Characterization of a receptor for insect tachykinin-like peptide agonists by functional expression in a stable Drosophila Schneider 2 cell line

STKR is an insect G protein-coupled receptor, cloned from the stable fly Stomoxys calcitrans. It displays sequence similarity to vertebrate tachykinin [or neurokinin (NK)] receptors. Functional expression of the cloned STKR cDNA was obtained in cultured Drosophila melanogaster Schneider 2 (S2) cells. Insect tachykinin-like peptides or "insectatachykinins," such as Locusta tachykinin (Lom-TK) III, produced dose-dependent calcium responses in stably transfected S2-STKR cells. Vertebrate tachykinins (or neurokinins) did not evoke any effect at concentrations up to 10(-5) M, but an antagonist of mammalian neurokinin receptors, spantide II, inhibited the Lom-TK III-induced calcium response. Further analysis showed that the agonist-induced intracellular release of calcium ions was not affected by pretreatment of the cells with pertussis toxin. The calcium rise was blocked by the phospholipase C inhibitor U73122. In addition, Lom-TK III was shown to have a stimulatory effect on the accumulation of both inositol 1,4,5-trisphosphate and cyclic AMP. These are the same second messengers that are induced in mammalian neurokinin-dependent signaling processes.     

Pharmacological characterization of STKR, an insect G protein-coupled receptor for tachykinin-like peptides

STKR is a G protein-coupled receptor that was cloned from the stable fly, Stomoxys calcitrans. Multiple sequence comparisons show that the amino acid sequence of this insect receptor displays several features that are typical for tachykinin (or neurokinin, NK) receptors. Insect tachykinin-related peptides, also referred to as "insectatachykinins," produce dose-dependent calcium responses in Drosophila melanogaster Schneider 2 cells, which are stably transfected with this receptor (S2-STKR). These responses do not depend on the presence of extracellular Ca(2+)-ions. A rapid agonist-induced increase of inositol 1,4,5-trisphosphate (IP(3)) is observed. This indicates that the agonist-induced cytosolic Ca(2+)-rise is caused by a release of Ca(2+) ions from intracellular calcium stores. The pharmacology of STKR is analyzed by studying the effects of the most important antagonists for mammalian NK-receptors on STKR-expressing insect cells. The results show that spantide II, a potent substance P antagonist, is a real antagonist of insectatachykinins on STKR. We have also tested the activity of a variety of natural insectatachykinin analogs by microscopic image analysis of calcium responses in S2-STKR cells. At a concentration of 1 microM, almost all natural analogs produce a significant calcium rise in stable S2-STKR cells. Interestingly, Stc-TK, an insectatachykinin that was recently discovered in the stable fly (S. calcitrans), also proved to be an STKR-agonist. Stc-TK, a potential physiological ligand for STKR, contains an Ala-residue (or A) instead of a highly conserved Gly-residue (or G). Arch.     

Ca<Superscript>2+</Superscript>-activated nonselective cation channels in rat neonatal atrial myocytes

A nonselective cation channel activated by intracellular Ca²⁺ was identified in inside-out membrane patches taken from cultured rat atrial myocytes. Ca²⁺ (0.01–1.00 mM) reversibly activated the channel in a concentration-dependent manner. The channel often showed a quick and irreversible rundown within a few minutes after patch excision. The I-V relationship of the channel was linear between –100 and +100 mV. The single channel conductance was 26.0 ± 0.5 pS and its open probability was weakly voltage-dependent. Ion-substitution experiments showed that the channel was permeable to monovalent cations (P_x/P_Cs: Li⁺ (1.5) = K⁺ (1.5)> Na⁺ (1.2) > Rb⁺ (1.1) > Cs⁺ (1.0)) but not to Cl^– (P_Cl/P_Cs < 0.01) and Ca²⁺ (P_Ca/P_Cs =0.02 ± 0.01). Present address: A.B. Zhainazarov UF Center for Smell and Taste, McKnight Brain Institute, University of Florida, 100 S Newell Dr., L1-131, P.O. Box 100015, Gainesville, FL 32610-0015, USA     

Rapid Kinetic Studies of the Light Emitting Protein Aequorin

SHIMUMURA et al.^1,2 isolated from luminescent jelly fish (Aequora forskalea) a protein (aequorin) which on addition of Ca²⁺ emits light. In contrast to other bioluminescent compounds, aequorin luminescence requires neither oxygen nor FMNH₂, ATP nor long chain aldehydes, but only Ca²⁺. Recently, other bioluminescent proteins reacting only with Ca²⁺ have been isolated and termed Ca²⁺-activated photoproteins^3,4. Since the intensity of the emitted light varies with the Ca²⁺ concentration, aequorin and related proteins can serve as useful tools for detecting small changes in Ca²⁺ concentration in biological systems^5,6. Little is known, however, about the mechanism of this reaction. Since light emission from aequorin occurs within a few milliseconds of rapid mixing with Ca²⁺, kinetic studies are possible only with rapid mixing instruments, stopped and continuous flow. Hastings et al. studied the kinetics of aequorin using stopped flow and double stopped flow apparatuses⁷. Because both have a dead time of about 2 ms, which is in the same range as the rise time of the light emission from aequorin, they were unable to establish a Ca²⁺ dependence for the rate of rise of the light emission. Ca²⁺ dependence of this rate might be very important for delineation of the reaction mechanism.     

Inhibitors of Na+/Ca2+ exchanger prevent oxidant-induced intracellular Ca2+ increase and apoptosis in a human hepatoma cell line

Oxidative stress appears to be implicated in the pathogenesis of various diseases including hepatotoxicity. Although intracellular Ca²⁺ signals have been suggested to play a role in the oxidative damage of hepatocytes, the sources and effects of oxidant-induced intracellular Ca²⁺ increases are currently debatable. Thus, in this study we investigated the exact source and mechanism of oxidant-induced liver cell damage using HepG2 human hepatoma cells as a model liver cellular system. Treatment with 200 μM of tert-butyl hydroperoxide (tBOOH) induced a sustained increase in the level of intracellular reactive oxygen intermediates (ROI) and apoptosis, assessed by 2′,7′-dichlorofluorescein fluorescence and flow cytometry, respectively. Antioxidants, N-acetyl cysteine (NAC) or N,N′-diphenyl-p-phenylenediamine significantly inhibited both the ROI generation and apoptosis. In addition, tBOOH induced a slow and sustained increase in intracellular Ca²⁺ concentration, which was completely prevented by the antioxidants. An intracellular Ca²⁺ chelator, bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid/cetoxymethyl ester significantly suppressed the tBOOH-induced apoptosis. These results imply that activation of an intracellular Ca²⁺ signal triggered by increased ROI may mediate the tBOOH-induced apoptosis. Both intracellular Ca²⁺ increase and induction of apoptosis were significantly inhibited by an extracellular Ca²⁺ chelator or Na⁺/Ca²⁺ exchanger blockers (bepridil and benzamil), whereas neither Ca²⁺ channel antagonists (verapamil and nifedipine) nor a nonselective cation channel blocker (flufenamic acid) had an effect. These results suggest that tBOOH may increase intracellular Ca²⁺ through the activation of reverse mode of Na⁺/Ca²⁺ exchanger. However, tBOOH decreased intracellular Na⁺ concentration, which was completely prevented by NAC. These results indicate that ROI generated by tBOOH may increase intracellular Ca²⁺ concentration by direct activation of the reverse mode of Na⁺/Ca>²⁺ exchanger, rather than indirect elevation of intracellular Na⁺ levels. Taken together, these results suggest that the oxidant, tBOOH induced apoptosis in human HepG2 cells and that intracellular Ca²⁺ may mediate this action of tBOOH. These results further suggest that Na⁺/Ca²⁺ exchanger may be a target for the management of oxidative hepatotoxicity.     

Annexin 1 regulates the H2O2‐induced calcium signature in Arabidopsis thaliana roots

Hydrogen peroxide is the most stable of the reactive oxygen species (ROS) and is a regulator of development, immunity and adaptation to stress. It frequently acts by elevating cytosolic free Ca²⁺ ([Ca²⁺]_cyt) as a second messenger, with activation of plasma membrane Ca²⁺‐permeable influx channels as a fundamental part of this process. At the genetic level, to date only the Ca²⁺‐permeable Stelar K⁺ Outward Rectifier (SKOR) channel has been identified as being responsive to hydrogen peroxide. We show here that the ROS‐regulated Ca²⁺ transport protein Annexin 1 in Arabidopsis thaliana (AtANN1) is involved in regulating the root epidermal [Ca²⁺]_cyt response to stress levels of extracellular hydrogen peroxide. Peroxide‐stimulated [Ca²⁺]_cyt elevation (determined using aequorin luminometry) was aberrant in roots and root epidermal protoplasts of the Atann1 knockout mutant. Similarly, peroxide‐stimulated net Ca²⁺ influx and K⁺ efflux were aberrant in Atann1 root mature epidermis, determined using extracellular vibrating ion‐selective microelectrodes. Peroxide induction of GSTU1 (Glutathione‐S‐Transferase1 Tau 1), which is known to be [Ca²⁺]_cyt‐dependent was impaired in mutant roots, consistent with a lesion in signalling. Expression of AtANN1 in roots was suppressed by peroxide, consistent with the need to restrict further Ca²⁺ influx. Differential regulation of annexin expression was evident, with AtANN2 down‐regulation but up‐regulation of AtANN3 and AtANN4. Overall the results point to involvement of AtANN1 in shaping the root peroxide‐induced [Ca²⁺]_cyt signature and downstream signalling.     

Focal increases in [Ca]i may account for apparent low Ca sensitivity in swine carotid artery

Estimates of [Ca²⁺]_i sensitivity in intact smooth muscle are frequently obtained by measuring [Ca²⁺]_i with indicators such as aequorin or Fura-2. We investigated whether focal in increases in [Ca²⁺]_i could impair such measures of [Ca²⁺]_i sensitivity. Stimulation of swine carotid artery with 10 μM histamine increased aequorin estimated [Ca²⁺]_i, Fura-2 estimated [Ca²⁺]_i and Ca²⁺ sensitivity without significantly altering the aequorin/Fura-2 ratio (an estimate of [Ca²⁺]_i homogeneity). Subsequent inhibition of Na⁺/Ca²⁺ exchange by replacement of Na⁺ in the PSS with choline⁺ significantly increased aequorin-estimated [Ca²⁺]_i but only minimally increased Fura-2 estimated [Ca²⁺]_i, myosin light chain (MLC) phosphorylation and force. This resulted in a large increase in the aequorin/Fura-2 ratio, suggesting an increase in [Ca²⁺] inhomogeneity. Addition of 100 μM histamine to tissues in the choline⁺ buffer initially increased both aequorin and Fura-2 estimated [Ca²⁺]_i but after 10 min exposure both of the [Ca²⁺]_i estimates declined to pre-histamine levels. Histamine addition significantly increased MLC phosphorylation and force, indicating increased Ca²⁺ sensitivity, but the aequorin/Fura-2 ratio remained elevated and uncharged from pre-histamine values. These data show that under certain conditions, aequorin and Fura-2 can yield widely differing estimates of [Ca²⁺]_i, and thus can cause misleading assessments of Ca²⁺ sensitization mechanisms. These discrepancies may arise from inhomogeneous or focal increases in [Ca²⁺]_i which can be evaluated with the aequorin/Fura-2 ratio.     

THE ROLE OF CALCIUM IN FLOW‐STIMULATED BIOLUMINESCENCE OF THE RED TIDE DINOFLAGELLATE LINGULODINIUM POLYEDRUM

Many marine planktonic dinoflagellates emit flashes of light in response to either laminar or turbulent flows as well as direct mechanical stimulation. The production of a flash of light is known to be mediated by a proton‐mediated action potential across the vacuolar membrane; the mechanotransduction process initiating this action potential is unknown. Here we report on an investigation into the role of Ca⁺² in the mechanotransduction process regulating bioluminescence in the red tide dinoflagellate Lingulodinium polyedrum. Calcium ionophores and low concentrations of the membrane‐disrupting agent digitonin stimulated bioluminescence only when calcium was present in the media or added with the agent, indicating that the flash‐triggering vacuolar action potential is specifically stimulated by a calcium influx. A variety of known calcium channel blockers or antagonists inhibited mechanically stimulated bioluminescence but did not affect cellular bioluminescent capacity. In many cases the inhibitory affect occurred after only a brief exposure. In addition, gadolinium (Gd⁺³), a blocker of many stretch‐activated ion channels, caused potent inhibition of mechanically stimulated bioluminescence. The order of potency of the transition metals tested was La⁺³ > Gd⁺³ > Co⁺² > Mn⁺² > Ni⁺², similar to their potency as blockers of known calcium channels. Experiments with a quantified shear flow demonstrated that flow‐stimulated bioluminescence depended on the level of extracellular calcium. Future work will elucidate the signaling pathway involving calcium‐mediated flow‐stimulated mechanotransduction. Our goal is to use bioluminescence as a proxy for the initial cellular mechanotransduction events triggered by fluid flow.     

Intracellular targeting of the photoprotein aequorin: A new approach for measuring,in living cells,Ca<Superscript>2+</Superscript> concentrations in defined cellular compartments

We here present a novel method, based on the targeting of the photoprotein aequorin, for measuring the concentration of Ca²⁺ ions in defined cellular compartments of intact cells. In this contribution we will discuss the application to mitochondria. A chimaeric cDNA was constructed by fusing in frame the aequorin cDNA with that for a mitochondrial protein. The cDNA encoded a “mitochondrially-targeted” aequorin, composed of a typical mitochondrial targeting signal at the N-terminus and the photoprotein at the C-terminus. The cDNA, inserted in the expression vector pMT2, was co-transfected into bovine endothelial and HeLa cells together with the selectable plasmid pSV2-neo and stable transfectants, selected for high aequorin production, were analyzed. In subcellular fractionations, aequorin was shown to be localized in mitochondria; in intact cells, the first direct measurement of mitochondrial free Ca²⁺, [Ca²⁺]_m, were obtained, which showed that [Ca²⁺]_m is low at rest (<0.5 μM), but rapidly increases to the micromolar range upon cell stimulation [1]. These data indicate that mitochondria “sense” very accurately the cytosolic Ca²⁺ concentration ([Ca²⁺]_i), and after cell stimulation [Ca²⁺]_m rises to values capable of activating the Ca²⁺-sensitive mitochondrial dehydrogenases.     

Mitochondrial free [Ca2+] levels and the permeability transition

Mitochondrial Ca²⁺ activates many processes, from mitochondrial metabolism to opening of the permeability transition pore (PTP) and apoptosis. However, there is considerable controversy regarding the free mitochondrial [Ca²⁺] ([Ca²⁺]_M) levels that can be attained during cell activation or even in mitochondrial preparations. Studies using fluorescent dyes (rhod-2 or similar), have reported that phosphate precipitation precludes [Ca²⁺]_M from increasing above 2–3 μM. Instead, using low-Ca²⁺-affinity aequorin probes, we have measured [Ca²⁺]_M values more than two orders of magnitude higher. We confirm here these values by making a direct in situ calibration of mitochondrial aequorin, and we show that a prolonged increase in [Ca²⁺]_M to levels of 0.5–1 mM was actually observed at any phosphate concentration (0–10 mM) during continuous perfusion of 3.5–100 μM Ca²⁺-buffers. In spite of this high and maintained (>10 min) [Ca²⁺]_M, mitochondria retained functionality and the [Ca²⁺]_M drop induced by a protonophore was fully reversible. In addition, this high [Ca²⁺]_M did not induce PTP opening unless additional activators (phenyl arsine oxide, PAO) were present. PAO induced a rapid, concentration-dependent and irreversible drop in [Ca²⁺]_M. In conclusion [Ca²⁺]_M levels of 0.5–1 mM can be reached and maintained for prolonged periods (>10 min) in phosphate-containing medium, and massive opening of PTP requires additional pore activators.     

Cuscuta reflexa invasion induces Ca2+ release in its host

Cuscuta reflexa induces a variety of reaction in its hosts. Some of these are visual reactions, and it is clear that these morphological changes are preceded by events at the molecular level, where signal transduction is one of the early processes. Calcium (Ca²⁺) release is the major second messenger during signal transduction, and we therefore studied Ca²⁺ spiking in tomato during infection with C. reflexa. Bioluminescence in aequorin‐expressing tomato was monitored for 48 h after the onset of Cuscuta infestation. Signals at the attachment sites were observed from 30 to 48 h. Treatment of aequorin‐expressing tomato leaf disks with Cuscuta plant extracts suggested that the substance that induced Ca²⁺ release from the host was closely linked to parasite haustoria.     

Comparative aspects of the calcium-sensitive photoproteins aequorin and obelin

1. The calcium-dependency of the process of light emission has been investigated for the photoproteins aequorin and obelin.2. The experimental curves of light production, expressed as a percentage of the maximal rate of utilisation, versus pCa are accurately predicted by the cooperative action of at least 2Ca²⁺ for aequorin and at least 3Ca²⁺ for obelin.3. At low total monovalent cation concentrations, a pH change from 6.8 to 7.1 shifts the light production vs pCa curve by approx. 0.2 pCa units to the right for aequorin, while that for obelin is shifted by some 0.37 pCa units.4. Other monovalent cations, such as Na⁺ are able to compete with Ca²⁺ for the active sites of aequorin and also shift the light production vs pCa curve to the right. There is no apparent change in the calcium stoichiometry for light production under these conditions.5. The same calcium stoichiometry for light emission was also obtained for aequorin or obelin in the presence of either unbuffered Ca²⁺ solutions or of calcium/EGTA buffers.     

Differential Involvement of Intracellular Ca2+ in 1-Methyl-4-phenylpyridinium- or 6-Hydroxydopamine-Induced Cell Viability Loss in PC12 Cells

1-Methyl-4-phenylpyridinium (MPP⁺) or 6-hydroxydopamine (6-OHDA) caused a nuclear damage, the mitochondrial membrane permeability changes, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in PC12 cells. Nicardipine (a calcium channel blocker), EGTA (an extracellular calcium chelator), BAPTA-AM (a cell permeable calcium chelator) and calmodulin antagonists (W-7 and calmidazolium) attenuated the MPP⁺-induced mitochondrial damage and cell death. In contrast, the compounds did not reduce the toxicity of 6-OHDA. Treatment with MPP⁺ or 6-OHDA evoked the elevation of intracellular Ca²⁺ levels. Unlike cell injury, addition of nicardipine, BAPTA-AM and calmodulin antagonists prevented the elevation of intracellular Ca²⁺ levels due to both toxins. The results show that the MPP⁺-induced formation of the mitochondrial permeability transition seems to be mediated by elevation of intracellular Ca²⁺ levels and calmodulin action. In contrast, the 6-OHDA-induced cell death seems to be mediated by Ca²⁺-independent manner.     

Ion Permeation and Block of P2X2 Purinoceptors: Single Channel Recordings

P2X₂ purinoceptors are cation-selective channels activated by ATP and its analogues. Using single channel measurements we studied the channel's selectivity for the alkali metal ions and organic monovalent cations NMDG⁺, Tris⁺, TMA⁺, and TEA⁺. The selectivity sequence for currents carried by alkali metal ions is: K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺, which is Eisenman sequence IV. This is different from the mobility sequence of the ions in free solution suggesting there is weak interaction between the ions and the channel interior. The relative conductance for alkali ions increases linearly in relation to the Stokes radius. The organic ions NMDG⁺, Tris⁺, TMA⁺ and TEA⁺ were virtually impermeant. The divalent ions (Mn²⁺, Mg²⁺, Ca²⁺ and Ba²⁺) induced a fast block visible as a reduction in amplitude of the unitary currents. Using a single-site binding model, the divalent ions exhibited an equilibrium affinity sequence of Mn²⁺ > Mg²⁺ > Ca²⁺ > Ba²⁺. Received: 3 May 1999/Revised: 23 August 1999     

A new short-term toxicity assay using <Emphasis Type="Italic">Aspergillus awamori</Emphasis> with recombinant aequorin gene

Background  

Most currently available short-term toxicity assays are based on bacterial cells. Therefore there is a need for novel eukaryotic microbial bioassays that will be relevant to higher eukaryotes such as animals and plants. Ca²⁺ is a universal intracellular signalling molecule found in all organisms from prokaryotes to highly specialized animal cells. In fungi calcium has been demonstrated to be involved in control of many important processes. The recombinant aequorin gene from the jellyfish Aequorea victoria responsible for the expression of the Ca²⁺-sensitive aequorin photoprotein has been cloned in the filamentous fungus Aspergillus awamori. This has allowed real life monitoring of [Ca²⁺]_c changes in living fungal cells. When subjected to different physico-chemical stimuli fungal cells respond by transiently changing the concentration of free Ca²⁺ in the cytosol ([Ca²⁺]_c) and the pattern of these changes (Ca²⁺ signature) is specific to each particular stimulus. Therefore it was interesting to investigate whether different environmental toxicants would be able to affect the pattern of [Ca²⁺]_c changes in a reproducible and dose dependant manner.     

A Comparison between Quin-2 and Aequorin as Indicators of Cytoplasmic Calcium Levels in Higher Plant Cell Protoplasts

Assessment of the regulation of plant metabolism by the calcium ion requires a knowledge of its intracellular levels and dynamics. Technical problems have prevented direct measurement of the concentration of intracellular Ca²⁺ in plant cells in all but a few cases. In this study we show that electropermeabilized protoplasts of Daucus carota and Hordeum vulgare took up the Ca²⁺ indicating fluorescent dye methoxyquinoline(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (Quin-2) and the Ca²⁺ indicating photoprotein, aequorin. These protoplasts subsequently recovered their plasma membrane integrity. However, up to 10% of intracellularly trapped Quin-2 was associated with a protoplast vacuolar fraction. Also, Quin-2 loading reduced total ATP levels by approximately 60% and inhibited subsequent protoplast division whereas aequorin loading reduced ATP content by only 20% and did not prevent division. Therefore, the basal cytoplasmic Ca²⁺ level measured with aequorin (less than 200 nanomolar) may more reliably reflect that found in vivo in the unperturbed protoplast than that measured with Quin-2 (120-360 nanomolar). However, measurements made with aequorin were found to be inaccurate at Ca²⁺ levels below 200 nanomolar, Quin-2 proving complementary in indicating these low Ca²⁺ concentrations. Cytosolic Ca²⁺ was observed to increase on treatment with azide and silver ions.     

Refined crystal structures of human Ca(2+)/Zn(2+)-binding S100A3 protein characterized by two disulfide bridges

S100A3, a member of the EF-hand-type Ca²⁺-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn²⁺ affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N,N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca²⁺/Zn²⁺ supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A + C68A) abolished the Ca²⁺ binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type III (PAD3), while SS2 disruption inversely increased both Ca²⁺ affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A + C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15−1.40 Å resolution shows that SS1 renders the C-terminal classical Ca²⁺-binding loop flexible, which are essential for its Ca²⁺ binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn²⁺ by (Cys)₃His residues that is compatible with SS2 formation in S100A3.     

Radial spread of aequorin Ca2+ signal in single frog skeletal muscle fibers

The Ca²⁺-sensitive photoprotein aequorin was injected into single frog skeletal muscle fibers, and the intracellular aequorin light intensity during muscle activation with different maneuvers was mapped with digital imaging microscopy. During 50 Hz electrical activation (tetanus), the aequorin light intensity from different locations in the muscle fiber rose with very similar time course. Caffeine (10 mM) application, on the other hand, caused aequorin light signals to show significantly different time courses, with an earlier increase in Ca²⁺ concentration near the surface of the fiber than near the core. The non-uniform rise of intracellular Ca²⁺ concentration with caffeine treatment is consistent with the slow inward diffusion of caffeine and subsequent Ca²⁺ release from sarcoplasmic reticulum.     

Na+ and Ca2+ channel expression in cultured newt retinal pigment epithelial cells: Comparison with neuronal types of ion channels

We cultured retinal pigment epithelial (RPE) cells dissociated from adult newt eye and analyzed their voltage-gated ion channels during culture using whole-cell patch-clamp techniques. The results were compared with those of retinal neurons under identical experimental conditions. After 6–9 days in culture (early stage), > 60% of RPE cells developed voltage-gated Na⁺ and Ca²⁺ channels that were not observed in freshly dissociated RPE cells. The number of cells expressing Na⁺ channels and Na⁺ current density were high after 12–15 days in culture (intermediate stage), while the number of Ca²⁺ channel-expressing cells and Ca²⁺ current density were high after 20–30 days in culture (late stage). The activation voltage of the Na⁺ current in the RPE cells was similar to that in neurons. The voltage dependence of Na⁺ current inactivation was somewhat different between two cell types. The steepness of the inactivation curve tended to be less in cultured RPE cells than in neurons, and the half-inactivation voltage was about −54 mV for the RPE cells and −45 mV for neurons. The Ca²⁺ current expressed in cultured RPE cells was too small to detect without replacement of external Ca²⁺ with Ba²⁺. The Ba²⁺ current, like Ca²⁺ current in neurons, was enhanced by Bay-K 8644 and blocked by nicardipine. These results suggest that the RPE cells, like neurons, expressed L-type Ca²⁺ channels in culture. The possibility that the development of both Na²⁺ and Ca²⁺ channels in cultured RPE cells is a manifestation of the transdifferentiation of RPE cells into neurons is discussed. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 377–390, 1997.