Neuropeptide Y Y2 receptor signalling mechanisms in the human glioblastoma cell line LN319

Peptides 22 (2001) 379 –386

Neuropeptide Y Y2 receptor signalling mechanisms in the human glioblastoma cell line LN319 Eric Grouzmann*, Christine Meyer, Ernst Bu¨rki, Hans Brunner Division of Hypertension, Lausanne University Hospital, 1011 Lausanne, Switzerland Received 26 May 2000; accepted 18 August 2000

Abstract Neuropeptide Y (NPY) regulates neurotransmitter release through activation of the Y2 receptor subtype. We have recently characterized a human glioblastoma cell line, LN319, that expresses exclusively NPY Y2 receptors and have demonstrated that NPY triggers transient decreases in cAMP and increases in intracellular calcium responses. The present study was designed to further characterize calcium signalling by NPY and bradykinin (BK) in LN319 cells. Both agonists elevated free intracellular calcium ([Ca2⫹]i) without soliciting calcium influx. NPY appeared to activate two distinct signalling cascades that liberate calcium from thapsigargin- and ryanodine-insensitive compartments. One pathway proceeded through phospholipase C (PLC)-dependent phosphatidylinositol turnover, while the other triggered calcium release through a so far unidentified mediator. Part of the response was sensitive to pertussis toxin (PTX) under conditions where the toxin totally abolished the NPY-mediated effects on cAMP. The calcium release induced by BK on the other hand was largely PTX-insensitive, PLC-dependent, and from both thapsigargin- and ryanodine-sensitive stores. Following stimulation with NPY, subsequent [Ca2⫹]i responses to NPY were strongly depressed. Partial heterologous desensitization occurred, when BK was used as the first agonist, whereas NPY had no effect on a subsequent stimulation with BK. These data suggest that NPY-induced calcium mobilization in LN319 cells involves two different G proteins and signalling mediators, and a hitherto unidentified calcium compartment. Homologous desensitization of NPY signalling might be explained by receptor-G protein uncoupling, while heterologous desensitization by BK could be the result of either transient depletion or inhibition of a mediator in the calcium signalling cascades activated by NPY. © 2001 Elsevier Science Inc. All rights reserved. Keywords: NPY; Y2 receptor; Bradykinin; Signal transduction; Desensitization; Inositol phosphates; Intracellular calcium; cAMP; Glioblastoma.

1. Introduction Neuropeptide Y is a 36-amino acid peptide that binds to G protein coupled receptors [5,10]. NPY stimulates at least 5 type of receptors, called Y1, Y2, Y4 and Y5 [22]. Exposure to a Y1 agonist causes an increase in blood pressure and potentiates post-synaptically the action of other vasoactive substances [33] whereas Y2 receptors are mainly located presynaptically and upon stimulation mediate the inhibition of neurotransmitter release [34]. Many studies have focused on the signalling systems linked to the Y1 receptor subtype. Inhibition of forskolin-stimulated cAMP accumulation [1,24] and in some cases an increase in free intracellular calcium [Ca2⫹]i have been demonstrated in cell lines and primary cell cultures that bear the Y1 receptor * Corresponding author. Tel.: ⫹41-21-314-0741; fax: ⫹41-21-3140761. E-mail address: [email protected] (E. Grouzmann).

[1,11,23,24]. Conflicting data have been reported on inositol 1,4,5-trisphosphate (InsP3) generation following stimulation of the Y1 receptor [6,24,26]. In some cell lines, calcium mobilization was from a thapsigargin-sensitive store [21]. The intracellular signalling pathways associated with Y2 receptors in rat dorsal root ganglion neurons have been extensively studied by Miller and collaborators [3]. In these cells NPY inhibited Ca2⫹ influx by voltage-dependent calcium channels coupled to PTX-sensitive G protein [32]. In the same cells, NPY and BK induced an increase in [Ca2⫹]i associated with InsP3 formation even in the absence of extracellular calcium [26]. NPY utilizes PTX-sensitive G proteins and BK a toxin-insensitive one [26]. However, the NPY-induced effects were partially mimicked by 100 nM of 13–36 NPY a Y2 agonist with partial agonistic effects on the Y1 receptor-subtype. Thus, it can not be excluded that in addition to Y2 receptors, these neurons also bear Y1 receptors. Shigeri et al. have used a human neuroblastoma cell line, SMS-KAN, that exhibits only Y2 receptors based on

0196-9781/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 1 ) 0 0 3 4 4 - 8

380

E. Grouzmann et al. / Peptides 22 (2001) 379 –386

binding studies [28]. In these cells, NPY inhibits not only forskolin-induced cAMP accumulation and ␻-conotoxinsensitive Ca2⫹ influx but also angiotensin II- and bradykinin-induced Ca2⫹ release from intracellular stores. In contrast to dorsal root ganglion neurons, SMS-KAN cells do not increase their [Ca2⫹]i after exposure to NPY [28]. In another neuroblastoma cell line, CHP234, Lynch et al. found that NPY increases [Ca2⫹]i via a calcium entry through receptor-operated channels without mobilization from intracellular stores [19]. Furthermore, the effect was insensitive to pertussis toxin, whereas the coupling of NPY to adenylate cyclase in CHP234 was inhibited by the toxin [19]. We have previously described a human glioblastoma cell line, LN319, that possesses exclusively Y2 receptors [2]. This cell line responds to NPY by an inhibition of forskolin-stimulated cAMP accumulation [2] and by a dosedependent increase in [Ca2⫹]i [14]. The blocking effect of a newly characterized Y2 antagonist on the NPY-induced calcium mobilization was also demonstrated [14]. Our goal was to characterize the Y2 receptor signalling in LN319 cells especially in term of calcium and cAMP pathways. In addition, we have performed studies on homologous and heterologous desensitization of NPY Y2 receptor. BK was used as the heterologous agent since a clear cross-talk between this peptide and NPY has already been established. Indeed, BK has been reported to inhibit the venoconstrictor effect of NPY [25]. Moreover, BK causes an increase in intracellular calcium in LN319 cells. In the present study we show that NPY transiently increased [Ca2⫹]i via PTX-sensitive and -insensitive G proteins from thapsigargin- and ryanodine-insensitive intracellular pools, involving inositol phosphates and a hitherto unknown mediator. In contrast, BK signalling appeared to follow the classical Gq-InsP3-thapsigargin/ryanodine-sensitive calcium mobilization. NPY strongly and BK significantly depressed further stimulation with NPY, demonstrating homologous and heterologous desensitization of NPYmediated intracellular effects in LN319 cells.

2. Material and methods 2.1. Materials Porcine NPY and Bradykinin were purchased from Novabiochem (La¨ufelfingen, Switzerland). Fluo-3 AM and pluronic acid were obtained from Molecular Probes (Eugen, OR,) and the calcium ionophore 4-bromo A-23187 from Sigma. Pertussis toxin, ryanodine, thapsigargin, the inhibitor of phospolipase C, U-73122, and its inactive isomer U-73343 were from Calbiochem (Lucerne, Switzerland), and Tween 20 from Pierce (Rockford, IL, USA). [3H] inositol was purchased from Anawa (Wangen, Switzerland) and Dowex AG1-X8 columns from Bio-Rad (Glattbrugg, Switzerland). BSA (RIA grade) was from Sigma.

2.2. Cell culture LN319 cells were isolated from a human glioblastoma as described earlier [2] and grown in DMEM supplemented with 5% FCS, glutamin, 100 IU of penicillin, and 100 ␮g/ml of streptomycin in a 5% CO2/95% air incubator at 37°C. Tissue culture media were purchased from Gibco Life Technologies (Basel, Switzerland) and fetal calf serum was obtained from Seromed (Berlin, Germany). The cells were used for experiments from passage 190 to 210. In order to get reproducible data cells were passaged at 70% confluency and seeded at a 1:3 dilution. Culture medium was changed every 3 days. 2.3. Determination of cAMP Six-well plates, containing confluent LN319 cell cultures, were washed and incubated at 37°C for 1 hour in Eagle’s minimum essential medium (EMEM) containing 0.5% BSA, 4 mM MgCl2, 10 mM Hepes, 100 ␮M papaverin and 10 ␮M forskolin and one of the peptides to be tested. Cells were washed once in sodium phosphate buffer (100 mM pH 7.5) and lysed with 0.75 ml of 0.1 M HCl. After centrifugation, the supernatant was recovered and lyophilized. cAMP concentration was measured by an RIA using a commercially available kit (Amersham, Buckinghamshire, UK). In studies investigating the effects of pertussis toxin, cells were pre-treated with 25 ng/ml of the toxin for 16 hours. 2.4. Determination of [Ca2⫹]i LN319 cells were seeded on glass coverslips 48 hours before [Ca2⫹]i measurements were performed. The free ion concentration was determined using the fluorescent probe fluo-3/AM. The dye was loaded into the cells by adding the acetoxymethyl ester fluo-3 AM (2.5 ␮M) from a 1 mM stock solution in dry dimethyl sulfoxide (DMSO) together with 2 ␮l/ml of 25% pluronic acid in DMSO to DMEM without serum and supplements. The cells were incubated for 30 min at room temperature in the dark. After loading, the cells were washed three times with serum-free medium and placed in a chamber with 0.5 ml physiological saline solution containing 140 mM NaCl, 2 mM CaCl2, 4.6 mM KCl, 1.0 mM MgCl2, 10 mM glucose and 10 mM Hepes, pH 7.4. Tween 20 (0.0008%) was added to the medium to prevent the sticking of NPY to the exposed surfaces. Fluorescence images of the intracellular calcium localisation were obtained with a laser-scanned confocal microscope (MRC 500 confocal imaging system, Biorad) equipped with an argon ion laser and a fluorescein (488 nm) filter cartridge. The scanner and detectors were attached to an inverted microscope (Diaphot, Nikon). Calibration of the fluorescent signal was performed after each experiment using the nonfluorescent calcium ionophore A-23187 (10 ␮M) to saturate the intracellular dye with calcium and thereby obtain max-

E. Grouzmann et al. / Peptides 22 (2001) 379 –386

381

imal fluorescence (Fmax). The minimal fluorescence (Fmin) was measured after addition of an excess of EDTA (5 mM). Each image acquisition lasted 5 seconds. Basal fluorescence was determined by the mean of 2 consecutive images. Fluorescent intensities were then translated into free calcium ion concentrations using the equation [Ca2⫹] ⫽ Kd (F ⫺ Fmin)/Fmax ⫺ F). The changes in [Ca2⫹] were evaluated in single cells on whole images containing 5–15 cells using the NIH Image 1.58 software. 2.5. Data analysis Results are expressed as means ⫾ SEM. Statistical analysis of the data were performed by a Student’s unpaired t test or a two-way analysis of variance when appropriate. 2.6. Inositol phosphates determination LN319 cells grown in 35-mm dishes were labelled with [3H] inositol at 3 ␮Ci/ml for 15–18 h in inositol-free DMEM supplemented with 1% fetal bovine serum. After labelling, cells were stimulated for 30 min with 200 nM NPY or 50 nM BK in the presence of 20 mM LiCl. Inositol phosphates were extracted as described by Lattion et al. [16] and separated on Dowex AG1-X8 columns. Total inositol phosphates were eluted with 1 M ammonium sulfate, 0.1 M formic acid and the radioactivity measured in a ␤-counter.

3. Results 3.1. NPY-induced calcium response We have previously shown that NPY is able to induce dose-dependently an increase in [Ca2⫹]i in LN319 cells with a threshold and a maximum effect at 0.1 nM and 100 nM, respectively [14]. The dose response curves were similar whether only responding cells or the whole cell population were analyzed. Preliminary studies showed that the addition of physiological saline solution to the cells elicited a negligible increase in fluorescence corresponding to a ⌬[Ca2⫹]i of 4.1 ⫾ 5 nM (mean ⫾ SD, n ⫽ 73). Therefore, we assessed as significant any increase in [Ca2⫹]i greater than 14 nM (mean ⫹ 2 SD) in all further studies. Here, we first examined the ability of NPY to raise [Ca2⫹]i in the absence of calcium in the extracellular medium to establish whether the increase in [Ca2⫹]i is of intraor extra-cellular origin. The coverslip was loaded with physiological saline solution containing calcium and when the cells were in focus, the medium was aspirated, replaced by a calcium-free solution, and confocal images acquired immediately. This procedure was used to avoid prolonged removal of extracellular calcium that might cause a depletion of free calcium from intracellular stores. The calibration was performed after adding back 2 mM calcium. As shown in Fig. 1, NPY was equally effective in calcium-

Fig. 1. The increase in intracellular calcium in response to 100 nM NPY in LN319 cells was independent of the presence of 2 mM extracellular calcium but partially blunted after inhibition of PLC by 10 ␮M U73122. The number of cells used was 250, 34, and 26, respectively (* p ⬍ 0.05).

supplemented and in calcium-free medium. NPY at 100 nM induced a mean ⌬[Ca2⫹]i in the total cell population of 56 ⫾ 4.0 nM (n ⫽ 250) in normal medium versus 59 ⫾ 9.3 nM (n ⫽ 73) in Ca2⫹ free medium. Mean ⌬[Ca2⫹]i for responding cells only was 93 ⫾ 5.0 nM in normal medium versus 96 ⫾ 12 nM in Ca2⫹ free medium. In addition, the fraction of responding cells was similar whether calcium was present in the medium or not (71 and 74% of cells, respectively). This observation indicates that NPY mobilizes [Ca2⫹]i from intracellular compartments and does not promote calcium influx. In order to investigate the mechanisms involved in intracellular Ca2⫹ release, we then analyzed the effects of phospholipase C inhibition on the NPY-induced calciumresponse. For this purpose, we pre-incubated LN319 cells for 2.5 minutes with the phospholipase C inhibitor, U73122. This agent, at a concentration of 10 ␮M, caused a 54% inhibition of the NPY-induced intracellular calcium response (Fig. 1), whereas its inactive isomer U73343 was devoid of any effect. In contrast to the blunting of the calcium response, U73122 had no effect on the ability of NPY to inhibit forskolin-stimulated cAMP accumulation (data not shown). To confirm the participation of phospholipase C in the NPY-activated calcium-pathway, we measured inositol phosphates accumulation. NPY stimulates the formation of inositol phosphates by 28% over control untreated cells. These data suggest that in LN319 cells, NPY caused mobilization of [Ca2⫹]i in part by the formation of inositol phosphates, while for the remainder of the response an additional second messenger appeared to be implicated. It has been reported that NPY responses were abolished by thapsigargin, an inhibitor of the endoplasmic reticulum

382

E. Grouzmann et al. / Peptides 22 (2001) 379 –386

Fig. 2. Thapsigargin (50 nM) and ryanodine (10 ␮M) had no significant effect on the intracellular free calcium increase induced by 100 nM NPY. The number of cells tested for each experimental condition was 250 (control), 24 and 72, respectively.

Ca2⫹-ATPase [1,6,21]. Thapsigargin may act on both inositol phosphates-sensitive and -insensitive compartments [20,30]. In LN319 cells, 50 nM thapsigargin produced a slow and prolonged increase in [Ca2⫹]i that peaked at 49 ⫾ 6 nM. When NPY (100 nM) was added after the signal had returned to the baseline level, the free calcium response was not different from the one in control cells (Fig. 2). It is thus, unlikely that [Ca2⫹]i responses to NPY are associated with thapsigargin sensitive stores. In addition to thapsigarginsensitive pools, LN319 cells appear to contain ryanodinesensitive compartments. Ryanodine, an opener of intracellular calcium channels, at 10 ␮M induced a small but consistent increase in [Ca2⫹]i (19 ⫾ 5 nM). As for thapsigargin, ryanodine was ineffective in inhibiting the NPYinduced response (Fig. 2). We therefore conclude that in LN319 cells, NPY activates a phospholipase C and an unidentified mediator. The subsequent increase in inositol phosphates and other second messengers appears to mobilize calcium from an unknown compartment(s) that is insensitive to thapsigargin and ryanodine. 3.2. Effect of pertussis toxin NPY effects are generally associated with pertussis toxin (PTX) sensitive Gi and GO proteins [9,17,31]. Thus, we investigated whether or not, in LN319 cells, cAMP and [Ca2⫹]i responses to NPY were affected by a pre-treatment with PTX. Fig. 3 shows that after exposure to 25 ng/ml of PTX for 16 hours, the effects of NPY on forskolin-induced cAMP accumulation was totally abolished and the effects on [Ca2⫹]i strongly, but not completely, inhibited. Only 4% of the cells exposed to the toxin were still able to increase their [Ca2⫹]i. However, the sensitivity to depolarization

Fig. 3. a) NPY dose-dependently inhibited forskolin stimulated cAMP accumulation. This effect was totally abolished by pre-incubation of the cells for 16 hours with pertussis toxin (25 ng/ml). These data are the mean of 3 separate experiments performed in triplicate. ***p ⬍ 0.001, compared to forskolin-induced cAMP levels in the absence of NPY. b) Pertussis toxin under the same conditions inhibited the average NPY-induced calcium response by approximately 70%, but only 4% of the cells were still able to respond. A total of 250 and 75 cells were analyzed, respectively (*** p ⬍ 0.001).

with 50 mM KCl was conserved (data not shown). The NPY Y2 receptor in LN319 cells, thus, couples to the cAMP pathway through a PTX-sensitive G protein, and to calcium mobilization through PTX-sensitive and -insensitive ones. 3.3. Homologous and heterologous desensitization experiments Tachyphilaxis of NPY Y1 receptors is well documented [6,21], however, it is unknown whether or not this phenomenon also occurs with Y2 receptors. The NPY concentration inducing a threshold calcium response was 0.1 nM [14].

E. Grouzmann et al. / Peptides 22 (2001) 379 –386

383

Table 1 Homologous desensitization of calcium mobilization by NPY and heterologous desensitization of the NPY response by BK Agonist 1

Agonist 2

% Responding Cells (n)

Mean ⌬[Ca2⫹]i in nM (⫾SEM) in the total cell population

Mean ⌬[Ca2⫹]i in nM (⫾SEM) in responding cells

Vehicle Vehicle NPY 0.1 nM BK 50 nM Vehicle NPY 100 nM

NPY 100 nM NPY 0.1 nM NPY 100 nM NPY 100 nM BK 50 nM BK 50 nM

59 (250) 6 (31) 48 (31) 54 (192) 36 (192) 29 (79)

56 ⫾ 4 8⫾1 20 ⫾ 3** 39 ⫾ 4** 26 ⫾ 3 19 ⫾ 4

93 ⫾ 5 23 ⫾ 2 33 ⫾ 5*** 69 ⫾ 6** 70 ⫾ 6 66 ⫾ 8

Threshold levels of NPY (0.1 nM) desensitize the signalling cascade to further stimulation by the same agonist, while even maximal effective concentrations (100 nM) do not modulate the response to BK. Cells become refractory to respond to NPY (100 nM) when they are first stimulated with BK (50 nM). The values represent the response to the second agonist when the cells were stimulated twice (** p ⬍ 0.01 and ***p ⬍ 0.001 when compared to the response to a single stimulation with 100 nM NPY).

Even though, at this concentration of NPY the increase in [Ca2⫹]i was barely detectable, it was sufficient to strongly depress the response to a subsequent dose of 100 nM NPY, the maximally effective concentration (Table 1). Therefore, homologous desensitization occurs for both Y1 and Y2 receptors. We examined the effect of BK on [Ca2⫹]i in LN319 cells, since this peptide has been shown to cause transient free calcium rises through InsP3 synthesis in other cells [7,26,28]. As shown in table 1, 50 nM BK elicited an increase of [Ca2⫹]i approximately one half of that triggered by 100 nM NPY. In order to determine whether desensitization to one peptide also desensitizes the response to the other, we added the second agonist after the response to the first agonist had returned to baseline (about 1.5 min). Following the response to 50 nM BK, the [Ca2⫹]i rise elicited by 100 nM NPY was significantly reduced (39 ⫾ 4 nM (n ⫽ 192) after BK vs 56 ⫾ 4 nM (n ⫽ 250), p ⬍ 0.004. On the other hand, NPY only slightly and non-significantly attenuated the [Ca2⫹]i response to BK (19 ⫾ 4 nM (n ⫽ 79) after 100 nM NPY vs 26 ⫾ 3 nM (n ⫽ 192), p ⫽ 0.19. Thus, NPY induced calcium signals are sensitive as well to previous Y2 receptor occupation as to activation of another G protein coupled receptor. The increase in [Ca2⫹]i in response to 50 nM BK was independent of extracellular calcium (Fig. 4). As observed for NPY, phospholipase C dependent inositol phosphates accumulation was clearly involved in the BK-activated calcium signalling pathway. The phospholipase C inhibitor U73122 almost completely abolished the increase in [Ca2⫹]i (Fig. 4). Inositol phosphates accumulated by 14% over control in response to 50 nM BK. Taken together these data argue for an intracellular origin of the BK-mediated [Ca2⫹]i response in LN319 cells. To determine whether BK can mobilize calcium from the same stores as NPY, cells were stimulated either with 50 nM thapsigargin or with 1 ␮M ryanodine and after return to basal calcium levels, 50 nM BK were applied; in contrast to NPY signalling, BK responses were strongly inhibited by

thapsigargin and ryanodine (42% and 62% decrease, respectively; Fig. 5). The BK-induced calcium increase was, conversely to NPY, not significantly attenuated by PTX (Fig. 6). However, the fraction of responders fell from 36 to 16% after PTX treatment. These data suggest that the responses to NPY and BK are mediated, to a large extent, by different G proteins. In addition, mobilization of free intracellular calcium appears to occur from different compartments. It seems, therefore, likely that the signal crosstalk responsible for the BK-induced desensitization of the NPY response is not due to reduced availability of stored [Ca2⫹]i.

Fig. 4. Bradykinin-induced calcium mobilization did not depend on the presence of extracellular calcium. Blocking PLC by U73122 almost completely blunted the response. The number of cells analyzed were 192, 46 and 53, respectively (** p ⬍ 0.01).

384

E. Grouzmann et al. / Peptides 22 (2001) 379 –386

Fig. 5. Thapsigargin (50 nM) and ryanodine (10 ␮M) each inhibited the intracellular free calcium rise induced by 50 nM BK by approximately 50%. The number of cells tested for each experimental condition were 192 (control), 70 and 95, respectively (* p ⬍ 0.05 and ** p ⬍ 0.01).

4. Discussion The present study was designed to characterize the signal transduction pathways activated by NPY and BK in the human glioblastoma cell line LN319. We used confocal microscopy to examine the NPY-induced intracellular calcium response in single cells. This procedure allows one not only to analyze responses in single cells but it further permits evaluation of the percentage of cells that, at a given time, are able to respond to external stimuli. It is, indeed, a current but so far poorly understood phenomenon that

Fig. 6. Lack of effect of pertussis toxin treatment on the BK-induced calcium response in LN319 cells. Experimental conditions were as described in figure 3b. 192 and 51 cells were analyzed, respectively.

within a cell population, a certain number of cells are refractory to stimulation [4]. Heterogeneity in cellular responsiveness actually also occurs in primary cell cultures and most likely in intact tissues [8,29]. Therefore, taking into account only responding cells might lead to incorrect interpretations. We expressed our data as the mean values of responding and non-responding cells to avoid this bias. In order to distinguish between responders and non-responders, we have arbitrarily set the threshold increase in [Ca2⫹]i at 14 nM (mean ⫹ 2 SD). The percentage of responding cells might then serve as an indicator in cases where alterations in the response do not reach statistical significance. We show here that NPY mobilizes [Ca2⫹]i from intracellular stores by PLC-dependent generation of inositol phosphates involving PTX-sensitive and -insensitive G proteins coupling to the Y2 receptor. Since blocking PLC with a specific inhibitor only partially abolished the calcium response, it is likely that an additional, so far unidentified second messenger is generated by Y2 receptor activation. PTX sensitivity of Y2 receptor mediated calcium signalling has also been observed in dorsal root ganglion neurons [9] but not in two other cellular models [5,19]. The ability of NPY to inhibit forskolin-stimulated cAMP accumulation and its PTX-sensitivity, however, has occurred in all cell types studied so far. The NPY effect on cAMP in LN319 cells is independent of the PLC-dependent calcium signal since inhibiting PLC prior to stimulation with NPY leaves the cAMP effect intact. The BK evoked [Ca2⫹]i increase in LN319 cells is not significantly affected by PTX. Yet, the percentage of responding cells after PTX treatment drops from 36% to 16%. Since LN319 cells do contain one or several PTX-sensitive G proteins (coupled to the Y2 receptor), we cannot a priori exclude participation of such proteins in BK-dependent intracellular calcium mobilization. Tachyphylaxis to NPY is a common feature in cells bearing Y1 receptors [6,21]. We here show that the calcium signalling pathway activated by the occupancy of Y2 receptors is equally desensitized to further stimulation by NPY. Further investigations would be required to unveil the mechanisms responsible for this desensitizing phenomenon, but the most likely appears to be Y2 receptor phosphorylation by a G protein-coupled receptor kinase [12]. In the dorsal root ganglion model, simultaneous addition of NPY and BK results in an additive accumulation of InsP3 [26]. This suggests that no direct crosstalk exists between the two— distinct—signalling pathways. In LN319 cells these pathways are also independent when BK is presented subsequently to NPY, resulting in an unaltered calcium response to BK. However, the calcium response to NPY in LN319 cells is significantly attenuated when the cells are previously stimulated with BK, suggesting a one-sided crosstalk between the two signal transduction mechanisms. This observation fits with one of the physiological effects of NPY since it has been reported that venoconstrictor effects of neuropeptide Y are readily reversed by bradykinin [25]. The mechanisms of this heterologous desensitization are

E. Grouzmann et al. / Peptides 22 (2001) 379 –386

unknown. On the one hand it could rely on depletion of mediators activated by both agonists. As mentioned above, the BK receptor in LN319 cells couples almost exclusively to PTX-insensitive G proteins (most probably of the subtype Gq/G11 [35]), while the response to NPY is strongly attenuated by the toxin. Provided the toxin-independent part of the NPY response is mediated by the same Gq/G11 pool, activation of part of these proteins by BK could lead to an attenuation of the calcium response to NPY. Both signal transduction pathways involve PLC-dependent generation of inositol phosphates. Yet, we are at the present unable to tell whether the same PLC isoform is activated by NPY and BK. Finally, BK and NPY release calcium from different intracellular storage sites. The calcium response to BK is inhibited to approximately 50% by emptying either the thapsigargin- or the ryanodine-sensitive compartments, suggesting that these are the only calcium pools solicited by BK. In contrast, NPY-induced calcium transients are not modulated by these agents. The signalling cross-talk, therefore, appears not to be due to reduction of ion concentration in a calcium pool shared by the two pathways. Alternatively to the depletion of common mediators, there is increasing and solid evidence that rapid heterologous desensitization is brought about by selective phosphorylation events mediated by second messenger-dependent kinases [12]. In the present case, BK potentially can activate protein kinase C (PKC) and other calcium-dependent kinases. Protein kinase A is an unlikely mediator in such a process, since BK does not elevate cAMP in LN319 cells (data not shown). There are numerous potential substrates for such kinases along the NPY-activated signalling cascade. One of the targets is the Y2 receptor itself, since its primary structure contains several serine/threonine phosphorylation motives [27]. Phosphorylation of Gi␣ subunits in response to receptor- or phorbol ester-induced PKC activation inhibits calcium mobilization by other receptor ligands in human platelets [13]. Furthermore, chemoattractant receptor activation has been reported to inhibit PLC through phosphorylation, rendering the lipase insensitive to further stimulation by other chemotactic peptides [18]. Finally, in vascular smooth muscle cells, cGMP-dependent inhibition of calcium mobilization has been reported to correlate with InsP3 receptor phosphorylation [15]. In conclusion, we have demonstrated that the calcium signalling cascade activated by the Y2 receptor in the astrocyte LN319 cell line involves PTX-sensitive and -insensitive G proteins, formation of inositol phosphates and of another, so far unidentified second messenger, and subsequent mobilization of intracellular calcium from an undefined thapsigargin- and ryanodine-insensitive pool. BK on the other hand activates mainly PTX-insensitive G proteins and induces inositol phosphates-dependent calcium release from thapsigargin- and ryanodine-sensitive storage sites. NPY-induced calcium transients are sensitive to prior activation with NPY or BK. The heterologous desensitization is

385

likely to involve either BK-induced depletion or phosphorylation-dependent inhibition of one or several mediators in the Y2 receptor-activated signal transduction.

Acknowledgment We wish to thank Drs. G. Milligan, Glasgow, UK, and S. Cotecchia, Lausanne, for helpful discussions. The expert technical assistance of M. Munoz for the measurements of inositol phosphates and of K. Bouzourene for the calcium measurements were greatly appreciated. This work was supported by a grant from the Swiss National Science Foundation (grant No 31.53256-98).

References [1] Aakerlund L, Gether U, Fuhlendorff J, Schwartz TW, Thastrup O. Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase. FEBS Lett 1990; 260:73– 8. [2] Beck-Sickinger AG, Grouzmann E, Hoffmann E, Gaida W, Van Meir EG, Waeber B, Jung G. A novel cyclic analog of neuropeptide Y specific for the Y2 receptor. Eur J Biochem 1992;206:957– 64. [3] Bleakman D, Colmers WF, Fournier A, Miller RJ. Neuropeptide Y inhibits Ca2⫹ influx into cultured dorsal root ganglion neurones of the rat via a Y2 receptor. Br J Pharmacol 1991;103:1781–9. [4] Civitelli R, Fujimori A, Bernier SM, Warlow PM, Goltzman D, Hruska KA, Avioli LV. Heterogeneous intracellular free calcium responses to parathyroid hormone correlate with morphology and receptor distribution in osteogenic sarcoma cells. Endocrinology 1992;130:2392– 400. [5] Colmers WF, Pittman QJ. Presynaptic inhibition by neuropeptide Y and baclofen in hippocampus: insensitivity to pertussis toxin treatment. Brain Res 1989;498:99 –104. [6] Daniels AJ, Lazarowski ER, Matthews JE, Lapetina EG. Neuropeptide Y mobilizes intracellular Ca2⫹ and increases inositol phosphate production in human erythroleukemia cells. Biochem Biophys Res Commun 1989;165:1138 – 44. [7] Dray A, Perkins M. Bradykinin and inflammatory pain. Trends Neurol Sci 1993;16:99 –104. [8] Dufy-Barbe L, Bresson L, Sartor P, Odessa MF, Dufy B. Calcium homeostasis in growth hormone (GH)-secreting adenoma cells: effect of GH-releasing factor. Endocrinology 1992;131:1436 – 44. [9] Edwald DA, Sternweis PC, Miller RJ. Guanine nucleotide-binding protein Go-induced coupling of neuropeptide Y receptors to Ca2⫹ channels in sensory neurons. Proc Natl Acad Sci USA 1988;85: 3633–7. [10] Ewald DA, Matthies HJG, Perney TM, Walker TM, Miller RJ. The effect of down regulation of protein kinase C on the inhibitory modulation of dorsal root ganglion neuron Ca2⫹ currents by neuropeptide Y. J Neurosci 1988;8:2447–51. [11] Fredholm BB, Jansen I, Edvinnson L. Neuropeptide Y is a potent inhibitor of cyclic AMP accumulation in feline cerebral blood vessels. Acta Physiol Scand 1985;124:467–9. [12] Freedman NJ, Lefkowitz RJ. Desensitization of G protein-coupled receptors. Rec Progr Hormone Res 1996;51:319 –51. [13] Gates LK, Ulrich CD, Miller LJ. Multiple kinases phosphorylate the pancreatic cholecystokinin receptor in an agonist-dependent manner. Am J Physiol 1993;264:G840 –7. [14] Grouzmann E, Buclin T, Martire M, Cannizzaro C, Do¨rner B, Razaname A, Mutter M. Characterization of a selective antagonist of

386

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

E. Grouzmann et al. / Peptides 22 (2001) 379 –386 neuropeptide Y at the Y2 receptor. J Biol Chem 1997;272:7699 –706 (published erratum appears in J Biol Chem 1998;273:27033). Komalavilas P, Lincoln TM. Phosphorylation of the inositol 1,4,5trisphosphate receptor by cyclic GMP-dependent protein kinase. J Biol Chem 1994;269:8701–7. Lattion AL, Diviani D, Cotecchia S. Truncation of the receptor carboxyl terminus impairs agonist-dependent phosphorylation and desensitization of the alpha 1B-adrenergic receptor. J Biol Chem 1994;269:22887–93. Linder ME, Ewald DA, Miller RJ, Gilman G. Purification and characterization of Go alpha and three types of Gi alpha after expression in Escherichia coli. J Biol Chem 1990;265:8243–51. Lipinsky D, Nussenzveig DR, Gershengorn MC, Oron Y. Desensitization of the response to thyrotropin-releasing hormone in Xenopus oocytes is an amplified process that precedes calcium mobilization. Eur J Physiol 1995;429:419 –25. Lynch JW, Lemos VS, Bucher B, Stoclet JC, Takeda K. A pertussis toxin-insensitive calcium influx mediated by neuropeptide Y2 receptors in a human neuroblastoma cell line. J Biol Chem 1994;269: 8226 –33. Lytton J, Westlin M, Hanley MR. Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps. J Biol Chem 1991;266:17067–71. Michel MC. Rapid desensitization of adrenaline- and neuropeptide Y-stimulated Ca2⫹ mobilization in HEL-cells. Br J Pharmacol 1994; 112:499 –504. Michel MC, Beck-Sickinger AG, Cox H, Doods HN, Herzog H, Larhammar D, Quirion R, Schwartz T, Westfall T. XVI. International union of pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol Rev 1998;50:143–50. Mihara S, Shigeri Y, Fujimoto M. Neuropeptide Y-induced intracellular Ca2⫹ increases in vascular smooth muscle cells. FEBS Lett 1989;259:79 – 82. Motulsky HJ, Michel MC. Neuropeptide Y mobilizes Ca2⫹ and inhibits adenylate cyclase in human erythroleukemia cells. Am J Physiol 1988;255:E880 –5.

[25] Peduzzi P, Simper D, Linder L, Strobel WM, Haefeli WE. Neuropeptide Y in human hand veins: pharmacologic characterization and interaction with cyclic guanosine monophosphate-dependent venodilators in vivo. Clin Pharmacol Ther 1995;58:675– 83. [26] Perney TM, Miller RJ. Two different G-proteins mediate neuropeptide Y and bradykinin-stimulated phospholipid breakdown in cultured rat sensory neurons. J Biol Chem 1989;264:7317–27. [27] Rose PM, Fernandes P, Lynch JS, Frazier ST, Fisher SM, Kodukula K, Kienzle B, Seethala R. Cloning and functional expression of a cDNA encoding a human type 2 neuropeptide Y receptor. J Biol Chem 1995;270:22661– 4. [28] Shigeri Y, Fujimoto M. Y2 receptors for neuropeptide Y are coupled to three intracellular signal transduction pathways in a human neuroblastoma cell line. J Biol Chem 1994;269:8842– 8. [29] Takeda M, Nelson DJ, Soliven B. Calcium signaling in cultured rat oligodendrocytes. Glia 1995;14:225–36. [30] Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca2⫹ stores by specific inhibition of the endoplasmic reticulum Ca2(⫹)-ATPase. Proc Natl Acad Sci USA 1990;87:2466 –70. [31] Wahlestedt C, Grundemar L, Hakanson R, Heilig M, Shen G, Zukowska-Grojec Z, Reis DJ. Neuropeptide Y receptor subtypes, Y1 and Y2. Ann NY Acad Sci 1990;611:7–26. [32] Walker MW, Ewald DA, Perney TM, Miller RJ. Neuropeptide Y modulates neurotransmitter release and Ca2⫹ currents in rat sensory neurons. J Neurosci 1988;8:2438 – 46. [33] Walker P, Grouzmann E, Burnier M, Waeber B. The role of neuropeptide Y in cardiovascular regulation. Trends Pharmacol Sci 1991; 12:111–5. [34] Westfall TC, Carpentier S, Chen X, Beinfeld MC, Naes L, Meldrum MJ. Prejunctional and postjunctional effects of neuropeptide Y at the noradrenergic neuroeffector junction of the perfused mesenteric arterial bed of the rat. J Cardiovasc Pharmacol 1987;10:716 –22. [35] Wilk-Blaszczak MA, Gutowski S, Sternweis PC, Belardetti F. Bradykinin modulates potassium and calcium currents in neuroblastoma hybrid cells via different pertussis toxin-insensitive pathways. Neuron 1994;121:109 –16.