Effects Of β₂ Adrenergic Receptor Agonist Clenbuterol On Neonatal Rat Cardiomyocytes

It is generally accepted that under physiological conditions catecholamines exert positive chronontropic, inotropic and lustropic response through β₁-adrenergic receptor (AR) selectively coupling to G_s protein, activating adenylyl cyclase and enhanceing cAMP production, leading to protein kinase A(PKA) activation, which phosphorylates a multitude of regulatory proteins.Studies during the past decade have demonstrated that coexisting cardiac β-AR, mainlyβ₁ AR and β₂ AR, activating different signaling pathway and fulfill strikingly distinct physiological and pathological roles in the heart. The role of β₂-AR in neonatal rat cardiac myocytes differs from adult rat cardiac myocyte due to the difference of age. To demonstrate the effect of β₂-AR signaling pathway in neonatal rat cardiomyocytes, this thesis investigated: (l)Effect ofβ₂-AR agonist clenbuterol on phospholamban phosphorylation in neonatal rat cardiomyocytes; (2) Effect of P2-AR agonist clenbuterol on MAPK signaling pathway in neonatal rat cardiomyocytes; (3) Effect of β₂-AR agonist clenbuterol on nitric oxide synthesis in neonatal rat cardiomyocytes. Section 1Effect of clenbuterol on phosphorylation of phospholamban in neonatal rat cardiomyocytesBack ground: Catecholamines hasten cardiac relaxation through βadrenergic receptors by phosphorylation of phospholamban and troponin I. β₂ AR stimulation in adult rat cardiomyocytes augments L-type Ca²⁺ current in a cAMP-dependent protein kinase (PKA)dependent manner but fails to the phosphorylation of phospholamban. We assessed the role of β₂ AR in phosphorylation of phospholamban in neonatal rat cardiomyocytes differed from adult rat cardiomyocytes.Methods: Increase in beat number and intracellular cAMP was measured in cardiomyocytes exposed to clenbuterol at diflferent time incubation. Phosphorylation of phospholamban at serine 16 and threonine 17 were determined by Western blot using phosphospecific antibodies.Results: Clenbuterol increased cardiomyocyte beat and intracellular cAMP accumulation in a time-dependent manner. Phosphorylation of phospholamban at serine 16 was increased by clenbuterol in a time-dose-dependent manner, and which was blocked by selective β₂ AR antagonist ICI 118551 and PKA inhibitor Rp-cAMP. Inhibition of G_i with pertussis toxin (PTX) slightly enhanced the phosphorylation of PLB at serine 16 induced by clenbuterol. Clenbuterol increased phosphorylation of Thr17 of phospholamban in a time-dependent manner. Conclusions: β₂ AR activation promotes phosphorylation of phospholamban at serine 16 and threonine 17, thereby potentially improving diastolic function in neonatal rat cardiomyocytes. Section 2Effect of clenbuterol on MAPKs signaling pathway in neonatal rat cardiomyocytes§1. Effect of clenbuterol on ERK1/2 signaling pathway in neonatal rat cardiomyocytesBackground: Several G protein-coupled receptors activate extracellular signal-regulated kinase 1/2 (ERK1/2) through G_i protein pathway. Accumulation of cAMP can either inhibit or stimulate ERKl/2 phosphorylation. Calcium mobilization is also involved in the phosphorylation and dephosphorylation of ERK1/2. β₂ adrenoceptor (AR) activate ERK1/2 in a G_i-dependent manner in cultured adult rat cardiomyocytes. However, β₂ AR agonist modulates the calcium mobilization and cAMP accumulation in neonatal rat cardiomyocytes but not adult rat cardiomyocytes. Therefore, we assumed that β₂ AR agonist mediated phosphorylation of ERKl/2 in neonatal rat cardiomyocytes differed from that observed in adult rat cardiomyocytes. To test this hypothesis, we investigated the effect of β₂ AR agonist clenbuterol on ERKl/2 phosphorylation in neonatal rat cardiomyocytes and explored underlying mechanisms. Methods: Cultured neonatal rat cardiomyocytes were pretreated with or without various agents of the β₂ AR signaling pathways. The phosphorylation of ERKl/2 was determined by Western blot using phospho-specific antibodies.Results: The addition of clenbuterol to the cells evoked a biphasic effect comprising an initial positive effect peaking at 2min, followed by a sustained negative effect leading to 40% decreases in basal phosphorylation of ERKl/2 after 30 min. Both the positive effect and negative effect are in a dose-dependent manner. The increase in phosphorylation was not altered in the presence or absence of extracellular calcium but was enhanced by ryanodine receptor agonist ryanodine. However, selective β₂ AR antagonist ICI 118551, pertussis toxin (PTX), calcium chelator BAPTA-AM and ryanodine receptor antagonist ruthenium red significantly inhibited the positive effect of clenbuterol, but nifedipine only slightly inhibited it. Whereas, protein phosphatases inhibitor okadaic acid or sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor thapsigargin pretreatment reversed the negative effect of clenbuterol. Rp-cAMP attenuated the effect of clenbuterol on ERK1/2 dephosphorylation. Both the positive and negative effects on ERK1/2 phosphorylation of clenbuterol were reproduced by caffeine. But only thapsigargin reversed the negative effect of caffeine, okadaic acid was failed to abrogate it. Caffeine also mimicked the persistent effect of clenbuterol on phospholamban phosphorylation. Conclusions: The present data provide evidence that clenbuterol has a time- and concentration-dependent biphasic effect on ERK phosphorylation. Intracellular calcium mobilization plays an important role in clenbuterol induced biphasic effect on ERK activation. These results indicate that phosphorylation of ERK induced by clenbuterol is via G_i signaling pathway and is involved with the release of calcium from sarcoplasmic reticulum (SR) Ca²⁺ store, whereas, clenbuterol negatively regulates ERK1/2 through restore calcium into SR via cAMP dependent PLB phosphorylation. §2. Effect of clenbuterol on p38 MAPK signaling pathway in neonatal rat cardiomyocytesBackground: Previous studies suggest an involvement of p38 MAPK (mitogen activated protein kinase, MAPK) in cardiac apoptosis. More recent studies proposed that p38 MAPK is activated by β-AR stimulation via a G_i dependent mechanism, protecting myocytes against P-AR/G_s-mediated apoptosis in cultured adult rat cardiomyocytes. Our previous study demonstrated the biphasic effect of clenbuterol on ERK1/2 phosphorylation via both G_s and G_i coupling in neonatal rat cardiomyocytes, but clenbuterol phosophorylated ERK1/2 via G_i coupling in adult rat cardiomyocytes. Therefore, we assumed that clenbuterol-midiated phosphorylation of p38 MAPK in neonatal rat cardiomyocytes differed from that observed in adult rat cardiomyocytes.Methods: Cultured neonatal rat cardiomyocytes were pretreated with or without various agents. The phosphorylation of p38 MAPK was determined by Western blot using phospho-specific antibodies.Results: Clenbuterol induced p38 MAPK phosphorylation in a time-dose-dependent manner in neonatal rat cardiomyocytes. The phosphorylation of p38 MAPK induced by clenbuterol was inhibited by selectiveβ₂ AR antagonist ICI 118551 and protein kinase A inhibitor Rp-cAMP, and which was enhanced by PTX. Calcium channel blocker nifedipine and calcium chelator BAPTA-AM attenuated the effect of p38 MAPK phosphorylation induced by clenbuterol.Conclusions: These data demonstrated that in neonatal rat cardiomyocytes, β₂ AR activates p38 MAPK via G_s/cAMP-dependent signaling pathway, and which correlates with calcium concentration increased by extracellular calcium entry. Section 3Effect of clenbuterol on nitric oxide synthesis in neonatal rat cardiomyocytesBackgound: Nitric oxide (NO) is currently considered an important intracellular messenger in cardiovascular, immune and neural systems. This messenger causes diverse biological actions including vasodilation and cytotoxic or cytoprotective effect in various cells. The inducible NO synthase (iNOS) is expressed in target tissuses (vascular smooth muscle cells and cardiac myocytes) after stimulation with endotoxin and cytokines, and synthesis of relatively much greater amounts of NO by this way. Previous studies have shown that iNOS gene expression occurs frequently in failing human cardiac myocytes of patients with dilated cardiomyopathy, ischemic heart disease and vascular heart disease.In previous studies, cAMP augments cytokine stimulated nitric oxide synthesis in rat cardiac myocytes and dietary clenbuterol have a persisitnet effect on nitric oxide synthesis in rat cultures smooth muscle cells. Our previous study demonstrates that clenbuterol increased cAMP accumulation in neonatal rat cardiomyocyes. The purpose of this study was to investigate the effect of clenbuterol on nitric oxide synthesis in neonatal rat cardiomyocytes.Methods: Using the Griess reagent, we measured the production of nitrite, a stable metabolite of NO, by cultured neonatal rat cardiomyocytes. The expression of inducible NO synthase (iNOS) protein was assayed by Western blot. Results: Incubation of cardiomyocytes for 24 hours with interleukin 1β(IL-1β) caused a significant increase in NO production. Clenbuterol significantly augmented NO synthesis in IL-1β-stimulated but not in unstimulated cells in a dose-dependent manner. The selective β₂ AR antagonist ICI118551 inhibited the effect of clenbuterol. The clenbuterol-induced NO production by IL-1βstimulated cells was accompanied by increase iNOS protein accumulation. The non-selectiveβ-AR antagonist propranolol and the selective β₂ AR antagonist ICI118551 blocked the effect of clenbuterol.Conclusions: These result indicate that clenbuterol upregulates IL-1β-induced iNOS expression and nitric oxide synthesis in neonatal rat cardiomyocytes, which is mediated throughβ₂ AR.