Fluorescence spectroscopic analysis of calpain II interactions with calcium and calmodulin antagonists

1. The intrinsic fluorescence of epoxysuccinyl-inhibited calpain II undergoes a Ca2(+)-dependent decrease which contrasts with the increase observed for calmodulin. 2. Calpain II was inhibited by the calmodulin antagonist toluidinylnaphthalenesulfonate (TNS), and a Ca2(+)-dependent increase in TNS fluorescence intensity was observed for epoxysuccinyl-inhibited calpain II. 3. The calmodulin antagonists calmidazolium CDZ and felodipine both caused decreases in the intrinsic fluorescence of epoxysuccinyl-inhibited calpain II. 4. Increasing concentrations of Ca2+ caused an increase in the fluorescence intensity of the inhibited enzyme in the presence of (CDZ), and a decrease in the presence of felodipine. 5. It is concluded from these studies that Ca2+ and calmodulin antagonists induce conformational changes in calpain II, and that changes occur in regions other than the Ca2(+)-binding domains.     

Influence of calcium and calcium and calmodulin antagonists on the cytokinin-induced amaranthin accumulation inAmaranthus tricolor

The concentration of kinetin and kinetinriboside plays an essential role in the induction of amaranthin accumulation in cotyledons ofAmaranthus tricolor during germination. The dose/effect ratio shows that kinetin induced 3- to 3.5-fold more amaranthin than kinetinriboside at the same molecular concentration. Various concentrations of exogenous Ca²⁺ did not influence the effects of kinetin on the betacyanin synthesis. However, when Ca²⁺ was applied together with kinetinriboside, the amaranthin production was stimulated. Time-course experiments show a lag phase of 16 h starting from the incubation with kinetin and a distinct increase of amaranthin thereafter. If the seedlings were treated simultaneously with kinetin and Ca²⁺, the increase of amaranthin started after 12 h. At 16 h of incubation in kinetin/Ca²⁺, the amount of amaranthin increased significantly compared to controls incubated with kinetin alone. If Ca²⁺ ions (16 h kinetin/Ca²⁺ incubation) were removed from the medium after 2 h, 4 h, and up to 14 h, the amaranthin content was enhanced compared to controls without Ca²⁺. The stimulating effect was highest in the presence of Ca²⁺ for 8 h. These data show that exogenous Ca²⁺ stimulated the amaranthin synthesis mainly during the first 12 h of incubation. The Ca²⁺ antagonists EGTA, chlorotetracycline, and CoCl₂ reduced the amaranthin content up to 80%. The calmodulin antagonists chloropromazine and trifluoperazine inhibited the betacyanin accumulation up to 97% when applied at the beginning of the incubation. Neither Co²⁺ nor trifluoperazine after 12 h of preincubation in kinetin had inhibiting effects on the amaranthin production. Therefore, we presume that a specific period of competence is required for calmodulin-mediated Ca²⁺ effects on the accumulation of amaranthin induced by cytokinins in the seedlings ofAmaranthus tricolor.     

Interaction of calcium antagonists with cyclic AMP phosphodiesterases and calmodulin

The calcium antagonists, nimodipine and nicardipine, competitively inhibited calmodulin-sensitive and calmodulin-insensitive forms of cyclic AMP phosphodiesterase, with IC_50's in the micromolar range. Verapamil showed similar inhibitory potency against calmodulin-insensitive phosphodiesterases, but in marked contrast, it was a very weak inhibitor (30–100 times less potent) against calmodulin-sensitive forms of the enzyme. Verapamil and nimodipine both antagonized the calmodulin stimulation of phosphodiesterase. Through use of hydrophobic fluorescent probes, verapamil, and another calmodulin antagonist, proadifen, were shown to interact directly with calmodulin in a manner that differed from the interaction of calmodulin with trifluoperazine.     

Effects of calcium antagonists, calmodulin antagonists, and methylated xanthines on polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta

We have studied the ability of fertilized eggs of Ilyanassa obsoleta to undergo polar lobe formation and cytokinesis in the presence of Ca2+ antagonists (Ca2+ channel blockers, Ca2+ uptake inhibitors). Earlier work had suggested little need for exogenous Ca2+ during these cellular shape changes. Again it appears that exogenous Ca2+ probably is not required, based on cell ability to undergo the shape changes with no, or only minor, delay in the presence of 50 mM La3+ at pH 6.5, 10 mM concentrations of Ni2+ or Co2+, 1 mM Cd2+, and 100 microM concentrations of Mn2+, papaverine, verapamil, D600, or diltiazem. In nominally Ca2+-free seawater (containing approximately 10 microM Ca2+) (CFSW), there still is no effect of Cd2+ (up to 100 microM), Ni2+, Co2+, Mn2+, or diltiazem; however, papaverine, verapamil, and D600 in CFSW cause longer delays in the shape changes than they do in the presence of normal levels of Ca2+ (SW). In 10-50 microM nifedipine, shape changes are progressively delayed to the same extent in both SW and CFSW, but more so in CFSW at concentrations above 50 microM nifedipine. Among calmodulin antagonists, trifluoperazine up to 100 microM was without effect, but chlorpromazine at 25-100 microM and calmidazolium at 50-100 microM caused substantial, concentration-dependent delays in the starting times for the shape changes. Methylxanthines caused a substantial speed-up in the starting times for both polar lobe formation and cytokinesis. The most effective of these, caffeine, at optimal concentrations of 0.7-10 mM in SW or CFSW caused shape changes to occur 12-15 min earlier than in controls undergoing a normal 50-min cycle. Caffeine is known to cause release of Ca2+ from muscle sarcoplasmic reticulum. A putative antagonist of intracellular Ca2+ mobilization, TMB-8, significantly inhibited the shape changes of the Ilyanassa cells, whereas a variety of inhibitors of exogenous Ca2+ uptake noted above did not inhibit. We conclude that Ca2+ may be necessary for polar lobe formation and cytokinesis in Ilyanassa cells, but that it may be released from intracellular, sequestered stores rather than derived from exogenous sources.     

Effects of calmodulin antagonists on human ovarian cancer cell proliferation in vitro

Examined were effects of calmodulin antagonists (W-5 and W-7) on proliferation of two kinds of human cell lines, designated HR and KF, derived from serous cystadecarcinoma of the ovary. Although both W-5 and W-7 inhibited their cell proliferation in vitro, the degree of inhibition was more marked with W-5 rather than with W-7. HR cells had higher sensitivity to cisplatinum than KF cells, while KF cells had higher sensitivity to adriamycin. Combinations of calmodulin antagonists and anti-cancer drugs resulted in adjuvant effects with regard to the inhibition of their cell proliferation in vitro.     

Effects of calmodulin antagonists on serine phospholipid base-exchange reaction in rabbit platelets

Effects of the calmodulin antagonists chlorpromazine, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide on phospholipid metabolism were examined in rabbit platelets using [3H]serine, [3H]ethanolamine, [3H]choline, and [3H]glycerol. All these drugs markedly stimulated the incorporation of [3H]serine into phosphatidylserine. On the other hand, these drugs had only a slight effect on the rate of incorporation of [3H]ethanolamine and [3H]choline into the corresponding phospholipid. When [3H]glycerol was used as a precursor of the phospholipids, 3H-labeled phospholipids were mainly composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Although the phosphorus content of phosphatidylserine was about 40% of that of phosphatidylcholine in rabbit platelets, the amount of phosphatidylserine labeled with [3H]glycerol was less than 2% of that of the labeled phosphatidylcholine, and calmodulin antagonists slightly stimulated the incorporation of [3H]glycerol into phosphatidylserine. Treatment with calmodulin antagonists caused a marked decrease in the content of endogenous free serine with concomitant increase in the contents of endogenous free ethanolamine and choline. On the other hand, the contents of other free amino acids, including essential and non-essential amino acids, were unchanged. These results suggest that the calmodulin antagonists we used did not affect de novo synthesis of phosphatidylserine, but did stimulate the serine phospholipid base-exchange reaction in rabbit platelets.     

Effect of calmodulin antagonists on auxin-induced elongation

Coleoptile segments of oat (Avena sativa var Cayuse) and corn (Zea mays L. var Patriot) were incubated in different concentrations of calmodulin antagonists in the presence and absence of α-naphthaleneacetic acid. The calmodulin antgonists (chlorpromazine (CP), trifluoperazine, and fluphenazine) inhibited the auxin-induced elongation at 5 to 50 micromolar concentrations. Chlorpromazine sulfoxide, an analog of chlorpromazine, did not have significant effect on the elongation of oat and corn coleoptiles. A specific inhibitor of calmodulin N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, a naphthalenesulfonamide derivative) inhibited coleoptile elongation, while its inactive analog N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) was ineffective at similar concentrations. During a 4-hour incubation period, coleoptile segments accumulated significant quantities of ³H-CP. About 85 to 90% of auxin-induced growth was recovered after 4 hours of preincubation with CP or 12 hours with W-7 and transferring coleoptiles to buffer containing NAA. Leakage of amino acids from coleoptiles increased with increasing concentration of CP, showing a rapid and significant increase above 20 micromolar CP. The amount of amino acids released in the presence of W-7 and W-5 was significantly lower than the amount released in the presence of CP. Both W-5 and W-7 increased amino acid release but only W-7 inhibited auxin-induced growth. Calmodulin activity measured by phosphodiesterase activation did not differ significantly between auxin-treated and control coleoptile segments. These results suggest the possible involvement of calmodulin in auxin-induced coleoptile elongation.     

Effects of chlorpromazine and other calmodulin antagonists on phosphatidylcholine-induced vesiculation of platelet plasma membranes

Dilauroylglycerophosphocholine (C12:0PC)-induced vesiculation of platelet plasma membranes (Kobayashi, T., Okamoto, H., Yamada, J.-I., Setaka, M. and Kwan, T. (1984) Biochim. Biophys. Acta 778, 210-218; Kobayashi, T., Yamada, J.-I., Satoh, N., Setaka, M. and Kwan, T. (1985) Biochim. Biophys. Acta 817, 307-312) was inhibited by chlorpromazine. Preincubation of platelets with chlorpromazine was required for inhibition but incorporation of chlorpromazine into C12:0PC liposomes was not necessary for it, indicating that the observed inhibition of vesiculation was mainly due to the effect of chlorpromazine on platelets and not that on liposomes. The change in platelet membrane fluidity caused by chlorpromazine was not the cause of inhibition of vesiculation. The inhibition of vesiculation by various other calmodulin antagonists was also observed. The inhibitory activities of these calmodulin antagonists and chlorpromazine correspond very well to their abilities to bind to calmodulin. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited vesiculation but a structural analogue of it, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), had no inhibitory activity. These results suggest the involvement of calmodulin in membrane vesiculation.     

Transglutaminase inhibitors,calmodulin antagonists,and calcium channel blockers: Influence on Arbacia sperm motility

Sea urchin spermatozoa react to transglutaminase inhibitors, which may antagonize the function of calmodulin, in dose-dependent fashion. The optimum concentration of diallyl-amino propionyl benzothiophenc (DAPBT), a potent, noncompetitive inhibitor of transglutaminase, 0.01 mM, causes a 3 1/2-fold increase in the forward swimming speed of Arbacia sperm. This effect apparently involves calcium-dependent enzymes since manipulation of both extracellular and intracellular calcium by means of chelating agents, calcium-channel blockers, and calmodulin antagonists depresses the stimulatory effect of DAPBT. These results suggest that in the spermatozoa the interaction of flagellar sliding filaments may be mediated by reversible cross-linking of the contractile proteins catalyzed by a calcium- dependent transglutaminase.     

Effects of calmodulin antagonists on auxin-stimulated proton extrusion in Avena sativa coleoptile segments

The effect of the 5 calmodulin (CaM) antagonists trifluoperazine (TFP). compound 48/80, N-(6-aminohexyl)-naphthalenesulfonamtde (W-5), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and calmidazolium on auxin-dependent medium acidification was investigated in abraded segments of Avena sativa L. cv. Victory I. Buffering capacity, Asn content, and changes in pH of bathing solutions were measured in the presence of these inhibitors. When coleoptiles were treated with TFP or compound 48/80, the Asn content and the buffering capacity increased, thus suggesting that plasma membrane permeability was modified. On the contrary. the effect of calmidazolium, W-5. and W-7 on Asn release and buffering capacity was rather low; only small effects being observable at the highest concentration employed. Calmidazolium and W-7 strongly inhibited auxin-dependent medium acidification. W-5 did not affect medium acidification. The specificity of these CaM antagonists and their effects on medium acidification are discussed. The data adduced is consistent with the working hypothesis which postulates an essential role for the Ca²⁺-CaM system on auxin-dependent medium acidification.     

Permissive role of calcium in the inhibition of T cell mitogenesis by calmodulin antagonists

The importance of Ca++ in the initiation of lymphocyte activation and mitogenesis has been supported by several studies. Because calmodulin functions as the intracellular mediator of the effects of Ca++, it likely plays a major role in the regulation of lymphocyte function. We have examined the effects of known calmodulin antagonists, the phenothiazines, on lectin-induced T cell mitogenesis and have shown a central role for Ca++ uptake in the expression of a phenothiazine-sensitive stage after lectin activation. The drug effects were observed only if the cells were previously activated by PHA or the ionophore A23187, and only in the presence of Ca++. These effects were restricted to a defined time period (5 hr) after lectin activation. The data support the concept that calmodulin is the target for the phenothiazine effects and demonstrate the permissive role of Ca++ in the mediation of these events.     

Inhibition of inositol trisphosphate-stimulated calcium mobilization by calmodulin antagonists in rat liver epithelial cells

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), an intracellular second messenger produced from the hydrolysis of phosphatidylinositol 4,5-bisphosphate, interacts with cytoplasmic membrane structures to elicit the release of stored Ca2+. Ins(1,4,5)P3-induced Ca2+ mobilization is mediated through high affinity receptor binding sites; however, the biochemical mechanism coupling receptor occupation with Ca2+ channel opening has not been identified. In studies presented here, we examined the effects of naphthalenesulfonamide calmodulin antagonists, W7 and W13, and a new selective antagonist, CGS 9343B, on Ca2+ mobilization stimulated by Ins(1,4,5)P3 in neoplastic rat liver epithelial (261B) cells. Intact fura-2 loaded cells stimulated by thrombin, a physiological agent that causes phosphatidylinositol 4,5-bisphosphate hydrolysis and Ins (1,4,5)P3 release, responded with a rise in cytoplasmic free Ca2+ levels that was dose dependently inhibited by W7(Ki = 25 microM), W13 (Ki = 45 microM), and CGS 9343B (Ki = 110 microM). Intracellular Ca2+ release stimulated by the addition of Ins(1,4,5)P3 directly to electropermeabilized 261B cells was similarly inhibited by pretreatment with anti-calmodulin agents. W7 and CGS 9343B, which potently blocked Ca2+/calmodulin-dependent protein kinase, had no significant effect on protein kinase A or C in dose range required for complete inhibition of Ca2+ mobilization. Ca2+ release channels and Ca2+-ATPase pump activity were also unaffected by calmodulin antagonist treatment. These results indicate that calmodulin is tightly associated with the intracellular membrane mechanism coupling Ins(1,4,5)P3 receptors to Ca2+ release channels     

Inhibition of phytoalexin biosynthesis in elicitor-treated tobacco cell-suspension cultures by calcium/calmodulin antagonists

A role for calcium/calcium-binding proteins in a mechanism of signaling elicitor-inducible phytoalexin biosynthesis was investigated. Two classes of calcium/calmodulin antagonists, phenothiazines and naphthalenesulfonamides, inhibited sesquiterpene phytoalexin accumulation in tobacco (Nicotiana tabacum) cell-suspension cultures when added 1 h before elicitor. The antagonists also inhibited the induction of sesquiterpene cyclase enzyme activity, a key regulatory enzyme for sesquiterpene biosynthesis. The antagonists suppressed the induction of sesquiterpene cyclase only if added before or simultaneously with elicitor. Additionally, the antagonists inhibited (a) accumulation of the cyclase protein as measured in immunoblots; (b) the in vivo synthesis rate of the cyclase protein, measured as the incorporation of [³⁵S]methionine into immunoprecipitable cyclase protein; and (c) the cyclase mRNA translational activity, measured as the incorporation of [³⁵S]methionine into immunoprecipitable cyclase protein synthesized by in vitro translation of RNA isolated from antagonist-treated, elicitor-induced cells. In contrast, elicitor-inducible phenylalanine ammonia lyase enzyme activity, the level of the enzyme protein, the in vivo synthesis rate, and the mRNA translational activity were not affected by any of the antagonist treatments. Uptake and incorporation of [³⁵S]methionine into total cellular proteins and total in vitro translation products were also not indiscriminately altered by the antagonist treatments. The current results suggest that calcium and/or calmodulin-like proteins may be elements of a signal transduction pathway mediating elicitor-induced accumulation of phytoalexins in tobacco.     

Effects of calcium antagonists on contraction of a holothurian muscle

Contractions evoked by acetylcholine or by tetanic stimulation differed from caffeine contractures, in muscle strips isolated from the longitudinal muscle bands of the body wall of Isostichopus badionotus (Selenka), an aspidochirote holothurian. Tetanic contractions and responses to acetylcholine remained reproducible for hours in sea water or artificial sea water. Caffeine contractures declined rapidly during a series of repetitions, carried out in a bath medium which had a calcium content equivalent to that of sea water. Manganese, lathanum, and dantrolene have been used as calcium antagonists, with the objective of differentiating the calcium stores used in reproducible contractions from the calcium stores used in rapidly extinguishing contractures. Muscle strips were pretreated with an ionophore (X-537A) to confer reproducibility in a series of caffeine contractures, before use of calcium antagonists. For reproducible caffeine contractures, the order of effectiveness of calcium antagonists was lanthanum greater than manganese greater than dantrolene. The order of reversibility was manganese greater than dantrolene greater than lanthanum. For acetylcholine contractions, or tetanic contractions, the order of effectiveness of calcium antagonists was lanthanum greater than manganese and the order of reversibility was manganese greater than lanthanum. Dantrolene reversibility enhanced contractions.     

Free calcium and calpain I activity

Activation of purified calpain I proceeds through a Ca(2+)-induced autolysis from the 80 kDa catalytic subunit to a 76 kDa form via an intermediate 78 kDa form, and from a 30 kDa form to a 18 kDa form as the result of two autocatalytic processes (intra and intermolecular). The minimum Ca2+ requirements for autolysis and proteolysis have been determined by physico-chemical and electrophoretic methods in the presence or absence of a digestible substrate. According to our results the activation process needs less free Ca2+ than the proteolysis of a digestible substrate, which means that proteolysis is really subsequent to activation. For very low Ca2+ levels, a digestible substrate does not initiate the calpain I activation process. In the presence of phospholipid vesicles, such as PI, PS or a mixture of PI (20%), PS (20%) and PC (60%), the apparent kinetic constants of activation are greatly increased without any change in the initial velocity of the substrate proteolysis. Thus, enzyme activation and substrate proteolysis are observed as independent phenomena. These results obtained from experiments using low free Ca2+ concentrations enable us to propose a hypothesis for the mechanism of regulation by which the enzyme could be activated in the living cell.     

Role of calcium ion in the excitability and electrogenic pump activity of theChara corallina membrane: II. Effects of La3+, EGTA,and calmodulin antagonists on the current-voltage relation

Summary The steady N shapeI/V curves were obtained by applying slow ramp hyper- and depolarization pulses toChara cells under the voltage-clamp condition. Application of calcium channel blocker, 20 m La³⁺, to theChara membrane caused, in about 30 min, a marked reduction of the transient inward current and later almost complete blocking of the pump current, while the steady outward current remained almost unaffected. Removal of external Ca²⁺ with 0.5mm EGTA caused similar results. Application of calmodulin antagonists, 10 m TFP or 20 m W-7, also gave very similar results, i.e., the decrease of the transient inward current and of H⁺-pump activity. These results suggest that not only the excitatory mechanisms but also the H⁺-pump activity ofChara membrane are regulated by calmodulin within a comparatively narrow range of internal Ca²⁺ level.     

Comparison of calpain I and calpain II from carp muscle

1. The content of calpain II is 3.4 times more than that of calpain I when estimated by the elution profiles from a column of DEAE-cellulose. 2. Calpain I required 1 mM Ca2+ and calpain II required 5 mM Ca2+ to show the full activities. These data demonstrated that Ca2+-sensitivities of both calpains were lower than those of mammalian calpains, respectively. 3. The optimum caseinolytic activity was pH 7.2 for calpain I and pH 7.5 for calpain II. 4. The molecular weight of calpain I was estimated to be 110 k and that of calpain II to be 120 k by gel filtration. 5. Calpain I was much more heat-stable than calpain II around 50-60 degrees C. 6. Both calpains were sensitive to calpastatin, an endogenous inhibitor for calpain.     

Effects of trifluoperazine on calcium binding by calmodulin. A microcalorimetric study

Microcalorimetric titrations of calmodulin with Ca2+ and trifluoperazine (TFP) at various molar ratios have been carried out at 25 degrees C and at pH 7.0. Ca2+ binding to calmodulin produces heat (-delta H) in the presence of TFP, while heat is absorbed in the absence of TFP. The total heat produced by Ca2+ binding to all four sites is increased at increasing TFP-to-calmodulin ratios, attaining a plateau at about 7. These results indicate that at the higher ratios, the enthalpy changes (delta H) associated with Ca2+ binding are affected by TFP molecules bound at both high- and low-affinity sites. In addition, the Ca2+ binding reaction of the calmodulin-TFP complex is driven solely by a favorable enthalpy change of -27 kJ/mol of site; the entropy change (delta S) is -35 J/mol/K. These thermodynamic changes are opposite to those for TFP-free calmodulin and distinctly different from other Ca2+ binding proteins such as skeletal and cardiac troponin C and parvalbumin, where the reaction is driven by favorable changes of entropy as well as enthalpy.     

The 240-kDa subunit of human erythrocyte spectrin binds calmodulin at micromolar calcium concentrations

The binding of the isolated alpha-subunit of human erythrocyte spectrin to calmodulin is demonstrated by partitioning in aqueous two-phase systems. The affinity of the alpha-subunit for calmodulin is slightly higher than that of the spectrin dimer, whereas the beta-subunit interacts only very weakly. The binding is in all cases calcium-dependent and is abolished on addition of chlorpromazine. At an ionic strength close to physiological conditions, about 1 microM free calcium is required to induce maximum binding of calmodulin to spectrin dimer.     

Autolysis of calpain large subunit inducing irreversible dissociation of stoichiometric heterodimer of calpain

Calpain, a calcium dependent cysteine protease, consists of a catalytic large subunit and a regulatory small subunit. Two models have been proposed to explain calpain activation: an autolysis model and a dissociation model. In the autolysis model, the autolyzed form is the active species, which is sensitized to Ca2+. In the dissociation model, dissociated large subunit is the active species. We have reported that the Ca2+ concentration regulates reversible dissociation of subunits. We found further that in chicken micro/m-calpain autolysis of the large subunit induces irreversible dissociation from the small subunit as well as activation. So we could propose a new mechanism for activation of the calpain by combining our findings. Our model insists that autolyzed large subunit remains dissociated from the small subunit even after the removal of Ca2+ to keep it sensitized to Ca2+. This model could be expanded to other calpains and give a new perspective on calpain activation.