Changes and role of adrenoceptors in PC12 cells after phenylephrine administration and apoptosis induction

Neurochemistry International 57 (2010) 884–892

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Neurochemistry International journal homepage: www.elsevier.com/locate/neuint

Changes and role of adrenoceptors in PC12 cells after phenylephrine administration and apoptosis induction Lubomira Lencesova a,b, Marta Sirova a, Lucia Csaderova b, Marcela Laukova c, Zdena Sulova a, Richard Kvetnansky c, Olga Krizanova a,* a b c

Institute of Molecular Physiology and Genetics, Center of Excellence for Cardiovascular Research, Slovak Academy of Sciences, Vlarska 5, 833 34 Bratislava, Slovak Republic Molecular Medicine Center, Slovak Academy of Sciences, Bratislava, Slovak Republic Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovak Republic

A R T I C L E I N F O

A B S T R A C T

Article history: Received 21 July 2010 Received in revised form 21 September 2010 Accepted 24 September 2010 Available online 1 October 2010

The present study addresses the hypothesis that adrenergic regulation modulates the effect of apoptosis. Therefore we studied, whether a1-adrenergic receptor’s agonist phenylephrine (PE) can affect or induce apoptosis in rat pheochromocytoma (PC12) cells. We have shown that PE treatment did not increase level of the apoptosis, or level of the caspase 3 mRNA. When apoptosis was induced in the presence of PE, caspase 3 mRNA was significantly increased, while the percentage of apoptotic cells remained unchanged compared to apoptotic group without PE. During this process, a1D-, b2- and b3-adrenergic receptors (ARs) were upregulated. Since all these three types of ARs are differently localized in the cell, we assume that mutual communication of all three ARs is crucial to participate in this signaling and during development of apoptosis, some of these systems might translocate. Another important system in handling noradrenaline during apoptosis might be noradrenaline transporter (NET), since it was downregulated in apoptotic cells treated with PE, compared to untreated apoptotic cells. However, precise mechanism of mutual communication among all these systems remains to be elucidated. ß 2010 Elsevier Ltd. All rights reserved.

Keywords: Adrenergic receptors Apoptosis Noradrenaline transporter Phenylephrine PC12 cells

1. Introduction Noradrenaline (NA) is an important adrenergic neurotransmitter. At large doses, NA has been shown to induce cell apoptosis in a variety of cell types, including neurons, cardiomyocytes and pheochromocytoma cells (Mao et al., 2006). Recent studies have shown that the proapoptotic effect of NA involves activation of caspases 3 and 9, but the exact cellular targets and related gene expressions induced by NA are still unknown (Fu et al., 2004). Both, NA and adrenaline (Adr) bind to adrenergic receptors (ARs). ARs are members of the G-protein coupled receptor family and they mediate physiological responses to the catecholamines noradrenaline and adrenaline. ARs are subdivided into three major families (a1, a2 and b) based on their structure, pharmacology and signaling mechanisms (Hieble et al., 1995). Lands et al. (1967) subdivided the b-AR mediated effects into b1 and b2 on the basis of the rank order of potency of NA and Adr in different tissues. Later, third type of b-ARs, b3-AR, was founded and characterized (for review see Skeberdis, 2004). ARs vary in the sensitivity to Adr/NA and also in the activation of downstream cascades (for review see Krizanova et al., 2007).

* Corresponding author. Tel.: +421 2 54772211; fax: +421 2 54773666. E-mail address: [email protected] (O. Krizanova). 0197-0186/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2010.09.007

Apoptosis, or programmed cell death, plays an indispensible role in embryonic development, maturation of the immune system, and maintenance of tissue and organ homeostasis (Martin, 1993). Involvement of catecholaminergic modulation in the process of apoptosis is still under investigation. Most of apoptotic effects of NA and Adr were shown on cardiomyocytes. In heart failure, apoptosis of cardiac myocytes in response to NA is believed to be an important component of the progression of cardiac fibrosis (Iwase et al., 1996; Communal et al., 1998). Stimulation of b1-AR in the rat heart can cause not only positive inotropic effects, but also can evoke cardiomyocyte apoptosis, while stimulation of b2-ARs appears to be antiapoptotic (Communal et al., 1999). Also, NA induces dose-dependent apoptosis in human and rat alveolar epithelial cells by a mechanism that involves the combination of aand b-ARs as well as autocrine angiotensin II production (Dincer et al., 2001). It was already shown that NA activated a mitochondrial apoptotic pathway, as evidenced by the increased cleaved 37 kDa caspase 9, as well as cytochrome c translocation from the mitochondria to the cytosol in PC12 cells (Mao et al., 2006). However, it is not clear, which ARs are involved in the activation of apoptotic pathways by NA in PC12 cells. Noradrenaline transporter (NET) is a member of gene family of Na+/Cl-dependent plasma membrane transporters, which removes NA from the synaptic space and quickly terminates the

L. Lencesova et al. / Neurochemistry International 57 (2010) 884–892

actions of extracellular NA on postsynaptic receptors (Blakely et al., 1994). It was already shown that decrease of NET density and NA uptake activity occurs also in PC12 cells after NA administration and these effects of NA are mediated via a posttranscription event caused by NA-derived oxidative stress (Blakely et al., 1994). Thus, NET plays a major role in the regulation of NA related actions at cellular level. Due to its early decrease in neurodegenerative diseases, NET appears to be a useful biomarker of these diseases. There are significant decreases in NET density in the LC and in Alzheimer’s disease brains as compared to age matched controls (Gulyas et al., 2010). The present study addresses the hypothesis that adrenergic modulation potentiated the effect of apoptosis in PC12 cell line. PC12 cells are derived from rat pheochromocytoma (Greene and Tischler, 1976). Pheochromocytomas are tumors of chromaffin cells that produce and often secrete catecholamines. Pharmacological control of the physiological and pathological effects of excess circulating catecholamines represents a continuing requirement in the treatment of metastatic or incompletely respectable invasive pheochromocytomas. Alpha adrenergic blockers and calcium channels antagonists often reduce hormone-mediated symptoms sufficiently. We studied, whether phenylephrine (PE), an a1-AR’s agonist can induce and/or affect apoptosis in rat PC12 cells and whether PE and induction of early stage of apoptosis (for 3 h) will affect gene expression and protein levels of ARs. Also, we used immunofluorescence staining and confocal laser scanning to determine the cellular and subcellular locations of selected ARs to propose the role of these receptors in the process of apoptosis. 2. Materials and methods 2.1. Cell culture PC12 cells were cultured in Minimal essential medium of Dulbecco (DMEM; Biochrom AG, Germany) with high glucose (4.5 g/l), supplemented with 15% fetal bovine serum (Biochrom AG, Germany) and antibiotics penicillin and streptomycin (0.5%; Calbiochem, Merck Biosciences, Germany). PC12 cells were cultured in a water-saturated atmosphere at 37 8C and 5% CO2. 2.2. Treatment of cells with PE and AIK PC12 cells were pretreated with 10 mM phenylephrine hydrochloride (PE; Sigma–Aldrich, Germany) for 3 h. Apoptosis inducer set I (AIK; Calbiochem, Merck Biosciences, Germany) was added to induce apoptosis in PC12 cells in the dilution 1:1000 as recommended by the provider. AIK is composed of following inducers – actinomycin D, camptothecin, cycloheximide, dexamethasone and etoposide. Apoptosis was induced for 3 h, afterwards cells were used for RNA isolation, Western blot analysis and immunofluorescence. 2.3. Measurement of pHi by fluorescence probe Intracellular pH (pHi) was measured using the fluorescent probe 20 ,70 biscarboxyethyl-5,6-carboxyfluorescein (BCECF; Sigma–Aldrich, USA). Cells plated onto 6-well plates were loaded with 8.2 mM BCECF and 5% pluronic acid in PBS buffer, pH 7.48 for 30 min at 37 8C, 5% CO2, in dark. Afterwards, cells were washed with PBS buffer and calibration was performed using PBS/HEPES buffers with different pH values (pH 7.51; pH 7.48; pH 7.03; pH 6.52; pH 6.01). The fluorescence was excited at 489 nm and measured at 525 nm on the fluorescence scanner BioTek (Germany). The pHi signal was calibrated to pH0 by adding 10 mM nigericin (Sigma–Aldrich, USA) with 130 mM KCl. These values were used for the calibration curve, from which different pHi values were calculated. 2.4. RNA preparation and relative quantification of mRNA levels by RT-PCR Population of total RNAs was isolated by TRI Reagent (MRC Ltd., OH, USA). Briefly, cells were scraped and homogenized by pipette tip in sterille water and afterwards TRI Reagent was added. After 5 min the homogenate was extracted by chloroform. RNAs in the aqueous phase were precipitated by isopropanol. RNA pellet was washed with 75% ethanol and stored in 96% ethanol at 70 8C. The purity and integrity of isolated RNAs was checked on GeneQuant Pro spectrophotometer (Amersham Biosciences, United Kingdom). Reverse transcription was performed using 1.5 mg of total RNAs and Ready-To-Go You-Prime First-Strand Beads (GE Healthcare-Life Sciences, UK) with pd(N6) primer. PCR specific for the type a1-AR

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was carried out afterwards using primers ALPHA1-a: 50 -CGA GTC TAC GTA GTA GCC-30 and ALPHA1-b: 50 -GTC TTG GCA GCT TTC TTC-30 , for the type a2-AR using primers ALPHA2-a: 50 -GCG CCT CAG AAC CTC TTC CTG GTG-30 and ALPHA2-b: 50 GAG TGG CGG GAA AAG GAT GAC GGC-30 . PCR specific for the type b1-AR was carried out afterwards using primers BETA1-a: 50 -GCC GAT CTG GTC ATG GGA-30 and BETA1-b: 50 -GTT GTA GCA GCG GCG CG-30 , for the b2-AR using primers BETA2a: 50 -ACC TCC TTC TTG CCT ATC CA-30 and BETA2-b: 50 -TAG GTT TTC GAA GAA GAC CG-30 and for b3-adrenergic receptor using primers BETA3-a: 50 -GCA ACC TGC TGG TAA TCA CA-30 , BETA3-b: 50 -GGA TTG GAG TGA CAC TCT TG-30 . Cyclophilin A (CYCLO) was used as a housekeeper gene control for semi-quantitative evaluation of PCR. Following primers for the cyclophilin A were used: CYCLO FW: 50 -CGT GCT CTG AGC ACT GGG GAG AAA-30 and CYCLO RE: 50 -CAT GCC TTC TTT CAC CTT CCC AAA GAC-30 (Gene ID: 203701). PCR specific for a1, a2A, b1, b2 and b3 started by initial denaturation at 94 8C and was followed by 38 cycles of denaturation at 94 8C for 1 min, annealing at 60 8C for 1 min and polymerization at 72 8C for 1 min. PCR specific for CYCLO started by initial denaturation at 94 8C and was followed by 22 cycles of denaturation at 94 8C for 1 min, annealing at 60 8C for 1 min and polymerization at 72 8C for 1 min. PCRs were terminated by final polymerization at 72 8C for 7 min. All PCR products were analyzed on 2% agarose gels. Intensity of individual bands was evaluated by measuring the optical density per mm2 and compared relatively to the CYCLO on PCBAS 2.0 software. 2.5. Western blot analysis The a1A-, a1D-, b1-, b2- and b3-AR proteins were determined in the crude membrane fraction from the cells. Cells were scraped and resuspended in 10 mM Tris–HCl, pH 7.5, 1 mM phenylmethyl sulfonylfluoride (PMSF, Serva, Germany), protease inhibitor cocktail tablets (Complete EDTA-free, Roche Diagnostics, Germany) and subjected to centrifugation for 10 min at 10,000  g and 4 8C. The pellet was resuspended in Tris-buffer containing the 50 mM CHAPS (3-[(3cholamidopropyl)dimethyl-ammonio] 1-propanesulfonate, Sigma, USA), and afterwards incubated for 10 min at 4 8C. The lysate was centrifuged for 10 min at 10,000  g at 4 8C. Protein concentration of supernatants was determined by the method of Lowry et al. (1951). 20 mg of protein extract from each sample was separated by electrophoresis on 10% SDS polyacrylamide gels and proteins were transferred to Hybond-P membrane using semidry blotting (Owl, Inc., USA). Membranes were blocked in 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBS-T) overnight at 4 8C and then incubated for 1 h with appropriate primary antibody. Following washing, membranes were incubated with secondary antibodies to mouse, rabbit or goat IgG conjugated to horseradish peroxidase, for 1 h at room temperature. An enhanced chemiluminiscence detection system (ECL Plus, Amersham Biosciences) was used to detect bound antibody. Optical density of individual bands was quantified using PCBAS 2.0 software. Antibodies: Antibodies raised against the following proteins were used: a1D-AR (goat, Santa Cruz Biotechnology, Inc., USA), a1A-AR (goat, Santa Cruz Biotechnology, Inc., USA), b1-AR (rabbit, Santa Cruz Biotechnology, Inc., USA), b2-AR (rabbit, Abnova, USA), b3-AR (rabbit, Alpha Diagnostic International, USA). 2.6. Immunofluorescence PC12 cells were plated on poly-L-lysine (10 mg/ml; Sigma–Aldrich, St. Louis, MD, USA) coated coverslips (Marienfeld GmbH & Co.KG, Germany) in 24-well plates in 0.5 ml of DMEM with 15% of fetal bovine serum and mixture of streptomycin and penicillin (Calbiochem, Merck Biosciences, Germany). Cells were incubated in a humidified atmosphere of 5% CO2 air at 37 8C. After the treatment procedures, cells were fixed in ice-cold methanol. Non-specific binding was blocked by incubation with phosphate-buffered saline (PBS) containing 3% BSA (Merck Biosciences, Germany) for 1 h at 37 8C. Afterwards, cells were incubated with corresponding primary antibodies diluted 1:250 for 60 min at 37 8C. We used primary rabbit polyclonal antibodies to b2-AR (Abnova, USA), b3-AR (Alpha Diagnostic International, USA), a1D-AR (Santa Cruz Biotechnology, Inc., USA) and NET (Chemicon International, USA), coverslips were washed in PBS and incubated with CFTM488 goat anti-rabbit IgG (H + L) (Biotium, Hayward, USA) and Alexa fluor 594 rabbit anti-goat IgG (Invitrogen, USA) secondary antibody for 60 min at 37 8C. Finally, cells were mounted onto slides in mounting medium with Citifluor (Agar Scientific Ltd., UK), analyzed by fluorescent microscope Leica DM450B with Leica DFC 480 and software Leica IM 500 also by inverted confocal microscope Zeiss Axiovert 200 M with LSM510 expert mode program. Micrographs were taken at 63 magnification with optical zoom 2. Micrographs were deconvolved in Huygens Essential software (SVI, Netherlands) and analyzed in ImageJ software Volume Viewer. 2.7. Detection of apoptosis with Annexin-V-FITC PC12 cells were washed with PBS and pelleted 200  g for 5 min. Cell pellet from each well was resuspended in 100 ml of Annexin-V-FITC labeling solution and incubated at room temperature in dark for 20 min. Labeling solution contained incubation buffer with 10 mM HEPES/NaOH pH 7.4, 140 mM NaCl and 5 mM CaCl2, 2 ml of Annexin-V-FITC (Roche Diagnostics, Germany) and 0.02 mg propidium iodide. After the incubation, cells were washed with 5 ml of PBS, pelleted at 200  g

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for 5 min, resuspended in 300 ml of PBS and measured on EPICS ALTRA flow cytometer (Beckman Coulter, Fullerton, USA). 2.8. Statistical analysis Each value represents the average of 9–15 wells from a least 5 independent cultivations of PC12 cells. Results are presented as means  S.E.M. Statistical differences among groups were determined by one-way analysis of variance (ANOVA). Statistical significance p < 0.05 was considered to be significant. For multiple comparisons, an adjusted t-test with p values corrected by the Bonferroni method was used (Instat, GraphPad Software, USA).

3. Results Intracellular pH decreased in AIK treated cells, compared to untreated controls (Fig. 1A). When cells were treated with combination of AIK and PE, there was still decrease in the intracellular pH compared to untreated control, but this pH was significantly higher than in AIK treated cells. Effect of PE did not reveal any significant increase in mRNA levels of caspase 3 compared to control (Fig. 1B). AIK significantly increased mRNA of caspase 3. This increase was even more pronounced, when both stimuli, AIK and PE were added to PC12 cells (Fig. 1B). The amount of apoptotic cells was measured by the binding of Annexin-V-FITC to PC12 cells (Fig. 1C). AIK and combined both stimuli, PE and AIK, significantly increased amount of apoptotic cells compared to control cells. PE for 3 h significantly increased mRNA of a1-AR (Fig. 2A) and also after AIK treatment significantly increased levels of a1-AR were observed. When effect of both these stimuli was combined, additional increase was not observed compared to AIK. PE and induction of apoptosis by AIK did not reveal any changes of mRNA of a2-AR compared to control (Fig. 2B). Whereas mRNA levels of the a1-AR were changed after PE and AIK treatment, protein levels of the individual types of a-ARs-a1A, a1B and a1D were performed by Western blot (Fig. 3). Protein levels of a1A-AR were not changed after PE and AIK treatment compared to control (Fig. 3A). Protein levels of the a1B-AR were under the detection limit (not shown). Protein expression of the a1D-AR was significantly increased after PE and AIK treatment and also in apoptotic cells after the exposure to PE (Fig. 3B). The mRNA levels of b1-AR were significantly increased only after the AIK treatment of PC12 cells compared to control (Fig. 4A). However, protein levels of the b1-AR were significantly increased after the exposure to PE or apoptotic stimulus for 3 h, but not in cells treated wit both, PE and AIK (Fig. 4B). Treatment of PC12 cells with PE combined with AIK resulted in significant increase in the mRNA (Fig. 4C and E) of b2- and b3-ARs. Protein expression (Figs. 4D and F) of the b2- and b3-ARs was increased after PE and also AIK treatment. When effect of both these stimuli was combined, additional increase in protein levels was visible (Fig. 4D and F). Subcellular distribution of the a1D-, b2- and b3-adrenergic ARs was shown by the immunofluorescence staining (Fig. 5). The a1D-ARs were present mostly in the cytoplasm. The b2-ARs were localized mostly in the nucleus, b3ARs and NET (Fig. 5D) were seen in the plasma membrane. Specificity of antibodies was verified by negative control, where primary antibody was omitted (not shown). As shown in Fig. 6, PE and AIK significantly increased mRNA (A) and protein levels (B) of NET in PC12 cells. Double labeled immunofluorescence (Fig. 7) revealed that in PC12 cells treated with combination of PE and AIK, b2-AR are localized mainly in the nucleus and a1D-ARs are mainly in the cytoplasm. Nevertheless, some of the b2-ARs translocates from the nucleus to plasma membrane (white arrows). 4. Discussion Adrenergic receptors mediate physiological responses of the catecholamines NA and Adr. It is not known whether a- and b-

Fig. 1. The intracellular pH (A), mRNA levels of the caspase 3 (B) and the amount of apoptotic cells (C) in PC12 cell culture after phenylephrine (PE) treatment for 3 h and/or induction of apoptosis (AIK) for 3 h. Intracellular pH (A) decreased in AIK treated cells, compared to untreated controls (KO). When cells were treated with combination of AIK and PE, there was still decrease in the intracellular pH compared to KO, but this pH was significantly higher than in AIK treated cells. AIK and also combination of both stimuli (PE + AIK) significantly increased mRNA levels of caspase 3 (B) compared to KO. The amount of apoptotic cells was measured by the Annexin-V-Fluos and necrosis by propidium iodide (C). AIK and combined PE and AIK stimuli significantly increased amount of apoptotic cells compared to KO. Necrosis was increased only in the cells with combined effect of AIK and PE. Each column is displayed as mean  S.E.M. and represents an average of 6 independent cultivations. Statistical significance was calculated by one-way ANOVA and t-test modified by Bonferroni‘s correction and represents *p < 0.05, **p < 0.01 and ***p < 0.001 (compared to KO) and ##p < 0.001 (compared to AIK).

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Fig. 2. The mRNA levels of a1- (A) and a2- (B) ARs after phenylephrine (PE) treatment for 3 h and induction of apoptosis (AIK) for 3 h. Both, PE and AIK for 3 h significantly increased mRNA of a1-AR compared to control cells (KO). When effect of both these stimuli (PE + AIK) was combined, increase in a1-AR was comparable to that after AIK treatment. PE and induction of apoptosis by AIK did not reveal any changes of mRNA of a2-AR compared to control cells. Each column is displayed as mean  S.E.M. and represents an average of 5 independent cultivations. Statistical significance was calculated by one-way ANOVA and t-test modified by Bonferroni‘s correction and represents *p < 0.05 and **p < 0.01.

adrenergic pathways regulate apoptosis in a coordinated or differential manner. We measured two parameters of apoptosis – caspase 3 mRNA and translocation of phosphatidylserine detected by annexin. Caspase 3 mRNA was twice as high in AIK + PE treated cells compared to AIK, while annexin measurements did not show this difference. Intracellular pH decreased in apoptosis, while in AIK + PE this increase was not so pronounced. It is known that caspases are activated by acidification (Fais, 2010). Therefore we propose that decrease in acidification due to addition of PE resulted in decrease of caspase 3 activity and this resulted in increased caspase 3 mRNA. Increased caspase 3 expression probably compensate the activity of caspase 3. It is well known that caspases, particularly caspase 3 cause phosphatidylserine to be exposed on the outside of the cellular membrane thus promoting phagocytosis by macrophages and neighboring cells. Our results with phosphatidylserine translocation strongly suggest

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Fig. 3. Protein levels of the a1A- (A) and a1D- (B) ARs after phenylephrine (PE) treatment for 3 h and induction of apoptosis (AIK) for 3 h. Protein levels of a1A-AR were not changed after PE, AIK treatment and combined effect both stimuli (PE + AIK) compared to control (KO). Protein expression of a1D-AR was significantly increased after the PE and AIK treatment and also in cells with combined treatment. Each column is displayed as mean  S.E.M. and represents an average of 5 independent cultivations. Statistical significance was calculated by one-way ANOVA and t-test modified by Bonferroni‘s correction and represents **p < 0.01 and ***p < 0.001.

that activity of caspase 3 was similar in AIK treated cells and AIK + PE treated cells. Also, the results observed suggest the involvement of catecholamines in the activation of endogenous (mitochondrial) pathway of apoptosis. It was already shown that NA caused dose-dependent apoptosis of human and rat alveolar epithelial cells that is mediated by a combined effect of a- and bARs and, indirectly, by angiotensin receptor activation via autocrine angiotensin II production (Dincer et al., 2001). We observed that in PC12 cells, a specific a1-AR agonist PE, when applied for 3 h, significantly increased mRNA of a1-ARs, and also protein expression of the a1D-ARs. We have shown that mRNA of a1-AR and also protein expression a1D-ARs were significantly increased after the PE treatment in apoptotic cells. The a1D-AR is a G-protein coupled receptor that is poorly

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Fig. 4. The mRNA and protein levels of b1- (A, B), b2- (C, D) and b3- (E, F) ARs after phenylephrine (PE) treatment for 3 h and induction of apoptosis (AIK) for 3 h. The mRNA levels of b1-AR (A) were significantly increased after AIK treatment of the cells compared to control (KO). Protein levels of b1-AR (B) were significantly increased after the exposure to PE or AIK. Combined effect of both stimuli did not reveal any additional increase (PE + AIK). Treatment of PC12 cells with PE combined with AIK resulted in significant increase in the mRNA (C and E) of b2- and b3-ARs. Protein expression (D and F) of the b2- and b3-ARs was increased after PE and also AIK stimulus. When effect of both these stimuli was combined, additional increase in protein levels was visible. Each column is displayed as mean  S.E.M. and represents an average of 5 independent cultivations. Statistical significance was calculated by one-way ANOVA and t-test modified by Bonferroni‘s correction and represents *p < 0.05, **p < 0.01 and ***p < 0.001.

expressed at the cell surface and largely nonfunctional when heterologously expressed alone in most cell types (Theroux et al., 1996; Chalothorn et al., 2002). In PC12 cells we observed the a1D-AR immunofluorescent signal mainly in the cytoplasm. Similar results were observed, when PE was replaced by NA. Up-regulation of several subtypes of ARs after agonist treatment was already reported in many papers. It was shown that after short-term exhaustive exercise, characterized by increased plasma catecholamines (CAs), increased b2-AR expression and density was observed on human lymphocytes (Graafsma et al., 1990; Murray et al., 1992). However, this parameter returned to control level after 30 min of rest. In addition, b3-ARs were

shown to be up-regulated in rat neonatal cardiomyocytes following chronic exposure to NA (Germack and Dickenson, 2006). Similarly, NA was found to selectively increase expression of a1A-AR in cardiac myocytes (Rokosh et al., 1996). Moreover, increase in a1A-AR was demonstrated in brown adipose tissue after 4 days of cold exposure (4 8C), typical by increased plasma NA, and this rise in a1A-AR was dependent on b3-AR stimulation (Granneman et al., 1997). The ability of b3-AR activation to fully induce a1A-AR mRNA and protein expression in brown adipose tissue indicates that this effect most likely involves generation of cyclic AMP (Granneman et al., 1997). Our observation is in the agreement with already published data, where the a1D-AR was primarily found in intracellular compartments when expressed in

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Fig. 5. Subcellular distribution of the a1D-, b2-, b3-ARs and norepinephrine transporter (NET) in the PC12 cells. Immunofluorescent staining was performed using primary antibody against a1D-, b2-, b3-ARs and NET and fluorescently labeled corresponding secondary antibody, as described in Section 2. Nuclei were labeled by DAPI. The a1DARs were localized primarily to cytoplasm, whereas b2-ARs were present in nucleus. The b3-ARs and NET were seen in the plasma membrane. Merged images show stained nuclei and labeled ARs and/or NET. Scale bar, 10 mm.

a variety of heterologous cells (Daly et al., 1998; McCune et al., 2000). The b-ARs mRNA was not changed after PE treatment. Nevertheless, protein levels of all, b1-, b2- and b3-ARs were increased after PE treatment for 3 h. This observation might suggest that translation of b-ARs is affected by PE rather than translation of a-ARs, probably by the indirect way. Induction of apoptosis increased all three types of b-ARs on both, mRNA and protein levels. However, when induction of apoptosis was done in the presence of PE, rapid increase of mRNA and protein of b2- and b3-ARs was observed. Immunofluorescent signal localized b2-ARs

mainly to the nucleus, while b3-ARs appeared at the plasma membrane. Previous studies on neuronal tissues showed that b2AR is localized not only in the membrane and cytoplasm, but also in the nucleus (Guo and Li, 2007; Qu et al., 2008). Qu et al. (2008) showed that b2-AR is localized not only in the membrane and cytoplasm, but also in nucleus of amygdala. The existence of b2ARs in both cytoplasm and nucleus strongly suggests that this receptor subtype may play a unique role in memory formation in the basolateral nucleus of amygdala. Other types of ARs, like b1-AR are distributed in the cell membrane and cytoplasm (Guo and Li, 2007).

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Fig. 6. The mRNA and protein levels of the NET in PC12 cells. Phenylephrine (PE) and apoptotic inducer set (AIK) significantly increased mRNA (A) and protein levels (B) of NET in PC12 cells. Each column is displayed as mean  S.E.M. and represents an average of 5 independent cultivations. Statistical significance was calculated by oneway ANOVA and t-test modified by Bonferroni‘s correction and represents *p < 0.05 and **p < 0.01.

Confocal imaging confirmed that coexpression with b2-AR resulted in translocation of a1D-AR from intracellular sites to the plasma membrane (Uberti et al., 2005). These authors also found that b2-AR promotes a1D-AR surface expression. Their data indicate that a1D-AR and b2-AR exhibit selective heterodimerization in a cellular content. The a1D-AR and b2-AR are both activated by the same endogenous ligands and are also known to be colocalized in many of the same cells in the cardiovascular, central nervous and immune systems (Young et al., 1990; Kavelaars, 2002). The interaction between a1D-AR and b2-AR could serve as a mechanism by which these receptors regulate each other’s function in native tissues. We observed that protein levels of both, the a1D-AR and b2-ARs are similarly increased by apoptosis, compared to controls. In both types, this increase was even more pronounced, when both PE and AIK were added. Double labeled immunofluorescence showed that in PC12 cells treated simultaneously with PE and AIK the b2-ARs are localized in the nucleus and a1D-ARs are mainly in cytoplasm. Some of the b2-ARs translocates from nucleus to cytoplasm. Localization of the b2-ARs in the nucleus was already shown by Guo and Li (2007), who localized b2-ARs in CA1 and CA3 regions of hippocampus. Authors suggested that the existence of b2-ARs in both nucleus and

cytoplasm may play a unique role in memory formation in the basolateral nucleus of amygdala. Translocation of the b2-AR to nucleus might regulate gene expression related to memory formation (Qu et al., 2008). The b3-AR was also upregulated by apoptosis and this upregulation was more pronounced in the presence of PE. Immunofluorescence with b3-AR antibody clearly localized this receptor to plasma membrane. Functional relevance of this observation is not clear. Until recently, b3-AR was shown to posses the same intracellular signaling pathway as b1- and b2ARs, i.e. activation of adenylyl cyclase and cAMP-dependent phosphorylation (Skeberdis, 2004). On the other hand, Rozec and Gauthier (2006) described that b3-ARs activate a NO pathway, resulting in the increased cGMP, activation of cGMP phosphodiesterase II and subsequent decrease in cAMP. It was already shown that b3-AR agonists, which relax bladder smooth muscle, are being developed to treat the urinary bladder hyperactivity induced by ovariectomy (Kullmann et al., 2009). Also, activation of neurogenic precursors and stem cells via b3-adrenergic receptors could be a potent mechanism to increase neuronal production, providing a putative target for the development of novel antidepressants (Jhaveri et al., 2010). Thus, we propose that importance of the increased expression of the b3-ARs after PE and AIK treatment might have a protective effect on PC12 cells, possibly through decreased cAMP levels. NET is a member of the gene family of Na+/Cl-dependent plasma membrane transporters, present in the noradrenergic cell type of the brain, peripheral sympathetic nerve terminals and some cell types outside of the nervous system such as PC12 cells (Kippenberger et al., 1999; Lorang et al., 1994). Primary function of NET is to remove NA from the synaptic space and quickly terminate the actions of extracellular NA on postsynaptic receptors (Blakely et al., 1994). Nevertheless, under certain conditions, serotonergic varicosities take up NA via serotonin transporter and might release NA in response to neuronal activity; thus, the serotonergic system might directly contribute to the regulation of extracellular NA concentration in the central nervous system (Vizi et al., 2004). However, NET expression is prominent in neoplastic derivatives of chromaffin cell, pheochromocytomas (Huynh et al., 2005). In addition, NET plays a key role in imaging and treatment modalities using agents such as 131iodo-metaiodobenzylguanidine (131I-MIBG; which is also used as therapeutic tool) and 18F-fluorodopamine (Pacak et al., 2001). We observed that both, PE and AIK increased significantly mRNA and also protein levels of NET. This would conform to previous observations of Armour et al. (1997), who reported that pretreatment with doxorubicin and/or cisplatin increased transcription of the transporter and thus improved the effectiveness of 131I-MIBG. Thus, upregulation of NET might be of therapeutic interest. We checked, whether the combination of PE and AIK may result in further upregulation of NET. In this setting we observed rapid down-regulation of NET compared to PE and AIK. Mechanism of this phenomenon remains to be clarified. Physiological relevance of increased ARs in apoptotic PC12 cells in the presence of apoptosis is not understood yet. Even the function of ARs in PC12 cell line is not clear. Nevertheless, in lymphocytes it was shown that endogenous catecholamines accelerated the apoptosis by altering the balance between proapoptotic and anti-apoptotic markers at both, mRNA and protein levels (Jiang et al., 2007). We assume that during apoptosis, PE can potentiate mitochondrial apoptotic pathway and cause dysbalance in NA signaling, which causes upregulation of b2- and b3-ARs and downregulation of NET. Taking together we assume that in PC12 cells the process of apoptosis is modulated by CAs, preferrentially through the a1D-, b2- and b3-ARs. Different localization of these receptors probably evokes their mutual communication and after

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Fig. 7. Co-localization of the a1D- and b2-ARs in PC12 cells. Double immunofluorescent staining was performed using primary antibody against a1D- (red) and b2-ARs (green) and corresponding fluorescently labeled secondary antibodies, as described in Section 2. Nuclei were labeled by DAPI. The a1D-ARs are localized mainly in the cytoplasm, while b2-ARs are localized maily in the nucleus. Nevertheless, some of the b2-ARs translocated from nucleus to plasma membrane (white arrows), as it is seen on the merged image. Scale bar, 10 mm.

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