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At least 19 recordsLinked to original sources
PubMed2001

Development of clenbuterol reference materials: lyophilized bovine eye samples free of clenbuterol (CRM 673) and containing clenbuterol (CRM 674). Part 1. Preparation, homogeneity and stability.

Within the EU Standards, Measurement and Testing Program (SMT) two clenbuterol reference materials (RMs) were developed. Since clenbuterol readily accumulates and is slowly depleted from pigmented tissues such as the retina, homogenized eye liquid content is the most sensitive tissue for the detection of clenbuterol misuse. Therefore, both of the RMs were produced from bovine eye matrix: a negative control--RM 673 eye reference material, clenbuterol free (<0.50 microg/kg eye matrix) and a positive--RM 674 eye reference material containing clenbuterol (approximately 10 microg/kg eye matrix). Eyes were sampled from 103 German Simmental cattle and the inner liquid content was homogenized to a wet homogenized liquid content (HLC). This clenbuterol negative pool was divided into two sub-pools, one of which was spiked with clenbuterol to a final concentration of 10 microg clenbuterol/kg HLC. Of each pool exactly 2.0 +/- 0.01 g (+/- 0.5%) portions were weighed into 790 containers. Lyophilization of the 1,580 containers was performed in one batch. Parameters for the filling of containers, dry matter content, and residual moisture were in accordance with EU requirements. A three-year stability study and two homogeneity studies at various storage temperatures (-60 degrees C, -20 degrees C, +4 degrees C, +20 degrees C, and +37 degrees C) were performed. Low variation was observed within all of the homogeneity studies, proving that each of the RMs were homogeneous and that this was independent of storage temperature and storage time. In the stability studies, measured clenbuterol concentrations remained constant for RM 673 under the detection limit at 0.15 +/- 0.01 microg clenbuterol equivalent/kg HLC (n = 110) and were also constant for RM 674 at 11.21 +/- 0.15 microg clenbuterol/kg HLC (n=150; measured as duplicates). These studies demonstrate that clenbuterol-containing and clenbuterol-free RMs in bovine eye matrix can be successfully produced. Based on the results described above, it is concluded that both RMs may be suitable as candidates for certification.

Animals↗
PubMed2001

Development of clenbuterol reference materials: lyophilized bovine eye samples free of clenbuterol (CRM 673) and containing clenbuterol (CRM 674). Part 2: certification.

The certification by inter-laboratory testing of two candidate reference materials (RMs) for the mass concentration of the anabolic agent clenbuterol in bovine eye material is described: RM 674 with ca 10 microg clenbuterol per kg of eye matrix and RM 673 clenbuterol-free eye matrix as the negative control (<0.50 microg kg(-1)). Both candidate RMs were certified by eleven EU laboratories, and sixty-six accepted replicate measurements were included in the "Certification Study". The precision of the measurement process was assessed by calculation of the standard variation determined within each laboratory during the certification step. The study was performed according to the "Guidelines for the production and certification of BCR reference materials" and to "ISO guide 31, 33, and 35". The certified clenbuterol mass concentration for clenbuterol-free eye material CRM 673 (calculated on the basis of clenbuterol as the free base) was <0.50 microg kg(-1). The corresponding concentration for clenbuterol-containing eye material CRM 674 was 9.42 +/- 0.88 microg kg(-1). These certified values are very close to the desired target concentration of <0.5 microg kg(-1) and ca 10 microg kg(-1). This study has demonstrated that successful certification of clenbuterol-containing and clenbuterol-free bovine eye materials is possible.

Analysis of Variance↗
PubMed1998

Total radioactive residues and clenbuterol residues in edible tissues, and the stereochemical composition of clenbuterol in livers of broilers after exposure to three levels of dietary [14C]clenbuterol HCl and three preslaughter withdrawal periods.

Thirty-six broiler chickens were randomly assigned to .5, 1.0, or 2.0 ppm dietary [14C]clenbuterol HCl for a 2-wk period starting at 5 wk of age. Four birds from each treatment were slaughtered after withdrawal periods of 0, 7, or 14 d. Total radioactive residues (TRR; clenbuterol HCl equivalents) were measured in adipose tissue, kidney, liver, skin with adhering adipose tissue, bile, blood, brain, gastrointestinal tract, heart, lung, spleen, and testes; parent clenbuterol was measured in liver and kidney. In edible tissues, TRR were roughly proportional to dietary [14C]clenbuterol level and inversely proportional to duration of the withdrawal period; kidney TRR ranged from nondetectable (14 d of withdrawal, .5 and 1.0 ppm treatments) to 211.5 ppb for the 2.0 ppm treatment at zero withdrawal. Liver TRR were detectable for all treatment and withdrawal periods. Rapid depletion of TRR from edible tissues occurred during the first 7 d of the withdrawal period, but depletion of TRR was much slower thereafter. Parent clenbuterol was below the limit of detection (1 ppb) or was undetectable in liver and kidney for all dietary levels after 7 and 14 d of withdrawal, but it represented 22 to 48% of the total radioactive residues at 0 withdrawal. The inactive S (+) stereoisomer constituted approximately 73% of the total clenbuterol residue in livers of chickens slaughtered with no withdrawal period, and the active R (-) stereoisomer accounted for the remainder. These data indicate that radioactive residues of clenbuterol were present well after parent clenbuterol had depleted from edible tissues in chickens, and the predominant stereoisomer remaining in livers at slaughter was the inactive isomer.

Adipose Tissue↗
PubMed1997

Effects of active immunization against clenbuterol on the growth-promoting effect of clenbuterol in rats.

We examined the effect of active immunization against clenbuterol on the growth-promoting effect of clenbuterol in rats in two experiments. Six-week-old female Sprague-Dawley rats were immunized against clenbuterol conjugated to histone by diazotization and then received clenbuterol approximately 4 wk after the initiation of immunization. Antibody titers were determined using indirect ELISA with diazotized clenbuterol-BSA conjugate as an antigen in coating the microwells. Antibody titer increased during booster injections. No significant difference in titer value was observed between two doses of immunogen (.1 vs .5 mg). Competitive ELISA showed that terbutaline cross-reacted with anti-clenbuterol antibodies, and the cross-reactivity was 12%. Alprenolol, propranolol, phentolamine, epinephrine, norepinephrine, and L644,969 showed no affinity for anti-clenbuterol antibodies. The rats immunized against clenbuterol-histone conjugate had 11% lower body weight gain during the 23-d immunization period than the rats immunized against histone only. When clenbuterol was administered after the immunization, no significant difference in growth rate was observed between the rats immunized against clenbuterol-histone conjugate and rats immunized against histone only. No significant difference in muscle weight was observed between the two groups at the termination of the experiment. Results indicate that active immunization against clenbuterol before clenbuterol administration did not modify the growth-promoting effects of clenbuterol in rats.

Adrenergic beta-Agonists↗
PubMedDate not supplied

Clenbuterol in the horse: confirmation and quantitation of serum clenbuterol by LC-MS-MS after oral and intratracheal administration.

Clenbuterol is a beta2 agonist/antagonist bronchodilator, and its identification in post-race samples may lead to sanctions. The objective of this study was to develop a specific and highly sensitive serum quantitation method for clenbuterol that would allow effective regulatory control of this agent in horses. Therefore, clenbuterol-d9 was synthesized for use as an internal standard, an automated solid-phase extraction method was developed, and both were used in conjunction with a multiple reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to allow unequivocal identification and quantitation of clenbuterol in 2 mL of serum at concentrations as low as 10 pg/mL. Five horses were dosed with oral clenbuterol (0.8 microg/kg, BID) for 10 days, and serum was collected for 14 days thereafter. Serum clenbuterol showed mean trough concentrations of approximately 150 pg/mL. After the last dose on day 10, serum clenbuterol reached a peak of approximately 500 pg/mL and then declined with a half-life of approximately 7 h. Serum clenbuterol declined to 30 and 10 pg/mL at 48 and 72 h after dosing, respectively. By 96 h after dosing, the concentration was below 4 pg/mL, the limit of detection for this method. Compared with previous results obtained in parallel urinary experiments, the serum-based approach was more reliable and satisfactory for regulation of the use of clenbuterol. Clenbuterol (90 microg) was also administered intratracheally to five horses. Peak serum concentrations of approximately 230 pg/mL were detected 10 min after administration, dropping to approximately 50 pg/mL within 30 min and declining much more slowly thereafter. These observations suggest that intratracheal administration of clenbuterol shortly before race time can be detected with this serum test. Traditionally, equine drug testing has been dependent on urine testing because of the small volume of serum samples and the low concentrations of drugs found therein. Using LC-MS-MS testing, it is now possible to unequivocally identify and quantitate low concentrations (10 pg/mL) of drugs in serum. Based on the utility of this approach, the speed with which new tests can be developed, and the confidence with which the findings can be applied in the forensic situation, this approach offers considerable scientific and regulatory advantages over more traditional urine testing approaches.

Administration, Oral↗
PubMed2000

Total radioactive residues and clenbuterol residues in swine after dietary administration of [14C]clenbuterol for seven days and preslaughter withdrawal periods of zero, three, or seven days.

Nine barrows (23.8 +/- 0.9 kg) and 9 gilts (23.1 +/- 0.9 kg) were used to determine the disposition of radiocarbon after oral [14C]clenbuterol (4-amino-alpha-[t-butylaminomethyl]-3,5-dichlorobenzyl [7-(14)C]alcohol hydrochloride) administration and to determine total and parent residues in edible tissues. Three barrows and three gilts, housed in metabolism crates, were fed 1 ppm [14C]clenbuterol HCl for seven consecutive days in three separate trials; a single barrow and gilt from each trial was slaughtered after 0-, 3-, or 7-d preslaughter withdrawal periods. Urine and feces were collected during the dosing and the withdrawal period; edible and inedible tissues were collected at slaughter. Total recovery of radiocarbon was 94.2 +/- 6.5%. Total clenbuterol absorption was greater than 75% for barrows and 60% for gilts. Total radioactive residues in tissues were not different (P > 0.05) between barrows and gilts. Concentrations of parent clenbuterol in liver, kidney, skeletal muscle, adipose tissue, and lung did not differ between barrows and gilts (P > 0.05). Total radioactive and parent residues declined in tissues as withdrawal period increased. After the 0-d withdrawal period, total liver residues (286 ppb) were approximately equal to lung residues, twice those of the kidney, and about 15 times those of adipose tissue and skeletal muscle. After a 7-d withdrawal period, total radioactive residues in liver (15 ppb) were roughly three times greater than lung, kidney, and adipose tissue total residues and about 13 times those of skeletal muscle total residues. Parent clenbuterol represented 79, 63, 42, 67, and 100% of the total radioactive residue in adipose tissue, kidney, liver, lung, and skeletal muscle, respectively, in hogs slaughtered with a 0-d withdrawal period. With increasing withdrawal period, the percentage of total radioactive residue present as parent clenbuterol within edible tissues (including lung) decreased, so that after a 7-d withdrawal period, 7, 16, and 29% of the total residue was composed of parent clenbuterol in kidney, liver, and lung, respectively. After a 7-d withdrawal period, parent clenbuterol exceeded the European maximum residue limit (0.5 ppb) 4.6-fold in liver and 2.4-fold in lung. In muscle, clenbuterol was approximately 40 times the limit after a 0-d withdrawal period but had dropped below 0.5 ppb after a 3-d withdrawal period. Results from this study indicate that clenbuterol HCl is well absorbed in swine and that the use of clenbuterol in this species in an off-label manner is inconsistent with human food safety standards used in developed countries.

Animals↗
PubMed1989

Effects of clenbuterol and propranolol on muscle mass. Evidence that clenbuterol stimulates muscle beta-adrenoceptors to induce hypertrophy.

1. A single subcutaneous injection of clenbuterol hydrochloride (0.125 mg/kg body wt.) to female Wistar rats produced a rapid increase in muscle cyclic AMP and lactate concentrations and a decrease in muscle glycogen concentrations. These changes are characteristic of muscle beta-adrenoceptor stimulation and were abolished by intraperitoneal injection of propranolol (12.5 mg/kg) 15 min before clenbuterol administration. 2. When this dose of clenbuterol was injected twice daily, the changes in muscle metabolite concentrations which followed its acute administration persisted until day 7 of treatment, and were accompanied by increases in muscle mass, body weight and muscle protein synthesis rate (ks). When the clenbuterol injections were preceded by propranolol injections (12.5 mg/kg administered according to the protocol described above), or if animals were treated with propranolol only, the values of these variables were not significantly different from those of sham-injected controls. 3. In rats fed on a semi-synthetic diet (PW3) supplemented with 2 mg of clenbuterol/kg of diet for 7 days, the muscle mass was greater than that of rats fed on unsupplemented PW3. The increased muscle mass was accompanied by increased muscle lactate and decreased muscle glycogen concentrations. When PW3 was supplemented with 2 mg of clenbuterol/kg and 200 mg of propranolol/kg, the increase in muscle mass remained, but decreased muscle glycogen concentrations and increased muscle lactate concentrations were also observed. 4. These data are consistent with the hypothesis that clenbuterol influences muscle growth via beta-adrenoceptor stimulation.

Animals↗
PubMed2000

Evaluation of commercial immunoassays for cross-reactivity to clenbuterol stereoisomers and bovine metabolites.

Several commercially available immunoassay kits have been developed to detect the beta-adrenergic agonist clenbuterol HCl. Technical materials supplied with the kits do not generally report cross-reactivity with clenbuterol metabolites. Use of such kits to quantitate clenbuterol might lead to an overestimation of parent drug if metabolites were present. The objective of this study was to measure the cross-reactivity of clenbuterol metabolites with several commercially available clenbuterol immunoassays. Three clenbuterol-glucuronide conjugates, clenbuterol-sulphamate, 4-amino-3,5-dichloro-hippuric acid (clenbuterol-hippurate), and purified clenbuterol-stereoisomers were tested for cross-reactivity. The clenbuterol-sulphamate metabolite showed significant cross-reactivity (42-77%), but clenbuterol-hippurate showed very little competition (< 0.2%) towards clenbuterol. Clenbuterol-glucuronides had little (0.1-1.6%) cross-reactivity. In addition, (R)-, (S)-, and racemic clenbuterol were used to determine the stereospecificity of the kits. Both (R)- and (S)-clenbuterol competed for binding in two of the kits, however, in one kit the (S)-clenbuterol stereoisomer had an affinity 100 times greater than the (R)-stereoisomer. The presence of significant quantities of the sulphamate metabolite of clenbuterol in a biological matrix would cause an overestimation of the amount of parent clenbuterol. This study illustrates the inherent problems of using unvalidated immunoassays for quantitation purposes.

Adrenergic beta-Agonists↗
PubMed1996

Clenbuterol protects mouse cerebral cortex and rat hippocampus from ischemic damage and attenuates glutamate neurotoxicity in cultured hippocampal neurons by induction of NGF.

It has been shown previously that clenbuterol, a beta 2-adrenergic receptor agonist, enhances NGF synthesis in adult rat brain. Since NGF is able to protect neurons against damage, we tried to find out whether clenbuterol can rescue cultured hippocampal neurons from excitotoxic damage by induction of NGF. The neuroprotective activity of clenbuterol on neurons in the vulnerable CA1 subfield of the hippocampus was tested in a rat model of transient forebrain ischemia. Additionally, in the mouse model of focal cerebral ischemia the ability of clenbuterol to reduce the infarct size was examined. Exposure of mixed neuronal/glial hippocampal cultures to clenbuterol (1 to 100 microM) enhanced significantly the content of NGF measured in the culture medium by two-site ELISA. The excitotoxic injury was induced in the same type of cells after 14 days in vitro by exposure to 1 mM L-glutamate for 1 h in serum-free medium. NGF itself (0.15 to 100 ng/ml) added to the growth medium 4 h before until 18 h after induction of injury (the point of glutamate-toxicity measurement), protected hippocampal neurons from excitotoxic damage. Clenbuterol (1 to 100 microM) provided similar neuroprotection as NGF under the same experimental conditions. The neuroprotective activity of clenbuterol (100 microM) against glutamate-induced damage in hippocampal cultures was blocked by anti-NGF monoclonal antibodies (0.5 microgram/ml) added to the medium during the clenbuterol exposure, demonstrating that the neuronal rescue is mediated by NGF. Propranolol, a beta-adrenergic receptor antagonist (10 microM) added 20 min before and kept in the medium during exposure of the cultures to clenbuterol (1 microM) reversed the neuroprotective activity, suggesting that the induction of NGF and neuroprotection caused by clenbuterol are mediated via beta-adrenergic receptor activation. The capacity of clenbuterol to protect hippocampal neurons was also demonstrated in vivo in a rat model of transient forebrain ischemia. Clenbuterol (4 x 1 mg/kg) administered intraperitoneally increased the number of viable neurons in CA1 subfield of the rat hippocampus. Furthermore, clenbuterol (0.3 and 1 mg/kg, i.p. and 1 mg/kg, s.c.) reduced significantly the infarct area on the mouse brain surface after occlusion of the middle cerebral artery. The present data demonstrate that clenbuterol induces NGF synthesis in cultured hippocampal cells and protects hippocampal neurons from excitotoxic damage. The neuroprotective activity of clenbuterol is also demonstrated in vivo in two rodent models of cerebral ischemia. The results offer strong evidence that the neuroprotective activity of clenbuterol is caused by activation of beta-adrenergic receptors and the subsequent increased expression of NGF.

Animals↗
PubMed2001

Clenbuterol in the horse: urinary concentrations determined by ELISA and GC/MS after clinical doses.

Clenbuterol is a beta2 agonist/antagonist bronchodilator marketed as Ventipulmin and is the only member of this group of drugs approved by the US Food and Drug Administration (FDA) for use in horses. Clenbuterol is a class 3 drug in the Association of Racing Commissioners International (ARCI) classification system; therefore, its identification in postrace samples may lead to sanctions. Recently, the sensitivity of postrace testing for clenbuterol has been substantially increased. The objective of this study was to determine the 'detection times' for clenbuterol after administration of an oral clinical dose (0.8 g/kg, b.i.d.) of Ventipulmin syrup. Five horses received oral clenbuterol (0.8 g/kg, b.i.d.) for 10 days, and urine concentrations of clenbuterol were determined by an enhanced enzyme-linked immunoabsorbent assay (ELISA) test and gas chromatography/mass spectrometric (GC/MS) analysis by two different methods for 30 days after administration. Twenty-four hours after the last administration, urine concentrations of apparent clenbuterol, as measured by ELISA, averaged about 500 ng/mL, dropping to about 1 ng/mL by day 5 posttreatment. However, there was a later transient increase in the mean concentrations of apparent clenbuterol in urine, peaking at 7 ng/mL on day 10 postadministration. The urine samples were also analysed using mass spectral quantification of both the trimethylsilyl (TMS) and methane boronic acid (MBA) derivatives of clenbuterol. Analysis using the TMS method showed that, at 24 h after the last administration, the mean concentration of recovered clenbuterol was about 22 ng/mL. Thereafter, clenbuterol concentrations fell below the limit of detection of the TMS-method by day 5 after administration but became transiently detectable again at day 10, with a mean concentration of about 1 ng/mL. Derivatization with MBA offers significant advantages over TMS for the mass spectral detection of clenbuterol, primarily because MBA derivatization yields a high molecular weight base peak of 243 m/z, which is ideal for quantitative purposes. Therefore, mass spectral analyses of selected urine samples, including the transient peak on day 10, were repeated using MBA derivatization, and comparable results were obtained. The results show that clenbuterol was undetectable in horse urine by day 5 after administration. However, an unexpected secondary peak of clenbuterol was observed at day 10 after administration that averaged approximately 1 ng/mL. Because of this secondary peak, the detection time for clenbuterol (0.8 g/kg, b.i.d. x 10 days) is at least 11 days if the threshold for detection is set at 1 ng/mL.

Administration, Oral↗
PubMed1976

[Profile of pharmacological actions of NAB 365 (clenbuterol), a novel broncholytic agent with selective activity on adrenergic beta2-receptors (author's transl)].

Effects of 4-amino-alpha-[(tert.-butylamino)methyl]-3,5-dichlorobenzyl alcohol hydrochloride (clenbuterol, NAB 365) on the adrenergic beta-receptors were investigated and compared with those of isoproterenol and salbutamol. The beta2-mimetic activity of clenbuterol on the smooth muscle of bronchi, uterus and vessels after i.v. injection corresponds to that of salbutamol in all laboratory animals. When given subcutaneously or as an aerosol clenbuterol is even somewhat more effective than isoproterenol. Clenbuterol differs from the known beta-mimetic drugs in its much longer duration of action. Therefore the integral of activity of single doses of clenbuterol, which are equally effective in the period of their maximal action, is remarkably greater than that of the other beta-mimetic substances. Clenbuterol differs from known beta-mimetic drugs used as bronchodilators in its efficacy after oral administration and in its mode of action on the heart. In the isolated auricle of the rabbit it has proved to be a weak partial agonist. In conscious rabbits, anesthetised guinea-pigs, dogs and cats the maximum of tachycardia obtainable by clenbuterol is lower than that of salbutamol. The higher degree of tachycardia in conscious dogs provoked by clenbuterol is a result of a reflex reaction to the vasodilation analogous to that of salbutamol. In higher doses clenbuterol shows beta1-blocking properties. Like other beta-blocking agents it owns qualities of a local-anesthetic and prolongs refractory period of the heart of guinea-pigs. In contrast to other beta-mimetic substances, clenbuterol causes only slight mobilization of heart muscle glycogen by doses higher than those which have broncholytic effects. The lipolytic and lactacidemia inducing activity of clenbuterol in rabbits corresponds to that of isoproterenol. The blood sugar is only slightly increased by clenbuterol as well as by other beta-mimetic agents. Degree and duration of action of clenbuterol and the other sympathomimetic amines on skeletal muscle of the cat shows parallelism with that of the broncholytic effect. In the rat clenbuterol inhibits the gastric secretion more than does isoproterenol. In contrast to other broncholytic substances, a very small dosage of clenbuterol is sufficient to protect rats against the liberation of histamine and serotonin caused by the anaphylactic reaction.

Adrenergic beta-Agonists↗
PubMed2000

Intratracheal clenbuterol in the horse: its pharmacological efficacy and analytical detection.

Clenbuterol, a beta2 agonist/antagonist, is the only bronchodilator approved by the US Food and Drug Administration for use in horses. The Association of Racing Commissioners International classifies clenbuterol as a class 3 agent, and, as such, its identification in post-race samples may lead to sanctions. Anecdotal reports suggest that clenbuterol may have been administered by intratracheal (IT) injection to obtain beneficial effects and avoid post-race detection. The objectives of this study were (1) to measure the pharmacological efficacy of IT dose of clenbuterol and (2) to determine the analytical findings in urine in the presence and absence of furosemide. When administered intratracheally (90 microg/horse) to horses suffering from chronic obstructive pulmonary disease (COPD), clenbuterol had effects that were not significantly different from those of saline. In parallel experiments using a behavior chamber, no significant effects of IT clenbuterol on heart rate or spontaneous locomotor activity were observed. Clenbuterol concentrations in the urine were also measured after IT dose in the presence and absence of furosemide. Four horses were administered i.v. furosemide (5 mg/kg), and four horses were administered saline (5 mL). Two hours later, all horses were administrated clenbuterol (IT, 90 microg), and the furosemide-treated horses received a second dose of furosemide (2.5 mg/kg, i.v.). Three hours after clenbuterol dose (1 h after hypothetical 'post-time'), the mean specific gravity of urine samples from furosemide-treated horses was 1.024, well above the 1.010 concentration at which furosemide is considered to interfere with drug detection. There was no interference by furosemide with 'enhanced' ELISA screening of clenbuterol equivalents in extracted and concentrated samples. Similarly, furosemide had no effect on mass spectral identification or quantification of clenbuterol in these samples. These results suggest that the IT dose of clenbuterol (90 microg) is, in pharmacological terms, indistinguishable from the dose of saline, and that, using extracted samples, clenbuterol dose is readily detectable at 3 h after dosing. Furthermore, concomitant dose of furosemide does not interfere with detection or confirmation of clenbuterol.

Administration, Inhalation↗
PubMed1999

The beta2-adrenoceptor agonist clenbuterol modulates Bcl-2, Bcl-xl and Bax protein expression following transient forebrain ischemia.

It is well known that proteins encoded by the Bcl-2 gene family play a major role in the regulation of apoptosis. We have demonstrated previously that neuronal apoptosis can be induced in the hippocampus and striatum after global ischemia. Clenbuterol, a beta2-adrenoceptor agonist, showed considerable activity against neuronal apoptosis. In the present study, we attempted to find out whether the members of the Bcl-2 family are induced after ischemia, and whether expression of these genes could be altered by clenbuterol. Transient forebrain ischemia was performed in male Wistar rats by clamping both common carotid arteries and reducing the blood pressure to 40 mmHg for 10 min. Clenbuterol (0.5 mg/kg, i.p.) or vehicle were injected 3 h before onset of ischemia or in non-ischemic rats. The hippocampus and striatum were taken from non-ischemic rats 3, 6 and 24 h after injection of clenbuterol, as well as from drug-treated and untreated rats 6 and 24 h after ischemia. Eighty micrograms/lane total protein were loaded on a 15% sodium dodecyl sulfate-polyacrylamide gel for western blotting. Bcl-2, Bax and Bcl-xl proteins were detectable in the non-ischemic hippocampus and the striatum. Clenbuterol up-regulated the expression of Bcl-2 protein at 3, 6 and 24 h after administration. Enhanced Bcl-xl signals were found in the non-ischemic striatum 3, 6 and 24 h after clenbuterol treatment, but no change of Bcl-xl expression by clenbuterol was seen in the non-ischemic hippocampus. Bax expression was not altered by clenbuterol in the non-ischemic hippocampus and striatum. Bcl-2 was up-regulated in both detected regions at 24 h after ischemia, while the increase in Bax and Bcl-xl protein expression had appeared already at 6 h and also 24 h after ischemia. Clenbuterol further increased the expression of Bcl-2 at 6 and 24 h after ischemia. In contrast, Bax protein level was down-regulated by clenbuterol at 6 and 24 h after ischemia. Clenbuterol also increased Bcl-xl level in the ischemic striatum. The results suggest that global ischemia induces proto-oncogenes which are associated with apoptosis. Clenbuterol not only increased Bcl-2 expression in the non-ischemic hippocampus and striatum, but also up-regulated Bcl-2 and down-regulated Bax expression in the ischemic hippocampus and striatum. The increase in the ratio of Bcl-2 and Bax may contribute to the anti-apoptotic effect of clenbuterol. The present study indicates that pharmacological modulation of Bcl-2 family member expression could become a new strategy to interfere with neuronal damage.

Adrenergic beta-Agonists↗
PubMed2006

Molecular impact of clenbuterol and isometric strength training on rat EDL muscles.

Clenbuterol, a beta2-adrenergic-receptor agonist, is known to provoke muscle hypertrophy and a slow-to-fast phenotype change. A more glycolytic phenotype should be paralleled by changes in muscle glycolytic metabolism. Two groups (n=16 for each) of 3-month-old male Wistar rats (UCL: untrained clenbuterol, and ECL: exercised clenbuterol) received a chronic administration of clenbuterol (2 mg/kg body weight/day). Two other groups of animals (U: untrained and E: exercised), were given a 0.9% NaCl solution instead of clenbuterol. E and ECL animals followed an 8-week progressive isometric force strength-training program. Both clenbuterol administration and training resulted in an increase in extensor digitorum longus (EDL) mass despite the fact that this muscle was indirectly mobilised during isometric force strength training. Clenbuterol and training induced a consistent slow-to-fast phenotype change without drastically increasing specific activities of glycolytic enzymes. Except for GAPDH and hexokinase, modifications in glycolytic-enzyme-specific activities were not explained by transcriptional changes. Lactate dehydrogenase activity was not affected by clenbuterol but was strongly augmented by training. In EDL of ECL rats, both treatments presented an opposite effect compensating each other. GLUT1 mRNA expression was augmented in EDL of UCL and ECL animals, whereas monocarboxylate transporter 1 mRNA amounts were decreased in EDL of UCL rats. Citrate synthase activity was reduced by clenbuterol treatment but remained unchanged in EDL of E animals. Creatine kinase activity was enhanced only by clenbuterol alone. These data show that clenbuterol-induced muscle hypertrophy and slow-to-fast phenotype changes are not associated with a glycolytic-enzyme-activity increase. They also suggest that in EDL isometric force strength training can reverse clenbuterol-induced molecular adaptations.

Adrenergic beta-Agonists↗
PubMed1998

Effect of clenbuterol on non-endothelial nitric oxide release in rat mesenteric arteries and the involvement of beta-adrenoceptors.

1. The aim of the present study was to explore the contribution of adrenergic, sensory and nitrergic innervations to the inhibitory effects of the beta2-adrenoceptor agonist clenbuterol on responses to electrical field stimulation (EFS, 200 mA, 0.3 ms, 1-16 Hz, for 30 s, at 1 min interval) in rat mesenteric artery segments without endothelium and the possible involvement of adrenergic, sensory and nitrergic innervations. 2. Clenbuterol (1 microM) reduced EFS-induced contractile responses, and this effect was reversed by the beta-antagonist propranolol (1 microM) (contraction at 16 Hz expressed as % of 75 mM K+-induced contraction was: control, 69+/-9, clenbuterol, 31+/-6, n=13, P<0.001; control, 83+/-5, clenbuterol+propranolol 70+/-7, n=11, P>0.05). 3. In arteries preincubated with [3H]-noradrenaline (NA), clenbuterol did not modify the tritium overflow evoked by EFS (200 mA, 0.3 ms, 4 Hz, for 60 s; ratio between tritium release in the second and first stimuli was: control, 0.80+/-0.05 and clenbuterol added before second stimulus, 0.91+/-0.11, n=5, P>0.05). 4. The nitric oxide (NO) synthase inhibitors NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME) (10 and 100 microM), and the guanylate cyclase inhibitor methylene blue (10 microM) increased the contractions caused by EFS (% contraction at 16 Hz, control, 81+/-7, n=26; 10 microM L-NMMA, 109+/-12, n=8, P<0.05; methylene blue, 119+/-6, n=6, P<0.05). However, these contractions were decreased by the NO synthase substrate L-arginine 10 microM (14+/-6%, n=6, P<0.001), but not modified by either the sensory neurones toxin capsaicin (0.5 microM, 75+/-6%, n=6, P>0.05) or the protein synthesis inhibitor cycloheximide (10 microM, 83+/-6%, n=8, P>0.05). None of these drugs altered the concentration-response curves to exogenous NA (n=7). 5. Pretreatment with capsaicin or cycloheximide did not modify the reduction of the EFS-evoked contraction provoked by clenbuterol. However the presence of L-NMMA (or L-NAME) or methylene blue did decrease the effect of clenbuterol (% contraction at 16 Hz, clenbuterol, 31+/-6, n=13; clenbuterol+10 microM L-NMMA, 93+/-11, n=8, P<0.05; clenbuterol+methylene blue, 90+/-7, n=6, P<0.05). 6. These results suggest that the reduction caused by clenbuterol in the contraction induced by EFS in rat mesenteric arteries seems to be mediated by NO release, through the activation of beta2-adrenoceptors probably present on nitrergic nerves.

Adrenergic beta-Agonists↗
PubMed2001

Anticonvulsant and sodium channel blocking activity of higher doses of clenbuterol.

Clenbuterol, a lipophilic beta2-adrenoceptor agonist, was investigated in various seizure models of experimental epilepsy. In the maximal electroshock seizure threshold test, clenbuterol (> or =4 mg/kg i.p.) increased the electroconvulsive threshold for tonic seizures in mice. In the traditional maximal electroshock seizure (MES) test in mice, ED50 values of 11 mg/kg i.p. or s.c. were determined. In both models, the beta2-receptor antagonist ICI 118.551 did not antagonize the anticonvulsant activity of clenbuterol. Combinations of clenbuterol with standard antiepileptics revealed additive anticonvulsant effects. Repeated administration of clenbuterol (5 mg/kg s.c., twice daily for 14 days) to mice did not significantly influence its anticonvulsant potency or the effectiveness of phenobarbital in the MES test. In various chemically-induced seizure tests with tonic convulsions, clenbuterol inhibited or tended to suppress the tonic phase. However, this drug was not effective in preventing clonic seizures in the pentylenetetrazol (85 mg/kg s.c.) seizure threshold test. In the rotarod ataxia test (mice), a minimal "neurotoxic" dose (TD50) of 41 mg/kg i.p. was determined. In unrestrained rats with chronically implanted electrodes in the dorsal hippocampus, clenbuterol (2 mg/kg and 4 mg/kg i.p.) significantly reduced the duration of electrically evoked hippocampal afterdischarges. In amygdala-kindled rats, clenbuterol (5 mg/kg and 10 mg/kg i.p.) reduced the seizure severity to stage 3. Additional studies indicated that clenbuterol (6 mg/kg i.p.) increased the heart rate and decreased the blood pressure, but this drug did not alter the plasma level of the two tested antiepileptics phenobarbital and carbamazepine. Furthermore, in whole-cell voltage-clamp experiments on cultured neonatal rat cardiomyocytes, clenbuterol (1-100 microM) depressed the fast sodium current in a concentration- and frequency-dependent manner. In conclusion, the anticonvulsant effects of higher doses of clenbuterol against generalized tonic-clonic and complex partial seizures seem to be related to the inhibition of voltage-dependent sodium channels and not to the modulation of beta-adrenoceptors.

Adrenergic beta-Agonists↗
PubMed2004

Clenbuterol treatment affects myosin heavy chain isoforms and MyoD content similarly in intact and regenerated soleus muscles.

AIMS: Pharmacological treatment with the beta2-adrenoceptor agonist clenbuterol is known to induce a slow-to-fast fibre type and myosin heavy chain (MHC) isoform transition in intact muscle. This study examined the sensitivity of regenerated soleus muscle to 4 weeks of clenbuterol treatment (2 mg kg-1 day-1). METHODS: Female Wistar rats were divided into two groups: vehicle treated (n = 8) and clenbuterol treated (n = 8). The clenbuterol effects on MHC and MyoD expression were examined in soleus muscles either intact, or previously degenerated by venom of the Notechis scutatus scutatus snake. RESULTS: Post-treatment body weights and skeletal muscle weights were not affected by clenbuterol treatment. Muscle protein concentration was higher, and body fat lower in clenbuterol-treated rats than in vehicle-treated animals (P < 0.05). Polyacrylamide gel electrophoresis of soleus myofibrillar protein indicated a clenbuterol-induced decrease in the relative percentage of type I MHC with a concomitant increase in type IIa MHC (31%, P < 0.001). No degeneration effect was observed after 28 days of recovery on the MHC isoform content, and regenerated soleus muscles exhibited the same phenotypical profile as intact soleus muscles, whether or not they were treated with clenbuterol. In intact and in regenerated soleus muscles, MyoD protein levels were significantly increased by clenbuterol treatment (90 and 77%, respectively, P < 0.001). CONCLUSION: These results show that regenerated soleus muscles, comprising a homogeneous population of fibres deriving from satellite cells, have a similar response to clenbuterol as intact muscle arising from at least two discrete populations of myotubes; it is suggested that the activity of signalling pathways involved in the effects of clenbuterol on MHC transitions is not related to the developmental history of myofibres.

Adrenergic beta-Agonists↗
PubMed2006

Rapamycin inhibits the growth and muscle-sparing effects of clenbuterol.

Clenbuterol and other beta2-adrenergic agonists are effective at inducing muscle growth and attenuating muscle atrophy through unknown mechanisms. This study tested the hypothesis that clenbuterol-induced growth and muscle sparing is mediated through the activation of Akt and mammalian target of rapamycin (mTOR) signaling pathways. Clenbuterol was administered to normal weight-bearing adult rats to examine the growth-inducing effects and to adult rats undergoing muscle atrophy as the result of hindlimb suspension or denervation to examine the muscle-sparing effects. The pharmacological inhibitor rapamycin was administered in combination with clenbuterol in vivo to determine whether activation of mTOR was involved in mediating the effects of clenbuterol. Clenbuterol administration increased the phosphorylation status of PKB/Akt, S6 kinase 1/p70(s6k), and eukaryotic initiation factor 4E binding protein 1/PHAS-1. Clenbuterol treatment induced growth by 27-41% in normal rats and attenuated muscle loss during hindlimb suspension by 10-20%. Rapamycin treatment resulted in a 37-97% suppression of clenbuterol-induced growth and a 100% reduction of the muscle-sparing effect. In contrast, rapamycin was unable to block the muscle-sparing effects of clenbuterol after denervation. Clenbuterol was also shown to suppress the expression of the MuRF1 and MAFbx transcripts in muscles from normal, denervated, and hindlimb-suspended rats. These results demonstrate that the effects of clenbuterol are mediated, in part, through the activation of Akt and mTOR signaling pathways.

Adrenergic beta-Agonists↗