating drug and enzyme together for at least 15 min before addition of substrate. In the former case a complete concentration-effect curve was obtained from a single cuvette. In the latter case each concentration was For each drug the determined in a separate cuvette. apparent potency against the CaM-activated enzyme in PC when were vesicles was considerably greater drugs preincubated with the enzyme than when they were added cumulatively. For example, vinblastine was over 5 times as potent, and TFP about 10 times, in this comparison.
550
DRUG
Fig.
3.
(104)
Drug-induced inhibition of ca2+-pump ATPase reconstituted in PC. The reconstituted enzyme was preincubated in a 2 ml assay system for 15 min at 37O C in the absence (solid lines) or presence (dashed lines) of 0.16 rg/ml of CaM and TFP (0-O) or imipramine (n-0) or dibucaine (0-O). Assay method was as described by Raess and Vincenzi (17). ATPase activity was initiated by the addition of ATP and carried out for 120 min at 37' C. TFP was apparently more potent against the CaM-activated ATPase in PC under these conditions than without preincubation (Fig. 1). All drugs inhibited basal activity at high concentrations. None inhibited the basal activity at concentrations less than those which were antagonistic to CaM. Values represent the mean of two experiments
Similar results were obtained using the Ca'+-pump ATPase in isolated REX membranes as the test system (not shown). In contrast to the above results, the potency of drugs against the asolectin-activated enzyme was not very dependent on whether they were preincubated or added cumulatively. In most cases tested, the drugs were even more potent when added cumulatively. For example, the respective preincubation and cumulative IC50 values for TFP were 113 x 2 and 91 ?: 1 pM. For vinblastine the respective values were 489 f 1 and 257 f 10 FM.
551
DRUG
Fig.
4.
(uM)
Drug-induced inhibition of ca2+-pump ATPase reconstituted in asolectin vesicles. Assay was carried out as in Fig. 3. in the absence (solid lines) and presence (dashed lines) of 0.16 rg/ml of CaM and TFP (0-O) or imipramine (m-m) or Addition of CaM resulted in a dibucaine (+-+I. modest increase in enzyme activity. TFP was the most potent inhibitor, both in the absence and presence of CaM. In contrast to the results in inhibited the basal Fig. 3., all three dF_LU+4s (asolectin-activated) Ca -pump ATPase activity at than which were concentrations less those antagonistic to CaM. Values represent the mean of two experiments.
Purified and reconstituted enzyme was assayed under conditions previously reported for REX membrane ATPase assays (21, 22). All the drugs tested were inhibitory under these conditions. In this assay procedure enzyme and buffer with or without CaM or drugs was incubated together at 37' C for 15 min before addition of ATP. The enzyme reaction was also carried out at 37' C. Aliquots were removed at various times and the reaction was terminated by addition.of SDS. The reaction was linear for at least 120 min. Fig. 3 shows the effects of three drugs, Tg+P, imipramine and dibucaine on the activity of purified Ca -pump ATPase reconstituted in PC vesicles. As expected, activity was very low in the absence of added CaM. Addition of 0.16 rg/ml CaM resulted in an approximately eight-fold increase
552
in activity. All three drugs inhibited the CaM-activated portion of the activity, with TFP being the most potent and imipramine and dibucaine being less potent, respectively. All these drugs also inhibited the basal activity of the enzyme but there was some selectivity against the CaM-dependent fraction of the activity. This was not very evident in the reconstituted enzyme which had very low basal activity at high concentrations. When reconstituted in asolectin, the enzyme had higher activity (Fig. 4) but the addition of 0.16 pg/ml of CaM resulted in an approximately 25% increase in activity. Evidently, incorporation into asolectin was less than optimal in this case. In any event, each of the three drugs tested inhibited the enzyme either in the presence or absence of CaM. TFP was the most potent inhibitor, but all the drugs inhibited both the asolectin-dependent and CaM-dependent activities. In particular it should be noted that all of the drugs significantly inhibited the asolectin-dependent enzyme activity at concentrations considerably lower than those needed to inhibit either the basal PC-dependent activity or the CaM-dependent activity. This curious result is not explained by these data but corresponds to the results shown in Figs. 1 and 2. Discussion
The present results show that a wide variety of rmacological agents antagonized the CaM-dependent ;;9+-pump ATPase of human RBC membranes both in the absence and in the presence of CaM. This was true for the enzyme activity measured in membranes and for the purified, reconstituted enzyme. Similar results were obtained using both a spectrophotometric coupled-enzyme ATPase assay and a more traditional calorimetric phosphate assay. The itative nature of the results (i.e., apparent drug potencies) were somewhat dependent on the ATPase assay system. As might be expected, drug potency depends on the lipid content of the system (19). On the other hand, the tative nature of the results (i.e., the finding that anti-CaM drugs inhibit the enzyme even in the absence of CaM) was not dependent on the ATPase assay system. The purified enzyme has been shown to consist of essentially a single polypeptide of approximately 138,000 M, (8). From the present result there seems to be little question that presumed anti-CaM drugs inhibit this enzyme in the absence of endogenous CaM. The specificity of both the "calmodulinomimetic" (17) effect and the anti-CaM effect questioned. Thus, for example, t;~ed;a2ZJ?pur$$zzst; human RBC membranes is activated by CaM (3,4), by acidic phospholipids (10, 24) and by simple amphipathic anions such as oleate (18). Similar activation by such agents has been 553
demonstrated for another CaM-dependent enzyme, cyclic nucleotide phosphodiesterase (25). It has been reported that putative anti-CaM drugs such as trifluoperazine antagonize the activation of these enzymes not only by CaM but by the calmodulinomimetic anions such as acidic phospholipids and fatty acids (16-18). One possible interpretation is that calmodulinomimetic anions bind at the same site as CaM to activate these enzymes. Another possible interpretation is that calmodulinomimetic anions interact with CaM-dependent enzymes, probably at different site(s), but in a manner which produces a state which is very much like that produced when CaM binds. This interpretation fits the observed data in many ways and seems a fair assumption. If this is so, then the effects of anti-CaM drugs can probably be clarified. One need only discard the notion that anti-CaM drugs simply act only as CaM-binding drugs. While this is a useful and perhaps applicable interpretation in certain systems under some conditions, it is, quite obviously, not generally applicable. If one assumes instead, or in addition, that anti-CaM drugs have the capacity to interact directly with CaM-dependent enzymes even (or perhaps only) in the absence of bound CaM then the present data fit into and contribute to a comprehensive pattern. The finding that higher concentrations of anti-CaM drugs are Cas$:;s,a,ry,,,;;E inhibition of the basal activity of the reconstituted in PC than for comparable inhibition of the enzyme activated by CaM or acidic phospholipids may indicate that the drugs interact preferentially with the "activated" A similar differentiation might be form of the enzyme. observed with other CaM-dependent enzymes. The present results would not be expected if TFP acted only by binding to the CaM(Ca2+) complex and preventing its binding to the enzyme, but the? might be expected if TFP exerted a direct inhibitory effect on the enzyme. This could occur by the binding of TFP directly to the enzyme and/or by insertion into the phospholipid vesicle. The not distinguish between these present results do possibilities. The fact that TFP and other anti-CaM drugs are amphipathic and that the concentrations employed were very high makes disruption of the phospholipid environment a very likely possibility, in any event. It has been shown elsewhere that a wide variety of amphipathic cationic dr s are non-competitive antagonists of the activation of Ca" -pump ATPase by CaM (16,18). This finding led to the idea of a direct interaction of drugs with the ATPase and to several related predictions. Among these was the prediction that such drugs would antagonize CaM-activated enzymes, no matter whether activated by CaM z &, by-acidic phospholipids, or by proteolysis (26, 27), Preliminary results in our laboratorp+s indicate that a f;agment of the proteolyzed, purified Ca -pump ATPase is 554
retained by a phenothiazine affinity chromatography column. In addition, some of the presumed anti-CaM drugs described here prevent activation by acidic phospholipids even when the enzyme is in the micellar form (19). Also, these drugs inhibit the enzyme reconstituted in PC after activation by limited proteolysis in the absence of CaM or acidic phospholipids.
This work was supported in part by a grant from the Swiss Nationalfonds.
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