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Keywords:

  • β-arrestin;
  • β1-adrenoceptor;
  • β2-adrenoceptor;
  • EGF receptor;
  • Src

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

J. Neurochem. (2010) 115, 1007–1023.

Abstract

Stimulation of β-adrenoceptors activates the canonical adenylate cyclase pathway (via Gs protein) but can also evoke phosphorylation of extracellular-regulated kinases 1 and 2 (ERK1/2) via Gs/Gi switching or β-arrestin-mediated recruitment of Src. In primary cultures of mouse astrocytes, activation of the former of these pathways required micromolar concentrations of the β12-adrenergic agonist isoproterenol, that acted on β1-adrenoceptors, whereas the latter was activated already by nanomolar concentrations, acting on β2 receptors. Protein kinase A activity was required for Gs/Gi switching, which was followed by Ca2+ release from intracellular stores and G- and metalloproteinase-dependent transactivation of the epidermal growth factor receptor (EGFR; at its Y1173 phophorylation site), via its receptor-tyrosine kinase, β-arrestin 1/2 recruitment, and MAPK/ERK kinase-dependent ERK1/2 phosphorylation. ERK1/2 phosphorylation by Src activation depended on β-arrestin 2, but not β-arrestin 1, was accompanied by Src/EGFR co-precipitation and phosphorylation of the EGFR at the Src-phosphorylated Y845 site and the Y1045 autophosphorylation site; it was independent of transactivation but dependent on MAPK/ERK kinase activity, suggesting EGFR phosphorylation independently of the receptor-tyrosine kinase or activation of Ras or Raf directly from Src. Most astrocytic consequences of activating either pathway (or both) are unknown, but morphological differentiation and increase in glial fibrillary acidic protein in response to dibutyryl cAMP-mediated increase in cAMP depend on Gs/Gi switching and transactivation.

Abbreviations used:
dBcAMP

dibutyryl cAMP

DMEM

Dulbecco’s Minimum Essential Medium

EGF

epidermal growth factor

EGFR

EGF receptor

ERK

extracellular-regulated kinase

HRP

horseradish peroxidase

MEK

MAPK/ERK kinase

PKA

protein kinase A

PTX

pertussis toxin

There are three subtypes of β-adrenergic receptors, β1, β2 and β3. All of them are Gs protein-coupled receptors. Stimulation of these receptors increases the activity of adenylate cyclase and causes accumulation of intracellular cAMP. Both β1 and β2 receptors are abundantly expressed in mammalian brain, perhaps in a ratio of 2 : 1 (Nahorski et al. 1978; Sastre et al. 2001). In contrast, the density of β3 receptor expression in rodent cerebral cortex amounts to only 3% of that in brown adipose tissue (Summers et al. 1995).

The canonical pathway for β-adrenergic receptor stimulation is protein kinase A (PKA) activation via Gs and cAMP. Nevertheless, β-adrenergic phosphorylation (activation) of the MAPK extracellular-regulated kinases 1 and 2 (ERK1/2) has repeatedly been demonstrated. ERK phosphorylation via the MAPK cascade Ras, Raf, MAPK/ERK kinase (MEK) classically occurs in response to stimulation of the epidermal growth factor (EGF) receptor (EGFR) by growth factors or transactivation of a Gi/o or Gq protein-coupled receptors. In transactivation of the EGFR, activation of a Gi/o or Gq protein-coupled receptor (Pierce et al. 2001; Peng 2004) or increase in [Ca2+]i (Zwick et al. 1997; Piiper et al. 2003) leads to metalloproteinase-catalyzed shedding of an agonist at the EGFR, for example, heparin binding EGF, which then stimulates EGFRs (Prenzel et al. 2001). All three subtypes of β-adrenergic receptors can induce ERK1/2 phosphorylation after coupling with Gi protein in a PKA-dependent ‘Gs/Gi switching,’ as shown in Fig. 1 (Zamah et al. 2002; Martin et al. 2004). As a result the phosphorylation becomes inhibitable by pertussis toxin (PTX) (Gerhardt et al. 1999; Zou et al. 1999; Gosmanov et al. 2002; Hu et al. 2002, 2003; Zamah et al. 2002; Martin et al. 2004), an inhibitor of G (Moss et al. 1984). In HEK293 cells activation of this pathway by the β2-adrenergic receptor required that the receptor be phosphorylated by PKA, and it was blocked by H-89, a PKA inhibitor (Daaka et al. 1997).

image

Figure 1.  Schematic illustration of stimulation of ERK phosphorylation by β-adrenergic receptors in astrocytes. Isoproterenol (ISO) binds to β-adrenergic receptors. At high concentrations (≥ 1 μM), the activation of the receptors induces β1-adrenergic (red arrows), PKA-dependent ‘Gs/Gi switching,’ which in turn induces an enhancement of intracellular Ca2+ concentration by Ca2+ release from intracellular stores. The latter activates Zn-dependent metalloproteinases (MMPs) and leads to shedding of growth factor(s). The released EGF ligand stimulates autophosphorylation of EGFR at Y1173 site in the same and adjacent cells. The downstream target of EGFR, ERK (shown in blue) is phosphorylated via the Ras/Raf/MEK pathway, contingent upon recruitment of β-arrestins 1 and 2. The ERK phosphorylation by isoproterenol at high concentration can be inhibited by H-89, an inhibitor of PKA, by PTX, an inhibitor of Gi protein, by BAPTA/AM, an intracellular Ca2+ chelator, by GM6001, an inhibitor of Zn-dependent metalloproteinase, by AG1478, an inhibitor of the receptor-tyrosine kinase of the EGFR, by siRNA against β-arrestin 1 and less completely by siRNA against β-arrestin 2, and by U0126, a MEK inhibitor (all inhibitors shown in yellow). In contrast, at low concentration (≤ 100 nM) β2-adrenergic (green arrows) activation of the receptors activates Src via recruitment of β-arrestin 2. Src in turn stimulates ERK phosphorylation and phosphorylates EGFR at Y845 and Y1045 sites without involvement of the receptor-tyrosine kinase. ERK1/2 phosphorylation is secondary to MEK activation, which may be induced by direct activation of Raf or Ras by Src, or by Src-mediated phosphorylation of the EGFR. The ERK phosphorylation by isoproterenol at low concentration can be inhibited by siRNA against β-arrestin 2, by PP1, a Src inhibitor, and by U0126, a MEK inhibitor.

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Stimulation of β-adrenergic receptors can also cause ERK1/2 phosphorylation independently of Gs/Gi switching and transactivation. After G-protein-coupled receptor stimulation and activation of heterotrimeric G proteins, the receptors are phosphorylated by specialized serine-threonine kinases, called G protein-coupled receptor kinases. This phosphorylation is dependent on the βγ subunits of the G protein (Koch et al. 1993). It is an important determinant for efficient plasma-membrane recruitment of β-arrestin proteins to the receptor cytoplasmic domains and for desensitization of G protein signaling (Lefkowitz 1998). Recent evidence has shown that β-arrestins also can activate signaling cascades by serving as multiprotein scaffolds that bring together elements of specific signaling pathways, including ERK1/2 phosphorylation (DeWire et al. 2007). The binding of β-arrestin to the activated β-adrenoceptor can recruit Src, a non-receptor tyrosine kinase inhibited by PP1 (Igishi and Gutkind 1998), and initial signaling in the MAPK cascade, leading to ERK1/2 phosphorylation (Luttrell et al. 1999). In Src-over-expressing cells stimulated with EGF, Stokoe and McCormick (1997) found that Src co-localized and co-internalized with the activated EGFR. However, the presence of an antibody preventing EGF access to its receptor failed to prevent EGFR activation, indicating that EGFR activation was not because of the action of a conventional EGFR ligand. Src activation in response to angiotensin II in CHO-K1 cells can lead directly to activation of Ras (Seta et al. 2002). Also, Raf can be activated via Src by vascular endothelial growth factor in human endothelial cells (Alavi et al. 2003) and by a Src analog in drosophila (Xia et al. 2008). Moreover, there is evidence that EGF receptor phosphorylation at the Y845 site by Src promotes EGFR signaling (Donepudi and Resh 2008; Samarakoon et al. 2008). These different possibilities are shown by dotted green lines in Fig. 1. Nevertheless, Src-mediated ERK1/2 phosphorylation after β2-adrenergic agonist application can also occur without the involvement of β-arrestins and independently of G proteins (Sun et al. 1975, 2007).

Src-independent β-adrenergic ERK1/2 phosphorylation can be mediated via the G/adenylate cyclase/cAMP pathway. This has been shown in kidney COS-7 cells transfected with β1-adrenergic receptors, where PTX-sensitive Gi protein, Src family tyrosine kinase, receptor transactivation and EGF receptors were not involved (Zheng et al. 2010). Also, as shown in Fig. 1, instead of activating PKA, cAMP can bind to cAMP-regulated guanine exchange factors, known as Epacs (exchange proteins directly activated by cAMP). This leads to activation of a GTPase, Rap, in a PKA-independent fashion (De Rooij et al. 1998). Because Rap can interact with the Ras/ERK cascade, Epac-mediated β2-adrenergic signaling can modulate ERK-dependent processes in eukaryotic cells (Keiper et al. 2004).

Although some β-adrenergic effects in brain are clearly evoked by classical cAMP/PKA-mediated signaling, for example, stimulation of glycogenolysis (Sorg and Magistretti 1991), others are not. Glycogenolysis almost exclusively occurs in astrocytes, a type of glial cells, not in neurons (Ibrahim 1975). Astrocytes are densely equipped with β-adrenergic receptors (Ebersolt et al. 1981; Maderspach and Fajszi 1982, 1983; Aoki 1997; Hertz et al. 2010). It has long been known that exposure of primary cultures of astrocytes to either noradrenaline or dibutyryl cAMP (dBcAMP), a cell membrane-permeable cAMP precursor, causes morphological differentiation of the cells (Kimelberg et al. 1978; Narumi et al. 1978; Meier et al. 1991). More recently, it has been shown that β-adrenoreceptor-mediated morphological differentiation is associated with a delayed but sustained induction of ERK activity, not with the initial increase in PKA activity (Gharami and Das 2004).

The signaling pathways leading to ERK phosphorylation in astrocytes have not been determined. In the present work, we found that the pathways used in isoproterenol-induced ERK phosphorylation in cultured mouse astrocytes are concentration-dependent. At lower concentrations (≤ 100 nM), our results show that isoproterenol stimulates ERK phosphorylation by β-arrestin-mediated Src activation, without involvement of the EGF receptor tyrosine kinase (green lines in Fig. 1). At higher isoproterenol concentrations (≥ 1 μM), a PKA-mediated Gs/Gi switch leads to an increase of [Ca2+]i which, in turn, stimulates metalloproteinases and induces shedding of growth factor(s), causing transactivation of the EGF receptor (red lines in Fig. 1).

Experimental procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Materials

Chemicals for preparation of medium and most other chemicals were purchased from Sigma (St. Louis, MO, USA). These included isoproterenol, the β1-adrenergic receptor antagonist, betaxolol (4-(2-cyclopropyl-methoxyethyl)-1-phenoxy-3-isopropylaminopropan-2-ol) (Bianchetti et al. 1979), the β2-adrenergic receptor antagonist ICI118551 (erythro-(±)-1(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol) (MacDonald and Lamont 1993), PTX, the PKA inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide) (Kessels et al. 1993) and BAPTA/AM (bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl)-ester), a chelator of cytosolic free calcium concentration ([Ca2+]i) (Gordon et al. 1993). The Epac-Selective Cyclic AMP analog, 8-pCPT-2′-O-Me-cAMP (Holz et al. 2008) was purchased form Biolog Life Science Institute (Bremen, Germany). Tyrphostin AG1478 (N-[(2R)-2-(hydroxamido-carbonymethyl)-4-methylpentanoyl]-l-tryptophan methylamide), GM6001 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene) and PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) were obtained from Calbiochem (La Jolla, CA, USA). Santa Cruz Biotechnology (Santa Cruz, CA, USA) supplied first antibodies, rabbit polyclonal antibody raised against ERK (K-23) : sc-94, mouse monoclonal antibody against phosphorylated ERK (E-4) : sc-7383, rabbit polyclonal antibody against Src and the second antibodies goat anti-rabbit IgG horseradish peroxidase (HRP) conjugate. The sheep polyclonal antibody used for immunoprecipitation of EGFR (06–129), as well as Protein G agarose bead slurry (16–266) were purchased from Upstate Biotechnology (Lake Placid, NY, USA). The rabbit polyclonal antibody against EGFR (2232) and the EGFR site-specific rabbit polyclonal antibodies, against p-Y1173, p-Y845, p-Y1045, p-Y992 or p-Y1068 used for Western blotting were purchased from Cell Signaling Technology (Danvers, MA, USA) and the second antibody goat anti-mouse IgG HRP conjugate from Promega (Madison, WI, USA). ECL detection reagents were purchased from Amersham Biosciences, Buckinghamshire, UK. Oligofectamine™ Reagent for RNA interference, Opti-MEMI and fura-2 were obtained from Invitrogen Corp. (Carlsbad, CA, USA). Random Hexamer and Taq-polymerase for RT-PCR, were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China), and Superscript II from Gibco Life Technology Invitrogen (Grand Island, NY, USA).

Cell cultures

Primary cultures of astrocytes were prepared from the neopallia of the cerebral hemispheres of newborn CD-1 mice as previously described (Hertz et al. 1978, 1998) and grown in Dulbecco’s Minimum Essential Medium (DMEM) with 7.5 mM glucose. After the age of 2 weeks, 0.25 mM dBcAMP was included in the medium. Such cultures have been used in our laboratories for more than 30 years (Hertz et al. 1978) and they are highly enriched in astrocytes (> 95% purity of astrocytes expressing glutamine synthetase and glial fibrillary acidic protein). Addition of dBcAMP leads to a morphological and functional differentiation as evidenced by the extension of cell processes, increases in several metabolic activities and expression of voltage sensitive L-channels for calcium (Ca2+) (Hertz et al. 1989; Meier et al. 1991; Zhao et al. 1996), features characteristic of astrocytes in situ.

Immunoprecipitation and Western blotting for EGFR and specific phosphorylation sites

After homogenization, immunoprecipitation was performed with anti-EGFR antibody (Upstate Biotechnology) and Protein G agarose bead. Whole cell lysates (1000 μg) were incubated with 8 μg of anti-EGFR antibody for 12 h at 4°C. The supernatant was collected and the entire immunoprecipitates were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The membranes were incubated with the first antibody, specific to one of the phosphorylation sites, or rabbit anti-EGFR antibody at 1 × 1000 dilution for 2 h at 25°C as previously described (Li et al. 2008a; Du et al. 2009). The second antibody goat anti-rabbit IgG HRP conjugate at 1 × 1000 dilution was also used for 2 h at 25°C.

Co-immunoprecipitation of EGFR and Src

Immunoprecipitation of EGFR was performed as described above except that cells were lysed in non-denaturing lysis buffer [50 mM Tris–HCl (pH 7.4), 5 mM EDTA, 250 mM NaCl, 50 mM NaF, 0.1% Triton X-100, 0.1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, 4 μg/mL aprotinin, and 40 μg/mL pepstatin] (Garriga et al. 1998). The nitrocellulose membranes were incubated with the first antibodies, rabbit anti-v-Src antibody or rabbit anti-EGFR antibody, at 1 × 1000 or at 1 × 800 dilution for 2 h at 25°C.

Western blotting for ERK

Samples containing 50 μg protein were applied on slab gels of 12% polyacrylamide. The nitrocellulose membranes were incubated with the first antibody, specific to either p-ERK or ERK at 1 × 1000 or at 1 × 3000 dilution for 2 h at 25°C, and specific binding was detected by goat-anti-mouse or goat-anti-rabbit horseradish peroxidase-conjugated secondary antibody at 1 × 1500 or at 1 × 3000 dilution as previously described (Gu et al. 2007; Li et al. 2008a). Ratios were determined between scanned p-ERK1/2 and scanned ERK1/2.

RT-PCR for Epac1/2 and β-arrestin 1 and 2

For determination of mRNA expression of Epac1/2 or β-arrestin 1 and 2, a cell suspension was prepared, the RNA pellet was precipitated, and RT was performed as previously described (Kong et al. 2002).

PCR amplification was performed in a Robocycler thermocycler with sense (5′-GCTCTCCCCTCCTGTCATCC-3′) and antisense (5′-GTTCCCGCTGGTTGTCAATG-3′) for Epac1, with sense (5′-CATGAGGGGAACAAGACGTT-3′) and antisense (5′-GGCCTT-CGAGGCTCTAATCT-3′) for Epac2 (Moon and Pyo 2007), with sense (5′-AAGGGACACGAGTGTTCAAGA-3′) and antisense (5′-CCCGCTTTCCCAGGTAGAC-3′) for β-arrestin 1, with sense (5′-GGCAAGCGCGACTTTGTAG-3′) and antisense (5′-GTGAG-GGTCACGAACACTTTC-3′) for β-arrestin 2 (Luan et al. 2009), and with sense (5′-CCACGGACAACTGCGTTGAT-3′) and antisense (5′-GGCTCATAGCTACTGAACTG-3′) for TATA-binding protein (TBP) (Marjou et al. 2000), used as a housekeeping gene. Initially the template was denatured by heating to 94°C for 2 min, followed by thirty 2-min amplification cycles, each consisting of three 45-s periods, the first at 94°C, the second at 58.3°C for Epac1 and 2 or at 60.8°C for β-arrestin 1 and 2 and TATA-binding protein, and the third at 72°C. The final step was extension at 72°C for 10 min. The PCR products were separated by 1% agarose gel electrophoresis, and captured by Fluorchem 5500 (Alpha Innotech Corporation, San Leandro, CA, USA).

Knock-down of Epac1 and 2 and β-arrestin 1 and 2 expression

Duplex of Epac1 and 2 siRNA was purchased from Santa Cruz Biotechnology. Duplex of β-arrestin 1 siRNA (sense 5′-AAAGCCUUCUGUGCUGAGAAC-3′, and antisense 5′-GUUCUCAGCACAGAAGGCUUU-3′) and β-arrestin 2 siRNA (sense 5′-AAGGACCGGAAAGUGUUCGUG-3′, and antisense 5′-CACGAACACUUUCCGGUCCUU-3′) (Ahn et al. 2003) were synthesized by Sangon Co., Ltd. (Shanghai, China). To allow incorporation of siRNAs into astrocytes, 3-week-old astrocytes grown in 24-well plates were incubated in DMEM without serum for 24 h on the day before transfection. Transfection solution contained 2 μL Oligofectamine and 40 μL Opti-MEMI, and 2.5 μL siRNA (666 ng) was added to the culture for 8 h. In siRNA (−) control cultures, transfection solution without siRNA was added instead. Thereafter, 87.5 μL DMEM with 37.5 μL serum was added to the cultures.

Cytosolic Ca2+ concentrations

For determination of cytosolic free Ca2+ concentrations ([Ca2+]i) an Olympus IX71 live cell imaging fluorescence microscope (Tokyo, Japan) was used to record fluorescence intensity of fura-2 introduced in astrocyte cultures grown on coverslips coated with polylysine. For fura-2 loading the growth medium was replaced with saline solution (137 mM NaCl, 5 mM KCl, 0.44 mM KH2PO4, 4 mM NaHCO3, 1.3 mM CaCl2, 0.8 mM MgSO4, and 0.5 mM MgCl2 with 10 mM glucose) containing 5 μM fura-2 for 30 min at 37°C. After two times wash with similar saline, 200 μL pre-heated saline was pipetted onto the coverslip, and readings were made at 340 and 380 nm excitation and 510 nm emission at 15-s intervals for 60 s (four cycles) to establish a baseline. Subsequently saline solution (control) or isoproterenol to a final concentration of 100 nM or 1 μM was added. Readings were continued for another 75 s (five cycles) with similar settings as before. Twenty cells were selected in each coverslip, and three coverslips were used in each experimental group.

Statistics

The differences between individual groups were analyzed by one-way anova followed by Fisher’s LSD test. The level of significance was set at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Time course, concentration dependence, and receptor subtype

The effect of 1 μM isoproterenol on ERK phosphorylation is shown in Fig. 2(a). The phosphorylation occurred quite slowly, with no effect after 2, 5 (Figure S1) and 10 min of drug treatment. A statistically significant increase of ERK phosphorylation was seen after 20 min of drug treatment, and it was maintained at the same level after 40 min. Thereafter, the level of p-ERK1/2 in the drug treated cultures declined to a level after 60 min which was not statistically significantly different from that in control cultures. As there was no significant difference between stimulation of ERK1 and ERK2, the bands of ERK1 and ERK2 were scanned and quantified together as p-ERK/ERK (Fig. 2a-ii). In the presence of the β2-adrenergic antagonist ICI118551, the stimulation was significantly reduced, indicating that at 1 μM isoproterenol β2-adrenergic stimulation does not account for the entire effect. A time course was also determined at 100 nM, but it showed no significant difference from that seen at 1 μM (results not presented).

image

Figure 2.  Time course, concentration dependence, and subtype specificity for isoproterenol effects on extracellular-signal regulated kinase 1 and 2 (ERK1/2) phosphorylation in astrocytes. (a) Time course of isoproterenol-induced ERK1/2 phosphorylation. Bands of 44 and 42 kDa represent p-ERK1 (phosphorylated ERK1) and p-ERK2 (phosphorylated ERK2), respectively (upper rows), or total ERK1 and ERK2 (lower rows) in primary cultures of astrocytes. Cells were incubated for 0 (Control) or 10, 20, 40 or 60 min at 1 μM isoproterenol in the absence or presence of 0.1 μM ICI118551, the β2-adrenergic antagonist. (a-i) Immunoblots from a representative experiment. Similar results were obtained from three independent experiments. Average ERK1/2 phosphorylation was quantitated as ratios between p-ERK1/2 and ERK1/2 (a-ii). SEM values are indicated by vertical bars. *Statistically significant (< 0.05) difference from isoproterenol group at the same incubation time. (b and c) Dose-dependent effects of isoproterenol and effects of β-adrenergic subtype-specific antagonists (the β1-adrenergic antagonist betaxolol and the β2-adrenergic antagonist ICI118551 [ICI]) on ERK1/2 phosphorylation. Cells were incubated for 20 min at an isoproterenol concentration of 0 (Control), 10 nM, 100 nM, 1 μM or 10 μM either under control conditions (no other drug) or in the additional presence of 30 μM betaxolol (following initial pre-incubation with the antagonist for 15 min) (b) or of 0.1 μM ICI118551 (following initial pre-incubation with the antagonist for 15 min) (c). (b-i and c-i) Immunoblots from a representative experiment. Similar results were obtained from three independent experiments. Average ERK phosphorylation was quantitated densitometrically as ratios between p-ERK1/2 and ERK1/2 (b-ii and c-ii). SEM values are indicated by vertical bars. *Statistically significant (< 0.05) difference from control group; #statistically significant (< 0.05) difference from isoproterenol group at the same concentration. (d) Combination of the β1-adrenergic antagonist betaxolol and the β2-adrenergic antagonist (ICI118551 [ICI]) abolishes the effect of 1 μM isoproterenol on ERK1/2 phosphorylation. Cells were incubated for 20 min at an isoproterenol concentration of 0 (Control) or 1 μM either under control conditions (no other drug) or in the presence of 30 μM betaxolol plus 0.1 μM ICI118551. (d-i) Immunoblots from a representative experiment. Similar results were obtained from three independent experiments. Average ERK phosphorylation was quantitated densitometrically as ratios between p-ERK1/2 and ERK1/2 (d-ii). SEM values are indicated by vertical bars. *Statistically significant (< 0.05) difference from control group.

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Based on the results in Fig. 2(a) the concentration dependence was measured after 20 min of exposure to isoproterenol. Ten nM isoproterenol caused a significant increase of ERK phosphorylation, that was further enhanced when the isoproterenol concentration rose to 100 nM, from there to 1 μM, and onwards to 10 μM. An inhibitor of the β1-adrenergic receptor, betaxolol, at 30 μM, had no significant effect at isoproterenol concentrations of 10 and 100 nM, but it partly inhibited isoproterenol induced ERK phosphorylation at 1 and 10 μM (Fig. 2b-i,ii). The β2-adrenergic antagonist ICI118551 inhibited at all concentrations, significantly at 0.1, 1 and 10 μM (Fig. 2c-i,ii). This pattern suggests that the effect of 10–100 nM isoproterenol is a β2-adrenergic effect, whereas there is an additional β1-adrenergic effect at 1 and 10 μM isoproterenol. As expected, the ERK phosphorylation induced by 1 μM isoproterenol was abolished in the combined presence of ICI118551 and betaxolol (Fig. 2d).

Inhibitors of transactivation and of Src have different effects at low and high isoproterenol concentrations

One μM of AG1478, an inhibitor of the EGF receptor-tyrosine kinase, had no effect on ERK phosphorylation induced by 10 and 100 nM isoproterenol (Fig. 3a). This is in spite of previous observations that this concentration of AG1478 abolishes ERK phosphorylation by direct stimulation of the EGF receptor with EGF as well as EGF receptor transactivation by dexmedetomidine, an α2-adrenergic agonist (Li et al. 2008b), or by fluoxetine, which in astrocytes acts as a subtype-specific 5-HT2B agonist (Li et al. 2008a). Consistent with the lack of EGF receptor stimulation, GM6001, an inhibitor of Zn-dependent metalloproteinases had also no effect (Fig. 3b), indicating that no shedding of growth factors was involved. However, both inhibitors partly inhibited ERK phosphorylation by 1 and 10 μM isoproterenol (Fig. 3a and b). Conversely, PP1, an inhibitor of Src, abolished the effect of isoproterenol at 10 and 100 nM, but inhibited it only partly at 1 and 10 μM (Fig. 3c).

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Figure 3.  ERK1/2 phosphorylation after exposure to 10 or 100 nM isoproterenol does not require EGFR activation and shedding of growth factor(s) in astrocytes, but does require Src activity, whereas the opposite is true at 1 and 10 μM isoproterenol. Cells were incubated for 20 min with same concentrations of isoproterenol as in Fig. 2 in the absence of any other drug (Control) or in the additional presence of 1 μM of the EGFR inhibitor AG1478 (following initial pre-incubation with the inhibitor for 15 min) (a), of 10 μM of the inhibitor of Zn-dependent metalloproteinase, GM 6001 (following initial pre-incubation with the inhibitor for 15 min) (b), or of 10 μM of the Src inhibitor, PP1 (following initial pre-incubation with the inhibitor for 15 min) (c). (a-i, b-i and c-i) Immunoblots from a representative experiment. Similar results were obtained from three independent experiments. Average ERK phosphorylation was quantitated as ratios between p-ERK1/2 and ERK1/2 (a-ii, b-ii and c-ii). SEM values are indicated by vertical bars. *Statistically significant (< 0.05) difference from control group; #statistically significant (< 0.05) difference from isoproterenol group at the same concentration.

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β-Arrestins are differently involved at low isoproterenol concentrations, high isoproterenol concentrations and stimulation with EGF

The level of mRNA expression of β-arrestins 1 and 2 in astrocytes was about one half of that in brain samples (Fig. 4a-i,ii). The expression of both was greatly reduced in cells treated with the corresponding siRNA [siRNA(+)], whereas transfection solution without siRNA [siRNA(−)] had no effect, and was similar to control. Knock-down by siRNA of the expression of β-arrestin 1 abolished the additional ERK1/2 phosphorylation normally seen when the concentration of isoproterenol was increased from 100 nM to 1 μM (Fig. 4b-i,ii), whereas siRNA against β-arrestin 2 abolished ERK1/2 phosphorylation by 100 nM isoproterenol (a statistically significant effect, although the stimulation by 100 nM isoproterenol was modest) and greatly reduced that in the presence of 1 μM isoproterenol, although a small but significant stimulation remained (Fig. 4b-iii,iv). That siRNA against β-arrestin 1 interferes with the micromolar effect of isoproterenol was confirmed by showing that it reduced ERK1/2 phosphorylation by 1 μM isoproterenol, when the β2-adrenergic effect was inhibited by ICI, whereas knock-down of β-arrestin 2 had no corresponding effect; however, the reduction of the stimulation by β-arrestin 2 was large enough to suggest that ERK phosphorylation by β1-mediated transactivation was also partly impaired. Analogously, a relatively small direct stimulation of the EGF receptor with EGF at 10 ng/mL was inhibited not only by siRNA against β-arrestin 1, but also by that against β-arrestin 2 (Fig. 4c-i,ii).

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Figure 4.  ERK1/2 phosphorylation induced by 100 nM isoproterenol requires β-arrestin 2, but not β-arrestin 1 in astrocytes. (a) Expression of β-arrestin 1 or β-arrestin 2 in adult mice brain and astrocytes, and astrocytes treated with either transfection solution without siRNA (siRNA (−) [Control]) or with siRNA specific to β-arrestin 1 or β-arrestin 2 [siRNA (+)] for 3 days. PCR product of β-arrestin 1 is 66 bp, β-arrestin 2 107 bp or TBP 236 bp. (a-i) Southern blot from a representative experiment. Similar results were obtained from three independent experiments. Average mRNA expression was quantitated as ratios between β-arrestin 1 or β-arrestin 2 and TBP, used for housekeeping (a-ii). SEM values are indicated by vertical bars. *Statistically significant (< 0.05) difference from brain sample for both β-arrestin 1 and β-arrestin 2; #statistically significant (< 0.05) difference from control astrocytes [siRNA (−)] for both β-arrestin 1 and β-arrestin 2. (b) Astrocytes were treated with either transfection solution without siRNA [siRNA (−)] or with siRNA specific to β-arrestin 1 [siRNA (+)] (b-i), or with siRNA specific to β-arrestin 2 [siRNA (+)] (b-iii). Three days later, cells were incubated for 20 min in the absence of any drug (Control) or in the presence of 100 nM isoproterenol. (b-i and b-iii) Immunoblots from a representative experiment. Similar results were obtained from three independent experiments. Average ERK phosphorylation was quantitated as ratios between p-ERK1/2 and ERK1/2 (b-ii and b-iv). SEM values are indicated by vertical bars. *Statistically significant (< 0.05) difference from other groups; #statistically significant (< 0.05) difference from 100 nM groups. (c) Astrocytes were treated with either transfection solution without siRNA [siRNA (−)], with siRNA specific to β-arrestin 1 [siRNA (+)] or with siRNA specific to β-arrestin 2 [siRNA (+)]. Three days later, cells were incubated for 20 min in the absence of any drug (Control) or in the presence of 1 μM isoproterenol, of the β2-adrenergic antagonist (ICI118551 [ICI]) at 0.1 μM, or of isoproterenol plus ICI or EGF at 10 ng/mL. (c-i) Immunoblots from a representative experiment. Similar results were obtained from three independent experiments. Average ERK phosphorylation was quantitated as ratios between p-ERK1/2 and ERK1/2 (c-ii). SEM values are indicated by vertical bars. *Statistically significant (< 0.05) difference from isoproterenol siRNA (−) in isoproterenol group, from isoproterenol plus ICI siRNA (−) and isoproterenol plus ICI with β-arrestin 2 siRNA (+) in ICI + isoproterenol group, or from EGF siRNA (−) in EGF group.

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Different phosphorylation sites of the EGF receptor are phosphorylated by low and high concentrations of isoproterenol and by EGF

Phosphorylation at sites Y1173, Y845, Y1045, Y992, and Y1068 of the EGF receptor was assayed with specific antibodies (Fig. 5a-i). In control samples, EGF receptors were phosphorylated at Y1173, Y845 and Y1045. Isoproterenol at 100 nM increased phosphorylation at Y845 and Y1045, but had no additional effect at Y1173, and Y992 and Y1068 remained non-phosphorylated (Fig. 5a-ii). EGF at 10 ng/mL stimulated phosphorylation at all five sites (Fig. 5a-iii) as previously reported (Peng et al. 2010). An increase of the isoproterenol concentration from 100 nM to 1 μM isoproterenol caused phosphorylation also of the Y1173 phosphorylation site, whereas sites Y845 and Y1045 showed no additional phosphorylation beyond that seen at 100 nM, and Y992 and Y1068 remained non-phosphorylated (Fig. 5b-i,ii). AG1478 and GM6001 inhibited phosphorylation of Y1173, but had no effect at Y845 and Y1045 (Fig. 5c). The Src inhibitor PP1 had no effect on phosphorylation of Y1173 by 1 μM isoproterenol (Fig. 5c-ii), but abolished that of Y845 and Y1045 (Fig. 5c-iii,iv). It had a similar effect on Y845 and Y1045 phosphorylation by 100 nM isoproterenol (results not presented), and co-immunoprecipitation of EGF receptors with Src showed that Src binding to the EGF receptor was almost tripled by 100 nM isoproterenol (Fig. 6a and b).

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Figure 5.  EGF and isoproterenol promote EGFR phosphorylation at specific tyrosine residues in astrocytes in primary cultures. Cells were incubated for 10 min in the absence of any drug (Control), in the presence of 100 nM isoproterenol, of 10 ng/mL EGF (a), in the presence of 1 μM isoproterenol (b), in the presence of one of the inhibitors (1 μM AG1478, an antagonist of EGFR, 10 μM GM 6001, an inhibitor of Zn-dependent metalloproteinase, or 10 μM PP1, an inhibitor of Src), or isoproterenol plus one of the inhibitors (c). (a-i, b-i and c-i) Immunoblots of immunoprecipitates from a representative experiments. Similar results were obtained from three independent experiments. Average tyrosine phosphorylation was quantitated as ratios between EGFR phosphorylated at each of the residues and non-phosphorylated EGF receptor (EGFR) (a-ii,iii, b-ii and c-ii,iii,iv). SEM values are indicated by vertical bars. *Statistically significant (< 0.05) difference from other groups at the same residue.

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Figure 6.  Isoproterenol induces recruitment of Src to EGFR. Cells were incubated for 10 min in the absence of any drug (Control) or in the presence of 100 nM isoproterenol. (a) Immunoblot of co-immunoprecipitates from a representative experiment. Similar results were obtained from three independent experiments. (b) Average Src co-immunoprecipitated with EGFR was quantitated as ratios between Src and EGFR. SEM values are indicated by vertical bars. *Statistically significant (< 0.05) difference from control group.

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Free cytosolic Ca2+ concentration ([Ca2+]i) is differently affected at low and high isoproterenol concentrations

Isoproterenol at 10 or 100 nM had no effect on free cytosolic Ca2+ concentration ([Ca2+]i), whereas 1 μM isoproterenol caused an increase (Fig. 7a). Following isoproterenol addition at 15 s, the increase in [Ca2+]i reached its peak 15 s later and returned to baseline within 1 min. It was completely inhibited by H-89, an inhibitor of protein kinase A (PKA) and by PTX, an inhibitor of the α subunit of Gi protein-coupled receptors (Fig. 7b and c), suggesting that the increase in [Ca2+]i was a response to Gs/Gi switching and PKA activity.

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Figure 7.  ERK1/2 phosphorylation induced by 1 μM isoproterenol requires ‘Gs/Gi switching,’ PKA activation and increase of intracellular Ca2+ concentration in astrocytes. (a, b and c) After the cells had been loaded with fura-2, they were incubated for 60 s in solution in the absence of any drug (only the last 15 s shown in the graph). Subsequently, either saline solution (Control) or isoproterenol solution to a final concentration of 100 nM or 1 μM was added (a), and the incubation was continued for another 75 s. In (b) H-89, an inhibitor of PKA to a final concentration of 5 μM, and in (c) PTX, an inhibitor of Gi protein, to a final concentration of 20 μg/mL, was added with isoproterenol. Results are averages of four individual experiments. *Statistically significant (p < 0.05) difference from all other groups at the same recording time. (d) After pre-treatment with H-89, BAPTA/AM or PTX for 15 min, cells were incubated for 20 min in the absence of any drug (Control) or in the presence of 1 μM of isoproterenol, of 500 nM H-89, an inhibitor of PKA, of 30 μM BAPTA/AM, the intracellular [Ca2+]i chelator, of 10 μM PTX, an inhibitor of Gi protein, or of isoproterenol plus one of the inhibitors. (d-i) Immunoblots from a representative experiment. Similar results were obtained from three independent experiments. Average ERK phosphorylation was quantitated as ratios between p-ERK1/2 and ERK1/2 (d-ii). SEM values are indicated by vertical bars. *Indicates statistically significant (< 0.05) difference from control and inhibitor groups and #statistically significant (< 0.05) difference from groups of isoproterenol plus one of the inhibitors. (e) β2-adrenergic antagonist (ICI118551 [ICI]) abolished the effect of 1 μM isoproterenol on ERK phosphorylation via the β-arrestin 2 and Src pathway. After pre-incubation with or without one of the inhibitors for 15 min, cells were incubated for 20 min in the absence of any drug (Control) or in the presence of 1 μM isoproterenol, of 500 nM H-89, an inhibitor of PKA, of 30 μM BAPTA/AM, the intracellular [Ca2+]i chelator, of 10 μM PTX, an inhibitor of Gi protein, or of isoproterenol plus one of the inhibitors. (e-i) Immunoblots from a representative experiment. Similar results were obtained from three independent experiments. Average ERK phosphorylation was quantitated as ratios between p-ERK1/2 and ERK1/2 (e-ii). SEM values are indicated by vertical bars. *Indicates statistically significant (< 0.05) difference from other groups.

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Gi-mediated signaling and PKA activity are differently involved at low and high isoproterenol concentrations

Pertussis toxin, H-89 or the [Ca2+]i chelator BAPTA/AM, which inhibits Ca2+ release from intracellular stores, also partly inhibited the phosphorylation of ERK in response to 1 μM isoproterenol (Fig. 7d-i,ii). In the additional presence of the β2-adrenergic antagonist ICI118551, the inhibition became complete (Fig. 7e-i,ii). In contrast, none of these inhibitors (Fig. 8a–c) was able to reduce the effect on ERK1/2 phosphorylation by 100 nM isoproterenol, indicating thatGs/Gi switching, PKA activity and Ca2+ release were not involved in the phosphorylation.

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Figure 8.  ERK1/2 phosphorylation induced by 100 nM isoproterenol does not require ‘Gs/Gi switching,’ PKA activation and increase of intracellular Ca2+ concentration in astrocytes. After pre-incubation with or without PTX, H-89 or BAPTA/AM for 15 min, cells were incubated for 20 min in the absence of any drug (Control) or in the presence of 100 nM isoproterenol, of 10 μM PTX, an inhibitor of Gi protein (a), of 500 nM H-89, an inhibitor of PKA (b), of 30 μM BAPTA/AM, the intracellular [Ca2+]i chelator (c), or of isoproterenol plus one of the inhibitors. (a-i, b-i and c-i) Immunoblots from a representative experiment. Similar results were obtained from three independent experiments. Average ERK phosphorylation was quantitated as ratios between p-ERK1/2 and ERK1/2 (a-ii, b-ii and c-ii). SEM values are indicated by vertical bars. *Indicates statistically significant (< 0.05) difference from other groups.

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Epac-mediated signaling is not involved in the effects of isoproterenol

Stimulation of PKA, required for ERK1/2 phosphorylation by 1 μM isoproterenol, occurs in response to elevation of cAMP, which might also stimulate the Epac pathway (Fig. 1). Although both Epac1 and Epac2 were expressed in the cultured astrocytes (reults not presented), Epac stimulation was not involved in the signaling pathway of isoproterenol, as Epac1/Epac2 siRNA had no effect on isoproterenol-induced ERK1/2 phosphorylation either at 100 nM or at 1 μM (Figure S2).

MEK activity is required for the effect of both low and high isoproterenol concentration

The inhibitor of MEK, U0126, abolished ERK1/2 phosphorylation in response to both 100 nM and 1 μM isoproterenol (Fig. 9a and b). This indicates a conventional mechanism for ERK phosphorylation rather than the direct interaction between Src and ERK described in Schwann cells (Tapinos and Rambukkana 2005).

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Figure 9.  ERK1/2 phosphorylation induced by isoproterenol requires MEK activation in astrocytes. After pre-incubation with or without U0126, an inhibitor of MEK, for 15 min, cells were incubated for 20 min in the absence of any drug (Control), in the presence of 100 nM or 1 μM isoproterenol, in the presence of 10 μM U0126, or in the presence of U0126 plus 100 nM or 1 μM isoproterenol. (a) Immunoblots from a representative experiment. Similar results were obtained from three independent experiments. Average ERK phosphorylation was quantitated as ratios between p-ERK1/2 and ERK1/2 (b). SEM values are indicated by vertical bars. *Indicates statistically significant (< 0.05) difference from other groups.

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Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Pathways activated by nanomolar and micromolar isoproterenol concentrations

Nanomolar isoproterenol concentrations

The inhibition by ICI118551, but not by betaxolol, identi-fies the nanomolar effect of isoproterenol as evoked by stimulation of β2-adrenoceptors. The exclusion of transactivation, Gs/Gi switching, PKA activation, increase in [Ca2+]i, and signaling via Epac, together with the requirement for β-arrestin 2 and Src and MEK activity, and the co-precipitation of Src and the EGF receptor suggest that Src may be activated via β-arrestin 2 and in turn might interact directly with the EGF receptor, independently of its receptor-tyrosine kinase, and thereby activate MEK and ERK phosphorylation (Fig. 1). An alternate possibility is that MEK activation occurs independently of EGF receptor activation as a direct response of Ras or Raf to Src activation, as previously reported by Seta et al. (2002) and Alavi et al. (2003). This possibility may be supported by the lack of inhibition by siRNA against β-arrestin 1, which inhibits the effect of EGF and apparently also of micromolar isoproterenol concentrations. However, it can also not be excluded that the dependence of the pathway from the EGF receptor to Ras may be influenced by the mode of activation of the EGF receptor.

The phosphorylation of the EGF receptor at Y845 and Y1045 reflects a direct interaction between Src and the EGFR. Y845 is known to be a major Src phosphorylation site (Biscardi et al. 1999; Tice et al. 1999; Wu et al. 2002), and it was also phosphorylated in astrocytes by dexmedetomidine, which required Src activity for ERK1/2 phosphorylation by transactivation (Li et al. 2008b). The Y845 site mediates binding to the mitochondrial cytochrome c oxidase subunit II (Boerner et al. 2004), which may be important for survival or death pathways. The demonstrated ERK1/2 phosphorylation is consistent with the finding by Donepudi and Resh (2008) that Src can induce EGFR activation and downstream signaling to Shc and ERK1/2 without EGF addition. In HEK293 cells, where β2-adrenergic activation stimulates ERK1/2 phosphorylation both after Gs/Gi switching and via arrestin-mediated Src stimulation, the former was rapid, peaking within 2–5 min and quite transient, whereas the Src-dependent activation was slower in onset, less robust, but more sustained (Shenoy et al. 2006). Such differences were not found in the present study. Y1045 is an autophosphorylation site (Carpenter 2000; Iordanov et al. 2002; Guo et al. 2003). It is important for receptor binding to the Cbl family of ubiquitin ligases (Oksvold et al. 2003; Grøvdal et al. 2004), a step towards endocytosis and degradation. Accordingly hypophosphorylation of this site leads to defective EGFR down-regulation (Han et al. 2006). Its co-phosphorylation during EGF receptor signaling is also essential for mitogenic activity (see below).

Micromolar isoproterenol concentrations

The inhibition by betaxolol, but not by ICI118551 identifies the micromolar effect as evoked by stimulation of β1-adrenoceptors. The inhibition by H-89, PTX, BAPTA/AM, GM6001, AG1478, U0126 and siRNA against β-arrestin 1 and apparently also against β-arrestin 2, but not by siRNA against Epac or by the inhibitor of Src, PP1, indicates that high isoproterenol concentrations signal via PKA, Gs/Gi switching, [Ca2+]i release from intracellular stores, metalloproteinase-mediated transactivation of the EGF receptor, and MEK-dependent MAPK activation, but not via Src (Fig. 1). β-Arrestin 1 and 2 recruitment during stimulation of the EGF receptor with EGF suggests that β-arrestin recruitment must be downstream of the EGF receptor. Although this pathway in many cases has been described after β2-adrenergic stimulation (Maudsley et al. 2000; Pierce et al. 2000; Yeh et al. 2005), there is precedence for its use after β1-adrenergic stimulation (Hu et al. 2003). It is also well established that EGF receptors, like many G protein-coupled receptors (GPCRs), specifically utilize β-arrestin proteins for their internalization (Vieira et al. 1996; Kim et al. 2003), suggesting that endocytosis is involved in ERK activation mediated by the EGFR. Moreover, a dominant inhibitory form of β-arrestin 1 significantly attenuated EGF receptor-mediated MAPK activation (Pierce et al. 2000).

The only additional site phosphorylated by micromolar isoproterenol concentrations was Y1173, whereas Y845 and Y1045 showed no further phosphorylation when the isoproterenol concentration was increased. Y1173 is a major interaction site for Shc (Ward et al. 1996; Saito et al. 2004), and thus for activation of the MAPK pathway Ras-Raf-MEK-ERK, whereas Y992 is a minor phosphorylation site (Bishayee et al. 1999), although its activation suffices to initiate the intracellular signaling pathways of EGFR (Sorkin et al. 1992).

Simultaneous activation of both pathways

Micromolar concentrations of isoproterenol activated both G protein- and β-arrestin-mediated signaling. Although we do not know what effect this has on signaling downstream from ERK1/2, simultaneous β2-adrenergic and EGF stimulation has downstream effects, which neither of the agonists has when administered alone, and which depend upon the co-phosphorylation of Y992, Y1173 and Y845 (Drube et al. 2006). DNA synthesis and mitogenic activity also require Y845 phosphorylation in addition to EGF or a transactivating G protein-coupled receptor agonist (Tice et al. 1999; Boerner et al. 2004, 2005). A dual pathway response to β2-adrenoceptor stimulation in mouse embryonic fibroblasts (Sun et al. 2007a,b) may provide ideal possibilities for DNA synthesis by a high adrenaline concentration.

Astrocytes and β-adrenoceptors

Astrocytes express a number of receptors for neurotransmitters (Hansson and Rönnbäck 2004) and play important roles in the normal function of the brain. β-Adrenergic receptors are densely expressed on astrocytes (Aoki 1997; Hertz et al. 2010) and their functions have been studied extensively during the last two decades (Hertz et al. 2004; Laureys et al. 2010). Until now especially Gs protein-mediated functions, for example, glycogenolysis, have been investigated and found to be of major importance both in brain energy metabolism (Hertz et al. 2007, 2010) and in astrocytic contributions to learning (Gibbs et al. 2008). However, astrocytes in the mouse brain in vivo respond to a high concentration of isoproterenol with an increase in [Ca2+]i (Bekar et al. 2008), indicating that Gs/Gi switching of β-adrenergic receptors occurs in the brain in situ. The importance of this effect in the adult brain is unknown but addition to astrocyte cultures of dBcAMP, which mimics isoproterenol by causing an increase in intracellular cAMP (Facci et al. 1987) leads to pronounced developmental changes (Meier et al. 1991). They include an extension of processes (stellation) and increase in the expression of glial fibrillary acidic protein. These effects are inhibited by H-89, GM6001 and AG1478 (Li et al. 2010; T. Du and L. Peng, unpublished results), indicating the involvement of Gs/Gi switch, release of EGF receptor agonist and activation of the receptor-tyrosine kinase of the EGF receptor. A lack of abolishment by the Src inhibitor PP1 shows that the β-arrestin/Src-mediated pathway is not essential for the responses. This is consistent with the β1-adrenergic pathway shown in Fig. 1.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This study was supported by Grants No. 30670651, No. 30770667 and No. 30711120572 from the National Natural Science Foundation of China.

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  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Figure S1. Isoproterenol does not induce ERK1/2 phosphorylation after 2 or 5 min incubation in astrocytes. Bands of 44 and 42 kDa represent p-ERK1 (phosphorylated ERK1) and p-ERK2 (phosphorylated ERK2), respectively (upper rows), or non-phosphorylated ERK1 and ERK2 (lower rows) in primary cultures of astrocytes. Cells were incubated for 2 or 5 min at 0 (Control), 100 nM or 1 µM isoproterenol. (a) Immunoblots from a representative experiment. Similar results were obtained from three independent experiments. Average ERK1/2 phosphorylation was quantitated as ratios between p-ERK1/2 and ERK1/2 (b). SEM values are indicated by vertical bars.

Figure S2. ERK1/2 phosphorylation induced by isoproterenol does not require Epac activation in astrocytes. (a) Expression of Epac1 and Epac2 mRNA in brain in vivo and in astrocytes or astrocytes treated with siRNA specific to Epac1/2 for 1 week. TBP is used as a house-keeping gene. The size of the PCR product of Epac1 is 823 bp, Epac2 230 bp and TBP 236 bp. (a-i) Southern blot from a representative experiment. Similar results were obtained from three independent experiments. Average mRNA expression was quantitated as ratios between Epac1or Epac2 and TBP (a-ii and a-iii). SEM values are indicated by vertical bars. *Indicates statistically significant (< 0.05) difference from brain sample for Epac1, and #statistically significant (< 0.05) difference from control astrocytes for both Epac1 and Epac2. (b, c and d) Astrocytes were treated with either transfection solution without siRNA {siRNA (−) [Control]} or with siRNA specific to Epac1/2 [siRNA (+)]. One week later, cells were incubated for 20 min in the absence of any drug (Control), in the presence of 1 µM 8-pCPT-2′-O-Me-cAMP (CPT), a specific stimulator of Epac (b), or in the presence of 100 nM isoproterenol (c), or in the presence of 1 µM isoproterenol (d). (b-i, c-i and d-i) Immunoblots from representative experiments. Similar results were obtained from four independent experiments. Average ERK phosphorylation was quantitated as ratios between p-ERK1/2 and ERK1/2 (b-ii, c-ii and d-ii). SEM values are indicated by vertical bars. *Indicates statistically significant (< 0.05) difference from other groups.

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