In vivo activation of cirral movement in Stylonychia by calcium

Summary Motor responses of cirri (= organelles consisting of bundles of cilia) in the protozoan Stylonychia are elicited by positive or negative shifts of the membrane voltage from its resting state. The same responses are evoked at voltages near the Ca²⁺ equilibrium potential (E_Ca) applying extremely positive steps under voltage clamp. Motor responses recorded at large positive voltages approaching E_Ca from the negative side corresponded to cirral activation following physiological depolarization from the resting potential (DCA). The hyperpolarization-induced activation of the cirri (HCA) was documented during step potentials positive to E_Ca, suggesting that the observed HCA of the cirri resulted from an efflux of Ca²⁺ from the ciliary space as compared with DCA, which is related to Ca²⁺ influx. The ciliary responses were graded functions of the rising outward or inward driving force for Ca²⁺. Slopes of reciprocal plots of response latencies near E_Ca as a function of membrane potential indicate a removal of Ca²⁺ during HCA which exceeds the free intraciliary Ca²⁺ content at rest. It is suggested that this excess Ca²⁺ is released from axonemal binding sites.     

Dephosphorylation of inner arm 1 is associated with ciliary reversals in Tetrahymena thermophila

In many organisms, depolarizing stimuli cause an increase in intraciliary Ca2+, which results in reversal of ciliary beat direction and backward swimming. The mechanism by which an increase in intraciliary Ca2+ causes ciliary reversal is not known. Here we show that Tetrahymena cells treated with okadaic acid or cantharidin to inhibit protein phosphatases do not swim backwards in response to depolarizing stimuli. We also show that both okadaic acid and cantharidin inhibit backward swimming in reactivated, extracted cell models treated with Ca2+. In contrast, treatment of whole cells or extracted cell models with protein kinase inhibitors has no effect on backward swimming. These results suggest that a component of the axonemal machinery is dephosphorylated during ciliary reversal. The phosphorylation state of inner arm dynein 1 (I1) was determined before and after cells were exposed to depolarizing conditions that induce ciliary reversal. An I1 intermediate chain is phosphorylated in forward swimming cells but is dephosphorylated in cells treated with a depolarizing stimulus. Our results suggest that dephosphorylation of Tetrahymena inner arm dynein 1 may be an essential part of the mechanism of ciliary reversal in response to increased intraciliary Ca2+.     

Visualization of calcium transients controlling orientation of ciliary beat

To image changes in intraciliary Ca controlling ciliary motility, we microinjected Ca Green dextran, a visible wavelength fluorescent Ca indicator, into eggs or two cell stages of the ctenophore Mnemiopsis leidyi. The embryos developed normally into free-swimming, approximately 0.5 mm cydippid larvae with cells and ciliary comb plates (approximately 100 microns long) loaded with the dye. Comb plates of larvae, like those of adult ctenophores, undergo spontaneous or electrically stimulated reversal of beat direction, triggered by Ca influx through voltage-sensitive Ca channels. Comb plates of larvae loaded with Ca Green dextran emit spontaneous or electrically stimulated fluorescent flashes along the entire length of their cilia, correlated with ciliary reversal. Fluorescence intensity peaks rapidly (34-50 ms), then slowly falls to resting level in approximately 1 s. Electrically stimulated Ca Green emissions often increase in steps to a maximum value near the end of the stimulus pulse train, and slowly decline in 1-2 s. In both spontaneous and electrically stimulated flashes, measurements at multiple sites along a single comb plate show that Ca Green fluorescence rises within 17 ms (1 video field) and to a similar relative extent above resting level from base to tip of the cilia. The decline of fluorescence intensity also begins simultaneously and proceeds at similar rates along the ciliary length. Ca-free sea water reversibly abolishes spontaneous and electrically stimulated Ca Green ciliary emissions as well as reversed beating. Calculations of Ca diffusion from the ciliary base show that Ca must enter the comb plate along the entire length of the ciliary membranes. The voltage-dependent Ca channels mediating changes in beat direction are therefore distributed over the length of the comb plate cilia. The observed rapid and virtually instantaneous Ca signal throughout the intraciliary space may be necessary for reprogramming the pattern of dynein activity responsible for reorientation of the ciliary beat cycle.     

Regulation of ciliary adenylate cyclase by Ca2+ in Paramecium.

In the ciliated protozoan Paramecium, Ca2+ and cyclic nucleotides are believed to act as second messengers in the regulation of the ciliary beat. Ciliary adenylate cyclase was activated 20-30-fold (half-maximal at 0.8 microM) and inhibited by higher concentrations (10-20 microM) of free Ca2+ ion. Ca2+ activation was the result of an increase in Vmax., not a change in Km for ATP. The activation by Ca2+ was seen only with Mg2+ATP as substrate; with Mn2+ATP the basal adenylate cyclase activity was 10-20-fold above that with Mg2+ATP, and there was no further activation by Ca2+. The stimulation by Ca2+ of the enzyme in cilia and ciliary membranes was blocked by the calmodulin antagonists calmidazolium (half-inhibition at 5 microM), trifluoperazine (70 microM) and W-7 (50-100 microM). When ciliary membranes (which contained most of the ciliary adenylate cyclase) were prepared in the presence of Ca2+, their adenylate cyclase was insensitive to Ca2+ in the assay. However, the inclusion of EGTA in buffers used for fractionation of cilia resulted in full retention of Ca2+-sensitivity by the ciliary membrane adenylate cyclase. The membrane-active agent saponin specifically suppressed the Ca2+-dependent adenylate cyclase without inhibiting basal activity with Mg2+ATP or Mn2+ATP. The ciliary adenylate cyclase was shown to be distinct from the Ca2+-dependent guanylate cyclase; the two activities had different kinetic parameters and different responses to added calmodulin and calmodulin antagonists. Our results suggest that Ca2+ influx through the voltage-sensitive Ca2+ channels in the ciliary membrane may influence intraciliary cyclic AMP concentrations by regulating adenylate cyclase.     

Cellular Behaviour Modulated by Ions: Electrophysiological Implications

. This essay considers the responses of Paramecium and other ciliates to the inorganic ion environment from an elec-trophysiological point of view. In reviewing data from published and unpublished sources it is shown that ions affect the cellular behaviour in multiple ways because the transmembrane potential can change due to the alteration of equilibrium potentials, ion conductances and surface charges of the membrane. Sensory input including effects from the ionic environment converge upon the membrane potential which has a temporal and spatial summing function. Hyperpolarizing and depolarizing potential shifts from the set point are near-simultaneously and omnidirectionally transmitted along the membrane including the ciliary boundaries. The membrane potential regulates ciliary motility via an intraciliary messenger, Ca²⁺, which can enter, and presumably leave, the cytosol directly adjacent to the ciliary motor. Integration of the responses of thousands of cilia occurs in accordance with the electrical and structural provisions of the cell. Potential-regulated motor and behavioural responses attenuate with time. This phenomenon, which has been loosely termed adaptation, has an electrophysiological basis in analogy to membrane accommodation following sustained stimulus input. The mechanisms of adaptation serve to restore, in principle, the membrane resting state and, thereby, the sensitivity to depolarizing and hyperpolarizing shifts of the membrane potential and the cell's responsiveness to environmental stimuli, respectively. For the inorganic ions involved in chemosensation the terms attractant and repellent are not applicable. They should be reserved to signalling substances which per se can define the behaviour of the cell.     

Inner arm dynein 1 is essential for Ca++-dependent ciliary reversals in Tetrahymena thermophila

Cilia in many organisms undergo a phenomenon called ciliary reversal during which the cilia reverse the beat direction, and the cell swims backwards. Ciliary reversal is typically caused by a depolarizing stimulus that ultimately leads to a rise in intraciliary Ca++ levels. It is this increase in intraciliary Ca++ that triggers ciliary reversal. However, the mechanism by which an increase in intraciliary Ca++ causes ciliary reversal is not known. We have previously mutated the DYH6 gene of Tetrahymena thermophila by targeted gene knockout and shown that the knockout mutants (KO6 mutants) are missing inner arm dynein 1 (I1). In this study, we show that KO6 mutants do not swim backward in response to depolarizing stimuli. In addition to being unable to swim backwards, KO6 mutants swim forward at approximately one half the velocity of wild-type cells. However, the ciliary beat frequency in KO6 mutants is indistinguishable from that of wild-type cells, suggesting that the slow forward swimming of KO6 mutants is caused by an altered waveform rather than an altered beat frequency. Live KO6 cells are also able to increase and decrease their swim speeds in response to stimuli, suggesting that some aspects of their swim speed regulation mechanisms are intact. Detergent-permeabilized KO6 mutants fail to undergo Ca++-dependent ciliary reversals and do not show Ca++-dependent changes in swim speed after MgATP reactivation, indicating that the axonemal machinery required for these responses is insensitive to Ca++ in KO6 mutants. We conclude that Tetrahymena inner arm dynein 1 is not only an essential part of the Ca++-dependent ciliary reversal mechanism but it also may contribute to Ca++-dependent changes in swim speed and to the formation of normal waveform during forward swimming.     

Ciliary reorientation is evoked by a rise in calcium level over the entire cilium

Internal Ca2+ levels control the pattern of ciliary and flagellar beating in eukaryotes. In ciliates, ciliary reversal is induced by a rise in intra-ciliary Ca2+, but the mechanism by which Ca2+ induces reversal is not known. We injected the fluorescent Ca2+ indicator Calcium Green into a ciliate Didinium nasutum and observed the intra-ciliary Ca2+ level during the initial reversed stroke preceding spontaneous cyclic reversed beating. In D. nasutum, Ca2+ rose throughout the length of the cilia undergoing initial reversed stroke. Electron microscopy with a combined oxalate-pyroantimonate method showed Ca2+ deposits distributed throughout the reversed cilia. We injected caged Ca2+ into D. nasutum and irradiated the base or mid region of the cilia with UV to locally increase Ca2+ concentration. Uncaging Ca2+ in the middle of the cilia produced reversal distally, but not proximally to the site of Ca2+ release. These results strongly suggest that not only Ca2+ influx sites, but also Ca2+ binding sites and vectoral bending machineries for ciliary reversal, are distributed throughout the cilium.     

Predicted profiles of ion concentrations in olfactory cilia in the steady state

The role of ciliary geometry for transduction events was explored by numerical simulation. The changes in intraciliary ion concentrations, suspected to occur during transduction, could thus be estimated. The case of a single excised cilium, having a uniform distribution of membrane channels, voltage clamped to -80 mV, was especially investigated. The axial profile of membrane voltage was that of a leaky cable. The Ca(2+) concentration profile tended to show a maximum in proximal segments, due to a preponderance of Ca(2+) inflow over Ca(2+) export at those locations. The local increase in Ca(2+) concentration activated Cl(-) channels. The resulting current caused a local drop in Cl(-) concentration, especially at the tip of the cilium and in distal segments, accompanied by a drop in ciliary K(+) concentration. In consequence, the membrane Cl(-) current was low in distal segments but stronger in proximal segments, where resupply was sufficient. The model predicts that the Cl(-) depletion will codetermine the time course of the receptor potential or current and the ciliary stimulus-response curve. In conclusion, when modeling with transduction elements presently known to participate, the ciliary geometry has large effects on ion distributions and transduction currents because ciliary ion transport is limited by axial electrodiffusion.     

Divalent cation affinity sites in Paramecium aurelia

Sites with high calcium affinity in Paramecium aurelia were identified by high calcium (5 mM) fixation and electron microscope methods. Electron-opaque deposits were observed on the cytoplasmic side of surface membranes, particularly at the basal regions of cilia and trichocyst-pellicle fusion sites. Deposits were also observed on some smooth cytomembranes, within the axoneme of cilia, and on basal bodies. The divalent cations, Mg2+, Mn2+, Sr2+, Ni2+, Ba2+, and Zn2+, could be substituted for Ca2+ in the procedure. Deposits were larger with 5 mM Sr2+. Ba2+, and Mn2+ at ciliary transverse plates and the terminal plate of basal bodies. Microprobe analysis showed that Ca and C1 were concentrated within deposits. In some analyses, S and P were detected in deposits. Also, microprobe analysis of 5 mM Mn2+-fixed P. aurelia showed that those deposits were enriched in Mn and C1 and sometimes enriched in P. Deposits were seen only when the ciliates were actively swimming at the time of fixation. Locomotory mutants having defective membrane Ca-gating mechanisms and ciliates fixed while exhibiting ciliary reversal showed no obvious differences in deposition pattern and intensity. Possible correlations between electron-opaque deposits and the locations of intramembranous particles seen by freeze-fracture studied, as well as sites where fibrillar material associate with membranes are considered. The possibility that the action sites of calcium and other divalent cations were identified is discussed.     

Calcium regulation of ciliary activity in rabbit tracheal epithelial explants and outgrowth

Ciliated epithelial cells from rabbit trachea were employed to examine the role of Ca2+ in the regulation of ciliary motility. Tracheal explants and outgrowths were maintained in culture, and ciliary frequency was determined using a photomultiplier interfaced with a spectrum analyzer capable of Fast Fourier transform analysis. Relative cellular Ca2+ levels were determined by measuring 45Ca2+ uptake and efflux. Elevated cellular Ca2+, from exposure to 10(-5) M calcium ionophore A23187, led to an increase in ciliary frequency followed by inhibition of motility after prolonged treatment. A decrease in ciliary frequency was observed upon lowering intracellular Ca2+ by exposing the epithelium to 1 mM EGTA. Exposure of ciliated cells to 10(-4) M trifluoperazine resulted in inhibition of ciliary motility, a result suggesting a possible role for calmodulin- or phospholipid-sensitive Ca2+-dependent protein kinases in ciliary function. These results support the hypothesis that intracellular Ca2+ is actively involved in modulating the frequency of ciliary beat.     

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.     

Olfactory response termination involves Ca2+-ATPase in vertebrate olfactory receptor neuron cilia

In vertebrate olfactory receptor neurons (ORNs), odorant-induced activation of the transduction cascade culminates in production of cyclic AMP, which opens cyclic nucleotide–gated channels in the ciliary membrane enabling Ca²⁺ influx. The ensuing elevation of the intraciliary Ca²⁺ concentration opens Ca²⁺-activated Cl⁻ channels, which mediate an excitatory Cl⁻ efflux from the cilia. In order for the response to terminate, the Cl⁻ channel must close, which requires that the intraciliary Ca²⁺ concentration return to basal levels. Hitherto, the extrusion of Ca²⁺ from the cilia has been thought to depend principally on a Na⁺–Ca²⁺ exchanger.In this study, we show using simultaneous suction pipette recording and Ca²⁺-sensitive dye fluorescence measurements that in fire salamander ORNs, withdrawal of external Na⁺ from the solution bathing the cilia, which incapacitates Na⁺–Ca²⁺exchange, has only a modest effect on the recovery of the electrical response and the accompanying decay of intraciliary Ca²⁺ concentration. In contrast, exposure of the cilia to vanadate or carboxyeosin, a manipulation designed to block Ca²⁺-ATPase, has a substantial effect on response recovery kinetics. Therefore, we conclude that Ca²⁺-ATPase contributes to Ca²⁺ extrusion in ORNs, and that Na⁺–Ca²⁺exchange makes only a modest contribution to Ca²⁺ homeostasis in this species.     

Regulation of axonemal Mg2+-ATPase from Paramecium cilia: effects of Ca2+ and cyclic nucleotides

Ciliary activity is regulated by Ca2+ and cyclic nucleotides, but the molecular mechanisms of the regulation are unknown. We have tested the ability of Ca2+ and cyclic nucleotides to alter ciliary Mg2+-ATPase or to stimulate phosphorylation of axonemal dynein. Mg2+-ATPase activity in cilia and axonemes from Paramecium was stimulated 2-fold by micromolar Ca2+, but this Ca2+ sensitivity was lost upon solubilization of the dyneins from the axoneme. The Ca2+-sensitive component of ciliary Mg2+-ATPase activity was inhibited by the dynein inhibitors vanadate and Zn2+, but was insensitive to the calmodulin antagonists calmidazolium and melittin. Dynein activity in the high-salt extract from axonemes was also insensitive to calmidazolium. Calmodulin did not sediment with 22 S or 12 S dyneins on sucrose gradients containing Ca2+, but it did sediment in the region from 19 S to 14 S. Mg2+-ATPase activity in ciliary fractions was unaltered in the presence of cAMP or cGMP. However, polypeptides associated with the 22 S and 12 S dyneins, as well as proteins of 19 S, 15 S, and 8 S, were substrates for endogenous ciliary kinases. High molecular weight polypeptides that sedimented at 22 S and 19 S were phosphorylated in a cyclic nucleotide-stimulated manner.     

Ca2+/calmodulin-dependent phosphorylation of ciliary beta-tubulin in Tetrahymena

The ciliary axoneme is the minimal structure responsible for Ca2+-dependent modulation of ciliary movement. We demonstrated that, in Tetrahymena ciliary axonemes, beta-tubulin was exclusively phosphorylated by an endogenous Ca2+/calmodulin-dependent protein kinase(s). The phosphorylation of beta-tubulin also occurred in the outerdoublet microtubule fraction, suggesting that the responsible enzyme(s) was tightly associated with outerciliary motility, Ca2+-dependent phosphorylation of beta-tubulin was also found to occur exclusively. From these results, it is inferable that the phosphorylation of beta-tubulin is involved in Ca2+-dependent ciliary reversal.     

Electrophysiological Properties of the Microstome and Macrostome Morph of the Polymorphic Ciliate Tetrahymena vorax

Polymorphic ciliates, like Tetrahymena vorax, optimize food utilization by altering between different body shapes and behaviours. Microstome T. vorax feeds on bacteria, organic particles, and solutes, whereas the larger macrostome cells are predators consuming other ciliates. We have used current clamp and discontinuous single electrode voltage clamp to compare electrophysiological properties of these morphs. The resting membrane potential was approximately ?30 mV in both morphs. The input resistance and capacitance of microstomes were approximately 350 MΩ and 105 pF, whereas the corresponding values for the macrostomes were 210 MΩ and 230 pF, reflecting the larger cell size. Depolarizing current injections elicited regenerative Ca²⁺ spikes with a maximum rate of rise of 7.5 Vs^?1 in microstome and 4.7 Vs^?1 in macrostome cells. Depolarizing voltage steps from a holding potential of ?40 mV induced an inward Ca²⁺ ‐current (I_ca) peaking at ?10 mV, reaching approximately the same value in microstome (?1.4 nA) and macrostome cells (?1.2 nA). Because the number of ciliary rows is the same in microstome and macrostome cells, the similar size of I_Ca in these morphs supports the notion that the voltage‐gated Ca²⁺ channels in ciliates are located in the ciliary membrane. In both morphs, hyperpolarizing voltage steps revealed inward membrane rectification that persisted in Na⁺‐free solution and was only partially inhibited by extracellular Cs⁺. The inward rectification was completely blocked by replacing Ca²⁺ with Co²⁺ or Ba²⁺ in the recording solution, and is probably due to Ca²⁺ ‐activated inward K⁺ current secondary to Ca²⁺ influx through channels activated by hyperpolarization.     

Vasodilator action of docosahexaenoic acid (DHA) in human coronary arteries in vitro

Coronary arterial tissues obtained from mammalian hearts are known to develop spontaneous phasic contractions. The aim of the present study was to investigate the vasodilatory effects of docosahexaenoic acid (DHA) on the rhythmic contractions of isolated human coronary arterial (HCA) preparations obtained from the recipient hearts of patients undergoing cardiac transplantation. Results from 8 hearts show that: (i) most HCA tissues displayed spontaneous rhythmic phasic contractions with a cycle length around 10 min in the absence or presence of PGF2alpha or elevated [K+]0 (20 mM); (ii) the rhythmic activity could be suppressed by a free fatty acid DHA (30 microM); (iii) high [K+]0 (20 and 80 mM) could induce sustained tonic contraction in addition to phasic contractions in HCA tissues, the tonic contraction could be antagonized by L-type Ca(2+) channel blockers or by DHA (depending on [K+]0); (iv) a digitalis substance ouabain also could induce tonic contraction and suppress phasic contraction; (v) in isolated HCA vascular smooth muscle cells, DHA increased the magnitude of outward voltage-gated K+ (IKV) currents and the inwardly rectifying IK1 currents. Enhancement of K+ currents could be related to vasorelaxation induced by DHA in HCA preparations. Further studies on the effects of DHA on various ionic currents and intracellular Ca(2+) transient are needed to clarify the Ca(2+)-dependent and the Ca(2+)-independent actions of DHA in HCA.     

Regulation of airway ciliary activity by Ca2+: simultaneous measurement of beat frequency and intracellular Ca2+.

Airway ciliary activity is influenced by [Ca2+]i, but this mechanism is not fully understood. To investigate this relationship, ciliary activity and [Ca2+]i were measured simultaneously from airway epithelial ciliated cells. Ciliary beat frequency was determined, for each beat cycle, with phase-contrast optics and high-speed video imaging (at 240 images s-1) and correlated with [Ca2+]i determined, at the ciliary base, by fast imaging (30 images s-1) of fura-2 fluorescence. As a mechanically induced intercellular Ca2+ wave propagated through adjacent cells, [Ca2+]i was elevated from a baseline concentration of 45 to 100 nM, to a peak level of up to 650 nM. When the Ca2+ wave reached the ciliary base, the beat frequency rapidly increased, within a few beat cycles, from a basal rate of 6.4 to 11.6 Hz at 20-23 degrees C, and from 17.2 to 26.7 Hz at 37 degrees C. Changes in [Ca2+]i, above 350 nM, had no effect on the maximum beat frequency. We suggest that airway ciliary beat frequency is 1) controlled by a low range of [Ca2+]i acting directly at an axonemal site at the ciliary base and 2) that a maximum frequency is induced by a change in [Ca2+]i of approximately 250-300 nM.     

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 degrees C, but not at 23 degrees 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 degrees C with 45Ca2+, Ca2+ efflux was rapid at 23 degrees C, but did not occur at 0 degrees C. This active Ca2+ efflux mechanism is probably responsible for maintaining the low internal Ca2+ levels in unstimulated cells.     

Evidence for a Ca2+-binding protein associated to non-actin microfilamentous systems in two ciliated protozoans

Electrophoretic distributions of proteins of isolated cortical cytoskeletons from two ciliated protozoans (Isotricha, Polyplastron) were compared in order to reveal any components common to non-actin microfilamentous structures. A low molecular weight protein (Mr approximately 22 kD) characterized by Ca2+-induced shifts in mobility on SDS-polyacrylamide gels was identified in both ciliates. Two-dimensional electrophoretic coordinates and peptide maps of Ca2+-binding proteins from Isotricha and Polyplastron are fairly similar, suggesting conservation of the same molecular species. In addition, an antiserum raised against two proteins (22-23 KD) from the filamentous ecto-endoplasmic boundary of Isotricha, one of which corresponds to the Ca2+-binding protein, cross-reacts specifically with that of Polyplastron. Using an immunogold staining procedure, the Ca2+-binding protein of Polyplastron was shown also to be located in a cortical microfilamentous layer. This protein is probably different from calmodulin. We postulate that it is involved in the control of the ordering of non-actin microfilaments within the cortex of ciliates.     

Pharmacological control of ciliary activity in the young sea urchin larva. Studies on the role of Ca2+ and cyclic nucleotides

The ciliary stimulation by monoamines is enhanced by adenylcyclase activators and a phosphodiesterase inhibitor indicating that cAMP is a mediator, a conclusion supported by the effects of db-cAMP. The role of cGMP is also examined. When stimulation exceeds certain levels, it is overpowered by some inhibitory feedback mechanism. The effects of altered Ca2+ concentrations, Ca2+ antagonists and a Ca2+ ionophore suggest that Ca2+ is involved in ciliary excitation as well as in the inhibitory mechanism. These suggestions are examined by experiments on the influence of altered Ca2+ concentrations and of the phosphodiesterase inhibitor on the response to various agents.