Phytohemagglutinin Induces Delays in Oral Regeneration in the Ciliate Stentor coeruleus1

Regenerating Stentor, exposed to the plant lectin phytohemagglutinin, are significantly delayed in completing oral regeneration. All of the delays are restricted to the earliest stages of regeneration prior to stage 3. The effects of phytohemagglutinin are reversible once the drug is removed. The addition of the presumed sugar receptor for phytohemagglutinin, N-acetylgalactosamine, at the start of oral regeneration could not reverse the effects of phytohemagglutinin, but the addition of excess (10 mM) extracellular calcium could. When the addition of the excess calcium was delayed for various times after phytohemagglutinin exposure, the effectiveness of the calcium in reversing the phytohemagglutinin-induced delays was reduced. Based on the mechanism of action of PHA in other cells, these results suggest that membrane proteins may be involved in controlling oral regeneration, possibly through mechanisms involving Ca⁺⁺.     

The presence of cyclic AMP, and its effects on oral regeneration and in situ ciliary regeneration in Stentor coeruleus.

We have found cyclic AMP in the large, heterotrichous ciliate Stentor coeruleus in amounts per milligram protein similar to those found in another ciliate, Tetrahymena pyriformis. The possible function of cyclic AMP in Stentor was first examined by determining its effects on oral regeneration, the process by which Stentor can replace a missing oral apparatus in eight to ten hours. Once begun (by brief exposure to a 15% sucrose solution, causing shedding of the oral apparatus) regeneration follows eight specific morphological stages visible with the dissecting microscope. Continuous exposure of regenerating cells to either N⁶, 2′-0-dibutyryl adenosine cyclic 3′:5′-monophosphate (DBC) or theophylline begun at the onset of oral regeneration (stage 0) caused delays in the completion of regeneration. The delays induced by DBC occurred in the early stages prior to stage 5. Regenerating cells exposed to DBC or theophylline at various stages of development were delayed, even at stages 5 and 6. Both DBC and theophylline reversibly bleached the cortical pigment of the cells. Guanosine 3′:5′-cyclic monophosphate (cyclic GMP), AMP, GMP, and sodium butyrate neither delayed oral regeneration nor bleached the cortical pigment. Excess extracellular calcium ions alone had no effect on oral regeneration, but 10 mM calcium and DBC caused more delay than DBC alone. Thus, the delay of oral regeneration in Stentor caused by cyclic AMP may involve calcium ions. To determine if cyclic AMP can retard in situ ciliary regeneration by Stentor, as it does in Tetrahymena, a new technique, more accurate than past methods, was developed to monitor ciliary regrowth. Using this procedure we found that both DBC and theophylline significantly delayed the in situ ciliary regeneration by Stentor.     

Pharmacological evidence for cell surface control of oral regeneration in Stentor coeruleus

This study suggests that membrane perturbations can affect oral morphogenesis in Stentor, possibly by a mechanism involving calcium ions. Exposure of regenerating Stentor to micromolar concentrations of the membrane active local anesthetics dibucaine, tetracaine, or procaine greatly delayed the progress of oral regeneration. In the case of tetracaine and dibucaine the greatest delays were observed in the early stages of regeneration prior to stage 4, when the majority of essential synthetic activity is occurring. The effects of dibucaine were generally readily reversible upon removal of the cells from the drug, with some residual effects occurring at higher dibucaine concentrations. Regenerating cells in the presence of dibucaine and excess extracellular calcium were not delayed, suggesting that the effects of dibucaine were reversible by calcium ions. The effects of tetracaine were not reversible by calcium ions, however. Exposure of regenerating cells to medium either lacking in, or containing an excess of, extracellular calcium had no effect on the time required to complete oral regeneration. The plant lectin, phytohemagglutinin, can also delay oral regeneration. The possible implications of these findings on the control of oral regeneration are discussed.     

The role of calcium in the initiation of superoxide release from alveolar macrophages

The role of calcium in the release of superoxide anion (O2-) was examined in alveolar macrophages after stimulation with the soluble stimuli: concanavalin A (Con A), N-formyl methionyl phenylalanine (FMP), and the calcium ionophore. A23187. The release of O2- by Con A was unaffected over a wide range of extracellular calcium concentrations (20 microM to 3 mM), whereas increasing the extracellular calcium above 2 mM inhibited FMP-stimulated O2- release. In contrast, A23187 did not stimulate O2- release in calcium-free medium (less than or equal to 30 microM). The addition of EGTA (50 microM) to calcium-free medium had no effect on Con A stimulation of O2- release or FMP-stimulated O2- release. These results suggest that, for the three soluble stimuli, there are different roles for Ca+2 in the activation and transmission of stimulatory signals across the cell membrane. Con A- or FMP-stimulated calcium efflux from calcium-loaded cells in either calcium-free medium or 0.5 mM calcium-containing medium. In calcium-free medium, FMP transiently retarded 45Ca+2 uptake, while in 0.5 mM calcium-containing medium, FMP transiently stimulated 45Ca+2 uptake. For either Con A or FMP, calcium efflux preceded O2- release by 30-45 sec. Quinine, an agent that blocks membrane hyperpolarization in macrophages, completely blocked O2- release by concanavalin A or FMP and inhibited 45CA+2 efflux by 50% or more for both agents. These results support the hypothesis that redistribution of cellular Ca+2 is one of the initial steps leading to the release of O2-.     

Calcium channels mediate phase shifts of the Bulla circadian pacemaker

1. Light-induced phase advances of the activity rhythm of the Bulla ocular circadian pacemaker are blocked when the extracellular calcium concentration is reduced with EGTA to 0.13 microM. Phase advances are also blocked in low calcium solutions without EGTA [( Ca] less than 50 microM). 2. The dependence of light-induced phase delays on extracellular calcium concentration in EGTA-free seawater was determined. Phase delays are blocked at calcium concentrations below 400 microM, and reduced at concentrations of 1 mM and 3.5 mM (relative to shifts in normal ASW, [Ca] = 10 mM). Phase delays are also reduced and blocked at calcium concentrations higher than normal (60 mM and 110 mM, respectively). 3. Low calcium EGTA also blocked both phase delays and phase advances induced by pulses of depolarizing high K+ seawater. Low calcium EGTA pulses presented alone at the same times did not generate significant phase shifts. 4. The organic calcium channel antagonists verapamil, diltiazem and nitrendipine as well as the inorganic calcium channel antagonists La3+, Co2+, Cd2+, and Mn2+ were applied along with light pulses, however, the treated eyes were either phase shifted by these substances, or these substances were found to be toxic. 5. The inorganic calcium channel antagonist Ni2+ blocked both light-induced phase delays and advances at a concentration of 5 mM. Ni2+ applied alone did not generate significant phase shifts. Phase delays induced by high K+ seawater were blocked in the presence of 50 mM Ni2+ but not in 5 mM Ni2+. The light-induced CAP activity of the putative pacemaker cells was not inhibited by Ni2+, suggesting that its blocking action was probably via its known role as a calcium channel antagonist.     

Concanavalin-Mediated Attachment to Membranes of a Cellular Slime Mould Cyclic AMP Phosphodiesterase

In the cellular slime mould Dictyostelium discoideum , a membrane-bound cyclic AMP phosphodi-esterase undergoes a tenfold increase in activity when amoebae reach the aggregation stage of development. Our previous studies had shown that when non-aggregating cells, which produce extracellular and intracellular forms of the enzyme, are treated with the lectin Concanavalin A (Con A), they exhibit prematurely high levels of the membrane bound enzyme. The present results indicate that this effect may be largely due not to the induction of the enzyme by Con A but rather to the binding of the intracellular form of the enzyme to membranes by Con A. This conclusion is based on the findings that: a) the enzyme activity associated with membranes from Con A treated cells can be decreased by treatment with the haptenic sugar α-methyl mannoside; b) membranes from untreated cells having only low membrane-bound phosphodiesterase activity can acquire increased activity after incubation with Con A and intracellular phosphodiesterase; c) the intracellular phosphodiesterase binds to Sepharose-Con A and is eluted with α-methyl mannoside. These results raise the possibility that some of the effects attributed to Con A in the literature may not be due directly to Con A but to glycoproteins attached to membranes by Con A.     

Concanavalin-mediated attachment to membranes of a cellular slime mould cyclic AMP phosphodiesterase.

In the cellular slime mould Dictyostelium, a membrane-bound cyclic AMP phosphodiesterase undergoes a tenfold increase in activity when amoebae reach the aggregation stage of development. Our previous studies had shown that when non-aggregating cells, which produce extracellular and intracellular forms of the enzyme, are treated with the lectin Concanavalin A (Con A), they exhibit prematurely high levels of the membrane bound enzyme. The present results indicate that this effect may be largely due not to the induction of the enzyme by Con A but rather to the binding of the intracellular form of the enzyme to membranes by Con A. This conclusion is based on the findings that: a) the enzyme activity associated with membranes from Con A treated cells can be decreased by treatment with the haptenic sugar alpha-methyl mannoside: b) mambranes from untreated cells having only low membrane-bound phosphodiesterase activity can acquire increased activity after incubation with Con A and intracellular phosphodiesterase; c) the intracellular phosphodiesterase binds to Sepharose-Con A and is eluted with alpha-methyl mannoside. These results raise the possibility that some of the effects attributed to Con A in the literature may not be due directly to Con A but to glycoproteins attached to membranes by Con A.     

Study of real-time lectin-carbohydrate interactions on the surface of a quartz crystal microbalance

A quartz crystal microbalance (QCM) biosensor system for lectin-carbohydrate interactions has been developed. Yeast mannan was immobilised on polystyrene-coated quartz crystals, and interactions tested with the lectin concanavalin A (Con A). The biosensor could be easily operated, where mannan immobilisation and all binding analyses were performed in real-time using a flow-through system. The apparent binding constant for yeast mannan to Con A was estimated to be 0.4 microM, well in accordance to reported literature values. In addition, the effective concentration values (EC50-values) for a series of mannose/mannoside ligands, acting as competitors to the mannan/Con A interaction, were determined to range from 0.18 to 5.3 mM, in good correlation with a related enzyme-labelled lectin assay (ELLA) protocol.     

Con A Binds to the Membranellar and Somatic Cilia of Stentor and to the Developing Oral Primordium During Oral Regeneration1

Binding sites for Concanavalin A have been located in the ciliate Stentor coeruleus by utilizing FITC-Con A and fluorescence microscopy. When both nonregenerating and regenerating Stentor are fixed prior to FITC-Con A exposure, FITC-Con A binds intensely to the cilia of the membranellar band and to the somatic cilia that cover much of the cell surface. No binding is observed between the ciliary rows. The FITC-Con A also binds to the developing oral primordia of regenerating cells. Binding of FITC-Con A in the early stages of regeneration (prior to stage 4) appears to be less intense than that in the later stages. Additional FITC-Con A binding appeared as a granular fluorescence in the area of the developing buccal cavity beginning at about stage 4 and disappearing around stages 6–7. The presence of α-D-methyl mannoside prevented the binding of FITC-Con A to either regenerating or nonregenerating cells. If nonregenerating Stentor are exposed to FITC-Con A prior to fixation, the binding pattern is entirely different with the fluorescence primarily in the form of random, granular patches spread over much of the cell but with no binding to either type of cilia. These results demonstrate that membrane glycoproteins capable of binding Con A are located primarily in the membranellar and somatic cilia and in the developing oral primordia during oral regeneration in Stentor. Concanavalin A binding to these sites may be involved in the Con A-induced inhibition of oral regeneration observed in earlier studies.     

Participation of calcium in flagellar shortening and regeneration in Chlamydomonas reinhardii

Cation chelators cause flagellar shortening in Chlamydomonas reinhardii. Most effective are EDTA and EGTA (1 mM) but pyrophosphate (10 mM) also is effective. Addition of 5 mM Ca2+ after shortening caused by 4 mM EGTA results in flagellar regeneration. Other divalent cations can replace Ca2+ with the following relative activities: Ca2+ greater than Sr2+ = Mn2+ much greater than Ba2+ = Mg2+. Although the specific ion requirement to reverse shortening is not clear, it is possible that all of the ions act by displacing one bound cation, presumably Ca2+. A specific requirement for Ca2+ in flagellar regeneration could be demonstrated, however, because as little as 50 microM EGTA in the presence of 500 microM Mg2+ delayed regeneration and prevented full regeneration. Ca2+ at 100 microM overcame this inhibition.     

Stimulation of human polymorphonuclear leukocyte phosphatidylinositol-4-phosphate kinase by concanavalin A and formyl-methionyl-leucyl-phenylalanine is calcium-independent. Correlation with maintenance of actin assembly.

Chemoattractants directly stimulate the enzyme activity that synthesizes phosphatidylinositol-4,5-bisphosphate (PIP2), phosphoinositol-4-monophosphate (PIP) kinase. The present study determined whether stimulation of this enzyme correlates with actin assembly by assessing the calcium dependence of this reaction. Incubation of neutrophils with 5 to 100 micrograms/ml Con A caused a concentration-dependent increase in PIP kinase activity ranging from 1.38- to 3.4-fold. The effective concentration which stimulated PIP kinase by 50% (17 micrograms/ml, EC50) corresponded with the EC50 for Con A-induced superoxide production (32 micrograms/ml). Like chemoattractants, the increase in PIP kinase by Con A was characterized by a 2.6-fold increase in the maximum velocity (Vmax) of the enzyme, and no change in the Km for ATP. The kinetics of FMLP- and Con A-induced filamentous actin formation preceded stimulation of PIP kinase and was sustained over the same time period that this increased enzyme activity was noted. Although transmembrane signaling by FMLP and Con A requires an increase in intracellular calcium for some polymorphonuclear leukocyte (PMN) functional responses, calcium depletion of PMN by incubation with 100 microM Quin 2 A/M and 5 mM EGTA did not prevent the stimulation of PIP kinase by FMLP or Con A. In addition, calcium depletion did not prevent the increase in filamentous actin formation by FMLP and Con A in PMN. These findings demonstrate that Con A increases PIP kinase activity in human PMN and that PIP kinase stimulation and maintenance of actin assembly are independent of calcium fluxes in these cells. Because PIP2 controls the function of the actin-regulatory proteins, profilin and gelsolin, changes in the synthetic rate of PIP2 through regulation of PIP kinase may provide a molecular basis for the prolonged stimulation of actin assembly in human PMN by agonists such as Con A and FMLP.     

Characteristics of angiotensin II-, K+- and ACTH-induced calcium influx in adrenal glomerulosa cells. Evidence that angiotensin II, K+, and ACTH may open a common calcium channel

The characteristics of angiotensin II-, K+-, and adrenocorticotropin (ACTH)-induced calcium influx were studied in isolated adrenal glomerulosa cells. Basal calcium influx rate is 0.64 +/- 0.09 nmol/min/mg of protein. Addition of angiotensin II (1 nM) causes a rapid 230% increase in calcium influx rate. This angiotensin II-induced calcium influx is sustained and is rapidly reversed by angiotensin II antagonist, [Sar1,Ala8]angiotensin II. Addition of either K+ or ACTH (1 nM) causes a 340 or 160% increase, respectively, in the rate of calcium influx. The effect of either angiotensin II, K+, or ACTH on calcium influx is dependent on extracellular calcium. The apparent Km for calcium is 0.46, 0.35, and 0.32 mM, respectively. When the extracellular concentration of K+ is 2 mM, neither angiotensin II nor ACTH stimulates calcium influx. Conversely, when extracellular K+ is increased to 6 mM, both angiotensin II and ACTH cause a greater stimulation of calcium influx than at 4 mM K+. When extracellular K+ is increased to 10 mM, calcium influx is 360% of the basal influx seen at 4 mM K+, and neither angiotensin II nor ACTH further stimulates the influx rate. Nitrendipine (1 microM) blocks both angiotensin II- and K+-induced calcium influx completely. In contrast, 10 microM nitrendipine does not completely block ACTH-induced calcium influx. The calcium channel agonist, BAY K 8644, also stimulates calcium influx; 10 nM BAY K 8644 leads to a rate of calcium influx which is 185% of basal. This BAY K 8644-induced increase in calcium influx and that caused by either angiotensin II or ACTH are additive. In contrast, BAY K 8644 has more than an additive effect on the calcium influx when paired with 6 mM K+. These results suggest that angiotensin II, K+, and ACTH stimulate calcium influx via a common calcium channel but act by different mechanisms to alter its function.     

Influence of extracellular calcium on cell permeabilization and growth regulation by the lymphokine leukoregulin

Permeablization of human K562 leukemia cells was measured in the presence and absence of extracellular ionic calcium to examine the relationship of ionic calcium to increased membrane permeability and the inhibition of cell proliferation by this lymphokine. In the absence of extracellular calcium, the ability of leukoregulin to permeabilize the cell membrane is diminished but is fully restored by addition of 1 mM extracellular Ca++ as shown flow cytometrically by loss of intracellular fluorescein. Membrane permeability is also increased by calcium ionophore A23187 but permeablization is completely blocked in calcium-free medium despite the intramembrane presence of the calcium ionophore. Membrane permeablization by the lectin phytohemagglutinin, in contrast, is independent of extracellular calcium. A similar divergence in cell proliferation activity of the three modulators of calcium flux and membrane permeability occurs in the absence of extracellular calcium. Leukoregulin inhibition of cell proliferation is abolished, inhibition by calcium ionophore A23817 is greatly reduced, and inhibition by phytohemagglutinin is unchanged. Leukoregulin permeabilized K562 cells isolated by fluorescence activated cell sorting resume proliferation after 72 h. In contrast cells permeablized by calcium ionophore A23187 or phytohemagglutinin fail to resume proliferation by 7 days. The membrane permeablizing action of leukoregulin is, therefore, partially dependent upon extracellular calcium. It is also effected through a mechanism other than calcium ionophore transport or lectin type transmembrane signaling, and is accompanied by a reversible inhibition of cell proliferation.     

Ethanol inhibition of cell-mediated lysis of antibody-sensitized target cells at a calcium-dependent step

Ethanol inhibits antibody-dependent cell-mediated cytotoxicity in a dose-dependent manner. The inhibitory effect of ethanol was reversed by the addition of excess calcium or calcium ionophore A23187. Excess calcium at 4-8 mM concentrations was required to reverse 50% of the inhibition caused by ethanol. In seven of nine experiments, 16 mM excess calcium completely reversed the inhibition and produced greater lysis than the control. Excess calcium in the absence of ethanol induced a dose-dependent increase in lytic activity by the spleen cells. However, the reversal of inhibition by ethanol could not be attributed to a simple additive effect resulting from the increased cytolytic capacity of the lymphocytes in the presence of excess Ca2+. Ionophore A23187 at 1 microM also partially reversed the inhibitory effect caused by ethanol. Ionophore alone did not potentiate lytic activity. When target cells were not sensitized with antibody, excess calcium had no effect on the lysis of target cells in the presence of ethanol-treated or untreated lymphocytes. These data suggest that ethanol inhibits antibody-dependent cell-mediated cytotoxicity at a calcium-dependent step.     

Calcium regulation of cell-cell contact and differentiation of epidermal cells in culture. An ultrastructural study

Calcium modulation of keratinocyte growth in culture was studied by both transmission (TEM) and scanning electron microscopy (SEM). Under standard culture conditions (1.2-1.8 mM calcium), cells were connected by desmosomes and stratified to 4-6 cell layers. Many aspects of in vitro epidermal maturation were analogous to the in vivo process, with formation of keratohyalin granules, loss of nuclei, formation of cornified envelopes and shedding of cornified cells containing keratin filaments. When the medium calcium concentration was lowered to 0.02-0.1 mM, the pattern of keratinocyte growth was strikingly changed. Cells grew as a monolayer with no desmosomal connections and proliferated rapidly, shedding largely non-cornified cells into the medium. Large bundles of keratin filaments were concentrated in the perinuclear cytoplasm. The elevation of extracellular calcium to 1.2 mM induced low calcium keratinocytes to stratify, keratinize and cornify in a manner analogous to that seen when plated in standard calcium medium. The earliest calcium-induced ultrastructural change was the asymmetric formation of desmosomes between adjacent cells. Desmosomal plaques with associated tonofilaments were observed 5 min after calcium addition; symmetric desmosomes were formed within 1-2 h. This system is presented as a useful model for the study of the regulation of desmosome assembly and disassembly.     

Evidence on the role of three calcium pools in Ca-ionophore A23187-stimulated rat blood platelet aggregation.

The effect of Ca-ionophore A23187 on activation of rat blood platelets was investigated to elucidate the involvement of extracellular and intracellular Ca2+ ions. Platelet aggregation induced by 10 concentrations of the stimulus was studied in Ca-free medium as well as in the presence of EGTA and/or calcium. In Ca-free medium, A23187 induced platelet aggregation in a dose-dependent way; the mean effective concentration was 1.43 +/- 0.08 mumol/l. The stimulatory effect of ionophore was potentiated by addition of 0.01 and 0.1 mM calcium and inhibited when the calcium concentration was increased to 1 mmol/l. In the presence of EGTA, A23187-stimulated aggregation of isolated rat platelets was recorded only at a 10-times higher ionophore concentration and was then reduced to 30% in comparison with aggregation in Ca-free medium. The inhibitory effect of 1 mM EGTA was abolished by addition of 2 mM calcium. We suggest the participation of at least three calcium pools in the stimulation of rat platelets by A23187, i.e. the extracellular pool, the membrane-associated pool and the pool displacing calcium intracellularly.     

Cytoskeletal and DNA synthesis modification by concanavalin A in embryonic fibroblasts maintained in serum-free and serum-added medium

The administration of lectin concanavalin A (Con A) to in vitro cultures of chick embryo fibroblasts caused dose-dependent changes in cell shape, cytoskeleton network and DNA synthesis. After 6 hrs. even a low doses of Con A produced evident effects in serum-free cultures, whereas higher doses were required to cause alterations in cells cultured in serum-added medium. As the sugar competitor mannoside abolishes the effects, it would seem that the lectin acts by binding to transmembrane receptors and that the fibronectin present in the serum engages the receptors so that they are not available to Con A.     

Effects of trifluoperazine and mitogenic lectins on calcium ATPase activity and calcium transport by human lymphocyte plasma membrane vesicles

The phenothiazine, trifluoperazine, and the mitogenic lectins, phytohemagglutinin (PHA) and Concanavalin A (Con A), were tested for their effects on human lymphocyte plasma membrane Ca-activated Mg-ATPase and ATP-dependent calcium uptake. Trifluoperazine completely inhibited Ca-uptake when present from the start of the assay at concentrations of 100 microM or more. When added during measurement of calcium uptake, trifluoperazine reduced the rate of vesicular calcium accumulation but was unlike the calcium ionophore, A23187, which caused a rapid release of accumulated calcium from the vesicles. Trifluoperazine also inhibited membrane vesicle Ca-ATPase activity, but this inhibition was non-specific since the Mg-ATPase and Na,K-ATPase activities were inhibited to similar extents at the same concentration of the phenothiazine. In contrast, concentrations of PHA and Con A, which are mitogenic for lymphocytes, did not cause any change in Ca-uptake when added to suspensions of membrane vesicles. Con A had no effect and PHA had a weak inhibitory effect on Ca-ATPase activity.     

Extracellular Calcium-Induced Neuroblastoma Cell Differentiation: Involvement of Phosphatidylinositol Turnover

The rat CNS neuroblastoma B50 cell line is known to differentiate on addition of 1 mM dibutyryl cyclic AMP or on withdrawal of serum. In this report it is shown that high levels of extracellular calcium (10-25 mM) cause neurite extension, an important component of morphological differentiation. Stimulation of calcium influx with the ionophore A 23187 or blockade of calcium efflux with lanthanum are less efficient than extracellular calcium in stimulating neurite extension. These data suggest that intracellular calcium is not sufficient to cause full expression of a calcium-dependent differentiated state. Furthermore, phosphatidylinositol turnover is sharply altered as early as 1 h after addition of calcium to the medium while cyclic nucleotide levels remain unaffected. This suggests that activation of the phosphatidylinositol second-messenger system by calcium at the level of the cell membrane is the initial step in the cascade of events leading to neurite extension. Later events include a decrease in DNA synthesis (6-10 h after addition of calcium), and increase in intracellular calcium levels (12-24 h after calcium addition) concurrent with neurite extension. The intracellular increase in calcium levels is facilitated by synergistic action of 1 mM dibutyryl cyclic AMP with high external calcium (10-25 mM). This combined treatment results in a more complex pattern of neurite formation characterized by many synaptic-like junctions; this pattern is not obtained when either dibutyryl cyclic AMP or calcium is used as sole inducer.