Xyloglucan antibodies inhibit auxin-induced elongation and cell wall loosening of azuki bean epicotyls but not of oat coleoptiles

Polyclonal antibodies were raised in rabbits against isoprimeverose (Xyl₁Glc₁), xyloglucan heptasaccharides (Xyl₃Glc₄), and octasaccharides (Gal₁Xyl₃Glc₄). Antibodies specific for hepta- and octasaccharides suppressed auxin-induced elongation of epicotyl segments of azuki bean (Vigna angularis Ohwi and Ohashi cv Takara). These antibodies also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time and the relaxation rate of the cell walls) of azuki segments. However, none of the antibodies influenced auxin-induced elongation or cell wall loosening of coleoptile segments of oat (Avena sativa L. cv Victory). Auxin caused a decrease in molecular mass of xyloglucans in the cell walls of azuki epicotyls and oat coleoptiles. The antibodies inhibited such a change in molecular mass of xyloglucans in both species. Preimmune serum exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosening, or breakdown of xyloglucans. The results support the view that the breakdown of xyloglucans is associated with the cell wall loosening responsible for auxin-induced elongation in dicotyledons. The view does not appear to be applicable to poaceae, because the inhibition of xyloglucan breakdown by the antibodies did not influence auxin-induced elongation or cell wall loosening of oat coleoptiles.     

Inhibitory Effect of Calmodulin Antagonists and Calcium Antagonists on Fertilization-Induced Cyanide-Insensitive Respiration in Sea Urchin Eggs

During initial several minutes after fertilization, sea urchin eggs exhibited high rate of respiration which was only slightly inhibited by cyanide. This cyanide-insensitive respiration was inhibited by calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide hydrochloride (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and chlorpromazine, which were added within 1 min after insemination. The inhibitory effect of W-7 on cyanide-insensitive respiration was higher than that of W-5. Cyanide-sensitive respiration of fertilized eggs observed after this initial period was not inhibited by these compounds. Ca²⁺ influx in eggs just after fertilization was inhibited by calcium antagonists but was rather enhanced by calmodulin antagonists. Fertilization-induced stimulation of cyanide-insensitive respiration probably results from calmodulin-dependent reactions which are activated by Ca²⁺ influx.     

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 auxin-induced cell elongation by galactose

Galactose at concentrations higher than 3 m M inhibited specifically auxin-induced elongation of oat, wheat and rice coleoptile segments but not of pea and mung bean stem segments. Glucose, arabinose, rhamnose, xylose, mannose and glucosamine did not inhibit auxin-induced elongation of coleoptile segments. Galactose inhibited auxin-induced but not hydrogen ion-induced growth.     

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.     

Inhibition by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide,a calmodulin inhibitor,of dopamine release from rat brain synaptosomes

The release of preloaded [³H]dopamine by the synaptosomal fraction prepared from rat forebrain was examined in the presence and absence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor. The release induced by high K⁺ was blocked by W-7 in a concentration-dependent manner after the pretreatment with and in the presence of the inhibitor. The inhibition by W-7 may specifically involve calmodulin, because little effects were seen with N-(6-aminohexyl)-naphthalenesulfonamide, an analog of W-7 with only a low affinity for calmodulin. W-7 may not affect the voltage-dependent Ca²⁺ channel of synaptosomal plasmalemma, since the inhibitor produced no change in the synaptosomal ⁴⁵Ca²⁺ uptake induced by high K⁺ depolarization. Thus, calmodulin may play a role in transmitter release and may function at the step(s) after the increase of free Ca²⁺ concentration in the cytosol of the nerve terminal. W-7 affected only to a small extent [³H]dopamine release in the presence of A23187 plus Ca²⁺.     

A fluorescence polarization-based assay for the identification and evaluation of calmodulin antagonists

A fluorescence polarization (FP) assay was developed to identify calmodulin (CaM) antagonists. A fluorescent tracer was newly designed by covalently labeling N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which is a well-known CaM antagonist, with the Cy5 dye. In the FP assay, the tracer (Cy5-W-7) was bound to CaM with a dissociation constant (K_d) of 6.5 μM and demonstrated efficient competitive activity with other CaM antagonists, including W-7, chlorpromazine, trifluoperazine, W-5, and clozapine, indicating that Cy5-W-7 binds to the ligand-binding site of CaM in a specific manner. The inhibitory activities of Cy5-W-7 and CaM antagonists were subsequently measured by the CaM-dependent calcineurin phosphatase assay, and the results were confirmed with those of the FP assays. In addition, assay optimization for high-throughput screening was performed, and a Z′ factor of 0.7 was achieved in a 1536-well format. The FP assay was found to be a simple and reliable alternative to conventional assays for evaluating CaM antagonists.     

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.     

Possible involvement of calmodulin and the cytoskeleton in electrofusion of plant protoplasts

Calmodulin antagonists, trifluoperazine, chlorpromazine, calmidazolium, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), strongly inhibited the electrofusion of barley (Hordeum vulgare L. cv Moor) protoplasts with a marked increase of broken fusion products, after 60 minutes of incubation. W-5, a dechlorinated analog of W-7, was found less effective for the inhibition than W-7. Ethyleneglycol-bis(β- aminoethylether)-N,N′-tetraacetic acid a Ca²⁺ chelator, La³⁺, a surface Ca²⁺ antagonist, and verapamil, a Ca²⁺ channel blocker, also inhibited electrofusion. Dielectrophoresis was inhibited by La³⁺. A microtubule inhibitor, vinblastine, inhibited electrofusion strongly while colchicine, slightly. A microfilament inhibitor, cytochalasin B, promoted fused cells to become spherical while phalloidin did not affect electrofusion.     

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.     

Galactose inhibition of auxin-induced cell elongation in oat coleoptile segments

Auxin-induced cell elongation in oat coleoptile segments was inhibited by galactose; removal of galactose restored growth. Galactose did not appear to affect the following factors which modify cell elongation: auxin uptake, auxin metabolism, osmotic concentration of cell sap, uptake of tritium-labeled water, auxin-induced wall loosening as measured by a decrease in the minimum stress-relaxation time and auxininduced glucan degradation. Galactose markedly prevented incorporation of [¹⁴C]-glucose into cellulosic and non-cellulosic fractions of the cell wall. It was concluded that galactose inhibited auxin-induced long-term elongation of oat coleoptile segments by interfering with cell wall synthesis.     

Calmodulin regulates the Ca2+-dependent slow-vacuolar ion channel in the tonoplast of Chenopodium rubrum suspension cells

The patch-clamp technique was applied to vacuoles isolated from a photoautotrophic suspension cell culture of Chenopodium rubrum L. and vacuolar clamp currents, which are predominantly carried by the previously identified Ca²⁺-dependent slow vacuolar (SV) ion channels, were recorded. These currents, which were activated by 1-s voltage pulses of -100 mV (vacuolar interior negative) in the presence of 100 M Ca²⁺ (cytosolic side), could be blocked completely and reversibly by the calmodulin antagonist W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and its chlorine-deficient analogue W-5; half-maximum inhibition was found at approx. 6 M for W-7 and 70 M for W-5. Inhibition was reversed by addition of 1 g · ml^–1 calmodulin purified from Chenopodium cell suspensions; reversal by bovine brain calmodulin was scarcely appreciable. We conclude that cytosolic calmodulin mediates the Ca²⁺ dependence of the SV-channel in the Chenopodium tonoplast.Abbreviations SV-channel slowly activated, vacuolar ion channel - W-5 N-(6-aminohexyl)-1-naphthalenesulfonamide - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide We acknowledge support by the Deutsche Forschungsgemeinschaft and the Bundesminister für Forschung und Technologie, Bonn, and by the Justus-Liebig-Universität Giessen (to W.B.)     

Comparison of the outer and inner epidermis : inhibition of auxin-induced elongation of maize coleoptiles by glucan antibodies

Polyclonal antibodies, raised against β-d-glucans prepared from oat (Avena sativa L.) caryopses, cross-reacted specifically with (1→3),(1→4)-β-d-glucans when challenged in a dot blot analysis of related polymers bound to a cellulose thin layer chromatography plate. The antibodies suppressed indoleacetic acid (IAA)-induced elongation of segments from maize (Zea mays L.) coleoptiles when the outer surface was abraded. However, IAA-induced elongation of nonabraded segments or segments with abrasion restricted to the interior of the cylinder was not influenced by the antibodies. Fab fragments prepared from the antibodies gave similar results. The capacity for IAA to overcome outward curvature of split coleoptile segments was partially reversed by treatment of the segments with the antibodies. Fluorescence microscopy revealed that antibody penetration was largely restricted to the epidermal cell wall region. These results support the view that the degradation of (1→3),(1→4)-β-d-glucans in the outer epidermal cell wall serves an essential role in auxin-induced elongation of Poaceae coleoptiles.     

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist,also inhibits phospholipid sensitive calcium-dependent protein kinase

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), commonly regared as a calmodulin antagonist, inhibted phospholipid-sensitive Ca²⁺-dependent protein kinase and to a lesser extent cyclic GMP- and cyclic AMP-dependent protein kinases. Kinetic studies of the inhibition of the homogenous spleen phospholipid-sensitive Ca²⁺-dependent protein kinase indicated that W-7 inhibited the enzyme activity competitively with respect to phospholipid (K_i = 60 μM). N-(6-Aminohexyl)-1-naphthalenesulfonamide (W-5) was found to be musch less potent than W-7. The findings indicate that W-6 was able to inhibit a variety of protein kinases, in addition to those requiring calmodulin previously reported.     

RIBONUCLEIC ACID METABOLISM AND CELL EXPANSION IN OAT COLEOPTILE

RNA metabolism in oat coleoptiles was studied using physiologicalresponses to 5-FU and actinomycin D; autoradiographic detectionof RNA and protein synthesis; and estimation of ribosomal concentrationby analytical ultracentrifugation. 5-FU failed to inhibit growthof either intact coleoptiles or isolated coleoptile segmentsbut completely blocked cell division in roots. Actinomycin Dmarkedly inhibited auxin-induced expansion of coleoptile segments.When supplied to isolated segments from coleoptiles of variouslengths the RNA precursors cytidine, adenine and adenosine allshowed weak incorporation into RNA of nuclei and in some cases,to a lesser extent, RNA of cytoplasm. IAA did not affect thisRNA synthesis but it was considerably reduced by actinomycinD. A proportion of the label incorporated from RNA precursorswas not removable with either RNase, PCA or hot TCA but wasextracted by trypsin. The amount of this spurious incorporationincreased with coleoptile age, as did the ability to incorporatelabelled amino acids. The concentration of both free and boundribosomes does not increase in growing coleoptiles and may evendecline. Free ribosomes decline markedly in fully grown coleoptileswhile the proportion of bound ribosomes increases. It is concludedthat young coleoptiles contain a full complement of ribosomesnecessary for subsequent growth but normal growth is dependenton continued production of an actinomycin D-sensitive messenger-typeRNA. No evidence for auxin mediation of RNA synthesis was found. ¹Present address: Laboratory of Cell Biology, Faculty of Science,Osaka City University, Sugimoto-cho, Sumiyoshi-ku, Osaka, Japan.     

Possible participation of calmodulin in stimulation of leucine transport by concanavalin A in human lymphocytes

Stimulation of leucine uptake by addition of concanavalin A, mediated by increase of intracellular free Ca2+ concentration [( Ca2+]), in lymphocytes (Mitsumoto, Y., Sato, K. and Mohri, T. (1988) Biochim. Biophys. Acta 968, 353-358) was abolished by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and chlorpromazine, which inhibited membrane hyperpolarization induced by the mitogen. Quinine (0.5-1 mM) completely inhibited the concanavalin A-induced hyperpolarization and extensively inhibited the induced stimulation of leucine uptake. Based on these results, we suggest that the stimulation of leucine uptake by concanavalin A is largely due to activation of the Ca2+-dependent K+ channel which reinforces negative potential of the plasma membrane and is regulated by calmodulin.     

Inhibition of the acrosome reaction of sea urchin spermatozoa by a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7)

A calmodulin antagonist, W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), inhibited induction of the acrosome reaction of sea urchin spermatozoa by egg jelly at 20-50 microM, but W-5 did not. The latter, a chlorine-deficient derivative of W-7, has a lower affinity for calmodulin than does W-7. These results suggest that a Ca2+-calmodulin-dependent process plays an important role in the triggering process of the acrosome reaction of sea urchin spermatozoa.     

In vitro and in vivo protein phosphorylation in Avena sativa L. coleoptiles: effects of Ca2+, calmodulin antagonists, and auxin

In vitro and in vivo protein phosphorylations in oat (Avena sativa L.) coleoptile segments were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. In vitro phosphorylation of several polypeptides was distinctly promoted at 1 to 15 micromolar free Ca2+ concentrations. Ca2(+)-stimulated phosphorylation was markedly reduced by trifluoperazine, chlorpromazine, and naphthalene sulfonamide (W7). Two polypeptides were phosphorylated both under in vitro and in vivo conditions, but the patterns of phosphorylation of several other polypeptides were different under the two conditions indicating that the in vivo phosphorylation pattern of proteins is not truly reflected by in vitro phosphorylation studies. Trifluoperazine, W7, or ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) + calcium ionophore A23187 treatments resulted in reduced levels of in vivo protein phosphorylation of both control and auxin-treated coleoptile segments. Analysis by two-dimensional electrophoresis following in vivo phosphorylation revealed auxin-dependent changes of certain polypeptides. A general inhibition of phosphorylation by calmodulin antagonists suggested that both control and auxin-treated coleoptiles exhibited Ca2+, and calmodulin-dependent protein phosphorylation in vivo.     

Ca^2＋-Calmodulin is Involved in Betacyanin Accumulation Induced by Dark in C3 Halophyte Suaeda salsa

The C3 halophyte Suaeda salsa was used to investigate the roles of Ca^2＋, Ca^2＋ channels, and calmodulin （CAM） in betacyanin metabolism. Seeds of S. salsa were cultured in both the dark and light for 3 days. The fresh weight and betacyanin content were much higher in S. salsa seedlings formed in the dark than in seedlings formed in the light. The addition of Ca^2＋ to the half-strength MS nutrient solution promoted betacyanin accumulation in the dark, whereas Ca^2＋ depletion by EGTA suppressed the dark-induced betacyanin accumulation in shoots of S. salsa. The Ca^2＋ channel blocker LaCl3 also inhibited dark-induced betacyanin accumulation. The highest activity of CaM and the maximum betacyanin content decreased by 51% and 45%, respectively, in shoots of S. salsa seedlings treated with the potent CaM antagonist chlorpromazine in the dark. Furthermore, the other CaM antagonist N-（6-aminohexyl）-5-chloro-l-naphthalenesulfonamide （W-7） also inhibited the activity of CaM and dark-dependent betacyanin accumulation, whereas its less active structural analog N-（6-aminohexyl）- 1-naphthalenesulfonamide （W-5） had little effect on the responses to dark of S. salsa seedlings. These results suggest that Ca^2＋, Ca^2＋-regulated ion channels, and CaM play an important role in dark-induced betacyanin accumulation in the shoots of the C3 halophyte S. salsa.     

Auxin-induced growth of Avena coleoptiles involves two mechanisms with different pH optima

Although rapid auxin-induced growth of coleoptile sections can persist for at least 18 hours, acid-induced growth lasts for a much shorter period of time. Three theories have been proposed to explain this difference in persistence. To distinguish between these theories, the pH dependence for auxin-induced growth of oat (Avena sativa L.) coleoptiles has been determined early and late in the elongation process. Coleoptile sections from which the outer epidermis was removed to facilitate buffer entry were incubated, with or without 10 micromolar indoleacetic acid, in 20 millimolar buffers at pH 4.5 to 7.0 to maintain a fixed wall pH. During the first 1 to 2 hours after addition of auxin, elongation occurs by acid-induced extension (i.e. the pH optimum is <5 and the elongation varies inversely with the solution pH). Auxin causes no additional elongation because the buffers prevent further changes in wall pH. After 60 to 90 minutes, a second mechanism of auxin-induced growth, whose pH optimum is 5.5 to 6.0, predominates. It is proposed that rapid growth responses to changes in auxin concentration are mediated by auxin-induced changes in wall pH, whereas the prolonged, steady-state growth rate is controlled by a second, auxin-mediated process whose pH optimum is less acidic.