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Clinical and Experimental Pharmacology and Physiology (2009) 36, 312– 318
DOPAMINE D2 RECEPTOR STIMULATION INHIBITS ANGIOTENSIN
II-INDUCED HYPERTROPHY IN CULTURED NEONATAL RAT
Hong Li,* Sa Shi,* Yi-Hua Sun,‡ Ya-Jun Zhao,* Quan-Feng Li,* Hong-Zhu Li,* Rui Wang†
and Chang-Qing Xu*
Departments of *Pathophysiology and ‡Clinical Laboratory, Second Affiliated Hospital of Harbin Medical University, Harbin, China and †Department of Biology, Lakehead University, Thunder Bay, Ontario, Canada Key words: cardiomyocytes, dopamine D2 receptor,
1. Myocardial hypertrophy is a common pathological
change that accompanies cardiovascular disease. Dopamine D2
receptors have been demonstrated in cardiovascular tissues.
However, the pathophysiological involvement of D2 receptors
Dopamine is a very important catecholamine neurotransmitter in the in myocardial hypertrophy is unclear. Therefore, the effects of
mammalian brain1 that has multiple roles in peripheral tissues.2–4 Its the D2 receptor agonist bromocriptine and the D2 receptor
effects are exerted via stimulation of dopamine D1–D5 receptors.5–7 antagonist haloperidol on angiotensin (Ang) II- or endothelin
Binding of dopamine to D1 and D5 receptors stimulates adenylyl (ET)-1-induced hypertrophy of cultured neonatal rat ventricular
cyclase and phospholipase C (PLC), as well as activating calcium myocytes were investigated in the present study.
channels.8,9 Conversely, stimulation of D2, D3 and D4 receptors 2. Protein content and protein synthesis, determined by
inhibits adenylyl cyclase and calcium channels and activates the examining [3H]-leucine uptake, were used as estimates of car-
opening of single K channels, resulting in an increase in K con- diomyocyte hypertrophy. The expression of D2 receptor protein
ductance and associated membrane hyperpolarization.10,11 in neonatal rat ventricular myocytes was determined using
Myocardial hypertrophy, which is an adaptive response to various western blotting. Changes in [Ca2] in cardiomyocytes were
mechanical changes and humoral stimuli, eventually leads to heart observed by laser scanning confocal microscopy.
failure.12,13 Because cardiomyocytes rapidly lose their ability to 3. Angiotensin II and ET-1, both at 10 nmol/L, induced
divide under basal conditions both in vivo and in culture, their myocyte hypertrophy, as demonstrated by increased protein
growth response to various stimuli primarily involves the hypertrophy content and synthesis, [Ca2] levels, protein kinase C (PKC)
of individual cells.14 Many studies have demonstrated that angio- activity and phosphorylation of extracellular signal-regulated
tensin (Ang) II is a potent growth promoter of cardiomyocytes by kinase, c-Jun N-terminal kinase and mitogen-activated protein
stimulating different signal transduction pathways,12,15 including the kinase (MAPK) p38 (p38). Concomitant treatment of cells with
activity of G , which facilitates activation of the PLC/protein kinase 10 nmol/L AngII plus 10 mol/L bromocriptine significantly
C (PKC) pathway.16 Activated mitogen-activated protein kinase inhibited cardiomyocyte hypertrophy, MAPK phosphorylation
(MAPK) may stimulate various transcriptors and induce increased and PKC activity in the membrane, as well as [Ca2] signalling
gene expression and protein synthesis.17,18 pathways, compared with the effects of AngII alone. In addition,
In a previous study, we detected D2 receptor mRNA and protein 10 mol/L bromocriptine significantly inhibited cardiomyocyte
expression in normal rat cardiac tissues and reported, for the first hypertrophy induced by 10 nmol/L ET-1. However, pretreatment
time, that expression decreased in an animal model of cardiac hyper- with haloperidol (10 mol/L) had no significant effects on
trophy.19 Furthermore, we showed that the D2 receptor is also present cardiomyocyte hypertrophy induced by either AngII or ET-1.
in cultured neonatal rat ventricular myocytes.20 Thus, the aim of the 4. In conclusion, D2 receptor stimulation inhibits AngII-
present study was to determine the effects of D2 receptor activation/ induced hypertrophy of cultured neonatal rat ventricular
inhibition on the hypertrophic response of cardiomyocytes to AngII myocytes via inhibition of MAPK, PKC and [Ca2] signalling
and the mechanisms involved.
Cells and treatment
Correspondence: Chang-Qing Xu, Department of Pathophysiology of Harbin Medical University, 194 XueFu Road, NanGang District, Harbin 150086, Neonatal rat ventricular myocyctes were isolated from 2-day-old Wistar rats by enzymatic digestion with 0.25% trypsin, as described previously.21 Received 10 June 2007; revision 17 August 2008; accepted 21 August 2008.
The culture medium was changed to serum-free medium for 24 h before 2008 The Authors treatment. Dishes from each culture preparation were randomly assigned to Journal compilation 2008 Blackwell Publishing Asia Pty Ltd one of the following experimental groups, each comprising eight dishes:

D2 inhibits myocardial hypertrophy (i) untreated controls; (ii) cells treated with 10 nmol/L AngII (Sigma, St Louis,MO, USA), 10 nmol/L ET-1 (Sigma), 10 mol/L bromocriptine (a D2 receptoragonist; Sigma) or 10 mol/L haloperidol (a D2 receptor antagonist; Sigma)alone; and (iii) cells pre-incubated with 10 mol/L bromocriptine or10 mol/L haloperidol for 10 h prior to the addition of 10 nmol/L AngII or10 nmol/L ET-1. All experiments were performed after cells had beenincubated for a further 72 h.
Cardiomyocyte purity and cell viability
Cardiomyocyte purity was monitored using an antibody to cardiac sarco-meric -actin (Boster Biological Technology, Wuhan, China) according to themanufacturer's instructions. Cell viability was analysed by the 3-(4,5-dimethyl- Immunohistochemical demonstration of the purity of cardiomyocytes.
2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma) assay, (a) Cardiomyocyte purity was monitored using an antibody to cardiac performed in triplicate. Cells and dye crystals were solubilized with 200 L sarcomeric -actin at 1:100 dilution. (b) The brown granule in the cytoplasm dimethylsulphoxide and absorbance was measured at 490 nm, using a model showed that the cell is a myocardial cell. The percentage of cardiomyocytes ELX-800 microplate assay reader (One Lambda, Canoga Park, CA, USA).
was greater than 95%. (Original magnification 400.) p-extracellular signal-regulated kinase (ERK; 1 : 2000 dilution; Promega), Measurement of beating rate, myocyte diameter, protein
anti-p-c-Jun N-terminal kinase (JNK; 1 : 1000 dilution; Promega), anti-p-p38 content and protein synthesis
(1 : 1000 dilution; Neomarker, Fremont, CA, USA) or anti-actin (1 : 200dilution; Boster) antibodies overnight at 4C. Alkaline phosphatase- The beating rate of myocardial cells was recorded with a JVC (Kanagawa-ku, conjugated goat anti-mouse IgG (1 : 2500 dilution; Promega) antibodies Japan) GZ-MG505AC digital recorder. Ten fields were chosen at random were added and membranes were incubated at 37C for 1 h. Immunodetection for every group and 10 cells were evaluated in each field. The number of was performed using a BI2000 Imaging Analysis System (Chengdu Taimeng beats over a 60 s period was counted manually.
Sci-Tec, Chengdu, China). -Actin was used as an internal control for Total cell protein of 5  105 cells was measured using a modification of the method of Lowry et al. and bovine serum albumin as a standard.22The total DNA content of each plate was quantified using ultraviolet spectro-photometry (DU-65 Spectrophotometer; Beckman, Woodland Hills, CA, Measurement of intracellular Ca2
USA). Each experiment was repeated 12 times.
[3H]-Leucine uptake was used as an index of protein synthesis, as Free intracellular calcium concentrations ([Ca2] ) in myocardial cells was described previously.23 To correct for any minor differences in cell number determined using the Fluo-3/AM (Dojindo Laboratories, Kumamoto, Japan) between treatment groups, protein synthesis and protein content were probe as follows. After treatment with AngII, bromocriptine or haloperidol analysed by [3H]-leucine uptake/DNA content.
alone, cells were incubated with 5 mol/L Fluo-3/AM for 40 min at 37Cunder a 95 : 5 air : CO atmosphere, washed three times with phosphate- buffered saline (PBS) and further incubated for 20 min in Dulbecco's Preparation of PKC reagents and PKC activity assay
modified Eagle's medium (DMEM; Gibco Invitrogen, Carlsbad, CA, USA)in the presence of 10 nmol/L AngII. Dynamic changes in [Ca2] in myocardial Cells were scraped into cool protein lysate (Hangzhou Sijiqing Biological cells were measured after stimulation with AngII for 30 min by measuring Engineering Materials, Hangzhou, China) containing 1% phenylmethyl- Fluo-3 fluorescence (excitation at 488 nm and emission at 525 nm). Fluor- sulphonyl fluoride (Amresco, Solon, OH, USA). Samples were centrifuged escence intensity was measured to determine changes in [Ca2] . The at 10 303 g for 15 min at 4C and the supernatant, representing the cytosolic fluorescence intensity was observed in eight randomly chosen cells using a fraction, collected. Pellets were suspended in protein lysate containing 0.1% laser scanning confocal microscope (TCS SP2; Leica, Mannheim, Germany) Triton X-100 (Amresco). After homogenization, samples were kept at 4C to calculate average fluorescence intensity for all cells.
for 1 h, agitated every 20 min for 15 s and then centrifuged at 10 303 g forfor 20 min at 4C. Protein kinase C activity from cytosolic and membranefractions was determined according to the methods provided with the Protein Kinase C Assay System (Promega, Madison, WI, USA). The incorporationof [-32P]-ATP (111 GBq/mmol, 0.37 MBq/L; Beijing Furei Biotechnology, Data are presented as the meanSEM and were analysed using spss v. 11.5 Beijing, China) was determined by liquid scintillation and the activity of software (SPSS, Chicago, IL, USA), with the number of observations PKC was determined by subtracting the activity of the enzyme in the absence indicated. Statistical significance was tested by post hoc analysis following of phospholipids (control buffer) from that of the enzyme in the presence one-way repeated-measures anova. Significance was set at P  0.05.
of phospholipids (activation buffer). In the present study, PKC activity wascalculated as pmol phosphate radioactivity transferred/min per mg protein.
Sodium dodecyl sulphate–polyacrylamide gel
Cardiomyocyte purity, viability and diameter
electrophoresis and western blotting
The percentage of -actin-positive cells was  95% of the total Samples (50 g protein) from different experimental groups were separated number of cells (Fig. 1). In preliminary experiments performed to by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis determine the optimum concentration of all drugs, viability decreased (SDS-PAGE) and transferred to polyvinylidene difluoride membranes after treatment of cells with 100 and 1000 nmol/L AngII or ET-1, but (Bio-Rad, Hercules, CA, USA) by electroblotting (100 V for 1.5 h). Mem- no adverse effects were observed after treatment of cells with branes were then blocked at 37C for 1 h in 5% (w/v) skimmed milk powderin TBS (Tris 10 mmol/L, NaCl 150 mmol/L, pH 8.0; Beijing Chemical 10 mol/L bromocriptine or haloperidol (Fig. 2a). At 10 nmol/L, Reagents Factory, Beijing, China) and incubated with mouse anti-D2 receptor AngII and ET-1 both increased cell diameter and there were no (1 : 200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti- obvious differences in the hypertrophy induced by 10, 100 and 2008 The Authors Journal compilation 2008 Blackwell Publishing Asia Pty Ltd

H Li et al. Expression of the D2 receptor decreases in myocardial hypertrophy induced by angiotensin (Ang) II or endothelin (ET)-1 (both at 10 nmol/L).
(a) Western blot analysis of D2 receptors. Each lane was loaded with 50 mgprotein. (b) Levels of D2 receptor protein were quantified by densitometryanalysis. Representative results from six different experiments are shown.
*P  0.05 compared with the control group.
At 10 nmol/L, treatment of cardiomyocytes with AngII and ET-1 alone increased cell diameter compared with the control group.
Pretreatment with 10 mol/L bromocriptine reduced the increasein cell diameter induced by both 10 nmol/L AngII and ET-1(Fig. 4b).
Protein content and synthesis were determined for each well (containing 5  105 cells). Compared with the control group, theprotein content and synthesis of ventricular myocytes were signifi-cantly higher following treatment with AngII and ET-1 (both at Cardiomyocyte (a) diameter and (b) viability. The effects of different 10 nmol/L). The D2 receptor agonist bromocriptine (10 mol/L) concentrations of angiotensin (Ang) II and endothelin (ET)-1 were examined: significantly decreased the AngII- or ET-1-induced increase in (ⵧ), 1 nmol/L; ( ), 10 nmol/L; ( ), 100 nmol/L; (䊏), 1000 nmol/L. In [3H]-leucine incorporation and, thus, protein content. In contrast, the addition, the effects of different concentrations of bromocriptine (Bro) and D2 receptor specific antagonist haloperidol had no significant effect haloperidol (Hal) were evaluated: (ⵧ), 1 mol/L; ( ), 10 mol/L; ( ), on the AngII- or ET-1-induced increases in protein content and 100 mol/L; (䊏), 1000 mol/L. Data are the meanSEM. *P  0.05 compared synthesis (Fig. 4c,d).
with control; †P  0.05.
Phosphorylation of ERK1/2, JNK and p38
1000 nmol/L AngII or ET-1. At this concentration (10 nmol/L), neitherAngII nor ET-1 had any significant toxic effects, so this concentra- There was no difference in non-phosphorylated ERK between tion was used in subsequent exeperiments. Bromocriptine and the different treatment groups. There was very little phosphorylation haloperidol alone (both at 1, 10, 100 and 1000 mol/L) had no effect of ERK, JNK and p38 MAPK in normal neonatal ventricular myo- on cardiomyocyte dimater (Fig. 2b). Therefore, in all subsequent cytes. When 10 nmol/L AngII was added to the medium, there was experiments, 10 mol/L bromocriptine and haloperidol was used.
a marked increase in phosphorylation of ERK1/2, JNK and p38MAPK. However, in the bromocriptine-pretreated group, the AngII-induced phosphorylation of ERK and JNK was decreased signifi- D2 receptor protein expression in neonatal rat
cantly (P  0.01; Fig. 5).
Expression of D2 receptor protein in neonatal rat ventricular myocytes Measurement of [Ca2] and PKC activity
was detected with western blotting. Protein levels of the D2 receptor were significantly lower in AngII- and ET-1-treated groups compared From fluorescence images taken by a laser scanning confocal with the control group (Fig. 3a,b).
microscope, we found that AngII markedly increased [Ca2] and that bromocriptine significantly inhibited this increase. However, the D2receptor antagonist haloperidol had no effect on AngII-induced Hypertrophy of neonatal rat ventricular myocytes
increase in [Ca2] (Fig. 6).
Cardiomyocytes were pretreated for 10 h with bromocriptine or Treatment of cardiomyocytes with AngII increased PKC activity haloperidol, then treated with AngII or Et-1; the beating rate was in the membrane and cytosolic fractions compared with athe control measured 72 h later. The beating rate of AngII-treated cardiomyo- group. There was no difference in PKC activity in the cytosolic fraction cytes was significantly higher than that of the control group. In contrast, between the AngII alone, AngII  bromocriptine and AngII  bromocriptine, haloperidol and ET-1 alone had no effect on the beating haloperidol groups. However, PKC activity in the membrane fraction rate of cardiomyocytes. Pretreatment of cells with bromocriptine decreased in the bromocriptine-pretreated AngII-treated group prior to AngII decreased the beating rate of myocardial cells (Fig. 4a).
2008 The Authors Journal compilation 2008 Blackwell Publishing Asia Pty Ltd D2 inhibits myocardial hypertrophy we demonstrated that stimulation of dopamine D2 receptors inhibitedthe hypertrophic response. This inhibition was associated withactivation of MAPK and [Ca2] signalling pathways.
Both D1 and D2 receptors have been identified in cardiac muscle.25–27 In a previous study, we detected the expression of D1and D2 receptor mRNA and protein in normal rat cardiac tissues;19interestingly, expression of D2 receptor mRNA and proteindecreased in an animal model of cardiac hypertrophy induced byexperimental aortic coarctation.19 In vivo, receptor downregulationmay be related to many humoral and neural factors. Culturedneonatal cardiac myocytes have been used extensively as an experi-mental model in which to investigate the mechanisms of myocytehypertrophy, avoiding interference from humoral and neural factors.
In this system, adrenoceptor stimulation, AngII, endothelin andpeptide growth factors cause myocyte hypertrophy without hyper-plasia. Some studies have shown that AngII plays an importantrole in the initiation of proto-oncogene expression and growth inmyocardial cells.28 In addition, angiotensin-converting enzymeinhibitors have been shown to prevent the development of cardiacremodelling after myocardial injury.29 In the present study, we observed D2 receptor mRNA and protein expression in neonatal rat ventricular myocytes in vitro and foundthat expression decreased following treatment of cells with AngII.
In order to determine whether decreases in D2 receptor mRNA andprotein expression are unique to AngII, we examined D2 receptorexpression ventricular myocytes after treatment with ET-1 andobserved similar results. Therefore, we speculate that the decreasein the expression of D2 receptors is related to the reduction in therelative density of D2 receptors (as a proprotion of the increased cellsurface area resulting from hypertrophy).
Dopaminergic ligands easily discriminate between the different dopamine receptor subtypes.30 In the present study, we chosebromocriptine and haloperidol as specific D2 receptor agonists andantagonists, respectively. The results showed that bromocriptinesignificantly inhibited hypertrophy of ventricular myocytes,with a decrease in protein content, cellular protein synthesis and celldiameter. These findings are consistent with recent observationsreported by Mejia-Rodriguez et al.24 In patients with end-stage renaldisease treated with continuous ambulatory peritoneal dialysis, therewas a 24.4% decrease in left ventricular mass index after treatmentwith bromocriptine compared with the control group. The authors Dopamine D2 receptor receptor stimulation inhibits myocardial conlcuded that bromocriptine inhibits noradrenaline release, ant- hypertrophy induced by angiotensin (Ang) II or endothelin (ET)-1 (both at agonizes aldosterone and downregulates angiotensin AT receptors, 10 nmol/L). (a) Beating rate, (b) cell diameter, (c) relative protein content which may lead to regression of left ventricular hypertrophy.24 The and (d) relative protein synthesis (normalized against DNA content) of results of the present study indicate that D2 receptor activation cardiomyocytes in the different experimental group. Data are the meanSEM.
*P  0.05. Bro, 10 mol/L bromocriptine; Hal, 10 mol/L haloperidol.
in vitro can also stimulate other pathways to inhibit myocardialhypertrophy, in addition to humoral and neural factors. Massonet al.31 have reported that CHF-1024, a D2 receptor agonist, bluntscardiac fibrosis in pressure overload and has no effect on cardiac mass. One possible explanation for this is that CHF-1024 is not able Cardiac hypertrophy is an independent risk factor for the development to reduce levels of AngII, a major hypertrophic factor. Hussain et of ischaemia, arrhythmia and sudden death.12,13 Evidence from al.32 have proposed that bromocriptine (1 mmol/L) alone stimulates in vivo studies suggests that both dopamine and its receptors are Na/K-ATPase activity. They have also suggested that pre-activation implicated in cardiac hypertrophy. The D2 receptor agonist bro- of D2, D3 and D4 receptors by bromocriptine prior to AngII mocriptine induces regression of left ventricular hypertrophy in peri- treatment abolishes AngII-mediated stimulation of Na/K-ATPase toneal dialysis patients.24 However, the mechanisms invovled after D2 activity and inhibition of cAMP accumulation.32 receptor activation were not determined in any of these previous studies.
In vivo, bromocriptine induces tachycardia.33 The results of In the present study, using an experimental model of AngII- or the present study show that the beating rate of isolated myocytes ET-1-mediated hypertrophy of neonatal rat cardiac myocytes in vitro, in vitro was not changed after treatment with bromocriptine, suggesting 2008 The Authors Journal compilation 2008 Blackwell Publishing Asia Pty Ltd

H Li et al. Western blot amplification of phosphorylated (a,b) extracellularsignal-regulated kinase (ERK) 1/2,(c) c-Jun N-terminal kinase (JNK) and(d) p38 protein in neonatal ventricularmyocytes. Levels of ERK1/2, JNKand p38 phosphorylation protein werequantified by densitometric analysis.
The results are representative of sixexperiments. Data are the meanSEM.
*P  0.05 compared with the controlgroup. AngII, 10 nmol/L angiotensinII; ET-1, 10 nmol/L endothelin-1;Bro, 10 mol/L bromocriptine; Hal,10 mol/L haloperidol.
Continuous record of [Ca2] in neonatal ventricular myocytes.
(a) Dynamic changes in [Ca2] in myocardial cells were measured 1 h after stimulation with 10 nmol/L angiotensin (AngII) II. (b) Fluorescence intensitychanges in [Ca2] were recorded continuously with a laser scanning confocal Protein kinase C (PKC) activity in the (a) membrane and (b) cytosolic microscope in the different treatment groups. Angiotensin II increased the fraction of neonatal ventricular myocytes. Data are the meanSEM.
intracellular concentration of calcium. Bro, 10 mol/L bromocriptine; Hal, *P  0.05. AngII, 10 nmol/L angiotensin II; Bro, 10 mol/L bromocriptine; 10 mol/L haloperidol.
Hal, 10 mol/L haloperidol.
2008 The Authors Journal compilation 2008 Blackwell Publishing Asia Pty Ltd D2 inhibits myocardial hypertrophy that bromocriptine increases the beating rate of cardiomyocytes cells. Further studies are needed to determine whether our findings in vivo by changing cardiac vagal or sympathetic tone.
are relevant to pressure overload in other species, as well as in Ganguly et al.34 have found evidence of a relationship between humans with chronic cardiac hypertrophy.
D1 receptors and hypertrophy. They have shown that SCH 23390, In summary, D2 receptor stimulation partly inhibited AngII-induced a D1 receptor antagonist, partially regresses cardiac hypertrophic hypertrophy in cultured neonatal rat ventricular myocytes via changes after aortic constriction. D1 and D2 receptors have different inhibition of MAPK, PKC and [Ca2] signalling pathways.
pharmacological characteristics in the PKC pathway: D1 receptor-stimulated adenylyl cyclase strongly stimulates cAMP accumulation, whereas activation of D2 receptors inhibits adenylyl cyclase.35 Thefact that blockade of D1 receptors and stimulation of D2 receptors This study was supported by grants from the Education Office inhibit hypertrophy suggests that suppression of the PKC signalling Project of Heilongjiang Province (No. 11511225) and the National pathway may cause inhibition of cardiac hypertrophy in vivo and Natural Science Foundation of China (No. 30470688).
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