Beta2-adrenergic signaling affects the phenotype of human cardiac progenitor cells through EMT modulation.

Human cardiac progenitor cells (CPCs) offer great promises to cardiac cell therapy for heart failure. Many in vivo studies have shown their therapeutic benefits, paving the way for clinical translation. The 3D model of cardiospheres (CSs) represents a unique niche-like in vitro microenvironment, which includes CPCs and supporting cells. CSs have been shown to form through a process mediated by epithelial-to-mesenchymal transition (EMT). ß2-Adrenergic signaling significantly affects stem/progenitor cells activation and mobilization in multiple tissues, and crosstalk between ß2-adrenergic signaling and EMT processes has been reported. In the present study, we aimed at investigating the biological response of CSs to ß2-adrenergic stimuli, focusing on EMT modulation in the 3D culture system of CSs. We treated human CSs and CS-derived cells (CDCs) with the ß2-blocker butoxamine (BUT), using either untreated or ß2 agonist (clenbuterol) treated CDCs as control. BUT-treated CS-forming cells displayed increased migration capacity and a significant increase in their CS-forming ability, consistently associated with increased expression of EMT-related genes, such as Snai1. Moreover, long-term BUT-treated CDCs contained a lower percentage of CD90+ cells, and this feature has been previously correlated with higher cardiogenic and therapeutic potential of the CDCs population. In addition, long-term BUT-treated CDCs had an increased ratio of collagen-III/collagen-I gene expression levels, and showed decreased release of inflammatory cytokines, overall supporting a less fibrosis-prone phenotype. In conclusion, ß2 adrenergic receptor block positively affected the stemness vs commitment balance within CSs through the modulation of type1-EMT (so called "developmental"). These results further highlight type-1 EMT to be a key process affecting the features of resident cardiac progenitor cells, and mediating their response to the microenvironment.

Calpastatin overexpression in the skeletal muscle of mice prevents clenbuterol-induced muscle hypertrophy and phenotypic shift.

Accumulating evidence suggests that the calpain/calpastatin system is involved in skeletal muscle remodelling induced by ß(2) -adrenoceptor agonist treatment. In addition to other pathways, the Akt/mammalian target of rapamycin (mTOR) pathway, controlling protein synthesis, and the calcium/calmodulin-dependent protein kinase 2 (CamK2) and AMP-activated protein kinase (AMPK) pathways, recently identified as calpain substrates, could be relevant in ß(2) -adrenoceptor agonist-induced skeletal muscle remodelling. In the present study we investigated muscle hypertrophy and phenotypic shifts, as well as the molecular response of components of the Akt/mTOR pathway (i.e. Akt, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), ribosomal protein S6 (rpS6), CamK2 and AMPK), in response to calpastatin overexpression in the skeletal muscle of mice treated with 1 mg/kg per day clenbuterol for 21 days. Using gene electrotransfer of a calpastatin expression vector into the tibialis anterior of adult mice, we found that calpastatin overexpression attenuates muscle hypertrophy and phenotypic shifts induced by clenbuterol treatment. At the molecular level, calpastatin overexpression markedly decreased calpain activity, but was ineffective in altering the phosphorylation of Akt, 4E-BP1 and rpS6. In contrast, calpastatin overexpression increased the protein expression of both total AMPK and total CamK2. In conclusion, the results support the contention that the calpain/calpastatin system plays a crucial role in skeletal muscle hypertrophy and phenotypic shifts under chronic clenbuterol treatment, with AMPK and CamK2 probably playing a minor role. Moreover, the calpastatin-induced inhibition of hypertrophy under clenbuterol treatment was not related to a decreased mTOR-dependent initiation of protein translation.

Endotoxin-induced liver damage in rats is minimized by beta 2-adrenoceptor stimulation.

OBJECTIVE AND DESIGN: To investigate the effects of beta(2)-adrenoceptor (beta(2)-AR) stimulation on endotoxin-induced liver damage and systemic cytokine levels in rats. SUBJECTS: Standard male Wistar rats. TREATMENT: A disease-model of lipopolysaccharide (LPS)-induced acute systemic inflammation was used. The beta(2)-selective AR agonist clenbuterol was administered before, during, and after LPS-challenge to investigate its effects on the acute inflammatory response and associated liver-failure. METHODS: The following parameters have been measured in plasma: TNF alpha, IL-1 beta, IL-6, IL-10, AST, ALT, and Bilirubin. Liver histological examination was performed to look for changes in tissue morphology. RESULTS: Administration of clenbuterol (p.o.) one hour before, or intravenous at the same time as LPS-challenge resulted in a marked reduction of plasma levels of TNF alpha, IL-1 beta, and IL-6. A change both in plasma-level and in time-concentration profile of the anti-inflammatory cytokine IL-10 was found. Clenbuterol minimized LPS-induced liver damage, as represented by significantly lowered concentrations of several parameters for liver-failure (AST, ALT, Bilirubin), and improved hepatic tissue morphology. Clenbuterol administration after LPS challenge failed to inhibit TNF alpha-release but reduced liver-damage. Simultaneous use of the beta(2)-AR antagonist propranolol augmented LPS-induced liver failure, suggesting a role of endogenous adrenoceptor-agonists in prevention of organ-failure during systemic inflammation. CONCLUSIONS: The results indicate that a selective beta(2)-AR agonist might be used as an additional therapeutic agent in the clinic for the treatment of (acute) systemic inflammatory disorders in order to reduce or prevent subsequent liver failure.

Regulation of equine lymphocyte beta-adrenoceptors under the influence of clenbuterol and dexamethasone.

In 12 healthy horses, the effects of the beta2-agonist clenbuterol and the glucocorticoid dexamethasone on the lymphocyte beta2-adrenoceptor density and affinity (determined by (-)-[125I]-iodocyanopindolol binding) as well as its responsiveness (assessed by lymphocyte cyclic AMP [cAMP] responses to 10 micromol/l (-)-isoprenaline) were studied. Clenbuterol treatment, 2 x 0.8 microg/kg/day i.v. for 12 days, decreased significantly ICYP binding sites by approximately 30-40%; concomitantly, lymphocyte cAMP response to (-)-isoprenaline was reduced. After withdrawal of clenbuterol, beta2-adrenoceptor density and responsiveness gradually increased, reaching predrug levels after 4 days. The effects of dexamethasone on clenbuterol-induced desensitisation were further investigated. Administration of dexamethasone (1 x 0.1 mg/kg/day, i.v. for 5 days) immediately after clenbuterol withdrawal accelerated beta2-adrenoceptor recovery: only 24 h after administration dexamethasone restored the number of binding sites and cAMP response to (-)-isoprenaline to levels statistically indistinguishable from values before clenbuterol treatment. Three days after dexamethasone administration, lymphocyte beta2-adrenoceptors were further increased about 2-fold the pretreatment values, and this increase declined gradually after dexamethasone withdrawal, reaching baseline values after 4 days. Furthermore, in groups exposed simultaneously to both drugs, dexamethasone completely prevented clenbuterol-induced decrease in lymphocyte beta2-adrenergic receptor density and responsiveness. No significant change was observed in the dissociation constant for ICYP in any of the situations. We conclude that dexamethasone (glucocorticoids) can reverse and prevent Clenbuterol-induced desensitisation (down-regulation) of the lymphocyte beta2-adrenoceptors and therefore, a combined therapy with clenbuterol and dexamethasone may be potentially beneficial in horses suffering from chronic obstructive pulmonary disease (COPD).

[Detection of circulating autoantibodies to beta 2-adrenergic receptors in patients with asthma].

To understand the roles of autoantibodies to beta 2 adrenergic receptors in the pathogenesis of asthma, we investigated the positive chronotropic action of beta 2-selective adrenergic agonist, clenbuterol, on cultured neonatal rat cardiomyocytes. Thereafter, we detected the autoantibodies to beta 2-adrenergic receptors in the sera from patients with asthma as it could inhibit the positive chronotropic action of clenbuterol. In the sera of all patients with asthma (16 cases) there were autoantibodies to beta 2-adrenergic receptors, but not in the normal controls (20 cases). Further study showed that the inhibitory autoantibodies were IgG type. This experiment suggests that the autoantibodies to beta 2-adrenergic receptors may play an important role in the pathogenesis of asthma.

Effects of digoxin on the anabolic response to clenbuterol.

The possible involvement of increased cation exchange in the anabolic response to the beta 2-selective adrenergic agonist clenbuterol was investigated using dietary admixtures of clenbuterol and the Na,K-adenosine triphosphatase (ATPase) inhibitor digoxin. In a rat feeding trial to assess the effects on body composition, it was found that the higher of two levels (5 and 30 mg/kg diet) of digoxin had an inhibitory effect on the repartitioning effects (ie, increased body weight and fat-free mass) of clenbuterol (2 mg/kg diet). In two further experiments using 30 and 60 mg digoxin/kg diet, it was found that the anabolic effects of clenbuterol on gastrocnemius muscle protein deposition were inhibited by digoxin, but the effects of clenbuterol on soleus muscle protein were more resistant to inhibition. Given the observed dose-dependent inhibition by digoxin of gastrocnemius muscle protein deposition in the three experiments, it was concluded that at least part of clenbuterol's anabolic actions on skeletal muscle may depend on increased Na,K-ATPase activity. However, different mechanisms or a different time course of Na,K-ATPase activation may occur in different muscle fiber types.

Effects of clenbuterol and prenalterol on behavior maintained under a multiple fixed-interval, fixed-ratio schedule.

The results of a number of studies have implicated beta adrenergic receptors in the brain in the actions of proven antidepressant drugs. This suspected involvement of central beta adrenergic receptors made it of interest to characterize the behavioral effects of centrally acting beta adrenergic agonists. Clenbuterol and prenalterol, unlike most beta adrenergic agonists, penetrate into the central nervous system after peripheral administration. In the present study, the effects of these agonists on behavior maintained under a multiple fixed-interval 5-min, fixed-ratio 30-response schedule were determined. Both compounds, in a dose-dependent manner, reduced response rate under both components of the multiple schedule. Under the fixed-interval component, clenbuterol and prenalterol altered the temporal pattern of responding. At no dose tested was there evidence for any stimulant action of either of the drugs. The effects of clenbuterol and prenalterol on behavior maintained under the multiple schedule appeared to be a result of an interaction of the agonists with beta adrenergic receptors. This was evidenced by the ability of the beta adrenergic antagonist propranolol to block the effects of the agonists. The behavioral effects of clenbuterol and prenalterol appear, in general, to be similar to effects reported previously for tricyclic and monoamine oxidase inhibiting antidepressant drugs.

Central beta-adrenoceptors can modulate 5-hydroxytryptamine-induced tremor in rats.

The effects of two beta 2-adrenoceptor agonists with different lipophilicities were studied on tremor induced by L-5-hydroxytryptophan (L-5-HTP) in pargyline- and carbidopa-pretreated rats. Tremor was recorded and analysed by an objective method based on accelerometry. Clenbuterol, a lipophilic beta 2-selective agonist, dose-dependently enhanced tremor intensity, whereas the hydrophilic beta 2-agonist terbutaline had no effect. The clenbuterol-induced enhancement of tremor was completely abolished by the beta 2-selective antagonist ICI 118,551 but unchanged by the beta 1-selective antagonist metoprolol. The results suggest that centrally located beta 2-adrenoceptors can mediate a modulation of 5-hydroxytryptamine-induced tremor in rats.