Binding of Ca ions by Paramecium caudatum

Binding of ⁴⁵Ca by live Paramecium caudatum was determined under various external ionic conditions. It was found that calcium uptake was separable into at least two components, a rapid and a slow one. The rapid component was influenced by the presence of certain other ions in a manner which agrees with the law of mass action. It appears that an ion exchange system may be involved in a binding equilibrium established between Paramecium, Ca⁺⁺, and certain other ions. K⁺, Rb⁺, and Ba⁺⁺ in the equilibrium medium are among those ions which inhibit calcium uptake. It is proposed that liberation of Ca⁺⁺ from binding sites on Paramecium by an exchange reaction with competing ions is the first step in the mechanism of ciliary reversal in the response to external application of these ions.     

Control of the Orientation of Cilia by Adenosinetriphosphate,Calcium, and Zinc in Glycerol-Extracted Paramecium caudatum

The predominant orientation of cilia in glycerol-extracted Paramecium is toward the posterior of the specimen in a KCl solution. The cilia became reoriented toward the anterior shortly after transfer of the extracted cell to a mixture of ATP, calcium, and zinc. The degree of response was graded as a function of the concentration of each of the three essential factors. Minimum concentrations for the maximum response were 0.2 mM in ATP, 0.8 mM in calcium, and 0.0002 mM in zinc. The observations support the hypothesis that cation-induced ciliary reversal in live specimens is initiated by calcium ions which become displaced from an inferred cellular cation exchanger system.     

Electrophysiological Control of Reversed Ciliary Beating in Paramecium

Quantitative relations between ciliary reversal and membrane responses were examined in electrically stimulated paramecia. Specimens bathed in 1 mM CaCl₂, 1 mM KCl, and 1 mM Tris-HCl, pH 7.2, were filmed at 250 frames per second while depolarizing current pulses were injected. At current intensities producing only electrotonic shifts the cilia failed to respond. Stimuli which elicited a regenerative response were followed by a period of reversed ciliary beating. With increasing stimulus intensities the latency of ciliary reversal dropped from 30 to 4 ms or less, and the duration of reversal increased from 50 ms to 2.4 s or more; the corresponding regenerative responses increased in amplitude and rate of rise. With progressively larger intracellular positive pulses, electric stimulation became less effective, producing responses with a progressive increase in latency and decrease in duration of reversed beating of the cilia. When 100-ms pulses shifted the membrane potential to +70 mV or more, ciliary reversal was suppressed until the end of the pulse. "Off" responses then occurred with a latency of 2–4 ms independent of further increases in positive potential displacement. These results suggest that ciliary reversal is coupled to membrane depolarization by the influx of ions which produces the regenerative depolarization of the surface membrane. According to this view suppression of the ciliary response during stimulation occurs when the membrane potential approaches the equilibrium potential of the coupling ion, thereby retarding its influx. Previous data together with the present findings suggest that this ion is Ca²⁺.     

Effect of Extracellular pH on Motility and K+-Induced Ciliary Reversal in Paramecium caudatum1

ABSTRACT. Paramecium caudatum, reared on bacterized hay infusions at pH 6.5 to 6.9, were washed into various buffered solutions containing 0.016 mM CaCl₂ and a pH of 3.5 to 10.4. Solutions of pH 4.5 to 9.5 support strong swimming of the cells for at least 24 h. At pH values acid to the culture medium, cells show an increasing frequency of spontaneous ciliary reversal episodes (“avoiding reactions”). Uninterrupted forward swimming is usually observed over the pH range of 7.1 to 8.5, and above pH 8.5, forward motion is interrupted by circular swimming. For all pH values tested, transfer of cells to a more acidic test solution than the solutions into which they were washed (adaptation solution) usually induced short duration, periodic ciliary reversal behavior. With transfer to a more alkaline test solution than the adaptation solution, the cells shift from forward left spiralling motion to forward right spiralling motion. With decreasing pH, the cells show progressively less sensitivity to KC1 stimulation, and at pH values of less than 5.0, cells fail to show significant ciliary reversal response to any KC1 concentration tested (1 - 128 mM). At alkaline pH values and higher KC1 concentrations, the cells show very pronounced ciliary reversal behaviors but usually fail to regain forward swimming behavior.     

Localization of Thermoreceptor Systems that Induce Step-Up and Step-Down Thermophobic Responses and Switching in the Dominance of These Systems in Blepharisma

The distribution of thermoreceptor systems that initiate step-up and step-down thermophobic responses in bisected cells of Blepharisma was examined. Anterior cell fragments responded by ciliary reversal to a step-down in temperature and by repression of spontaneous ciliary reversal to a step-up. Posterior fragments responded by ciliary reversal to a step-up in thermal stimulation and by repression of spontaneous ciliary reversal in response to step-down stimulation. Results indicate that two kinds of thermoreceptor systems exist in the anterior half of each cell; one is responsible for ciliary reversal induced by step-down stimulation, and the other is responsible for repression of the ciliary reversal caused by step-up thermal stimulation. Likewise, there are also two kinds of thermoreceptor systems in the posterior half of the cell; one is responsible for ciliary reversal in response to a step-up in temperature, and the other is responsible for the repression of ciliary reversal on a step-down in thermal stimulation. Below about 27°C, intact cells showed ciliary reversal only when a step-down in thermal stimulation occurred, while above about 27°C cells only responded to a step-up in thermal stimulation. At about 27°C there was a switch in the dominant response from the anterior to the posterior half of an individual cell.     

Inactivation of Ca2+-induced ciliary reversal by high-salt extraction in the cilia of Paramecium

Intracellular Ca²⁺ induces ciliary reversal and backward swimming in Paramecium. However, it is not known how the Ca²⁺ signal controls the motor machinery to induce ciliary reversal. We found that demembranated cilia on the ciliated cortical sheets from Paramecium caudatum lost the ability to undergo ciliary reversal after brief extraction with a solution containing 0.5 M KCl. KNO₃, which is similar to KCl with respect to chaotropic effect; it had the same effect as that of KCl on ciliary response. Cyclic AMP antagonizes Ca²⁺-induced ciliary reversal. Limited trypsin digestion prevents endogenous A-kinase and cAMP-dependent phosphorylation of an outer arm dynein light chain and induces ciliary reversal. However, the trypsin digestion prior to the high-salt extraction did not affect the inhibition of Ca²⁺-induced ciliary reversal caused by the high-salt extraction. Furthermore, during the course of the high-salt extraction, some axonemal proteins were extracted from ciliary axonemes, suggesting that they may be responsible for Ca²⁺-induced ciliary reversal.     

Relations Between Photobehavior and Ionically Stimulated Swimming Behavior in Paramecium bursaria1

ABSTRACT. Cells of Paramecium bursaria, which harbor a symbiotic alga of the genus Chlorella, reverse the effective beat of their cilia and swim backward when stimulated in either of two ways. Ionic stimulation is introduction of cells into a solution high in K⁺ while step-down photostimulation is a sharp reduction in the intensity of light falling on the culture. Much is known about the mechanisms of ionic stimulation of ciliary reversal, but little is known about step-down photobehavior. Inhibitors of ionically stimulated ciliary reversal were applied to cells undergoing step-down photobehavior; Ca-channel inhibitors, neomycin and W-7, inhibit both behaviors. Activation of Ca-channels in the ciliary membrane is involved in step-down photobehavior, suggesting that the algae may alter the Paramecium membrane to make it more excitable.     

Role of cell wall bound calcium in Neurospora crassa

Cell wall bound calcium constitutes a significant fraction (25%) of total mycelia calcium in Neurospora crassa. Wall bound calcium increases as a function of growth and calcium concentration, while cell wall bound calcium decreases in Ca-free medium. Removal of wall bound calcium causes its rapid replacement from intracellular pool, inhibited by verapamil, nifedipine, concanamycin A, and wortmanin in a vacuolar mutant (Vma-5), but is unaffected by trifluoropyrazine, and calmidizoluim in a calcineurin mutant (Cnb-1) of N. crassa. Ca²⁺ removal from surface with EGTA resulted in leakage of periplasmic enzymes invertase and alkaline phosphatase. Scanning and transmission electron microscopy revealed gross abnormalities represented by giant vacuoles. Toxic metal ions bound to wall fraction by displacing calcium. Our data underline the physiological importance of wall bound calcium in N. crassa.     

Studies on the size,location and turnover of calcium pools accessible to growing Tetrahymena cells

Tetrahymena pyriformis cells in the logarithmic phase of growth accumulate 2.5–3.75 times as much calcium per unit volume as is present in the growth medium. It appears that most of this calcium is stored in a non-ionic form, with approximately 30% existing in the cilia, near its site of action in effecting ciliary reversal. The exchange of extracellular ⁴⁵Ca²⁺ with the major internal pools is extremely rapid, exhibiting a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of less than 0.5 h. Sites located on the cilia are responsible for 35–50% of Ca²⁺ influx, with the remainder entering through other positions on the cell surface.     

The effect of monovalent cations on calcium efflux in yeasts

The properties of the calcium efflux system in the yeast Saccharomyces cerevisiae were investigated. After growing the cells overnight in medium containing ⁴⁵Ca, the cells were transferred to medium containing glucose, Hepes buffer (pH 5.2) and monovalent cations. The presence of potassium or sodium in the medium induced efflux of calcium from the cells. The magnitude of the efflux was dependent on the concentration of these cations in the medium. The time course of calcium efflux was analyzed, and two types of exchangeable calcium pools, which turned over at different rates, were detected: ‘Fast turnover’ and ‘slow turnover’. Increase in the concentration of monovalent cations in the medium caused an increase in the fraction of cellular calcium which turned over at a fast rate, and activation of calcium efflux from the ‘slow turnover’ calcium pool. The specific changes in the parameters of calcium efflux induced by monovalent cations were different from those reported previously to be induced by divalent cations. Both processes, i.e. activation of calcium efflux by monovalent and by divalent cations, were found to be additive, indicating that they operate via different mechanisms. Experiments using the respiratory inhibitor Antimycin A, showed that stimulation of calcium efflux by monovalent cations is energy dependent. Lanthanum ions which are known to inhibit calcium influx into yeast cells, inhibitted the activation of calcium efflux by both divalent and monovalent cations. Determination of the cationic composition of the cells indicated that the stimulation of calcium efflux was accompanied by influx of potassium or sodium into the cells.     

Certain components of lettuce juice modify the thermoresponse of the ciliated protozoan Blepharisma japonicum

When the cells of Blepharisma cultured in lettuce juice were transferred to media containing lettuce juice with temperature gradient of 20–30°C, they accumulated in a region corresponding to about 25°C. The cells washed in a saline solution, however, accumulated in the region above 30°C. The results indicate that certain components contained in lettuce juice change the thermosensitivity of the cells. When the temperature was suddenly changed, a transient decrease or increase in the frequency of ciliary reversal was observed. The response of the cells incubated in a saline solution without lettuce juice was different from those in the saline solution containing lettuce juice above 25°C. Above 25°C, the cells incubated in a saline solution without lettuce juice responded by increased frequency of ciliary reversal only to step-down in temperature and by repression of ciliary reversal to a step-up in temperature, while the cells incubated in the same saline solution containing lettuce juice responded by increased frequency of ciliary reversal to a step-up in temperature and by repression of ciliary reversal to a step-down in temperature. The thermal response was examined in bisected fragments of the cells. The difference in response between the saline solution without lettuce juice and the solution containing lettuce juice was detected only in posterior fragments above 25°C. Above 25°C, the posterior fragments incubated in the solution without lettuce juice did not respond to sudden temperature changes, whereas the fragments kept in the medium containing lettuce juice responded (step-up thermophobic response and step-down repression of ciliary reversal) in the presence of lettuce juice.     

Specific adsorption of phosphate ions on proteins evidenced by capillary electrophoresis and reversed-phase high-performance liquid chromatography

Specific adsorption of phosphate ions at pH=7.0 was studied on different proteins, either counter-ions of phosphate (lysozyme, lactoferrin) or co-ion of phosphate (α-lactalbumin). The theoretical electrophoretic mobility of globular proteins lysozyme and α-lactalbumin (apo and holo (+1 calcium per molecule) forms) was compared with those measured by capillary electrophoresis in phosphate at pH 7.0, versus the ionic strength (I) in the range 0–0.775 mol L⁻¹. The specific adsorption of phosphate ions was evidenced by difference. From the experimental charge number (Z^eff) of protein in phosphate medium, a phosphate content per protein molecule was determined at pH=7.0.

• •For lactoferrin (pI=8–9), the electrophoretic mobility (μ) was constant and negative, highlighting a charge reversal due to phosphate adsorption.
• •For α-lactalbumin (holo form) experimental μ was roughly constant and more negative than predicted. Z^eff increased continuously from −4 to −11 in the ionic strength range from 0.005 to 0.775 mol l⁻¹, respectively. Accordingly, one to six phosphates were bound per molecule, respectively.
• •For lysozyme, experimental electrophoretic mobility was positive but lower than predicted. Z^eff was only discrete values +5 for I in the range 0.001–0.020 mol l⁻¹ and about +3 in the range 0.050–0.500 mol l⁻¹, whereas the theoretical Z value was +7 at pH=7.0. Lysozyme bounds one phosphate at low ionic strength and about two — three at higher ionic strength.

Reversed-phase HPLC confirms that adsorption of phosphate is different for the three proteins.     

Sequential mechanism of calcium binding and translocation in sarcoplasmic reticulum adenosine triphosphatase

Cooperative calcium binding (apparent Kd = 1.04 X 10(-6) M) to the ATPase of sarcoplasmic reticulum vesicles occurs with a maximal stoichiometry of 2 mols of divalent cation/mol of enzyme in the absence of ATP. The bound calcium is distributed into two pools which undergo fast or slow isotopic exchange, respectively. The two pools retain a 1:1 molar ratio under various conditions and are both located within a protein crevice, as suggested by their cooperative interaction and exchange kinetics. Following enzyme phosphorylation by ATP, both pools of bound calcium are "internalized" (cannot be displaced by quench reagents). If following 45Ca2+ binding, isotopic dilution is obtained in the medium by adding 40Ca2+ with ATP, internalization of both pools of bound 45Ca2+ (2 mol/mol of phosphoenzyme) is still observed within the first enzyme cycle. When the cycle is reversed by addition of excess ADP soon after ATP, only half of the internalized 45Ca2+ is released from the enzyme into the medium outside the vesicles, while the other half remains with the vesicles. If half of the bound 45Ca2+ is exchanged (fast exchange) with 40Ca2+ previous to the addition of ATP, none of the remaining 45Ca2+ is released outside the vesicles upon reversal of the enzyme cycle. Therefore, the pool of bound calcium which undergoes slower exchange with the outside medium, is the first to be released inside the vesicles upon enzyme phosphorylation. A sequential mechanism of calcium binding and translocation is proposed, that accounts for binding cooperativity and exchange kinetics, presteady state transients following addition of ATP, and the Ca2+ concentration dependence of ATPase activity in steady state.     

Effect of some mineral ions on pollen tube growth and release of proteins in culture

In the absence of cations, the release of proteins from pollen tubes ofNicotiana tabacum in culture is greatly dependent on boron concentration and inversely related to growth stimulation. The minimum of proteins in the medium occurs at 100 mg 1^? of boric acid, which is the optimum concentration for growth. The shift of boron level to this optimum further increases the proportion of proteins bound to the insoluble pollen tube fraction; on the other hand the amount of soluble proteins is not affected inside pollen tubes, but greatly decreased in the medium. The loss of proteins into the medium is considerably reduced by oalcium and also at the optimal boron concentration and in the presence of K⁺ and Mg²⁺ ions. The rate of tube elongation, however, is slightly decreased and the duration of pollen growth activity is not extended. The release of proteins is not affected by potassium and is slightly reduced by magnesium. The possible mechanism of calcium and boron action on protein release is discussed in relation to the exocytotic function of secretory vesicles and it is suggested that boron supports the incorporation of proteins transported to the growing tip into the pollen tube wall structures.     

On the significance of the influx of calcium ions into stimulated human blood platelets

Blood platelets, upon stimulation with various substances, take up calcium ions from the suspending medium. This influx occurs simultaneously with the release reaction, i.e. the specific secretion of a variety of substances from storage organelles and the second wave of aggregation. Various inhibitors of the release reaction inhibit this Ca²⁺ influx. Platelets previously loaded with ⁴⁵Ca show an increased efflux of the cation upon stimulation by thrombin. These results suggest that the plasma membrane acquires an increased permeability to Ca²⁺ only in a later phase of platelet activation, in most cases after the earlier release of Ca²⁺ into the cytoplasm from Ca-storing organelles. Rapid shape change and release proceed independently of external calcium, whereas clot retraction depends upon a prolonged increased permeability of the plasma membrane to this cation.     

Studies on the Germination of Pine Pollen (Pinus mugo) in vitro. II. Effects of Different Ions

Studies on the initial germination of pollen of Pinus mugo showed no significant influence of ions on O₂ uptake and uptake of ³²P-labelled phosphate. At the onset of tube growth O₂ uptake decreased in the absence of calcium. In inorganic media tube growth and ³²P uptake were reduced in the absence of calcium or boric acid. In the absence of calcium a requirement for magnesium was observed. When the medium was deprived of polyvalent ions with EDTA, growth and ³²P uptake ceased. The presence of calcium in the medium was found to be essential for the maintenance of the structural and functional integrity of the cell membranne. — The ion requirement was more pronounced when tube growth was stimulated with sucrose. Calcium, magnesium, boric acid, and nitrate (as nitrogen source) were essential constitutents of the medium. The stimulation due to calcium required either magnesium or boric acid. — A density effect was observed which can be related to diffusible substances from the pollen into the medium. This was not observed when calcium and magnesium were present in the medium. The phenomenon is explained as an enrichment of the medium with diffusible substances from non-germinated dead pollen. — Germination and the tube growth were found to be greatly dependent on a short period of equilibration of pollen at room temperature before sowing.     

Biophysical effects of the natural product euplotin C on the <Emphasis Type="Italic">Paramecium</Emphasis> membrane

The effect of euplotin C—a cytotoxic secondary metabolite produced by the protist ciliate Euplotes crassus—on the voltage-dependent Ca²⁺ channel activity was studied in a single-celled system by analyzing the swimming behavior of Paramecium. When the intraciliary Ca²⁺ concentration associated with plasma membrane depolarization increases, a reversal in the direction of ciliary beating occurs, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca²⁺ influx. The present study demonstrates that the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, is longer in euplotin C-treated cells. Using selective Ca²⁺ channel blockers, we demonstrate that euplotin C modulates Ca²⁺ channels similar to the T- and L-types that occur in mammalian cells. Indeed, the increase of CCR duration significantly decreased when flunarizine and nimodipine-verapamil blockers were employed. Membrane fluidity measurements using a fluorescent dye, 6-lauroyl-2-dimethylaminonaphtalene (laurdan), indicated that membranes in euplotin C-treated cells are more tightly packed and ordered than membranes in control cells. Our data suggest that euplotin C enhances backward swimming in our unicellular model system by interacting with the ciliary Ca²⁺ channel functions through the reduction of cell membrane fluidity.     

pH dependence of the effect of adenosine triphosphate and ethylenediaminetetraacetate on sodium and magnesium binding by cellular membrane fragments

The pH dependence of previously reported effects of adenosine triphosphate (ATP) and ethylenediaminetetraacetate (EDTA) on cation binding by rat liver microsomes was studied by an equilibration and washing procedure. Equilibration of microsomes in media containing 95 mM NaCl and 4 mM MgCl₂ with pH varied from 4 to 8 resulted in an increase in bound cations from zero below pH 4 to 0.90 mmoles Mg and 0.34 mmoles Na/g N at pH 8; the ratio of bound Na/bound Mg increased from 0.15 at pH 5 to 0.38 at pH 8. Addition of 5 mM EDTA to the equilibration media produced striking changes in cation binding such that bound Na/bound Mg increased from 0.30 at pH 5 to 3.90 at pH 7 and decreased to 3.55 at pH 8. In the presence of added 10 mM ATP, bound Na/bound Mg increased from 0.10 at pH 5 to a maximum of 0.80 at pH 7. The observed changes could generally be correlated with known mass law relationships, although the system containing added ATP was complicated considerably by the hydrolysis of ATP. Results demonstrate that environmental pH is an important factor in determining the effect of ATP and EDTA on the cation binding pattern of cellular membranes. Because hydrogen ion is a product of ATP hydrolysis as well as of other metabolic reactions, the described interactions may be of particular significance in the molecular mechanisms of ATP effects on cation binding and transport in living cells.     

One-way calcium spill-over during signal transduction in Paramecium cells: from the cell cortex into cilia, but not in the reverse direction

We asked to what extent Ca(2+) signals in two different domains of Paramecium cells remain separated during different stimulations. Wild-type (7S) and pawn cells (strain d4-500r, without ciliary voltage-dependent Ca(2+)-channels) were stimulated for trichocyst exocytosis within 80 ms by quenched-flow preparation and analysed by energy-dispersive X-ray microanalysis (EDX), paralleled by fast confocal fluorochrome analysis. We also analysed depolarisation-dependent calcium signalling during ciliary beat rerversal, also by EDX, after 80-ms stimulation in the quenched-flow mode. EDX and fluorochrome analysis enable to register total and free intracellular calcium concentrations, [Ca] and [Ca(2+)], respectively. After exocytosis stimulation we find by both methods that the calcium signal sweeps into the basis of cilia, not only in 7S but also in pawn cells which then also perform ciliary reversal. After depolarisation we see an increase of [Ca] along cilia selectively in 7S, but not in pawn cells. Opposite to exocytosis stimulation, during depolarisation no calcium spill-over into the nearby cytosol and no exocytosis occurs. In sum, we conclude that cilia must contain a very potent Ca(2+) buffering system and that ciliary reversal induction, much more than exocytosis stimulation, involves strict microdomain regulation of Ca(2+) signals.     

Affinities or Apparent Affinities of the Transport Adenosine Triphosphatase System

The interactions of potassium ions and ATP on transport ATPase activity are discussed, and the interpretation of these interactions is shown to be often ambiguous. Caldwell''s (1968) Physiological Review model is discussed with particular reference to the observed kinetics of sodium: sodium exchange in red cells. Recent experimental work on the properties of the ouabain-sensitive component of potassium efflux from red cells is described. This component of efflux occurs only if either sodium or potassium are present in the external medium, but the effects of external sodium and potassium are not additive. The relation between ouabain-sensitive potassium efflux and the external concentration of sodium (in a potassium-free medium) or of potassium (in low- and high-sodium media) are described. When starved sodium-poor red cells are poisoned with iodoacetamide, loaded with phosphate, and incubated in high-sodium potassium-free media, the ouabain-sensitive efflux of potassium appears to be accompanied by the reversal of the entire ATPase system. About two to three potassium ions leave by the ouabain-sensitive route for each molecule of ATP synthesized. If potassium is present in the external medium, no ouabain-sensitive synthesis of ATP occurs and the ouabain-sensitive efflux of potassium presumably involves the reversal of only the last part of the ATPase system.