Evidence for a specific recognition site for tiflucarbine on calmodulin

European Journal of Pharmacology - Molecular Pharmacology Sectiol:. 189 (1990) 411-4t8

411

Elsevier EJPMOL 90125

Evidence for a specific recognition site for tiflucarbine on calmodulin B e r n a r d H. S c h m i d t t, T h o m a s G l a s c r i P e t e r - R u d o l f Seide! 2 a n d J~Srg T r a b e r 1 t Troponwerke, Institute for Neurobiolo,~', Department of Bic=hemical Pharmacol.. D-5000 KiJtn. F.R.G., and 2 Chemical Research Laboratories, Pharrna Div.. Bayer AG, D-5600 Wuppertal L ER. G.

Received 3 May 1990, revised MS received 24 July 1990. accepted 28 AttguM19~3

The putative antidepressant drug tiflucarbine (BAY P ,1495) has previously been shov, n to inhibit calmodutia-dependent cyclic nucleotide phosphodiesterase competitively with respect to calmodulin. In order to determ;ne whether this effect is mediated by a direct interaction with calmodulim we measured the effects of radiolabelIed tifluearbine in a direct ligand binding assay, using agarose-immobilized cahnodulin. [JH]Tiflucarbine associated with low micromolar affinity with an apparently homogeneous class of recognition sites on catmodulin-agarose. No binding could be observed on ea!modulin-deficient agarose. The effect was specific, saturable and reversible. Tifiucarbine was the most potent ealmodulin antagonist from a variety of structural analogues examined. The potenci~ of these derivatives to inhibit calmodulin-stimulated phosphodiesterase significantly correlated with their affinities towards the tiftucarbine binding site on calmodulin. No such correlation was evident when structnraily unrelated reference compounds were tested. The association of tiflucarbine with ealmodulin thus appe::rs pharmacologically specific and selective and possibly contributes to the potent antidepres:,ant activity of the drug. Tiflucarbine; Calmodulin (binding to); Cyclic nucleotide phosphodiesterase

1. Introduction Tiflucarbine (fig. 1) is a tetrahydrothieno-7carboline derivative showing potent activity in animal models indicative for antidepressant, anxiolytic and antiaggressive properties (Glaser and Seidel, 1987; S c h u u r m a n et al., 1986). On the other hand, the drug is virtually devoid of anticholinergic or antihistaminergic side effects, which are a c o m m o n drawback in tke use of a great n u m b e r of antidepressant therapeutics today. This interesting profile in behavioral studies is thought to be related to the drug's ability to selectively interact with central 5-HT 2 receptors (Glaser and

Seidel, 1987: Schuurman et aL. !986) and to downregulate both 5-.HT2 receptors and fi-adrenoceptors in rat brain following subchronic treatment (Glaser and SeideL 1987: Schmidt and Schultz, 1986). In a previous study (Schmidt and Schultz, t986), we observed that the/3-adrenergic adaptations in rat cerebral cortex during a 10-day oral treatment with tiflucarbine were closely paralleled by an increase in the activity of cahnodulin-dependent

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412 cyclic nucleotide phosphodiesterase in the same tissue. Both effects were prevented by the destruction of bi-ain adrenergic nerve terminals via intracerebroventricular injections of the neurotoxin 6-OHDA prior to drug treatment, indicating a functional linkage between these effects. Indeed, tiflucarbine was shown to selectively inhibit the calmodulin-dependent isozyme(s) of phosphodiesterases from rat and pig brain, competitively with exogenous caimodulin (Schmidt and Schultz, 1986). However, it remains to be established whether tiflucarbine acts by a direct interaction with the Ca2+-trigger protein itself or with the calmodulin-recognition site on the enzyme. For this purpose, we developed a ligand binding assay, using caimodulin covalently immobilized on agarose, to detect possible specific binding sites for the radiolabelled compound, [3H]tiflucarbine, on calmodulin.

2. Materials and methods

Tiflucarbine and structural variations of tiflucarbine were synthetized in our chemical facilities at the Bayer Pharma Division (Wuppertal, F.R.G.). [3H]cyclic 3' : 5"-adenosine monophosphate ([3H]cAMP) and [3H]tiflucarbine (custom synthetized) were obtained from Amersham-Buehler (Braunsehw~lg, F.R.G.). Due to its chemical instability, [3H]tiflucarbine had to be parified before use by thin-layer chromatography on silica gel 60 F254 (Merck, Darmstadt, F.R.G.). The solvent consisted of 25 ml dichlorethane, 25 ml cyclohexane, 25 ml isopropanol and 2 ml ammonium hydroxide 25% in 0.1 mM ascorbic acid. Bovine heart calmodulin-dependent phosphodiesterase was from Boehringer (Mannheim, F.R.G.). All other drugs and chemicals including calmodulin and calmodulin-agarose were purchased from Sigma (Munich, F.R.G.). Standard binding experiments were conducted at 25°C; 40 #1 of calmodulin-agarose, corresponding to 25/~g of calmodulin, were diluted in a f'mal volume of 200 tzl of buffer containing TrisHC! 10 mM, CaCI 2 1 mM and MgCI 2 3 mM (pH 7.4). For competition experSments, drugs under study were included in this mixture. The reaction

was started by the addition of [3H]tiflucarbine (0.5 #M), incubated for 30 min at room temperature and stopped by addition of 3 ml of ice-cold incubation buffer followed by immediate suction through Whatman G F / C filters. Filters were rinsed three times with 3 mi of cold buffer, placed into scintillation vials and measured for radioactivity in a liquid scintillation counter. Determination of nonspecific binding was performed in the presence of 100 # M of nnlabelled tiflucarbine. Phosphodiesterase measurements were carried out by the method of PiSch (1971). Briefly, the incubation medium (500 /~1) contained Tns-HCI 10 mM pH 7.4, CaCI 2 1 mM, MgCI 2 3 mM, A M P 1 mM, calmodulin-dependent phosphodiesterase 50 gg, calmodulin 10 ng a~d drugs at the concentrations indicated. The reaction was started by the additic,a of 20 ItM [3H]cAMP (50 nCi), incubated for 15 rain at 3 7 ° C and stopped with 200 #1 of 0.2 M ZnSO4. Under these assay conditions, about 10-15% of the substrate were used up during the reaction in the absence of inhibitory drugs. Following coprecipitation and centrifugation of the reaction product, [3H]AMP, with Ba(OH)2, the remaining substrate was quantitated in the supernatant by liquid scintillation counting. If not stated otherwise, data in the figures and text are the means + S.E.M. of three independent experiments, each carried out in triplicates. Statistics were calculated using the Student's t-test for independent measures.

3. Results If 25/~g of calmodulin that was immobilized on agarose was incubated with [3H]tiflucarbine for 30 rain, filtered and washed, radioactivity was dosedependently recovered on the filter. The curve (not shown) was linear up to a concentration of 100 nM [3H]tiflucarbine, with a mean recovery rate of 24.5 + 0.8% of the total radioactivity (N = 17). In the presence of 100 ttM unlabelled tiflucarbine, this value decreased to 7.3 + 0.3% (N = 17) of the total radioactivity. This displacement by unlabelled tifluearbine was dose dependent (fig. 2) with an ICso value of 8.6 + 0.9 /LM (N = 3). By

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Fig. 2. Effect of increasingisotopicdilution of 13Hltiftt~carbine (3,8 riM, 75026 epm) by unlabelled tiflucarbineon total radioligand binding to 25 /zg calmodulin, covalently attached to agarose. Incubationtime 30 rain. calculation of the dispiacable, spcdfic binding from fig. 2 we obtained the saturation isotherm (fig. 3), which demonstrates that the specific association of tiflucarbine to calmodulin-agarose is concentration-dependent and saturable. From the Seatchard analysis (insert in fig. 3)" it is obvious that tiflucsrbine binds to an apparently homogeneous class of binding sites. The equilibrium dis-

sociation constant K D is calculated to 9.9 _+0,78 /LM, the density of accessible binding sites B,),~ is 23 _+ 1.8 n m o l / m g protein (N = 3). According to the results depicted in figs. 2 and 3, the tiflucarbine concentration in all following experiments was fixed to 0.5/*M and 100 ~M for the oetermination of total and unspecific binding, respectively. The association of [3H]tiflucarbine to calmodulin-agarosc at 25°C was rapid with a half lifetime of 12 s (fig. 4). To determine the reversibility of the binding, 100 FM of unlabelled tiflucarbine was added to the incubation medium after equilibrium had been reached. The radioligand dissociated with a half lifetime of about 3.5 s (fig. 4). Half lifetime values about twice as high were obtained when the same experiment was carried out at 4 ° C (t8 s for association lind 6 s for dissociation, data not shown). From these kinetic data, a K D value of 3.4 /~M can be calculated, which is in the same range a~ the value obtained from the Scatchard andlysis of the saturation isotherm. Although from the point of reproducibility of the re.suits there was no evidence for a toss of

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Fig. 4. Association and dissociation kinetics of specific [SH]fiflucarbinebinding to calmodulin-agarose at 25 o C. 45000 cpm of the purified [3H]tiflucarbine preparation were adjusted to a final concentration of 0.5/zM with uniabefied tiflncarbine and incubated with 40 #1 of calmodulin-agaros¢ (corresponding to 25 .ag of calmodulin) for selected time periods. Unspecific binding was determined in parallel tubes containing 100 taM of uniabelled tiflucarbine. After a 30-min incubation period, dissociation was initiated by addition of 100 FM unlabelled tiflucarbine (arrow); and the remaining binding measured at the time points indicated. Each point of the curve represents a single measurement of [aH]tiflucarbine binding at a given incubation time. The experiment was replicated two times with identical results.

binding due to filtration, we performed a series of experiments to validate this assumption. Under our experimental cenditions we did not observe any significant ( > 1%) loss during the stop and filtration procedure. On the other hand, when the dilution volume was enhanced to 200 times the incubation sample~ partial dissociation occurrexi with a p p r o x i m a t e l y the seane h a l f lifetime (10 s) as t h a t o b s e r v e d in the cold following a 2G0-fold isotopic dilution. T o v e r i f y w h e t h e r t i f l u c a r b i n e b i n d s to c a l m o d u f i n o r to the a g a r o s e m a t r i x , w e also perf o r m e d b i n d i n g e x p e r i m e n t s using a g a r o s e free o f c a l m o d u l i n (fig. 5). T h e r e w a s n o specific b i n d i n g o f [ 3 H ] t i f l u c a r b i n e to the calmodu!in-deficient

support up to gel concentrations of 30~ in the assay. On the other hand, the amount of [3H]tiflucarbine specifically bound to calmodulinagarose was clearly dependent on the gel concentration and was saturable with half maximal values of 2.2% of calmodulin-agaros¢ in the test. ~ u m i n g an M, of calmodulin of about 17 kDa, ::i i!

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Fig. 5. Comparison of specific [3H]tiflucarbine binding to agarose with and without covalendy attached ca]moduILn. [3HITiflucarbine (65696 cpm) was assayed at a final concentration of 0.5 .aM. Incubation time was 30 min. Unspecific binding was estimated as binding in the presence of 100 .aM ,.mlabelled tiflucarbine.

this EDho value corresponds to a calmodulinconcentration of about 140 p g / m l . Competition experiments with bovine serum albumin were carried out to exclude the possibifty that the selective binding of [ aH]tiflucarbine to the calmodalin-moiety of calmodulin-agarose was due to a rather unselective affinity to protein (fig. 6). However, bovine serum albumin did not compete for [3H]tiflucarbine binding to calmodulin-agarose except at high concentrations (ICs0 7.5 mg/mi), indicating an at least 50-fold greater selectivity of tiflucarbine for calmodulin than for bovine serum albumin. Calmodulin is a Ca2+-binding protein which binds up to 4 mol of Ca z+ per tool of protein. It

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~,15 thereby undergoes a switch in conformation resuiting in the orientation of hydrophobic sites to the outer surface of the molecule. We evaluated the dependency of specific [3H]tiflucarbine binding to eaimodulin on the presence of the metal ion in the incubation medium in order to see whether these hydrophobic regions are of major importance for drug binding. Addition of 10 mM of the Ca 2+ chelator EGTA to test tubes containing 100 /~M Ca 2+ completely abolished specific [3H]tiflucarbine binding. The effect was concentration-dependent with an ECs0 value of 3.6 _+ 0.2 mM (fig. 7). This value was further decreased to 0,9+0.1 mM when ~.Le EGTA-dose response curve was performed starting from an initial concentration of 1 0 / t M Ca 2+, thus excluding a direct competifive displacement of [~H]tiflucarbtae by EGTA (data not shown). It remains to be demonstrated that the reported inhibition of calmodulin-stimulated p h o s phodiesterase (Sehmidt and Schultz, 1986) by tifluearbine is indeed mediated by the observed binding of the drug to calmodulin. For this purpose we compared the potencies of a number of structural derivatives of tifluearbine to inhibit calmodulin-dependent phosphodiesterase from bovine heart and to displace [3H]tiflucarbine binding to calmodulin-agarose (fig, 8). Amongst the compounds studied, tiflucarbine was the most potent drug in either paradigm. A significant correlation (R = 0.757, 2P < 0.01) of the parameters examined indicated a similar structure-depend-

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Fig. g. Correlation of the ability of tiflucarbine and analogu~ to inhibit calmodulin-dependentphosphodiesteraseand to bind to caImodulin-agarose.R = 0.757: 2P < 0.01.

ency for both effects. However, no such correlation was obse~,ed when tiflucarbine was compared to other, structurally unrelated calmodulin inhibitors such as compound 48/80, calmidazolium, trifiuoperazine, chlorpromazine and W-7 (fig. 9). In this way pharmacological .selectivity and specificity of tifluearbine binding to calmodulin could be demonstrated.

4. Discussion By means of an interaction vntn calmodulin-dependent enzymes, a great variety of structurally

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Fig. 9. Effects of re~erenee cm'modutin-antagonists on calmodulin-sfimulat~ pbosphod~ester.~ and on ~pecific 3Hlfi•ucatbine hi.halingto calmodulin-agarose.R = 0.355; nol significant. 1, compound47,/80; 2. calmi'dazedium; 3, tritluc, perazine: 4, tiflucarh~ne: 5, cMorpromazine: 6, W-7.

416

very different drugs have been discovered to inhibit calmodulin functions in vitro and in situ (for review see Vogel, 1988), and the number is still growing (Jefferson and Schulman, 1988; Sch~ichtele et al., 1989). With such enzyme inhibition tests it is, however, very difficult to unequivocally define the site of drug interaction to calmodulin, since an effect on the calmodulin-recognizing regulatory site of the respective enzyme can hardly be excluded. An alternative way to solve this problem is to measure drug binding to calmodulin directly. Furtherrnore, such an approach allows the characterization of the drug binding site on the Ca2+-trigger protein. To date, two techniques have been used in the literature to quantitatively determine an association of drugs with calmodulin: the equilibrinm dialysis method (Levin and Weiss, 1979) and a filter binding test using ~25J-labelled calmodulin to measure recovery (Morgan et al., 1987). The numerous advantages of the latter method, e.g., higher sensitivity, short procedure of protocol or high signal/noise ratio led us to further develop the technique, introducing the use of agaroseoimmobilized ealmodulin instead of free calmodulin in order to circumvent the necessity of recovery experiments. With this method we were able to demonstrate that ~iz'ivcarbine binds with low mic,,,~olar affinity to an apparently homogeneous class of binding sites on calmodulin-agarose. Binding is saturable, reversible and structure-dependent, whereby the tbJophen residue ~s well as an unsubstituted nitrogen h,. the indole structure seem to be of crucial importance for the binding affinity (data not shown). The finding that no specific binding was observed on agarose devoid of calmodulin, together with the lack of ability of bovine serum albumin to displace tiflucarbine binding to the gel at low concentrations, leads us to speculate that the binding is indeed to calmodulin. The selectivity for calmodulin with respect to other functional Ca2+-binding proteins, however, remains to be established. According to the manufacturer's indication that ! ml of the gel material used throughout this study contai~s 1.3 nag of lyophilized ealmodulin, the B ~ value estimated from the Scatchard plot of t h e specific tiflucarbine binding to calmodulin-

agarose can be calculated to a theoretical stoichiometric ratio of 0.39 tool of tiflucarbine binding sites per tool of cahnodulin. However, a calculation on a weight basis may be somewhat mislead~ ing, since a purified calmodulin lyophilisate was reported to contain up to 52% of impurities by weight, consisting mainly of buffer salts (Morgan et al., 1987). Moreover, it cannot be assumed that all drug binding sites on the calmodulin molecule attached to the agarose gel are ecuaV- ~eessi,:le. Despite these limitations, there io . i i i a ~xiking similarity of the Bmax value obtained by Moigan et al. (1978) for [3H]Ro 5-4864 binding to car~modulin and that of [3H]tiflucarbine (28 vs. 23 p m o l / # g of calmodulin). With respect to the Ca 2+ and structure dependency, reversibility and micromolar affinity of tiflucarbine binding to ealmodulin, very similar characteristics have been reported for the inhibition of ea!modulin-dependent enzymes or calmodulin-binding, not only by peripheral benzodiazepine receptor ligands (Morgan et al., 1987), but also by phenothiazines (Levin and Weiss, 1979; Prozialeck and Weiss, 1982), naphthalene sulfonamides (Hidaka et al., 1981), and phenylethylamines (Schiichtele et ai., 1989). Although, all these drugs vary significantly in their structure and chemical properties, they all share the common principle for calmoduk;n antagonists of a hydrophobic moiety and basic residue as postulated by Prozialeck and Weiss (1982). Thus, the question arises whether aH these drug interactions with calmodulin are finally mediated by a common drug recognition site on the Ca2+-trigger protein. Indeed, a number of selected well known calmodulin antagonists are able to compete for [3H]tiflucarbh~e binding to calmodulin-agarose with similar K i values ranging from 10 to 100 #M. However, this effect was not reflected by their potency to inhibit calmodulin-stimulated phosphodiesterase from bovine heart. Here the Ka values range from 40 nM up to 10 #M, the rank order of potency being: compound 48/80 > calmidazolium > trifluoperazine > tiflucarbine > chlorpromazine > W-7. In contrast, there is a significant correlation between the potency of structural analogues of tii'lucarbine to displace [3H]tifluearbine binding to calmodulin and to block calmodulin-dependent phosphodiesterase.

417 Taken together, these observations indicate that there is a specific and selective binding site for tiflucarbine on calmodulin which is pharmacologically different from that of known ealmodulinantagonists and that multiple drug recogmtion sites may exist on calmodulin. Accordingly, it is well possible that one or even more of the reference ealmoctufin-antagonists could act on the same drug recognition epitope on calmodulin as tiflucarbine. However, much more experiments involving structural analogues of the respective compounds are required to elaborate this assumption. For this purpose, the method of radioligand binding experiments to immobilized calmodulin as presented in this study may be a suitable approach. In view of the low affinity of the compounds known today to inhibit calmodulin functions in vitro, it is tempting to question the physiological importance of such effects. On the other hand, however, numerous recent reports demonstrated the efficacy of calmodulin antagonists in in situ pharmacological models, such as protein phosphorylation in and hormone release from cultured G H 3 cells (Jefferson and Schulman, 1988; Sletholt et al., 1987), steroidogenesis in a mouse adrenal tumor cell line (Hall et al., 1981), renin release in anesthetized rats (Shinyama et al., 1987), low density lipoprotein receptor mRNA-formation in cultured human fibroblasts (Eckardt et al., 1988), contractile responses of blood vessels (Kostrzeska et at., 1988), and human malaria parasite growth in vitro (Scheibel et at., 1987). In line with these e~amples of an in vivo effectiveness of calmodulin-antagonists, it seems justified to relate our previous finding of m~ enhanced acz~vity of calmodulin-dependent phosphodiesterase isozyme(s) in rat cerebral cortex following a 10-day treatment with pharmacologically relevant doses (I0 m g / k g / d p.o.) of tiflucarbine (Schmidt and Schultz, 1986), to the drug's ability to bind to calmodulin and to inhibit its stimulating effect on phosphodiesterase. Furthermore, it is tempting to hypotLesize that this effect may also have contributed to the tiflucarbine-induced downregulation at least of the ,8-adrenoceptors in rat brain, because both the phosphodiesterase induction and the B-receptor downregulation were dependent on

intact adrenergic nerve terminals (Schmidt and Schuhz, 1986). Indeed, tiflucarbine (unpublished results) as well as W-7 (Reimann et aL, 1988) at catmodulin-antagonistie concen,~aqons have been observed to facilitale neurotra~-~smitter release from rat brain slices. On the basis of the present data, however, it is still premature to conclude that tiflucarbine-mediated calmodulin-antagonism is involved in the induction of neurotransmitter receptor 8ownregulation, which is an important common feature of many clinically effective antidepressant drugs (Green, 1987, Sulser el aL, 1978). In conclusion, this study has shown that tiflucarbine associates with a pharmacologically novel and previously not characterized class of binding sites and thus mediates an inhibition of the activity of calmodulin-dependent phosphodiesterase. This effect adds to the reported selective serotonin re-uptake inhibition and ser~,tonin-2-receptor binding. Further data are required to veri~ whether it possibly contributes to the d r u g s tongterm effects indicative for antidepressant properties.

Ac~owledgments We gratefully acknowledge the excdten~ technical assis:.ance of Mrs. An,~a Scher~kra~an and the experfi~ of Mrs. Helga Wodarz a~d Ms. HeAkeBastgen in the preparation of ~his manuscript. Thanks al.~o go io Dr. Jo~i Bockaert for critical discussions.

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418 Jefferson, A.B. and H. Schulman, 1988, Sphingosine inhibits calmodulin-dependent enzymes, J. Biol. Chem. 263, 15241. Kostrzeska, A., T. Laadansld and ~. Batra, 1988, Effect of calcium and calmodulin antagnnists on contractile responses of the human uterine artery, Br. J. Pharmacol. 94, 1037. Levin, R.M. and B. Weiss. 1977, Binding of trifluoperazine to the calcium-dependent activator of cyclic nuclcotide phosphodiesterase, Mol. Pharmacol. 13, 690. Levin. R.M. and B. Weiss, 1979, Selective binding of antipsychotics and other psychoactive agents to the calcium-dependent activator of cyclic nuclcotide phosphodiesterase, J. PharmacoL Exp. "l'her. 208, 454. Morgan. P.E, J. Patel and P.J. Marangos, 1987, Characterization of [3H]-Ro 5-4864 binding to calmcMulin using a rapid filtration technique, Bioehem. Pharmacol. 36, 4257. Pbch, G., 1971, Assay of phosphodiesterase with radioactively labeled cyclic 3',5'-AMP as substrate. Naunyr.-Sehmiedeb. Arch. Pharmacol. 268, 272. Prozialeck, W.C. and B. Weiss, 1982, Inhibition of calmodulin by phenothiazines and related drugs: structure-activity relationships, J. Pharmacol. Exp. Ther. 222, 509, Reimann, W., U. K611hofer and B. Wagner. 1988, W-7 ai calmodulin-antagonistic concentrations facilitates noradrenaline release from rat brain cortex slices, European J Pharm~,,~ol. 147, 481. Sehiichtele, C., B. Wagner and C. RtMolph, 1989, Effect of

Ca -'+ entry blockers on myosin light-chain kinase and protein kinase C, European J. Fharmacol. 163, 151. Scheibel, L.W., P.M. Colomhani, A.D. Hess, M, Aikawa. C,T. Atkinson and W.K. Milhous, 1987, Calcium and calmodu lir~ antagonists inhibit human malaria parasites (plasmodium falciparum): implications for drug design. Proc. Natl Aead. Sci. U.S.A. 84. 7310. Schmidt, B.H. and 3.E. Sehultz, ~986, The potential antidepressant tiflucarbine down-regnlates/~-adrenoceptors in rat brain, European J. Pharmacol. 130, 27. Schumman, T., W.U. Dompert, T. Giaser and 2. Traber. 1986, Biochemical and behavioral pharmacology of the pWative antidepressant tff?.ucarbme ('I~'/X P 4495), Nau,aynSchraiedeb. Arch. Ph~.rmacol. 332 (Suppl.), R 87. Shinyama, H., Y. Matsumura, Y. Sasaki, F. Ichihar= and S. Morimoto. 1987. Renin release in s nesthetized rats is enhanced by the ealmodulin antagonist V¢-7, Life S.'i. 40, 1687. Sletholt. K.. E. Hang, J. Gordeladze, O. Sand and K.M. Oautv:.k, 1987. Effec!s of calmedulirt antagonists on hormone re!ease a, ld cyclic AMP le',e!s in G H s pituitary cells, Acta Physiol. Scand. 13~. 333. Sulser, F.. J. Ve;ularti and P.I,. Mobley, 1978, Mode of action cf antidepressant drugn, Biochem, Pharmacol. 27, 257. Vogel. N.J., t988, Ligand binding sites o~ calmodulin, in: Calcium in Drug Actions, ed. P.F. Baker (Springer-Verlag, Berlin) p. 57.