Crosstalk between β1 -adrenoceptor downregulation and β3 -adrenoceptor upregulation following β1 - or β3 -agonist stimulation in neonatal rat cardiomyocytes: role of map kinases and PI3 kinase/akt pathway

R. Germack and J.M. Dickenson Nottingham Trent University, Nottingham, UK

In the heart, β1 and β2-adrenergic receptors (β1-/β2-ARs) increase cardiac function through Gs/PKA dependent pathway whereas β3-adrenergic receptor (β3-AR) activation induces a decrease in cardiac contractility involving G i/o protein (Gauthier et al, 2000). We have shown that β-ARs continuously stimulated with noradrenaline induces an increase in β3 -ARs and a decrease in β1 -ARs at the functional, genomic and protein levels (Germack et al, 2006). This compensatory modification of β -ARs induced by noradrenaline is probably one of the consequences of cardiac depression observed in heart disease. The aim of this work was to investigate the possible interaction between β1 - and β3 -ARs by studying the effect of dobutamine (Dob, β1 agonist) and Cl 316243 (Cl31, β3 ) on cAMP response in neonatal rat cardiomyocytes.

Cardiomyocytes were isolated from 1-4 day old Wistar rats. Measurement of [3H]-cAMP accumulation in presence or absence of 1.5 μM forskolin was performed using cells pre-labelled with [3H]-adenine. The cells were untreated or treated with 10μM Dob and 1μM ICI 118551 ( b 2 antagonist; Dob/ICI) or 2 m M Cl31 for 24h in absence and presence of 50μM PD98059 (PD, MEK1 inhibitor), 100nM wortmannin (wort, PI3K inhibitor), 10μM SB 20380 (SB, p38 inhibitor) and 10μM SP 600125 (SP, JNK inhibitor). We also determined Akt, ERK1/2, p38 and SAPK/JNK phosphorylation by Western blotting. Results are expressed as mean ± s.e.m for 5-6 experiments.

Dob increased cAMP response (Emax, % over the basal: 234±11%; pEC50: 6.46±0.18). Pretreatment with Dob/ICI and Cl31 decreased significantly β1 response by 67% (P<0.001) and 52% (P<0.001), respectively. Cl31 inhibited forskolin response in Dob/ICI and Cl31 treated cells (Imax: 23±6% and 30±5%, respectively; pIC50: 8.04±0.45 and 8.45±0.23). No β3 response was observed in untreated cells. Wort and SB inhibited downregulation of β1 response in cells treated with Dob/ICI (60%, P<0.05 and 30%, P<0.01, respectively) or Cl31 (47%, P<0.05 and 22%, P<0.01). No modification of β1 response was observed with PD and SP following both treatments. PD and SB decreased Dob/ICI (~ 22%, P<0.05) and Cl31 (~ 40%, P<0.01) treatment-induced β3- AR upregulation. Wort and SP had no effect on b 3 response in treated cardiomyocytes. ERK1/2 and p38 were activated by Dob/ICI (% of untreated control: 117±6, P<0.05; 163±17, P<0.01) and Cl31 (149±17%, P<0.001; 164±23%, P<0.05) treatments. SAPK/JNK was activated in Dob/ICI treated cardiomyocytes (341±70%, P<0.01) whereas Cl31 treatment induced Akt phosphorylation (249±54%, P<0.01).

In conclusion these data indicate that the compensatory interaction between β1 - and β3 -ARs involved p38, PI3K and MEK/ERK1/2 pathway following chronic activation of β1 - or β3 -ARs. In addition, chronic β3 -AR stimulation activated PI3K/Akt pathway suggesting that β3 -ARs might play an important role in cardioprotection.

Gauthier et al (2000) Trends Pharmacol Sci, 21:426-431
Germack and Dickenson (2006), J Pharmacol Exp Ther, 316:392-402.

Work supported by the British Heart Foundation Grant FS/03/095/16317.