Injected cyclic AMP increases ciliary beat frequency in conjunction with membrane hyperpolarization

Injections of cyclic AMP (cAMP) and 8-Br-cAMP into Paramecium and external application of isobutylmethylxanthine (IBMX), an inhibitor of cAMP breakdown, to these cells increased the frequency of ciliary beating and hyperpolarized the membrane potential. When the membrane potential was held equal to the resting potential under voltage clamp, the same experimental conditions which serve to increase intracellular cAMP did not raise the ciliary frequency. We conclude that cAMP is presumably not the direct mediator of the hyperpolarization-induced ciliary activation, although it may be associated with this motor response.     

Control of the ciliary beat by cyclic nucleotides in intact cortical sheets from Paramecium

The locomotor behavior of Paramecium depends on the ciliary beat direction and beat frequency. Changes in the ciliary beat are controlled by a signal transduction mechanism that follows changes in the membrane potential. These events take place in cilia covered with a ciliary membrane. To determine the effects of second messengers in the cilia, cortical sheets were used with intact ciliary membrane as a half-closed system in which each cilium is covered with a ciliary membrane with an opening to the cell body. Cyclic nucleotides and their derivatives applied from an opening to the cell body affected the ciliary beat. cAMP and 8-Br-cAMP increased the beat frequency and the efficiency of propulsion and acted antagonistically to the action of Ca(2+). cGMP and 8-Br-cGMP increased the efficiency of propulsion accompanying clear metachronal waves but decreased the beat frequency. These results indicate that the cyclic nucleotides affect target proteins in the ciliary axonemes surrounded by the ciliary membrane without a membrane potential and increase the efficiency of propulsion of the ciliary beat. In vitro phosphorylation of isolated ciliary axonemes in the presence of cyclic nucleotides and their derivatives revealed that the action of cAMP was correlated with the phosphorylation of 29-kDa and 65-kDa proteins and that the action of cGMP was correlated with the phosphorylation of a 42-kDa protein.     

Protein phosphatase 2C is involved in the cAMP-dependent ciliary control in Paramecium caudatum

Forward swimming of the Triton-extracted model of Paramecium is stimulated by cAMP. Backward swimming of the model induced by Ca(2+) is depressed by cAMP. Cyclic AMP and Ca(2+) act antagonistically in setting the direction of the ciliary beat. Some ciliary axonemal proteins from Paramecium caudatum are phosphorylated in a cAMP-dependent manner. In the presence of cAMP, axonemal 29- and 65-kDa polypeptides were phosphorylated by endogenous A-kinase in vitro. These phosphoproteins, however, were not dephosphorylated after in vitro phosphorylation, presumably because of the low endogenous phosphoprotein phosphatase activity associated with isolated axonemes. We purified the protein phosphatase that specifically dephosphorylated the 29- and 65-kDa phosphoproteins from Paramecium caudatum. The molecular weight of the protein phosphatase was 33 kDa. The protein phosphatase had common characteristics as protein phosphatase 2C (PP2C). The characteristics of the protein phosphatase were the same as those of the PP2C from Paramecium tetraurelia (PtPP2C) [Grothe et al., 1998: J. Biol. Chem. 273:19167-19172]. We concluded that the phosphoprotein phosphatase is the PP2C from Paramecium caudatum (PcPP2C). The PcPP2C markedly accelerated the backward swimming of the Triton-extracted model in the presence of Ca(2+). On the other hand, the PcPP2C slightly depressed the forward swimming speed. This indicates that the PP2C plays a role in the cAMP-dependent regulation of ciliary movement in Paramecium caudatum through dephosphorylation of 29- and/or 65-kDa regulatory phosphoproteins by terminating the action of cAMP.     

Outer dynein arm light chain 1 is essential for controlling the ciliary response to cyclic AMP in Paramecium tetraurelia

The individual role of the outer dynein arm light chains in the molecular mechanisms of ciliary movements in response to second messengers, such as Ca(2+) and cyclic nucleotides, is unclear. We examined the role of the gene termed the outer dynein arm light chain 1 (LC1) gene of Paramecium tetraurelia (ODAL1), a homologue of the outer dynein arm LC1 gene of Chlamydomonas reinhardtii, in ciliary movements by RNA interference (RNAi) using a feeding method. The ODAL1-silenced (ODAL1-RNAi) cells swam slowly, and their swimming velocity did not increase in response to membrane-hyperpolarizing stimuli. Ciliary movements on the cortical sheets of ODAL1-RNAi cells revealed that the ciliary beat frequency was significantly lower than that of control cells in the presence of ≥ 1 mM Mg(2+)-ATP. In addition, the ciliary orientation of ODAL1-RNAi cells did not change in response to cyclic AMP (cAMP). A 29-kDa protein phosphorylated in a cAMP-dependent manner in the control cells disappeared in the axoneme of ODAL1-RNAi cells. These results indicate that ODAL1 is essential for controlling the ciliary response by cAMP-dependent phosphorylation.     

Differential regulation of Paramecium ciliary motility by cAMP and cGMP

cAMP and cGMP had distinct effects on the regulation of ciliary motility in Paramecium. Using detergent-permeabilized cells reactivated to swim with MgATP, we observed effects of cyclic nucleotides and interactions with Ca2+ on the swimming speed and direction of reactivated cells. Both cAMP and cGMP increased forward swimming speed two- to threefold with similar half-maximal concentrations near 0.5 microM. The two cyclic nucleotides, however, had different effects in antagonism with the Ca2+ response of backward swimming and on the handedness of the helical swimming paths of reactivated cells. These results suggest that cAMP and cGMP differentially regulate the direction of the ciliary power stroke.     

Membrane control of ciliary movement in ciliates

Ciliary movement is generated in the axoneme by the unidirectional sliding of the outer doublets of microtubules produced by the adenosine triphosphate (ATP)-energized dynein arms. It is composed of an effective stroke phase and a passive recovery stroke phase. Two parameters are modulated to determine swimming characteristics of the cell (speed and direction): beat frequency; direction of the effective stroke. They are linked to the internal Ca++ level and to the membrane potential. The membrane governs the internal Ca++ level by regulating Ca++ influx and efflux. It contains voltage-sensitive Ca++ channels through which a passive Ca++ influx, driven by the electrochemical gradient, occurs during step depolarization. The rise of the Ca++ level, up to 6.10-7M triggers ciliary reversal and enhances beat frequency. Ca+ is extruded from cilia by active transport. Ca++ also activates a multistep enzymatic process, the first component of which is a membrane calmodulin-dependent guanylate cyclase. cGMP interacts with Ca++ to modulate the parameters of the ciliary beat. The phosphorylation-dephosphorylation cycle of axoneme and membrane proteins seems to play a major role in controlling ciliary movement. Hyperpolarization of the membrane enhances beat frequency by an unknown mechanism. It could be a modification of the ratio of axonemal bound Ca++ and Mg++, or activation by cyclic adenosine monophosphate (cAMP) produced by a membrane adenylate cyclase. The ciliary membrane behaves as a receptor able to detect modifications of external parameters, and as a transductor transmitting the detected signal by a second or third messengers toward the interior of the cilia. These messengers. acting at different levels, modulate the parameters of the mechanism that generates ciliary movement.     

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.     

Okadaic acid, an inhibitor of protein phosphatase 1 in Paramecium, causes sustained Ca2(+)-dependent backward swimming in response to depolarizing stimuli.

Backward swimming is a stereotypic behavioural response of Paramecium. It is triggered by depolarizing stimuli, which open calcium channels in the excitable ciliary membrane. The influx of Ca2+ causes the reversal of ciliary beat and initiates backward swimming. Here, we demonstrate that the protein phosphatase inhibitor okadaic acid does not affect the normal forward swimming pattern of Paramecium, but greatly extends the duration of backward swimming as initiated by depolarization caused by a rise in extracellular K+. Chelation of external Ca2+ results in an immediate resumption of forward swimming. The results suggest that the voltage-operated calcium channel is inactivated by a dephosphorylation event, and that okadaic acid blocks this dephosphorylation without any effect on the motile apparatus of the cilia. In addition, Paramecium is unique among eukaryotic cells, in that okadaic acid inhibits just one protein phosphatase, namely a type 1 enzyme, 75% of which is tightly associated with the excitable ciliary membrane. The type 2A protein phosphatases in Paramecium are unaffected by okadaic acid. The results indicate that protein phosphatase 1 is the enzyme responsible for the dephosphorylation and closure of the calcium channel in Paramecium.     

Variable acceleration influences cyclic AMP levels in Paramecium biaurelia

Paramecium is used as a model system to analyse the gravity signal transduction pathway, that leads to gravitaxis and gravikinesis. In order to prove whether gravistimulation is coupled with second messenger production (cyclic AMP: hyperpolarization, cyclic GMP: depolarization) Paramecium was fixated under variable accelerations (1 x g, 9 x g and 10(-4) x g) on a centrifuge and during a sounding rocket flight (TEXUS 39). The analysis of cAMP and cGMP levels revealed an acceleration-dependent change in cAMP, while cGMP-levels showed gravity-independent variations. Hypergravity did not only induce an amplification of gravitaxis and gravikinesis, but also an increase in cAMP compared to the 1 x g-data. We conclude that the increased pressure of the cytoplasm on the lower membrane of upward swimming cells enhance the number of open K+(-)channels, thus causing hyperpolarization and change in cAMP concentration. Consequently, transition to microgravity declines gravitaxis and gravikinesis, and decreases cAMP concentration due to the loss of pressure on the cell membrane.     

Effects of calcitonin gene-related peptide on airway epithelial functions in dogs

We studied the effects of calcitonin gene-related peptide (CGRP) on ciliary beat frequency (CBF) and electrical properties of canine tracheal epithelium by a photoelectric method and Ussing's short-circuit technique, respectively. CGRP dose dependently increased CBF, an effect that was accompanied by elevation of intracellular cyclic AMP but not affected by blockade of either Ca2+-influx or arachidonic acid metabolism. In contrast, CGRP elicited only a small and transient increase in short-circuit current without significant alterations in transepithelial potential difference or tissue conductance. These results suggest that CGRP may play a role in regulating airway mucociliary transport function.     

Swimming Speed of Larval Snail Does Not Correlate with Size and Ciliary Beat Frequency

Many marine invertebrates have planktonic larvae with cilia used for both propulsion and capturing of food particles. Hence, changes in ciliary activity have implications for larval nutrition and ability to navigate the water column, which in turn affect survival and dispersal. Using high-speed high-resolution microvideography, we examined the relationship between swimming speed, velar arrangements, and ciliary beat frequency of freely swimming veliger larvae of the gastropod Crepidula fornicata over the course of larval development. Average swimming speed was greatest 6 days post hatching, suggesting a reduction in swimming speed towards settlement. At a given age, veliger larvae have highly variable speeds (0.8–4 body lengths s⁻¹) that are independent of shell size. Contrary to the hypothesis that an increase in ciliary beat frequency increases work done, and therefore speed, there was no significant correlation between swimming speed and ciliary beat frequency. Instead, there are significant correlations between swimming speed and visible area of the velar lobe, and distance between centroids of velum and larval shell. These observations suggest an alternative hypothesis that, instead of modifying ciliary beat frequency, larval C. fornicata modify swimming through adjustment of velum extension or orientation. The ability to adjust velum position could influence particle capture efficiency and fluid disturbance and help promote survival in the plankton.     

Calcium regulates independently ciliary beat and cell contraction in Paramecium cells

Intracellular Ca(2+) concentration is a well-known signal regulator for various physiological activities. In many cases, Ca(2+) simultaneously regulates individual functions in single cells. How can Ca(2+) regulate these functions independently? In Paramecium cells, the contractile cytoskeletal network and cilia are located close to each other near the cell surface. Cell body contraction, ciliary reversal, and rises in ciliary beat frequency are regulated by intracellular Ca(2+) concentration. However, they are not always triggered simultaneously. We injected caged calcium into Paramecium caudatum cells and continuously applied weak ultraviolet light to the cells to slowly increase intracellular Ca(2+) concentration. The cell bodies began to contract just after the start of ultraviolet light application, and the degree of contraction increased gradually thereafter. On the other hand, cilia began to reverse 1.4s after the start of ultraviolet application and reversed completely within 100ms. Ciliary beat frequency in the reverse direction was significantly higher than in the normal direction. These results indicate that cell body contraction is regulated by Ca(2+) in a dose-dependent manner in living P. caudatum. On the other hand, ciliary reversal and rise in ciliary beat frequency are triggered by Ca(2+) in an all-or-none manner.     

Swimming behavior regulation by GABAB receptors in Paramecium

In Paramecium, internal Ca(2+) concentration increase coupled to membrane depolarization induces a reversal in the direction of ciliary beating and, consequently, a reversal in swimming direction. The ciliary reversal (CR) duration is correlated to Ca(2+) influx, and the addition of drugs that block the Ca(2+) current leads to a reduction in the backward swimming duration. In this study we have examined the possible function of GABA(B) receptors in P. primaurelia swimming control. The presence of GABA(B) immunoanalogue in Paramecium was evidenced using SDS-PAGE, Western blotting, and confocal laser scanning microscopy. By applying the specific GABA(B) receptor agonist baclofen, a dose-dependent inhibition of the membrane depolarization-induced CR duration was observed. This inhibition was antagonized by phaclofen, persisted when K(+) channel blockers were applied, and disappeared after treatment with nifedipine and verapamil. Moreover, the action of baclofen on depolarization-induced CR was suppressed by treatment with pertussis toxin. Therefore, these experiments suggest that baclofen modulates CR by a G protein (G(0) or G(1)) mediated inhibition of dihydropyridine-sensible calcium channels. Finally, synthesis and release of GABA in the environment by Paramecium have been demonstrated by HPLC. Possible correlations between GABA(B) receptor activation and the regulation of intracellular Ca(2+) levels are discussed.     

Ionic regulation of cyclic AMP levels in Paramecium tetraurelia in vivo

cAMP levels in Paramecium increased dose dependently after a step increase of [Ca] or [Sr] in the incubation, provided K was present. Two mM Ca or Sr tripled cAMP concentrations within 3 s and induced an increase in forward swimming speed. The increase in cAMP formation was strictly dependent on the Donnan ratio [K]: square root [Ca]. Na, Li, or tetraethylammonium could not replace K. The data provide evidence for regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the K: Ca ratio.     

Identification of a 42 kDa protein as a substrate of protein phosphatase 1 in cilia from Paramecium.

Okadaic acid, a specific inhibitor of protein phosphatase 1 in Paramecium causes sustained backward swimming in response to depolarising stimuli (S. Klumpp et al. (1990) EMBO J. 9, 685). Here, we employ okadaic acid, tautomycin, microcystin LR and inhibitor 1 as phosphatase inhibitors to identify a 42 kDa protein in the excitable ciliary membrane that is dephosphorylated by protein phosphatase 1. Identification of the 42 kDa protein was facilitated by the finding that the protein kinase responsible for its phosphorylation uses Ca-ATP as a substrate just as effectively as Mg-ATP. Notably, dephosphorylation of the 42 kDa protein is specifically inhibited by cyclic AMP; cyclic GMP has no effect.     

Effect of in vitro cultivation and Mycoplasma pneumoniae infection on intracellular cyclic AMP levels in hamster tracheal organ cultures.

Exogenous cyclic AMP and dibutyryl cyclic AMP decreased the relative ciliary activity values of tracheal organ cultures. In contrast, theophylline and cholera toxin were not ciliostatic. The use of a radioimmunoassay for cyclic AMP indicated that all of the tested substances increased intracellular cyclic AMP levels to some extent (from 3-fold for cholera toxin to almost 40-fold for dibutyryl cyclic AMP). Physical inactivation of explants by either freeze-thaw or heat destroyed all ciliary activity and greatly decreased intracellular cyclic AMP levels. Cyclic AMP levels of explants remained relatively constant during in vitro cultivation. Three strains of Mycoplasma pneumoniae were found to contain extremely low amounts of cyclic AMP. Infection of tracheal explants produced a significant decrease in relative ciliary activity, but only a slight decline in organ-culture cyclic AMP levels.     

Control of ciliary orientation through cAMP-dependent phosphorylation of axonemal proteins in paramecium caudatum

Ciliary reorientations in response to cAMP do not take place after a brief digestion with trypsin in ciliated cortical sheets from Triton-glycerol-extracted Paramecium. In this study, we examined the effects of tryptic digestion on the cAMP-dependent phosphorylation of axonemal proteins to clarify the relationship between phosphorylation and ciliary reorientation. As reported for Paramecium tetraurelia, cAMP stimulated phosphorylations of the 29 kDa and 65 kDa axonemal polypeptides also in Paramecium caudatum. After a brief digestion of axonemes by trypsin, none of the cAMP-dependent phosphorylations occurred. On the other hand, the 29 kDa polypeptide still remained to be labeled after a brief digestion of axonemes that had previously been labeled with (32)P in the presence of cAMP, which indicates that this brief digestion breaks down endogenous cAMP-dependent protein kinases but not phosphorylated proteins. This must be the reason that trypsin-treated cilia on the sheets cannot reorient towards the posterior part of the cell. Our results indicate that cAMP regulates not only the beat frequency but also the ciliary orientation via phosphorylation of dynein subunits in Paramecium.     

Control of ciliary beat frequency in the gill of Mytilus--I. Activation of the lateral cilia by cyclic AMP

The effect of cyclic AMP on the beat frequency of the lateral cilia on the gill of Mytilus edulis was investigated. Dibutyryl cyclic AMP (0.1 mM) weakly stimulated the cilia to beat. The activation was augmented by 1 mM theophylline. The cilia were hardly activated by cyclic AMP (0.1 mM) but a strong activation was observed when it was applied with saponin (0.0025-0.01% w/v) and theophylline (1 mM). The results suggest that cyclic AMP is involved in the mechanism of ciliary activation by 5-hydroxytryptamine.     

The role of cortical orientation in the control of the direction of ciliary beat in Paramecium

The swimming behavior of many ciliate protozoans depends on graded changes in the direction of the ciliary effective stroke in response to depolarizing stimuli (i.e., the avoiding reaction of Paramecium). We investigated the problem of whether the directional response of cilia with a variable plane of beat is related to the polarity of the cell as a whole or to the orientation of the cortical structures themselves. To do this, we used a stock of Paramecium aurelia with part of the cortex reversed 180 degrees. We determined the relation of the orientation of the kineties (ciliary rows) to the direction of beat in these mosaic paramecia by cinemicrography of particle movements near living cells and by scanning electron microscopy of instantaneously fixed material. We found that the cilia of the inverted rows always beat in the direction opposite to that of normally oriented cilia during both forward and backward swimming. In addition, metachronal waves of ciliary coordination were present on the inverted patch, travelling in the direction opposite to those on the normal cortex. The reference point for the directional response of Paramecium cilia to stimuli thus resides within the cilia or their immediate cortical surroundings.     

The involvement of cyclic adenosine monophosphate in the control of schistosome miracidium cilia

This study examined the possible involvement of cyclic adenosine monophosphate (cAMP) in the control of ciliary action of Schistosoma mansoni miracidia. Miracidia immobilized in hypertonic NaCl solution were treated with 3 compounds that are known to increase intracellular cAMP concentrations. Forskolin, at a concentration of 50 microM, induced 50.1% of the miracidia to swim in hypertonic solution. The corresponding values obtained for 3-isobutyl-1-methylxanthine (IBMX) at 1 mM and 8-bromo-cAMP at 10 mM were 42.2 and 50.4%, respectively. The motility-enhancing effect of these compounds was dose dependent. Nevertheless, the swimming speed of miracidia activated in this way was only 10% of that observed in artificial pond water (APW). Cholera toxin had no apparent effect on miracidia swimming in hypertonic NaCl solution. Likewise, swimming in APW treated with forskolin at 50 microM, IBMX at 1 mM, or 8-bromo-cAMP at 10 mM did not induce any apparent change in motility. Miracidia swimming in APW were then treated with 3 compounds that decrease the intracellular concentration of cAMP. MDL-12,330A, at a concentration of 250 microM, caused a dramatic decrease in swimming over a period of 1 hr. Likewise, SQ22536 and imidazole, at concentrations of 20 and 50 mM, respectively, caused 36.5 and 73.4% decreases in swimming under the same conditions. Finally, inhibitors of cAMP-dependent protein kinase, i.e., PKI(14-22)amide, H89, and H88, completely inhibited miracidia swimming in APW at concentrations of 25, 50, and 100 microM, respectively. These results suggest that cAMP and cAMP-dependent protein kinase are involved in osmosis-controlled ciliary motion of schistosome miracidia.