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.     

Veratridine induces a Ca2+ influx,cyclic GMP formation,and backward swimming inParamecium tetraurelia wildtype cells and Ca2+ current-deficient pawn mutant cells

Summary Veratridine opens voltage-dependent Na⁺ channels in many metazoans. InParamecium, which has voltage-dependent Ca²⁺ channels and a Ca/K action potential, no such Na⁺ channels are known. A Ca-inward current is correlated to an intracellular increase in cGMP. The addition of veratridine toParamecium wildtype and to pawn mutant cells, which lack the Ca-inward current, transiently increased intracellular levels of cGMP about sevenfold to 40 pmol/mg protein. A half-maximal effect was obtained with 250 m veratridine. The increase in cGMP was maximal about 15 sec after the addition of veratridine and declined rapidly afterwards. Intracellular cAMP levels were not affected. The effect of veratridine on cGMP was dependent on the presence of extracellular Ca²⁺. The time dependence and extent of stimulation closely resembled the effects observed after stimulation by Ba²⁺, which causes the repetitive firing of action potentials, Ca-dependent ciliary reversal, and cGMP formation. The effects of Ba²⁺ and veratridine were not additive. Wildtype cells and, surprisingly, also pawn mutant cells showed avoiding reactions upon addition of veratridine indicating that it induced a Ca²⁺ influx into the cilia, which causes ciliary reversal. The potency of veratridine to stimulate cGMP formation was little affected by Na⁺ in wildtype cells, three pawn mutant strains, and in the cell line fast-2, which is defective in a Ca-dependent Na-inward current. Divalent cations (Ca²⁺, Mg²⁺, and Ba²⁺) inhibited the effects the veratridine similar to metazoan cells. The results indicate that veratridine can open the voltage-operated Ca²⁺ channels inParamecium wildtype and, most interestingly, in pawn mutant cells. The pawn mutation is suggested to represent a defect in the activation of the Ca²⁺ channel. This explains the lack of differences in ciliary proteins between wildtype and pawn cells reported earlier.     

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⁺.     

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.     

Photosensory transduction in the flagellated alga,Euglena gracilis

Euglena were cultured under 3 W m^-2 constant white light. In culture medium, cells show immediate and long lasting step-down photophobic responses and photoaccumulation behavior to blue light if dim red light-adapted for 30 min. However, if cells are suspended in buffered, saltcontaining solutions (adaptation buffers), strong step-down photobehavior and photoaccumulation responses are not observed for several hours. These behaviors gradually increase in strength to reach a maximum after 6–12 h; after which a stable response is maintained. The relative rates of appearance and the relative strengths of the responses are influenced by the concentrations of Ca²⁺ and K⁺, but not H⁺ or Na⁺ ions, in the adaptation buffers. Expression of the stepdown photobehavior thus requires that the cells adapt to the chemical environment in which they are suspended.Abbreviations Hepes N-2-hydroxypiperazine-N-2-ethanesulfonic acid - Mes 2(N-morpholino)-ethanesulfonic acid - Pipes piperazine-N,N-bis (2-ethanesulfonic acid) - Taps tris(hydroxymethyl) methylaminopropanesulfonic acid This work was supported, in part, by grant No. PCM-79-05320 from the U.S. National Science Foundation to B.D.     

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.     

Anion sensitivity of motility and step-down photophobic responses of Euglena gracilis

The motility and step-down photophobic responses of Euglena are influenced by inorganic and organic anions. Persistent motility (with Ca²⁺, Mg²⁺ and K⁺ present) is supported with chloride or sulfate but not with acetate, nitrate or propionate as the only added anions. Cells in media containing acetate displayed a cell aggregation (clumping) behavior that was both red light sensitive and, under some conditions, was accompanied by suppression of the step-down photophobic response. Addition of sodium salts (Cl^-, SO ₄ ^2- , acetate or propionate) to cells in Cl^- or SO ₄ ^2- based media had differential effects on the duration of the step-down photophobic responses induced by blue light removal: anions alter the response. In addition, cells in all Cl^- containing media showed constant photophobic response duration following repeated stimulation. Cells in some SO₄ ^2- containing media, however, showed response summation to repeated stimulation. This latter effect was reversible and was overcome by the addition of chloride anions.     

Biochemical studies of the excitable membrane of Paramecium aurelia. I. 45Ca2+ fluxes across resting and excited membrane

The characteristics of Ca²⁺ transport across the excitable membrane of Paramecium aurelia were studied by measuring ⁴⁵Ca²⁺ influx and efflux. The intracellular concentration of free Ca²⁺ in resting P. aurelia was at least ten times less than the extracellular concentration. Ca²⁺ influx was easily measurable at 0°C, but not at 23°C. The influx of ⁴⁵Ca²⁺ was stimulated by the same conditions which cause membrane depolarization and ciliary reversal. Addition of Na⁺ and K⁺ (which stimulate ciliary reversal) resulted in a 10-fold increase in the rate of Ca²⁺ influx. An externally applied, pulsed, electric field (1–2 mA/cm² of electrode surface), caused the rate of Ca²⁺ influx to increase 3–5 times, with the extent of stimulation dependent on the current density and the pulse width Ca²⁺ influx had the characteristics of a passive transport system and was associated with the chemically or electrically triggered Ca²⁺ “gating” mechanism, which has been studied electrophysiologically. In contrast, Ca²⁺ efflux appeared to be catalyzed by an active transport system. With cells previously loaded at 0°C with ⁴⁵Ca²⁺, Ca²⁺ efflux was rapid at 23°C, but did not occur at 0°C. This active Ca²⁺ efflux mechanism is probably responsible for maintaining the low internal Ca²⁺ levels in unstimulated cells.     

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.     

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.     

Photosensory transduction in the flagellated alga, Euglena gracilis. III. Induction of Ca2+-dependent responses by monovalent cation ionophores

(1) The effects of monovalent cation ionophores (valinomycin and gramicidin), a protonophore (nigericin) and extracellular pH change on the motility and blue light-induced photobehavior (step-down photophobic response) of Euglena were investigated. (2) Monovalent cation ionophores, but not the protonophore, can both partially suppress photobehavior and, under appropriate conditions, induce a change in flagellar activity (and thus cell movement) that appears identical to that associated with the photobehavior. (3) Valinomycin, at low extracellular KCl, delays the induction of photobehavior and also induces a light-independent elevation in the frequency of directional changes in the cells' swimming path. Both effects are suppressed by elevation in extracellular KCl. (4) Gramicidin, in the presence of the anion tetraphenylborate, suppresses photobehavior. The same combination, if applied in the presence of elevated extracellular NaCl, induces a light-independent cell tumbling and elevation in the frequency of directional changes in the cells' swimming path. The induced behavior is dependent on the extracellular Na concentration, requires the presence of extracellular Ca²⁺ and is blocked by La³⁺. (5) Photobehavior is observed over the pH range 3.5–8.2 and fluence/response relationships for photobehavior are not significantly different over the pH range 5.5–8.2. (6) The results provide a link between the previously reported effects of Ca²⁺ ionophores, and the effects of monovalent cations and monovalent cation-transport inhibitor on motility and photobehavior.     

Cationic inhibition of Ca current and Ca-dependent ciliary responses in Paramecium

The Paramecium cell membrane was voltage-clamped under K current suppression conditions. Ciliary beating was registered using high-speed video microscopy. Depolarizing step pulses activated a transient inward current and induced reversed ciliary beating. Very strong positive steps inhibited ciliary reversal during the pulse suggesting inhibition of the Ca influx. We call the potential, which is sufficiently positive to induce transition from reversed to normal ciliary beating, the transition potential. The transition potential rose with increasing external Ca²⁺ showing saturation beyond 1 mM Ca²⁺. Addition of Mg²⁺, Ba²⁺ or K⁺ to the 1 mM Ca²⁺ bathing solution depressed the transition potential in a concentration-dependent manner. The depolarization-activated inward Ca current increased with rising external Ca concentration, and addition of either Mg²⁺, Ba²⁺ or K²⁺ diminished the inward Ca current. The diverging results of Ca²⁺-dependent positive shifts, and Mg²⁺-(Ba²⁺-, K⁺-) dependent negative shifts in transition potential are compared with shifts of V_Imax. It is concluded that external cations bind competitively — in addition to membrane surface charges — to affinity sites of Ca channel, where they specifically modulate permeation of calcium.     

Photolysis of caged Ca2+ induces trichocyst discharge inParamecium caudatum

Summary Trichocyst discharge, ciliary reversal, and cell body contraction inParamecium spp. have all been claimed to be regulated by the intracellular Ca²⁺ concentration ([Ca²⁺]_i) at the cortical region of the cell. We injected caged Ca²⁺ intoP. caudatum cells and applied ultraviolet (UV) light to the cell for 125 ms. This did not induce trichocyst discharge but did induce both ciliary reversal and cell body contraction. A re-application of UV for 125 ms triggered trichocyst discharge. These results demonstrate that (1) trichocyst discharge and ciliary reversal and cell body contraction are controlled by [Ca²⁺]_i and (2) the threshold of [Ca²⁺]i for trichocyst discharge is higher than those for the other two functions.Abbreviations DTT dithiothreitol - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ICL infraciliary lattice - [Ca²⁺]_i intracellular Ca²⁺ concentration - NP-EG o-nitrophenyl EGTA - PMT photomultiplier tube - UV ultraviolet     

Modulation of concanavalin A stimulation of hamster lymphoid cells by lithium chloride.

Addition of LiCl (1–25 mM) to serum-free cultures of MHA hamster thymocytes, lymph node cells, or splenocytes stimulated with concanavalin A had a biphasic effect on [³H]thymidine incorporation. These concentrations of LiCl enhanced stimulation of [³H]thymidine incorporation by suboptimal levels of concanavalin A but inhibited stimulation of optimal and supraoptimal concentrations of concanavalin A. This effect was specific for Li⁺ since it was not observed when similar concentrations of Na⁺, K⁺, or Mg²⁺ were added to cultures stimulated by concanavalin A. The inhibitory effect of LiCl on concanavalin A stimulation was not reversed by addition of Na⁺, Ca²⁺, Mg²⁺, or Ca²⁺ + Mg²⁺ to the cultures. Significant reversal of LiCl inhibition of stimulation was observed when KCl was added to the cultures. However none of the ions tested blocked the Li-induced enhancement of [³H]thymidine incorporation in the presence of suboptimal concentrations of concanavalin A.     

Trichocysts—Paramecium's Projectile‐like Secretory Organelles

This review summarizes biogenesis, composition, intracellular transport, and possible functions of trichocysts. Trichocyst release by Paramecium is the fastest dense core‐secretory vesicle exocytosis known. This is enabled by the crystalline nature of the trichocyst “body” whose matrix proteins (tmp), upon contact with extracellular Ca²⁺, undergo explosive recrystallization that propagates cooperatively throughout the organelle. Membrane fusion during stimulated trichocyst exocytosis involves Ca²⁺ mobilization from alveolar sacs and tightly coupled store‐operated Ca²⁺‐influx, initiated by activation of ryanodine receptor‐like Ca²⁺‐release channels. Particularly, aminoethyldextran perfectly mimics a physiological function of trichocysts, i.e. defense against predators, by vigorous, local trichocyst discharge. The tmp's contained in the main “body” of a trichocyst are arranged in a defined pattern, resulting in crossstriation, whose period expands upon expulsion. The second part of a trichocyst, the “tip”, contains secretory lectins which diffuse upon discharge. Repulsion from predators may not be the only function of trichocysts. We consider ciliary reversal accompanying stimulated trichocyst exocytosis (also in mutants devoid of depolarization‐activated Ca²⁺ channels) a second, automatically superimposed defense mechanism. A third defensive mechanism may be effectuated by the secretory lectins of the trichocyst tip; they may inhibit toxicyst exocytosis in Dileptus by crosslinking surface proteins (an effect mimicked in Paramecium by antibodies against cell surface components). Some of the proteins, body and tip, are glycosylated as visualized by binding of exogenous lectins. This reflects the biogenetic pathway, from the endoplasmic reticulum via the Golgi apparatus, which is also supported by details from molecular biology. There are fragile links connecting the matrix of a trichocyst with its membrane; these may signal the filling state, full or empty, before and after tmp release upon exocytosis, respectively. This is supported by experimentally produced “frustrated exocytosis”, i.e. membrane fusion without contents release, followed by membrane resealing and entry in a new cycle of reattachment for stimulated exocytosis. There are some more puzzles to be solved: Considering the absence of any detectable Ca²⁺ and of acidity in the organelle, what causes the striking effects of silencing the genes of some specific Ca²⁺‐release channels and of subunits of the H⁺‐ATPase? What determines the inherent polarity of a trichocyst? What precisely causes the inability of trichocyst mutants to dock at the cell membrane? Many details now call for further experimental work to unravel more secrets about these fascinating organelles.     

Membrane potentials inStentor coeruleus

Summary The membrane potential ofStentor coeruleus was measured by means of glass microelectrodes. The tips of the electrodes had a diameter of 2 , and were filled with 0.1 M KCl. The maximum value of the potential is –30.8±8.6 mV and is reached within 600–800 msec after electrode insertion; the potential then decreases to a negative value of a few mV within 5–10 sec. Contraction initiated by mechanical stimulation or by rapid cooling is correlated with a positive potential jump of about 3–5 mV. Similarly, a fully reversible potential shift develops in close correlation to the reversal of ciliary beat in oral membranelles.     

Use of a Novel Cell Adhesion Method and Digital Measurement to Show Stimulus‐dependent Variation in Somatic and Oral Ciliary Beat Frequency in Paramecium

When Paramecium encounters positive stimuli, the membrane hyperpolarizes and ciliary beat frequency increases. We adapted an established immobilization protocol using a biological adhesive and a novel digital analysis system to quantify beat frequency in immobilized Paramecium. Cells showed low mortality and demonstrated beat frequencies consistent with previous studies. Chemoattractant molecules, reduction in external potassium, and posterior stimulation all increased somatic beat frequency. In all cases, the oral groove cilia maintained a higher beat frequency than mid‐body cilia, but only oral cilia from cells stimulated with chemoattactants showed an increase from basal levels.     

Membrane permeabilities determining resting,action and mechanoreceptor potentials inStentor coeruleus

Summary In response to mechanical stimuli the protozoan,Stentor coeruleus, contracts in an all-or-none fashion and simultaneously reverses the direction of its ciliary beat. These behaviors have previously been shown to be correlated with the presence of a mechanoreceptor potential and all-or-none action potential (Wood 1970, 1973a). In the studies reported below the ionic bases of the resting, receptor and action potentials ofStentor were determined by use of intracellular microelectrodes penetrating animals chilled to 8.5–10 °C. The resting potential is most dependent on the extracellular concentration of KCl but some dependence on CaCl₂ concentration was also observed. If allowance is made for the large increases in membrane conductance observed in solutions containing 2–8 mM KCl it is found that the resting potential data are well described by a modified form of the Goldman equation whereP _Ca/P _K = 0.068 andP _Cl/P _K = 0.072. The intracellular ionic activities (K _i ⁺ = 13.1 mM, Cl _i ^– = 9.9 mM, Ca _i ⁺ = 0 mM) which provide the best fit of this equation to the resting potential data are in close agreement with the intracellular concentration values measured by flame microspectrophotometry (K_i=12.4 mM, Cl_i = 9.4 mM) except in the case of Ca_i where most of the intracellular concentration is presumed to be bound. 65 to 75 mV action potentials are produced by suprathreshold depolarizations but contractions were not generally seen in these chilled animals, only ciliary reversals. The action potential peak varies with CaCl₂ concentration with a slope of 12.6 mV/10 fold change but varies only slightly with KCl or Cl^– concentration. These peak potentials are well described by assuming that theP _Ca/P _k = 7.9 andP _Cl/P _K=1.0 at the time of the action potential peak. Depolarizing receptor potentials and brief inward receptor currents were observed for all forms of punctate and gross bodily mechanical stimulation employed. No evidence was found for any form of hyperpolarizing mechanoreceptor potentials as observed in some other ciliates. The reversal potential of the mechanoreceptor current varied with CaCl₂ concentration in a manner similar to that of the action potential peak. As in the case of the action potential both theP _Ca/P _k andp _cl/p _k ratios appear to increase as a result of mechanical stimulation to 9.3–15 and 1.2–1.95 respectively. Mechanoreceptor currents are voltage dependent being increased when the membrane is depolarized above resting potential and decreased when the membrane is hyperpolarized. In general the electrophysiological characteristics ofStentor appear similar to those ofParamecium andStylonychia, but its resting membrane appears more selectively permeable to K⁺, it produces only depolarizing receptor potentials when mechanically stimulated and the initial action potential elicited by depolarizing current pulses can be all-or-none even in culture medium.     

Roles of Adenylate Cyclases in Ciliary Responses of Paramecium to Mechanical Stimulation

Paramecium shows rapid forward swimming due to increased beat frequency of cilia in normal (forward swimming) direction in response to various kinds of stimuli applied to the cell surface that cause K⁺‐outflow accompanied by a membrane hyperpolarization. Some adenylate cyclases are known to be functional K⁺ channels in the membrane. Using gene‐specific knockdown methods, we examined nine paralogues of adenylate cyclases in P. tetraurelia to ascertain whether and how they are involved in the mechanical stimulus‐induced hyperpolarization‐coupled acceleration of forward swimming. Results demonstrated that knockdown of the adenylate cyclase 1 (ac1)‐gene and 2 (ac2)‐gene inhibited the acceleration of forward swimming in response to mechanical stimulation of the cell, whereas that spared the acceleration response to external application of 8‐Br‐cAMP and dilution of extracellular [K⁺] induced hyperpolarization. Electrophysiological examination of the knockdown cells revealed that the hyperpolarization‐activated inward K⁺ current is smaller than that of a normal cell. Our results suggest that AC1 and AC2 are involved in the mechanical stimulus‐induced acceleration of ciliary beat in Paramecium.     

Ionic Control of the Reversal Response of Cilia in Paramecium caudatum : A calcium hypothesis

The duration of ciliary reversal of Paramecium caudatum in response to changes in external ionic factors was determined with various ionic compositions of both equilibration and stimulation media. The reversal response was found to occur when calcium ions bound by an inferred cellular cation exchange system were liberated in exchange for externally applied cations other than calcium. Factors which affect the duration of the response were (a) initial amount of calcium bound by the cation exchange system, (b) final amount of calcium bound by the system after equilibration with the stimulation medium, and (c) concentration of calcium ions in the stimulation medium. An empirical equation is presented which relates the duration of the response to these three factors. On the basis of these and previously published data, the following hypothesis is proposed for the mechanism underlying ciliary reversal in response to cationic stimulation: Ca⁺⁺ liberated from the cellular cation exchange system activates a contractile system which is energized by ATP. Contraction of this component results in the reversal of effective beat direction of cilia by a mechanism not yet understood. The duration of reversal in live paramecia is related to the time course of bound calcium release.