SEARCH

SEARCH BY CITATION

Keywords:

  • calcium response;
  • desensitization;
  • okadaic acid;
  • protein phosphatase;
  • purinergic receptor;
  • receptor endocytosis

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

De- and re-sensitization and trafficking of P2Y nucleotide receptors modulate physiological responses of these receptors. Here, we used the rat brain P2Y1 receptor tagged with green fluorescent protein (P2Y1-GFP receptor) expressed in HEK293 human embryonic kidney cells. Ca2+ release was used as a functional test to investigate ATP-induced receptor de- and re-sensitization. By confocal laser scanning microscopy (CLSM), endocytosis of P2Y1-GFP receptor was visualized in live cells. Stimulation of the cells with ATP induced complete receptor endocytosis within 30 min and appearance of the P2Y1 receptor in small vesicles. Removal of the agonist resulted in reappearance of the receptor after 60 min on the plasma membrane. Exposure of the cells to KN-62 and KN-93, inhibitors of the calmodulin dependent protein kinase II (CaMKII), prevented receptor internalization upon stimulation with ATP. However, the receptor which was still present on the plasma membrane was desensitized, seen by decreased Ca2+ response. The decreased Ca2+ response after 30-min exposure to ATP can be attributed to desensitization and is not as a result of depletion of internal stores, as the cells exposed to ATP for 30 min exhibited a normal Ca2+ response upon stimulation with thrombin. However, okadaic acid, an inhibitor of protein phosphatase 2A (PP2A), did not affect ATP-induced P2Y1 receptor endocytosis, but delayed the reappearance of the P2Y1 receptor on the plasma membrane after ATP withdrawal. Consistently, in okadaic acid-treated cells the ATP-induced Ca2+ response observed after the 30-min exposure to ATP recovered only partially. Thus, CaMKII seems to be involved in P2Y1 receptor internalization, but not desensitization, whereas protein phosphatase 2A might play a role in recycling of the receptor back to the plasma membrane.

Abbreviations used
ADP

adenosine diphosphate

ATP

adenosine triphosphate

CaMKII

calmodulin-dependent protein kinase II

[Ca2+]i

intracellular free calcium concentration

CLSM

confocal laser scanning microscope

DAG

diacylglycerol

DMEM

Dulbecco's modified Eagles's medium

FCS

fetal calf serum

GFP

green fluorescent protein

GPCR

G protein-coupled receptor

HEK293

cells, human embryonic kidney cells

InsP3

inositol 1,4,5-trisphosphate

KN-62

(S)-5-isoquinolinesulfonic acid 4-[2-[(5-isoquinolinylsulfonyl) methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl) propyl] phenyl ester

2-Me-S-ADP

2-methylthio adenosine diphosphate

2-Me-S-ATP

2-methylthio adenosine triphosphate

PBS

phosphate-buffered saline

PLC

phospholipase C

PP2A

protein phosphatase 2A

P2Y1-GFP

P2Y1, receptor with the GFP tag at the carboxy terminus

ROI

region of interest

Plasma membrane P2 receptors mediate the actions of extracellular nucleotides in cell signalling. These receptors have great clinical potential (Ralevic and Burnstock 1998; Agteresch et al. 1999). P2X receptors are ligand-gated ion channels, whereas P2Y receptors, seven transmembrane domain receptors, are coupled to G proteins. At present, the P2Y receptor family comprises eight cloned and functionally defined subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14), and all these subtypes are found in human tissue (von Kügelgen and Wetter 2000; Nicholas 2001).

P2Y1 receptors are activated most potently by the physiological agonist ATP, ADP and through the selective agonists 2-methylthioadenosine diphosphate (2-Me-S-ADP) and 2-Me-S-ATP. They are coupled both to pertussis toxin-insensitive Gq/G11 and pertussis toxin-sensitive Gi/G0 G proteins (Brown et al. 2000). P2Y receptors activate phospholipase C (PLC), leading to increased levels of inositol 1,4,5-trisphosphate (InsP3), diacylglycerol (DAG), cytosolic free Ca2+ ([Ca2+]i) and stimulation of protein kinase C, which in turn may activate phosphatidylcholine-specific PLC and phospholipase D (Ralevic and Burnstock 1998). Moreover, P2Y receptors were shown to regulate phospholipase A2, adenylyl cyclase, mitogen-activated protein kinase pathway and K+ and Ca2+ influx via voltage-operated channels (Bofill-Cardona et al. 2000; Powell et al. 2000; Wirkner et al. 2004).

After binding the activating ligands, G protein-coupled receptors (GPCRs) undergo a complex series of reactions to turn off the signal transduction. Part of this process is receptor desensitization to attenuate the response to stimulation. This was studied in detail in cell systems expressing β-adrenergic receptors (Ferguson and Caron 1998). After removal or degradation of the agonist, the GPCRs re-sensitize and the receptors regain their ability to respond to the ligands. Prolonged or repetitive stimulation of the cells can also result in a reduction of the number of receptors at the plasma membrane by receptor internalization. Sequestered receptors are either recycled back to the plasma membrane or sorted for degradation into lysosomes (Luzio et al. 2000; Oksche et al. 2000). The latter occurs most likely after prolonged agonist exposure.

Receptor internalization might be involved in processes leading to re-sensitization of the receptors (Ferguson and Caron 1998). Internalized receptors might be connected to further signalling pathways (Pierce et al. 2000). GPCRs that are coupled to adenylyl cyclase are believed to generally follow the scheme of receptor phosphorylation, desensitization, endocytosis and re-sensitization clarified in detail for the β-adrenergic receptors (Ferguson and Caron 1998; Milligan 1999). However, for Gq/PLC-coupled receptors, like the P2Y1 receptor, the mechanisms underlying agonist-induced desensitization and endocytosis are less well understood (Firestein et al. 1996).

We have previously generated an HEK293 cell line stably expressing rat brain P2Y1 receptors and another cell line expressing a chimera of P2Y1 receptor and green fluorescent protein (GFP) at the carboxy terminus. In fura-2AM-loaded cells, the Ca2+ response evoked through receptor activation has been examined. Pharmacological characterization of these heterologously expressed receptors with Ca2+ imaging technique showed a marked increase in high-affinity ligand recognition of adenosine di- and triphosphates in comparison with untransfected cells (Vöhringer et al. 2000; Zündorf et al. 2001). The EC50 values for the agonists 2-Me-S-ADP and 2-Me-S-ATP were 50 and 70 nm, respectively, which were substantially lower than in untransfected cells, where the EC50 values were 450 and 630 nm, respectively (Vöhringer et al. 2000). In the present study, these HEK293 cells transfected with the P2Y1-GFP receptor were used for investigation of receptor trafficking by confocal laser scanning microscopy (CLSM) in live cells with concomitant determination of de- and re-sensitization by measuring the Ca2+ response.

We investigated the effect of inhibition of protein kinase and phosphatase on receptor trafficking. CLSM was employed to visualize the trafficking of the receptor in live cells after stimulation of the cells with ATP. We also used the Ca2+ response to measure the functionality of the receptor on the plasma membrane. It was observed that inhibition of calmodulin dependent kinase II (CaMKII) by KN-62 inhibited internalization of the activated receptor, but did not prevent its functional desensitization. Treatment of the cells with okadaic acid, an inhibitor of protein phosphatase 2A (PP2A), delayed the recycling of the endocytosed receptor back to the plasma membrane and reduced the degree of functional recovery.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

Poly-l-lysine, heparin, thrombin were from Sigma (Deisenhofen, Germany); Geneticin (G 418), KN-62, KN-92, KN-93 and okadaic acid from Calbiochem (Bad Soden, Germany); Ham's F12, Dulbecco's modified Eagles's medium (DMEM), penicillin/streptomycine, fetal calf serum (FCS), Seromed (Berlin, Germany); cell culture dishes from Nunc (Wiesbaden, Germany); fura-2AM, LysoTracker Red from Molecular Probes (Eugene, OR, USA); 2-MeSADP, ATP, from Biotrend (Köln, Germany); enhanced GFP vector plasmid pEGFPN3 from Clontech (Heidelberg, Germany).

Cell culture and transfection

Transfection of HEK293 cells was carried out as described earlier (Vöhringer et al. 2000). In brief, HEK293 cells were transfected with the plasmid pEGFPN3 alone (as a control) or with a pEGFPN3 plasmid containing the full-length cDNA of the rP2Y1 receptor. Transfected cells were grown in medium consisting of DMEM/Ham's F12 (1 : 1), supplemented with 10% FCS, 100 IU/mL penicillin and 100 IU/mL streptomycin in a 5% CO2/95% air, humidified atmosphere at 37°C. The cells were plated on round coverslips (Ø = 22 mm) that were treated with poly-l-lysine (0.01%), before being placed into culture dishes (Ø = 50 mm) at a density of 5 × 105 to 1 × 106 cells/dish.

Functional characterization by measuring Ca2+ release

Intracellular Ca2+ release was determined in single cell measurements. HEK293 cells stably expressing P2Y1-GFP receptor were loaded with 2 μm fura-2AM for 30 min at 37°C in Na-HBS (HEPES-buffered saline solution: 145 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 25 mm glucose and 20 mm HEPES, pH 7.4). Only green fluorescent cells were used for evaluation within regions of interest (ROIs) by an imaging system (Till-Photonics, Martinsried, Germany) attached to a ZEISS Axioscope using a dichroic mirror LP490UV and an LP 515 emitter exciting at 488 nm. Intracellular Ca2+ release was measured corresponding to the equation described (Grynkiewicz et al. 1985) during alternate excitation at 340 and 380 nm. The cells were pretreated for 30 min with inhibitor and then stimulated with 100 μm ATP at 37°C, as described in the figures, at constant perfusion and the amplitude of the Ca2+ responses elicited was recorded.

Visualization of endocytosis of the rP2Y1-GFP receptor in live cells via confocal laser scanning microscopy (CLSM)

For imaging of agonist-induced endocytosis, HEK293 cells expressing P2Y1-GFP receptor were observed on coverslips at 37°C in a superfusion chamber under 5% CO2 in culture medium. After pre-incubation without or with 10 μm KN-62 or 5 nm okadaic acid and 100 nm LysoTracker for 30 min, 100 μm ATP was added. After a further 40 min, the cells were washed carefully with warm culture medium free of agonist and then observed for a further period of 120 min under the same conditions. Pictures were taken at the indicated time points with a Zeiss inverted LSM 510 confocal laser scanning microscope (CLSM) equipped with a Plan-Apochromat 63 × objective. Fluorescence of GFP was observed by using a 488-nm argon/krypton laser for excitation, and emitted fluorescence was detected with 505–530 nm band pass filter. For LysoTracker Red a 543-nm helium/neon laser was used for excitation and fluorescence was detected with a 560-nm long pass filter. Images were processed with Zeiss confocal microscopy software, release 3.2.

Data analysis

Unless stated otherwise results are presented as means ± SD and statistical analysis was achieved by Student's unpaired t-test using SigmaPlot (Jandel Scientific, Erkrath, Germany).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Regulation of P2Y1 receptor de- and re-sensitization

A cDNA encoding a modified form of GFP (eGFP) was linked to the C-terminus of the rat P2Y1 receptor cDNA. The fusion protein P2Y1-GFP receptor was stably expressed in HEK293 cells (Vöhringer et al. 2000; Zündorf et al. 2001). In these cells, the P2Y1-GFP receptor is mainly localized at the plasma membrane (Fig. 1a, inset). In the present work, we used the P2Y1-GFP receptor-expressing HEK293 cells to investigate the ATP-induced regulation of trafficking and functional activity of the receptor.

image

Figure 1. Functional de- and re-sensitization of P2Y1-GFP receptors, stably expressed in HEK293 cells. (a) In HEK293 cells transfected with P2Y1-GFP, the receptor is visualized in live cells by CLSM (inset; scale bar 20 μm). ATP-induced Ca2+ response by HEK293 cells expressing P2Y1-GFP receptor (average from 12 cells). The fura-2 ratio value was normalized to 1, referring to the value measured before applying the agonist ATP. Cells were stimulated with ATP (100 μm) for 30 min, then cells were allowed to recover for 60 min and stimulated for the second time with 100 μm ATP for 1 min. (b) Quantification of ATP-induced desensitization and following re-sensitization in experiments as depicted in (a). Cells were pre-incubated with 10 μm KN-62 or 5 nm of okadaic acid for 30 min, and the inhibitor was present in the buffer for the entire duration of the experiment. Stimulation of the cells by ATP with initial Ca2+ rise (i), Ca2+ level after 30 min of stimulation (ii), return to baseline Ca2+ value 10 min after withdrawal of the agonist (iii). At 90 min (60 min after agonist withdrawal) the Ca2+ response amplitude was tested by a 1-min ATP pulse (iv). Bars represent the mean values of Ca2+ responses. Increases in normalized fura-2 ratio (F340nm/F380nm) values are shown. Each value is the mean ± SD of 3–5 experiments, which comprise measurement of more than 70 single cells.

Download figure to PowerPoint

P2Y1 receptor desensitization, apparent by a loss of Ca2+ response, depends on the duration and concentration of the agonist stimulus applied. Desensitization was studied in experiments, where the Ca2+ responses were measured in cells without or with pretreatment with protein kinase and phosphatase inhibitors, KN-62 and okadaic acid. The cells were stimulated firstly with 100 μm ATP for 30 min (trace in Fig. 1a), and the initial Ca2+ response [Fig. 1a and (i) ] was determined. In the continued presence of ATP, the Ca2+ response declined, but the Ca2+ level remained still above the baseline [Fig. 1a (ii)]. After withdrawal of the agonist, the Ca2+ level dropped back to the baseline [Fig. 1a (iii)]. The cells were allowed to recover for a period of 60 min and were then stimulated again with a short 1-min pulse of 100 μm ATP. The Ca2+ rise that was observed in response to this stimulus [Fig. 1a (iv)] was analyzed to quantify re-sensitization. Under control conditions, i.e. without any pretreatment of the cells, the first 100 μm ATP stimulus raised the fura-2 fluorescence ratio by 1.61 times above the basal value (Fig. 1b black bar), which declined after 30 min to 0.104 times above basal value (6% of the initial response value). After withdrawal of the agonist, the Ca2+ concentration returned to the basal level. After the recovery period of 60 min, the second ATP stimulus of 1 min caused a fura-2 response of 1.21 times above basal value, amounting to 75% of the initial response value, as seen in the statistical analysis in Fig. 1(b). This indicates that the receptor was partially re-sensitized.

Similar desensitization experiments were carried out by using the P2Y1 receptor specific agonist 2-Me-S-ADP. With this agonist we found a comparable temporal profile of desensitization. The initial Ca2+ response to 2-Me-S-ADP (Table 1, response no. 1) was higher than that obtained with 100 μm ATP (Table 1, response no. 2). The Ca2+ level seen after 30 min of continuous stimulation with 100 μm 2-Me-S-ADP (Table 1, response no. 3) was not different from that obtained in the case of ATP (Table 1, response no. 4). After withdrawal of 2-Me-S-ADP, the Ca2+ level returned to basal values (Table 1, response no. 5). This is similarly observed in ATP-stimulated cells (Table 1, response no. 6).

Table 1.  Ca2+ responses obtained upon stimulation of HEK293 cells stably expressing rP2Y1-GFP receptor
Response no.Agonist, and mode of applicationTime of observation (min)Response amplitude (increase in ratio)
  1. Cells were stimulated with the two different P2Y1 receptor-specific agonists 2-Me-S-ADP and ATP (100 μm each), or for comparison with thrombin (5 U/mL). The values for the response amplitude give the mean increase of the ratio of fura-2 fluorescence (obtained at 340 and 380 nm). Each value is the mean ± SD of 3–5 experiments, which comprise measurement from more than 70 single cells. The time of observation gives the time point, at which the amplitude was determined.

12-Me-S-ADP, continuous for 30 min0 (initial)1.83 ± 0.18
2ATP, continuous for 30 min0 (initial)1.61 ± 0.11
32-Me-S-ADP, continuous for 30 min300.15 ± 0.02
4ATP, continuous for 30 min300.17 ± 0.03
52-Me-S-ADP, after continuous 2-Me-S-ADP application for 30 min with subsequent removal of agonist400.04 ± 0.03
6ATP, after continuous ATP application for 30 min with subsequent removal of agonist400.05 ± 0.024
7ATP, after 30 min of ATP applied in 1-min pulses at intervals of 2 min300.11 ± 0.09
8ATP, 1-min pulse at 32 min after continuous ATP application for 30 min with intermittent removal of agonist320.26 ± 0.082
9ATP, 1-min pulse at 32 min after 30 min of ATP applied in 1-min pulses at intervals of 2 min320.34 ± 0.136
10Thrombin, 1-min pulse at 34 min, after no. 8, after continuous ATP application for 30 min with intermittent removal of agonist340.74 ± 0.152
11Thrombin, 1-min pulse at 34 min, after no. 9 after 30 min of ATP applied at intervals of every 2 min340.71 ± 0.153
12Thrombin, 1-min pulse0 (initial)0.64 ± 0.056

A commonly used protocol for desensitization of GPCRs is application of repetitive stimuli at a given concentration of agonist to the cells at defined intervals of time. We repetitively stimulated the cells every 2 min for 1 min with 100 μm ATP. At the end of 30 min of this repetitive stimulation, we found that the Ca2+ level (Table 1, response no. 7) was similar to that obtained when the cells were exposed to 100 μm ATP continuously for 30 min [Fig. 1b (ii) and Table 1, response no. 4]. This result confirms that continuous stimulation of the cells and repeated stimulation of the cells result in a similarly desensitized Ca2+ response at the end of 30 min.

To test whether the receptor desensitization was complete, we stimulated the cells that were continuously exposed to 100 μm ATP for 30 min, at 32 min with 100 μm ATP for 1 min. This response (Table 1, response no. 8) was not significantly different from that obtained when the cells had been stimulated repeatedly before with 100 μm ATP (Table 1, response no. 9).

To confirm that the decrease in the Ca2+ signal is because of receptor desensitization and not as a result of depletion of the internal Ca2+ stores, we stimulated the cells with thrombin (5 U/mL) at 34 min. Thrombin was used as a stimulus for the PAR receptors, which are also GPCRs. PARs cause an elevation in intracellular Ca2+ via a mechanism like that elicited by the P2Y1 receptor (Wang and Reiser 2003). Previously we have characterized PARs in astrocytes (Wang et al. 2002) and in brain (Rohatgi et al. 2004). We observed that the cells did exhibit a robust Ca2+ response (Table 1, response no. 10). This demonstrates that the internal stores are not depleted. The response obtained with 5 U/mL thrombin in the case of cells that were repeatedly stimulated with 100 μm ATP was not different (Table 1, response no. 11) from that observed in the case of cells that were continuously exposed to 100 μm ATP. The rise in intracellular Ca2+ that was obtained with 5 U/mL thrombin in the ATP-pretreated cells was comparable with the control response obtained on stimulating P2Y1-GFP receptor-expressing HEK293 cells with 5 U/mL thrombin (Table 1, response no. 12). This indicates that the stores are not depleted and the decrease in the Ca2+ signal after treatment with ATP is as a result of receptor desensitization. In the following experiments we used ATP (100 μm), which is a naturally occurring agonist of the P2Y1 receptor.

In order to find candidate mechanisms that might be involved in the regulation of the P2Y1 receptor, we searched within the receptor sequence for consensus sequences for possible phosphorylation by protein kinases. We could identify by ProSite one potential CaMKII site in the third intracellular loop. This sequence that we obtained, RRKS (amino acids 255–258), was identically found in the sequence of the human P2Y1 receptor. To test if this protein kinase was involved in the regulation of the P2Y1 receptors, we performed Ca2+ measurements after pretreatment of the HEK293 cells with 10 μm KN-62, an inhibitor of CaMKII (Davies et al. 2000). Stimulation of the pretreated cells with 100 μm ATP gave an initial calcium peak with an amplitude of 1.17 (Fig. 1b, hatched bar). Continued stimulation with ATP resulted in a decrease in the response to 0.108 (9% of the initial response) after 30 min [Fig. 1b, hatched bar in (ii)]. With removing the agonist, the value returned to 0.062, which is the same as in control cells (Fig. 1b). After a further recovery period of 60 min without agonist, a short 1-min pulse of 100 μm ATP elicited a fura-2 response of 1.3 times above basal value [Fig. 1b, hatched bar in (iv)], which is 111% of the initial response seen in the presence of KN-62.

We also tested the effect of 5 nm okadaic acid, an inhibitor of protein phosphatase 2A. The concentration of 5 nm is specific for inhibiting PP2A only, as the isoform PP2B is inhibited at concentrations greater than 5000 nm and PP2C is not inhibited by it at all (Schonthal 1998; Zolnierowicz 2000). The IC50 value for okadaic acid with respect to PP2A is 1 nm. Pretreating the cells with 5 nm okadaic acid gave a reduced initial Ca2+ response [Fig. 1b, white bar in (i)]. The initial response to 100 μm ATP of 0.97 times above basal value is 40% lower than that obtained in the absence of okadaic acid. This did not affect the loss in Ca2+ response. It dropped to 0.19 (19% of the initial response in the presence of okadaic acid) upon continued exposure to ATP [Fig. 1b, white bar in (ii)]. After removal of the agonist, the value returned to baseline level of 0.04 [Fig. 1b, white bar in (iii)]. After a further 60 min without agonist, the 1-min pulse of ATP resulted in a Ca2+ response of 0.38 times above basal value. This response is only 39% of the initial Ca2+ response obtained in the case of okadaic acid-treated cells. Thus, pretreatment of the cells with okadaic acid strongly inhibited the recovery of the Ca2+ response. Representative traces of the Ca2+ response exhibited by the inhibitor-pretreated cells to ATP (100 μm) are found in the supplementary material. To get a clue about the mechanism underlying these differences in the Ca2+ response regulation, we also investigated the effect of these two substances on the trafficking of the receptor upon stimulation of the cells with ATP, in order to correlate de- and re-sensitization with internalization and recycling of the receptor.

Visualization of receptor trafficking

To visualize agonist-induced receptor trafficking, P2Y1-GFP receptor-transfected HEK293 cells were pre-incubated with LysoTracker Red and observed by two-channel CLSM (Fig. 2). In unstimulated P2Y1-GFP receptor-expressing cells, green fluorescence detection corresponds to the cell shape, consistent with a localization of the P2Y1-GFP receptor fusion protein mainly at the plasma membrane, whereas the LysoTracker signal is localized in vesicles in the cytoplasm (Fig. 2a). In control cells without exposure to stimuli, the distribution of the receptor and of the LysoTracker was unchanged for at least 150 min (data not shown). The following data of P2Y1 receptor translocation are shown, firstly, in Fig. 2 in three series of exemplifying pictures and, secondly, in the graph in Fig. 3, which gives a quantitative analysis derived from evaluation of respective ROIs.

image

Figure 2. Agonist-induced trafficking of rP2Y1-GFP receptor detected by live cell imaging. Transfected cells were pre-incubated with LysoTracker Red and visualized before, during 30 min of stimulation with ATP (100 μm) and during a following 120-min re-sensitization period. The pictures displayed were taken at the respective time points, which are indicated at the right side of each horizontal row of pictures. 2-channel confocal imaging allowed localization of GFP (green) and LysoTracker (red), which was performed at 37°C in culture medium in an atmosphere containing 5% (v/v) CO2. Control (a–e): in unstimulated cells (a) P2Y1-GFP receptor is on the plasma membrane. After 30 min of stimulation (b), endocytotic vesicles appear (asterisk), but no co-localization with LysoTracker even after 40 min (c). Removing the agonist results in disappearance of the small vesicles and reappearance of membrane fluorescence (d, e). Treatment with KN-62 does not affect the control localization of the receptor (f), but blocks receptor internalization (g, h). Treatment with okadaic acid has no influence on the control distribution of the receptor (i) and on the agonist-induced internalization (j) and there is no co-lcocalization with lysososmes (k). However, it impedes reappearance of the receptor on the plasma membrane (l, m). Results shown are representative of at least three individual experiments. In each case, the cell marked by the arrow is shown at the enlarged scale in the inset. Scale bar is 10 μm.

Download figure to PowerPoint

image

Figure 3. Quantification of fluorescence intensity in cells. The change in the fluorescence quantitated in user-defined regions of interest (ROI) that encompass either the plasma membrane or cytoplasmic regions of the same cell, using Zeiss software. The fluorescence intensities at the plasma membrane (a) and in the cytosol (b) demonstrate endocytosis of the P2Y1-GFP receptor and a corresponding rise in the fluorescence intensity in the cytoplasm. The changes in the fluorescence intensity under the different conditions, stimulation with ATP (circles), KN-62 treatment plus ATP (squares) and okadaic acid treatment plus ATP (triangles), are representative of at least three individual experiments.

Download figure to PowerPoint

The amount of receptor that was endocytosed by stimulation of the cells with ATP was quantified by measuring the change in fluorescence intensity at the plasma membrane and in the cytoplasm. A simultaneous measurement in three channels (green, red and transmission) was performed at each time point for the figures indicated. The measurement also included a Z-scan. Green fluorescence showed the P2Y1 receptor coupled to GFP and the red fluorescence detected the lysosomes in the cytoplasm. The Z-scan was used to identify the upper plasma membrane via an overlay between the transmission picture and the GFP channel. The position of the ROIs for the plasma membrane was adjusted for every time point with reference to the transmission picture to encompass the plasma membrane and not the cytoplasm. To confirm that the ROI on the plasma membrane did not comprise minimal or any of the cytoplasmic regions, a co-localization analysis was performed between the green and the red channel (Weisshart et al. 2004). The fluorescence was determined in ROIs that were marked on the plasma membrane and in the cytoplasm, respectively. During the course of the experiment transmission pictures were also obtained. The position of the ROIs for the plasma membrane was adjusted for every time point with reference to the transmission picture to encompass the plasma membrane and not the cytoplasm. The changes in the fluorescence which were observed in the ROIs over the time period of the experiment were normalized to the fluorescence value which was seen in the respective ROI at the beginning of the experiment.

The series of pictures in Fig. 2(a to e) shows an example of live cell imaging, where the same group of cells is studied for 150 min during exposure to ATP. After incubation with ATP for 10 min at 37°C, we observed a marked decrease of the fluorescence signal at the cell surface and the appearance of a vesicular pattern nearby the plasma membrane, which represents endocytotic vesicles of P2Y1-GFP receptor (data not shown). After 30 min of incubation, there was complete endocytosis of the receptor (Fig. 2b), as can be seen by the aggregates marked by an asterisk. This can be quantified as a decrease in membrane fluorescence, as shown by the respective curve in Fig. 3(a), and a corresponding increase in fluorescence in the cytoplasm (Fig. 3b). After the 30-min incubation with ATP there was no co-localization of the receptor (green) with lysosomes (red), as shown in Fig. 2(b). A further stimulation of the cells for another 30 min was required to detect a clear co-localization of the receptor with lysosomes (data not shown). When we removed the agonist after the 30-min stimulus, the membrane fluorescence gradually reappeared and a smooth fluorescence signal on the plasma membrane was again detectable. A reappearance of the receptor was observed at the end of 60 min (Fig. 2d). The intracellular cluster in the cell disappeared. This membrane fluorescence was still increased after 120 min of agonist removal (Fig. 2e).

Exposure of cells to KN-62 inhibited the ATP-induced internalization of the receptor with 30-min stimulation with ATP (Figs 2g and h). After removal of the agonist and further observation there was no change in the distribution of the receptor or fusion with lysososmes (data not shown). In KN-62-treated cells, the complete inhibition of endocytosis of the receptor was also reflected in the quantification of the fluorescence intensity at the plasma membrane (Fig. 3a, curve given by squares). There was also almost no change in the fluorescence intensity in the cytoplasm (Fig. 3b, curve given by squares).

In addition, to confirm the role of CaMKII in endocytosis of the P2Y1 receptor and the specificity of KN-62, we repeated the above experiments with KN-93, a water-soluble analogue of KN-62. In cells that were pre-incubated for 30 min with 10 μm KN-93, we observed an inhibition of endocytosis of the P2Y1 receptor after 30 min of stimulation with 100 μm ATP (data not shown). For control, the cells were pre-incubated with the inactive analogue of KN-93, 10 μm KN-92 for 30 min and then challenged with 100 μm ATP for 30 min. In that case, normal endocytosis of the receptor was observed. KN-62, KN-93 and KN-92 did not affect the basal fluorescence of the cells. In addition to this, in vitro experiments indicate that, at this concentration, other kinases are not inhibited except for GSK3β, which has a residual activity of 38% (Davies et al. 2000).

Treatment with 5 nm okadaic acid did not influence the localization of the receptor or the basal fluorescence (Fig. 2i). Importantly, the ATP-induced endocytosis of the receptor was not significantly affected. Thus, complete endocytosis of the receptor was seen after 30 min of stimulation with ATP (Fig. 2j, as indicated by the asterisk). This could be confirmed by the quantification of the loss of the fluorescence from the plasma membrane. The temporal profile of decrease of fluorescence from the plasma membrane in the presence of okadaic acid (Fig. 3a, curve given by triangles) was very similar to that seen for untreated cells (Fig. 3a, curve given by circles). After 40 min there was no co-localization of the receptor with lysosomes (Fig. 2k). After removal of the agonist the receptor reappeared slowly on the plasma membrane, but a large fraction of the endocytosed receptor remained in vesicular structures in the cytoplasm (Fig. 2l, as indicated by the asterisk). Even 2 h after withdrawal of the agonist, the receptor did not completely return to the plasma membrane (Fig. 2m). In the case of okadaic acid, the reappearance of the receptor to the plasma membrane was delayed. This time course could be determined by the quantification of the fluorescence intensity on the plasma membrane and in the cytoplasm (Fig. 3, curve given by triangles). The value for the fluorescence intensity on the plasma membrane did not return to the initial level that was observed before stimulation of the cells with ATP.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

P2Y receptors, which belong to the family of GPCRs, mediate the actions of extracellular nucleoside di- and triphosphates. P2Y receptor regulation is of growing interest, as nucleotides have been shown to be involved in processes leading to protection and degeneration of neural and immune cells (Amadio et al. 2002; Kannan 2002; Volonte et al. 2003; Chorna et al. 2004). Therefore, understanding P2Y receptor regulation should provide clues for pharmacological treatment of various disorders. Previously, we have described the stable expression of a P2Y1 receptor as well as a P2Y1-GFP chimera in HEK293 cells and proved functional coupling to Ca2+ release (Vöhringer et al. 2000; Zündorf et al. 2001). A receptor modification using GFP as a fusion partner is a valuable biochemical tool to monitor localization for investigating receptor internalization and recycling in live cells (Kallal and Benovic 2000). Moreover, HEK293 cells are a most suitable system to study P2Y receptor regulation, because these cells endogenously express the P2Y1, P2Y2, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14 (Van der Weyden et al. 2000; Fischer et al. 2003; Moore et al. 2003).

Agonist-induced internalization has been reported until now only for one other member of the P2Y receptor family, the P2Y2 receptor, using a hemagglutinin A epitope at the N-terminus of the human P2Y2 receptor (Sromek and Harden 1998). Recently the mechanism of endocytosis of the GFP-tagged P2Y2 receptor has been reported, analysing the clathrin and actin cytoskeleton dependence (Tulapurkar et al. 2005). Furthermore, it has been shown that C-terminal phosphorylation of the P2Y2 receptor mediates agonist-induced desensitization (Garrad et al. 1998; Otero et al. 2000), suggesting that several kinases and phosphatases are involved in the regulation of the P2Y2 receptor. Until now nothing is known about the type of kinases, which take part in this process for the P2Y1 receptor. Pharmacological experiments proved that various GPCRs are phosphorylated by CaMKII (Zamani and Bristow 1996), protein kinase A (Post et al. 1996), protein kinase C (Chen and Lin 1999), G protein-coupled receptor kinases (Bünemann and Hosey 1999) or casein kinase 1α (Budd et al. 2000), thereby directly regulating their activity.

We used here the stably transfected HEK293 cells expressing the rP2Y1 receptor tagged with GFP which is potently activated by ATP (Vöhringer et al. 2000; Tulapurkar et al. 2005). With these cells, we were able to both visualize P2Y1 receptor trafficking and detect the functional response by monitoring the agonist-induced Ca2+ release. This enabled us to analyse receptor internalization together with desensitizing events (Garland et al. 1996; Garrad et al. 1998; Szekeres et al. 1998; Ferguson 2001). Thus, we investigated the receptor responsiveness and receptor translocation under the influence of kinase and phosphatase inhibitors.

Stimulation of the cells with ATP induced an elevation of the intracellular Ca2+ level which is caused by P2Y1-GFP receptor-induced Ca2+ release (Vöhringer et al. 2000). With continued stimulation of the cells, the Ca2+ response declined. Removal of the agonist resulted in a return of the intracellular Ca2+ concentration to the basal level. A second ATP stimulus after an interval of 60 min gave a Ca2+ response with 75% recovery of the amplitude. The cells were stimulated for 30 min with 100 μm ATP by two different methods, namely continuous presence of agonist or repeated short applications of the agonist. By both these methods the calcium response that was observed at the end of 30 min stimulation declined to a similarly low value. This indicates that both protocols induce a similar desensitization of the receptor. We also verified that the desensitization profile observed with ATP is similar to that obtained with 2-Me-S-ADP, a highly specific agonist for the P2Y1 receptor. This indicates that the stimulation with ATP, the naturally occurring agonist of the P2Y1 receptor, is not influenced by the presence of the endogenous P2Y2 receptor, which can also be activated by ATP.

The question was whether the decrease in the Ca2+ signal that we obtained after prolonged stimulation is as a result of desensitization of the receptor and not because of depletion of the internal stores. This was verified by stimulation of the cells with the agonist thrombin. We chose thrombin as an agonist, as HEK293 cells endogeneously express PAR receptors [Amadesi et al. (2004) and our own unpublished data]. PAR receptors mediate a rise in intracellular Ca2+ via a mechanism similar to that induced by P2Y receptors. We observed that cells that were pretreated for 30 min with 100 μm ATP exhibited a Ca2+ response upon stimulation with thrombin. The pretreatment with ATP did not affect the amplitude of the thrombin-mediated rise in intracellular Ca2+. Thus, we were able to show that the decrease in Ca2+ response was because of desensitization of the P2Y1 receptor and not as a result of the emptying of the stores.

By CLSM we visualized agonist-induced endocytosis in live cells under the same conditions, using the GFP tag to localize the P2Y1 receptor and LysoTracker to label lysosomes. The inhibitors used did not influence the basal fluorescence observed in the cells. A time-dependent receptor endocytosis was observed. The receptors were not yet co-localized with lysosomes after a 30-min exposure to agonist. Endocytosis was quantified as a decrease in the fluorescence intensity on the plasma membrane and an increase in the fluorescence intensity in the cytoplasm. After removal of the agonist, the receptor reappeared on the plasma membrane after 60 min. At this time point, we observed that the Ca2+ response was back to 75% of the value of the initial response. These results show that the cells are desensitized concomitant with endocytosis of the receptor from the plasma membrane. After removal of the agonist the cells are then re-sensitized together with receptor reappearance.

When the cells were pre-incubated with the CaMKII inhibitor KN-62 and then stimulated with ATP, the de- and re-sensitization pattern of the P2Y1 receptors was similar to that in the absence of the inhibitor. In the presence of KN-62 there was almost complete re-sensitization. The Ca2+ response reached again 111% of the first response obtained upon stimulation with ATP of the KN-62-pretreated cells. This recovery of the Ca2+ response was even higher than in cells that were not pretreated with KN-62.

However, treatment with KN-62 completely inhibited the endocytosis of the receptor. To confirm the specificity of the effects of KN-62 on the endocytosis of the receptor, we repeated the experiments with KN-93, a most recently available and even more potent inhibitor of CaMKII. We observed similar effects. Thus, CaMKII seems to be involved in P2Y1 receptor endocytosis. To further underpin our results we used KN-92, an inactive form of KN-93. The cells that were pretreated with KN-92 exhibited normal endocytosis of the P2Y1 receptor upon challenge with 100 μm ATP. Obviously, P2Y1 receptor desensitization does not depend upon endocytosis.

After treatment of the cells with the protein phosphatase inhibitor okadaic acid, the initial Ca2+ response was slightly reduced, similar to that observed with KN-62. The desensitization proceeded in a manner similar to that seen in untreated cells. However, exposure to okadaic acid greatly inhibited the re-sensitization of the P2Y1 receptor, as the Ca2+ response observed at 60 min after the 30-min long ATP stimulus amounted only to 39% of the initial response.

Okadaic acid did not affect the endocytosis kinetics in P2Y1 receptor trafficking. After removal of the agonist, however, the reappearance of the receptor on the plasma membrane was considerably delayed compared with untreated cells. There was only reduced fluorescence that reappeared on the plasma membrane. Thus, the fact that the Ca2+ response did not return to the initial level seems to be caused by incomplete reappearance of the receptor on the plasma membrane. The complete reappearance of the P2Y1 receptor was apparently suppressed by inhibition of the enzyme PP2A by okadaic acid. This may prevent dephosphorylation of the endocytosed receptor or downstream effectors. Similarly, with β2-adrenergic receptor heterologously expressed in HEK293 cells (Oakley et al. 1999), treatment of the cells with okadaic acid caused a reduced stimulation of adenylate cyclase activity. This was attributed to the reduction in the reappearance of the receptor on the plasma membrane. De-phosphorylation of the receptor seems to be important for the reappearance of the receptor on the plasma membrane (Spampinato et al. 2002).

In summary, our results indicate that endocytosis of the P2Y1 receptor is controlled by the activity of CaMKII. Inhibition of CaMKII suppresses endocytosis of the receptor. However, the receptor nevertheless undergoes desensitization. This prevents continued activation of the cell because of lingering ligand in the extracellular compartment. Inhibition of the activity of okadaic acid-sensitive phosphatase does not affect internalization and desensitization but delays the reappearance of the P2Y1 receptor on the plasma membrane. With partial reappearance of the P2Y1 receptor back on the plasma membrane, there is only partial recovery of the ATP-induced Ca2+ response in the cells. These observations underline that phosporylation and de-phosporylation of the P2Y1 receptor play important roles in the functionality of this receptor.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The work was supported by Graduiertenkolleg of Deutsche Forschungsgemeinschaft (GRK253/5), and Land Sachsen-Anhalt 1896A and 0012KS. We thank K. Christoph for expert technical assistance with the cell cultures and Dr F. Sedekizade for help with the generation of the stably transfected cells.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • Agteresch H. J., Dagnelie P. C., Van Den Berg J. W. and Wilson J. H. (1999) Adenosine triphosphate: established and potential clinical applications. Drugs 58, 211232.
  • Amadesi S., Nie J., Vergnolle N. et al. (2004) Protease-activated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia. J. Neurosci. 24, 43004312.
  • Amadio S., D'Ambrosi N., Cavaliere F., Murra B., Sancesario G., Bernardi G., Burnstock G. and Volonte C. (2002) P2 receptor modulation and cytotoxic function in cultured CNS neurons. Neuropharmacology 42, 489501.
  • Bofill-Cardona E., Vartian N., Nanoff C., Freissmuth M. and Boehm S. (2000) Two different signaling mechanisms involved in the excitation of rat sympathetic neurons by uridine nucleotides. Mol. Pharmacol. 57, 11651172.
  • Brown D. A., Filippov A. K. and Barnard E. A. (2000) Inhibition of potassium and calcium currents in neurones by molecularly-defined P2Y receptors. J. Auton Nerv. Syst. 81, 3136.
  • Budd D. C., McDonald J. E. and Tobin A. B. (2000) Phosphorylation and regulation of a Gq/11-coupled receptor by casein kinase 1α. J. Biol. Chem. 275, 19 66719 675.
  • Bünemann M. and Hosey M. M. (1999) G-protein coupled receptor kinases as modulators of G-protein signalling. J. Physiol. 517, 523.
  • Chen B. C. and Lin W. W. (1999) PKCβI mediates the inhibition of P2Y receptor-induced inositol phosphate formation in endothelial cells. Br. J. Pharmacol. 127, 19081914.
  • Chorna N. E., Santiago-Perez L. I., Erb L., Seye C. I., Neary J. T., Sun G. Y., Weisman G. A. and Gonzalez F. A. (2004) P2Y receptors activate neuroprotective mechanisms in astrocytic cells. J. Neurochem. 91, 119132.
  • Davies S. P., Reddy H., Caivano M. and Cohen P. (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95105.
  • Ferguson S. S. (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol. Rev. 53, 124.
  • Ferguson S. S. and Caron M. G. (1998) G protein-coupled receptor adaptation mechanisms. Semin. Cell Dev. Biol. 9, 119127.
  • Firestein B. L., Xing M., Hughes R. J., Corvera C. U. and Insel P. A. (1996) Heterogeneity of P2u- and P2y-purinergic receptor regulation of phospholipases in MDCK cells. Am. J. Physiol. 271, F610F618.
  • Fischer W., Wirkner K., Weber M. et al. (2003) Characterization of P2X3, P2Y1 and P2Y4 receptors in cultured HEK293-hP2X3 cells and their inhibition by ethanol and trichloroethanol. J. Neurochem. 85, 779790.
  • Garland A. M., Grady E. F., Lovett M., Vigna S. R., Frucht M. M., Krause J. E. and Bunnett N. W. (1996) Mechanisms of desensitization and resensitization of G protein-coupled neurokinin1 and neurokinin2 receptors. Mol. Pharmacol. 49, 438446.
  • Garrad R. C., Otero M. A., Erb L., Theiss P. M., Clarke L. L., Gonzalez F. A., Turner J. T. and Weisman G. A. (1998) Structural basis of agonist-induced desensitization and sequestration of the P2Y2 nucleotide receptor. Consequences of truncation of the C terminus. J. Biol. Chem. 273, 29 43729 444.
  • Grynkiewicz G., Poenie M. and Tsien R. Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 34403450.
  • Kallal L. and Benovic J. L. (2000) Using green fluorescent proteins to study G-protein-coupled receptor localization and trafficking. Trends Pharmacol. Sci. 21, 175180.
  • Kannan S. (2002) Amplification of extracellular nucleotide-induced leukocyte(s) degranulation by contingent autocrine and paracrine mode of leukotriene-mediated chemokine receptor activation. Med. Hypotheses 59, 261265.
  • Von Kügelgen I. and Wetter A. (2000) Molecular pharmacology of P2Y-receptors. Naunyn Schmiedebergs Arch. Pharmacol. 362, 310323.
  • Luzio J. P., Rous B. A., Bright N. A., Pryor P. R., Mullock B. M. and Piper R. C. (2000) Lysosome–endosome fusion and lysosome biogenesis. J. Cell Sci. 113, 15151524.
  • Milligan G. (1999) Exploring the dynamics of regulation of G protein-coupled receptors using green fluorescent protein. Br. J. Pharmacol. 128, 501510.
  • Moore D. J., Murdock P. R., Watson J. M., Faull R. L., Waldvogel H. J., Szekeres P. G., Wilson S., Freeman K. B. and Emson P. C. (2003) GPR105, a novel Gi/o-coupled UDP-glucose receptor expressed on brain glia and peripheral immune cells, is regulated by immunologic challenge: possible role in neuroimmune function. Brain Res. Mol. Brain Res. 118, 1023.
  • Nicholas R. A. (2001) Identification of the P2Y12 receptor: a novel member of the P2Y family of receptors activated by extracellular nucleotides. Mol. Pharmacol. 60, 416420.
  • Oakley R. H., Laporte S. A., Holt J. A., Barak L. S. and Caron M. G. (1999) Association of β-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization. J. Biol. Chem. 274, 32 24832 257.
  • Oksche A., Boese G., Horstmeyer A., Furkert J., Beyermann M., Bienert M. and Rosenthal W. (2000) Late endosomal/lysosomal targeting and lack of recycling of the ligand-occupied endothelin B receptor. Mol. Pharmacol. 57, 11041113.
  • Otero M., Garrad R. C., Velazquez B. et al. (2000) Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor. Mol. Cell Biochem. 205, 115123.
  • Pierce K. L., Maudsley S., Daaka Y., Luttrell L. M. and Lefkowitz R. J. (2000) Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and non-sequestering G protein-coupled receptors. Proc. Natl Acad. Sci. USA 97, 14891494.
  • Post S. R., Aguila-Buhain O. and Insel P. A. (1996) A key role for protein kinase A in homologous desensitization of the β2-adrenergic receptor pathway in S49 lymphoma cells. J. Biol. Chem. 271, 895900.
  • Powell A. D., Teschemacher A. G. and Seward E. P. (2000) P2Y purinoceptors inhibit exocytosis in adrenal chromaffin cells via modulation of voltage-operated calcium channels. J. Neurosci. 20, 606616.
  • Ralevic V. and Burnstock G. (1998) Receptors for purines and pyrimidines. Pharmacol. Rev. 50, 413492.
  • Rohatgi T., Sedehizade F., Reymann K. G. and Reiser G. (2004) Protease-activated receptors in neuronal development, neurodegeneration, and neuroprotection: thrombin as signaling molecule in the brain. Neuroscientist 10, 501512.
  • Schonthal A. H. (1998) Role of PP2A in intracellular signal transduction pathways. Front. Biosci. 3, D1262D1273.
  • Spampinato S., Di Toro R., Alessandri M. and Murari G. (2002) Agonist-induced internalization and desensitization of the human nociceptin receptor expressed in CHO cells. Cell Mol. Life Sci. 59, 21722183.
  • Sromek S. M. and Harden T. K. (1998) Agonist-induced internalization of the P2Y2 receptor. Mol. Pharmacol. 54, 485494.
  • Szekeres P. G., Koenig J. A. and Edwardson J. M. (1998) Involvement of receptor cycling and receptor reserve in resensitization of muscarinic responses in SH-SY5Y human neuroblastoma cells. J. Neurochem. 70, 16941703.
  • Tulapurkar M. E., Schäfer R., Hanck T., Flores R. V., Weisman G. A., Gonzalez F. A. and Reiser G. (2005) Endocytosis mechanism of P2Y2 nucleotide receptor tagged with green fluorescent protein: clathrin and actin cytoskeleton dependence. Cell Mol. Life Sci. 62, 13881399.
  • Van der Weyden L., Adams D. J., Luttrell B. M., Conigrave A. D. and Morris M. B. (2000) Pharmacological characterisation of the P2Y11 receptor in stably transfected haematological cell lines. Mol. Cell Biochem. 213, 7581.
  • Vöhringer C., Schäfer R. and Reiser G. (2000) A chimeric rat brain P2Y1 receptor tagged with green-fluorescent protein: high-affinity ligand recognition of adenosine diphosphates and triphosphates and selectivity identical to that of the wild-type receptor. Biochem. Pharmacol. 59, 791800.
  • Volonte C., Amadio S., Cavaliere F., D'Ambrosi N., Vacca F. and Bernardi G. (2003) Extracellular ATP and neurodegeneration. Curr. Drug Targets CNS Neurol. Disord. 2, 403412.
  • Wang H. and Reiser G. (2003) Thrombin signaling in the brain: the role of protease-activated receptors. Biol. Chem. 384, 193202.
  • Wang H., Ubl J. J. and Reiser G. (2002) Four subtypes of protease-activated receptors, co-expressed in rat astrocytes, evoke different physiological signaling. Glia 37, 5363.
  • Weisshart K., Jungel V. and Briddon S. J. (2004) The LSM 510 META–ConfoCor 2 system: an integrated imaging and spectroscopic platform for single-molecule detection. Curr. Pharm. Biotechnol. 5, 135154.
  • Wirkner K., Schweigel J., Gerevich Z., Franke H., Allgaier C., Barsoumian E. L., Draheim H. and Illes P. (2004) Adenine nucleotides inhibit recombinant N-type calcium channels via G protein-coupled mechanisms in HEK293 cells; involvement of the P2Y13 receptor-type. Br. J. Pharmacol. 141, 141151.
  • Zamani M. R. and Bristow D. R. (1996) The histamine H1 receptor in GT1-7 neuronal cells is regulated by calcium influx and KN-62, a putative inhibitor of calcium/calmodulin protein kinase II. Br. J. Pharmacol. 118, 11191126.
  • Zolnierowicz S. (2000) Type 2A protein phosphatase, the complex regulator of numerous signaling pathways. Biochem. Pharmacol. 60, 12251235.
  • Zündorf G., Schäfer R., Vöhringer C., Halbfinger E., Fischer B. and Reiser G. (2001) Novel modified adenosine 5′-triphosphate analogues pharmacologically characterized in human embryonic kidney 293 cells highly expressing rat brain P2Y1 receptor: biotinylated analogue potentially suitable for specific P2Y1 receptor isolation. Biochem. Pharmacol. 61, 12591269.