Direct interaction of calmodulin antagonists with Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase

Trypsin-treated Ca2+/calmodulin-dependent phosphodiesterase (CA2+-PDE), which had lost its sensitivity to Ca2+-calmodulin, was inhibited by various calmodulin antagonists, trifluoperazine, chlorpromazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and aminoalkyl chain analogues of W-7 (A-3, A-4, A-5, I-240, A-6, A-7). These inhibitory effects were less than those on calmodulin-activated Ca2+-PDE. The ability of these compounds to inhibit trypsin-treated Ca2+-PDE correlated well with the inhibitory effect on calmodulin-activated Ca2+-PDE. W-7 inhibited trypsin-treated Ca2+-PDE in a competitive fashion with respect to cyclic GMP and the Ki value was 300 microM. The inhibition of trypsin-treated Ca2+-PDE by W-7 (300 microM) or A-7 (100 microM) was overcome by the addition of excess calmodulin. Trypsin-treated Ca2+-PDE can bind to W-7-coupled cyanogen bromide-activated Sepharose 4B in the presence of 1 mM EGTA. These results suggest that Ca2+-PDE possesses a binding site for calmodulin antagonists and that the binding site for these antagonists on this enzyme may be structurally similar to the binding site on calmodulin itself.     

Calmodulin antagonists differentiate between Ni(2+)- and Mn(2+)-stimulated phosphatase activity of calcineurin.

The interaction of calmodulin antagonists with a phosphoprotein phosphatase, calcineurin, was investigated using para-nitrophenyl phosphate (pNPP) as a substrate. Calmidazolium, a potent calmodulin antagonist, inhibited the Ni(2+)-stimulated calmodulin-independent phosphatase activity to much the same extent as it did the Ca2+/calmodulin-stimulated activity. Other calmodulin antagonists, such as trifluoperazine, thioridazine, and W-7, also inhibited the Ni(2+)-stimulated phosphatase activity. On the other hand, calmidazolium only weakly and partially inhibited the Mn(2+)-stimulated phosphatase activity and the other calmodulin antagonists examined increased the Mn(2+)-stimulated activity, in the absence of calmodulin. With the addition of an equimolar amount, as to the inhibited holoenzyme, of the purified B subunit of calcineurin, the Ni(2+)-stimulated phosphatase activity recovered from 38 to 63% of the control level in the presence of 5 microM calmidazolium. When the amount of additional B subunit was increased, the phosphatase activity recovered to 94% of the control level, thereby implying that calmidazolium inhibits the Ni(2+)-stimulated phosphatase activity by interacting with the B subunit, in the absence of calmodulin. The Mn(2+)-stimulated phosphatase activity also recovered from the inhibition by calmidazolium, but a much larger amount of the B subunit was necessary for the recovery. These results indicate that the Ni(2+)- and Mn(2+)-stimulated activities of calcineurin are differentially affected by calmodulin antagonists and that the B subunit plays a crucial role in the expression of the Ni(2+)-stimulated phosphatase activity.     

Calmodulin-independent inhibition of platelet phospholipase A2 by calmodulin antagonists

We tested the effects of calmodulin, two types of calmodulin antagonists, and various phospholipids on the phospholipase A2 activities of intact platelets, platelet membranes, and partially purified enzyme preparations. Trifluoperazine, chlorpromazine (phenothiazines) and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), at concentrations which antagonize the effects of calmodulin, significantly inhibited thrombin- and Ca2+ ionophore-induced production of arachidonic acid metabolites by suspensions of rabbit platelets and Ca2+-induced arachidonic acid release from phospholipids of membrane fractions, but not phospholipase A2 activity in purified enzyme preparations. The addition of acidic phospholipids, but not calmodulin, stimulated phospholipase A2 activity in purified enzyme preparations while decreasing its Km for Ca2+. The dose-response and kinetics of inhibition by calmodulin antagonists of acidic phospholipid-activated phospholipase A2 activity in purified preparations were similar to those of Ca2+-induced arachidonic acid release from membrane fractions. Calmodulin antagonists were also found to inhibit Ca2+ binding to acidic phospholipids in a similar dose-dependent manner. Our results suggest that the platelet phospholipase A2 is the key enzyme involved in arachidonic acid mobilization in platelets and is regulated by acidic phospholipids in a Ca2+-dependent manner and that calmodulin antagonists inhibit phospholipase A2 activity via an action on acidic phospholipids.     

Functional characteristics of calcium-sensitive adenylyl cyclase of ciliate Tetrahymena pyriformis

Calcium-sensitive forms of adenylyl cyclase (AC) were revealed in most vertebrates and invertebrates and also in some unicellular organisms, in particular ciliates. We have shown for the first time that calcium cations influence the AC activity of ciliate Tetrahymena pyriformis. These cations at the concentrations of 0.2-20 microM stimulated the enzyme activity, and maximum of catalytic effect was observed at 2 microM Ca2+. Calcium cations at a concentrations of 100 microM or higher inhibited the AC activity. Calmodulin antagonists W-5 and W-7 at the concentrations of 20-100 microM inhibited the catalytic effect induced by 5 microM Ca2+ and blocked the effect at higher concentrations of Ca2+. Chloropromazine, another calmodulin antagonist, reduced Ca2+-stimulated AC activity only at the concentrations of 200-1000 microM. AC stimulating effects of serotonin, EGF and cAMP increased in the presence of 5 microM Ca2+. AC stimulating effects of EGF, cAMP and insulin decreased in the presence of 100 microM Ca2+, and AC stimulating effect of cAMP decreased also in the presence of calmodulin antagonists (1 mM). At the same time, stimulating effect of D-glucose in the presence of Ca2+ and calmodulin antagonists did not change essentially. The data obtained speak in favor of the presence of calcium-sensitive forms of AC in ciliate T. pyriformis which mediate enzyme stimulation by EGF, cAMP, insulin, and serotonin.     

A possible role for calmodulin in Ca2+-induced swelling of mitochondria

Ca2+-induced mitochondrial swelling was inhibited by a low concentration of calmodulin antagonists. Two affinities of Ca2+ to mitochondrial swelling were observed: high (2-5 microM) and low (more than 100 microM) systems. The high-affinity change was inhibited by micromolar level of trifluoperazine and W-7, but not by W-5. The possible mechanism of this inhibition and the role of CaM in mitochondria are discussed.     

The effects of several ion channel blockers and calmodulin antagonists on fertilization-induced acid release and 45Ca2+ uptake in sea urchin eggs

The effects of calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, chlorpromazine, N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), were tested on two responses of the sea urchin egg to insemination: (1) H+ release; (2) Ca2+ uptake. It was found that calcium antagonists inhibited both processes, while calmodulin antagonists only inhibited H+ release but not Ca2+ uptake. Verapamil and diltiazem were effective to inhibit H+ release when added to the egg suspension up to 120 sec and W-7 was effective around 150 sec after insemination. Calcium antagonists became ineffective earlier than W-7 in inhibiting H+ release. A calmodulin-dependent step may thus occur linking the Ca2+ uptake and H+ release. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion channel blocker, also inhibited both Ca2+ uptake and H+ release. This result suggests that an uptake of anion(s) occurs along with Ca2+ uptake.     

Inhibition of the phosphatase activity of the red cell membrane Ca2+ pump by acidic phospholipids.

The effect of phospholipids was tested on the p-nitrophenylphosphatase activity of the Ca2+ pump. Acidic phospholipids like phosphatidylserine and phosphatidylinositol inhibited the phosphatase activity, while neutral phospholipids like phosphatidylcholine did not. This result contrasts sharply with the known activating effect of acidic phospholipids on the Ca2(+)-ATPase activity of the pump. It is known that the phosphatase activity of the Ca2+ pump can be elicited either by calmodulin and Ca2+ or by ATP and Ca2+. Unlike calmodulin, acidic phospholipids failed to stimulate the phosphatase activity. Furthermore, calmodulin-activated phosphatase was completely inhibited by acidic phospholipids. Maximal inhibition of the ATP-activated phosphatase was only 70%. Inhibition by acidic phospholipids was non-competitive regarding to calmodulin, suggesting that acidic phospholipids and calmodulin do not bind to the same domain of the pump. The presence of Ca2+ was essential for the inhibition, and the apparent affinity for Ca2+ for this effect was increased by acidic phospholipids. Results are consistent with the idea that acidic phospholipids stabilize an enzyme-Ca complex lacking phosphatase activity.     

Calcium ions and glycogen act synergistically as inhibitors of hepatic glycogen-synthase phosphatase.

We investigated the inhibitory effect of Ca2+ in the micromolar range on the activation of glycogen synthase in crude gel-filtered liver extracts [van de Werve (1981) Biochem. Biophys. Res. Commun. 102, 1323-1329]. The magnitude of the inhibition was highly dependent on the glycogen concentration in the final liver extract. Ca2+ inhibited the activation of purified hepatic synthase b by the G-component of synthase phosphatase, as present in the isolated glycogen-protein complex. The cytosolic S-component was not inhibited. Maximal inhibition of the crude G-component occurred at 0.3 microM-Ca2+. The inhibition was not influenced by the addition of either calmodulin or calmodulin antagonists, or by various proteinase inhibitors. The use of purified G-component revealed that the inhibition by 0.3 microM-Ca2+ increased from 45% to 85% when the concentration of glycogen was raised from 1.5 to 20 mg/ml. Muscle glycogen synthase, extensively phosphorylated in vitro, was also used as substrate for purified G-component. Activation and dephosphorylation were similarly inhibited by 0.3 microM-Ca2+, but the magnitude of the inhibition was much greater with the hepatic substrate. No effect of 0.3 microM-Ca2+ was found on the activity of phosphorylase phosphatase in various liver preparations. We conclude that the inhibition of synthase activation by Ca2+ is one of the mechanisms by which cyclic AMP-independent glycogenolytic hormones promote the inactivation of glycogen synthase in the liver, especially in the fed state.     

Novel effects of calmodulin and calmodulin antagonists on the plasma membrane (Ca2+ + Mg2+)-ATPase from rabbit kidney proximal tubules.

In this work we report an unusual pattern of activation by calmodulin on the (Ca2+ + Mg2+)-ATPase from basolateral membranes of kidney proximal tubule cells. The activity of the ATPase depleted of calmodulin is characterized by a high Ca2+ affinity (Km = 2.2-3.4 microM) and a biphasic dependence on ATP concentration. The preparation responded to the addition of calmodulin by giving rise to a new Ca2+ site of very high affinity (Km less than 0.05 microM). Calmodulin antagonists had diverse effects on ATPase activity. Compound 48/80 inhibited calmodulin-stimulated activity by 70%, whereas calmidazolium did not modify this component. In the absence of calmodulin, 48/80 still acted as an antagonist, increasing the Km for Ca2+ to 5.7 microM and reducing enzyme turnover by competing with ATP at the low affinity regulatory site. Calmidazolium did not affect Ca2+ affinity, but it did displace ATP from the regulatory site. At fixed Ca2+ (30 microM) and ATP (5 mM) concentrations, Pi protected against 48/80 and potentiated inhibition by calmidazolium. At 25 microM ATP, Pi protected against calmidazolium inhibition. We propose that the effects of ATP and Pi arise because binding of the drugs to the ATPase occurs mainly on the E2 forms.     

Altered calmodulin activity in fluphenazine-resistant mutant strains. Pleiotropic effect on development and cellular organization in Volvox carteri

Genetically altered calmodulin activity in spontaneously derived mutant strains, which were selected for resistance to the toxic effect of a specific inhibitor, the phenothiazine drug fluphenazine, is demonstrated. Partially purified calmodulin preparations from wild-type and fluphenazine-resistant strains of the multicellular alga Volvox carteri, were tested for the ability to activate Ca2+-ATPase of the erythrocyte membranes, and the inhibition of this stimulatory activity by fluphenazine. Unlike the preparation obtained from wild-type cells, mutant calmodulin is shown to be insensitive to fluphenazine inhibition, in one case, and calmodulin from another strain was found to be inactive in vitro, i.e. it did not activate Ca2+-ATPase. The pleiotropic phenotype of the spontaneously derived mutant strains involved aberrant multicellular organization and hormone-independent commitment of the multipotent asexual reproductive cells, gonodia, to sexual development. These results clearly implicate calmodulin in the control of development and morphogenesis in this simple multicellular eukaryote. In addition, intracellular inhibition of calmodulin in wild-type cells is shown to block the morphogenic process of embryo inversion and to arrest motility. The availability of mutant calmodulin will facilitate further investigation of the role of this ubiquitous regulatory protein in the control of development and differentiation in multicellular eukarytes, as well as the fine structure/function relationship with regard to calmodulin modulation of a wide variety of cellular processes.     

Studies on the mechanism of cortisol inhibition of human natural killer cell activity: effects of calcium entry blockers and calmodulin antagonists

The role of Ca2+ in mediating the inhibition by glucocorticoids of human natural killer (NK) activity was investigated using Ca2+ entry blockers (verapamil and its desmethoxy-derivatives LU46973 and LU47093) and calmodulin antagonists (pimozide and two naphthalenesulfopamide derivatives, W-7 and W-13). Peripheral blood mononuclear (PBM) cell preparations were incubated for 20 h with 1 x 10(-6) M cortisol and these agents in various combinations (concentration range: 1 x 10(-7) - 1 x 10(-5) M) and then assayed in a direct 4-h cytolytic assay using 51Cr-labeled K 562 target cells. Exposure to cortisol led to a significant reduction of NK cell activity (about 50% with respect to the spontaneous activity). Ca2+ entry blockers displayed per se a dose-dependent depressive effect on cytotoxicity and gave significant enhancement of cortisol-dependent inhibition. Calmodulin antagonists were per se minimally effective but clearly amplified the cortisol-mediated inhibition. Raising extracellular Ca2+ by CaCl2 or intracellular Ca2+ by the ionophore A23187 yelded an appreciable reduction of these effects. Our data are compatible with the view that extracellular and intracellular Ca2+ play a role in the control of human NK cell activity. Moreover, it is conceivable that the mechanisms involved in glucocorticoid inhibition of NK cell activity involve Ca2+-dependent pathways.     

Induction of ornithine decarboxylase in guinea-pig lymphocytes. Synergistic effect of diacylglycerol and calcium

Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5) and trifluoperazine inhibited ornithine decarboxylase induction in lymphocytes activated with phytohemagglutinin or inophore A23187. W-7, a more potent calmodulin antagonist than W-5, suppressed ornithine decarboxylase induction in a higher extent than did W-5. These results suggest that calmodulin may play an important role in ornithine decarboxylase induction in the activated lymphocytes. However, the extent of ornithine decarboxylase induction was greater in cells pretreated with Clostridium phospholipase C and then incubated with ionophore A23187 than in cells incubated with ionophore A23187 without the pretreatment. Moreover, combined treatment of cells with ionophore A23187 and tumor promotor, phorbol 12-myristate 13-acetate, caused synergistic induction of ornithine decarboxylase activity. These results, taken together, suggest that both activations of Ca2+-activated phospholipid-dependent protein kinase by diacylglycerol and of calmodulin-dependent function resulted from an elevation of cytosolic Ca2+ concentration may operate in the induction of ornithine decarboxylase in the activated lymphocytes.     

Inhibition of macrophage activation by isoquinolinesulfonamides, phenothiazines, and a napthalenesulfonamide

The influence of isoquinolinesulfonamides (H-7 and H-8), phenothiazines(trifluoperazine and fluphenazine), and a naphthalenesulfonamide (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on stimulated superoxide anion production and phosphatidyl inositol (PI) cycle activity was investigated in the guinea pig alveolar macrophage. All five drugs were able to inhibit superoxide anion production stimulated by n-formyl-nel-leu-phe (FNLP), leukotriene B4 (LTB4), and phorbol-12,13-dibutyrate (PDB). The order of potency was trifluoperazine greater than or equal to fluphenazine greater than H-7 = W-7 greater than H-8. The dose response curves could be shifted to less efficacy by increasing extracellular calcium. By itself, W-7 markedly stimulated 45Ca+2 efflux, fluphenazine and trifluoperazine slightly stimulated 45Ca+2 efflux, while H-7 and H-8 had no effect on 45Ca+2 efflux from macrophages preloaded with 45Ca+2. Consistent with these results, W-7 markedly stimulated PI cycle activity, fluphenazine and trifluoperazine slightly stimulated PI cycle activity, while H-7 and H-8 had no significant effects on PI cycle activity. In addition, W-7 by itself was able to stimulate a weak and short-lived "burst" of superoxide anion production. In order to evaluate whether a site of action of the inhibitors was at protein kinase C and whether protein kinase C was involved in terminating the normally short-lived FNLP- and LTB4-stimulated macrophage activation, fluphenazine and H-7 were used to evaluate the duration of FNLP- and LTB4-stimulated PI cycle activity, at concentrations of the inhibitors that significantly blocked stimulated superoxide anion production. In all cases, FNLP and LTB4 still stimulated PI cycle activity, which still terminated even though protein kinase C was inhibited. These results suggest that all five drugs block protein kinase C, but H-7 was the most specific in its action at protein kinase C, while the phenothiazines and W-7 have multiple sites of action. In addition, these results suggest that protein kinase C may not function to cause the termination of FNLP- and LTB4-stimulated PI cycle activity and subsequent superoxide anion production.     

Effects of calcium antagonists and calmodulin inhibitors on angiotensin II-induced prostaglandin productions in the isolated dog renal arteries

Angiotensin II markedly potentiated both PGE2 and PGI2 productions in the isolated dog renal arteries. This angiotensin II-induced response was significantly reduced by the treatments of EGTA and calcium antagonists such as verapamil, nifedipine and 8-(N,N'-diethylamino)-octyl-3,4,5,-trimethoxybenzoate (TMB-8). Calmodulin inhibitors, trifluoperazine and W-7 also inhibited the angiotensin II-induced PG productions while an inactive analogue of W-7, W-5 did not have any effect. The results suggest that angiotensin II may enhance the intracellular Ca2+ level through the influx of extracellular Ca2+ and then, calmodulin activated with Ca2+ will stimulate both PGE2 and PGI2 productions via its activation of phospholipase A2 in the dog renal arteries.     

Activation by calmodulin of inositol-1,4,5-trisphosphate 3-kinase in guinea pig peritoneal macrophages

The activity of inositol-1,4,5-trisphosphate 3-kinase in the cytosol fraction of guinea pig macrophages was assayed with special reference to the dependence on the free Ca2+ concentration. The enzyme activity, as assessed by the production of inositol 1,3,4,5-tetrakisphosphate was reversibly activated by free Ca2+ concentrations ranging from 10(-7) to 10(-6)M. The calmodulin antagonists, W-7 and chlorpromazine, inhibited the Ca2+-activated enzyme activity in a dose-dependent fashion, thereby indicating that calmodulin may be involved in the activation by Ca2+. The content of calmodulin in the cytosol fraction (about 2.8 micrograms/mg of cytosol protein) was markedly reduced to less than 0.03 microgram/mg of proteins by subfractionation by ammonium sulfate, followed by an anion-exchange chromatography. The subfraction obtained by the chromatography showed no Ca2+ dependence in the enzyme activity, while an exogenous addition of calmodulin with 10(-6)M Ca2+ increased the enzyme activity. The enzyme activity was retained on a calmodulin-affinity column in the presence of Ca2+, and was eluted from the column by lowering the free Ca2+ concentration by adding ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. These results clearly indicate that calmodulin activates the inositol-1,4,5-trisphosphate 3-kinase activity.     

Anti-calmodulins and tricyclic adjuvants in pain therapy block the TRPV1 channel

Ca(2+)-loaded calmodulin normally inhibits multiple Ca(2+)-channels upon dangerous elevation of intracellular Ca(2+) and protects cells from Ca(2+)-cytotoxicity, so blocking of calmodulin should theoretically lead to uncontrolled elevation of intracellular Ca(2+). Paradoxically, classical anti-psychotic, anti-calmodulin drugs were noted here to inhibit Ca(2+)-uptake via the vanilloid inducible Ca(2+)-channel/inflamatory pain receptor 1 (TRPV1), which suggests that calmodulin inhibitors may block pore formation and Ca(2+) entry. Functional assays on TRPV1 expressing cells support direct, dose-dependent inhibition of vanilloid-induced (45)Ca(2+)-uptake at microM concentrations: calmidazolium (broad range) > or = trifluoperazine (narrow range) chlorpromazine/amitriptyline>fluphenazine>W-7 and W-13 (only partially). Most likely a short acidic domain at the pore loop of the channel orifice functions as binding site either for Ca(2+) or anti-calmodulin drugs. Camstatin, a selective peptide blocker of calmodulin, inhibits vanilloid-induced Ca(2+)-uptake in intact TRPV1(+) cells, and suggests an extracellular site of inhibition. TRPV1(+), inflammatory pain-conferring nociceptive neurons from sensory ganglia, were blocked by various anti-psychotic and anti-calmodulin drugs. Among them, calmidazolium, the most effective calmodulin agonist, blocked Ca(2+)-entry by a non-competitive kinetics, affecting the TRPV1 at a different site than the vanilloid binding pocket. Data suggest that various calmodulin antagonists dock to an extracellular site, not found in other Ca(2+)-channels. Calmodulin antagonist-evoked inhibition of TRPV1 and NMDA receptors/Ca(2+)-channels was validated by microiontophoresis of calmidazolium to laminectomised rat monitored with extracellular single unit recordings in vivo. These unexpected findings may explain empirically noted efficacy of clinical pain adjuvant therapy that justify efforts to develop hits into painkillers, selective to sensory Ca(2+)-channels but not affecting motoneurons.     

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.     

Calcium ion regulates the release of lipase of Fusarium oxysporum.

The lipase production of a plant pathogenic fungus, Fusarium oxysporum f. sp. lini SUF 402, was induced by fat as the carbon source, and its release was stimulated by the infusion of intracellular free calcium ion with a calcium ionophore, A23187. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7, a calmodulin inhibitor) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl- L-tyrosyl]-4-phenylpiperazine (KN-62, a Ca2+/calmodulin dependent protein kinase II inhibitor) reduced the extracellular release of lipase in vivo. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7, a protein kinase C inhibitor) did not have this ability. After K2H32PO4 had been incorporated into the cells, they were treated with W-7 or KN-62 and stimulated by Ca2+ ionophore. On SDS-PAGE of intracellular proteins followed by autoradiography, W-7- and KN-62-treated cells showed inhibition of the incorporation of 32Pi into the 20 kDa protein resulting from Ca2+ stimulation. F. oxysporum had calmodulin (CaM)-dependent protein kinase activity in the cytoplasmic fraction and had the ability to phosphorylate of syntide 2, a specific substrate of CaM kinase II. The partially purified CaM-dependent protein kinase was inhibited by 10 microM KN-62 in vitro. Increase of the intracellular Ca2+ concentration of F. oxysporum activated CaM and CaM-dependent protein kinase, resulting in the extracellular lipase release. These results suggest the existence of a Ca2+ signalling system in F. oxysporum like those observed in higher eucaryotes.     

Effects of ruthenium red on activation of Ca2(+)-dependent cyclic nucleotide phosphodiesterase.

Ruthenium red inhibited Ca2(+)-dependent phosphodiesterase (Ca2(+)-PDE) selectively with an IC50 value of 15 microM. Increasing calmodulin concentration in the presence of both 100 microM and 4000 microM Ca2+ completely reversed the inhibition of Ca2(+)-PDE activity by ruthenium red. Ruthenium red-induced inhibition of Ca2(+)-PDE activity was also overcome by increasing the concentration of Ca2+ in the presence of both 200 ng and 2000 ng calmodulin, in sharp contrast to fluphenazine-induced inhibition of Ca2(+)-PDE. These results indicate that ruthenium red has distinct inhibitory mechanism which differs from that of calmodulin antagonists previously reported.