Neurochemistry International 57 (2010) 884–892

Changes and role of adrenoceptors in PC12 cells after phenylephrineadministration 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 Institute of Molecular Physiology and Genetics, Center of Excellence for Cardiovascular Research, Slovak Academy of Sciences, Vlarska 5, 833 34 Bratislava, Slovak Republicb Molecular Medicine Center, Slovak Academy of Sciences, Bratislava, Slovak Republicc Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovak Republic

A R T I C L E I N F O

Article history:

Received 21 July 2010

Received in revised form 21 September 2010

Accepted 24 September 2010

Available online 1 October 2010

Keywords:

Adrenergic receptors

Apoptosis

Noradrenaline transporter

Phenylephrine

PC12 cells

A B S T R A C T

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.

� 2010 Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

Neurochemistry International

journa l homepage: www.e lsev ier .com/ locate /neuint

1. Introduction

Noradrenaline (NA) is an important adrenergic neurotransmitter.At large doses, NA has been shown to induce cell apoptosis in avariety of cell types, including neurons, cardiomyocytes andpheochromocytoma cells (Mao et al., 2006). Recent studies haveshown that the proapoptotic effect of NA involves activation ofcaspases 3 and 9, but the exact cellular targets and related geneexpressions induced by NA are still unknown (Fu et al., 2004). Both,NA and adrenaline (Adr) bind to adrenergic receptors (ARs). ARs aremembers of the G-protein coupled receptor family and they mediatephysiological responses to the catecholamines noradrenaline andadrenaline. ARs are subdivided into three major families (a1, a2 andb) based on their structure, pharmacology and signaling mechan-isms (Hieble et al., 1995). Lands et al. (1967) subdivided the b-ARmediated effects into b1 and b2 on the basis of the rank order ofpotency 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 activationof downstream cascades (for review see Krizanova et al., 2007).

* Corresponding author. Tel.: +421 2 54772211; fax: +421 2 54773666.

E-mail address: olga.krizanova@savba.sk (O. Krizanova).

0197-0186/$ – see front matter � 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuint.2010.09.007

Apoptosis, or programmed cell death, plays an indispensiblerole in embryonic development, maturation of the immunesystem, and maintenance of tissue and organ homeostasis (Martin,1993). Involvement of catecholaminergic modulation in theprocess of apoptosis is still under investigation. Most of apoptoticeffects of NA and Adr were shown on cardiomyocytes. In heartfailure, apoptosis of cardiac myocytes in response to NA is believedto be an important component of the progression of cardiac fibrosis(Iwase et al., 1996; Communal et al., 1998). Stimulation of b1-AR inthe rat heart can cause not only positive inotropic effects, but alsocan evoke cardiomyocyte apoptosis, while stimulation of b2-ARsappears to be antiapoptotic (Communal et al., 1999). Also, NAinduces dose-dependent apoptosis in human and rat alveolarepithelial cells by a mechanism that involves the combination of a-and b-ARs as well as autocrine angiotensin II production (Dinceret al., 2001). It was already shown that NA activated amitochondrial apoptotic pathway, as evidenced by the increasedcleaved 37 kDa caspase 9, as well as cytochrome c translocationfrom the mitochondria to the cytosol in PC12 cells (Mao et al.,2006). However, it is not clear, which ARs are involved in theactivation of apoptotic pathways by NA in PC12 cells.

Noradrenaline transporter (NET) is a member of gene family ofNa+/Cl�-dependent plasma membrane transporters, whichremoves NA from the synaptic space and quickly terminates the

L. Lencesova et al. / Neurochemistry International 57 (2010) 884–892 885

actions of extracellular NA on postsynaptic receptors (Blakely et al.,1994). It was already shown that decrease of NET density and NAuptake activity occurs also in PC12 cells after NA administrationand these effects of NA are mediated via a posttranscription eventcaused by NA-derived oxidative stress (Blakely et al., 1994). Thus,NET plays a major role in the regulation of NA related actions atcellular level. Due to its early decrease in neurodegenerativediseases, NET appears to be a useful biomarker of these diseases.There are significant decreases in NET density in the LC and inAlzheimer’s disease brains as compared to age matched controls(Gulyas et al., 2010).

The present study addresses the hypothesis that adrenergicmodulation potentiated the effect of apoptosis in PC12 cell line.PC12 cells are derived from rat pheochromocytoma (Greene andTischler, 1976). Pheochromocytomas are tumors of chromaffincells that produce and often secrete catecholamines. Pharmaco-logical control of the physiological and pathological effects ofexcess circulating catecholamines represents a continuing re-quirement in the treatment of metastatic or incompletelyrespectable invasive pheochromocytomas. Alpha adrenergicblockers and calcium channels antagonists often reduce hor-mone-mediated symptoms sufficiently. We studied, whetherphenylephrine (PE), an a1-AR’s agonist can induce and/or affectapoptosis in rat PC12 cells and whether PE and induction of earlystage of apoptosis (for 3 h) will affect gene expression and proteinlevels of ARs. Also, we used immunofluorescence staining andconfocal laser scanning to determine the cellular and subcellularlocations of selected ARs to propose the role of these receptors inthe 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

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 BETA2-

a: 50-ACC TCC TTC TTG CCT ATC CA-30 and BETA2-b: 50-TAG GTT TTC GAA GAA GAC

CG-30and 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 8Cfor 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-[(3-

cholamidopropyl)dimethyl-ammonio] 1-propanesulfonate, Sigma, USA), and after-

wards 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 Biotechnolo-

gy, 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

[()TD$FIG]

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).

L. Lencesova et al. / Neurochemistry International 57 (2010) 884–892886

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 tountreated controls (Fig. 1A). When cells were treated withcombination of AIK and PE, there was still decrease in theintracellular pH compared to untreated control, but this pH wassignificantly higher than in AIK treated cells. Effect of PE did notreveal any significant increase in mRNA levels of caspase 3compared to control (Fig. 1B). AIK significantly increased mRNA ofcaspase 3. This increase was even more pronounced, when bothstimuli, AIK and PE were added to PC12 cells (Fig. 1B). The amountof apoptotic cells was measured by the binding of Annexin-V-FITCto PC12 cells (Fig. 1C). AIK and combined both stimuli, PE and AIK,significantly increased amount of apoptotic cells compared tocontrol cells. PE for 3 h significantly increased mRNA of a1-AR(Fig. 2A) and also after AIK treatment significantly increased levelsof a1-AR were observed. When effect of both these stimuli wascombined, additional increase was not observed compared to AIK.PE and induction of apoptosis by AIK did not reveal any changes ofmRNA of a2-AR compared to control (Fig. 2B). Whereas mRNAlevels of the a1-AR were changed after PE and AIK treatment,protein levels of the individual types of a-ARs-a1A, a1B and a1Dwere performed by Western blot (Fig. 3). Protein levels of a1A-ARwere not changed after PE and AIK treatment compared to control(Fig. 3A). Protein levels of the a1B-AR were under the detectionlimit (not shown). Protein expression of the a1D-AR wassignificantly increased after PE and AIK treatment and also inapoptotic cells after the exposure to PE (Fig. 3B). The mRNA levelsof b1-AR were significantly increased only after the AIK treatmentof PC12 cells compared to control (Fig. 4A). However, protein levelsof the b1-AR were significantly increased after the exposure to PEor apoptotic stimulus for 3 h, but not in cells treated wit both, PEand AIK (Fig. 4B). Treatment of PC12 cells with PE combined withAIK resulted in significant increase in the mRNA (Fig. 4C and E) ofb2- and b3-ARs. Protein expression (Figs. 4D and F) of the b2- andb3-ARs was increased after PE and also AIK treatment. When effectof both these stimuli was combined, additional increase in proteinlevels was visible (Fig. 4D and F). Subcellular distribution of thea1D-, b2- and b3-adrenergic ARs was shown by the immunofluo-rescence staining (Fig. 5). The a1D-ARs were present mostly in thecytoplasm. The b2-ARs were localized mostly in the nucleus, b3-ARs and NET (Fig. 5D) were seen in the plasma membrane.Specificity of antibodies was verified by negative control, whereprimary antibody was omitted (not shown). As shown in Fig. 6, PEand AIK significantly increased mRNA (A) and protein levels (B) ofNET 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 mainlyin the cytoplasm. Nevertheless, some of the b2-ARs translocatesfrom the nucleus to plasma membrane (white arrows).

4. Discussion

Adrenergic receptors mediate physiological responses of thecatecholamines NA and Adr. It is not known whether a- and b-

[()TD$FIG]

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.

[()TD$FIG]

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.

L. Lencesova et al. / Neurochemistry International 57 (2010) 884–892 887

adrenergic pathways regulate apoptosis in a coordinated ordifferential manner. We measured two parameters of apoptosis– caspase 3 mRNA and translocation of phosphatidylserinedetected by annexin. Caspase 3 mRNA was twice as high inAIK + PE treated cells compared to AIK, while annexin measure-ments did not show this difference. Intracellular pH decreased inapoptosis, while in AIK + PE this increase was not so pronounced. Itis known that caspases are activated by acidification (Fais, 2010).Therefore we propose that decrease in acidification due to additionof PE resulted in decrease of caspase 3 activity and this resulted inincreased caspase 3 mRNA. Increased caspase 3 expressionprobably compensate the activity of caspase 3. It is well knownthat caspases, particularly caspase 3 cause phosphatidylserine tobe exposed on the outside of the cellular membrane thuspromoting phagocytosis by macrophages and neighboring cells.Our results with phosphatidylserine translocation strongly suggest

that activity of caspase 3 was similar in AIK treated cells andAIK + PE treated cells. Also, the results observed suggest theinvolvement of catecholamines in the activation of endogenous(mitochondrial) pathway of apoptosis. It was already shown thatNA caused dose-dependent apoptosis of human and rat alveolarepithelial cells that is mediated by a combined effect of a- and b-ARs and, indirectly, by angiotensin receptor activation viaautocrine 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 shownthat mRNA of a1-AR and also protein expression a1D-ARs weresignificantly increased after the PE treatment in apoptotic cells.The a1D-AR is a G-protein coupled receptor that is poorly

[()TD$FIG]

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.

L. Lencesova et al. / Neurochemistry International 57 (2010) 884–892888

expressed at the cell surface and largely nonfunctionalwhen heterologously expressed alone in most cell types(Theroux et al., 1996; Chalothorn et al., 2002). In PC12 cellswe observed the a1D-AR immunofluorescent signal mainly inthe cytoplasm. Similar results were observed, when PE wasreplaced by NA.

Up-regulation of several subtypes of ARs after agonisttreatment was already reported in many papers. It was shownthat after short-term exhaustive exercise, characterized byincreased plasma catecholamines (CAs), increased b2-AR expres-sion and density was observed on human lymphocytes (Graafsmaet al., 1990; Murray et al., 1992). However, this parameter returnedto control level after 30 min of rest. In addition, b3-ARs were

shown to be up-regulated in rat neonatal cardiomyocytesfollowing chronic exposure to NA (Germack and Dickenson,2006). Similarly, NA was found to selectively increase expressionof a1A-AR in cardiac myocytes (Rokosh et al., 1996). Moreover,increase in a1A-AR was demonstrated in brown adipose tissueafter 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 fullyinduce a1A-AR mRNA and protein expression in brown adiposetissue indicates that this effect most likely involves generation ofcyclic AMP (Granneman et al., 1997). Our observation is in theagreement with already published data, where the a1D-AR wasprimarily found in intracellular compartments when expressed in

[()TD$FIG]

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 a1D-

ARs 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.

L. Lencesova et al. / Neurochemistry International 57 (2010) 884–892 889

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 wereincreased after PE treatment for 3 h. This observation mightsuggest that translation of b-ARs is affected by PE rather thantranslation of a-ARs, probably by the indirect way. Induction ofapoptosis increased all three types of b-ARs on both, mRNA andprotein levels. However, when induction of apoptosis was done inthe presence of PE, rapid increase of mRNA and protein of b2- andb3-ARs was observed. Immunofluorescent signal localized b2-ARs

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

[()TD$FIG]

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 one-

way ANOVA and t-test modified by Bonferroni‘s correction and represents *p < 0.05

and **p < 0.01.

L. Lencesova et al. / Neurochemistry International 57 (2010) 884–892890

Confocal imaging confirmed that coexpression with b2-ARresulted in translocation of a1D-AR from intracellular sites to theplasma membrane (Uberti et al., 2005). These authors also foundthat b2-AR promotes a1D-AR surface expression. Their dataindicate that a1D-AR and b2-AR exhibit selective heterodimer-ization in a cellular content. The a1D-AR and b2-AR are bothactivated by the same endogenous ligands and are also known tobe 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-ARcould serve as a mechanism by which these receptors regulate eachother’s function in native tissues. We observed that protein levelsof both, the a1D-AR and b2-ARs are similarly increased byapoptosis, compared to controls. In both types, this increase waseven more pronounced, when both PE and AIK were added. Doublelabeled immunofluorescence showed that in PC12 cells treatedsimultaneously with PE and AIK the b2-ARs are localized in thenucleus and a1D-ARs are mainly in cytoplasm. Some of the b2-ARstranslocates from nucleus to cytoplasm. Localization of the b2-ARsin the nucleus was already shown by Guo and Li (2007), wholocalized b2-ARs in CA1 and CA3 regions of hippocampus. Authorssuggested that the existence of b2-ARs in both nucleus and

cytoplasm may play a unique role in memory formation in thebasolateral nucleus of amygdala. Translocation of the b2-AR tonucleus might regulate gene expression related to memoryformation (Qu et al., 2008).

The b3-AR was also upregulated by apoptosis and thisupregulation was more pronounced in the presence of PE.Immunofluorescence with b3-AR antibody clearly localized thisreceptor to plasma membrane. Functional relevance of thisobservation is not clear. Until recently, b3-AR was shown toposses the same intracellular signaling pathway as b1- and b2-ARs, i.e. activation of adenylyl cyclase and cAMP-dependentphosphorylation (Skeberdis, 2004). On the other hand, Rozec andGauthier (2006) described that b3-ARs activate a NO pathway,resulting in the increased cGMP, activation of cGMP phosphodies-terase II and subsequent decrease in cAMP. It was already shownthat b3-AR agonists, which relax bladder smooth muscle, are beingdeveloped to treat the urinary bladder hyperactivity induced byovariectomy (Kullmann et al., 2009). Also, activation of neurogenicprecursors and stem cells via b3-adrenergic receptors could be apotent mechanism to increase neuronal production, providing aputative target for the development of novel antidepressants(Jhaveri et al., 2010). Thus, we propose that importance of theincreased expression of the b3-ARs after PE and AIK treatmentmight have a protective effect on PC12 cells, possibly throughdecreased cAMP levels.

NET is a member of the gene family of Na+/Cl�-dependentplasma membrane transporters, present in the noradrenergic celltype of the brain, peripheral sympathetic nerve terminals andsome cell types outside of the nervous system such as PC12 cells(Kippenberger et al., 1999; Lorang et al., 1994). Primary functionof NET is to remove NA from the synaptic space and quicklyterminate the actions of extracellular NA on postsynapticreceptors (Blakely et al., 1994). Nevertheless, under certainconditions, serotonergic varicosities take up NA via serotonintransporter and might release NA in response to neuronal activity;thus, the serotonergic system might directly contribute to theregulation of extracellular NA concentration in the centralnervous system (Vizi et al., 2004). However, NET expression isprominent in neoplastic derivatives of chromaffin cell, pheochro-mocytomas (Huynh et al., 2005). In addition, NET plays a key rolein imaging and treatment modalities using agents such as131iodo-metaiodobenzylguanidine (131I-MIBG; which is alsoused as therapeutic tool) and 18F-fluorodopamine (Pacak et al.,2001). We observed that both, PE and AIK increased significantlymRNA and also protein levels of NET. This would conform toprevious observations of Armour et al. (1997), who reported thatpretreatment with doxorubicin and/or cisplatin increased tran-scription of the transporter and thus improved the effectiveness of131I-MIBG. Thus, upregulation of NET might be of therapeuticinterest. We checked, whether the combination of PE and AIK mayresult in further upregulation of NET. In this setting we observedrapid down-regulation of NET compared to PE and AIK. Mecha-nism of this phenomenon remains to be clarified.

Physiological relevance of increased ARs in apoptotic PC12 cellsin the presence of apoptosis is not understood yet. Even thefunction of ARs in PC12 cell line is not clear. Nevertheless, inlymphocytes it was shown that endogenous catecholaminesaccelerated the apoptosis by altering the balance between pro-apoptotic and anti-apoptotic markers at both, mRNA and proteinlevels (Jiang et al., 2007). We assume that during apoptosis, PE canpotentiate mitochondrial apoptotic pathway and cause dysbalancein NA signaling, which causes upregulation of b2- and b3-ARs anddownregulation of NET. Taking together we assume that in PC12cells the process of apoptosis is modulated by CAs, preferrentiallythrough the a1D-, b2- and b3-ARs. Different localization of thesereceptors probably evokes their mutual communication and after

[()TD$FIG]

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.

L. Lencesova et al. / Neurochemistry International 57 (2010) 884–892 891

the induction of apoptosis altering the balance between pro-apoptotic (i.e. caspase 3) and anti-apoptotic markers.

Acknowledgements

This work was supported with scientific grants APVV 51/0397and VEGA 2/0049/10.

References

Armour, A., Cunningham, S.H., Gaze, M.N., Wheldon, T.E., Mairs, R.J., 1997. The effectof cisplatin pretreatment on the accumulation of MIBG by neuroblastoma cellsin vitro. Br. J. Cancer 75, 470–476.

Blakely, R.D., De Felice, L.J., Hartzell, H.C., 1994. Molecular physiology of norepi-nephrine and serotonin transporters. J. Exp. Biol. 196, 263–281.

Chalothorn, D., McCune, D.F., Edelmann, S.E., Garcia-Cazarin, M.L., Tsujimoto, G.,Piascik, M.T., 2002. Differences in the cellular localization and agonist-mediatedinternalization properties of the a1-adrenoceptor subtypes. Mol. Pharmacol.61, 1008–1016.

Communal, C., Singh, K., Pimentel, D.R., Colucci, W.S., 1998. Norepinephrine sti-mulates apoptosis in adult rat ventricular myocytes by activation of the beta-adrenergic pathway. Circulation 98, 1329–1334.

Communal, C., Singh, K., Sawyer, D.B., Collucci, W.S., 1999. Opposing effects ofbeta1- and beta2-adrenergic receptors on cardiac myocyte apoptosis: role of apertussis toxin-sensitive G protein. Circulation 100, 2210–2212.

Daly, C.J., Milligan, C.M., Milligan, G., Mackenzie, J.F., McGrath, J.C., 1998. Cellularlocalization and pharmacological characterization of functioning alpha-1 adre-noceptors by fluorescent ligand binding and image analysis reveals identicalbinding properties of clustered and diffuse populations of receptors. J. Phar-macol. Exp. Ther. 286, 984–990.

Dincer, H.E., Gangopadhyay, N., Wang, R., Uhal, B.D., 2001. Norepinephrine inducesalveolar epithelial apoptosis mediated by alpha-, beta-, and angiotensin recep-tor activation. Am. J. Physiol. Lung. Cell. Mol. Physiol. 281, 624–630.

Fais, S., 2010. Proton pump inhibitor-induced tumour cell death by inhibition of adetoxification mechanism. J. Intern. Med. 267, 515–525.

Fu, Y.C., Chi, C.S., Yin, S.C., Hwang, B., Chiu, Y.T., Hsu, S.L., 2004. Norepinephrineinduces apoptosis in neonatal rat endothelial cells via down-regulation of Bcl-2and activation of beta-adrenergic and caspase-2 pathways. Cardiovasc. Res. 61,143–151.

Germack, R., Dickenson, J.M., 2006. Induction of beta3-adrenergic receptor func-tional expression following chronic stimulation with noradrenaline in neonatalrat cardiomyocytes. J. Pharmacol. Exp. Ther. 316, 392–402.

Graafsma, S.J., Hectors, M.P., van Tits, L.J., Rodrigues de Miranda, J.F., Thien, T., 1990.The relationship between adrenaline and beta2-adrenoceptors on human lym-phocytes. Br. J. Clin. Pharmacol. 30, 145S–147S.

Granneman, J.G., Zhai, Y., Lahners, K.N., 1997. Selective up-regulation of alpha1a-adrenergic receptor protein and mRNA in brown adipose tissue by neural andbeta3-adrenergic stimulation. Mol. Pharmacol. 51, 644–650.

Greene, L.A., Tischler, A.S., 1976. Establishment of a noradrenergic clonal line of ratadrenal pheochromocytoma cells which respond to nerve growth factor. Proc.Natl. Acad. Sci. U.S.A. 73, 2424–2428.

Gulyas, B., Brockschnieder, D., Nag, S., Pavlova, E., Kasa, P., Beliczai, Z., Legradi, A.,Gulya, K., Thiele, A., Dyrks, T., Halldin, Ch., 2010. The norephinephrine trans-porter (NET) radioligand (S, S)-[18F]FMeNER-D2 shows significant decreases inNET density in the human brain in Alzheimer’s disease: a post mortem autora-diographic study. Neurochem. Int. 56, 789–798.

Guo, N.N., Li, B.M., 2007. Cellular and subcellular distributions of b1- and b2-adrenoceptors in the CA1 and CA3 regions of the rat hippocampus. Neurosci-ence 146, 298–305.

Hieble, J.P., Bondinell, W.E., Ruffolo Jr., R.R., 1995. Alpha- and beta-adrenoceptors:from the gene to the clinic 1. Molecular biology and adrenoceptor subclassifi-cation. J. Med. Chem. 38, 3415–3444.

Huynh, T.T., Pacak, K., Brouwers, F.M., Abu-Asab, M.S., Worrell, R.A., Walther, M.M.,Elkahloun, A.G., Goldstein, D.S., Cleary, S., Eisenhofer, G., 2005. Different ex-

L. Lencesova et al. / Neurochemistry International 57 (2010) 884–892892

pression of catecholamine transporters in phaeochromocytomas from patientswith von Hippel-Lindau syndrome and multiple endocrine neoplasia type 2.Eur. J. Endocrinol. 153, 551–563.

Iwase, M., Bishop, S.P., Uechi, M., Vatner, D.E., Shannon, R.P., Kudej, R.K., Wight, D.C.,Wagner, T.E., Ishikawa, Y., Homcy, C.J., Vatner, S.F., 1996. Adverse effects ofchronic endogenous sympathetic drive induced by cardiac GS alpha overex-pression. Circ. Res. 78, 517–524.

Jhaveri, D.J., Mackay, E.W., Hamlin, A.S., Marathe, S.V., Nandam, L.S., Vaidya, V.A.,Bartlett, P.F., 2010. Norepinephrine directly activates adult hippocampal pre-cursors via beta3-adrenergic receptors. J. Neurosci. 30, 2795–2806.

Jiang, J.L., Peng, Y.P., Qiu, Y.H., Wang, J.J., 2007. Effect of endogenous catecholamineson apoptosis of Con A-activated lymphocytes of rats. J. Neuroimmunol. 192, 79–88.

Kavelaars, A., 2002. Regulated expression of alpha-1 adrenergic receptors in theimmune system. Brain Behav. Immun. 16, 799–807.

Kippenberger, A.G., Palmer, D.J., Comer, A.M., Lipski, J., Burton, L.D., Christie, D.L.,1999. Localization of the noradrenaline transporter in rat adrenal medulla andPC12 cells: evidence for its association with secretory granules in PC12 cells. J.Neurochem. 73, 1024–1032.

Krizanova, O., Myslivecek, J., Tillinger, A., Jurkovicova, D., Kubovcakova, L., 2007.Adrenergic and calcium modulation of the heart in stress: from molecularbiology to function. Stress 10, 173–184.

Kullmann, F.A., Limberg, B.J., Artim, D.E., Shah, M., Downs, T.R., Contract, D., Wos, J.,Rosenbaum, J.S., de Groat, W.C., 2009. Effects of beta3-adrenergic receptoractivation on rat urinary bladder hyperactivity by ovariectomy. J. Pharmacol.Exp. Ther. 330, 704–717.

Lands, A.M., Arnold, A., McAuliff, J.P., Luduena, F.P., Brown Jr., T.G., 1967. Differen-tiation of receptor systems activated by sympathomimetic amines. Nature 214,597–598.

Lorang, D., Amara, S.G., Simerly, R.B., 1994. Cell-type-specific expression of cate-cholamine transporters in the rat brain. J. Neurosci. 14, 4903–4914.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurementwith the Folin phenol reagent. J. Biol. Chem. 193, 265–275.

Mao, W., Iwai, C., Keng, P.C., Vulapalli, R., Liang, C.S., 2006. Norepinephrine-induced oxidative stress causes PC-12 cell apoptosis by both endoplasmicreticulum stress and mitochondrial intrinsic pathway: inhibition of phos-phatidylinositol 3-kinase survival pathway. Am. J. Physiol. Cell. Physiol. 290,1373–1384.

Martin, S.J., 1993. Apoptosis: suicide, execution or murder? Trends Cell. Biol. 3,141–144.

McCune, D.F., Edelmann, S.E., Olges, J.R., Post, G.R., Waldrop, B.A., Waugh, D.J., Perez,D.M., Piascik, M.T., 2000. Regulation of the cellular localization and signalingproperties of the alpha(1B)- and alpha(1D)-adrenoceptors by agonists andinverse agonists. Mol. Pharmacol. 57, 659–666.

Murray, D.R., Irwin, M., Rearden, C.A., Ziegler, M., Motulsky, H., Maisel, A.S., 1992.Sympathetic and immune interactions during dynamic exercise Mediation via abeta 2-adrenergic-dependent mechanism. Circulation 86, 203–213.

Pacak, K., Linehan, W.M., Eisenhofer, G., Walther, M.M., Goldstein, D.S., 2001. recentadvances in genetics, diagnosis, localization, and treatment of pheochromocy-toma. Ann. Intern. Med. 134, 315–329.

Qu, L.L., Guo, N.N., Li, B.M., 2008. Beta1- and beta2-adrenoceptors in basolateralnucleus of amygdala and their roles in consolidation of fear memory in rats.Hippocampus 18, 1131–1139.

Rokosh, D.G., Stewart, A.F., Chang, K.C., Bailey, B.A., Karliner, J.S., Camacho, S.A.,Long, C.S., Simpson, P.C., 1996. Alpha1-adrenergic receptor subtype mRNAs aredifferentially regulated by alpha1-adrenergic and other hypertrophic stimuli incardiac myocytes in culture and in vivo Repression of alpha1B and alpha1D butinduction of alpha1C. J. Biol. Chem. 271, 5839–5843.

Rozec, B., Gauthier, Ch., 2006. Beta3-adrenoceptors in the cardiovascular system:putative roles in human pathologies. Pharmacol. Therap. 111, 652–673.

Skeberdis, V.A., 2004. Structure and function of beta3-adrenergic receptors. Med-icina (Kaunas) 40, 407–413.

Theroux, T.L., Esbenshade, T.A., Peavy, R.D., Minneman, K.P., 1996. Coupling effi-ciencies of human alpha 1-adrenergic receptor subtypes: titration of receptordensity and responsiveness with inducible and repressible expression vectors.Mol. Pharmacol. 50, 1376–1387.

Uberti, M.A., Hague, C., Oller, H., Minneman, K.P., Hall, R.A., 2005. Heterodimeriza-tion with beta2-adrenergic receptors promotes surface expression and func-tional activity of alpha1D-adrenergic receptors. J. Pharmacol. Exp. Ther. 313, 16–23.

Vizi, E.S., Zsilla, G., Caron, M.G., Kiss, J.P., 2004. Uptake and release of norepinephrineby serotonergic terminals in norepinephrine transporter knock-out mice:implications for the action of selective serotonin reuptake inhibitors. J. Neu-rosci. 24, 7888–7894.

Young, M.A., Vatner, D.E., Vatner, S.F., 1990. Alpha- and beta-adrenergic control oflarge coronary arteries in conscious calves. Basic Res. Cardiol. 85, 97–109.