Cross-regulation between β₁- and β₃-adrenoceptors following chronic β-adrenergic stimulation in neonatal rat cardiomyocytes

Abstract

Background and purpose:

We have previously shown that β-adrenoceptors continuously stimulated with noradrenaline induces an increase in β₃-adrenoceptors (Gα_iPCRs) and a decrease in β₁-adrenoceptors (Gα_sPCRs) at functional, genomic and protein levels. This compensatory modification induced by noradrenaline is probably one of the consequences of cardiac depression observed in heart disease. Therefore, we investigated further the interaction between β₁- and β₃-adrenoceptors in neonatal rat cardiomyocytes.

Experimental approach:

Functional studies were performed by cyclic adenosine monophosphate (cAMP) accumulation assays in cells untreated or treated with dobutamine and ICI 118551 (β₁-adrenoceptor) or CL-3162436243 (β₃-adrenoceptor) for 24 h in the presence or absence of protein kinase inhibitors. β-adrenoceptor and protein kinase expression was monitored by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and by Western blotting, respectively.

Key results:

Chronic β₁- or β₃-adrenoceptor stimulation reduced β₁-adrenoceptor-mediated cAMP accumulation in association with a decrease in β₁-adrenoceptor mRNA and protein levels through protein kinase C (PKC), phosphoinositide 3-kinase (PI3K) and p38 mitogen-activated protein kinase (p38MAPK) activation. In contrast, both treatments induced an increase in β₃-adrenoceptor expression and β₃-adrenoceptor-inhibited forskolin response through PKC, extracellular-signal-regulated kinases 1 and 2 (ERK1/2) and p38MAPK phosphorylation, although no β₃-adrenoceptor response was observed in untreated cells. ERK1/2 and p38MAPK were activated by both treatments. The modulation of β₁- or β₃-adrenoceptor function did not require stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) although chronic β₁-adrenoceptor stimulation activated SAPK/JNK. β₃-adrenoceptor treatment activated Akt although PI3K was not involved in β₃-adrenoceptor up-regulation.

Conclusion and implications:

We show for the first time that chronic β₁- or β₃-adrenoceptor stimulation leads to the modulation of β₁- and β₃-adrenoceptors by a cross-regulation involving PKC, PI3K p38MAPK and MEK/ERK1/2 pathway, and through protein kinase A when β₁-adrenoceptors are chronically activated.

Introduction

Three β-adrenoceptors (β₁, β₂ and β₃-adrenoceptors; nomenclature follows Alexander et al., 2008) are expressed in heart (Dzimiri, 1999; Rozec and Gauthier, 2006). These receptor subtypes belong to the G-protein coupled receptor (GPCR) superfamily and modulate cardiac function after stimulation by the catecholamines, noradrenaline and adrenaline (Dzimiri, 1999). Increases in heart rate and force of contraction are mediated mainly by β₁-adrenoceptors and to a lesser extent by β₂-adrenoceptors through a coupling to the G_s protein pathway (Dzimiri, 1999). In contrast, the β₃-adrenoceptor subtype coupled to G_i protein decreases cardiac contractility (Dzimiri, 1999; Rozec and Gauthier, 2006). It is well known that heart failure induces an increase in circulating cathecholamines, which leads to an alteration of cardiac function through a decrease in β₁-adrenoceptor density (Cohn et al., 1984; Dzimiri, 1999; Tilley and Rockman, 2006). In contrast, β₃-adrenoceptors were found up-regulated in failing human and animal hearts (Cheng et al, 2001; Moniotte et al., 2001; Morimoto et al., 2004; Zhang et al., 2005). Similarly, β₁-adrenoceptor expression was decreased and β₃-adrenoceptor level increased both in hearts from diabetic and from physically trained rats (Dinçer et al., 2001; Barbier et al., 2007). It is noteworthy that catecholamines, particularly noradrenaline, increase during physical exercise, and likewise during heart failure (Pagliari and Peyrin, 1995; Dzimiri, 1999; Tilley and Rockman, 2006). We have shown previously that chronic stimulation of cardiomyocytes with noradrenaline decreases β₁-adrenoceptor function and expression, and enhances β₃-adrenoceptor function (Germack and Dickenson, 2006). Interestingly, Thomas et al (1992) have reported a similar opposite regulation in adipocytes chronically stimulated with isoprenaline. In addition, β₁-adrenoceptor expression was lower in transgenic mice specifically overexpressing human β₃-adrenoceptor in the heart (Kohout et al., 2001) whereas β₃-adrenoceptor knockout mice exhibited an increase in β₁-adrenoceptor mRNA in adipose tissues (Susulic et al., 1995).

It is well known that β₁ and β₂-adrenoceptors are subject to desensitization, internalization and down-regulation following chronic exposure to agonists leading to a decrease in β-adrenoceptor responsiveness, whereas β₃-adrenoceptors are resistant to desensitization (Dzimiri, 1999; Rozec and Gauthier, 2006). Indeed, the β₁ and β₂-adrenoceptors contain consensus sites mainly in the C-terminal region that can be phosphorylated by G-protein receptor kinases (GRKs), mainly GRK2, and cAMP protein kinase A (PKA) that are involved in desensitization of the receptors. In contrast, the β₃-adrenoceptor subtype lacks phosphorylation sites preventing its desensitization by this mechanism (Dzimiri, 1999; Tilley and Rockman, 2006). β-adrenoceptor internalization requires the recruitment of β-arrestins and phosphoinositide 3-kinase (PI3K), which bind to phosphorylated β-adrenoceptors (Dzimiri, 1999; Naga Prasad et al., 2001; Nienaber et al., 2003). The down-regulation of GPCRs following persistent stimulation also involves transcriptional and post-transcriptional mechanisms leading to a decrease in mRNA and protein synthesis (Dzimiri, 1999). Headley et al. (2004) demonstrated that extracellular signal-regulated kinase (ERK1/2), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein kinase (p38MAPK) pathways can modulate β₁-adrenoceptor mRNA stability. Finally, Li et al. (1998) showed that protein kinase C (PKC) induced the down-regulation of β₁-adrenoceptors at protein and mRNA levels in rat C6 glioma cells. Similarly, isoproterenol-induced adenylyl cyclase activity was impaired in human embryonic kidney (HEK) 293 cells overexpressing constitutively active PKC isoforms (α, βII, ε and ζ) and transfected with β₁- or β₂-adrenoceptors (Guimond et al., 2005).

Overall, these observations suggest that compensatory interactions between β₁- and β₃-adrenoceptors take place during chronic stimulation with the nonselective agonists, noradrenaline or isoprenaline. In addition, β₁-adrenoceptor desensitization and down-regulation involve PI3K and protein kinases whereas the implication of these kinases in β₃-adrenoceptor up-regulation is unknown. Therefore, we investigated the possible interaction between β₃-adrenoceptor up-regulation and β₁-adrenoceptor down-regulation at the functional and expression levels, and identified kinases implicated in the functional receptor modulation following chronic β₁- or β₃-adrenoceptor selective stimulation in neonatal rat cardiomyocytes.

In this study, we demonstrate for the first time that a cross-regulation at the expression and functional level between β₁- and β₃-adrenoceptors occurs following chronic receptor stimulation via the activation of PKC, PI3K, p38 MAPK and MEK/ERK1/2 pathways.

Methods

Cell culture

All animal care and experimental procedures complied with the UK Home Office Policy and approved by the Nottingham Trent University Ethical Committee. Neonatal ventricular myocytes were prepared from 1–4 day-old Wistar rats using the Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corporation, Lornes laboratories, Reading, UK). As described previously (Germack and Dickenson, 2006), the cells were preplated three times for 30 min in a humidified incubator (95% air/5% CO₂ at 37°C) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 10% (v/v) foetal calf serum and penicillin/streptomycin (100 U·mL⁻¹) in order to minimize fibroblast contamination. Cardiomyocyte-rich cultures (>90%) were plated onto fibronectin-coated plates at a final density of 1.25 × 10⁵ cells·cm⁻² in supplemented DMEM. For cAMP accumulation assay, the cells were plated onto 24-well plates and for RT-PCR and Western blotting analysis onto 6-well plates. After 3 days, confluent and spontaneously beating cells were serum-starved and untreated or treated with 10 µM dobutamine (a β₁-adrenoceptor selective agonist) and 1 µM ICI118551 (ICI, β₂-adrenoceptor selective antagonist) or 10 µM procaterol (a β₂-adrenoceptor selective agonist) and 1 µM CGP 20712A (CGP, β₁-adrenoceptor selective antagonist) or 2 µM CL-316243 (a β₃-adrenoceptor selective agonist) for 24 h. Because we did not observe any difference in the functional β₁-adrenoceptor down-regulation induced by chronic β₃-adrenoceptor stimulation in the presence and absence of propranolol at the concentration, which inhibits β₁ and β₂-adrenoceptors (data not shown), we did not combine CL-316243 treatment with an antagonist such as nadolol or propranolol to block β₁- and β₂-adrenoceptors. Similarly, Yoshida et al. (1994) showed that the treatment of obese diabetic mice with CL-316243 induces anti-obesity and anti-diabetic effects through specifically β₃-adrenoceptors without involving β₁- and β₂-adrenoceptors (potency β₁ : β₂ : β₃= 0:1:100 000). Finally, the CL-316243-inhibited forskolin response was only observed in noradrenaline-treated neonatal rat cardiomyocytes and not modified by the presence of propranolol (Germack and Dickenson, 2006). Therefore, these studies indicate that the treatment of cardiomyocytes with CL-316243 in the present investigation mediates its effects only through β₃-adrenoceptor stimulation. The signalling pathway involved in the regulation of β₁- and β₃-adrenoceptor cAMP responses following dobutamine/ICI or CL-316243 treatments was investigated in the absence or presence of protein kinase inhibitors (50 µM PD 98059, MEK1 inhibitor; 10 µM SB 203580, p38MAPK inhibitor; 10 µM SP 600125, SAPK/JNK inhibitor; 100 nM wortmannin, PI3K inhibitor; 1 µM KT 57201, PKA inhibitor; or 10 µM GF 109203, PKC inhibitor).

cAMP accumulation assay

Following the treatment of cardiomyocytes with dobutamine/ICI or procaterol/CGP or CL-316243 for 24 h, assays were carried out in serum-free DMEM in a humidified incubator (95% air/5% CO₂ at 37°C). Agonists as required by the experiments were added as described in the figure legends. The cells were incubated for 3 h in a humidified incubator (95% air/5% CO₂ at 37°C) with 500 µL of serum-free DMEM containing [³H]adenine (2 µCi per well). [³H]adenine-labelled cells were washed twice with Hanks/Hepes buffer and then incubated in 500 µL per well serum-free DMEM containing the cAMP phosphodiesterase inhibitor, rolipram (10 µM) for 15 min at 37°C in a humidified incubator. Agonists were added 5 min prior to the incubation with 1.5 µM forskolin (10 min). Antagonist or inhibitors were added 30 min before agonist. Incubations were terminated by the addition of 500 µL 5% trichloroacetic acid after removing the medium. [³H]cAMP was isolated by sequential Dowex-alumina chromatography as previously described (Germack and Dickenson, 2006). After elution, the levels of [³H]cAMP were determined by liquid scintillation counting.

Quantitative RT-PCR

Total RNA was extracted from untreated or treated cardiomyocytes for 24 h with dobutamine/ICI or procaterol/CGP or CL-316243 combining RNA isolation reagent, RNABee (AMS Biotechnology, Oxon, UK) and RNeasy Mini Kit (Qiagen Ltd, Crawley, UK) according to the manufacturer's instructions. Reverse transcription was performed with 3 µg total RNA for the synthesis of cDNA using oligo d(T)15 (Promega, Southampton, UK) and Superscript II reverse transcriptase (Invitrogen Ltd, Praisley, UK) according to the manufacturer's instructions. Quantitative PCR was carried out with a Bio-Rad iCycler system using iQ SYBR green Supermix kit from Bio-Rad (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK). The PCR protocol used was 3 min initial denaturation at 95°C followed by 45 cycles of denaturation (20 s at 95°C), annealing (30 s at 65°C) and elongation (30 s at 72°C). For exact quantification, standard curves were generated by serial dilutions (1 × 10⁶–1 × 10² single stranded cDNA molecules) for each target gene used as external standards. Standard cDNAs were prepared as followed: initial PCR was conducted with 1 µg cDNA in the presence of deoxyribonucleotides triphosphate (dNTPS) (1.25 mM), 200 ng of respective primers (Table 1) and 1.5 U Taq DNA polymerase (Promega, Southampton, UK). Thirty five cycles of the amplification steps involved 1 min denaturation at 94°C and 1 min annealing at 65°C and 1 min extension at 72°C. The PCR products were analysed using 1.5% agarose gel electrophoresis and extracted from the gel using QIAquick gel extraction kit (Qiagen Ltd, Crawley, UK). The purified products were again subjected to PCR and purified using QIAquick PCR purification kit (Qiagen Ltd, Crawley, UK) followed by quantification by spectrophotometry (λ:260/280). β-actin mRNA was used as an internal standard to normalize β₁-, β₂- and β₃-adrenoceptor mRNA.

Table 1.

Sequences of the specific primers for the three rat β-adrenoceptors (β₁-AR, β₂-AR, β₃-AR) and for β-actin

	Primer	PCR product Base pairs	Reference
β1-AR		368
Forward	5′-TTCGGTAGACGTGCTATGTGTGAC-3′
Reverse	5′-TCTTCTTCACCTGTTTCTGGGCCT-3′
β2-AR		328
Forward	5′-TGCGTGATTGCAGTGGATCGCTAT-3′
Reverse	5′-CTATCTTCTGCAGCTGCCTTTTGG-3′
β3-AR		202	Bensaid et al. (1993)
Forward	5′-AACTCTGCCTTCAACCCGCTCAT-3′
Reverse	5′-TTCATGTGGGAAATGGACGCTCAC-3′
β-actin		301	Germack and Dickenson (2006)
Forward	5′-CGTAAAGACCTCTATGCCAA-3′
Reverse	5′-GGTGTAAAACGCAGCTCAGT-3′

Western blot analysis

Cardiomyocytes untreated or treated with dobutamine/ICI or procaterol/CGP or CL-316243 for 24 h were washed with ice-cold phosphate-buffered saline (PBS) and lysed in ice-cold hypotonic buffer [30 mM Tris-HCl, 5 mM MgCl2, 100 mM NaCl, 1 mM EGTA, 0.1 mM phenylmethylsulphonylfluoride (PMSF), 5 µg·mL⁻¹ leupeptin] for G protein and β-adrenoceptor investigation. Cell membranes were collected by centrifugation for 30 min at 100 000 g and 4°C. The resulting plasma membrane pellets were dissolved in ice-cold detergent buffer [150 mM NaCl, 50 mM Tris.HCl, 5 mM ethylediaminetetraacetic acid (EDTA), 1% (v/v) IGEPAL CA-630, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulphate (SDS), 1 mM Na₃VO₄, 1 mM NaF, 1 mM benzamidine, 0.1 mM PMSF, 5 µg·mL⁻¹ leupeptin]. For the study of the cell signalling, cardiomyocytes untreated or treated with dobutamine/ICI or CL-316243 for 24 h in the presence or absence of appropriate kinase inhibitors were washed with ice-cold PBS and lysed in ice-cold detergent buffer. Cell membrane homogenates or cell lysates were clarified by centrifugation (5 min; 9500×g) in an Eppendorf microcentrifuge. 100 µL of the cell lysate removed and stored at −20°C until required. Protein concentration was determined using Bio-Rad DC Protein assay (Bio-Rad laboratories, Hertfordshire, UK) with bovine serum albumin as the standard. Samples in lysis buffer were heated at 95°C in SDS-polyacrylamide gel electrophoresis (SDS/PAGE) sample buffer (v/v). Aliquots of the cell lysate (20–30 µg protein) were separated by SDS/PAGE (12% acrylamide gel), using a Bio-Rad Mini-Protean II system (1 h at 200 V). Proteins were transferred to nitrocellulose membranes using a Bio-Rad Trans-Blot system (1 h at 100 V in 25 mM Tris, 192 mM glycine and 20% MeOH). Following transfer, the membranes were washed with Tris-buffered saline (TBS) and blocked for 1 h at room temperature in blocking buffer (TBS, 5% (w/v) skimmed milk powder, 0.1% Tween-20). Blots were then incubated for 24 h at 4°C with primary antibody in blocking buffer against Gα_i-protein, Gα_s-protein, phosphorylated ERK1/2, P38 MAPK, JNK or PKB at 1:1000 dilution or β₁-adrenoceptor at 1/200 dilution or β₂- or β₃-adrenoceptors at 1/100. The primary antibody was removed and the blot was extensively washed three times for 5 min in TBS/Tween 20. Blots were then incubated for 1 h at room temperature with the goat anti-rabbit secondary antibody coupled to horseradish peroxidase (DAKO Ltd, Cambridge, UK) at 1:1000 dilution in blocking buffer. Following removal of the secondary antibody, blots were extensively washed as previously described and developed using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Little Chalfont, UK) and quantified by densitometry using GeneGenius BioImaging System (Syngene, Synoptics Ltd, UK). The uniform transfer of proteins to the nitrocellulose membrane was routinely monitored by transiently staining the membranes with Ponceau S stain (Sigma Chemical Co.) prior to application of the primary antibody. In addition, replicate samples from each experiment were analysed on separate blots using an antibody (1:1000) that recognizes β-actin or unphosphorylated (total) ERK1/2, P38 MAPK, JNK and PKB.

Statistical analysis

Results are expressed as means ± SEM. Concentration-response curves were analysed by computer-assisted iteration using the GraphPad Prism (GraphPad software, San Diego, CA). Statistical significance was determined by analysis of variance (ANOVA) followed by Dunnett's test, and P < 0.05 was considered as the limit of statistical significance.

Materials

Bovine serum albumin, DMEM, foetal calf serum, fibronectin, forskolin, CL-316243, deoxynucleotide mix, IGEPAL CA-630 and leupeptin were all obtained from Sigma Chemical Co. (Poole, Dorset, UK). Trichloroacetic acid was purchased from Calbiochem (Nottingham, UK). Dobutamine, procaterol hydrochloride, ICI 118551, CGP 20712A, SB 203580, PD 98059, SP 600125, wortmannin, KT 5720 and GF 109203X were all from Tocris (Bristol, UK). β-actin, phospho-ERK1/2 (Thr²⁰²/Tyr²⁰⁴), ERK1/2, phospho-SAPK/JNK (Thr¹⁸³/Tyr¹⁸⁵), SAPK/JNK, phospho-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸²), p38 MAPK, phospho-PKB (Ser⁴⁷³) and PKB antibodies were purchased from New England Biolabs (Hitchin, UK). Mouse β₁- (sc-568) and β₂- (sc-570) adrenoceptor antibodies were provided by Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Mouse β₃-adrenoceptor antibody was purchased from Alpha Diagnostic international (San Antonio, TX). G protein α_i-2antibody was provided by Abcam (Cambridge, UK) and G protein α_s was from Chemicon (Eastleigh, UK). ([8-³H]adenine was obtained from Amersham (Bucks, UK).

Results

Effect of chronic stimulation of β₁, β₂ or β₃-adrenoceptors on the functional response induced by the β-adrenoceptor subtypes in neonatal rat cardiomyocytes

We have shown previously that β₁-, β₂- and β₃-adrenoceptors are expressed in neonatal rat cardiomyocytes, and following chronic stimulation with noradrenaline, β₁- and β₂- are down-regulated whereas β₃-adrenoceptors are up-regulated (Germack and Dickenson, 2006). Therefore, we investigated the functional response induced by β₁-, β₂- and β₃-adrenoceptor stimulation following chronic stimulation of each subtype for 24 h (Figure 1, Table 2). Chronic stimulation of β₁-adrenoceptors was performed using 10 µM dobutamine (a β₁-adrenoceptor selective agonist) in the presence of 1 µM ICI118551 (ICI, a β₂-adrenoceptor selective antagonist). Continuous activation of β₂-adrenoceptors was carried out with 10 µM procaterol (a β₂-adrenoceptor selective agonist) and 1 µM CGP 20712A (CGP, a β₁-adrenoceptor selective antagonist). Finally, the pretreatment of cardiomyocytes with 2 µM CL-316243 (a β₃-adrenoceptor selective agonist) stimulated chronically β₃-adrenoceptors. The concentrations of agonists and antagonists were chosen according to their potency and maximal responses previously published (Germack and Dickenson, 2006). Following dobutamine/ICI (β₁) or CL-316243 (β₃) treatments, dobutamine-induced cAMP accumulation was decreased by 67% and 52%, respectively (Figure 1A) indicating an interaction between both β-adrenoceptor subtypes. In addition, the decrease in β₁-adrenoceptor response was significantly higher when the cells were treated with dobutamine/ICI. Chronic stimulation of β₂-adrenoceptors did not modify β₁-adrenoceptor maximal response although the sensitivity to dobutamine was shifted to the right and decreased by around 10 times suggesting an interaction also between β₁- and β₂-adrenoceptors. Indeed, pretreatment with dobutamine/ICI resulted in a significant decrease in β₂-adrenoceptor response by 69% and a decrease in sensitivity by 35 times whereas β₃-adrenoceptors pretreatment had no effect on procaterol-induced cAMP accumulation (Figure 1B). Chronic stimulation of β₂-adrenoceptors abolished procaterol response. As shown previously and in Figure 1C, β₃-adrenoceptors are not functional in normal physiological conditions in neonatal rat cardiomyocytes (Germack and Dickenson, 2006). Chronic stimulation of β₂-adrenoceptors did not induce functional β₃-adrenoceptor expression. In pretreated cardiomyocytes with dobutamine/ICI or CL-316243, CL-316243 induced a significant inhibition of forskolin-mediated cAMP accumulation without changing the potency between both treatments strengthening the assumption of a compensatory regulation between β₁- and β₃-adrenoceptors (Table 2). In addition, CL-316243 treatment induced a higher β₃-adrenoceptor response than β₁-adrenoceptor treatment.

Figure 1.

Activation of cAMP accumulation induced by dobutamine, β₁-selective agonist (A) and procaterol, β₂-selective agonist (B), and effect of the selective β₃-adrenoceptor agonist CL-316243 on forskolin (Forsk)-induced cAMP accumulation (C) in neonatal rat cardiomyocytes untreated, treated with 10 µM dobutamine in the presence of 1 µM ICI 118551, 10 µM procaterol in the presence of 1 µM CGP 20712A or 2 µM CL-316243 for 24 h. cAMP accumulation was measured as described under Methods. Data are expressed as percentage of the basal level of cAMP accumulation (100%, A, B) or as the percentage of 1.5 µM forskolin response in the absence of agonist (100%, C). Each point represents the mean ± SEM of four to six experiments performed in duplicate.

Table 2.

Effect of dobutamine/ICI118551, procaterol/CGP 20712A and CL-316243 treatments on dobutamine and procaterol-induced cAMP accumulation, and CL-316243-mediated inhibition of forskolin-induced cAMP response

Agonist		Untreated	Treatment
			β1 treatment	β2 treatment	β3 treatment
Dobutamine	Emax	234 ± 11	77 ± 4***	219 ± 28	112 ± 6***, #
β1 response	pD2	6.46 ± 0.18	6.15 ± 0.30	5.05 ± 0.32***	6.65 ± 0.30
Procaterol	Emax	156 ± 10	49 ± 2***	NR	162 ± 12###
β2 response	pD2	7.67 ± 0.27	6.16 ± 0.29***	–	7.71 ± 0.31
CL-316243	Imax	NR	23 ± 6	NR	30 ± 5#
β3 response	pIC50	–	8.04 ± 0.45	–	8.45 ± 0.23

Values are means ± SEM of four to six experiments performed in duplicate. The potencies of the agonists were evaluated by their EC₅₀, concentration of agonist inducing 50% of the maximal cAMP accumulation expressed as –log₁₀ EC₅₀ (pD₂) or their IC₅₀, concentration of agonist inducing 50% of inhibition expressed as –log₁₀ IC₅₀ (pIC₅₀). Emax is the maximal response expressed in % over basal response. Imax is the maximal percentage of inhibition.

^*P < 0.05 and

^***P < 0.001 versus untreated cells and

^#P < 0.05 and

^###P < 0.001 versus β₁ treatment. NR means no response.

Effect of chronic stimulation of β₁, β₂ or β₃-adrenoceptors on mRNA and protein expression of the β-adrenoceptor subtypes in neonatal rat cardiomyocytes

The functional studies suggest that a potential interaction exists between β₁- and β₂-adrenoceptors as well as β₁- and β₃-adrenoceptors. Therefore, we investigated whether this cross-regulation involves the modulation of the receptor expression by measuring the mRNA and protein level of the three receptors following the different treatments ( Figures 2 and 3). The expression of the β-adrenoceptors in untreated cells by real time PCR (Figure 2A) displayed a β₁/β₂/β₃ ratio of 0.39/0.61/0.0008 indicating that β₂-adrenoceptors are predominantly expressed in neonatal rat cardiomyocytes as recently reported by Morisco et al. (2008) using binding studies. The β₁/β₂/β₃ mRNA ratio was 0.25/0.745/0.0017 or 0.24/0.75/0.0017 following β₁- or β₃-adrenoceptor treatments respectively. Although an increase in β₃-adrenoceptors was observed, β₃-adrenoceptor level remained very low, which may explain the absence of functional response in physiological conditions and a low functional activity (30%) following chronic stimulation. Chronic β₁- or β₃-adrenoceptor stimulation induced a decrease in β₁-adrenoceptor mRNA level by 47% and 50% respectively (Figure 2B). At protein level, β₁-adrenoceptor was reduced by 58% following β₁-treatment and 42% subsequent to β₃-adrenoceptor treatment (Figure 3A). β₂-adrenoceptor treatment had no effect on β₁-adrenoceptor expression (Figures 2B and 3A). A decrease in β₂-adrenoceptor expression by around 40% occurred at mRNA and protein level when cardiomyocytes were treated with procaterol/CGP whereas β₁- or β₃-adrenoceptor treatments had no effect (Figures 2C and 3B). No effect of chronic β₂-adrenoceptor stimulation on β₃-adrenoceptor mRNA and protein level was observed (Figures 2D and 3C). The amount of β₃-adrenoceptor transcripts was increased by 65% and 72%, and β₃-adrenoceptor protein level by 43% and 68% following chronic stimulation of β₁- or β₃-adrenoceptors respectively (Figures 2D and 3C). The observed bands corresponded to the expected molecular weight (Diebold et al., 2001) and were modulated similarly to β-adrenoceptor mRNAs suggesting specific antibody binding. Overall, these data show that the modulation of receptor expression correlated well with the changes in β₁- and β₃-adrenoceptor functions.

Figure 2.

Quantitative expression of β-adrenoceptors in untreated neonatal rat cardiomyocytes (A) and expression of β₁- (B), β₂- (C) and β₃-adrenoceptors (D) mRNA obtained from untreated (control), treated with 10 µM dobutamine in the presence of 1 µM ICI 118551 (β₁ treatment), 10 µM procaterol in the presence of 1 µM CGP 20712A (β₂ treatment) or 2 µM CL-316243 (β₃ treatment) for 24 h. Total RNA was prepared, reverse transcription and real time PCR were carried out as described under Methods. Values were expressed as β-adrenoceptor molecules × 10⁻³β actin molecules in A and normalized to β-actin molecules and expressed as the percentage of untreated cardiomyocytes (100%) in B, C and D. Each point represents the mean ± SEM of five to six independent experiments. **P < 0.01 and ***P < 0.001 versus untreated control.

Figure 3.

Expression of β₁- (A), β₂- (B) and β₃-adrenoceptors (C) obtained from neonatal rat cardiomyocytes untreated (control), treated with 10 µM dobutamine in the presence of 1 µM ICI 118551 (β₁ treatment), 10 µM procaterol in the presence of 1 µM CGP 20712A (β₂ treatment) or 2 µM CL-316243 (β₃ treatment) for 24 h. Cell membrane homogenates were monitored by Western blotting for β₁-, β₂- and β₃-adrenoceptors as described under Methods. The same samples were also analysed on separate blots using an antibody that recognizes β actin to confirm equal loading on each lane. Representative immunoblots for each proteins are shown in panel C. Data are expressed as the percentage of untreated cardiomyocytes (100%) following the calculation of β-adrenoceptor/β-actin ratio for each lane. The combined results (panel A, B and C) obtained from densitometric analysis of blots represent the mean ± SEM of five to seven independent experiments. *P < 0.05 and **P < 0.01 versus untreated control.

Effect of chronic stimulation of β₁, β₂ or β₃-adrenoceptors on Gα_s and Gα_i-2 protein in neonatal rat cardiomyocytes

Both, β₁- and β₃-adrenoceptor treatments induced a similar modulation of β₁-adrenoceptor or β₃-adrenoceptor expression, whereas the decrease in β₁-adrenoceptor and increase in β₃-adrenoceptor functional responses were higher in cardiomyocytes treated with dobutamine/ICI and CL-316243 respectively. These results indicate that the modulation of β₁- and β₃-adrenoceptor function may also involve modifications in receptor and G protein coupling.

Therefore, we investigated the expression of Gα_s and Gα_i-2 proteins in neonatal rat cardiomyocytes following β₁-, β₂- or β₃-adrenoceptor treatment using Western blotting (Figure 4). Gα_s protein level was not significantly modified in cardiomyocytes following the three treatments (Figure 4A). Inversely, Gα_i-2 protein was increased by 44% when β₁-adrenoceptors were chronically stimulated and by 94% and 112% following β₂- or β₃-adrenoceptor treatments respectively (Figure 4B). These results suggest that the higher inhibition of the response to forskolin induced by β₃-adrenoceptor stimulation following CL-316243 treatment, compared with dobutamine/ICI treatment involves a better coupling to Gα_i-2 protein.

Figure 4.

Expression of Gα_s and Gα_i proteins obtained from neonatal rat cardiomyocytes untreated (control), treated with 10 µM dobutamine in the presence of 1 µM ICI 118551 (β₁ treatment), 10 µM procaterol in the presence of 1 µM CGP 20712A (β₂ treatment) or 2 µM CL-316243 (β₃ treatment) for 24 h. Cell membrane homogenates were monitored by Western blotting for Gα_s (A) and Gα_i (B) proteins as described under Methods. The same samples were also analysed on separate blots using an antibody that recognizes β actin to confirm equal loading on each lane. Representative immunoblots for each proteins are shown in panel C. Data are expressed as the percentage of untreated cardiomyocytes (100%) following the calculation of β-adrenoceptor/β-actin ratio for each lane. The combined results (panels A and B) obtained from densitometric analysis of blots represent the mean ± SEM of four to six independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, (a) versus untreated control and (b) versus β₁-adrenoceptor treatment.

Effect of kinase inhibitors on the down-regulation of β₁-adrenoceptor functional response induced by chronic stimulation of β₁ or β₃-adrenoceptors in neonatal rat cardiomyocytes

PI3K, PKA, PKC have been shown to be involved in the process of β₁-adrenoceptor down-regulation (Li et al., 1998; Dzimiri, 1999; Naga Prasad et al., 2001; Nienaber et al., 2003). In addition, ERK1/2, SAPK/JNK and p38 seem to be also involved in the regulation of β-adrenoceptor expression following chronic stimulation (Headley et al., 2004). As β₂-adrenoceptor treatment did not show any interaction at both expression and functional levels between β₂- and β₁-adrenoceptors, we investigated the signalling pathway involved in the down-regulation of the β₁-adrenoceptor following chronic stimulation of β₁- and β₃-adrenoceptors. In order to determine the mechanisms implicated in β₁-adrenoceptor down-regulation, cardiomyocytes were treated for 24 h with dobutamine/ICI or CL-316243 in the presence or absence of inhibitors of MAP kinase (MEK1, 50 µM, PD 98059), PI3K (100 nM, wortmannin), p38 MAPK (10 µM, SB 203580), SAPK/JNK (10 µM, SP 600125), PKA (1 µM, KT 57201) and PKC (10 µM, GF 109203). As shown in Figure 5, wortmannin, SB 203580 and GF 10920 produced a decrease in dobutamine-induced cAMP accumulation by 25%, 30% and 24% in untreated cells respectively. The inhibition of β₁-adrenoceptor response in untreated cardiomyocytes was likely to occur following dobutamine/ICI and CL-316243 treatments. Therefore, to consider only the effect of both treatments on the modulation of β₁-induced cAMP accumulation, the functional values obtained in the treated groups were corrected from the inhibitory effect of wortmannin, SB203580 or GF 10920 observed in untreated cells (decrease in untreated cells + value of treated cardiomyocytes for each individual experiments), as explained in the legend of Figure 5. PD 98059 and SP 600125 had no effect on the functional down-regulation induced by both β₁- or β₃-adrenoceptor treatments indicating that MEK and SAPK/JNK are not implicated in this process. Wortmannin, SB 203580 and GF 109203 reversed the inhibition of dobutamine response following β₁-adrenoceptor treatment by 64%, 32% and 58%, and by 44%, 20% and 34% when cardiomyocytes were treated with CL-316243 (β₃-adrenoceptor treatment) respectively. The PKA inhibitor, KT 57201 counteracted the decrease in β₁-adrenoceptor function by 26% in the cells treated with dobutamine/ICI and did not modify β₁-induced cAMP accumulation in CL-316243 treated cardiomyocytes.

Figure 5.

Effect of kinase inhibitors on dobutamine-induced cAMP accumulation in neonatal rat cardiomyocytes untreated, treated with 10 µM dobutamine in the presence of 1 µM ICI 118551 (β₁ treatment), or 2 µM CL-316243 (β₃ treatment) for 24 h. In addition, untreated and treated cells were incubated in panel A with 50 µM PD 98059 (MEK inhibitor) or 100 nM wortmannin (phosphoinositide 3-kinase inhibitor), in panel B with 10 µM SB 203580 (p38 MAPK inhibitor) or 10 µM SP 600125 (JNK inhibtor) and in panel C with 1 µM KT 57201 (PKA inhibitor) or 10 µM GF 109203 (PKC inhibitor). cAMP accumulation was measured as described under Methods. Data are expressed as the percentage of the basal level of cAMP accumulation (set at 100%, striped bars). Black and open bars represent cAMP accumulation induced by dobutamine in the absence and presence of protein kinase inhibitors in all groups respectively. Wortmanin (A), SB 203580 (B) and GF 109203 (C) elicited a significant decrease in cAMP accumulation induced by dobutamine in untreated cardiomyocytes. Grey filled bars represent the data corrected from the effect of these inhibitors on dobutamine response in untreated cardiomyocytes in order to consider only the effect of β₁- or β₃-adrenoceptor treatments on the modulation of β₁-adrenoceptor response (decrease in untreated cells + value of treated cardiomyocytes for each individual experiments). Each bar represents the mean ± SEM of six to seven independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, (a) versus basal level, (b) versus dobutamine response in untreated cells, (c) versus dobutamine response in β₁-adrenoceptor-treated cells and (d) versus dobutamine response in β₃-adrenoceptor-treated cells.

Effect of kinase inhibitors on the up-regulation of β₃-adrenoceptor functional response induced by chronic stimulation of β₁ or β₃-adrenoceptors in neonatal rat cardiomyocytes

β₃-Adrenoceptor agonist-induced inhibition of responses to forskolin was investigated when cardiomyocytes were treated for 24 h with dobutamine/ICI or CL-316243 in the presence or absence of inhibitors of MEK1 (50 µM, PD 98059), PI3K (100 nM, wortmannin), p38 (10 µM, SB 203580), SAPK/JNK (10 µM, SP 600125), PKA (1 µM, KT 57201) and PKC (10 µM, GF 10920). As shown in Figure 6, PD 98059, SB 203580 and GF 10920 inhibited β₁- or β₃-adrenoceptor treatment-induced up-regulation of β₃-adrenoceptor functional response indicating the involvement of MEK, p38 and PKC in the regulation of the β₃-adrenoceptor subtype. KT 57201 also abolished the increase in β₃-adrenoceptor response when the cells were treated with dobutamine/ICI but not with CL-316243. No effect of wortmannin and SP 600125 was observed indicating that PI3K and SAPK/JNK do not regulate β₃-adrenoceptor up-regulation.

Figure 6.

Effect of kinase inhibitors on CL-3162436243-inhibited forskolin response in neonatal rat cardiomyocytes untreated, treated with 10 µM dobutamine in the presence of 1 µM ICI 118551 (β₁ treatment), or 2 µM CL-316243 (β₃ treatment) for 24 h. In addition, untreated and treated cells were incubated in panel A with 50 µM PD 98059 (MEK inhibitor) or 100 nM wortmannin (phosphoinositide 3-kinase inhibitor), in panel B with 10 µM SB 203580 (p38 MAPK inhibitor) or 10 µM SP 600125 (JNK inhibtor) and in panel C with 1 µM KT 57201 (PKA inhibitor) or 10 µM GF 109203 (PKC inhibitor). cAMP accumulation was measured as described under Methods. Data are expressed as the percentage of 1.5 µM forskolin (FSK) response in the absence of agonist (100%, striped bars). Black and open bars represent the inhibition of FSK-mediated cAMP accumulation induced by CL-316243 in the absence and presence of protein kinase inhibitors in all groups respectively. Each bar represents the mean ± SEM of six to seven independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, (a) versus CL-316243 response in untreated cells, (b) versus CL-316243 response in β₁-adrenoceptor treated cells and (c) versus CL-316243 response in β₃-adrenoceptor treated cells.

Effect of chronic stimulation of β₁ or β₃-adrenoceptors on ERK1/2, p38, SAPK/JNK and Akt activation in neonatal rat cardiomyocytes

The functional study indicates that PI3K, MEK, p38 but not SAPK/JNK are involved in the modulation of the receptors following the different treatments. Consequently, chronic stimulation of β₁- and β₃-adrenoceptors should lead to the activation of p38, extracellular signal regulated-kinase (ERK1/2, down-stream MEK target) and Akt (down-stream PI3K target). Therefore, we investigated the activation of ERK1/2, p38, SAPK/JNK and Akt by either dobutamine/ICI or CL-316243 treatments. Chronic stimulation of β₁- or β₃-adrenoceptor subtypes induced ERK1/2 phosphorylation by 17% and 49% respectively (Figure 7A). Significantly, lower ERK1/2 activation was obtained when the cells were treated with dobutamine/ICI compared with chronic β₃-adrenoceptor stimulation. Both treatments activated p38 by around 60%, which correlated well with the involvement of this kinase in the regulation of β₁- and β₃-adrenoceptor functional response following dobutamine/ICI or CL-316243 treatments (Figure 7B). Chronic β₁-adrenoceptor stimulation produced an increase in SAPK/JNK activation by 241% whereas β₃-adrenoceptor treatment had no effect (Figure 7C). Interestingly, Akt was phosphorylated by 149% when cardiomyocytes were treated with CL-316243 and not with dobutamine/ICI (Figure 7D) indicating a possible involvement of the β₃-adrenoceptor subtype in cell survival.

Figure 7.

Effect of β₁-adrenoceptor and β₃-adrenoceptor treatments on ERK1/2, p38 MAPK, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and Akt activation in neonatal rat cardiomyocytes. The cells were untreated or treated with 10 µM dobutamine in the presence of 1 µM ICI 118551 (β₁-AR treatment), or 2 µM CL-316243 (β₃-AR treatment) for 24 h. Following the treatments, cell lysates were analysed by Western blotting as described under Methods using phospho-specific antibodies. The same samples were also analysed on separate blot using an antibody that recognizes both unphosphorylated (total) and phosphorylated kinases to confirm equal loading on each lane. Data are expressed as percentage of the untreated control following the calculation of the phosphorylated/total ratio for each lane. The combined results (panel A for ERK1/2, panel B for p38 mitogen-activated protein kinase, panel C for SAPK/JNK and panel D for Akt) obtained from densitometric analysis of blots, represent the mean ± SEM of for to six independent experiments. *P < 0.05 and **P < 0.01, (a) versus untreated control, (b) versus β₁-adrenoceptor treated cells.

Discussion and conclusions

We have previously shown that chronic β-adrenoceptor stimulation with noradrenaline induces a decrease in β₁-adrenoceptors and an increase in β₃-adrenoceptors at the functional, genomic and protein levels (Germack and Dickenson, 2006). For the first time, the present study provides evidence of a cross-regulation at the expression and functional level between β₁- and β₃-adrenoceptors following chronic receptor stimulation via PI3K, PKC, p38 MAPK and MEK/ERK1/2 pathway in neonatal rat cardiomyocytes.

Changes in cardiac rate and force are triggered by β₁- and β₂-adrenoceptors acting via the G_s/cAMP/PKA signalling pathway whereas β₃-adrenoceptors mediate a decrease in contractility through a coupling to G_i protein (Dzimiri, 1999; Rozec and Gauthier, 2006). It is well established that β₁- and β₂-adrenoceptors are down-regulated following chronic stimulation (Dzimiri, 1999). As expected, in this study, chronic β₁-adrenoceptor treatment led to a decrease in β₁-adrenoceptor-induced cAMP accumulation and continuous β₂-adrenoceptor stimulation abolished β₂-adrenoceptor response, which was well correlated to the receptor expression. Interestingly, β₂-adrenoceptor treatment decreased sensitivity to dobutamine without changing β₁-adrenoceptor expression. Similarly, chronic β₁-adrenoceptor stimulation reduced β₂-adrenoceptor maximal response and efficiency with no alteration in β₂-adrenoceptor level. These results indicate that the modification of β₁- and β₂-adrenoceptor functional response without modification of receptor expression may reflect an interaction between both subtypes at the membrane level. Indeed, Zhu et al. (2005) have demonstrated that adult cardiomyocytes elicit not only a colocalization and physical association between β₁- and β₂-adrenoceptors, but also a functional synergy in cAMP and contractile responses. The disruption of β₁- and β₂-adrenoceptor heterodimerization may also contribute to the decrease in cardiac function in addition to receptor down-regulation in heart diseases where catecholamines are elevated (Dzimiri, 1999; Tilley and Rockman, 2006). Chronic β₃-adrenoceptor stimulation also produced a decrease in β₁-adrenoceptor response and expression. In contrast, this treatment induced an increase in β₃-adrenoceptor function and expression as observed in cardiomyocytes continuously exposed to β₁-adrenoceptor treatment. It is noteworthy that the relative abundance of the β₃-adrenoceptor in untreated neonatal rat cardiomyocytes was similar to the relative β₃-adrenoceptor level observed in healthy human hearts using also real-time PCR (β₁/β₂/β₃ ratio: neonatal rat 0.39/0.61/0.0008 vs. human 0.71/0.28/0.0012; Moniotte et al., 2007). Sepsis is also associated with impairment in β-adrenoceptor response and myocardial dysfunction (Silverman et al., 1993; Muller-Werdan et al., 2006). Interestingly, the relative proportion of the β₃-adrenoceptor increased in human hearts from septic patients, from 0.0012 to 0.0024 (Moniotte et al., 2007) as observed in our study in neonatal rat cardiomyocytes following β₁- or β₃-adrenoceptor treatments (0.0008 to 0.0017). In addition, Moniotte et al. (2007) when using cytokines in murine cardiomyocytes as a model of sepsis showed a decrease in contractility by around 45% following β₃-adrenoceptor stimulation. All together, these data indicate that even when it is up-regulated but expressed at low level, the β₃-adrenoceptor may regulate cardiac function in pathological situation. Overall, our results clearly indicate that changes in β₁- and β₃-adrenoceptor regulation following chronic stimulation involves a cross-talk between both subtypes as observed in failing human and animal hearts (Cheng et al, 2001; Moniotte et al., 2001; Morimoto et al., 2004; Zhang et al., 2005).

The up-regulation of β₃-adrenoceptor function following β₃-adrenoceptor treatment was higher by 30% compared with chronic β₁-adrenoceptor activation. In addition, both treatments did not produce a significant difference in the increase in β₃-adrenoceptor expression suggesting an enhancement in the coupling between β₃-adrenoceptors and Gα_i protein. Indeed, the overexpression of Gα_i protein in neonatal cardiomyocytes reduced isoprenaline-activated adenylyl cyclase, which was restored in the presence of Pertussis toxin (PTX) (Rau et al, 2003). Furthermore, the increase in Gα_i protein level in addition to β-adrenoceptor down-regulation led to the desensitization of functional response, which was improved with PTX in failing rat heart (Kompa et al., 1999; Xiao et al., 2003). These works indicate that the function mediated by β-adrenoceptor is also regulated by coupling to Gα_i protein when this protein is highly expressed. Although three isoforms Gα_{i-1, 2 and 3} have been identified, Gα_i-2 is mainly expressed in the heart and up-regulated during heart failure (El-Armouche et al., 2003). In addition, the inhibition of cardiac Gα_i-2 increased infarct size and apoptosis in transgenic mice expressing a Gα_i-2 inhibitor peptide, when the mice were subjected to ischemia/reperfusion indicating an important role of this isoform in cardioprotection. In our study, Gα_i-2 expression was increased by both, β₁- or β₃-adrenoceptor treatments as observed in neonatal cardiomyocytes chronically treated with noradrenaline and in failing rat heart (Reithmann et al., 1990; Kompa et al., 1999; Xiao et al., 2003). Interestingly, cardiomyocytes chronically stimulated with CL-316243 displayed a further increase in Gα_i-2 protein level compared with β₁-adrenoceptor treated cells by 32%. We can assume that an enhancement in β₃-adrenoceptor coupling to Gα_i protein may explain the difference in β₃-adrenoceptor functional response between both, β₁- and β₃-adrenoceptor treatments. It is noteworthy that β₂-adrenoceptor treatment also increased Gα_i-2 protein as much as β₃-adrenoceptor treatment without inducing β₃-adrenoceptor functional response. This implies that the up-regulation of β₃-adrenoceptor function requires an enhancement of the receptor expression taking place at the genomic level and an increase in Gα_i protein as observed with chronic β₁-adrenoceptor stimulation. In contrast, chronic β₁-adrenoceptor stimulation led to a lower decrease in β₁-adrenoceptor response by 31% compared with chronic β₃-adrenoceptor activation suggesting that the increase in Gα_i protein did not affect β₁-adrenoceptor response as previously reported in cardiomyocytes from failing heart (Xiao et al., 2003).

β₁-Adrenoceptor desensitization requires receptor phosphorylation by GRK2 (agonist dependent, homologous regulation) and PKA (agonist independent, heterologous regulation), which alters the coupling between the receptor and Gα_s protein, leading to the reduction of β₁-adrenoceptor functional response (Dzimiri, 1999; Tilley and Rockman, 2006). In the present study, the PKA inhibitor, KT 57 201 partially blocked the functional β₁-adrenoceptor down-regulation following chronic β₁-adrenoceptor stimulation, whereas this inhibitor had no effect on β₃-adrenoceptor treatment-induced decrease in β₁-adrenoceptor response. β₁-adrenoceptors desensitization by PKA may explain the difference in β₁-adrenoceptor down-regulation observed between chronic β₁- and β₃-adrenoceptor stimulation. Interestingly, the up-regulation of β₃-adrenoceptor response was counteracted by KT 57201 only following chronic β₁-AR activation. These data confirm previous reports that β₃-adrenoceptors do not activate the Gα_s/PKA signalling pathway in cardiomyocytes (Germack and Dickenson, 2006; Rozec and Gauthier, 2006). In our study, the PKC inhibitor, GF 109203, also reversed β₁-adrenoceptor responsiveness following both, β₁- or β₃-adrenoceptor treatments. As previously shown, PKC mediates heterologous desensitization of β-adrenoceptors. Indeed, pretreatement of rat C6 glioma cells with phorbol 12-myristate 13-acetate (PMA, a PKC activator) produced β₁-adrenoceptor down-regulation at protein and mRNA levels (Li et al., 1998). Guimond et al. (2005) have reported that overexpression of PKC isoforms (α, βII, ε and ζ) in HEK 293 cells transfected with β₁-adrenoceptors resulted in the decrease in β₁-adrenoceptor-induced adenylyl cyclase activity. Finally, PKC produced by the activation of angiotensin II receptors and overexpression α_1B-ARs reduced heart rate and inotropic response induced by β₁-ARs respectively (Schwartz and Naff, 1997; Lemire et al., 1998). The PKC inhibitor also impaired the functional β₃-adrenoceptor up-regulation in chronic β₁- or β₃-adrenoceptor-treated cardiomyocytes. It is noteworthy that Gβγ subunits from GPCRs activate PI3K (Salazar et al., 2007) that in turn targets PKC ε, η, ζ and λ (Toker and Cantley, 1997). These observations may explain PKC activation mediated by chronic β₁- or β₃-adrenoceptor stimulation observed in our investigation.

We found in the present study that β₁- or β₃-adrenoceptor treatments in the presence of the PI3K inhibitor wortmannin restored β₁-adrenoceptor function in cardiomyocytes indicating the recruitment of PI3K by either chronic β₁- or β₃-adrenoceptor stimulation in the regulation of β₁-adrenoceptors. Indeed, β-adrenoceptor desensitization and down-regulation require PI3Kγ recruitment through the interaction with GRK2 (Naga Prasad et al., 2001; Ribas et al., 2007). In addition, cardiac overexpression of inactive PI3Kγ in mice prevented β-adrenoceptor down-regulation and desensitization to chronic isoprenaline administration, and preserved β-adrenoceptor-induced left ventricle contractility and adenylyl cyclase activity (Nienaber et al., 2003). Although the heart expressed two PI3K isoforms, PI3Kα and PI3Kγ the former form is mainly involved in the regulation of GPCRs (Salazar et al., 2007). In contrast to β₁-adrenoceptor down-regulation, wortmannin had no effect on β₃-adrenoceptor up-regulation induced by both, β₁- or β₃-adrenoceptor treatments. As shown previously, short term β₃-adrenoceptor stimulation induced ERK1/2 activation through G_i/o/PI3K pathway in β₃-adrenoceptor transfected CHO/K1 cells and human lung epithelial-derived cell line A549 (Gerhardt et al., 1998; Robay et al., 2005). In addition, Akt was also activated by the G_i/o/PI3K pathway in β₃-adrenoceptor transfected cells (Gerhardt et al., 1998) as observed in our study where chronic β₃-adrenoceptor but not β₁-adrenoceptor treatments induced Akt phosphorylation. Liu et al. (2005) showed that Akt binds to GRK2 and this association inhibits Akt activity. This regulation may explain why chronic β₁-adrenoceptor stimulation did not induce Akt phosphorylation while PI3K was activated. Overall, these data indicate that the chronic stimulation of both, β₁- or β₃-adrenoceptors, activates PI3K leading to β₁-adrenoceptor down-regulation. In addition, continuous β₃-adrenoceptor treatment triggered Akt activation suggesting the involvement of this subtype in cardioprotection. Indeed, the PI3K/Akt pathway is well known to induce cell viability through an antiapoptotic effect (Matsui and Rosenzweig, 2005).

Chronic β-adrenoceptor stimulation activates several members of the MAPK family including p38 MAPKs, SAPK/JNK and ERK1/2, which may be involved in the regulation of the β-adrenoceptor receptors (Palfi et al., 2005; Kim et al., 2006). The p38 MAPK inhibitor, SB 203580 inhibited the functional β₁- and β₃-adrenoceptor changes induced by β₁- or β₃-adrenoceptor treatments. In addition, both treatments activated p38 MAPK as previously suggested by Peter et al. (2007) regarding β₁-adrenoceptors. Indeed, chronic β₁-adrenoceptor stimulation enhances p38 MAPK activation in left ventricle from transgenic mice overexpressing β₁-adrenoceptors. In contrast to SB 203580, the SAPK/JNK inhibitor, SP 6000125 did not modify the functional β₁- and β₃-adrenoceptor response modulated by both β₁- or β₃-adrenoceptor treatments. However, chronic β₁-adrenoceptor stimulation activated SAPK/JNK as observed in rat heart following continuous isoprenaline administration (Palfi et al., 2005). Chronic β₁- or β₃-adrenoceptor stimulation also mediated ERK1/2 activation. Soeder et al. (1999) have shown that continuous β₃-adrenoceptor stimulation leads to ERK1/2 activation through the Gα_i pathway in 3T3F442A adipocytes as observed in the present study in neonatal cardiomyocytes. However, Robidoux et al. (2006) showed that β₃-adrenoceptors activated ERK1/2 through a PKA-dependent and PKA-independent pathway in 3T3-L1 cells, the former involving src and epidermal growth factor receptor kinase. This seems to indicate that different pathways inducing ERK1/2 activation may be cell type dependent. However, only β₃-adrenoceptor up-regulation was sensitive to PD 98059 (MEK inhibitor). It is well known that MAPKs are involved in the regulation of transcription factors and gene expression (Edmunds and Mahadevan, 2004). The modulation of β₁- and β₃-adrenoceptor mRNA levels suggests the involvement of a transcriptional regulation in the change of receptor expression following chronic β-adrenoceptor stimulation. Therefore, β₁-adrenoceptor down-regulation and β₃-adrenoceptor up-regulation should involve the activation of transcription factors like ICER or cAMP response element binding protein via MAPK stimulation. Further studies are required to investigate possible transcriptional mechanism involved in the cross-regulation between β₁- and β₃-adrenoceptors.

In conclusion, we have presented novel data showing that chronic stimulation of β₁- or β₃-adrenoceptors leads to an opposite regulation of the receptors at the expression and functional levels. The cross-talk between β₁- and β₃-adrenoceptors occurs via the activation of PKC, PI3K, p38 MAPK and MEK/ERK1/2 pathway, and through PKA when β₁-adrenoceptors are continuously stimulated.

Glossary

Abbreviations:

DMEM

Dulbecco's modified Eagle's medium

ERK1/2

extracellular responsive kinase

GPCR

G-protein coupled receptor

PKA

protein kinase A

PKC

protein kinase C

PMA

phorbol 12-myristate 13-acetate

p38MAPK

p38 mitogen-activated protein kinase

PTX

Pertussis toxin

SAPK/JNK

stress-activated protein kinase/c-Jun N-terminal kinase

Conflict of interest

The authors state no conflict of interest.