The P2U Purinoceptor Obligatorily Engages the Heterotrimeric G Protein G16 to Mobilize Intracellular Ca2+ in Human Erythroleukemia Cells*

(Received for publication, September 9, 1996, and in revised form, January 29, 1997)

Kurt Baltensperger Dagger and Hartmut Porzig

From the Institute of Pharmacology, University of Bern, CH-3010 Bern, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

To assess the role of G16, a trimeric G protein exclusively expressed in hematopoietic cells, Galpha 16 antisense RNA was stably expressed in human erythroleukemia (HEL) cells. Western blot analysis showed that in transfected cell lines, the expression of endogenous Galpha 16 protein was suppressed, but the expression of Galpha q/11, Galpha i2, and Galpha i3 remained unaffected. Suppression of Galpha 16 in transfected HEL cells did not interfere with transient elevations of intracellular free Ca2+ concentrations induced by prostaglandin E1 (PGE1), platelet-activating factor, or thrombin. In parental HEL cells, UTP and ATP mobilized Ca2+ from intracellular stores with half-maximum effective concentrations of 3.6 ± 0.7 and 4.7 ± 1.6 µM, respectively, apparently by stimulating P2U purinoceptors. By contrast, Ca2+ mobilization by UTP or ATP was completely abrogated in Galpha 16-suppressed cells, indicating specific coupling of G16 to P2U purinoceptors. Pertussis toxin inhibited the effect of UTP in parental HEL cells by 57.6 ± 4.9%. These data indicate that signaling by the P2U purinoceptor obligatorily requires G16 but may be modulated further by activation of Gi. Priming of HEL cells with UTP or ATP prior to stimulation with PGE1 markedly enhanced the PGE1-induced intracellular Ca2+ release. This indirect, potentiating effect of UTP and ATP was not impaired in Galpha 16-suppressed cells but was inhibited by pertussis toxin, indicating that functional P2U purinoceptors are present on these cells and that the potentiating effect primarily depends on Gi. The data demonstrate (i) that Galpha 16 antisense RNA selectively inhibits endogenous Galpha 16 protein expression in HEL cells; (ii) that stimulation of endogenous P2U (P2Y2) purinoceptors leads to the mobilization of intracellular Ca2+ by a mechanism that strictly depends on Galpha 16; and (iii) that P2U purinoceptors in HEL cells can communicate with two distinct signaling pathways diverging at the G protein level.


INTRODUCTION

G proteins, a group of heterotrimeric membrane-associated proteins, composed of a GTP-binding alpha -subunit and a beta gamma -heterodimer, transduce the signals of hormones and neurotransmitters that bind to a large family of receptors with seven transmembrane domains. Effector systems for G protein-coupled receptors include adenylyl cyclases, phosphoinositide-specific phospholipases C (PLC),1 or ion channels. Based on amino acid sequence homology and sensitivity to bacterial toxins, the GTP-binding alpha -subunits of G proteins are grouped into four subfamilies, Gs, Gi, Gq, and G12/13 (1, 2). Two subfamilies (Gs and Gi) activate or inhibit adenylyl cyclases, respectively, and are sensitive to ADP-ribosylation catalyzed by either cholera toxin or pertussis toxin (PTX). Although many G protein subtypes do not show any tissue specificities, the alpha -subunit of G16, a member of the Gq subfamily, is expressed exclusively in hematopoietic cells (3). Members of this subfamily, which lack an ADP-ribosylation site, activate PLCs and thus mobilize Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores.

In some cases, the expression pattern of G proteins is subject to changes during cellular differentiation (4-8). In the hematopoietic cell line HL-60, Galpha 16 is down-regulated during terminal differentiation (3). Analysis of Galpha 16 expression in patients with acute lymphoid leukemia suggests that Galpha 16 is expressed in progenitor B cell-derived malignant cells but not in leukemic cells that are derived from more mature differentiation stages (7). Interestingly, Galpha 16 displays growth inhibitory effects when overexpressed as a constitutively active (GTPase-deficient) form in Swiss 3T3 fibroblasts or in small cell lung carcinoma cells (9, 10). Thus it appears that Galpha 16 expression is regulated during cellular differentiation and that its expression levels may be critical for controlling proliferation rates in slowly cycling stem and progenitor cells.

The signaling pathways that engage G16 in hematopoietic cells are still poorly defined. Recent studies suggest that G16 can interact indiscriminately with a multitude of G protein-coupled receptors when receptors and Galpha 16 were overexpressed in COS-7 cells (11, 12). On the other hand, some receptors for chemotactic factors such as C5a, formyl-methionyl-leucyl-phenylalanine, or interleukin-8 appear to interact with G16 but not with Gq or G11, to stimulate the production of InsP3 (13-16). Thus, some receptors may utilize G16, preferentially, to mobilize intracellular Ca2+. However, these studies could not address the question of which receptors may couple specifically to G16 in native cells. Overexpression of wild type alpha -subunits in transfected cells potentially shifts the balance between signal transduction through beta gamma - and alpha -subunits because of a redistribution of beta gamma -subunits bound to the overexpressed versus the endogenous G proteins. In transient transfection assays, competition between alpha -subunits for common beta gamma -dimers was indeed demonstrated (17). Moreover, altering the stoichiometric relationships between G protein and effector systems may result in spillover effects in transfected cells which do not occur in wild type cells. Other studies employed cells overexpressing GTPase-deficient mutants as tools to define the role of individual Gq subfamily members (9, 10, 18). However, uncertainties remain in the interpretation of results from such studies because these mutants act independently of beta gamma -heterodimers. As a more gentle alternative, an antisense-based approach with oligodeoxynucleotides or expression of antisense RNA to reduce the expression of specific alpha -subunits has proven to be a powerful tool in defining the roles of specific G protein isoforms of various subfamilies (19).

In the present study, we sought to identify signaling pathways that obligatorily require G16 in a hematopoietic cell line, human erythroleukemia (HEL) cells. Full-length antisense RNA expression was used to suppress selectively the endogenous biosynthesis of Galpha 16. In Galpha 16-suppressed cells, a specific deficit of the P2U (P2Y2)2 purinoceptor to stimulate the release of intracellular Ca2+ was detected, whereas other functions triggered by the same receptor remained intact.


EXPERIMENTAL PROCEDURES

Tissue Culture Media and Reagents

Tissue culture media and media supplements were purchased from Life Technologies, Inc. DOTAP, ATP, and UTP were from Boehringer Mannheim, G418 sulfate was from Promega Corp. Fura-2/AM was from Molecular Probes. PGE1, PGE2, and thrombin were obtained from Sigma. PAF was purchased from Calbiochem-Novabiochem. UK14304 and thapsigargin were from Research Biochemicals International (RBI). Antibodies against G protein alpha - and beta -subunits were a generous gift from Dr. K. Spicher and Dr. B. Nürnberg, both from the laboratory of Prof. G. Schultz, Dept. of Pharmacology, Free University of Berlin, Germany (see below for details). All other reagents were from Fluka Chemie or from Merck Darmstadt.

Complementary DNA and Plasmids

The cDNA for human Galpha 16 including 5'- and 3'-flanking regions (224 and 60 base pairs, respectively) (3) was inserted into the mammalian expression vector pcDNA3 (Invitrogen Corp.) in antisense orientation with respect to the direction of transcription from the cytomegalovirus promoter, thus allowing the expression of Galpha 16 antisense RNA in transfected cells. The resulting plasmid, pG16AS, also harbors a copy of the aminoglycoside phosphotransferase gene, allowing selection of transfected cells with G418 sulfate.

Cell Culture

HEL cells (22) and K-562 cells were obtained from the American Type Culture Collection. The cells were maintained at densities between 5 × 104 and 5 × 105 cells/ml in a humidified atmosphere of 95% air and 5% CO2 in RPMI medium that was supplemented with 10% fetal bovine serum, 2 mM Glutamax I (Life Technologies, Inc.), 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 µg/ml streptomycin.

Stable Transfection of HEL Cells

HEL cells (2.5 × 107 cells at a density of 2 × 106 cells/ml) were transfected in Opti-MEM (Life Technologies, Inc.) with 50 µg of the plasmid pG16AS, using DOTAP as the transfection reagent according to the manufacturer's instructions. Sixteen h later, the medium was replaced with RPMI medium plus supplements, and the cells were seeded at a density of 106/ml. Twenty-four h later the cells received fresh medium that included 400 µg/ml medium of active G418 sulfate. The following day, the cells (8.6 × 107) were transferred into 96-well dishes (105 cells/well) to select independent G418 sulfate-resistant clones. Fourteen days after transfection, 47 of 864 wells contained viable, G418 sulfate-resistant clones, which were picked and expanded. Six clonal cell lines out of 21 that showed sustained growth were selected randomly and characterized further.

Measurement of Intracellular Free Calcium

HEL cells were loaded with fura-2/AM (9 µM) as described previously (6). The loaded cells were washed, and a stock suspension was prepared which contained 5 × 106 cells/ml medium A (phosphate-buffered saline supplemented with 1 mM MgCl2, 5 mM KCl, 22 mM glucose, and 5% fetal bovine serum). The Ca2+ concentration of medium A was around 90 µM as measured by atomic absorption spectrophotometry. Prior to use, cells from the stock suspension were diluted to a density of 2.5 × 105/ml into medium A and preincubated for 5 min at 25 °C. Agonist-induced changes in [Ca2+]i were measured in a thermostatted (25 °C) cuvette in a Perkin Elmer LS-50B dual wavelength fluorescence spectrophotometer, which was equipped with the fast-fura measuring device (Perkin-Elmer). Emission intensities (at 485 nm) at excitation wavelengths of 340 and 380 nm were recorded. At the end of each experiment, maximum and minimum fluorescence ratios were determined by adding Ca2+ (1 mM) plus digitonin (12.5 µM) followed by Tris-buffered EGTA (20 mM, pH 8.5), respectively, to the incubation medium. Intensity ratios were converted to intracellular Ca2+ concentrations using the FL data manager software (version 3.5) and were based on an apparent Kd of 224 nM for the fura-2·Ca2+ complex (23).

Extraction of G Protein alpha -Subunits

Cells were harvested and washed once in phosphate-buffered saline. Cells were then disrupted by three freeze/thaw cycles (liquid nitrogen) in buffer 1 (137 mM NaCl, 20 mM Hepes, pH 7.4, 2 mM MgCl2, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). Membranes were collected by centrifugation (14,000 × g for 2 min), and membrane-bound proteins were solubilized with Triton X-100 lysis buffer (137 mM NaCl, 20 mM Tris, pH 7.4, 1 mM MgCl2, 1% Triton X-100, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). Insoluble material was removed by centrifugation (14,000 × g for 2 min).

Immunoblotting and Antibodies

Solubilized membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% acrylamide) and electrophoretically transferred to nitrocellulose filters (Bio-Rad). The alpha -subunits of G16, Gi3, Gi2, and Gq/11 were detected by immunostaining with affinity-purified antibodies (AS 339, AS 86, AS 269, and AS 368, respectively) that were raised in rabbits against sequence-specific peptides (24). G protein beta -subunits in cellular extracts and in a preparation of purified Gbeta gamma from porcine brain (a gift from Dr. B. Nürnberg and Dr. G. Schultz) were detected with an antibody (AS 11) that was raised against a peptide corresponding to a conserved sequence within Gbeta 1, Gbeta 2, and Gbeta 4 (25). Bound antibodies were visualized by enhanced chemiluminescence (Amersham Corp.), using horseradish peroxidase-conjugated anti-rabbit IgG (Sigma) as the secondary antibody. In some instances, nitrocellulose filters were incubated twice for 90 min each with stripping buffer (100 mM glycine, pH 2.2, 20 mM magnesium acetate, 50 mM KCl) to remove antibodies. Filters were then blocked again and used for reprobing with a different antibody.

Data Analysis

Statistical significance was calculated by unpaired t tests using StatView for the Macintosh, version 4.0 (Abacus Concepts, Inc.). Half-maximum effective concentrations (EC50) were calculated by fitting the Hill equation to the data with the Levenberg-Marquardt fit algorithm of Kaleidagraph, version 3.0 (Abelbeck Software).


RESULTS

Depletion of Endogenous Galpha 16 by Antisense RNA Expression

Individual G418 sulfate-resistant HEL cell lines that were isolated following transfection with a plasmid expressing Galpha 16 antisense RNA (pG16AS) were analyzed by protein immunoblot to assess the extent of suppression of endogenous Galpha 16 expression. In three G418 sulfate-resistant cell lines (1E3, 3D4, and 1G3), Galpha 16 expression was greatly diminished compared with parental HEL cells (Fig. 1A). In a fourth cell line (7H6) and two others (data not shown), Galpha 16 levels were not decreased, although these cell lines were resistant to the antibiotic. Thus, the cell line 7H6 was used further as a nonsuppressed control along with parental HEL cells. The specificity of the antibody (AS 339) for Galpha 16 was assessed using wild type K-562 cells, which do not express detectable levels of Galpha 16 (3), and two K-562-derived cell lines (K-562-D5 and K-562-A4) that were stably transfected with a plasmid containing the full-length clone of Galpha 16 under the transcriptional control of the cytomegalovirus promoter. A protein immunoblot with cell extracts from K562-D5 and -A4, but not from parental cells, showed a unique major immunoreactive band demonstrating that the antibody (AS 339) is highly specific for Galpha 16 (Fig. 1A). The electrophoretic mobility of overexpressed Galpha 16 was identical to that of the major immunoreactive protein detected in HEL cells.


Fig. 1. Selective suppression of endogenous Galpha 16 in HEL cell lines that were stably transfected with Galpha 16 antisense RNA. Membrane extracts (20 µg of protein) from HEL cells (in duplicate) or transfected cell lines were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then analyzed for the expression of various G protein alpha -subunits and of beta -subunits by protein immunoblot. Panel A, extracts were analyzed for the expression of Galpha 16 with the antibody AS 339. To indicate the specificity of the antibody to Galpha 16, membrane extracts (20 µg) of wild type K-562 cells, which do not express detectable levels of Galpha 16 (3), and of two K-562-derived cell lines (K-562-D5, K-562-A4), which were stably transfected with Galpha 16, were also analyzed for Galpha 16. Panel B, the nitrocellulose filter from panel A was reprobed for Galpha i3, using the subtype-specific antibody AS 86. Additionally, membrane extracts were probed for Galpha q/11 and Galpha i2 using antibodies specific to each alpha -subunit subtype (AS 368, and AS 269, respectively). Panel C, membrane extracts (20 µg) of parental or transfected HEL cells were analyzed for the expression of beta -subunits with the antibody AS 11. As a standard, 100 ng of purified Gbeta gamma heterodimer from porcine brain was also analyzed. This preparation consists of Gbeta 1 (major component) and Gbeta 2, which are associated with at least three different Ggamma isoforms. Bound antibodies were visualized using the enhanced chemiluminescence technique (for details see "Experimental Procedures"). Representative experiments of at least three independent experiments with similar results are shown.
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To assess the specificity of Galpha 16 down-regulation in the transfected cell lines, the expression levels of other G protein alpha -subunits were also examined in transfected and parental cells. A major band was detected with the antibody raised against a common peptide of Galpha q and Galpha 11, two phylogenetically close congeners of Galpha 16, which comigrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two alpha -subunits (collectively termed Galpha q/11) were expressed at comparable levels in Galpha 16-suppressed and nonsuppressed cells as demonstrated in Fig. 1B. Galpha i3 and Galpha i2 expression also remained unchanged. Since the expression level of G protein alpha -subunits and Gbeta gamma may be coordinately regulated (26), Galpha 16-suppressed and nonsuppressed HEL cells were analyzed for the level of beta -subunit protein, with an antibody (AS 11) that recognizes Gbeta 1, Gbeta 2, and Gbeta 4 (25). However, no differences in Gbeta protein expression were observed (Fig. 1C). In conclusion, the data indicate that Galpha 16 antisense RNA expression inhibits the expression of endogenous Galpha 16 in HEL cells, but the expression of other G protein subunits is not affected.

UTP-dependent Ca2+ Mobilization Is Abrogated in Galpha 16-suppressed HEL Cell Lines

Experimental data from transiently transfected COS cells showed that heterologously expressed Galpha 16 could mediate the mobilization of intracellular Ca2+, after stimulation of diverse G protein-coupled receptors that were cotransfected into these cells (11-13, 27). Being a member of the Gq subfamily of G protein alpha -subunits, a role of Galpha 16 in mediating Ca2+ mobilization is expected. However, it is unclear whether Galpha 16 is indeed required in hematopoietic cells to promote Ca2+ mobilization. Galpha 16-suppressed HEL cell lines thus provided a possibility of identifying signaling pathways that depend strictly on the presence of Galpha 16.

A variety of ligands are known to induce a transient increase in [Ca2+]i in HEL cells in a G protein-dependent fashion (28). Fig. 2A summarizes the peak increments of [Ca2+]i over resting levels after adding different agonists at maximum effective concentrations. In Galpha 16-suppressed and nonsuppressed cell lines, PGE1 and thrombin gave rise to similar peak increases in [Ca2+]i, indicating that Galpha 16 suppression did not inhibit signaling pathways utilized by these two ligands. Changes in [Ca2+]i upon stimulation with PAF appeared to be more heterogeneous between the individual cell lines, but down-regulation of Galpha 16 certainly did not impair PAF-dependent signaling.


Fig. 2. Galpha 16-suppressed HEL cells retain responsiveness to PGE1, thrombin, and PAF and show sensitivity to PTX similar to that of parental cells. Panel A, parental HEL cells, or Galpha 16-suppressed cell lines (1E3, 3D4), or a nonsuppressed control cell line (7H6) were challenged with PGE1 (7 µM), thrombin (2 units/ml), or with PAF (50 nM), and changes in [Ca2+]i were monitored in a dual wavelength fluorescence spectrophotometer using the fura-2 method. For each experiment, the peak level of [Ca2+]i was determined, and the level of [Ca2+]i prior to stimulation was subtracted to obtain the peak increase in [Ca2+]i. Peak increments of five (PGE1, PAF) or three (thrombin) measurements were determined. Panel B, HEL and 3D4 cells were treated with PTX (50 ng/ml) for 16 h. Cells were then challenged with thrombin (2 units/ml), PGE1 (7 µM), or thapsigargin (10 µM). In the figure, the effects in PTX-treated cells are given as percentage of the effects observed in the respective controls that were not exposed to PTX. The absolute Ca2+ changes in the controls were (cells/agonist): HEL/thrombin, 118.7 ± 3.7 nM; 3D4/thrombin, 81.9 ± 1.7 nM; HEL/PGE1, 113.0 ± 7.7 nM; 3D4/PGE1, 99.8 ± 4.8 nM; HEL/thapsigargin, 125.0 ± 2.1 nM; 3D4/thapsigargin, 87.7 ± 2.1 nM. Resting levels of [Ca2+]i were not affected by the PTX treatment. The figure shows mean values ± S.E. of at least five experiments (for details, see "Experimental Procedures").
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Agonist-stimulated changes in [Ca2+]i are transduced by pathways with different sensitivity to PTX treatment, depending on the extent of coupling of receptors to Gq type or Gi type G proteins. The downstream effectors, isoforms of PLC, are either activated by GTP-bound Galpha released from Gq or by Gbeta gamma released from Gi, whereby the different isoforms of the enzyme show varying sensitivities toward the G protein subunits (29). The sensitivity to PTX can help to identify the subclass of G proteins and, consequently, the possible subtypes of PLC-beta which may be involved in intracellular Ca2+ mobilization. Schwaner et al. (28) reported that in HEL cells, the transient increase of [Ca2+]i induced by thrombin is rather insensitive to PTX, whereas the effects of PGE1 and PGE2 are partially or completely inhibited by the toxin, respectively. In our experiments in parental HEL and Galpha 16-suppressed 3D4 cells, PTX inhibited thrombin-induced changes in [Ca2+]i by 23 and 14%, respectively (Fig. 2B). These data indicate that the thrombin receptor couples primarily, although not exclusively, to a PTX-insensitive G protein, probably of the Gq type. Under the same conditions, the toxin inhibited the effect of PGE1 in HEL and 3D4 cells by 93 and 86%, respectively, suggesting a major role of Gi type G proteins in PGE1-induced Ca2+ signaling. To exclude that PTX treatment simply reduced the filling state of intracellular Ca2+ stores, PTX-treated cells and nontreated controls were incubated with thapsigargin, a potent and specific inhibitor of the endoplasmic reticulum Ca2+ pump which blocks reuptake of cytosolic Ca2+ into the lumen of the endoplasmic reticulum (30). Parental and Galpha 16-suppressed cells responded with similar transient, thapsigargin-induced increments in [Ca2+]i, whether or not they were treated with PTX (Fig. 2B). PGE2, which acts via Gi-coupled receptors (28), was completely ineffective after PTX treatment, demonstrating that PTX treatment was exhaustive (not shown). The similar relative inhibitions of agonist-induced Ca2+ changes that were observed after PTX treatment in parental and Galpha 16-suppressed cells make it unlikely that the expression pattern of Galpha - and Gbeta gamma -sensitive PLC-beta isoforms differed considerably between Galpha 16-suppressed cells and parental HEL cells.

In HEL cells, the concentration of intracellular Ca2+ also increased transiently when cells were incubated with UTP (100 µM) and reached a maximum between 40 and 60 s after adding the agonist (Fig. 3A, inset). The mean peak increase in [Ca2+]i in parental HEL cells amounted to 78.8 ± 4.8 nM (mean ± S.E.) (Fig. 3A). In contrast, UTP failed to increase [Ca2+]i in any of the three Galpha 16-suppressed cell lines (1E3, 3D4, 1G3). Mean changes in [Ca2+]i were 3.4 ± 0.7 nM for 1G3 cells and even smaller in the other two cell lines. Similarly, ATP was no longer able to increase [Ca2+]i in Galpha 16-suppressed cell lines (data not shown). These observations indicate a critical role of Galpha 16 in UTP- and ATP-induced Ca2+ changes. To substantiate further the requirement of Galpha 16 expression for UTP-dependent Ca2+ signaling, [Ca2+]i was also measured in the G418 sulfate-resistant control cell line 7H6, which expressed wild type levels of Galpha 16. In this cell line, UTP-induced Ca2+ changes (74.3 ± 2.2 nM) were not significantly different from those evoked by the agonist in parental HEL cells (Fig. 3A). These data strongly suggest that inhibition of Galpha 16 expression in transfected HEL cells is accompanied by a severe defect in UTP-dependent Ca2+ signaling.


Fig. 3. Disruption of UTP-stimulated Ca2+ increase in Galpha 16-suppressed cell lines. Panel A, UTP-induced peak increments in [Ca2+]i were determined using the fura-2 method as described in the legend of Fig. 2. Parental HEL cells, or Galpha 16-suppressed cells (1E3, 3D4, 1G3), or control cells (7H6) were stimulated with UTP (100 µM). The figure shows the result of a typical experiment with five measurements/cell line (mean ± S.E.). Inset, representative traces from parental cells (HEL) or from Galpha 16-suppressed cells (3D4). Panel B, HEL cells were incubated with 50 ng/ml PTX for 16 h or left untreated (Control). PTX-treated cells and controls were then challenged with UTP (100 µM) or UK14304 (5 µM). Changes in [Ca2+]i were determined as described above. The figure shows changes in [Ca2+]i as proportions of the signal obtained in untreated HEL cells with UTP alone. Resting levels of [Ca2+]i were not affected by the PTX treatment. Mean values ± S.E. of five independent measurements/condition are given. * denotes a significant difference from nontreated cells (p0 < 0.001).
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If UTP-dependent Ca2+ signaling in HEL cells requires Galpha 16, then no effect of PTX on UTP-induced Ca2+ changes would be expected. However, PTX inhibited the UTP-induced increase in [Ca2+]i by 57.6 ± 4.9% (n = 6) compared with nontreated HEL cells (Fig. 3B). Inhibition by the toxin was exhaustive because the effect of UK14304, a selective agonist for the Gi-coupled alpha 2-adrenoreceptor, was inhibited completely under these conditions. Thus, a Gi type G protein may also be involved in the propagation of UTP-dependent Ca2+ signaling. However, the role of Gi seems to be modulatory rather than obligatory because its action absolutely depends on the costimulation of Galpha 16.

In HEL Cells, UTP and ATP Stimulate a P2U Purinoceptor, Which Triggers the Mobilization of Intracellular Ca2+ from Thapsigargin-sensitive Stores

The UTP-dependent increase in [Ca2+]i was mainly due to the mobilization of intracellular Ca2+ as demonstrated in Fig. 4, A-C. In the presence as well as in the absence of free extracellular Ca2+, incubation of HEL cells with UTP led to an increase in [Ca2+]i (Fig. 4A, left and right panel, respectively). This indicates that the UTP-dependent increase in [Ca2+]i is mainly due to the release of Ca2+ from intracellular stores. A slightly larger increase in [Ca2+]i was observed when Ca2+ was present in the extracellular medium compared with Ca2+-free conditions (mean Ca2+ changes of 55.5 ± 0.6 nM versus 48.8 ± 2.0 nM (n = 4), respectively). The small, but statistically significant difference (p0 < 0.02) was consistently observed and may be due to the contribution of a capacitative Ca2+ influx that follows the mobilization of intracellular Ca2+ in HEL cells (31). Additional experiments with thapsigargin confirmed these results. Incubation of HEL cells with thapsigargin prior to stimulation with UTP rendered the cells unresponsive to UTP (Fig. 4B). Furthermore, incubation of HEL cells and Galpha 16-suppressed cell lines (3D4 and 1E3) with thapsigargin led to similar transient increases in [Ca2+]i, thus reflecting similar filling states of intracellular Ca2+ stores (Fig. 4C).


Fig. 4. UTP mobilizes Ca2+ from internal, thapsigargin-sensitive stores via the P2U purinoceptor. Fura-2-loaded cells were incubated under various buffer conditions and/or in the presence of agonists prior to stimulation with UTP (100 µM) as indicated in the figure. Panel A, HEL cells were treated with UTP (100 µM) in the presence (90 µM) (left panel) or in the absence (right panel) of extracellular Ca2+ (i.e. after adding 3 mM EGTA to chelate extracellular Ca2+), and changes in [Ca2+]i were monitored. Panel B, HEL cells were incubated with thapsigargin (10 µM). After [Ca2+]i had returned to a new plateau, cells were challenged with UTP (100 µM). Panel C, HEL cells and two Galpha 16-suppressed cell lines (1E3, 3D4) were incubated with thapsigargin (10 µM), and peak increments of [Ca2+]i were determined as described in the legend of Fig. 2. Mean values of three experiments (± S.E.) are shown. Panel D, HEL cells were incubated with UTP (100 µM) or ATP (100 µM) as the first agonist. After [Ca2+]i had returned to resting levels, cells were challenged with ATP (100 µM) or UTP (100 µM), respectively, as the second agonist. Panel E, HEL cells were stimulated with UTP (100 µM) or mock-treated (Control) and subsequently challenged with ADP (5 µM). Panels A, B, D, and E, representative traces of three to five experiments are shown.
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UTP may activate at least three different receptor subtypes of the P2 purinoceptor class, which are stimulated by UTP, ATP, and ADP with different potencies (20, 32). The P2U (P2Y2) subtype shows a potency order of UTP >=  ATP > ADP (33), whereas the potency order for the "novel P2" (P2Y6) subtype is UTP > ADP > ATP (34). At the uridine nucleotide (P2Y4) receptor, UTP is a full agonist, whereas ATP and ADP are only partial agonists (35, 36). All of these receptor subtypes are capable of stimulating PLC-beta and hence are able to mobilize intracellular Ca2+ via an InsP3-dependent pathway. Therefore, UTP-, ATP-, and ADP-dependent changes in [Ca2+]i of HEL cells were monitored to determine the subtype of P2 purinoceptors through which UTP acted in HEL cells. UTP and ATP were equally potent in stimulating a rise in [Ca2+]i with EC50 values of 3.6 ± 0.7 and 4.7 ± 1.6 µM, respectively (mean values of three independent experiments ± S.E.). When cells were incubated sequentially with the two agonists, either nucleotide showed cross-desensitization with respect to its capacity to increase [Ca2+]i (Fig. 4D). This suggests that both nucleotides act through a common nucleotide receptor, most likely of the P2U purinoceptor subtype. Consistently, the presence of receptors in HEL cells for UTP and ATP has been shown in earlier studies (28). As shown above, UTP did not depend on extracellular Ca2+ to evoke its effects, suggesting that the receptor for UTP is not ionotropic (such as P2 receptors of the X subtype). HEL cells also responded to the P2Y (P2Y1) receptor agonist ADP, with a transient rise in [Ca2+]i, but the maximum response to ADP was not affected by prior incubation with UTP (Fig. 4E). These data rule out a contribution of this receptor in the UTP-stimulated increase in [Ca2+]i. Taken together, these results demonstrate that in HEL cells, UTP and ATP stimulate a P2U purinoceptor, which leads to the mobilization of Ca2+ from intracellular, thapsigargin-sensitive stores. The inability of UTP (and ATP) to mobilize Ca2+ in Galpha 16-suppressed cell lines appears to be a consequence of the interruption of P2U purinoceptor-dependent cellular signaling.

UTP Generates a Potentiating Effect on PGE1-induced Changes in [Ca2+]i Which Is Not Affected by Galpha 16 Suppression

In addition to activating an effector system directly, receptor-mediated stimulation of hematopoietic cells may also result in potentiating the action of a second ligand that is administered simultaneously with, or subsequently to, the primary agonist (31, 37, 38). Thus, the stimulation of HEL cells with UTP (100 µM) prior to challenging with PGE1 substantially enhanced the mobilization of intracellular Ca2+ by the second agonist (Fig. 5A, left panel). This potentiating effect of UTP on PGE1-induced Ca2+ release was not impaired in 3D4 cells (Fig. 5A, right panel), nor in any other of the Galpha 16-suppressed cell lines, even though the mean absolute PGE1-dependent increase in [Ca2+]i following preincubation with UTP was higher in nonsuppressed cells than in Galpha 16-suppressed cells (Fig. 5B). Similar to UTP, ATP also enhanced the response to PGE1 in parental and Galpha 16-suppressed cell lines (data not shown).


Fig. 5. Incubation of HEL cells and Galpha 16-suppressed cells with UTP enhances the Ca2+ response to PGE1. Fura-2-loaded parental or transfected HEL cells were incubated in the presence of UTP (100 µM) or mock treated. When [Ca2+]i had returned to resting levels, PGE1 (7 µM) was added, and changes in [Ca2+]i were monitored further. Panel A, individual traces from HEL and 3D4 cells are shown. Panel B, peak increments in [Ca2+]i after treatment with PGE1 were determined as described in the legend of Fig. 3. Mean values ± S.E. of four measurements that were conducted as shown in panel A are plotted in the figure. Statistically significant differences were observed between UTP-treated cells and their respective, mock-treated controls at the levels of *p0 < 0.01 and **p0 < 0.001. Panel C, HEL cells were treated with UTP in the presence (90 µM) or absence of free extracellular Ca2+ (i.e. after adding 3 mM EGTA to chelate extracellular Ca2+) as indicated in the figure. After [Ca2+]i had returned to resting levels, cells were challenged with PGE1 (7 µM). PGE1-induced peak increments of [Ca2+]i from four measurements for each condition were averaged. Pairwise comparisons as indicated by identical symbols in Fig. 5C showed statistically significant differences between treatments at the levels of *p0 < 0.001, **,§p0 < 0.0001, and #p0 < 0.0005.
[View Larger Version of this Image (31K GIF file)]


The effect of PGE1 on [Ca2+]i as well as the potentiating effect of UTP (and ATP) could be caused either by an influx of extracellular Ca2+ or by a Ca2+ release from internal stores or by both mechanisms. Therefore, PGE1-dependent Ca2+ signaling was examined in the presence of different concentrations of extracellular Ca2+ ([Ca2+]o). When [Ca2+]o was lowered from 90 µM to < 10-7 M (i.e. after chelating extracellular Ca2+ with EGTA), the PGE1-dependent rise in [Ca2+]i was reduced by 41.0 ± 7.7% (Fig. 5C). Similarly, when UTP-treated cells were stimulated with PGE1, the subsequent rise in [Ca2+]i was reduced by 39.2 ± 3.6% under Ca2+-free conditions. Irrespective of the level of [Ca2+]o, UTP treatment of HEL cells always potentiated the PGE1 response compared with the respective controls (1.7 ± 0.1-fold versus 1.8 ± 0.1-fold, in Ca2+-containing versus Ca2+-free medium, Fig. 5C). Comparable data were also obtained with the Galpha 16-suppressed cell line 3D4 (not shown). We conclude that PGE1 primarily induces Ca2+ release from internal stores, which subsequently triggers a capacitative Ca2+ influx that depends on [Ca2+]o. The relative contributions of Ca2+ release and influx to the PGE1-induced overall change in [Ca2+]i were similar with or without UTP treatment. UTP seems to enhance both the PGE1-dependent cellular Ca2+ mobilization and the Ca2+ influx component.

The potentiating effect of UTP on intracellular Ca2+ release was not restricted to cells stimulated with PGE1, nor did it appear to be a general phenomenon elicited by UTP. Potentiation was also observed when UTP-treated cells were stimulated subsequently with adenosine or neuropeptide Y, rather than with PGE1. On the other hand, ADP- or PAF- induced rises in [Ca2+]i were not enhanced by pretreatment with UTP (Fig. 4E and data not shown, respectively). PTX inhibited the effect of UTP on the PGE1-induced rise in [Ca2+]i by 78.8 ± 8.1% (mean ± S.E., n = 3), suggesting a major, although not exclusive, role of Gi in potentiation.

The Potentiating Effect of UTP Is Also Mediated by the P2U Purinoceptor

To assess whether the receptor subtype that triggered the UTP-dependent mobilization of intracellular Ca2+ was identical to that provoking the UTP-mediated potentiating effect on PGE1-induced Ca2+ mobilization, the potencies of UTP to induce these two effects were compared with those of ATP. ATP also potentiated PGE1-induced Ca2+ mobilization (Fig. 6A). Exposing cells to maximum effective concentrations of ATP and UTP together had no additive effect on PGE1-induced Ca2+ mobilization, as would be expected for a P2U purinoceptor-mediated effect (Fig. 6A). As stated above, ATP stimulated the mobilization of intracellular Ca2+ with a mean EC50 value that was similar to the one for UTP. If the receptors inducing the direct and indirect (potentiating) effects were indeed identical, the EC50 value for UTP for enhancing the PGE1 signal should also be similar to that for ATP. To test this hypothesis experimentally, HEL cells were stimulated with different concentrations of UTP or ATP to determine their potencies for direct Ca2+ mobilization. Subsequently, the same cells were challenged with PGE1, and increments of PGE1-induced changes in [Ca2+]i over control values (from cells that were not previously treated with nucleotides) were determined to estimate EC50 values for this potentiating effect. UTP mobilized Ca2+ with an EC50 value of 2.4 ± 0.2 µM, whereas the EC50 value for ATP was 4.0 ± 0.6 µM (Fig. 6, B and C). The EC50 values for the potentiating effect were lower by more than 1 order of magnitude for both nucleotides, but their relative potencies (UTP versus ATP) again were similar (EC50 = 0.10 ± 0.02 and 0.38 ± 0.07 µM, respectively) (Fig. 6, B and C). Furthermore, UDP, an agonist with a potency similar to that of UTP at the uridine nucleotide receptor (P2Y4), neither stimulated Ca2+ mobilization nor enhanced the PGE1-induced Ca2+ signal at concentrations that would have been fully effective for UTP (not shown).


Fig. 6. UTP and ATP are equally effective in potentiating the PGE1-induced rise in [Ca2+]i. Panel A, HEL cells were treated with ATP (100 µM), UTP (100 µM), or both or mock-treated as indicated (agonists 1). After [Ca2+]i had returned to resting levels, cells were challenged with PGE1 (7 µM) as the second agonist. Representative traces are shown. Panels B and C, dose-response relationships for the direct and potentiating effects of UTP or ATP were obtained by treating cells with varying concentrations of UTP or ATP prior to stimulation with PGE1 (7 µM) as described for panel A. The direct (closed symbols) and indirect, potentiating (open symbols) effects of UTP (panel B) and ATP (panel C) are shown. The data are presented as percentages of maximum changes as determined by fitting the data to the Hill equation assuming a Hill coefficient of 1. For the potentiating effect, nucleotide-dependent increments of PGE1-induced changes in [Ca2+]i after subtraction of the PGE1-induced changes in [Ca2+]i of mock-treated cells are given. The data points give the results from a single experiment. Similar results were obtained in an additional independent experiment for each nucleotide.
[View Larger Version of this Image (17K GIF file)]


Taken together, these data strongly suggest that direct Ca2+ mobilization by UTP and its indirect, potentiating effect on the Ca2+ release by other agonists are both mediated through P2U purinoceptors. We conclude that in HEL cells, P2U purinoceptors are coupled to two different signaling pathways. One pathway leads to the direct mobilization of intracellular Ca2+ and is strictly dependent on the presence of Galpha 16, the second pathway is independent of Galpha 16, mediates the potentiation of Ca2+ signals induced by some but not all agonists that raise [Ca2+]i, and involves Gi as a major signal transducer.


DISCUSSION

In the present study, we have used Galpha 16 antisense RNA expression to create Galpha 16-suppressed HEL cell sublines, which allowed us to assess the role of this protein by analyzing the resulting defects in receptor-effector coupling. We conclude from our results that endogenous purinoceptors of the P2U subtype interact specifically with the Galpha 16 protein to trigger intracellular Ca2+ release. It seemed obvious from the data in Fig. 1 that the antisense technique, although causing a major reduction, did not result in a complete elimination of Galpha 16 immunoreactivity. Since the residual activity could not prevent the loss of a partial function of the P2U purinoceptor, this function appears to require a threshold level of Galpha 16. Alternatively, the amount of immunoreactive material may significantly exceed the amount of functionally intact G protein. Similar discrepancies between residual G protein levels after antisense RNA expression or antisense oligodeoxynucleotide treatment and the associated functional defects have also been noted by others (39-42).

A number of recent studies indicated that Galpha 16 has the potential to couple to a broad spectrum of seven transmembrane domain receptors (11-14, 27). In all cases, PLC-beta isoforms seem to represent the primary effector system. These receptor-G protein interactions were detected when different receptors together with the alpha -subunits of various G proteins were overexpressed in COS-7 cells. However, whether all of these potential interactions indeed exist, and more importantly, are functionally relevant in hematopoietic cells, remains elusive. In HEL cells, the endogenous P2U purinoceptor productively interacts with G16 but apparently is unable to trigger Ca2+ release via Gq/11. Hematopoietic cell lines, including HEL cells, contain high levels of the latter G proteins, which share the same G protein family with Galpha 16 and are also linked to intracellular Ca2+ release (43-45). The selective functional defect resulting from the reduction in Galpha 16 expression clearly indicates that Gq and G16 are functionally not equivalent. In HEL cells, receptors for thrombin and PAF appear to engage Gq/11 (Ref. 28 and this study). Unlike P2U purinoceptor responses, the cellular response in HEL cells to the activation of these latter receptors is not affected significantly by the suppression of Galpha 16 (present data). Hence, in HEL cells Galpha 16 plays a distinct role specifically for the transduction of signals triggered by the P2U purinoceptor. Interestingly, signal transduction through the thrombin receptor was not affected by suppression of Galpha 16 in HEL cells, although thrombin receptors may couple to Galpha 16 in COS cells (11).

The inability of Galpha 16-suppressed cells to respond to UTP by increasing [Ca2+]i could have been the result of unintended clonal selection, which might have led to cell lines that no longer expressed P2U purinoceptors. We have not formally established the presence of P2U purinoceptors on the various cell lines in binding studies because there is still a lack of selective ligands with sufficiently high affinity. The analysis using the potentiating effect of the P2U purinoceptor agonist UTP to stimulate the Ca2+ response induced by PGE1 was used instead to demonstrate the presence of P2U purinoceptors in Galpha 16-suppressed cell lines. Since UTP may also stimulate the uridine nucleotide receptor and the novel P2 receptor, these receptors have also to be considered for explaining the potentiating effect. However, ATP is approximately 1,000-fold less potent than UTP at the novel P2 receptor (34). At the uridine receptor, ATP is at best a partial agonist (35, 36). In our study in HEL cells, ATP always showed the same efficacy as UTP. Thus, the distinct properties of the uridine and the novel P2 nucleotide receptors regarding UTP and ATP rule out a contribution of these receptors under our experimental conditions. As an alternate possibility, the lack of response to UTP might have been caused by altered expression of Galpha - and Gbeta gamma -stimulated isoforms of PLC-beta , favoring signaling by receptors that stimulate Gbeta gamma -sensitive isoforms such as PLC-beta 2 or -beta 3. Galpha -mediated stimulation of PLC requires the activation of Gq type G proteins; Gbeta gamma -mediated activation of the enzyme is initiated by the stimulation of Gi-coupled receptors and the subsequent liberation of Gbeta gamma subunits from Gi (29). A potential shift in the expression of PLC isoforms in Galpha 16-suppressed cells would therefore have resulted in enhanced sensitivity of these cells toward PTX when challenged with agonists that stimulate Gq- and Gi-dependent pathways. However, the relative inhibition of thrombin- or PGE1-stimulated changes in [Ca2+]i in HEL and in Galpha 16-suppressed cells was similar after PTX treatment. This indicates that the contributions of Gi- versus Gq-dependent pathways to the overall Ca2+ signal are similar in parental and transfected cell lines when challenged with agonists that stimulate both pathways.

The potentiating effects of UTP (or ATP) as outlined in Fig. 5 were also detected when PGE1 was added at the same time as the nucleotides and persisted for at least 15 min after the addition of the nucleotides (data not shown). Thus, potentiation does not appear to be subject to the same rapid desensitization process that was observed for Ca2+ mobilization (Fig. 4D). In rat basophilic leukemia RBL-2H3 cells, a similar potentiating ("priming") effect of thrombin on the chemoattractant-induced release of InsP3 was observed which also lasted several minutes (38). These cross-talk events between two different receptor-effector systems may thus require signaling components distal to receptors and their associated G proteins. Such a model would also predict that the dose-response relationships of agonists that stimulate direct and indirect effects through the same receptor may be different. The striking differences of EC50 values which were observed for the direct, G16-dependent Ca2+-mobilizing effect, and the indirect, Gi-mediated potentiating effect of UTP or ATP thus support the concept that two distinct signaling pathways originate at the P2U purinoceptor.

The observation that PTX inhibited P2U purinoceptor-stimulated Ca2+ signaling only partially, while suppression of Galpha 16 completely abrogated it, suggests strongly that Galpha 16 is obligatory for stimulation, whereas stimulation by Gbeta gamma represents a positively modulatory function. An analogous mechanism has been reported in the literature for types II and IV adenylyl cyclases involving Galpha s as the obligatory and Gi-derived Gbeta gamma as the modulatory G protein subunits (46). In HEL cells, the molecular site for integrating Galpha - and Gbeta gamma -mediated signaling pathways cannot be deduced from the present studies, but the beta 2 and beta 3 isoforms of PLC, which can be activated by both G protein subunits, may represent possible candidates for such a role. The priming effect of thrombin on chemoattractant signaling may indeed critically involve PLC-beta 2 (38). However, the activation of PLC-beta 2 by the two G protein subunits appears to be independent rather than conditional, as has been concluded from transient transfections or in vitro reconstitution studies (29, 47-49). The molecular basis for the conditional, Galpha 16-dependent priming event observed in this study will need further characterization of the phospholipases and perhaps other downstream components required in UTP-dependent Ca2+ mobilization.

The potential of the endogenous P2U purinoceptor to initiate two distinct signaling pathways that diverge at the level of the engaged G proteins is shared by many other receptors in various tissues (for a review, see Ref. 19). In native cells, multiple G protein coupling has usually been assumed if PTX inhibited signaling to some but not all effector systems, or if effector systems were only partially inhibited by the toxin (28, 50, 51). Such results could best be explained by alternative coupling of the receptor to PTX-sensitive (Gi, Go) and PTX-insensitive (Gq/11, G12/13) G protein subfamilies. Bifunctional coupling of the P2U purinoceptor has also been observed in the DDT1 MF-2 smooth muscle cell line (51). In this system, P2U purinoceptors activate a PLC-beta via a PTX-resistant pathway and inhibit adenylyl cyclase activity via a PTX-sensitive pathway. Since non-hematopoietic cells lack Galpha 16, PTX-resistant effects of P2U purinoceptor stimulation must be mediated by other members of the Gq subfamily. Moreover, in epithelial cells, activation of P2U purinoceptors resulted in a PTX-sensitive release of InsP3, suggesting interaction with a Gi type protein (52). In hematopoietic cells, coupling of the P2U purinoceptor to G16 and to Gi was observed (this study). These results indicate that P2U purinoceptors in native cells (as in the cotransfection studies mentioned above) are capable of interacting with G proteins from different subfamilies. The mechanisms determining which of the G proteins are specifically recruited by individual receptors in a given cell type are largely unknown. The work of Kleuss et al. (53, 54) suggests that the expression of specific isoforms of beta - and gamma -subunits of the trimeric G proteins together with a given alpha -subunit may confer cell-specific receptor-effector coupling.

It is well established that G protein-dependent signals may promote cell proliferation on their own or by modulating growth factor receptor responses (55, 56). Recent reports demonstrated that extracellular UTP and ATP are potent mitogens in rat renal mesangial and aortic smooth muscle cells (57-59). The nucleotides seem to act via stimulation of P2U purinoceptors. In addition, diadenosine tetraphosphate, which is released from neurons, chromaffin cells, and platelets, may also represent a physiological ligand at the P2U purinoceptor (60). The expression of P2U purinoceptors in a variety of tissues (33), including endothelial and hematopoietic cells, makes the P2U purinoceptor an interesting candidate for the hormonal (as opposed to growth factor-dependent) regulation of cell proliferation in the hematopoietic and vascular systems.


FOOTNOTES

*   This project was supported by Swiss National Science Foundation Grant 3100-039678.93/1 (to H. P.).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. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Pharmakologisches Institut, Universität Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland. Tel.: 41-31-632-3290; Fax: 41-31-632-4992; E-mail: baltensperge{at}pki.unibe.ch.
1   The abbreviations used are: PLC, phosphoinositide-specific phospholipase C; PTX, pertussis toxin; InsP3, inositol 1,4,5-trisphosphate; HEL, human erythroleukemia; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate; PGE1, prostaglandin E1; PGE2, prostaglandin E2; PAF, platelet-activating factor; [Ca2+]i, intracellular Ca2+ concentration; EC50, half-maximum effective concentration; [Ca2+]o, extracellular Ca2+ concentration.
2   The nomenclature for P2 purinoceptor subtypes is according to Watson and Girdlestone (20). The provisional nomenclature for purinoceptors as proposed by the IUPHAR Committee on Receptor Nomenclature and Drug Classification is given in parentheses (21).

ACKNOWLEDGEMENTS

We thank Dr. M. I. Simon (California Institute of Technology, Pasadena, CA) for providing the cDNA for Galpha 16. We are grateful to Dr. K. Spicher and Dr. B. Nürnberg, both from the laboratory of Prof. G. Schultz, Dept. of Pharmacology, Free University of Berlin, Germany, for antibodies and for purified Gbeta gamma heterodimer, respectively.


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