Cholecystokinin Activates CCKB-Receptor-Mediated Ca-Signaling in Hippocampal Astrocytes
Wolfgang Müller,
Uwe Heinemann, and
Karin Berlin
Institut für Physiologie der Charité, Abteilung Neurophysiologie, Arbeitsgruppe Molekulare Zellphysiologie, D-10117 Berlin, Germany
 |
ABSTRACT |
Müller, Wolfgang, Uwe Heinemann, and Karin Berlin. Cholecystokinin activates CCKB-receptor-mediated Ca-signaling in hippocampal astrocytes. J. Neurophysiol. 78: 1997-2001, 1997. Cholecystokinin-8S (CCK-8S) is the most abundant neuropeptide in the mammalian cortex and the limbic system; however, its physiological functions remained largely obscure. We studied effects of CCK on astrocytic Ca signaling, which has met considerable interest as a second messenger in astrocytic-neuronal signaling, by digital ratio-imaging of Fura-2/AM loaded rat and mouse hippocampal astrocytes in dissociated culture. Superfusion of CCK-8S (5-50 nM for 2 min) evoked repetitive Ca increases of several hundred nanomolar in a subpopulation of astrocytes. Mouse astrocytes appeared to be more responsive to CCK than rat cells with respect to the fraction of cells responding as well as to the amplitudes of Ca increases. The Ca responses persisted in the absence of extracellular Ca, indicating that release of Ca from intracellular stores is the primary source of these Ca increases. The CCK-8S-induced Ca increases were blocked by the CCKB receptor antagonist PD135158 (100 nM) but not by the CCKA antagonist lorglumide (100 nM). We surmise that astrocytes might be a major primary target for CCK in the CNS.
 |
INTRODUCTION |
Neuropeptides have met increased interest as possible cotransmitters in the CNS, being released primarily during strong neuronal activity. They are candidates for modulatory, long-lasting effects on neuronal information processing. There are many examples where effects on neurons are associated with effects on glial cells, thereby possibly affecting glial-neuronal communication. With respect to cholecystokinin (CCK), the most abundant neuropeptide in the mammalian cortex and the limbic system, effects on neuronal behavior appear to be variable and complex (Branchereau et al. 1993
; Davidowa et al. 1995
; Hösli et al. 1993
). Excitatory as well as inhibitory effects have been observed. The latter appear to be, at least in some cases, due to indirect inhibition via excitation of inhibitory neurons (Branchereau et al. 1992
). In contrast, long-term effects like the neuroprotective effect of CCK against NMDA-receptor-mediated glutamate cytotoxicity (Akaike et al. 1991
) may well involve activation of glia. Autoradiographic studies indeed confirmed binding of cholecystokinin to astrocytes (Hösli and Hösli 1994
).
Astrocytes have long been considered to play a rather passive role in information processing in the CNS before fast glial calcium (Ca) signaling, and plasticity in this signaling has been demonstrated. Astrocytes have been shown to respond to various neurotransmitters and drugs with a rich variety of spatiotemporal Ca signaling patterns (Cornell-Bell et al. 1990
; Dani et al. 1992
). Little is known, however, about CCK effects on glial Ca signaling. In the present study, we demonstrate that CCK evokes oscillations in intracellular free Ca in confluent astrocytic cultures, suggesting astrocytes as a major target for cholecystokininergic transmission in the hippocampus.
 |
METHODS |
Cell culture
Hippocampal cultures were prepared from embryonic day 17 (E17)- to E19-day-old Wistar rats or from E16/17-day-old NMRI mice according to standard methods (Banker and Cowan 1977
). In brief, the pregnant animal was decapitated under deep ether anesthesia. The embryos were removed and decapitated. The hippocampi were mechanically dissociated without prior enzymatic treatment. Cells were plated onto poly-D-lysine-coated coverslips in 35-mm plastic culture dishes at a density of 60,000 cells per coverslip in 1 ml Eagles basal medium per dish (BME, GIBCO, Karlsruhe, Germany) supplemented with 2 mM glutamine, 10% glucose, and 10% horse serum (GIBCO). Penicillin (25 IU/ml, Sigma) and streptomycin (25 µg/ml, Sigma) were added to the medium. Cells were kept at 37°C (5% CO2, moist atmosphere). After 2 days in vitro, horse serum was reduced to 2%. Culture medium was changed twice a week. Exchange with cool medium (8°C) resulted in a rapid and complete loss of all neurons in the cultures. The remaining cells were flat polygonal cells. A vast majority of these cells (>98%) was labeled with antibodies to the astroglial marker GFAP. Cultures were washed for at least three times with recording saline (see below) before recording.
Recording conditions and data analysis
Ca was imaged with an upright microscope (Zeiss Axioskop FS) and a ×20 or a ×40 water immersion objective. A charge coupled device camera system (Photometrics, Tucson, AZ) similar to ones described previously (Connor 1986
; Müller and Connor 1991a
,b
) was used to acquire digitized images of Fura-2. Astrocytic hippocampal cultures were loaded with Fura-2/AM (3 µM) for 20-30 min at 37°C. Depending on the power of the objective used and the local density of the astrocytes, Ca could be monitored simultaneously in 5-50 astrocytes. Free Ca concentrations were determined from background corrected image pairs at 350 and 380 nm excitation with the ratio method (Grynkiewicz et al. 1985
). Mean ± SD are given in the results.
The external solution contained (in mM) 124 NaCl, 3 KCl, 1.6 CaCl2, 1.8 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, and 10 glucose, pH 7.4. Experiments were performed at room temperature (20-22°C) at a perfusion rate of 1 ml/min. Drugs (all from RBI, Natick, MA, except 6-cyano-7-nitroquinoxaline-2,3-dione, which was from Tocris, Bristol, UK) were applied by the bath by switching the perfusion to drug-containing saline. In this way, drug concentration starts to rise in the vicinity of the cells within 0.9-1.3 min, depending on the position of cells in the chamber. All bars given in the figures are not corrected for this delay but indicate the actual times of switching valves. After switching back to control saline, wash out begins after ~1 min. Fifty percent wash out time is ~5 min. This implies that after a 20-min wash out of a CCK-8S application activating >90% of the receptor capacity (5 nM; to obtain a close to maximum response), the residual concentration of ~0.3 nM is still sufficient to maintain ~50% of receptors in the active configuration (cf. Müller et al. 1988
). Antagonists were tested with respect to reversal of ongoing activity in the presence of a diminished CCK-8S concentration during wash out as well as with respect to blocking responses to CCK-8S applied in the presence of the antagonist. According to the kinetics of our system described above, a good recovery from bath-applied CCK receptor antagonists required wash out for at least 15 min.
 |
RESULTS |
CCK: effects on astrocytic Ca concentration
Fura-2/AM-loaded astrocytes in neuron-free primary culture (10-25 days in vitro) showed homogenous fluorescence signals across the cytosol with above average fluorescence, as seen previously in neurons in the slice preparation, in the nuclear area at 350 as well as at 380 nm excitation (Fig. 1A: ×, arrow). The astrocytes, perfused with oxygenated Ringer saline, had resting levels of free intracellular Ca in the range of 50-150 nM. Free Ca appeared to be largely homogenous across an individual cell with apparently slightly lower levels in the nuclear area, presumably due to dye trapped in the organellar component of the cytoplasm and a mixed signal from this area (Connor 1993
; cf. Stehno-Bittel et al. 1995
). In this control condition, intracellular free Ca levels were stable within the mean resting level ±30 nM. In our cultures, we did not observe spontaneous Ca increases of 150-2,000 nM like those described by Fatatis and Russel (1992). To facilitate the on-line judgement of Ca responses, CCK-8S was applied at a minimum concentration of about the 30-fold of the Kd and up (5-50 nM for 2 min). CCK-8S (5 nM) evoked within 2-5 min (3.8 ± 1.3 min, mean ± SD, n = 108) transient increases or oscillations of free intracellular calcium of up to several hundred nM (480 ± 190 nM) in 28 ± 19% of astrocytes studied (n = 108 in 8 cultures). After such an application the Ca signaling activities fairly recovered within 6-12 min during wash out of CCK. Even after 20-30 min of wash out, however, we still observed transient increases of [Ca]i. This is most likely due to a residual CCK-8S concentration of about the Kd(cf. METHODS). Application of higher concentrations ofCCK-8S resulted in even longer lasting responses, as expected for an exponential drug wash out. The Ca responses showed no signs of desensitization with repeated applications of CCK-8S (up to 5 applications with intervals of ~20 min were tested). Spread of Ca increases to adjacent cells occurred in <5% of cells and were not included in the analysis. Such responses are considered secondary, presumably due to some cell coupling by gap junctions. The variable and slow response times for CCK-induced Ca increases were not significantly reduced by application of higher concentrations of CCK and indicate presumably a variable and probably rather slow transduction mechanism, e.g., due to an individual intracellular accumulation of an intracellular messenger like inositol trisphosphate (IP3). This conclusion was confirmed by fast responses of cells to perfusion with a high potassium saline (50 mM, response time 2.1 ± 0.3 min).

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| FIG. 1.
Gray scale-coded images of free [Ca]i in rat hippocampal astrocytes in neuron-free culture in control (B1), during a moderate increase of [Ca]i in almost all cells in response to cholecystokinin-8S (CCK-8S, 5 nM for 2 min, B2, note slight increase in overall brightness) and partial recovery (B3). In the raw fluorescence image at 380 nm excitation (A), nuclei are slightly brighter than cytosols (cf. ×, arrow). The strong Ca response shown in B2 appears to be localized largely to the nuclear region (bottom part, cf. B2, A, ).
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Figure 1B shows a moderate response to CCK-8S in almost all cells shown in a sequence of gray scale coded Ca images of rat astrocytes. During the response to CCK-8S, we observed in ~20% of responding cells strong Ca increases in the nuclear region like in the cell marked with an arrow (Fig. 1). In these cells Ca increases in the nuclear region apparently exceeded those in the cytosol by 180 ± 120 nM (n = 17). In weakly responding cells, free Ca in the nuclear area did not exceed cytosolic levels.
The plots of Fig. 2A demonstrate time courses and amplitudes of [Ca]i responses to 5 nM CCK-8S in the cytosol of individual cells and the average for the responding cells (top thick line and right scales). The data were extracted from ratio image pairs like those depicted in Fig. 1. These graphs demonstrate that CCK-8S induced Ca spikes of up to 700 nM (cf. Fig. 3) lasting 5-120 s. In addition, CCK evoked a long-lasting (5-10 min) slight increase in basal [Ca]i by 10-70 nM (40 ± 25 nM, n = 108, cf. Fig. 2A).

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| FIG. 2.
Graphs of cytosolic [Ca]i responses of individual astrocytes to 5 nM CCK-8S for 2 min (- - -). CCK-8S evokes Ca spikes as well as a long-lasting (5-10 min) slight increase in basal [Ca]i (A). In 0 Ca (0.1 mM ethylene glycol-bis( -aminoethyl ether)-N,N,N ,N -tetraacetic acid) responses to CCK-8S were neither blocked nor reduced, indicating that CCK-8S increases [Ca]i via release of Ca from intracellular stores (B). Data were extracted from ratio image pairs like those depicted in Fig. 1. Top fat lines and right scales give the average of [Ca]i for responding cells in this experiment.
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| FIG. 3.
Cytosolic Ca graphs of 3 selected astrocytes showing that the CCKB receptor antagonist PD 135158 (100 nM) reverses still ongoing Ca signaling responses during the wash out phase of CCK-8S (5 nM for 2 min, dashed line) and completely blocks responses to subsequent applications of CCK-8S (A). The blockade of the CCK-8S effect is reversible with wash out of PD 135158 (B). Top thick lines and right scales give the more representative average of [Ca]i for the responding cells (n = 8).
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CCK-induced Ca signaling depends on Ca release secondary to CCKB receptor activation
To study whether CCK-evoked increases in [Ca]i are mediated by transmembrane Ca influx or release from intracellular stores, cultures were perfused for 5 min with Ca-free saline containing 100 µM ethylene glycol-bis(
-aminoethyl ether)-N,N,N
,N
-tetraacetic acid (EGTA). Figure 2B shows that, in this condition, responses to CCK-8S were neither blocked nor reduced (n = 200 cells in 4 cultures). This result indicates that CCK-induced Ca signaling depends primarily on release of Ca from intracellular Ca stores, most likely controlled by the second messenger inositol trisphosphate (IP3).
CCK exerts its effects in the CNS by acting on at least two different receptor subtypes, CCKA and CCKB receptors (Davidowa et al. 1995
; Innis and Snyder 1980
). The availability of highly specific receptor subtype antagonists (Hughes et al. 1990
) allowed us to test for the involvement of the respective CCK receptor subtypes. Figure 3 shows that 100 nM PD 135158, a concentration expected to block CCKB receptors by >95% but CCKA receptors by <5%, reduced still ongoing Ca signaling responses during the wash out phase of CCK-8S within 3-5 min. The continued presence of 100 nM PD 135158 completely blocked responses to subsequent applications of CCK-8S (Fig. 3, n = 250 cells in 4 cultures). Similarly, CCK-8S applications were ineffective in the presence of PD 135158, but evoked Ca responses similar to control after 15-20 min wash out of PD 135158. Glutamate (100 mM) and serotonin (10 mM) evoked Ca responses similar to CCK in our cultured astrocytes. PD 135158 (100 nM) did neither affect glutamate- nor serotonin-evoked Ca responses, indicating absence of nonspecific side effects of PD 135158 on the cellular Ca signaling mechanisms (data not shown). The effect of PD 135158 on CCK-induced Ca signaling was essentially reversible after wash out of the antagonist for at least 15 min (Fig. 3B).

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| FIG. 4.
Ca graphs showing that the CCKA receptor antagonist Lorglumide (100 nM) is ineffective in antagonizing the effects of CCK-8S (5 nM for 2 min, - - -) on astrocytic signaling in free [Ca]i.
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In contrast, 100 nM lorglumide, expected to block >97% of CCKA receptors but <15% of CCKB receptors, did neither reverse ongoing Ca signaling during the wash out phase of CCK-8S nor did it antagonize effectively astrocytic Ca responses when 5 nM CCK-8S was applied in the presence of the antagonist. This has been confirmed in the very same astrocytes that did not respond in the presence of PD 135158 (Fig. 4, n = 250 cells in 4 cultures) as well as in cells not treated previously with PD 135158.
The comparison of the effects of PD 135158 and lorglumide strongly suggests that astrocytic increases in free intracellular calcium are mediated primarily by CCKB receptors.
 |
DISCUSSION |
The present study demonstrates that CCK has strong effects on the [Ca]i in hippocampal astrocytes. Intracellular free Ca can transiently increase by several hundred nanomolar. To our knowledge we are the first to report CCK evoked Ca increases in CNS cells in the absence of extracellular Ca, indicating that these responses are caused primarily by a release of Ca from intracellular Ca stores. The primary mechanism for receptor-activated intracellular release of Ca is activation of phospholipase C (PLC) causing subsequent formation of IP3 that in turn gates Ca release from intracellular stores.
Indeed, CCK has been shown to stimulate phosphoinositide hydrolysis in several peripheral cell types (Kelley et al. 1995
; Wakui et al. 1991
; Yu et al. 1994
), probably via activation of CCKA receptors. We find that astrocytic Ca increases are mediated exclusively by CCKB receptors and subsequent release of Ca from intracellular stores. Release of Ca from intracellular stores is well known to be a highly nonlinear process (e.g., Bezprozvanny et al. 1991
) and appears to follow often an all-or-nothing rule in astrocytes, probably because of secondary triggering of Ca-induced Ca release (Langley and Pearce 1994
; Shao and McCarthy 1995
). This tends to complicate functional receptor discrimination. Nevertheless, the relative effects of both CCK receptor antagonists, highly specific for either CCKA or CCKB receptors, strongly support a primary involvement of CCKB receptor activation. With respect to the release of Ca from intracellular stores one has to consider that CCKB receptors in the CNS differ with respect to the agonist/antagonist binding site from the primarily peripheral CCKA receptors (Innis and Snyder 1980
). Nevertheless, coupling of the CCKB receptor to PLC seems the most likely explanation for the release of intracellular Ca reported here. This statement finds further support in the notion that the CCKB receptor is linked to a G protein and is very similar to or identical with another PLC-activating receptor, i.e., the gastrin receptor (Gut et al. 1989
; Hunter et al. 1993
; Schnefel et al. 1988
).
The observation of strong Ca increases in the nuclear region of some cells might be of significance with respect to possible control of gene expression by CCK (Morgan and Curran 1988
). With respect to the relative levels of free Ca observed in the nucleus as compared with the cytosol, one should keep in mind a well-known problem. Compartmentation of Fura-2 into cytosolic organelles with low Ca levels give underestimates of free Ca for the cytosol due to a mixed Fura-2 signal (Al-Mohanna et al. 1994
; Connor 1993
).
Activation of PLC causes, in addition to Ca mobilization, release of diacylglycerol and activation of protein kinase C. In this way a large variety of targets is controlled by phosphorylation. Such mechanisms usually have considerable impact on the behavior of a cell, both, short term and rather long term. The observed strong Ca signals in the vicinity or within the nucleus are likely to represent signals involved in the control of transcriptional processes and, hence, represent another class of processes with a long-lasting impact.
The recent discovery of neurotransmitter-induced Ca waves in astrocytic syncytium has met strong interest as a possible long-range signaling system possibly involved directly in CNS information processing (Charles et al. 1991
; Cornell-Bell et al. 1990
). The observation of various forms of glial-neuronal signaling adds further support to this notion (Charles 1994
; Nedergaard 1994
; Parpura et al. 1994
). Based on our observations, we suggest astrocytes as a primary target for CCK in the CNS. Signaling events downstream of Ca signaling and liberation of diacylglycerol could cause short- and long-term effects on all brain cells in spatial proximity to these signals. From the intimate relation between astrocytic processes and neuronal synapses in situ, strong Ca signals localized to postsynaptic spines (Müller and Connor 1991b
; Yuste and Denk 1995
) and the release of freely diffusable "retrograde" messengers like arachidonic acid or nitric oxide (Dumuis et al. 1988
; Snyder and Bredt 1991
), a complex interaction of the biochemical signaling in the synaptic, astrocytic and neuronal compartments can be anticipated.
It is conceivable that many CCK responses reported in nonastrocytic cells might be secondary to Ca increases in astrocytes adjacent to these cells. For example, Ca increases in response to CCK have been reported to occur in ~20% of striatal neurons in culture (Miyoshi et al. 1991
). These increases were considerably smaller than the ones reported here in astrocytes and are mediated by a voltage-dependent Ca channel. It is therefore conceivable that these Ca increases might have been evoked, at least in part, indirectly by glial release of glutamate.
Cortical neurons can be protected against glutamate neurotoxicity by concomitant application of CCK (Akaike et al. 1991
). It has been suggested that inhibition of nitric oxide formation by CCK may mediate this protection. Our results suggest the possibility that activation of astrocytes by CCK might be involved in the therapeutic benefit in excitotoxicity, too. Interestingly, the neuroprotective CCK effects are mediated, like the effects on Ca signaling in astrocytes, exclusively by CCKB receptors. In contrast, neostriatal neurons in vivo have been shown to be excited via both CCKA and CCKB receptors.
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ACKNOWLEDGEMENTS |
We thank R. Schneider and A. Düerkop for excellent technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft, Bonn (Heisenberg-Stipendium and research Grant C4, SFB 507 to W. Müller) and by the Charité, Berlin Germany.
 |
FOOTNOTES |
Address for reprint requests: W. Müller, Institut für Physiologie der Charité, AG Molekulare Zellphysiologie, Tucholskystr. 2, D-10117 Berlin, Germany.
Received 15 January 1997; accepted in final form 17 June 1997.
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