Adrenoceptor Agonist Clenbuterol Research Articles

Perfusion imaging of spinal cord contusion: injury-induced blockade and partial reversal by β2-agonist treatment in rats

Traumatic injury to the spinal cord results in considerable delayed tissue loss. The authors investigated the extent to which ischemia occurs following contusion-induced spinal cord injury and whether ischemia exacerbates tissue damage that leads to the loss of locomotor function. They also determined if ischemia is reversed with β2-adrenoceptor agonist treatment, which has been established to be neuroprotective following contusion injury. The extent and role of circulation loss in spinal cord injury was determined in an established experimental model of contusion injury. The spinal cord dura mater of Wistar rats was exposed by performing a laminectomy at T-8 to T-11. Laser Doppler perfusion imaging was then used to measure microcirculation in the exposed spinal cord. After imaging, a moderately severe contusion injury was produced using a weight-drop device unto the exposed dura at T-10. Perfusion imaging was again performed, scans were quantitated, and integrated intensities were compared. Postinjury imaging revealed an 18%-27% reduction in perfusion in regions rostral and caudal to the injury site, and a 68% reduction was observed at the contusion epicenter. These perfusion losses persisted for at least 48 hours. At 24 hours after injury, some rats were intraperitoneally injected with 2 mg/kg of the β2-adrenoceptor agonist clenbuterol, which has been shown to promote the partial recovery of locomotor function and spare spinal cord tissue when administered within 2 days after contusion injury. Clenbuterol injection caused a gradual increase in perfusion, which was detectable at 30 minutes postinjection and continued over time, resulting in an 127% overall increase in perfusion at the epicenter 24 hours after treatment. These results suggest that the occurrence of chronic perfusion loss after contusion contributes to delayed damage and tissue loss. In contrast, β2-adrenoceptor agonist treatment may exert neuroprotection by restoring perfusion, thereby preventing ischemic neurodegeneration. The ability of laser Doppler imaging to measure the loss of perfusion and its restoration upon treatment suggests that it may have clinical utility in the assessment and treatment of spinal cord injury.

The β 2-adrenoceptor agonist clenbuterol elicits neuroprotective, anti-inflammatory and neurotrophic actions in the kainic acid model of excitotoxicity

Excitotoxicity is a mechanism of neuronal cell death implicated in a range of neurodegenerative conditions. Systemic administration of the excitotoxin kainic acid (KA) induces inflammation and apoptosis in the hippocampus, resulting in neuronal loss. Evidence indicates that stimulation of glial β 2-adrenoceptors has anti-inflammatory and neurotrophic properties that could result in neuroprotection. Consequently, in this study we examined the effect of the β 2-adrenoceptor agonist clenbuterol on KA-induced inflammation, neurotrophic factor expression and apoptosis in the hippocampus. Clenbuterol (0.5 mg/kg) was administered to rats one hour prior to KA (10 mg/kg). Epileptic behaviour induced by KA was assessed for three hours following administration using the Racine scale. Twenty-four hours later TUNEL staining in the CA3 hippocampal subfield and hippocampal caspase-3 activity was assessed to measure KA-induced apoptosis. In addition, expression of inflammatory cytokines (IL-1β and IFN-γ), inducible nitric oxide synthase (iNOS), kynurenine pathway enzymes indolamine 2,3-dioxygenase (IDO) and kynurenine monooxygenase (KMO), the microglial activation marker CD11b, and the neurotrophins BDNF and NGF were quantified in the hippocampus using real-time PCR. Whilst clenbuterol treatment did not significantly alter KA-induced epileptic behavior it ameliorated KA-induced apoptosis, and this neuroprotective effect was accompanied by reduced inflammatory cytokine expression, reduced expression of iNOS, IDO, KMO and CD11b, coupled with increased BDNF and NGF expression in KA-treated rats. In conclusion, the β 2-adrenoceptor agonist clenbuterol has anti-inflammatory and neurotrophic actions and elicits a neuroprotective effect in the KA model of neurodegeneration.

β2‐adrenoceptor agonist clenbuterol reduces infarct size and myocardial apoptosis after myocardial ischaemia/reperfusion in anaesthetized rats

Considerable evidence indicates that the beta(2)-adrenoceptor agonist clenbuterol decreases apoptosis in a rodent model of ischaemic cardiomyopathy. In this study, we investigated the effects of clenbuterol on infarct size caused by myocardial ischaemia/reperfusion (I/R) in anaesthetized rats. Rats were randomly assigned to the following groups: (i) sham (ii) I/R (iii) clenbuterol + I/R (iv) ICI 118551 + clenbuterol + I/R (v) metoprolol + clenbuterol + I/R (vi) metoprolol + I/R (vii) pertussis toxin + clenbuterol + I/R. Under anaesthesia, left anterior descending coronary artery was occluded for 30 min followed by reperfusion for 2 h. Compared with the control I/R group,the clenbuterol (0.5 mg.kg(-1), i.p.) group had reduced infarct size, improved diastolic function and sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) activity, increased superoxide dismutase activity, and decreased malondialdehyde (MDA) level and LDH, CK release. Clenbuterol increased the phosphorylation of ERK1/2, which resulted in inhibition of myocardial apoptosis as indicated by the reduction of terminal deoxynucleotidyltransferase end labelling-positive staining, Bax/Bcl-2 mRNA and caspase-3 protein expression. The G(i)-protein inhibitor pertussis toxin blocked the clenbuterol-induced improvement in cardiac function and infarct size. Pretreatment with ICI 118551(a selective beta(2)-adrenoceptor antagonist) inhibited the effects of clenbuterol mentioned above. The beta(1)-adrenoceptor agonist metoprolol had similar effects to clenbuterol but failed to reduce MDA and improve SERCA activity. When administered together, metoprolol and clenbuterol did not induce synergistic effects. Clenbuterol pretreatment provides significant cardioprotection against ischaemia/reperfusion injury and this is mediated by the beta(2)-adrenoceptor-G(i)-protein signalling. A combination of the beta(2)-adrenoceptor agonist clenbuterol and the beta(1)-antagonist metoprolol did not lead to a synergistic anti-apoptotic effect.

Enantio-selective effects of clenbuterol in cultured neurons and astrocytes, and in a mouse model of cerebral ischemia

Neuroprotective effects of the lipophilic β 2-adrenoceptor agonist clenbuterol have been established in neuronal cultures and in various rodent models of stroke. In previous studies, however, clenbuterol was always applied as a racemate, while it has not been established whether the enantiomers differ in their neuroprotective activities. Here, we demonstrate that R,S-clenbuterol and S(+)-clenbuterol, but not the R(−)-enantiomer protect cultured neurons against glutamate-mediated excitotoxicity and staurosporine-induced apoptosis. Similar to previous findings with clenbuterol racemate, the neuroprotective effect of S(+) -clenbuterol correlated well with morphological changes of astrocytes which transformed into dense stellate cells with dendritic processes indicating β 2-adrenoceptor-mediated activation. Most importantly, the S(+)-enantiomer but not R(−)-clenbuterol reduced ischemic brain damage similar to the effect of the racemate. The selective β 2-adrenoceptor antagonist butoxamine blocked this neuroprotective effect of S(+)-clenbuterol. In addition, S(+)-clenbuterol significantly reduced blood pressure, enhanced blood glucose levels and increased glucocorticoid levels compared to vehicle-or R(−)-clenbuterol-treated controls. These results clearly demonstrate that S(+)-clenbuterol is the eutomer that mediates neuroprotective effects of the β 2-adrenoceptor agonist but also according changes of physiological parameters.

β2-Adrenergic receptor responsiveness of the calpain–calpastatin system and attenuation of neuronal death in rat hippocampus after transient global ischemia

In the CNS, where Ca 2+ overload has been established as a mechanism contributing to neuronal damage associated with excitotoxicity, stroke and ischemia, there is interest in understanding the role of calpain inhibition in rescuing neurons from death. In these settings, the activation of large stores of latent calpain may rapidly lead to the demise of the neuron within hours. The activity of calpain is strictly regulated by calcium concentrations and interactions with calpastatin (endogenous calpain inhibitor). The interaction between calpains and calpastatin is calcium dependent, and little is known about the regulation of the neuronal calpain–calpastatin system in vivo. It has been postulated that calpastatin can be modulated by nerve growth factors (NGFs). We have demonstrated in vitro as well as in vivo a neuroprotective effect of the β 2-adrenoceptor agonist clenbuterol (CLN) mediated through an increased NGF expression. In this study we attempt to find out whether CLN is capable (1) of modulating proteolysis regulated by the calpain–calpastatin system and (2) of attenuating DNA-fragmentation induced by cerebral ischemia. Rats received CLN daily for 1 week, were then subjected to ischemia and finally perfused at different times post-ischemia. The proteolytic activity of calpain was measured by the immunolocalisation of calpastatin and spectrin-breakdown products (SBP). The time course of apoptosis was assessed by terminal dUTP nick end-labeling (TUNEL)-staining. CLN reduced CA1-hippocampal cell damage by 23%, attenuated DNA-laddering and decreased proteolysis of spectrin by enhancing calpastatin activity. These results provide evidence that CLN is a potent neuroprotective substance, which through the enhancement of calpastatin synthesis attenuates the apoptotic machinery and modulates proteolysis.

Stimulation of β-adrenoceptors activates astrocytes and provides neuroprotection

Our previous studies established that induction of growth factor synthesis and neuroprotection by the β 2-adrenoceptor agonist clenbuterol in vitro and in vivo was associated with the activation of astrocytes, the major source of trophic factors in the brain. In the present study, we further investigated the specificity of β 2-adrenoceptor-mediated effects on astrocyte activation and neuroprotection. In mixed hippocampal cultures neuroprotection against glutamate-induced cell death by clenbuterol (1 μM) was blocked by the β 1/2-adrenoceptor antagonist propranolol and the specific β 2-adrenoceptor antagonists 1-[2,3-(Dihydro-7-methyl-1 H-inden-4-yl)-oxy]-3-[(1-methylethyl)-amino]-2-butanol (ICI 118,551, 10 μM) and butoxamine (10 μM), while the β 1-adrenoceptor-selective antagonist metoprolol (10 μM) showed no effect. The β 2-adrenoceptor agonists clenbuterol (1–100 μM) and salmeterol (0.01–1 μM) induced profound morphological changes of cultured astrocytes which transformed into activated astroglia with pronounced dendrite-like processes. This phenomenon was blocked by butoxamine (1 mM) and propranolol (10 μM), but not by metoprolol (10 μM). However, similar morphological changes in astrocytes were also observed after stimulation of β 1-adrenoceptors by dobutamine (1–10 μM) and norepinephrine (1–10 μM). This effect was blocked by propranolol (10 μM) and metoprolol (10 μM) but not by butoxamine (1 mM), suggesting that stimulation of either β 1- or β 2-adrenoceptors was sufficient to induce activation of astrocytes. In addition, β 1-adrenoceptor stimulation by dobutamine (1–10 μM) protected hippocampal neurons against glutamate toxicity. In a model of focal cerebral ischemia in mice the cerebroprotective effect of clenbuterol (0.3 mg/kg) was blocked by propranolol (5 mg/kg) and butoxamine (5 mg/kg). Interestingly, the infarct size was reduced after co-treatment with clenbuterol (0.3 mg/kg) and metoprolol (5 mg/kg) as compared to clenbuterol treatment (0.3 mg/kg) alone. In conclusion, activation of astrocytes and neuroprotection can be achieved by stimulation of either β 1- or β 2-adrenoceptors in vitro, whereas in vivo neuroprotection is preferentially mediated through β 2-adrenoceptors.

Elevated IGF-II mRNA and phosphorylation of 4E-BP1 and p70(S6k) in muscle showing clenbuterol-induced anabolism.

Muscle wasting affects large numbers of people, but few therapeutic approaches exist to treat and/or reverse this condition. The beta(2)-adrenoceptor agonist clenbuterol produces a muscle-specific protein anabolism in both normal and catabolic muscle and has been used to limit muscle wasting in humans. Because clenbuterol appears to interact with or mimic innervation, its effect on the expression of the neurotrophic agents insulin-like growth factor (IGF)-II and H19 and their putative pathways was examined in normal rat plantaris muscle. The results showed that the well-documented early effects of clenbuterol on protein metabolism were preceded by elevated levels of IGF-II and H19 transcripts together with increased phosphorylation of eukaryotic initiation factor (eIF)4E binding protein-1 (4E-BP1) and p70(S6k). By 3 days, transcript levels for IGF-II and H19 and 4E-BP1 and p70(S6k) phosphorylation had returned to control values. These novel findings indicate that clenbuterol-induced muscle anabolism is potentially mediated, at least in part, by an IGF-II-induced activation of 4E-BP1 and p70(S6k).

Skeletal muscle myosin heavy chain isoforms and energy metabolism after clenbuterol treatment in the rat.

Prolonged treatment with the beta(2)-adrenoceptor agonist clenbuterol (1-2 mg. kg body mass(-1). day (-1)) is known to induce the hypertrophy of fast-contracting fibers and the conversion of slow- to fast-contracting fibers. We investigated the effects of administering a lower dose of clenbuterol (250 microgram. kg body mass(-1). day (-1)) on skeletal muscle myosin heavy chain (MyHC) protein isoform content and adenine nucleotide (ATP, ADP, and AMP) concentrations. Male Wistar rats were administered clenbuterol (n = 8) or saline (n = 6) subcutaneously for 8 wk, after which the extensor digitorum longus (EDL) and soleus muscles were removed. We demonstrated an increase of type IIa MyHC protein content in the soleus from approximately 0.5% in controls to approximately 18% after clenbuterol treatment (P < 0.05), which was accompanied by an increase in the total adenine nucleotide pool (TAN; approximately 19%, P < 0.05) and energy charge [E-C = (ATP + 0.5 ADP)/(ATP + ADP + AMP); approximately 4%; P < 0.05]. In the EDL, a reduction in the content of the less prevalent type I MyHC protein from approximately 3% in controls to 0% after clenbuterol treatment (P < 0.05) occurred without any alterations in TAN and E-C. These findings demonstrate that the phenotypic changes previously observed in slow muscle after clenbuterol administration at 1-2 mg. kg body mass(-1). day(-1) are also observed at a substantially lower dose and are paralleled by concomitant changes in cellular energy metabolism.

K ATP channels mediate the β 2-adrenoceptor agonist-induced relaxation of rat detrusor muscle

We propose that ATP-sensitive K + (K ATP) channels are normally inactive but involved in β 2-adrenoceptor stimulated relaxation of the rat bladder. Spontaneous detrusor muscle contractions were unaffected by glibenclamide (K ATP channel blocker) but were reduced when pinacidil (K ATP channel opener) concentrations exceeded 10 −5 M. Inhibition by β 2-adrenoceptor agonist clenbuterol (10 −6 M) of 1 Hz electrical field stimulated contractions was abolished by glibenclamide (10 −6 M). Glibenclamide (10 −6 M) decreased forskolin-induced relaxation (10 −9–10 −4 M) in bladder muscle stimulated with 1 Hz electrical field. In the presence glibenclamide (10 −6 M) or myristoylated protein kinase A inhibitor (2×10 −6 M), clenbuterol (10 −9–10 −5 M) failed to inhibit bladder contraction in response to 1 Hz electrical field stimulation. Therefore, K ATP channel opening and the subsequent hyperpolarization of cell membranes in response to β 2-adrenoceptor activation is mediated by raised cyclic-AMP levels and activation of protein kinase A. This counteracts ATP-stimulated depolarization in bladder muscle, thereby reducing cell contraction.

Inhibition of the contractile response of the rat detrusor muscle by the β 2-adrenoceptor agonist clenbuterol

The action of clenbuterol, β 2-adrenoceptor agonist, on the contractile response of isolated rat detrusor muscle strips was investigated in vitro. Clenbuterol (10 −5 M) inhibited the detrusor muscle frequency response (1–40 Hz, p<0.02) with a more pronounced effect at 1 Hz than 40 Hz. Clenbuterol (10 −6 M) significantly inhibited the contractile response to exogenous ATP (10 −4 to 10 −2 M, p<0.05) but not to carbachol (10 −9 to 10 −4 M). The presence of 10 −5 M ICI 118, 551, β 2-adrenoceptor antagonist, shifted significantly the clenbuterol dose–response to 1 Hz electrical field stimulation (EC 50 3.4×10 −6 M (±2.2×10 −6 M) for clenbuterol alone, to 4.1×10 −4 M (±8.8 ×10 −5 M), P<0.05). In conclusion, clenbuterol inhibits electrical field and ATP-stimulated contractions of detrusor muscle. Reversal of the clenbuterol inhibition of detrusor muscle contraction by ICI 118, 551 shows that clenbuterol is probably acting through postsynaptic β 2-adrenoceptors, which modulate the response to ATP released from purinergic nerves.

NGF mediates the neuroprotective effect of the β2-adrenoceptor agonist clenbuterol in vitro and in vivo: evidence from an NGF-antisense study

Previous studies in our laboratory suggested that neuroprotective effects of the β 2-adrenoceptor agonist clenbuterol in vitro and in vivo occurred due to enhanced synthesis of nerve growth factor. The aim of the present study was to evaluate the effects of a phosphothioated NGF oligodeoxynucleotide on neuroprotection by clenbuterol in vitro and in vivo. After clenbuterol treatment (1–100 μM) an increase in nerve growth factor mRNA and protein levels (200–300% of control) was observed in primary cultures of rat cortical astrocytes. Nerve growth factor antisense oligonucleotide (0.3–1 μM for 3 days) reduced the content of nerve growth factor protein in the medium of the astrocytes concentration-dependently to 20% of control level. Nerve growth factor content in the medium of mixed hippocampal cells was reduced to 55% of sister cultures receiving the vehicle or a random control oligonucleotide. In mixed hippocampal cultures pretreated with random oligonucleotide (1 μM, 30 h), clenbuterol (10 μM) reduced the percentage of damaged neurons after glutamate exposure (0.5 mM, 1 h) to 17%. Pretreatment with nerve growth factor antisense oligonucleotide (1 μM) for 30 h before glutamate incubation blocked the protective effect of clenbuterol. In vivo, clenbuterol (0.01–0.1 mg/kg) reduced the infarct volume in a rat model of permanent focal cerebral ischemia dose-dependently. Nerve growth factor antisense oligonucleotides injected into the cortical tissue before ischemia abolished the cerebroprotective effect of clenbuterol. Our results indicate that the nerve growth factor antisense oligonucleotide presented in this study is a useful tool to investigate the effects of nerve growth factor knock down. By using the nerve growth factor antisense oligonucleotide we could demonstrate that nerve growth factor mediated the neuroprotective effects of the β 2-adrenoceptor agonist clenbuterol in vitro and in vivo.

Anabolic effects of clenbuterol after long-term treatment and withdrawal in the rat

Injection of rats with the β 2-adrenoceptor agonist clenbuterol (1 mg/kg/d for 15 days) stimulated an increase in body weight (9%) and protein (8%) and water (7%) content, but reduced food intake (4%) and epididymal fat pad mass (39%). Nine days after termination of treatment, ex-clenbuterol rats were heavier (5%) and had a greater protein (7%) and water (6%) content and lower fat pad mass (32%) than controls. Clenbuterol-fed rats (2 mg/kg diet for 10 days, providing an average of 0.04 mg clenbuterol/kg/d) increased body weight (7%), muscle mass (15% to 21%), and muscle protein content (9% to 26%), whereas epididymal fat pad weight and muscle glycogen content were reduced. During the withdrawal period, the greater body weight of ex-clenbuterol rats was sustained overall (ANOVA, P < .00005), but by day 10 this difference was no longer significant. At this point, gastrocnemius muscle mass was still higher (11%) when compared with that of control animals, but soleus muscle mass, muscle glycogen concentration, and epididymal fat pad weight had reverted to control values. These results were corroborated in a subsequent experiment using older rats. It was concluded that, unlike other β-adrenoceptor-mediated effects, muscle protein accumulated during clenbuterol treatment can be maintained in certain muscles after removal of the drug for a period of time that is at least equivalent to the duration of treatment. This could have implications for the potential therapeutic use of this class of compound, and differences in the response observed between muscle types may help to elucidate the mechanisms responsible for the muscle protein deposition induced by clenbuterol.

Increased association of ribosomes with myofibrils during the skeletal-muscle hypertrophy induced either by the β-adrenoceptor agonist clenbuterol or by tenotomy

Ribosome distribution in skeletal-muscle myofibres was investigated by immunohistochemistry and microdensitometry by using anti-(60 S ribosomal subunit) antibodies. Administration of the beta-adrenoceptor agonist clenbuterol caused an increase in the staining of the myofibrillar region with this antibody relative to that found in the subsarcolemmal cytoplasm. A similar effect was observed during hypertrophy of the plantaris muscle following severance of the tendon to the gastrocnemius. The results suggest that increased association of ribosomes with the myofibrils occurs during muscle hypertrophy.

Muscle wasting associated with endotoxemia in the rat: modification by the beta 2-adrenoceptor agonist clenbuterol.

A single injection of endotoxin (1 mg/kg, sc) in rats caused significant fever, body weight loss and reduction in gastrocnemius muscle mass, none of which was mimicked by pair-feeding. Infusion of endotoxin via osmotic minipump over five days caused transient fever and suppression of growth. Recovery of body weight was significantly enhanced by the administration of the beta 2-adrenoceptor agonist clenbuterol (added to the diet at 4 mg/kg). In a separate experiment, injections of endotoxin (day 0 and day 2) caused significant reductions in body weight gain (42%), mass (9%) and protein content (13%) of gastrocnemius muscle over 3 days. Addition of clenbuterol to the diet reversed all of these effects but did not alter food intake or the febrile response to endotoxin. Clenbuterol caused large (20%) increases in the ratio of RNA to protein in muscle indicating that it may have stimulated protein synthesis. beta 2-adrenoceptor agonists may therefore be of value in preventing or inhibiting muscle atrophy associated with infection or injury.

Effects of the β 2-adrenoceptor agonist clenbuterol on tyrosine and tryptophan in plasma and brain of the rat

The β 2-adrenoceptor agonist, clenbuterol (initially 5 mg/kg), was found to significantly reduce plasma tyrosine and raise brain tryptophan levels (P < 0.01). By comparison, decreases in plasma tryptophan and increases in brain tyrosine were small and often nonsignificant. Amino acid levels measured in different brain regions revealed that the elevations were similar among the cerebellum, striatum, and cortex. These effects were partially blocked by propanolol but not by atenolol. The ed 50 was estimated from dose-response curves to be about 0.05 mg/kg for both the decrease in plasma tyrosine and the increase in brain tryptophan. The effects of low doses of clenbuterol were prevented completely by propranolol. Peripheral organs displayed strikingly different patterns of change in amino acid concentrations. Only the spleen had any accumulation of tryptophan, but that was much less than in brain. In contrast, tyrosine and tryptophan were decreased in heart and unaltered in liver; tyrosine was decreased in lung. The elevation in brain tryptophan levels was attenuated by the β 2-antagonist, ICI 118,551, but not by the β 1-antagonist, betaxolol; but the reduction in plasma tyrosine was unaffected by either drug. The serotonin antagonist, methysergide, failed to block the effects of clenbuterol. We conclude that changes in amino acid concentrations produced by clenbuterol are mediated by β 2-adrenoceptor stimulation. Although the increases in brain tyrosine and tryptophan were similar to increases in the plasma ratios of these amino acids to the sum of the other large neutral amino acids competing for transport into the brain, the disparity between the effects of ICI 118,551 in brain and plasma suggests that clenbuterol may also have a direct action in brain to regulate levels of aromatic amino acids. Since clenbuterol has been purported to have an antidepressant effect and since other antidepressants also increase brain tryptophan, this may be a common feature of antidepressant drug action.

The β2-adrenoceptor agonists clenbuterol and salbutamol enhance the hypothermic action of 8-hydroxy-2-(di- n-propylamino)tetralin (8-oh-dpat) in mice by a central mechanism

The hypothermie response of mice to injection of 8-hydroxy-2-(di- n-propylamino)tetralin (8-OH-DPAT) was enhanced by injection of the β 2-adrenoceptor agonist clenbuterol with an ED 50 of 0.4 mg/kg. This effect of clenbuterol is through a central mechanism since salbutamol, a β 2-adrenoceptor agonist with poor penetration into the brain, had no effect at a dose of 2 mg/kg, whereas intra-cerebroventricular injection of clenbuterol (3 μg) or salbutamol (2 μg), produced a significant enhancement. The enhancing effect of clenbuterol was unaffected by pretreatment with the β 1-adrenoceptor antagonist metoprolol but was totally antagonised by the β 2-adrenoceptor antagonist ICI 118,551 and to a lesser extent by butoxamine. Clenbuterol therefore enhances the function of the presynaptic 5-HT 1 receptor through a β 2-adrenoceptor mechanism.