PHOTOSENSORY TRANSDUCTION IN CILIATES. II. POSSIBLE ROLE OF G-PROTEIN AND cGMP IN Stentor coeruleus

Abstract— The heterotrichous ciliate, Stentor coerulus , exhibits a welll defines photophobic response to a sudden increase in the intensity of visible light. the phobic reactions usually appear with a latency perios (i.e. a time delay between the onset of the stimulus and the stop response). This latency of phobic response was significatly increased when the cells werw incubated with 8-bromo-guanosine3',5'-cyclic monophospjhate. In the presence of this nucleotide, a reduction of cell responsiveness (i.e. the number of photophobically responding cells) was also observed. similar effects were observed when cells were treated with pertussis toxin, a G-protein activity modulator, and 3'-isobutyl-methylxanthine, an inhibitor of guanosine 3', 5'-cyclic monophosphate (cGMP) phosphodiesterase. the G-protein activator fluoroaluminate and 6-anilino-5,8-quinolinedione (LY 83583) (an effective agent for lowerin cellular cGMP levels) showed opposite effects on hte cell photophobic response. These result indirectly suggesnt that the level of cytoplamic cGMP, possibly modulated by a G-protein-coupled CGMP phosphodiesterase, plays a phototreasducing role in Stentor . In addition, using an antiserum raised against bovine transducin, a cross reacting protein with an apparent molecular mass of 39 kDa was detected on immunoblots. The α-subunits of a Stentor G-protein has also been partially cloned and sequenced. However, the possible coupling between the G-protein and the putative phosphodiesterase remains to be established.     

Additional evidence for the cyclic GMP signaling pathway resulting in the photophobic behavior of Stentor coeruleus.

We report that exo- and endogenous guanosine 3',5'-cyclic monophosphate (cGMP) specifically influenced the photophobic response. In behavioral experiments the slowly hydrolyzable and membrane-permeable analogs of cGMP (8-bromo-cGMP [Br-cGMP] and N6,2'-o-dibutyryl-cGMP) dramatically prolonged the time for ciliary stop response and decreased the duration of ciliary reversal in a dose-dependent manner. When analogs of adenosine 3',5'-cyclic monophosphate (cAMP) (8-bromo-cAMP or N6,2'-o-dibutyryl-cAMP) were used, no essential effects were detected on the kinetics of the photophobic response. Both nonspecific cyclic nucleotide phosphodiesterase (PDE) activity inhibitors (3-isobutyl-1-methylxanthine [IBMX] and 1,3-dimethylxanthine [theophylline]) and the highly specific cGMP-PDE activity inhibitor 1,4-dihydro-5-[2-propoxyphenyl]-7H-1,2,3-triazolo[4,5-d]pyrimidine-7-one (zaprinast) mimicked the effects of cGMP analogs. Treatment of cells with an inhibitor of guanylate cyclase activity (6-anilino-5,8-quinolinedione [LY 83583]) exerted an effect opposite to that of cGMP analogs and PDE activity inhibitors. The positive physiological effect of LY 83583 was significantly diminished in ciliates that were treated simultaneously with Br-cGMP. In an assay of cell cyclic nucleotide content, the exposure of dark-adapted Stentor to light evoked a transient decrease in the basal level of intracellular cGMP. Alterations in internal cGMP levels were more distinct when the intensity of applied illumination was increased. In the presence of IBMX or theophylline the basal content of cGMP was markedly enhanced, and the photoinduced changes in cGMP level were less pronounced. In this paper the possible whole molecular mechanism by which the ciliary orientation in Stentor is controlled by light is presented.     

Contribution of phosphoinositide-dependent signalling to photomotility of Blepharisma ciliate

The effect of experimental procedures designed to modify an intracellular phosphoinositide signalling pathway, which may be instrumental in the photophobic response of the protozoan ciliate Blepharisma japonicum, has been investigated. To assess this issue, the latency time of the photophobic response and the cell photoresponsiveness have been assayed employing newly developed computerized videorecording and standard macro-photographic methods. Cell incubation with neomycin, heparin and Li+, drugs known to greatly impede phosphoinositide turnover, causes evident dose-dependent changes in cell photomotile behaviour. The strongest effect on photoresponses is exerted by neomycin, a potent inhibitor of polyphosphoinositide hydrolysis. The presence of micromolar concentrations of neomycin in the cell medium causes both prolongation of response latency and decrease of cell photoresponsiveness. Neomycin at higher concentrations (> 10 microM) abolishes the cell response to light at the highest applied intensity. A slightly lower inhibition of cell responsiveness to light stimulation and prolongation of response latency are observed in cells incubated in the presence of heparin, an inositol trisphosphate receptor antagonist. Lithium ions, widely known to deplete the intracellular phosphoinositide pathway intermediate, inositol trisphosphate, added to the cell medium at millimolar level, also cause a slowly developing inhibitory effect on cell photoresponses. Mastoparan, a specific G-protein activator, efficiently mimics the effect of light stimulation. In dark-adapted ciliates, it elicits ciliary reversal with the response latency typical for ciliary reversal during the photophobic response. Sustained treatment of Blepharisma cells with mastoparan also suppresses the photoresponsiveness, as in the case of cell adaptation to light during prolonged illumination. The mastoparan-induced responses can be eliminated by pretreatment of the cells with neomycin. Moreover, using antibodies raised against bovine transducin, a cross-reacting protein with an apparent molecular mass of about 55 kDa in the Blepharisma cortex fraction is detected on immunoblots. The obtained results indirectly suggest that the changes in internal inositol trisphosphate level, possibly elicited by G-protein-coupled phospholipase C, might play a role in the photophobic response of Blepharisma. However, further experiments are necessary to clarify the possible coupling between the G-protein and the putative phospholipase C.     

Mass spectrometric identification of Rab23 phosphorylation as a response to challenge by cytidine 3',5'-cyclic monophosphate in mouse brain

While the functions and mechanisms of action of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) are well established and are the basis of the action of a large number of successful pharmaceuticals, the role of a third naturally occurring cyclic nucleotide, cytidine 3',5'-cyclic monophosphate (cCMP), remains to be elucidated. Immobilized metal affinity chromatography (IMAC) was used to selectively extract proteins phosphorylated in mouse brain in response to challenge by cAMP, cGMP and cCMP, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToFMS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) of tryptic digests to identify Rab23 as the first protein reported to be phosphorylated only in response to cCMP.     

Photosensitive signal transduction induces membrane hyperpolarization in Paramecium bursaria

The protozoan ciliate Paramecium bursaria exhibits membrane hyperpolarization in response to photostimulation, accompanied with an increased swimming speed. The external addition of cyclic nucleotide phosphodiesterase (PDE) inhibitors, either theophylline (1,3-dimethylxanthine) or 3-isobutyl-1-methylxanthin (IBMX), increased in both amplitudes of the membrane hyperpolarization and the increase in swimming speed. Moreover, the addition of membrane permeable cyclic nucleotide analogs, either 8-bromo-adenosine 3',5'-cyclic monophosphate (Br-cAMP) or 8-Br-guanosine 3',5'-cyclic monophosphate (Br-cGMP), increased these amplitudes. On the other hand, the addition of l-cis-diltiazem, known to block the conductance of cyclic nucleotide-gated channels, partially decreased both amplitudes of the membrane hyperpolarization and the increase in swimming speed. An enzyme immunoassay of cellular cyclic nucleotide contents showed that photostimulation induced a rapid increase in adenosine 3',5'-cyclic monophosphate (cAMP), but little increase in guanosine 3',5'-cyclic monophosphate (cGMP), raising the possibility that a rapid increase in cAMP mediates the light-induced hyperpolarization in P. bursaria.     

ACTION SPECTRA OF THE PHOTOPHOBIC RESPONSE OF BLUE AND RED FORMS OF Blepharisma japonicum

Abstract— When exposed, in the presence of molecular oxygen, to light intensities of the order of3–30 W m^-2, the ciliate Blepharisma japonicum changes its color from red to blue, because of the photooxidation of the photoreceptor pigment, blepharismin, to pxyblepharismin. Both red-and blue-pigmnentes cells show step-up photophobic responses. The action spectra f the light-dependent behaviour of the red and the blue form of Blepharisma have been determined; their structure is very similar to that the photosensing and phototransducing properties of blepharismin are maintained in its photooxidized form. oxyblepharismin.     

Photosensory transduction in unicellular eukaryotes: a comparison between related ciliates Blepharisma japonicum and Stentor coeruleus and photoreceptor cells of higher organisms

Blepharisma japonicum and Stentor coeruleus are related ciliates, conspicuous by their photosensitivity. They are capable of avoiding illuminated areas in the surrounding medium, gathering exclusively in most shaded places (photodispersal). Such behaviour results mainly from motile photophobic response occurring in ciliates. This light-avoiding response is observed during a relatively rapid increase in illumination intensity (light stimulus) and consists of cessation of cell movement, a period of backward movement (ciliary reversal), followed by a forward swimming, usually in a new direction. The photosensitivity of ciliates is ascribed to their photoreceptor system, composed of pigment granules, containing the endogenous photoreceptor -- blepharismin in Blepharisma japonicum, and stentorin in Stentor coeruleus. A light stimulus, applied to both ciliates activates specific stimulus transduction processes leading to the electrical changes at the plasma membrane, correlated with a ciliary reversal during photophobic response. These data indicate that both ciliates Blepharisma japonicum and Stentor coeruleus, the lower eukaryotes, are capable of transducing the perceived light stimuli in a manner taking place in some photoreceptor cells of higher eukaryotes. Similarities and differences concerning particular stages of light transduction in eukaryotes at different evolutional levels are discussed in this article.     

ADDITIONAL EVIDENCE FOR BLEPHARISMIN PHOTORECEPTOR PIGMENT MEDIATING STEP-UP PHOTOPHOBIC RESPONSE OF UNICELLULAR ORGANISM, Blepharisma

Abstract— In the ciliated protozoan, Blepharisma japonicum, the pink-colored pigment (blepharismin) contained in the pigment granules is believed to be the photoreceptor pigment responsible for the step-up photophobic response. When the cells partially bleached by extrusion of the pigment granules caused by cold shocks were subsequently cultured under illuminated conditions, the pigment-less granules regenerated and the cells were further bleached (pigment content below 0.5%). The photosensitivity of such colorless cells disappeared completely. In contrast, the blepharismin pigment regenerated gradually when such colorless cells were transferred to darkness. The photosensitivity of the cells also recovered with regeneration of the pigment. We found that blepharismin pigment was not photobleached in the absence of O₂. The step-up photophobic response was also completely repressed in the absence of O₂. These results strongly confirm that blepharismin is a photoreceptor pigment mediating photobehavior of Blepharisma and that O₂ is required for the early step in the phototransduction of the light-excited pigment.     

Primary photoprocesses involved in the sensory protein for the photophobic response of Blepharisma japonicum

We present new femtosecond transient-absorption and picosecond fluorescence experiments performed on OBIP, the oxyblepharismin-binding protein believed to trigger the photophobic response of the ciliate Blepharisma japonicum. The formerly identified heterogeneity of the sample is confirmed and rationalized in terms of two independent populations, called rOBIP and nrOBIP. The rOBIP population undergoes a fast photocycle restoring the initial ground state in less than 500 ps. Intermolecular electron transfer followed by electron recombination is identified as the excited-state decay route. The experimental results support the coexistence of the oxyblepharismin (OxyBP) radical cation signature with a stimulated-emission signal at all times of the evolution of the transient-absorption spectra. This observation is interpreted by an equilibrium being reached between the locally excited state and a charge-transfer state on the ground of a theory developed by Mataga and co-workers to explain the fluorescence quenching of aromatic hydrogen-bonded donor-acceptor pairs in nonpolar solvents. OxyBP is supposed to bind to an as yet unknown electron acceptor by a hydrogen-bond (HB) and the coordinate along which forward and backward electron transfer proceed is assumed to be the shift of the HB proton. The observed kinetic isotope effect supports this interpretation. Protein relaxation is finally proposed to accompany the whole process and give rise to the highly multiexponential observed dynamics. As previously reported, the fast photocycle of rOBIP can be interpreted as an efficient sunscreen mechanism that protects Blepharisma japonicum from continuous irradiation. The nrOBIP population, the transient-absorption of which strongly reminds that of free OxyBP in solution, might be proposed to actually trigger the photophobic response of the organism through excited-state deprotonation of the chromophore occurring in the nanosecond regime. Additional femtosecond transient-absorption spectra of OxyBP and peri-deprotonated OxyBP are also reported and used as a comparison basis to interpret the results on OBIP.     

IRRADIATION IN THE FROZEN STATE: A TECHNIQUE FOR DIRECT PHOTOAFFINITY LABELING

UV irradiation of rabbit muscle phosphofructokinase (PFK) in the presence of adenosine 3',5'-cyclic phosphate (cAMP) resulted in the covalent attachment of this ligand molecule to the enzyme protein. Irradiation in the frozen ice state enhanced the rate of this incorporation more than 10-fold above that achieved in aqueous solution, without significantly affecting the rate of photodestruction of the protein. [³H]-cAMP and [³²P]-cAMP were each incorporated into PFK at identical rates in the frozen state. Rates of photoincorporation in the frozen and liquid states were both half-maximal at a free ligand concentration approximately equal to the dissociation constant of cAMP and PFK. Adenosine diphosphate (ADP) and adenosine monophosphate (AMP), both of which are known to compete for cAMP binding to PFK, inhibited photoincorporation of cAMP. Guanosine monophosphate (GMP), inosine monophosphate (IMP), and guanosine 3',5'-cyclic phosphate (cGMP), which do not compete for cAMP binding, had no effect on photoincorporation of cAMP. Irradiation of [³H]-AMP or [³H]-ADP resulted in photoincorporation into PFK at 0°C, with enhancement at — 77°C similar to that noted with cAMP.     

A videomicroscopic study of the effect of l-cis-diltiazem on the photobehavior of Stentor coeruleus

The protozoan ciliate Stentor coeruleus displays a step-up photophobic response to an increase in light intensity in its environment. The motile response consists of a delayed stop of ciliary beating and transient ciliary reversal period. Such light-avoiding behavior was significantly influenced by an incubation of cells with l-cis-diltiazem, a common blocker of cyclic guanosine monophosphate (cGMP)-gated ion channel conductance. The introduction of l-cis-diltiazem to the medium induced ciliary reversal in control cells, mimicking the step-up photophobic response. In light-stimulated ciliates, the presence of this inhibitor caused a substantial decrease of the latency of ciliary stop response, prolongation of the ciliary reversal duration and also an increase of cell photoresponsiveness in a dose- and time-dependent manner. The obtained behavioral results support the suggestion that the photosensitive ciliate S. coeruleus possesses cGMP-gated channels, which may be involved in the process of light signal transduction for the motile photophobic response.     

Localization of Blepharismin Photosensors and Identification of a Photoreceptor Complex Mediating the Step-up Photophobic Response of the Unicellular Organism, Blepharisma

Abstract— Photosensitivity for the step-up photophobic response of Blepharisma is localized in the anterior 1/5 of the cell body. Blepharismin pigment, which is believed to be a photoreceptor pigment mediating the step-up photophobic response of the cells, was separated into five types of blepharismin (BL-1, -2, -3, -4 and -5). Blepharismin-1, -3, -4 and -5 were localized in the posterior 4/5, while BL-2 was located over the entire cell body; the anterior end, which is the photosensitive region, contained only BL-2. The results indicate that a functional photoreceptor pigment mediating the step-up photophobic response is BL-2. Hydroxylapatite chromatography revealed that BL-2 was bound to a 200 kDa membrane protein. We concluded that a photoreceptor mediating the step-up photophobic response was a BL-2/200 kDa protein complex.     

PHOTOMOVEMENT RESPONSES OF THE HETEROTRICHOUS CILIATE Blepharisma Japonicum

Light-induced movement responses of the heterotrichous ciliate Blepharisma japonicum were studied by physiological experiments. Two photosensory responses could be identified. A step-up photophobic response is observed as a very rapid backward movement. Microbeam irradiations of individual cells showed that only the anterior part of the ciliate is able to perceive the light stimulus that mediates the phobic reaction. The action spectrum peaks at approximately 400 nm, which indicates that a blue light receptor is involved.
Positive photokinesis of Blepharisma could be shown as a forward movement that is accelerated by increasing the applied photon fluence rate. The steady state level of the velocity depends highly on wavelength and photon fluence rate of the actinic light. After specific inhibition of the phobic reaction bv 1 m/W NH₄⁺, photokinesis can be induced by microbeam irradiation at any part of the cell.
We isolated two main pigments by thin layer chromatography and characterized them as hypericin-like compounds: a red pigment that is obviously responsible for the red color of the ciliates (= blepharismin). and a yellow one with maximal absorption near 420 nm. The possible photoreceptor functions of these pigments are discussed.
We could not find in Blepharisma a distinct phototactic behavior which is so typical for the related ciliate Stentor.     

Salidroside attenuates hydrogen peroxide-induced cell damage through a cAMP-dependent pathway

Salidroside, a major component of Rhodiola rosea L., has shown various pharmacological functions, including antioxidant effects, but the signal transduction pathway of its antioxidant effects is not very clear. In this study, we found that salidroside could attenuate hydrogen peroxide (H(2)O(2))-induced HL-7702 cell damage, inhibit H(2)O(2)-induced cytosolic free Ca2+ ([Ca2+]i) elevation, scavenge reactive oxygen species (ROS) and increase 3'-5'-cyclic adenosine monophosphate (cAMP) level in a dose-dependent manner, but it couldn't influence 3'-5'-cyclic guanosine monophosphate (cGMP) levels. Therefore, these results indicated that the antioxidant effects of salidroside were associated with down-regulation of [Ca2+]i, ROS occur via a cAMP-dependent pathway.     

Novel 3',5'-cyclic nucleotide analogue: adenosine 3',5'-cyclic boranomonophosphate

A general procedure for the first synthesis of a 3',5'-cyclic boranomonophosphate was established. Specifically, adenosine 3',5'-cyclic boranomonophosphosphate (cyclic AMPB, 4c), a P-borane (BH(3)) analogue of adenosine 3',5'-cyclic monophosphate (cAMP), was synthesized via a phosphite approach in good yield. The method is also applicable for syntheses of natural cAMP and its phosphorothioate analogue. The two diasteromers of cyclic AMPB 4c were separated, and their chemical structures were established via spectroscopic methods.     

Cytidylate cyclase activity: identification of cytidine 3',5'-cyclic monophosphate and four novel cytidine cyclic phosphates as biosynthetic products from cytidine triphosphate

Identification of cytidine 3',5'-cyclic monophosphate (cyclic CMP) as one of the products resulting from the incubation of dialysed cell-free preparations from rat brain, liver and kidney with cytidine 5'-triphosphate (CTP) is described. The non-acidic precipitable products after incubation of the tissue preparations with unlabelled, with 14C-single labelled, and with 14C- and 32P-dual labelled CTP were examined by thin-layer chromatography and high-pressure liquid chromatography, isotopic ratio determination, UV absorbance spectrophotometry, selective hydrolysis with nucleotidase, phosphodiesterase and acid, and by fast atom bombardment mass spectrometry with mass-analysed ion kinetic energy spectrum scanning. In addition to cyclic CMP and unchanged CTP, the products of the reaction were found to include cytidine monophosphate (CMP) and cytidine diphosphate (CDP) together with four novel cytidine compounds identified as cytidine 3',5'-cyclic pyrophosphate, cytidine 2'-monophosphate 3',5'-cyclic monophosphate, cytidine 2'-O-aspartyl-3',5'-cyclic monophosphate and cytidine 2'-O-glutamyl-3',5'-cyclic monophosphate. The evidence presented constitutes conclusive proof of the natural occurrence of cytidylate cyclase activity; the four novel cytidine cyclic phosphates described provide a feasible explanation of the discrepancies in previous reports which have led to the controversy which exists concerning the existence of cytidylate cyclase activity.     

ISOLATION OF BLEPHARISMIN-BINDING 200 kDa PROTEIN RESPONSIBLE FOR BEHAVIOR IN Blepharisma

Abstract— The ciliated protozoan, Blepharisma, shows an avoidance reaction (step-up photophobic response) in response to light stimulation. A profile of a gel-permeation of a crude detergent-solubilized sample of the cells resulted in several red-colored fractions. Among these blepharismin-containing fractions, the fractions III-V did not contain amino acids. The peak of fraction II monitored by 580 nm absorbance was much smaller. A prominent peak appeared in fraction I, which contained a large amount of amino acids. The absorption spectrum of fraction I was well fitted to the action spectrum of the step-up photophobic response, although free pigment (blepharismin) also fitted. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction resulted in a thicker band corresponding to molecular mass of 200 kDa. These results suggest that the 200 kDa chromoprotein (blepharismin-protein complex) is responsible for the step-up photophobic response in Blepharisma. The absorption spectrum of free chromophore dissociated from the chromophore-protein complex was identical to free red pigment termed blepharismin. The absorption spectrum of the other fractions agreed with that of thin-layer chromatography-purified red pigment, indicating that the pigments contained in these fractions are free pigment dissociated from the chromophore-protein complex.     

Predictive value of the analogy between hormone-sensitive adenylate cyclase and light-sensitive photoreceptor cyclic GMP phosphodiesterase: a specific role for a light-sensitive GTPase as a component in the activation sequence.

We report experiments which involve a light sensitive GTPase in the light dependent activation of retinal rod 3'5'-cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE). The data suggest that the light activated GTPase is intermediate between rhodopsin and PDE in the light-dependent activation sequence. We list the many striking similarities between hormone sensitive adenylate cyclase and light activated PDE in order to emphasize that the findings presented herein may have predictive value for ongoing studies of the hormone sensitive adenylate cyclase specifically regarding the role of the hormone activated GTPase in the activation sequence.     

Light Induces lnositol Trisphosphate Elevation in Blepharisma japonicum

Abstract— Photoinduced formation of inositol 1,4,5-trisphosphate (Ins[1,4,5]P₃) was examined using a specific radioimmu-noassay to investigate the molecular mechanisms of light signal transduction mediating photophobic responses in the ciliate Blepharisma japonicum. Application of light stimuli of moderate intensity to dark-adapted cells induced a rapid and significant increase in the basal level of Ins (1,4,5)P₃, with a peak at about 20 s. Thereafter, the level of Ins (1,4,5)P₃ declined to the resting value within the subsequent 100 s. Light stimuli of higher intensity raised the cell Ins (1,4,5)P₃ content to still higher levels within about 20 s, but the decaying time course was considerably prolonged. In ciliates incubated under dark conditions with agents interfering with the inositol signalling pathway, like neomycin and Li⁺ the basal levels of Ins (1,4,5)P₃ were lower than in control cells. A photoinduced rise of Ins (1,4,5)P₃, content in ciliates treated with neomycin or Li⁺ was significantly inhibited in a dose-dependent manner. Depolarizing ionic stimuli in dark-adapted ciliates induced no significant alterations of the resting Ins (1,4,5)P₃ level, indicating a lack of a contribution of this kind of stimulation to the inositol turnover. These studies are the first in vivo demonstration of a possible role for inositol trisphosphate as a second messenger in the light signal transduction process in the ciliate B. japonicum.     

PHOTOSENSORY TRANSDUCTION IN CILIATES. III. THE TEMPORAL RELATION BETWEEN MEMBRANE POTENTIALS AND PHOTOMOTILE RESPONSES IN Blepharisma japonic urn

Abstract— Blepharisma japonicum exhibits a step-up photophobic response when subjected to an increase in light stimulus intensity. This response is characterized by the stop reaction after a period of delay followed by backward swimming (lateral rotation). The latency of the stop response decreased and duration of the lateral rotation increased as the intensity of light stimuli was raised. A step-increase in light intensity elicited a graded membrane depolarization (photic receptor potential), as measured by intracellular microelectrode. When the amplitude of receptor potential exceeded a threshold depolarization for membrane excitation (15–25 mV), an all-or-none action potential of 50–65 mV in amplitude was evoked which also occurred with some latency. Light stimuli of higher intensity (suprathreshold) elicited action potential which was followed by a membrane after-depolarization. Increasing the intensity of stimuli caused generation of an action potential with shorter lag period and prolonged after-depolarization. The action spectra for the latency of stop reaction, receptor potential amplitude and cell photoresponsiveness showed maxima at 460, 530 and 580 nm. The analysis of temporal relationships between the electrophysiological responses and the motile events showed that latency of an action potential, induced by the receptor potential, correlates well with the latency of a cell stop response. Also the duration of membrane after-depolarization resembled the time period of the cell's backward swimming (cell rotation). The data obtained indicate that the primary reaction initiated by light absorption in the photoreceptor pigment (blepharismin) is converted into the observed electrical potential changes, which in turn results in the photomotile response of Blepharisma cells.