Cell Tissue Res (2002) 307:101–114 DOI 10.1007/s004410100466
REGULAR ARTICLE
S. Schöniger · M.D.A. Kopp · C. Schomerus E. Maronde · F. Dehghani · A. Meiniel E.M. Rodríguez · H.-W. Korf · F. Nürnberger
Effects of neuroactive substances on the activity of subcommissural organ cells in dispersed cell and explant cultures Received: 11 July 2001 / Accepted: 21 August 2001 / Published online: 31 October 2001 © Springer-Verlag 2001
Abstract The subcommissural organ (SCO), an ependymal (glial) circumventricular organ, releases glycoproteins into the cerebrospinal fluid; however, the regulation of its secretory activity is largely unknown. To identify neuroactive substances that may regulate SCO activity, we investigated immunocytochemically identified bovine SCO cells by means of calcium imaging. This analysis was focused on: (1) serotonin (5HT) and substance P (SP), immunocytochemically shown to be present in axons innervating the bovine SCO; and (2) ATP, known to activate glial cells. 5HT had no effect on the intracellular calcium concentration ([Ca2+]i), and its precise role remains to be clarified. SP elicited rises in [Ca2+]i in approx. 30% and ATP in even 85% of the analyzed SCO cells. These effects were dose-dependent, involved NK3 and P2Y2 receptors linked to G protein and phospholipase C (PLC) activation, and could not be mimicked by forskolin or 8-bromo-cAMP. In 50% of the SP-sensitive cells, the increases in [Ca2+]i comprised calcium release from thapsigargin-sensitive intracellular stores and an influx of extracellular calcium via protein kinase C (PKC)induced opening of L-type voltage-gated calcium channels (VGCCs). In the remaining SP-sensitive cells, the increase in [Ca2+]i was caused exclusively by influx of extracellular calcium via VGCCs of the L-type. In all Supported by the Volkswagen-Stiftung (grant I/74062) S. Schöniger · M.D.A. Kopp · C. Schomerus · E. Maronde F. Dehghani · H.-W. Korf · F. Nürnberger (✉) Institut für Anatomie II, Fachbereich Medizin, J.W. Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany e-mail:
[email protected] Tel.: +49-69-63016906, Fax: +49-69-63016017 A. Meiniel Laboratoire de Biochimie Médicale, Unité 384, Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine, 28 Place Henri Dunant, 63001 Clermont-Ferrand Cédex, France E.M. Rodríguez Instituto de Histología y Patología, Universidad Austral de Chile, Valdivia, Chile
ATP-sensitive cells the increase in [Ca2+]i involved calcium release from thapsigargin-sensitive intracellular stores and a PKC-mediated influx of extracellular calcium via L-type VGCCs. Our data suggest that SP and ATP are involved in regulation of the activity of SCO cells. Keywords ATP · Calcium · Reissner’s fiber · Serotonin · Signal transduction · Subcommissural organ · Substance P · Bovine
Introduction The subcommissural organ (SCO), a gland of ependymal (glial) origin, is located at the rostral border of the mesencephalic aqueduct beneath the posterior commissure and comprises ependymal and hypendymal cells (Oksche 1961; Oksche et al. 1993). The hypendymal cells release secretory products into the perivascular and subarachnoid spaces, while the ependymal cells secrete glycoproteins into the cerebral aqueduct, where they aggregate to form Reissner’s fiber (RF; Rodríguez et al. 1987; Nualart et al. 1991; Hein et al. 1993; Schoebitz et al. 1993; Gobron et al. 1996). These glycoproteins are most thoroughly studied in the bovine (Rodríguez et al. 1987; Meiniel et al. 1988; Nualart et al. 1991; Hein et al. 1993). The amino acid sequences of some of these proteins have been analyzed and one of them, SCO-spondin, has been shown to share homologies with proteins such as thrombospondin-1 and -2 that regulate cellular adhesion and neuritic outgrowth (Neugebauer et al. 1991; Adams et al. 1995; Gobron et al. 1996; Meiniel 2001). Nevertheless, the precise physiological function of the SCO-RF complex is unknown. In vitro experiments indicate a role during brain development, because RF and synthetic peptides derived from conserved domains of SCO-spondin modulate neuronal survival and aggregation as well as neuritic outgrowth (Monnerie et al. 1995, 1996, 1997, 1998; Gobron et al. 1996). RF also seems to regulate cerebrospinal fluid (CSF) circulation
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(Fernández-Llebrez et al. 1993; Cifuentes et al. 1994; Vio et al. 2000) and may trap neuroactive substances and metabolites from the CSF (Hess and Sterba 1973; Rodríguez et al. 1999). So far, the regulation of SCO secretory activity is largely unknown and effects of neurotransmitters present in SCO afferents have not been investigated at the cellular level. Thus, we performed experiments (1) to identify neuroactive substances that may be involved in the regulation of SCO activity, and (2) to characterize their intracellular signal transduction cascades in SCO cells. As an experimental model, we selected the bovine SCO, because it can be isolated readily and the glycoproteins secreted from the SCO have been extensively studied in this species. By using immunohistochemistry, we demonstrated that the bovine SCO is innervated by serotonin (5HT)and substance P (SP)-containing nerve fibers, as previously shown in the rat (Ljungdahl et al. 1978; Møllgard and Wiklund 1979; Léger et al. 1983; Bouchaud 1993; Knigge and Schock 1993; Mikkelsen et al. 1997). In subsequent experiments on dispersed cell and explant cultures, we investigated potential effects of 5HT and SP on the second-messenger calcium within the SCO by use of the fura-2 technique. In systems other than the SCO, SP has been shown to induce calcium-mediated secretion (Schamgochian and Leeman 1992; Mau et al. 1997; Moura et al. 1999). This may also hold true for the SCO and may involve the activation of calcium-dependent enzymes (e.g., PKC, Ca2+/calmodulin-dependent protein kinase), which stimulate the exocytotic machinery downstream of the [Ca2+]i elevation (for review, see Burgoyne and Morgan 1998; Hilfiker et al. 1999). We also analyzed calcium responses to ATP, which acts as a neurotransmitter in the central and peripheral nervous system (for review, see Zimmermann 1994). So far, the physiological role of ATP in the SCO has not been elucidated. Studies with cultured astrocytes have revealed that ATP stimulates the secretory activity (i.e., release of eicosanoids) via activation of the calcium signal transduction cascade (Pearce et al. 1989; Langley and Pearce 1998). Moreover, ATP participates in the local control of hormone release in the neural lobe of the pituitary gland (Sperlagh et al. 1999). It thus appears likely that ATP is also involved in the regulation of the secretory activity in the bovine SCO. Our results demonstrate that SP and ATP, but not 5HT, increase [Ca2+]i in bovine SCO cells. We further identified the receptor subtypes responsible for SP- and ATP-activated calcium signaling. Our data give specific insight into intracellular processes that may serve the regulation of SCO activity, and they provide general information on neuron-glia interactions, since SCO cells are thought to belong to the ependymal/astroglial cell lineage (Bouchard et al. 1999).
Materials and methods Immunocytochemical characterization of cells and nerve fibers in the bovine SCO Immunohistochemistry Bovine SCOs were obtained from male and female animals at an abattoir in the area of Frankfurt/Main, Germany, immersion-fixed in Zamboni’s solution (3.5% paraformaldehyde, 15% picric acid in phosphate-buffered saline, PBS) within 10 min after death, and sliced with a vibratome (50–100 µm). Immunhistochemistry was carried out as described (von Gall et al. 1998). Briefly, free-floating sections were washed in PBS, preincubated with 10% normal goat serum in PBS, and then incubated with the primary antibodies (polyclonal antibody against SP, 1:250; Chemicon, Hofheim, Germany; polyclonal antibody against 5HT, 1:2000; Incstar, Stillwater, Minn., USA; C1B8A8 monoclonal antibody, 1:50, specifically recognizing glycoproteins secreted by SCO cells; Meiniel et al. 1988, 1991) for 2 days at room temperature (RT). After repeated washing in PBS, the sections were incubated with the secondary antibodies (Cy3-conjugated goat anti-rabbit IgG; Dianova, Hamburg, Germany; and Alexa 488-conjugated goat anti-mouse IgG; Molecular Probes, Leiden, The Netherlands) for 1 day at RT. Thereafter, the sections were mounted on glass slides and coverslipped with fluorescence mounting medium (Dako, Hamburg, Germany). Confocal laser scanning microscopy A Zeiss LSM 510 confocal imaging system equipped with monochromatic argon and helium-neon laser light sources and an inverse Axiovert 100 microscope (Zeiss, Göttingen, Germany) was used for the optical analysis of the sections. The 488-nm line of the argon laser and the 543-nm line of the helium-neon laser were applied to excite the Alexa fluorophore and the Cy3 fluorophore, using a dichroic beam splitter FT (488/543) and emission bandpass filters BP 505–530 (Alexa 488) and BP 580–615 (Cy3), respectively. Immunofluorescence images of both channels were stored for further analysis as digitized images with an 8-bit resolution (1,024×1,024 pixels). Primary SCO cell culture and calcium imaging in immunocytochemically identified SCO cells SCO cell culture To analyze calcium signal transduction cascades, isolated SCO cells were prepared either from whole SCOs freshly dissected from the adjacent posterior commissure and cut into small pieces (~1 mm in diameter) or from SCO explant cultures that had initially been cultured for 1–2 months according to the method of Lehmann et al. (1993) and Schöniger et al. (2001). The tissue was dissociated by papain digestion as described (Schaad et al. 1993; Schomerus et al. 1995; Kopp et al. 1999). The dissociated cells were plated onto poly-L-lysine-coated coverslips with an internal grid and cultured in DMEM/F12 supplemented with 10 mM HEPES, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml ascorbic acid, 10% fetal calf serum, and 50 nM cytosine β-D-arabino-furanoside in an incubator at 37°C, in an atmosphere of 7% CO2 and 93% air for 3–4 days. Dye loading For the analysis of [Ca2+]i, cells were loaded with the fluorescent calcium indicator fura-2 pentacetoxymethylester (fura-2/AM, 3 µM; Molecular Probes, Eugene, Ore., USA) in supplemented DMEM/F12 for 20 min in the incubator and then rinsed with artificial cerebrospinal fluid (aCSF) containing: NaCl 140 mM; KCl
103 5 mM; CaCl2 2 mM; MgCl2 2 mM; HEPES 10 mM; glucose 5 mM. Thereafter, coverslips were transferred to a perfusion chamber on the heatable stage of an inverted microscope (Axiovert 100; Zeiss, Jena, Germany) and superfused with aCSF at a flow rate of 0.7 ml/min at a constant temperature of 37°C. Fluorescence microscopy The microscope was equipped with the Attofluor illumination and photomultiplier-camera system (Atto Instruments, Rockville, Md., USA), and a personal computer with appropriate software (Attofluor Ratio Vision; Atto Instruments) was used to control the optic equipment and to record, analyze, and store the images and data (for details, see Schomerus et al. 1995; Kroeber et al. 1997; Kopp et al. 1999).
an incubator at 37°C in an atmosphere of 7% CO2 and 93% air using supplemented DMEM/F12 as the culturing medium. They were immobilized on poly-L-lysine-coated coverslips 1 week before the experiments. For the analysis of [Ca2+]i, the explants were loaded with fura-2/AM (6 µM) for 3 h in the incubator. The calcium response of the explants was determined only in the periphery, comprising a single layer of SCO cells. Calcium measurements and data analysis were performed as described already. Materials Drugs and chemicals were obtained from the following sources: ●
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Drug application All drugs tested were stored frozen as stock solutions and diluted with prewarmed aCSF immediately before use. Drugs were applied by stopping the superfusion and adding them with a Pasteur pipette directly into the perfusion chamber. The drugs were washed out by starting the superfusion again, and complete washout of the chamber was achieved within 30 s.
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Data analysis
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In some experiments ratio data of the emission intensities at 334 nm and 380 nm were converted to estimate calcium concentrations as described previously (Grynkiewicz et al. 1985; Schomerus et al. 1995). However, this calibration procedure is an approximation, and the methods for calibrating emission ratios make many assumptions that cannot be readily tested experimentally (Leong 1989; D’Souza and Dryer 1994). Thus, most of the data of this study are presented semiquantitatively as 334 nm to 380 nm emission ratios. A positive response to a stimulation was defined by an increase in these values of more than 0.15. The data in the diagrams are presented as means ± standard error of the mean (SEM). Statistical analysis was performed using unpaired Student’s t-test with Welsh correction and confidence levels of 95%.
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Immunocytochemical characterization of the analyzed cells Following the fluorometric measurements, cells were fixed for 15 min in ice-cold methanol and washed in TRIS-buffered saline (TBS, pH 7.7) containing: Na2HPO4 8.4 mM; KH2PO4 3.5 mM; NaCl 119.8 mM; TRIS-base 41.3 mM; NaN3 1.5 mM. Cells were incubated overnight at room temperature with the primary antibody AFRU (1:1,000) that recognizes the secretory glycoproteins stored in SCO cells (Rodríguez et al. 1984). Binding of the antibody was visualized using: (1) swine anti-rabbit immunoglobulins (1:80; Dako, Hamburg, Germany) as secondary antibodies; (2) peroxidase-anti-peroxidase complexes developed in rabbit (1:50; Dako, Hamburg, Germany) as the third antibody solution; and (3) diaminobenzidine as the coloring reagent. All antibodies were diluted in TBS containing 0.7% carageenan lambda and 0.3% Triton X-100. Coverslips were finally mounted onto glass slides using Kaiser’s Glyceringelatine. Immunocytochemical and functional data were correlated at the level of single cells; the location of the cells was determined by means of the grid etched onto the coverslip (Schomerus et al. 1995; Kroeber et al. 1997; Kopp et al. 1999). Analysis of [Ca2+]i in SCO explant culture SCO explant cultures were prepared according to Lehmann et al. (1993) and Schöniger et al. (2001). The explants were cultured in
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NaCl, KCl, CaCl2, MgCl2, Triton X-100, KH2PO4, glucose, EGTA, and Kaiser’s Glyceringelatine from Merck, Darmstadt, Germany EBSS, DMEM/F12, fetal calf serum, penicillin, streptomycin, glutamine, and HEPES from Gibco Life Technologies, Eggenstein, Germany Papain, adenosine-5′-triphosphate (ATP), adenosine-5′-diphosphate (ADP), adenosine-5′-monophosphate (AMP), adenosine5′-O-(3-thiotriphosphate) tetralithium salt (ATPγS), and uridine-5′-triphosphate (UTP) from Boehringer Mannheim, Germany Substance P, neurokinin A, neurokinin B, forskolin, thapsigargin, GDPbS, U-73122, U-73343, calphostin C, and chelerythrine from Calbiochem-Novabiochem, Bad Soden, Germany 8-Bromo-cAMP (8-Br-cAMP) from BioLog, Bremen, Germany. All other drugs and chemicals from Sigma, Deisenhofen, Germany. Coverslips with the coordinate grid etched onto the glass from Eppendorf, Hamburg, Germany.
Results Immunocytochemical characterization of cells and innervation of the bovine SCO Virtually all SCO cells were immunostained with the monoclonal C1B8A8 antibody that specifically detects SCO-secreted glycoproteins. According to their location, these cells were characterized as ependymal and hypendymal elements which were surrounded by a dense plexus of 5HT-immunoreactive (IR) nerve fibers. Many of these fibers penetrated into the ependymal and hypendymal cell layers of the SCO and some of them terminated close to the lumen of the third ventricle (Fig. 1a). Furthermore, a small number of SP-IR nerve fibers was found in the hypendymal cell layer, but SPIR nerve fibers could not be observed in the ependymal portion of the SCO (Fig. 1b). Cell bodies immunoreactive for 5HT or SP were not observed in the bovine SCO. The analysis of [Ca2+]i was performed with cells that were later immunocytochemically identified as SCO cells by means of the AFRU immunoreaction (Fig. 2). This immunoreaction was found to be positive in approximately 95% of the dispersed cells; the intensity of the immunoreaction, however, varied from cell to cell. The responsiveness of the cells was very similar irrespective of whether they displayed a strong, moderate, or weak AFRU-IR. Most of the AFRU-immunonegative
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Fig. 2a–c Identification of SCO cells previously analyzed with the fura-2 technique by immunocytochemistry. a Transmission image of living cells located on a coverslip with an internal alphanumeric grid that facilitates the correlation of functional and immunocytochemical data of the analyzed cells. b Fluorescence image of the same cells loaded with fura-2 and illuminated with light of 334 nM wavelength. Ratio values are coded by pseudocolors. c Transmission image of the same cells fixed with ice-cold methanol and immunolabeled with the AFRU-antibody. Note fixationdependent shrinkage of the cells. The analysis of [Ca2+]i in this study was selectively performed with immunocytochemically identified SCO cells. Bar 20 µm Fig. 1a–c Immunocytochemical demonstration of cells and serotonin (5HT)- or substance P (SP)-immunoreactive nerve fibers in thick sections of the bovine subcommissural organ (SCO). a Dense plexus of 5HT-immunoreactive nerve fibers (red fluorescence) surrounding the SCO cells labeled with the C1B8A8 antibody that recognizes glycoproteins specifically secreted by SCO cells (green fluorescence). 5HT-immunoreactive nerve fibers within the SCO appear yellow by interference of colors. b Some SPimmunoreactive nerve fibers (red fluorescence) at the base of the SCO (green fluorescence). c High magnification of the framed area in b. (III third ventricle). Bars 20 µm
cells did not take up the fluorescent calcium indicator fura-2, and the fura-2 labeled AFRU-immunonegative cells never responded to any of the neurotransmitters/neuropeptides used in this study.
Calcium responses of immunocytochemically identified bovine SCO cells 5HT (1 nM to 100 µM; n=278) had no direct effect on [Ca2+]i in any of the dissociated SCO cells investigated (Fig. 3). In contrast, SP and ATP elicited dose-dependent calcium increases in SCO cells. Thirty percent of them responded to SP (100 nM to 1 µM; n=1,576) and even 85% to ATP (10–100 µM; n=2,020) with an increase in [Ca2+]i (Figs. 3, 8a, c, 9a, b). These effects of 5HT, SP, and ATP on [Ca2+]i were identical in cell cultures obtained by dissociation of (1) freshly dissected SCOs and (2) SCO explant cultures that had initially been cultured
105 Fig. 3a, b Response patterns of [Ca2+]i in bovine SCO cells after stimulation with 5HT (1 nM to 100 µM; n=278 cells in 48 experiments) previous to stimulation with SP (a) or ATP (b). 5HT had no effect on [Ca2+]i. SP (100 nM) and ATP (10 µM) caused elevation of [Ca2+]i. The duration of 5HT, SP, or ATP application is indicated by bars
Fig. 4a, b [Ca2+]i-response patterns in cultured explants of the bovine SCO to stimulation (a) with 5HT before stimulation with SP (four experiments) and (b) with ATP (five experiments). 5HT (10 µM) showed no effect on [Ca2+]i; SP (100 nM) and ATP (10 µM) induced increases in [Ca2+]i that were identical with those elicited in cultured dissociated SCO cells. The duration of 5HT, SP, or ATP application is indicated by bars Fig. 5a–d Approximation of [Ca2+]i by means of the Grynkiewicz equation. a, b Application of SP (100 nM; n=25) increased the base [Ca2+]i from ~100 nM to peak concentrations ranging from 700 nM to 1.9 µM. c, d ATP (10 µM; n=25) increased the [Ca2+]i from ~100 nM to peak concentrations ranging from 700 nM to 2.3 µM. The duration of SP or ATP application is indicated by bars. Note the high variability of SP- and ATP-induced increases in [Ca2+]i among individual SCO cells
for 1–2 months. Identical results were obtained by measurements of SCO explant cultures: No increase in [Ca2+]i could be elicited by 5HT (10 µM; 4 explants), whereas SP (100 nM; 4 explants) and ATP (10 µM; 5 explants) induced calcium rises that were comparable with those observed in the SCO cell culture (Fig. 4). Conversion of the ratio data to approximate intracellular calcium concentrations performed by in vitro calibrations subsequently to some experiments revealed that [Ca2+]i
showed a high variability between individual SCO cells. After application of SP (100 nM; n=25) [Ca2+]i increased from a basal concentration of ~100 nM to peak concentrations ranging from 700 nM to 1.9 µM (Fig. 5a, b). ATP (10 µM; n=25) elicited increases in [Ca2+]i from basal concentrations of ~100 nM to peak concentrations ranging from 700 nM to 2.3 µM (Fig. 5c, d). In the following subset of experiments, the response patterns evoked by SP (100 nM to 1 µM; n=265) and
106 Fig. 6a–d SP-induced calcium response patterns in bovine SCO cells. a Twenty-five percent of the SP-sensitive cells (n=66) showed short-lasting increases in [Ca2+]i (10–20 s). b–d Seventy-five percent of the SP-sensitive cells (n=199) showed longer-lasting calcium rises (~ 100 s). d Longer-lasting stimulation clearly indicated that the [Ca2+]i returned to base levels after ~ 100 s (see also a, b). The duration of SP application is indicated by bars
ATP (10 µM to 100 µM; n=496) were characterized. In 25% of the SP-sensitive cells (n=66), the neuropeptide induced a rapid increase in [Ca2+]i to a peak level followed by an immediate decrease to the basal concentration (Fig. 6a). In 75% of the responding cells (n=199), the application of SP caused an initial rapid increase in [Ca2+]i which declined to the basal concentration more slowly (Fig. 6b–d). About 15% of these cells showed a plateau phase at the highest level of [Ca2+]i with or without calcium oscillations (Fig. 6b). [Ca2+]i may return to the baseline in the presence of the stimulus (Fig. 6d). Interestingly, in one and the same measurement, neighboring SCO cells showed different response patterns. ATP (10–100 µM) induced in 55% of the responding cells (n=279) an initial rapid increase in [Ca2+]i, which slowly declined to the baseline (Fig. 7a). About 15% of the ATP-sensitive cells (n=74) showed a plateau phase at the highest level of [Ca2+]i with or without calcium oscillations (Fig. 7b, c). In the remaining 30% of the responsive cells (n=143), the initial maximum was followed by a decrease in [Ca2+]i, consisting of a first rapid phase and a second slower phase. In most of these cells, the second phase showed a high calcium level (Fig. 7d, f), but in some of them the calcium concentration was elevated just above the baseline (Fig. 7e). [Ca2+]i may return to the baseline in the presence of the stimulus (Fig. 7f). Like SP, ATP elicited different response patterns in neighboring SCO cells. Characterization of receptor subtypes In order to characterize the subtypes of tachykinin receptors that mediate the SP-induced increases in [Ca2+]i in
the bovine SCO, cells were treated with neurokinin B (NKB), SP, and neurokinin A (NKA). The effects of these treatments were dose-dependent (Fig. 8a). In concentrations of 1 nM (n=122) and 10 nM (n=156), NKB caused a rise in [Ca2+]i in ~10% of the cells, whereas 30% or 45% of the SCO cells reacted to 100 nM (n=150) or 1 µM (n=160) NKB, respectively. One nanomolar (n=78) SP had no effect on [Ca2+]i, but the number of responding cells increased with enhanced SP concentrations: 15% to 10 nM (n=221); 20% to 100 nM (n=591); 30% to 1 µM (n=545). NKA was ineffective in a concentration of 1 nM (n=80), whereas 5% of the cells responded to 10 nM (n=154), 15% to 100 nM (n=138), and 30% to 1 µM NKA (n=169). Representative dose-dependent calcium responses of single SCO cells to applications of NKB, SP, or NKA are shown in Fig. 8b–d. These data suggest that in bovine SCO cells the SP-induced increases in [Ca2+]i are primarily mediated by NK3 receptor activation. To characterize the purinoceptors involved in ATP-induced calcium signaling, the cells were treated with: (1) adenosine, which specifically activates P1 receptors, (2) the ATP-analog ATPγS, which is very slowly hydrolyzed by ATPases and phosphatases and specifically activates P2 receptors, or (3) suramin, which inhibits the activation of P2 receptors. The application of adenosine (10 µM; n=134) had no effect on [Ca2+]i in SCO cells. ATPγS (10 µM; n=308) evoked calcium increases in approximately 90% of the cells, and pretreatment with suramin (100 µM; n=222) significantly reduced the number of cells that responded to ATP (10 µM) with increases in [Ca2+]i (Fig. 9c). Thus, the ATP-induced increases in [Ca2+]i in SCO cells appear to be mediated via the activation of P2 purinergic receptors. For characterization of
107 Fig. 7a–f ATP-induced calcium response patterns in bovine SCO cells. a Fifty-five percent of the ATP-sensitive cells (n=279) showed a rapid increase in [Ca2+]i followed by a slow decrease to the baseline. b Fifteen percent of the responding cells (n=74) showed a plateau phase at the highest level of [Ca2+]i. c Sometimes calcium oscillations occurred at this plateau phase. d–f Thirty percent of the ATP-sensitive cells (n=143) displayed an initial peak followed by a plateau phase at an intermediate level. f [Ca2+]i may return to base levels in the presence of the stimulus. The duration of ATP application is indicated by bars
Fig. 8a–d Tachykinin-induced increases in [Ca2+]i and characterization of tachykinin receptors in bovine SCO cells. a Potency of different tachykinins to induce calcium increases (NKB>SP>NKA; NKB, n=588; SP, n=1435; NKA, n=541), indicated the presence of NK3 receptors in bovine SCO cells. b–d Representative calcium responses of single SCO cells to stimulations with increasing concentrations of neurokinin B (NKB), SP, or neurokinin A (NKA), respectively. The duration of tachykinin application is indicated by bars
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were repeatedly stimulated with SP (100 nM to 1 µM; n=262) or ATP (10–100 µM; n=661) in time intervals of 1–10 min. Even 10 min after a first SP application, about 75% of the SP-sensitive cells did not react to a second SP stimulation with increases in [Ca2+]i (Fig. 10a), indicating a long-lasting desensitization of SP-induced calcium signaling. In the remaining 25% of SCO cells, which responded to a second SP stimulation, both calcium responses were of similar amplitude and duration (Fig. 10b). Most of the ATP-sensitive cells showed desensitization of calcium signaling which lasted for a period of less than 5 min, and virtually all of the cells responded to a second ATP stimulus provided 5 min or 10 min after the first pulse (Fig. 10c). Notably, these responses were of identical amplitude and shape (Fig. 10d). Characterization of the calcium-activated signaling pathways
Fig. 9a–c ATP-induced increases in [Ca2+]i and characterization of purinoceptors in bovine SCO cells. a Dose-response curve of ATP on the [Ca2+]i in cultured, dispersed SCO cells. b Representative calcium responses of a single SCO cell to increasing concentrations of ATP. The duration of ATP application is indicated by bars. c SCO-cell responses to stimulation with ATP (10 µM, n=511), ATP analog ATPγS (10 µM; n=308), P1 receptor agonist adenosine (Ads, 10 µM; n=134), ATP after preincubation with P2receptor blocker suramin (Sur, 100 µM; n=222), UTP (10 µM; n=106), ADP (10 µM; n=115), or AMP (10 µM; n=70). ***PADP>AMP) indicates that activation of P2Y2 receptors mediates ATP-induced calcium signaling in SCO cells. To analyze the desensitization of SP- and ATP-induced calcium signaling in the bovine SCO, the cells
The activation of the adenylate cyclase by forskolin (10–100 µM; n=137) had no effect on [Ca2+]i in SCO cells and did not interfere with SP- or ATP-evoked calcium rises (Fig. 11). Identical results were obtained by application of the cAMP analog 8-bromo-cAMP (1–5 mM; n=83; data not shown). Thus, SP- or ATP-induced increases in [Ca2+]i are not mediated via activation of the cAMP signal transduction pathway. To characterize the mechanisms of [Ca2+]i elevation elicited by SP or ATP, the cells were first stimulated with SP (100 nM to 1 µM; n=233) or ATP (10–100 µM; n=133) in calcium-free and then in calcium-containing aCSF or vice versa. In calcium-containing aCSF, 30% of the analyzed cells responded to SP. When kept under calcium-free conditions, the response to SP was completely abolished in half of the SP-sensitive cells (Fig. 12a). In the remaining cells, SP induced either calcium increases with a lower amplitude and/or a faster decrease to the baseline (90% of the responding cells; Fig. 12b), or virtually identical responses (10% of the responding cells; graph not shown). ATP induced identical calcium responses in all cells kept in calcium-free buffer; these showed a rapid increase to a maximal level followed by an immediate decrease to the baseline; responses of cells stimulated under calcium-containing perfusion conditions were characterized by a very slow decrease in [Ca2+]i (Fig. 12c). These data indicate that calcium of intra- and extracellular origin is involved in SP- and ATPactivated calcium signaling in the bovine SCO. To characterize the intracellular calcium stores affected by SP- or ATP-signaling, cells were exposed to caffeine, which mobilizes calcium from ryanodine-sensitive stores, or to thapsigargin that releases calcium from intracellular stores by inhibiting the microsomal (endoplasmic reticular) Ca2+-ATPase. The application of caffeine (100 µM to 10 mM) neither increased [Ca2+]i nor interfered with the calcium responses elicited by SP (100 nM to 1 µM; n=143) or ATP (10–100 µM; n=128; Fig. 13). The treatment with thapsigargin (2 µM) caused
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Fig. 10a–d Desensitization of SP- and ATP-induced calcium signaling in bovine SCO cells. a SP (100 nM) caused a long-lasting desensitization of the SCO cells (n=262); a second SP stimulation after 10 min raised the [Ca2+]i in only ~25% of the SP-responsive cells. b Trace of calcium responses of a single SCO cell to repeated SP stimulations. The response to a second SP stimulus applied after 10 min was of similar amplitude and duration as the initial response. c The desensitization of the purinergic calcium signaling
observed after an initial ATP stimulation (10 µM) of SCO cells (n=661) lasted only a few minutes; nearly all ATP-sensitive cells responded to a second ATP stimulation provided 5–10 min after the first stimulus. d Calcium responses of a single SCO cell to repeated ATP stimulations. The response to a second ATP stimulus applied after 10 min was of similar amplitude and duration as the initial response. The duration of SP or ATP application is indicated by bars
Fig. 11a, b Influence of forskolin on [Ca2+]i. The activation of the adenylate cyclase by forskolin (Fk, 10–100 µM; n=137) neither increased base calcium levels nor interfered with SP- (a) or ATP-evoked (b) calcium rises. The duration of Fk, SP, or ATP application is indicated by bars
an increase in [Ca2+]i. After preincubation with thapsigargin, only 15% of the analyzed cells (tendency, low significance) responded to SP (100 nM to 1 µM; n=254; Fig. 13a). Increases in [Ca2+]i induced by ATP (10–100 µM; n=123) were completely abolished after preincubating the cells with thapsigargin (Fig. 13b). These experiments show the existence of two mechanisms in bovine SCO cells by which SP induces increases in [Ca2+]i: (1) an initial depletion of thapsigargin-sensitive intracellular calcium stores that is followed by a calcium-mediated calcium influx from the extracellular space (50% of the SP-sensitive cells=15% of all cells examined); and (2) a direct activation of calcium influx independent of the preceding calcium release from intracellular sources (another 50% of the SP-sensitive
cells=15% of all cells examined). The ATP-induced calcium increases involve a calcium release from thapsigargin-sensitive intracellular stores and a subsequent calcium influx. To analyze the signal transduction cascades that link the receptor activation to the depletion of intracellular thapsigargin-sensitive calcium stores, SCO cells were preincubated with GDP-β-S (1 mM), an inhibitor of G protein activation, with U-73122 (10 µM), an inhibitor of phospholipase C (PLC), or with U-73343 (10 µM), an analog of U-73122 that does not inhibit the PLC. After preincubation with GDP-β-S, only 7% of the cells responded to stimulation with SP (100 nM to 1 µM; n=227; Fig. 13a). In cells pretreated with U-73122, SP did not elicit a calcium response (n=126), whereas the
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Fig. 13a, b Effects of depletion of intracellular calcium stores and inhibition of G proteins and phospholipase C (PLC) on SP- and ATP-induced increases in [Ca2+]i. a [Ca2+]i response to stimulation with SP (100 nM), SP plus caffeine (Caff, 100 µM to 10 mM; n=143), SP plus thapsigargin (Tg, 2 µM; n=254), SP plus G protein inhibitor (GDP-β-S, 1 mM; n=227), SP plus PLC inhibitor (U73122, 10 µM; n=126), or SP stimulation plus specific negative control (U73433, 10 µM; n=114). b [Ca2+]i responses to stimulation with ATP (10 µM), ATP plus caffeine (100 µM to 10 mM; n=128), ATP plus thapsigargin (2 µM; n=123), ATP plus GDP-β-S (1 mM; n=190), ATP plus U73122 (10 µM; n=209), or ATP plus U73343 (10 µM; n=212). **PNKB; NK2: NKA>NKB>SP; NK3: NKB> NKA>SP; for review, see Krause et al. 1994). Bovine SCO cells responded most sensitively to NKB with calcium increases, suggesting that they possess NK3 receptors. In addition, they may also express NK1 and NK2 receptors, because SP and NKA elicited calcium increases when applied in nanomolar concentrations. The tachykinin receptors of the bovine SCO appeared to desensitize quickly, and most of the SP-sensitive cells did not respond to a second SP pulse within a period of at least 10 min. This desensitization can be mediated via phosphorylation of G proteins by a G protein receptor kinase or via internalization of the SP/NK receptor complex, followed by enzymatic SP degradation and receptor recycling (for review, see Grady et al. 1997). Notably, SP failed to enhance the intracellular cAMP concentration in the bovine SCO (S. Schöniger, unpublished observation). In 50% of the SP-sensitive cells, the calcium rises were mediated by G protein and subsequent PLC activation, resulting in an initial release of calcium from thapsigargin-sensitive intracellular stores. In most cells this increase in [Ca2+]i caused activation of PKC, which induced a calcium influx via opening of L-type VGCCs in the plasma membrane. Such an SP-activated signal transduction pathway has also been shown in the rat pituitary gland (Mau et al. 1997). In the remaining 50% of the responding cells, SP exclusively induced an influx of extracellular calcium ions. An SP-elicited and G proteinmediated modulation of ion channels has also been shown in human T-lymphocytes, where SP modulates potassium currents (Schuhmann and Gardner 1989), and in rabbit colon, where SP influences the activity state of chloride channels (Sun et al. 1993). The intracellular mechanisms that link tachykinin receptor activation to an opening of membrane-bound calcium channels in the bovine SCO are unclear and have to be elucidated in future studies. Our data indicate that activation of PLC contributes to these processes. In conclusion, SP appears to influence SCO activity by the activation of different calcium signal transduction cascades without exerting an in-
fluence on the cAMP signaling pathway. The physiological role of this mechanism has to be clarified in future studies. In addition to the neurotransmitters 5HT and SP, we tested the effects of ATP on calcium signaling in SCO cells, because (1) purinoceptors have been found on glial cells of the peripheral and central nervous system (Zimmermann 1994), and (2) ATP has been shown to activate calcium signal transduction cascades as well as secretory processes in glial cells (Pearce et al. 1989; Langley and Pearce 1998). Here we demonstrate that ATP is a very potent stimulus for bovine SCO cells which caused increases in [Ca2+]i in more than 80% of the cells. Notably, ATP failed to enhance the intracellular cAMP concentration in SCO cells (S. Schöniger, unpublished observation). ATP acting upon SCO cells may be derived from different sources. On the one hand, SCO afferents may contain ATP as a neurotransmitter. On the other hand ATP may be released from the SCO cells and serve their intercellular communication. This type of cell-to-cell signaling has been shown for astrocytes (Guthrie et al. 1999). In principle, ATP may act upon purinoceptors that are distinguished into P1 receptors, preferentially activated by adenosine, and P2 receptors showing a higher affinity for ATP and ADP (for review, see Zimmermann 1994). The P2 receptors are divided into ionotropic P2X and metabotropic P2Y receptors, and the latter are subdivided according to their sensitivity to different nucleotides (Fredholm et al. 1994; Burnstock 1996, 1997; Burnstock and King 1996). Our attempts to identify the receptor subtype responsible for the ATP-induced calcium rises in SCO cells revealed that: (1) adenosine was without any effect on [Ca2+]i; (2) ATPγS, an ATP analog that is very slowly hydrolyzed by ATPases and phosphatases, elicited identical calcium responses, as did ATP; (3) preincubation with the P2 receptor blocker suramin significantly reduced the number of cells responding to ATP; (4) the application of a G protein inhibitor significantly reduced the number of ATP-sensitive cells; and (5) ATP and UTP were equally effective, and both ATP and UTP were more potent than ADP or AMP to increase [Ca2+]i. These results indicate the existence of P2Y2 receptors in bovine SCO cells, which show desensitization within the first 5 min after the application of the first stimulus and gain sensitivity again after that period. The stimulation of P2Y2 receptors led to G protein-mediated PLC activation that induced an initial calcium release from thapsigarginsensitive intracellular stores. The increase in [Ca2+]i activated PKC, which mediated the opening of L-type VGGCs in the plasma membrane and a subsequent calcium influx, resulting in longer-lasting elevated [Ca2+]i. Very similar results were obtained with astrocytes (King et al. 1996; Centemeri et al. 1997) and enteric glial cells (Kimball and Mulholland 1996). In conclusion, our investigations have identified neuroactive substances that influence the activity of SCO cells via specific receptors. Previous studies have demonstrated that SCO cells secrete different species of gly-
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coproteins with molecular masses ranging from 10–540 kDa (Rodríguez et al. 1987; Meiniel et al. 1988; Nualart et al. 1991; Hein et al. 1993). Some of them have neurotrophic properties, in particular, SCO-spondin was shown to stimulate neuritic outgrowth, to promote the survival and to modulate the aggregation and proliferation of cultured neurons (Gobron et al. 1996; Monnerie et al. 1995, 1996, 1997, 1998). In future studies it must be clarified whether and how 5HT, SP, and ATP affect the release of SCO-specific glycoproteins that can be measured in the medium of SCO cultures by means of ELISA (Estivill-Torrus et al. 1998). Acknowledgements Drs. S. Schöniger and M.D.A. Kopp should be considered equally as first authors. The authors are grateful to R. Kühn, E. Laedtke, and S. Leslie for expert technical assistance and Dr. Q. Zhang for helpful advice.
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