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Am J Physiol Cell Physiol 277: C616-C627, 1999;
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Vol. 277, Issue 4, C616-C627, October 1999

EDITORIAL FOCUS
Distinct pharmacological properties of ET-1 and ET-3 on astroglial gap junctions and Ca2+ signaling

Fredrik Blomstrand1, Christian Giaume2, Elisabeth Hansson1, and Lars Rönnbäck1

1 Institute of Neurobiology and Institute of Clinical Neuroscience, Göteborg University, Göteborg, Sweden; and 2 Neuropharmacologie, Institut National de la Santé et de la Recherche Médicale U114, Collège de France, Paris, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Astrocytes represent a major target for endothelins (ETs), a family of peptides that have potent and multiple effects on signal transduction pathways and can be released by several cell types in the brain. In the present study we have investigated the involvement of different ET receptor subtypes on intercellular dye diffusion, intracellular Ca2+ homeostasis, and intercellular Ca2+ signaling in cultured rat astrocytes from hippocampus and striatum. Depending on the ET concentration and the receptor involved, ET-1- and ET-3-induced intracellular Ca2+ increases with different response patterns. Both ET-1 and ET-3 are powerful inhibitors of gap junctional permeability and intercellular Ca2+ signaling. The nonselective ET receptor agonist sarafotoxin S6b and the ETB receptor-selective agonist IRL 1620 mimicked these inhibitions. The ET-3 effects were only marginally affected by an ETA receptor antagonist but completely blocked by an ETB receptor antagonist. However, the ET-1-induced inhibition of gap junctional dye transfer and intercellular Ca2+ signaling was only marginally blocked by ETA or ETB receptor-selective antagonists but fully prevented when these antagonists were applied together. The ET-induced inhibition of gap junction permeability and intercellular Ca2+ signaling indicates that important changes in the function of astroglial communication might occur when the level of ETs in the brain is increased.

endothelins; cultured astrocytes; gap junctions; calcium waves


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELINS (ETs) constitute a peptide family composed of at least three isoforms termed ET-1, ET-2, and ET-3. The first and most thoroughly studied member, ET-1, was originally found in porcine endothelial cells as a very potent vasoconstrictor peptide (74). Subsequently, members of the ET family were found to have multiple biological activities in both vascular and nonvascular tissues. They have been implicated in a wide variety of physiological functions associated with the cardiovascular, endocrine, pulmonary, renal, and nervous systems. In the brain, ET-1 and ET-3 have been detected by autoradiography and by RT-PCR in rats and humans (36, 66), and they are known to be produced in some neurons (17) and in endothelial cells (75). Also, ETs are expressed in glial cells under certain circumstances, e.g., in activated astrocytes in vivo (35, 73), as well as in cultured astrocytes (13, 44). Although less documented than in peripheral systems, biological effects of ETs have also been investigated in the central nervous system. This includes the increase of glucose uptake (65) and glutamate efflux (60), the stimulation of proliferation (63) and mitogenesis (44, 64), the increase of c-fos and nerve growth factor expression, the regulation of ionic channels activity (64), the inhibition of gap junction-mediated intercellular communication (18), the mobilization of various transduction pathways, and the triggering of intercellular Ca2+ waves (see Ref. 19).

The ET activity in mammals is mediated via at least two ET receptor subtypes, ETA and ETB, which are coupled to heterotrimeric G proteins. In the vascular system, ETs have potent effects on cerebral blood flow, differing according to the class of ET receptor they stimulate: ETA receptors mediate vasoconstriction, whereas ETB receptors mediate vasodilatation. In the brain, ETA receptors are mostly expressed in vascular cells (31), whereas ETB receptors are mainly expressed on glial cells (31, 40). However, expression of both ETA and ETB receptor mRNA has been detected in astrocytes in culture (12). ET binding sites are widely distributed in the brain, with the highest levels in the hippocampal formation and in the cerebellum (see Ref. 58). The ET receptor subtypes can be pharmacologically distinguished by their different affinities for ET isoforms; the ETA receptors show the affinity ranking ET-1 >=  ET-2 >> ET-3, while they are equipotent at ETB receptors. Several antagonists and agonists for the ETA and ETB receptors have been developed. Among them, BQ-123 (32) is a selective antagonist for the ETA receptor, whereas BQ-788 (33) is a selective antagonist for the ETB receptor, IRL 1620 (67) is a selective agonist for the ETB receptor, and sarafotoxin S6b (SFTX) (37) is a nonselective ET agonist. However, there might be subtypes of these two receptors and possibly other ET receptors; the existence of ETA1, ETA2, ETB1, and ETB2 receptor subtypes as well as an ETC receptor has been postulated (see Ref. 51). Recently, measurements of ET-1 level in culture media, RT-PCR for ET-1 mRNA, and binding studies performed with selective ligands of ET receptors have lead to the proposal that cultured astrocytes could express an atypical ET receptor characterized by unusual binding and pharmacological properties (27, 34).

The physiological significance of the involvement of ET responses in active and passive functions fulfilled by astrocytes is still mostly unclear. ETs are known to be released by reactive astrocytes and have been implicated as having a role in various disorders (49). Indeed, ET immunoreactivity and ETB receptor expression are significantly increased in astrocytes after brain injury (9, 57, 59). ET levels were also shown to increase in neurological disorders, such as Alzheimer's disease (76), virus infection (43), subarachnoidal hemorrhage (45), and ischemia (1, 73). Interestingly, ET receptor antagonists have been shown to exert therapeutic effects in animal models of cerebrovascular diseases (for reviews see Refs. 1 and 54). From these studies it appears that a better understanding of the effect of ETs on astrocytic properties should help determine the role of these peptides in these pathological and experimental situations.

Two major properties of in vivo as well as in vitro astrocytes are their elaborated Ca2+ signaling feature, which can be activated by a great variety of stimuli (72), and the network organization they display due to the high degree of intercellular communication through gap junction channels (19). The combination of these two astrocytic features provides the basis for the initiation and propagation of intercellular Ca2+ waves that are proposed to represent a long-range signaling process between astrocytes and neurons (62). ETs are potent activators of Ca2+ signaling in astrocytes (14), and focal application of ET-1 is able to trigger Ca2+ waves (71). Furthermore, ETs have been reported to inhibit gap junctional permeability in astrocytes (18). These observations suggest that ETs could control the spread of Ca2+ waves in astrocytes. Moreover, the expression of ET receptors on astrocytes and the increase of ETs and ET receptors in several pathological conditions (49, 51) prompted us to determine which ET receptor subtype is involved in the inhibition of gap junctional permeability. In the present study, we report that ET-1 and ET-3 inhibit astrocytic gap junctional permeability and intercellular Ca2+ wave propagation induced by mechanical stimulation. The use of ETA and ETB receptor agonists and antagonists indicates that the two ET isoforms induce their inhibitory effect with distinct pharmacological properties. We further report that the Ca2+ signal pattern in single cells responding to ETs depends on the ET dose and on which receptor subtype is stimulated.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell cultures. Mixed astroglial-neuronal primary cultures containing ~5-10% neurons (7) and neuron-free astroglial primary cultures (26) were obtained from newborn Sprague-Dawley rat striatum and hippocampus (Charles River, Uppsala, Sweden). Astrocytes in the mixed astroglial-neuronal cultures were confluent and used at 7-11 days old, whereas the astrocytes in neuron-free cultures were confluent and used at 12-15 days.

Scrape loading/dye transfer. Gap junction permeability was determined at room temperature (20-22°C) by the scrape-loading/dye transfer technique. Control experiments were performed by preincubating the cells for 9 min in HEPES-buffered Hanks' balanced salt solution (HHBSS) (50) complemented with 0.1% BSA, pH 7.35. The cells were then washed thoroughly for 1 min in the same buffer in which Ca2+ was omitted to prevent uncoupling of the cells due to high Ca2+. Scrape loading was performed with a razor blade in Ca2+-free buffer containing 0.1% Lucifer yellow. The Lucifer yellow was rinsed away 1 min after the scrape, and standard buffer was reintroduced. Junctional permeability was measured 9 min after the scrape by taking five successive digital images per trial using a Hamamatsu C5810 chilled 3CCD camera.

ET-1 was used at 10-10 to 10-7 M for dose-response experiments and at 10-7 M in the following pharmacological experiments. SFTX and the ETB receptor agonists, ET-3 and IRL 1620, were used at 10-7 M. When the effects of the agonists were studied, they were present in the preincubation solution (9 min) and all other solutions until the images were captured. Dose-response experiments with BQ-123 (ETA receptor antagonist) and BQ-788 (ETB receptor antagonist) were performed in hippocampal cultures at 10-8 to 10-5 M in combination with ET-1 or ET-3 at 10-7 M, respectively. Elsewhere the antagonists were used at 10-6 M. The antagonists were added 10 min before the agonists and were then present for the whole experiment.

Quantification of the dye spreading was performed by subtraction of the background and computation of the fluorescent areas (18) using NIH Image (Scion). A non-gap-junction-permeable high-molecular weight rhodamine dextran (mol wt 11,000) was used to determine the area of the cells initially loaded with the dye (48). This area was similar to that seen after gap junction blockage using 18alpha -glycyrrhetinic acid (6) and was subtracted from the dye transfer area in all experiments to yield the operational dye transfer. The effect of the drugs on dye spreading was expressed as a percentage of the spreading in the control situation of a sister culture.

Ca2+ imaging. Confluent cultures were incubated at 37°C with 8 µM of the Ca2+-sensitive probe fluo 3-AM and 0.03% Pluronic acid for 45 min in HHBSS, pH 7.35. Thapsigargin, an inhibitor of Ca2+-ATPases on the endoplasmic reticulum, was used to study the Ca2+ refilling into intracellular Ca2+ pools after ET stimulation. Thapsigargin (10-5 to 10-7 M) experiments were made with fluo 3-AM but also with fura 2-AM (incubated in the same way) to perform ratio determinations. All experiments were performed at room temperature (20-22°C) using a SPEX fluoromax interfaced with a Nikon diaphot-inverted microscope, except the fura 2 experiments, which were performed in a Photon Technology International imaging system. Time-lapse images were captured (1-3 Hz, excitation at 485 nm and emission at 535 nm for fluo 3; excitation at 340/380 nm and emission at 510 nm for fura 2), and mechanically induced intercellular Ca2+ waves were initiated as described earlier (7). These Ca2+ waves were initiated in untreated cells and in cells treated with agonists for 10 min. In the mixed cultures, the Ca2+ waves were initiated in neuron-free fields, to avoid direct involvement of neurons. Repeated stimuli were mostly done in different fields on the same coverslip to avoid phototoxicity and damage to the stimulated cell. Images were sampled when the drugs were added to later determine the number of astrocytes displaying intracellular Ca2+ concentration ([Ca2+]i) transients. The [Ca2+]i response frequencies were determined to be the number of responding astrocytes divided by the total number of identified astrocytes present in the microscopic field. Furthermore, the Ca2+ response patterns were investigated according to the classification of Finkbeiner: monophasic peak, monophasic sustained plateau, biphasic response with peak and sustained plateau, and polyphasic (oscillating) responses (14). The percentage of astrocytes that responded with a certain Ca2+ response pattern were determined to be the number of astrocytes with this response divided by the total number of responding astrocytes for each stimulation type.

A ×10 or ×20 fluorescence dry lens was used to quantify the extent of propagation as previously described (7). Arrival of the spreading wave at each pixel was defined as 20% intensity increases in (F - F0)/F0 above basal fluorescence level (F0). Estimation of [Ca2+]i in fura 2 experiments was performed with in vitro calibrations using Molecular Probes' Ca2+ Calibration Buffer Kit 1 and according to the equation described in Ref. 23.

Chemicals and treatments. When using antagonists, they were preincubated 10 min before addition of the agonists and were present during the whole experiment. The pharmacological studies performed and concentrations used were the same as in the dye transfer experiments, except that no dose-response studies were performed for the antagonists.

The HHBSS-BSA was used in all dye transfer and Ca2+-imaging experiments and the BSA vehicle itself had no impact on the studied parameters. Antagonists themselves had no effect on dye transfer or [Ca2+]i or on the extent of Ca2+ wave propagation in any of the concentrations used. All ET receptor agonists and antagonists used were from RBI (Natick, MA), except SFTX (Sigma Chemical, St. Louis, MO). Fluo 3-AM, fura 2-AM, and Pluronic acid were from Molecular Probes (Leiden, Netherlands). All other chemicals were from Sigma.

Statistical methods. Statistical analyses were made using paired Student's t-test followed by Holm's sequentially rejective test for multiple comparisons (30) on loge-transformed raw data. The data in Figs. 1 and 5 are presented as the mean percent of control ± SE, while the pairing of selected groups of interest made statistical comparisons. These groups were A: ET-1 vs. control; B: BQ-123/ET-1 vs. ET-1; C: BQ-788/ET-1 vs. ET-1; D: mixed BQ-123 and BQ-788 (BQ-123,788)/ET-1 vs. BQ-123/ET-1 and BQ-788/ET-1 separate; E: ET-3 vs. control; and F: BQ-123/ET-3 vs. ET-3. The analyses were performed to investigate possible differences between these groups with agonists at 10-7 M and antagonists at 10-6 M.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pharmacological properties of ET-induced inhibition of gap junctional permeability. During the present study, mixed cultures of astrocytes and neurons were mainly used instead of pure cultures of astrocytes. Some additional control experiments, of astroglial primary cultures devoid of neurons and originating from the same brain structures as the mixed cultures, were performed. Unless otherwise indicated, the presented data were obtained from mixed cultures.

ET-1 inhibited intercellular dye transfer in cultured rat astrocytes from hippocampus and striatum in a dose-dependent manner. The dye transfer in hippocampal and striatal astrocytes was not significantly affected by 10-10 M ET-1 but was almost completely inhibited by 10-7 M ET-1 or by 10-7 M ET-3 (Fig. 1). Dye transfer (mean percent of control ± SE) was also reduced by IRL 1620 (ETB receptor agonist; 22.9 ± 2.8, n = 4; and 16.7 ± 7.0, n = 3) and by SFTX (nonselective agonist; 15.4 ± 3.5, n = 5; and 7.7 ± 5.9, n = 3) in hippocampal and striatal astrocytes, respectively. To determine which type of ET receptor was responsible for this inhibition, a series of dose-response experiments was performed in hippocampal cultures with selective antagonists BQ-123 or BQ-788 to evaluate their antagonistic potency on ET-1 or ET-3 at 10-7 M, respectively. The ETA receptor-selective antagonist BQ-123 at a range from 10-8 to 10-5 M had only minor effects on the ET-1- or ET-3-induced inhibition on intercellular dye transfer (Fig. 2). However, although ET-1 and ET-3 bind to the ETB receptor with similar affinity, only the ET-3-induced dye transfer inhibition was prevented with BQ-788 (ETB receptor-selective antagonist). The ET-3 effect was strongly antagonized at 10-6 or 10-5 M BQ-788 with a dye transfer of 96.0 ± 2.8% of control and 96.6 ± 8.1% of control, respectively. In contrast, the ET-1 inhibition was only marginally affected by BQ-788 in a range of 10-8 to 10-5 M (Fig. 2). However, coincubation of both antagonists (10-6 M each) fully prevented the ET-1 effect. This antagonism of ET-1-induced dye transfer inhibition through simultaneous ETA and ETB receptor blockage was more than additive (P < 0.01) compared with the blockages of either receptor (Fig. 1A). Similarly in striatal astrocytes, a blockage of both ETA and ETB receptors simultaneously inhibited the ET-1 effect in a more than additive manner (P < 0.05), whereas the ET-3 effect was completely inhibited by an ETB blockage alone but not by an ETA blockage (Fig. 1B).


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  		Fig. 1.   Endothelin-1 (ET-1) induced a concentration-dependent inhibition of dye transfer in hippocampal (A) and striatal (B) astrocytes. Incubation with ET-1 or ET-3 (10-7 M) for 10 min significantly decreased dye transfer in hippocampal (A) and striatal (B) astrocytes compared with control (P < 0.001). Note that data are presented as mean percent of control ± SE, while pairing selected groups of interest made statistical comparisons. ET-1-induced inhibition of dye transfer was only marginally affected by ETA (BQ-123) or ETB (BQ-788) receptor blockage (P < 0.05, except for BQ-123 in striatal astrocytes, which was not significant). However, the ET-1 effect was completely prevented after simultaneous blockage of both receptors. A synergistic antagonism (more than additive effect) of BQ-123 and BQ-788 when coincubated was seen in hippocampal (P < 0.01) and in striatal (P < 0.05) astrocytes. The ET-3 effect was not affected by ETA receptor blockage but was completely blocked by ETB receptor blockage in hippocampal (A) as well as in striatal (B) astrocytes. Numbers of separate experiments (cultures from different cell preparations) are indicated above each bar.



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  		Fig. 2.   Dose-response experiments of antagonistic efficacy of BQ-123 or BQ-788 (10-8 to 10-5 M, respectively) on ET-1- or ET-3-induced (10-7 M, respectively) dye transfer inhibition (mean percent of control) in hippocampal astrocytes. Number of separate experiments was n = 3, except for BQ-123 at 10-6 M, as well as for BQ-788 at 10-6 M (n =7 and n = 5, for ET-1 and ET-3, respectively).

The possibility of regional differences in junctional permeability was investigated by pairing the dye transfer raw data of hippocampal and striatal cultures from the same cell-culturing week. Striatal astrocytes displayed significantly lower dye transfer area, 82.0 ± 5.5% (percent of the dye transfer in hippocampal astrocytes ± SE) than hippocampal astrocytes (P < 0.05, n = 7, separate culturing weeks).

Analysis of intracellular Ca2+ transients induced by the stimulation of ET receptors. Ca2+-imaging experiments were performed in mixed hippocampal and striatal cultures using cells loaded with the Ca2+ indicators fluo 3-AM or fura 2-AM. In nontreated cells loaded with fura 2-AM, the mean 340/380 nm ratios were 0.77 ± 0.13 (n = 67 cells from 3 preparations) and 0.76 ± 0.14 (n = 59 cells from 3 preparations) for hippocampal and striatal astrocytes, respectively. These ratios corresponded to resting Ca2+ levels between 65 and 90 nM.

The pattern and the relative number of Ca2+ responses in astrocytes loaded with fluo 3-AM were investigated using several concentrations of ET-1. As shown in Table 1, the percentage of responding cells was found to be dose dependent in astrocytes derived from both brain structures. In addition, the analysis of the shape of the ET-1-induced increases in [Ca2+]i indicated that these responses were heterogeneous. Indeed, four different patterns were identified with two main ones that prevail in >99% of the responses to 10-7 M of ET-1 (Table 1). In hippocampal astrocytes, a biphasic response with an initial Ca2+ peak followed by a sustained plateau or a monophasic sustained plateau were frequently seen at high ET-1 doses (10-7 or 10-8 M). At 10-9 M, the most common response was polyphasic Ca2+ oscillations, whereas, at even lower doses (10-10 M), the most frequent response was a single peak (Table 1). Typical Ca2+ increases illustrating the major pattern of responses are shown in Fig. 3. The response patterns obtained from hippocampal and striatal astrocytes were comparable, although the monophasic peaks and oscillations were frequent (17.9% of the responding cells, respectively) already at 10-8 M of ET-1 stimulation in the striatal astrocytes. ETB receptor-selective agonists ET-3 or IRL 1620 or the nonselective agonist SFTX, all used at 10-7 M, induced similar Ca2+ response patterns, as did ET-1 (10-7 M) in astrocytes originating from both brain regions (Table 1). The frequency of response to ET agonists was also investigated. The relative number of responding astrocytes was higher in hippocampal than in striatal astrocytes for all ET receptor agonists tested (ET-1, ET-3, IRL 1620, or SFTX) at 10-7 M, respectively. In hippocampal mixed cultures, >95% of the astrocytes responded with [Ca2+]i rises to ET-1 stimulation performed at 10-7 M, and a similar proportion of responding astrocytes was found for ET-3, IRL 1620, or SFTX used at the same concentration. In striatal cultures >80% of the astrocytes responded to the two ET isoforms used at 10-7 M. Also, the ETB receptor-selective agonist IRL 1620 and the nonselective agonist SFTX (10-7 M, respectively) were potent inducers of [Ca2+]i rises in cultured astrocytes from hippocampus and striatum since similar percentages of response were monitored (Table 1).

                              
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  		Table 1.   Intracellular Ca2+ responses induced by ET receptor agonists and antagonists/agonists in hippocampal and striatal astrocytes



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  		Fig. 3.   Four main types of Ca2+ responses to ET peptides are described. Shown are typical examples of these Ca2+ response types. These relative amounts of cells responding with a typical pattern after various stimulations and blockades are presented in Table 1. Exemplifying curves are ET-1 stimulation at 10-7, 10-8, 10-9, and 10-10 M from top to bottom. Note typical time delay from ET-1 addition to Ca2+ response in the 10-10 M stimulation.

The pharmacological properties of ET-1- or ET-3-evoked Ca2+ responses were further evaluated using selective ETA receptor and/or ETB receptor antagonists. When the ETA or ETB receptors (BQ-123 or BQ-788 at 10-6 M, respectively) were blocked, the relative number of astrocytes responding to ET-1 (10-7 M) was only marginally lower than that measured without blockage, whereas a mix of them almost completely abolished ET-1-induced [Ca2+]i increases in hippocampal and striatal astrocytes (Table 1). However, only an ETB receptor blockage was needed to eliminate ET-3-induced [Ca2+]i responses in hippocampal astrocytes and to reduce the response frequency to 1.8% in striatal astrocytes. In contrast, the effect of ETA receptor blockage by BQ-123 (10-6 M) was minor. Interestingly, although the relative number of cells responding to ET-1 stimulation was as high or almost as high with or without BQ-788 preincubation, the relative number of cells displaying a certain Ca2+ signal pattern was markedly changed. After ET-1 (10-7 M) stimulation no hippocampal and only one striatal astrocyte out of 500 and 310, respectively, displayed oscillations, whereas ET-1 stimulation after ETB receptor blockage resulted in 42 hippocampal and 43 striatal astrocytes displaying Ca2+ oscillations out of a total of 166 and 137 astrocytes in each region. After ETA receptor blockade, however, almost no oscillations or monophasic peaks were seen in response to ET-1 addition, thus showing a response pattern similar to that after pure ET-1 (10-7 M) stimulation. After simultaneous ETA and ETB receptor blockage, no striatal astrocytes responded to ET-1 (10-7 M), whereas all of the few responding hippocampal astrocytes (11 of 177) showed the typical low-dose pattern with a single peak.

Data on [Ca2+]i responses in single astroglial cells in hippocampal and striatal mixed cultures are presented in Table 1. ET-1 dose-response experiments were also performed in astroglial primary cultures devoid of neurons. Based on measurements from between 98 and 769 astrocytes (3-6 separate cell culture preparations) for each treatment, the results showed a response frequency of 93.8%, 78.1%, 38.0%, and 3.3% in hippocampal cultures and 83.6%, 56.5%, 38.3%, and 3.3% in striatal cultures for 10-7, 10-8, 10-9, and 10-10 M ET-1 stimulation, respectively. The relative response pattern at different ET-1 doses was similar to astrocytes in mixed cultures (data not shown).

ETs inhibit the propagation of intercellular Ca2+ signaling. Mixed cultures of neurons and astrocytes were used routinely throughout this work (5-10% of the cells were neurons). The possibility for direct interactions between astrocytes and neurons on cellular Ca2+ homeostasis (47, 52) was avoided by studying the propagation of Ca2+ waves in neuron-free regions of the mixed cultures.

Mechanical targeting of a single astroglial cell induced a wave of increased [Ca2+]i, propagating out from the stimulated cell in all directions. Approximately 80% of the astrocytes in the area passed by the wave responded with [Ca2+]i increases. The mean velocity of Ca2+ wave propagation was initially 25-30 µm/s. The velocity decreased rapidly in the beginning of the wave propagation and then stabilized with a lower rate of the velocity decrease after 100-150 µm of propagation, until it was later abruptly annihilated. In hippocampal cultures, the Ca2+ waves propagated 150-250 µm radially from the stimulated cell. A thorough description of the wave characteristics in mixed hippocampal cultures is given in Ref. 7.

ET-1 inhibited astroglial intercellular Ca2+ wave propagation in a dose-dependent manner in hippocampal and striatal cultures (see Fig. 5). In Fig. 4, top sequence, a typical control Ca2+ wave propagation is shown in pseudo color. On superfusion with 10-7 M ET-1, almost all cells responded with increased [Ca2+]i (Fig. 4, middle sequence, and see Table 1). When restimulating the same cell mechanically after 10 min of ET-1 treatment, [Ca2+]i increased in this cell, but no wave propagation occurred (Fig. 4, bottom sequence). Occasionally its closest neighboring cells also displayed increased [Ca2+]i in cultures from hippocampus and striatum. Similar inhibitory effects on the extent of Ca2+ wave propagation were obtained with ET-3 (10-7 M, 10 min; Fig. 5). Furthermore, the ETB receptor-selective agonist IRL 1620 and the nonselective agonist SFTX used in identical conditions mimicked the effect of ET isoforms on the extent of Ca2+ wave propagation. The propagation areas were reduced to 16.5 ± 9.3% and 6.0 ± 3.3% in hippocampal and 25.9 ± 5.5% and 17.3 ± 3.1% in striatal astrocytes for IRL 1620 and SFTX, respectively (data in percent of control ± SE, n = 3 for each combination). The ET-1-induced (10-7 M) inhibition (percent of control ± SE) of Ca2+ wave propagation areas, 8.1 ± 1.8% in hippocampal and 5.7 ± 1.4% in striatal astrocytes, was only partially blocked by BQ-123 or BQ-788. The propagation areas (percent of control ± SE) in BQ-123/ET-1-treated astrocytes were 26.5 ± 7.6% and 14.8 ± 3.7% in hippocampal and striatal astrocytes, respectively. Similarly, in BQ-788/ET-1-treated cells the areas were 30.2 ± 5.7% and 12.3 ± 1.5% for hippocampal and striatal astrocytes, respectively (Fig. 5). However, a mix of BQ-123 and BQ-788 completely blocked the ET-1 effect and wave propagations were 106.1 ± 7.2% and 108.5 ± 5.9% of control for hippocampal and striatal astrocytes, respectively (Fig. 5). As in the case of dye transfer, the effect of the antagonists when coincubated was more than additive in the present study (P < 0.05 in striatal astrocytes, not significant in hippocampal astrocytes). The ET-3-induced inhibition of the Ca2+ waves was only marginally affected by BQ-123; however, it was completely blocked by BQ-788 alone in hippocampal and striatal astrocytes (Fig. 5). Thus, when ET-1 or ET-3 effects were blocked, a pharmacological profile for inhibition of Ca2+ wave propagation, similar to that for dye transfer, was seen. Although the relative inhibitory effect of ETs on Ca2+ wave propagation was similar in the two brain region cultures, striatal astrocytes displayed a lower extent of Ca2+ wave propagation [77.3 ± 9.5% (percent of the extent in hippocampal astrocytes ± SE)] than that for hippocampal astrocytes (P < 0.05, n = 5 separate culturing weeks).


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  		Fig. 4.   Pseudocolor images of 3 successive experiments in a part of a microscopic field (×20 lens). Shown under each time-lapse pseudocolor sequence are fluo 3 fluorescence traces [(F - F0)/F0] of 4 cells over time (x-axis in seconds). Trace 1 (red trace) is mechanically stimulated cell, and cells 2, 3, and 4 (green, yellow, and blue traces, respectively) are lying along the wave propagation direction of a typical control mechanically induced intercellular Ca2+ wave (top sequence). Cells were then bath stimulated with ET-1 (10-7 M; middle sequence), 10 min after the control wave. In bottom sequence, a new Ca2+ wave was elicited 10 min after addition of ET-1. This wave was induced from the same cell as shown in top sequence and with ET-1 from the middle sequence still on. Fluorescence changes over time are shown for the same 4 cells in all experiments. These cells are indicated with arrows in 1 frame in which they are all visually distinct. Sampling was done at 3 Hz, and then an averaging of 3 frames in line was performed. Thus the frames shown correspond to average fluorescence in 1 particular second, which is indicated at top left of each frame. Stimulation, either mechanical or with drug addition, was performed at time 0. The first frames in middle and bottom sequences show 2 s before each stimulation, respectively, to show that the fluorescence corresponding to intracellular Ca2+ concentration ([Ca2+]i) was returned to resting levels. Note time delay between [Ca2+]i increases in cells lying along Ca2+ wave direction (top sequence), more simultaneous and random order of [Ca2+]i increases after ET-1 bath stimulation (middle sequence), and failure of wave propagation after mechanical stimulation in ET-1-treated cells (bottom sequence). Shown is 1 typical experiment out of 4, with repeated mechanical stimulation on the same cell after 10 min of ET-1 incubation.



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  		Fig. 5.   Quantification and pharmacological study of ET-induced inhibitory effect on extent of intercellular Ca2+ wave propagation in hippocampal (A) and striatal (B) astrocytes. ET-1 induced a concentration-dependent inhibition of the extent in hippocampal (A) and striatal (B) astrocytes. Incubation with ET-1 or ET-3 (10-7 M) for 10 min significantly decreased dye transfer in hippocampal (A) and striatal (B) astrocytes, compared with control (P < 0.001). Note that data are presented as mean percent of control ± SE, while pairing selected groups of interest made statistical comparisons. ET-1-induced inhibition of Ca2+ wave propagation was not affected or was marginally affected by ETA (BQ-123) or ETB (BQ-788) receptor blockage (P < 0.05 for BQ-788 in hippocampal astrocytes). However, the ET-1 effect was completely prevented after simultaneous blockage of both receptors. A synergistic antagonism (more than additive effect) of BQ-123 and BQ-788 when coincubated was seen in hippocampal (not significant) and in striatal astrocytes (P < 0.05) in present experiments. ET-3 effect in hippocampal and striatal astrocytes was only partially affected by ETA receptor blockage (P < 0 .05). However, it was completely inhibited by ETB receptor blockage in hippocampal (A) as well as in striatal (B) astrocytes. Numbers of separate experiments (cultures from different cell preparations) are indicated above each bar.

As presented above, ET peptides induced rises in [Ca2+]i in hippocampal and striatal astrocytes. One possibility for blocking intercellular Ca2+ waves could be by emptying intracellular Ca2+ stores. To investigate this issue, we followed the fluo 3 fluorescent signal or fura 2 ratio corresponding to intracellular free Ca2+ levels for 10 min and then added thapsigargin (10-7 to 10-5 M). The results show that [Ca2+]i returned to resting levels within 3-8 min after the ET-1 addition. At the addition of thapsigargin, the fluorescent signal increased again, showing that the intracellular Ca2+ stores were refilled with Ca2+, although ET-1 was still present in the bath (Fig. 6). To further investigate intracellular Ca2+ homeostasis, after 10 min of ET-1 incubation extracellular Ca2+ was thoroughly rinsed away using an ET-1-containing Ca2+-free HHBSS-BSA buffer (with 1 mM EGTA), and mechanical stimulation was performed directly afterward. The fluorescent signal in the mechanically stimulated cells increased to control levels, but no intercellular waves were elicited (n = 4, not shown), further indicating filled intracellular Ca2+ stores but blocked intercellular Ca2+ signaling. Another argument against store depletion is the unaltered Ca2+ response amplitude in the mechanically stimulated cell after 10 min of ET incubation compared with control (see top sequence and bottom sequence in Fig. 4).


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  		Fig. 6.   ET-1 (10-7 M) elicited [Ca2+]i transients in hippocampal astrocytes in mixed astroglial-neuronal culture. [Ca2+]i level was returned to resting levels within 3-8 min after addition of ET-1. Filling state of intracellular Ca2+ stores was tested with addition of thapsigargin (Thap, 10-6 M) after 10 min of treatment with ET-1. This experimental setup with thapsigargin was tested using fluo 3 (n = 3) and fura 2 (n = 3). Each experiment involved 12-50 astrocytes. Shown is [Ca2+]i response in 1 astrocyte in a typical experiment with fura 2 ratio (340/380 nm) over time. [Ca2+]i levels were estimated from fura ratio (see MATERIALS AND METHODS). Shown is a typical experiment with resting [Ca2+]i estimated to 75 nM and an ET-1 peak at ~350 nM. Due to long sampling time in ultraviolet (UV) light and the importance of reliable [Ca2+]i levels, the light was turned off twice as indicated.

Astroglial primary cultures devoid of neurons and originating from the same brain structures as the mixed cultures were used to demonstrate that the effects of ETs on astroglial gap junctions and intercellular Ca2+ waves are due to the stimulation of astrocytic receptors. Indeed, these comparative experiments in neuron-free primary astroglial cultures showed a similar relative blocking effect on the extent of Ca2+ wave propagation by ET-1 at various concentrations. As in the case of mixed cultures, the Ca2+ waves were not significantly affected by 10 min of treatment with 10-10 M ET-1, where the extent of propagation was 106.9 ± 6.0% (n = 3) and 95.7 ± 6.3% (n = 5; mean percent of control ± SE) for hippocampal and striatal astrocytes, respectively. Furthermore, also in these cultures the intercellular astroglial Ca2+ signaling was strongly reduced by treatment with 10-7 M ET-1, where the extent of propagation was 4.8 ± 2.6% (n = 3) and 1.4 ± 0.72% (n = 3) (percent of control ± SE) for hippocampal and striatal astrocytes, respectively (not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this report, ET-1, ET-3, IRL 1620, and SFTX were shown to inhibit gap junction permeability and to block the propagation of intercellular Ca2+ waves. These observations confirm that, in the brain, astrocytes represent an important target for ETs. The effects of these peptides may be physiologically relevant, since they suppress one of the characteristic properties of this class of glial cells, which constitute >40% of the cell number in the brain. ET-induced inhibition of gap junctional permeability indicates that the level of intercellular communication between astrocytes can be switched off when the level of ETs in the brain is increased. Gap junctional communication is involved in multiple functions of astrocytes, such as the following: intracellular and extracellular ionic homeostasis (55), metabolic trafficking (20), proliferation (46), neuroprotection (5), propagation of death signals (42), and cell swelling (61). Consequently, it is likely that several biological effects of ETs in the brain could be related to their potent action on astroglial gap junction channels reported here. Several observations already argue in favor of this statement, since ET-1 was reported to control intercellular diffusion and the use of glucose (20) and since ETs stimulate astrocytic proliferation (44, 64) and are involved in cell volume regulation (4). Moreover, we observed that the propagation of intercellular Ca2+ waves is blocked by ETs with a pharmacological profile similar to that in the inhibition of gap junction permeability. Finally, the difference in the effect of selective antagonists for ET receptor subtypes on the inhibitions induced by ET-1 and ET-3 suggests that in cultured astrocytes ET isoforms act, at least in part, at different binding sites.

The mixed preparation was selected because the coexistence of neurons with astroglia has been shown to give a more in-vivo-like astroglial morphology (28) and to increase dye coupling between astrocytes (15). As described previously (7), the neurons and astrocytes in mixed astroglial-neuronal primary cultures can be morphologically and immunologically distinguished. Furthermore, astrocytes but not neurons are immunopositive for the major astroglial gap junction protein, connexin 43, in mixed hippocampal primary cultures (6). In scrape loading experiments, cells with neuronal morphology were occasionally loaded with Lucifer yellow; however, most of these neurons were in the calculated area of loaded astrocytes. The area occupied by the rare neurons pointing out from this area was below the sensitivity available in this method. No dye transfer was detected from these rare neurons to astrocytes outside the loaded astroglial area, as investigated after the first week of coculture. The Lucifer yellow-filled neurons seen here could have been loaded either by astroglia-neuron junctional coupling (16) or by direct loading through a cut neuronal cell process.

Gap junctional communication and intercellular Ca2+ waves. The similar effect of ETs on the passive intercellular diffusion of a dye and on the cell-to-cell propagation of Ca2+ signaling molecules indicates that, in our preparation, functional gap junctions play a critical role in astrocytic Ca2+ waves. This statement converges with several previous studies that have reported that nonphysiological uncoupling agents block the Ca2+ wave propagation process. In addition, receptor ligands, such as alpha 1-adrenergic agonist, ATP, and anandamide, which inhibit gap junctional permeability, were also shown to prevent the spread of Ca2+ waves in astrocytes. However, several studies have demonstrated that an external component is also involved in the propagation process of astrocytic Ca2+ waves (10, 24). The participation of these two alternative pathways seems to vary following the studies (see Refs. 8 and 21). These differences could be due, for instance, to a difference in the cell preparations used or in the stimuli used to trigger the waves. However, several data presented here argue for a participation of gap junctional communication in the spread of astrocytic Ca2+ waves. First, there is a very close correlation between the dose dependence of ET-1 in blocking the rate of dye diffusions and in inhibiting the extent of Ca2+ waves in response to mechanical stimulation. This suggests that the two phenomena might be linked. Second, the pharmacological profiles of the two sets of experiments are principally similar, which also argues for a common mechanism. Third, ET treatment that blocks gap junction permeability does not affect other parameters known to be important in the propagation process. Indeed, the integrity of the internal Ca2+ stores is restored and basal [Ca2+]i levels are normalized within 3-8 min of ET treatment. Finally, the comparison of astrocyte properties in cultures from the hippocampus and the striatum indicates that the brain region with the highest gap junctional permeability shows the most extended Ca2+ waves. Although this higher number of responding cells might be due to a difference in other steps involved in Ca2+ signaling, the more efficient gap junctional permeability in hippocampal astrocytes could be responsible for this difference. Indeed, it is noteworthy that cultured hippocampal astrocytes are highly coupled by gap junctions compared with several brain regions (6), whereas striatal astrocytes communicate less well through gap junctions compared with astrocytes originating from hypothalamus (3). These dye transfer observations were correlated with a higher level of expression of connexin 43, the major gap junction protein in astrocytes.

ETs and intercellular Ca2+ signaling in astrocytes. As a whole, astrocytic responses to ETs indicate that these peptides have multiple effects on the process of intercellular Ca2+ signaling in astrocytes. As shown by focal application of ET-1, the stimulation of ET receptors in a single astrocyte is sufficient to initiate a Ca2+ wave, which involves 20-30 adjacent cells (71). Besides that, we report here that ETs are also potent inhibitors of these waves, likely through the closure of gap junction channels. These two actions seem to be opposite; however, this is not the case if one considers that an important aspect of the action of ETs is their kinetics. As indicated by the duration of the initial peak of the increase in [Ca2+]i (<20 s) and the propagation speed of the ET-1-induced Ca2+ waves (15-20 µm/s) (71), the initiation and propagation of the waves is a rather fast phenomenon. Indeed, an increase in [Ca2+]i triggered by the stimulation of ET receptors in a single cell can be transmitted to >20 of its neighbors in <20 s. During that period of time it is unlikely that gap junctional channels are already closed by the peptides. An idea of the kinetics of this inhibition is given by double patch-clamp recordings of junctional current between pairs of astrocytes, which indicate that complete uncoupling occurs after a 120-s application of ET-1 (19). Accordingly, the effect of ETs on intercellular communication should be considered as a dynamic and sequential process. Activation of ET receptors in astrocytes first increases [Ca2+]i in the stimulated cell due to the production of inositol trisphosphate (IP3) and the release of Ca2+ from internal stores. Thanks to the passage of IP3 and/or Ca2+ through gap junctional channels, this rise in [Ca2+]i is transmitted to adjacent cells. Then, by a passive and/or active process of diffusion, the increase in [Ca2+]i propagates to other receiving cells. If the stimulation of ET receptors is long enough, the inhibition of gap junction channels occurs and leads to the uncoupling of astrocytes. Therefore, apparently ETs seem to have two opposite actions in astrocytes. First it generates communication between groups of cells, and then it isolates each cell from the others. In fact, these two events must be considered as a sequence and thus might account for a more subtle mode of action. ET-1 is one of the most powerful activators of the Ca2+ signaling in astrocytes (14). It is able to generate waves, which involves a large number of cells compared with neurotransmitters, such as glutamate and alpha 1-adrenergic and muscarinic receptor agonists (70). These ET-induced increases in [Ca2+]i have multiple intracellular targets and evoke biological responses that may develop with a different fate in communicating and noncommunicating astrocytes. Finally, since the two effects of ETs described here can occur with a different time scale, dissociation between the generation of Ca2+ waves and the uncoupling process is expected, depending on the duration of the ET stimulation. Indeed, the triggering of a wave requires only a transient increase in ET level, whereas the inhibition of gap junction channels and the block of Ca2+ waves need a much longer peptide exposure time (1-2 vs. 120 s). Thus it can be postulated that, depending on the mode of release and the reason for the increase in ET level, only one (the first) or both effects will occur.

Receptors responsible for the ET effects on gap junction permeability and Ca2+ waves. In the brain, in situ hybridization showed that ETA receptors are mostly expressed on vascular cells, whereas ETB receptors are abundant on glial cells (31). However, expression of both ETA and ETB receptor mRNA has also been detected on astrocytes (13). In addition, receptor autoradiography has indicated that the rat striatum contains essentially ETB receptors (68). More recently, binding studies have indicated single receptor kinetics with both ETA and ETB receptor-like pharmacology (34).

Intracellular Ca2+ responses were frequently seen when ET receptors were stimulated. The response pattern to ET-1 stimulation varied with the concentration, which is in line with an earlier report on ET-3 (22). Interestingly, although the response frequency to ET-1 stimulation after blockage of either ETA or ETB receptors was similar, the pattern of the Ca2+ response varied. It has been discussed that different spatiotemporal Ca2+ signal patterns might mediate signal-specific activation of various Ca2+-sensitive proteins and thereby differentiated responses to various stimuli (see Ref. 69). Indeed, recent experimental data show that the Ca2+ oscillation frequency can induce specificity in gene transcription (11, 41). Thus our results indicate that astrocytes, in some senses, might respond differently, depending on the ET receptor binding site that is stimulated.

Part of the results obtained with astrocytes cultured from hippocampus and striatum suggests that ETs inhibit gap junction permeability and Ca2+ wave propagation with a pharmacology indicating the stimulation of ETB receptors. Indeed, SFTX and the ETB receptor agonists ET-3 and IRL 1620 reproduced the ET-1 effects. Furthermore, the block of ET-3-induced responses in the presence of the selective ETB receptor antagonist BQ-788 strengthens the possible involvement of ETB receptors. However, another set of observations indicates that ET responses are not solely evoked through the activation of ETB receptors. Indeed, the use of ETA or ETB receptor antagonists BQ-123 and BQ-788, respectively, is only marginally efficient in blocking ET-1 inhibition of gap junction permeability and Ca2+ waves. In fact, these antagonists must be applied together to fully prevent the effect of ET-1.

To account for the differences observed in the action of BQ-123 and BQ-788 to antagonize the effect of ET-1 and ET-3, several possibilities can be advanced. First, these peptides could act on two different types of receptors linked by G proteins to the same transduction pathway. Alternatively, the stimulation of two distinct classes of receptors could lead to the activation of different signaling pathways, which induce separately the inhibition of intercellular communication. In both cases, ET-1 must act through both subtypes since blocking one of them is not sufficient to prevent its effect. According to previous works carried out in astrocytes, these two receptor subtypes should be ETA and ETB receptors. In this case, either a complete effect cannot be fulfilled by the stimulation of only one receptor or the partial blocking seen could be due to an unspecific weak antagonism at the respective other receptor for BQ-123 and BQ-788. However, the concentration of ET receptor antagonists used here has been shown to be specific in other systems (32, 33). A recent binding study described a competitive binding by ETB receptor ligands for ET-1 only in the presence of BQ-123 (ETA receptor antagonist) and a "cross talk" in terms of receptor dimerization was proposed (29). Such cross talk might explain presented data about blocking ET-1 function. Another possibility is that there is only one type of receptor, which is characterized by unusual binding properties for ET-1 and ET-3 due to the combination of two distinct binding sites. Such a proposal has recently been forwarded for cultured cortical astrocytes, with a model of an ETA/ETB hybrid receptor (34). Indeed, the presented synergism of ET receptor antagonism on ET-1 function is in line with a recent report on ET-1 function in cultured cortical astrocytes (27). If such a receptor is responsible for the effects seen here, it seems to inhibit junctional communication through the stimulation of either site but distinguishes between different bindings in terms on the Ca2+ signal pattern in single cells.

The use of ET receptor agonists and antagonists to discriminate between ETA and ETB receptors is still a problem, since so far there is no selective ETA receptor agonist (51). The future development of new pharmacological tools should help to explain the atypical pharmacology of the ET-induced inhibition of gap junction permeability and Ca2+ wave propagation. Another approach in addressing this question is to carry out a detailed analysis of the intracellular mechanism leading to the closure of gap junction channels. It is now well documented that several second messengers can inhibit the permeability of gap junctions. These messengers include arachidonic acid, nitric oxide, ATP depletion, and a large increase in [Ca2+]i (see Ref. 19). Several pathways that control the level of these second messengers are activated by ET-1 and/or ET-3 in astrocytes. Accordingly, the comparison of the intracellular mechanisms involved in ET-1 and ET-3 uncoupling should contribute to determining whether these isoforms act on the same class of receptor.

Functional implications. ET-1 and ET-3 have powerful effects on intercellular Ca2+ signaling in cultured astrocytes, most likely due to their effects on gap junctional permeability. This suggests that a number of astrocytic functions and neuroglial interactions may be altered when ET levels are increased in the brain. It will now be important to validate these observations in a more in-vivo-like preparation. There is already evidence for functional gap junctions in brain slices from striatum (25) and hippocampus (38) and Ca2+ wave propagation in astrocytes in hippocampal and neocortical slices (2, 53). In addition, functional astroglial gap junctions have been shown to contribute to redistribution of metabolites (65), to transfer toxic compounds after central nervous system (CNS) injury (42, 56), to provide neuronal protection to oxidative stress (5), and to provide intercellular Ca2+ signaling (21). Several other possible functions, e.g., a role in the regulation of extracellular ion and amino acid concentration, have been proposed for intercellular Ca2+ signaling in astrocytes (62). A possibility to selectively and differentially manipulate intercellular astroglial communication should be considered as one possible neuroprotective action, in which ET analogs could be useful. Indeed, ET antagonists have already been proposed as possible pharmacological tools in a variety of CNS dysfunction (1, 54), although the role of astrocytes has not been discussed.


    ACKNOWLEDGEMENTS

We are grateful to Drs. Elisabeth Svensson and Sture Holm for statistical advice. The skilful technical assistance of Ulrika Johansson and Barbro Eriksson is greatly appreciated.


    FOOTNOTES

This work was supported by the Swedish Institute, Göteborgs Kungliga Vetenskaps-och Vitterhets-Samhälle, the Swedish Society for Medical Research, and by grants from the Swedish Medical Research Council projects 14X-06005 and 04X-13015.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: F. Blomstrand, Institute of Neurobiology, Göteborg Univ., Box 420, SE-405 30 Göteborg, Sweden (E-mail: fbl{at}neuro.gu.se).

Received 1 April 1999; accepted in final form 17 June 1999.


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MATERIALS AND METHODS
RESULTS
DISCUSSION
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