The latency of the response of Limulus photoreceptors to inositol trisphosphate lacks the calcium-sensitivity of that to light

Summary The latent period before depolarization of Limulus ventral photoreceptors by light flashes was compared with that following brief, intracellular, pressure-injection of d-myo-inositol 1,4,5 trisphosphate. At temperatures between 18 °C and 22 °C and with an extracellular calcium concentration of 10 mM, the responses of 4 cells to light and to injections of 100 M inositol trisphosphate displayed average latencies of 71 and 56 ms, respectively. The latencies of responses to InsP₃ included an estimated 20 ms dead-time inherent in the injection method. Reducing the temperature lengthened the latency of the response to light (Q₁₀ approximately 3.2 between 7 and 22 °C) more than that to inositol trisphosphate (Q₁₀ approximately 2.3). Bathing the photoreceptors in seawater containing no added calcium and 1 mM of the calcium chelator EGTA greatly increased the latency of the light response at all temperatures, but did not increase the latency of the response to inositol trisphosphate. We conclude that the response to inositol trisphosphate lacks the calcium- and temperature-sensitive latent period which characterizes the response to light. If inositol trisphosphate acts, via the release of stored calcium, to stimulate an intermediate in the visual cascade, then that intermediate would appear to be downstream from the latency-generating mechanism.Abbreviations InsP ₃ D-myo-inositol 1,4,5 trisphosphate - ASW Artificial seawater - Ca _i Cytosolic free calcium ion concentration - Ca ₀ Extracellular calcium ion concentration     

Enhancement of sensitivity in photoreceptors of the honey bee drone by light and by Ca2+

Summary Deeply dark adapted (1 h) photoreceptor cells of the honey bee drone show a light-induced enhancement of sensitivity (facilitation) as an aftereffect of illumination or in the presence of dim backgrounds.The Ca²⁺-dependency of this effect was studied: Reduction of extracellular Ca²⁺ to 0.1 mM decreases the sensitivity of a dark adapted cell, and the light-induced increase in sensitivity due to repetitive, dim, 20 ms test flashes is slower than in normal saline. After a sensitizing conditioning light, the sensitivity drops faster in low-calcium saline. The light-induced enhancement of sensitivity is mimicked by pressure injections of low amounts of Ca²⁺ (Ca²⁺/EGTA-buffers; 0.15 M free Ca²⁺) into a dark adapted cell. Injection of EGTA alone decreases the sensitivity. Injection of a solution containing ca1 mM free Ca²⁺ sequentially decreases and later increases the sensitivity transiently.These results suggest a model in which a progressive increase in intracellular Ca²⁺ concentration by light first increases (facilitates), and, at higher concentrations, decreases (light adapts) the sensitivity of the cells. One possible site of action for this positive and negative feedback control of cell sensitivity by Ca²⁺ is the endoplasmic reticulum.     

Intracellular Ca2+ concentration and latency of light-induced Ca2+ changes in photoreceptors of the honeybee drone

We have measured Ca_i at rest and upon light stimulation in the photoreceptors of the honeybee drone microfluorometrically with the fluorescent Ca²⁺ indicator dyes fura-2, fluo-3 and Ca-green 5N.In darkness, Ca_i was 90 nM after 5 min of dark adaptation. A saturating light step caused Ca_i to rise in the bulk cytoplasm to 750 nM within 1 s. Our measurements with the low affinity dye Ca-green 5N showed that bright 1-s light flashes cause a rapid increase in Ca_i which was graded with stimulus intensity. Ca-green 5N fluorescence reached a peak in about 200 ms, and then decayed to a slightly lower sustained plateau. The fluorescence signal peaked, when the receptor potential was repolarizing from its peak to the plateau. This observation is in agreement with the proposal that the peak-to-plateau transition of the receptor potential is caused by the rise in Ca_i From our Fluo-3 measurements it appears that the latency of the Ca²⁺ increase is by 3–4 ms longer than the latency of the receptor potential elicited by bright 100-ms light flashes. This result provides no support for the proposal that Ca²⁺ mediates the opening of those membrane channels responsible for the upstroke of the receptor potential.Abbreviations ER endoplasmic reticulum - IP₃ Inositol 1,4,5-trisphosphate - SMC submicrovillar cisternae     

Calcium is necessary for light excitation in barnacle photoreceptors

Summary Illumination of barnacle (Balanus amphitrite) photoreceptors is known to increase the membrane permeability to sodium and Ca²⁺ ions resulting in a depolarizing receptor potential. In this report, we show that lanthanum (La³⁺), a known inhibitor of Ca-binding proteins, reversibly eliminates the receptor potential of barnacle photoreceptors when applied to the extracellular space. Similar reversible elimination of the light response was obtained by removing extracellular Ca²⁺ by application of the calcium chelating agent EGTA. Iontophoretic injection of Ca²⁺, but not K⁺ into the cells protected both the transient and the steady-state phases of the receptor potential from elimination by EGTA while only the transient phase was protected in the presence of La³⁺. The EGTA experiments suggest that internal Ca²⁺ is necessary for light excitation of barnacle photoreceptors while the La³⁺ experiments suggest that La³⁺-sensitive inward current is necessary to maintain excitation during prolonged light.Abbreviations EGTA ethylenglyol-bis-(-aminoethylether) N, N, N¹, N¹-tetraacetate - BAPTA bis-(0-aminophenoxy)-ethane-N, N, N¹, N¹-tetraacetic acid - DMSO dimethyl sulfoxide - trp transient receptor potential - nss no steady state - ASW artificial sea water     

Metabolism of inositol 1,4,5-trisphosphate in squid photoreceptors

Summary Inositol 1,4,5-trisphosphate (InsP₃) is rapidly formed in squid photoreceptors in response to light, where it is converted sequenctially into inositol bisphosphate (InsP₂) and inositol monophosphate (InsP₁). All of the InsP₃ appears to be degraded to inositol 1,4-bisphosphate via an InsP₃-phosphatase, which is characterized in this study. The enzyme is water-soluble and present in the light-transducing distal segments of squid photoreceptors. It has a K_m of 50 M for InsP₃, requires Mg⁺⁺ for its activity, is maximally active at neutral pH, specifically hydrolyses the 5-phosphate and is inhibited by 2,3-diphosphoglycerate. In these respects, InsP₃-phosphatase of squid is very similar to the enzymes of other cells. Since no InsP₄ or more highly phosphorylated inositols are found in squid photoreceptors, the InsP₃-phosphatase may be important in the regulation of InsP₃ concentration within these cells.Abbreviations InsP ₁ , InsP ₂ , InsP ₃ , InsP ₄ , InsP ₆ inositol monobis-, tris-, tetrakis-, hexakisphosphate, respectively - 2,3-DPG 2,3-diphosphoglycerate - EDTA ethylene diamine tetraacetic acid - DTT dithiothreitol - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PMSF phenylmethylsulfonyl fluoride     

TRP channels in Drosophila photoreceptors: the lipid connection

The light-sensitive current in Drosophila photoreceptors is mediated by transient receptor potential (TRP) channels, at least two members of which (TRP and TRPL) are activated downstream of phospholipase C (PLC) in response to light. Recent evidence is reviewed suggesting that Drosophila TRP channels are activated by one or more lipid products of PLC activity: namely diacylglycerol (DAG), its metabolites (polyunsaturated fatty acids) or the reduction in phosphatidylinositol 4,5-bisphosphate (PIP₂). The most compelling evidence for this view comes from analysis of rdgA mutants which are unable to effectively metabolise DAG due to a defect in DAG kinase. The rdgA mutation leads to constitutive activation of both TRP and TRPL channels and dramatically increases sensitivity to light in hypomorphic mutations of PLC and G protein.     

Transduction in photoreceptors with bistable pigments: Intermediate processes

The prolonged depolarizing after potential (PDA) in the R1–6 receptors of the fly was used to isolate intermediate processes in phototransduction which are not manifested directly in the voltage response. It is first demonstrated that a pigment shift by light from metarhodopsin to rhodopsin in four species of the flies: Drosophila, Calliphora, Chrysomya and Musca induces an independent antagonistic process to the PDA, which is manifested in a strong inhibitory effect on PDA induction and is called the anti-PDA.By using mutants of Drosophila the existence of processes underlying the PDA were examined. The norpA ^H52and the trp mutant were used in which the voltage response of the photoreceptors could be reversibly abolished by elavated temperature and long intense light respectively. It is shown that the excitatory process underlying the PDA could be induced and depressed in conditions that block the voltage response of the photoreceptors, thus indicating the existance of intermediate processes which link the pigment activation by light to the PDA voltage response.Based on material presented at the European Neurosciences Meeting, Florence, September 1978     

Actions of neomycin on electrical light responses, Ca2+ release, and intracellular Ca2+ changes in photoreceptors of the honeybee drone

Neomycin, known to inhibit phospholipase C-mediated IP3 formation, was applied in the bath or injected into cells and its effects on electrical light responses were analyzed. Neomycin effects on inositol 1,4,5-trisphosphate- and Ca2+-induced Ca2+ release from the endoplasmic reticulum and/or the light-induced Ca2+ elevation were also studied. Neomycin (0.5 mmol x l(-1)) blocked inositol 1,4,5-trisphosphate-, caffeine-, and Ca2+-induced Ca2+ release. Bath application of neomycin decreased the sensitivity to 20-ms light flashes by a factor of up to 100 and slowed the kinetics of dim flash responses. Intracellularly injected neomycin desensitized the photoreceptors more than 1 log unit, increased the latency, and slowed the rate of rise of the light response. Neomycin (0.5 mmol x l(-1)) in the bath delayed and reduced the transient component of responses to 1-s steps of light at intermediate intensities. It also decreased and slowed the light-induced, and it blocked the caffeine-induced intracellular Ca2+ elevation. The combined pharmacological effects of neomycin are suggested to decrease the Ca2+-mediated amplification of the phototransduction cascade and the Ca2+-mediated acceleration of processes determining the kinetics of light responses.     

Involvement of Ca2 + and flowing endoplasm in recovery of cytoplasmic streaming after K+-induced cessation

Summary When K⁺ of high concentration (50 mM) was applied toNitella cells, the cytoplasmic streaming stopped instantly as in the case of electrical stimulation. Recovery of the streaming after chemical stimulation was much slower than after electrical stimulation. When the endoplasm content was modified by centrifugation, streaming recovery was accelerated in the centrifugal cell fragments rich in endoplasm and deccelerated in those poor in it. The recovery was also accelerated either by permeabilizing the plasmalemma in the presence of EGTA in the external solution or by removing the tonoplast by vacuolar perfusion with the EGTA-containing medium. We concluded that the streaming was recovered due to decrease of the cytoplasmic Ca²⁺ concentration, which seems to be accelerated by sequestering of Ca²⁺ by endoplasmic components. The slow recovery of the streaming after KCl-stimulated cessation is assumed to be caused by continuous influx of Ca^{2 +} during the prolonged membrane depolarization.Abbreviations ATP adenosine 5-triphosphoric acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

Discrete intracellular Ca2+ pools coupled to two distinct Ca2+ influx pathways in human platelets

Ca(2+) influx is an important event associated with platelet activation and regulated by the content of intracellular Ca(2+). Previous studies have suggested two different Ca(2+) pools and two Ca(2+) influx pathways exist in platelets. In the present study, we have investigated the regulation of thrombin- and thapsigargin-induced Ca(2+) entry into human platelets, using fluorescent indicators to monitor Ca(2+) mobilization and membrane potential. It was found that depletion of thapsigargin-sensitive Ca(2+) stores was coupled to Ca(2+) influx through a Ca(2+)-selective pathway. Additional release of Ca(2+) from the thapsigargin-insensitive pool by thrombin caused the opening of a nonselective cation channel.     

Analysis of the Ca2+ domain in the Arabidopsis H+/Ca2+ antiporters CAX1 and CAX3

Ca²⁺ levels in plants are controlled in part by H⁺/Ca²⁺ exchangers. Structure/function analysis of the Arabidopsis H⁺/cation exchanger, CAX1, revealed that a nine amino acid region (87–95) is involved in CAX1-mediated Ca²⁺ specificity. CAX3 is 77% identical (93% similar) to CAX1, and when expressed in yeast, localizes to the vacuole but does not suppress yeast mutants defective in vacuolar Ca²⁺ transport. Transgenic tobacco plants expressing CAX3 containing the 9 amino acid Ca²⁺ domain (Cad) from CAX1 (CAX3-9) displayed altered stress sensitivities similar to CAX1-expressing plants, whereas CAX3-9-expressing plants did not have any altered stress sensitivities. A single leucine-to-isoleucine change at position 87 (CAX3-I) within the Cad of CAX3 allows this protein to weakly transport Ca²⁺ in yeast (less than 10% of CAX1). Site-directed mutagenesis of the leucine in the CAX3 Cad demonstrated that no amino acid change tested could confer more activity than CAX3-I. Transport studies in yeast demonstrated that the first three amino acids of the CAX1 Cad could confer twice the Ca²⁺ transport capability compared to CAX3-I. The entire Cad of CAX3 (87–95) inserted into CAX1 abolishes CAX1-mediated Ca²⁺ transport. However, single, double, or triple amino acid replacements within the native CAX1 Cad did not block CAX1 mediated Ca²⁺ transport. Together these findings suggest that other domains within CAX1 and CAX3 influence Ca²⁺ transport. This study has implications for the ability to engineer CAX-mediated transport in plants by manipulating Cad residues.     

Extracellular pH modulates the Ca2+ current activated by depletion of intracellular Ca2+ stores in human macrophages

Intracellular Ca²⁺ (Ca_i) signaling following the binding of surface receptors activates a Ca²⁺ permeable plasma membrane conductance which has been shown to be associated with store depletion in a number of cell types. We examined the activation of this conductance in human monocyte-derived macrophages (HMDMs) using whole-cell voltage-clamp techniques coupled with fura-2 microfluorimetry and characterized the importance of external pH (pH_o) as a modulator of current amplitude. Current activation was observed following experimental maneuvers designed to deplete intracellular Ca²⁺-stores including: (i) dialysis of the cell with 100 m inositol 1,4,5-triphosphate (IP₃), (ii) intracellular dialysis with high concentrations of the Ca²⁺ buffers EGTA and BAPTA, or (iii) exposure of the cell to the Ca²⁺-ATPase inhibitor thapsigargin (1 m). Currents associated with store depletion were inwardly rectifying with kinetics, inactivation, and selectivity that appeared similar irrespective of the mode of activation. Currents were Ca²⁺ selective with a selectivity sequence of Ca²⁺ > Sr²⁺ Mg²⁺ = Mn²⁺ = Ni²⁺. The Ca²⁺ influx current was modulated by changes in pH_o; modulation was not produced as a consequence of changes in internal pH (pH_i). External acidification led to a reversible reduction in current amplitude with a pKa at pH 8.2. Changes in pH_o alone failed to induce current activation. These observations are consistent with a scheme by which changes in pH_o, as would be encountered by macrophages at sites of inflammation, could change the time course and magnitude of the Ca_i transient associated with receptor activation by regulating the influx of Ca²⁺ ions.The authors wish to gratefully acknowledge the expert technical assistance of Weiwen Xie without whom the study could not have been completed. This work was supported by National Institutes of Health GM36823.     

Translocation of the Drosophila transient receptor potential-like (TRPL) channel requires both the N- and C-terminal regions together with sustained Ca2+ entry

In Drosophila photoreceptors the transient receptor potential-like (TRPL), but not the TRP channels undergo light-dependent translocation between the rhabdomere and cell body. Here we studied which of the TRPL channel segments are essential for translocation and why the TRP channels are required for inducing TRPL translocation. We generated transgenic flies expressing chimeric TRP and TRPL proteins that formed functional light-activated channels. Translocation was induced only in chimera containing both the N- and C-terminal segments of TRPL. Using an inactive trp mutation and overexpressing the Na(+)/Ca(2+) exchanger revealed that the essential function of the TRP channels in TRPL translocation is to enhance Ca(2+)-influx. These results indicate that motifs present at both the N and C termini as well as sustained Ca(2+) entry are required for proper channel translocation.     

Sarcolemmal Na+-Ca2+ exchange and Ca2+-pump activities in cardiomyopathies due to intracellular Ca2+-overload

In order to identify defects in Na⁺-Ca²⁺ exchange and Ca²⁺-pump systems in cardiomyopathic hearts, the activities of sarcolemmal Na⁺-dependent Ca²⁺ uptake, Na⁺-induced Ca²⁺ release, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase were examined by employing cardiomyopathic hamsters (UM-X7.1) and catecholamine-induced cardiomyopathy produced by injecting isoproterenol into rats. The rates of Na⁺-dependent Ca²⁺ uptake, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities of sarcolemmal vesicles from genetically-linked cardiomyopathic as well as catecholamine-induced cardiomyopathic hearts were decreased without any changes in Na⁺-induced Ca²⁺-release. Similar results were obtained in Ca²⁺-paradox when isolated rat hearts were perfused for 5 min with a medium containing 1.25 mM Ca²⁺ following a 5 min perfusion with Ca²⁺-free medium. Although a 2 min reperfusion of the Ca²⁺-free perfused hearts depressed sarcolemmal Ca²⁺-pump activities without any changes in Na⁺-induced Ca²⁺-release, Na⁺-dependent Ca²⁺ uptake was increased. These results indicate that alterations in the sarcolemmal Ca²⁺-efflux mechanisms may play an important role in cardiomyopathies associated with the development of intracellular Ca²⁺ overload.     

Inositol 1,4,5-trisphosphate may mediate closure of K+ channels by light and darkness in Samanea saman motor cells

Leaflet movements of Samanea saman (Jacq.) Merr. depend in part upon circadian-rhythmic, light-regulated K⁺ fluxes across the plasma membranes of extensor and flexor cells in opposing regions of the leaf-moving organ, the pulvinus. We previously showed that blue light appears to close open K⁺ channels in flexor protoplasts during the dark period (subjective night) (Kim et al., 1992, Plant Physiol 99: 1532–1539). In contrast, transfer to darkness apparently closes open K⁺ channels in extensor protoplasts during the light period (subjective day) (Kim et al., 1993, Science 260: 960–962). We now report that both these channel-closing stimuli increase inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] levels in the appropriate protoplasts. If extensor cells are given a pulse of red light followed by transfer to darkness, channels still apparently close (Kim et al. 1993) but changes in Ins(1,4,5)P₃ levels are complex with an initial decrease under red light followed by accumulation. Neomycin, an inhibitor of polyphosphoinositide hydrolysis, inhibits both blue-light-induced Ins(1,4,5)P₃ production and K⁺-channel closure in flexor protoplasts and both dark-induced Ins(1,4,5)P₃ production and K⁺ channel closure in extensor protoplasts. The G-protein activator, mastoparan, mimics blue light and darkness in that it both increases Ins(1,4,5)P₃ levels and closes K⁺ channels in the appropriate cell type at the appropriate time. These results indicate that phospholipase C-catalyzed hydrolysis of phosphoinositides, possibly activated by a G protein, is an early step in the signal-transduction pathway by which blue light and darkness close K⁺ channels in S. saman pulvinar cells.Abbreviations DiS-C₃-(5) 3,3-dipropylthiadicarbocyanine iodide - F measure change in Dis-C₃-(5) fluorescence - F_o initial Dis-C₃-(5) fluorescence - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PtdIns(4,5)P₂ phosphatidylinositol 4,5-bisphosphate - rbc red blood cell Supported by grants from NSF (IBN 9206179 and MCB 9305154) and U.S.-Israel Binational Agricultural Research and Development Fund (IS-1670-90RC) to R.C.C. We thank the University of Connecticut Biotechnology Center for the use of a fluorescent spectrophotometer.     

Specific inhibitors of intracellular Ca2+ transport ATPases

Support from the National Institutes of Health and the American Heart Association is gratefully acknowledged.     

Reversible inhibition of cytoplasmic streaming by intracellular Ca2+ in tonoplast-free cells ofChara australis

Summary The effect of the intracellular concentration of Ca²⁺ on the cytoplasmic streaming of tonoplast-free cells ofChara australis was studied by intracellular perfusion. The perfusion media contained 1 mM Mg · ATP. Both cell ends were cut and left open. Media of different Ca²⁺ concentrations were perfused through the cell and the rate of the cytoplasmic streaming just after perfusion was measured. The critical concentration of Ca²⁺ for inhibiting the streaming was 5 × 10^–4M, which was substantially higher than that found earlier byWilliamson (1975) andHayama et al. (1979). Recovery from the inhibition occurred, though not completely, by removing Ca²⁺.In tonoplast-free cells the Ca²⁺ sensitivity differed according to the culture conditions. Cells cultured indoors exhibited a higher sensitivity than those cultured outdoors. Theformer cells contained granule-rich endoplasm aggregates after loss of the tonoplast, while the latter cells did no such aggregates. The aggregates were fixed to the cortical gel with a high dosage of Ca²⁺ and freed by removing it.     

Identification of new functions of Ca2+ release from intracellular stores in central nervous system

Ca²⁺ release from intracellular stores regulates muscle contraction and a vast array of cell functions, but its role in the central nervous system (CNS) has not been completely elucidated. A new method of blocking IP₃ signaling by artificially expressing IP₃ 5-phosphatase has been used to clarify the functions of intracellular Ca²⁺ mobilization in CNS. Here I review two of such functions: the activity-dependent synaptic maintenance mechanism and the regulation of neuronal growth by spontaneous Ca²⁺ oscillations in astrocytes. These findings add new bases for better understanding CNS functions and suggest the presence of as yet unidentified neuronal and glial functions that are regulated by Ca²⁺ store-dependent Ca²⁺ signaling.     

A patch-clamp study of the Ca2+ mobilization from internal stores in bovine aortic endothelial cells. I. Effects of caffeine on intracellular Ca2+ stores

Summary The effects of agents known to interfere with Ca²⁺ release processes of endoplasmic reticulum were investigated in bradykinin (BK)-stimulated bovine aortic endothelial cells (BAE cells), via the activation of Ca²⁺-activated potassium channels [K(Ca²⁺) channels]. In cell-attached patch experiments, the external application of caffeine (1 mm) caused a brief activation of K(Ca²⁺) channels in Ca²⁺-free and Ca²⁺-containing external solutions. The application of BK (10 nm) during cell stimulation by caffeine (1–20 mm) invariably led to a drastic channel activation which was maintained during a recording period longer than that observed in caffeine-free conditions. In addition, the cell exposure to caffeine (20 mm) during the BK stimulation enhanced systematically the channel activation process. Since a rapid inhibition of BK-evoked channel activity was also produced by removing caffeine from the bath medium, it is proposed that the sustained single-channel response recorded in the concomittant presence of both agents was due to their synergic action on internal stores and/or the external Ca²⁺ entry pathway resulting in an increased [Ca²⁺]_i. In addition, the local anesthetic, procaine, depressed the initial BK-induced K(Ca²⁺) channel activity and completely blocked the secondary phase of the channel activation process related to the external Ca²⁺ influx into stimulated cells. In contrast, this blocking effect of procaine was not observed on the initial caffeine-elicited channel activity and could not suppress the external Ca²⁺-dependent phase of this channel activation process. Our results confirm the existence of at least two pharmacologically distinct types of Ca²⁺-release from internal stores in BAE cells: an inositol 1,4,5-triphosphate (InsP₃)-dependent and a caffeine-induced Ca²⁺-release process.The authors would like to thank Dr. A. Diarra for his contribution to the fluorescence measurements and Diane Vallerand for preparing cell cultures. These data were presented in part at the 14th Scientific Meeting of the International Society of Hypertension (Madrid, Spain, June 14–18, 1992), and have been published in abstract form in the Journal of Hypertension (1992). Dominique Thuringer is a fellow of the Heart and Stroke Foundation of Canada. Rémy Sauvé is a senior fellow from the Fonds de la Recherche en Santé du Québec. This work was supported by a grant from the Medical Research Council of Canada.     

Turgor regulation and cytoplasmic free Ca2+ in the algaLamprothamnium

Summary In internodal cells ofLamprothamnium succinctum, turgor regulation in response to hypotonie treatment is inhibited by lowering external Ca²⁺ concentration ([Ca²⁺]_e) from 3.9 (normal) to 0.01 (low) mM. In order to clarify whether a change in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_c) is involved in turgor regulation, the Ca²⁺ sensitive protein aequorin was injected into the cytoplasm of internodal cells. A large transient light emission was observed upon hypotonic treatment under normal [Ca²⁺]_e but not under low [Ca²⁺]_e. Thus hypotonic treatment induces a transient increase in [Ca²⁺]_c under normal [Ca²⁺]_e but not under low [Ca²⁺]_e.Abbreviations ASW artificial sea water - _i cellular osmotic pressure - [Ca²⁺]_c cytoplasmic free Ca²⁺ concentration - EDTA ethylenediamine-tetraacetic acid - EGTA ethylenglycol-bis(-aminoethyl ether(N,N-tetraacetic acid - [Ca²⁺]_e external Ca²⁺ concentration - _e external osmotic pressure - GM glass micropipette - GP glass plate - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid - MS microscope stage - OL objective lens - PIPES piperazine-N-N-bis(2-ethanesulfonic acid) - W Weight