Effect of the beta-adrenoceptor agonist flerobuterol on serotonin synthesis in the rat brain

The influence of 2- and 14-day treatments with flerobuterol, a preferential beta(2)-adrenoceptor agonist, on regional serotonin (5-HT) synthesis in the rat brain was studied by autoradiography using alpha-[(14)C]methyl-L-tryptophan. Flerobuterol was delivered at a rate of 0.5 mg/kg/day using osmotic pumps implanted s.c. The 2-day flerobuterol treatment significantly increased plasma Trp, both free and total, and decreased plasma Leu and Ile. This resulted in a significant increase in the facilitated transport of Trp. There was a significant increase in the synthesis of 5-HT in the 2-day treatment group in the dorsal and median raphe as well as in all postsynaptic structures, with the exception of the hypothalamus. In contrast, after a 14-day treatment, the enhanced facilitated transport of Trp was no longer present, and the increase in the rate of 5-HT synthesis persisted only in the parietal and occipital cortex and the superior colliculus. These data suggest that flerobuterol, similar to other beta-adrenergic agonists, acutely increases 5-HT synthesis, in part, through an elevation of brain Trp availability.     

Studies on the mechanisms by which clenbuterol, a beta-adrenoceptor agonist, enhances 5-HT-mediated behaviour and increases metabolism of 5-HT in the brain of the rat

The head twitch response in mice produced by injection of 5-hydroxytryptophan (100 mg/kg i.p.) and carbidopa (25 mg/kg i.p.) was enhanced by administration of clenbuterol (0.5 mg/kg i.p.), a beta-adrenoceptor agonist. Clenbuterol also enhanced the hyperactivity syndrome in rats produced by quipazine (25 mg/kg i.p.), a 5-hydroxytryptamine (5-HT) agonist. This enhancement was not prevented by depletion of 5-HT in brain with p-chlorophenylalanine or after pretreatment with prazosin. The behavioural responses of the rats to administration of the alpha 2-adrenoceptor agonist, clonidine, was unaltered by acute or longer-term administration of clenbuterol. Following chronic administration of clenbuterol (5 mg/kg daily for 14 days), a procedure resulting in down-regulation of central beta-adrenoceptors, a larger dose of clenbuterol was necessary to enhance the quipazine-induced hyperactivity, suggesting that the mechanism of enhancement involved central post-synaptic beta-adrenoceptors. Further evidence for this conclusion was that a lesion of central noradrenaline pathways produced by 6-hydroxydopamine did not abolish the clenbuterol-induced enhancement of the quipazine-mediated behaviour. The binding characteristics of 5-HT2-receptors were unchanged by acute or chronic administration of clenbuterol. Clenbuterol (5 mg/kg) increased the percentage of plasma free (non-albumin bound) tryptophan, plasma free fatty acid concentration and the concentration of tryptophan and 5-hydroxyindoleacetic acid (5-HIAA) in the brain. The increase in 5-HT turnover in brain was prevented by pretreatment with the beta 1-adrenoceptor antagonist atenolol, which enters the brain poorly. It is therefore suggested that the clenbuterol-induced increase in 5-HT metabolism results from the increase in the concentration of plasma free fatty acid which increases plasma free tryptophan and thus increases the concentration of tryptophan in brain and 5-HT synthesis in brain. The clenbuterol-induced enhancement of 5-HT-mediated behaviour is therefore not associated with its effect on 5-HT metabolism. The data are discussed in relation to that obtained after administration of antidepressant drugs.     

Inverse agonist activities of beta-adrenoceptor antagonists in rat myocardium.

1. Negative inotropic effects of several beta-adrenoceptor (betaAR) antagonists on electrically-stimulated right atria, left atria, right ventricles and left ventricular papillary muscles from reserpine-treated rats were used as a measure of their inverse agonist activities. 2. Beta1AR antagonists acebutolol, atenolol and metoprolol, beta2AR antagonist ICI-181,551 and nonselective betaAR antagonists alprenolol, nadolol, propranolol and timolol produced negative inotropic effects, which were most marked on the right atria. 3. The nonselective betaAR antagonist pindolol did not exhibit inverse agonist activity but inhibited the negative inotropic activities of ICI-118,551, atenolol and propranolol. 4. The negative inotropic effects of lidocaine, nifedipine and pentobarbitone were similar on all the four myocardial preparations. 5. The positive inotropic efficacy of salbutamol on right and left atria but not on right ventricles and papillary muscles was comparable to that of isoprenaline. The antagonist activity of ICI-118,551 against isoprenaline was greater on right atria than on other cardiac regions. 6. Beta1AR proteins were expressed in all regions of the heart but of beta2AR were primarily localized in the right atrium. 7. It is concluded that beta2AR play a greater role in right atria than in other cardiac regions and almost all betaAR antagonists behave as inverse agonists.     

Effect of ethanol metabolism on initiation of protein synthesis in rat hepatocytes

Rat liver parenchymal cells were incubated in the presence and absence of ethanol (80 mM). Polysomes were isolated and analysed on sucrose gradients. Ethanol was shown to (1) inhibit the incorporation of ¹⁴C-valine into proteins, (2) result in a shift in the distribution of polysomes towards smaller sizes, (3) inhibit the formation of 40S initiation complexes, and (4) diminish the concentration of glucose-6-phosphate in the hepatocytes. Addition of 4-methylpyrazole (0.5 mM) partially prevented the inhibition of protein synthesis and completely restored the polysomal distribution. It is concluded that ethanol inhibits protein synthesis partly by a mechanism linked to ethanol metabolism. This effect takes place at the level of initiation and may be mediated by a reduced gluconeogenesis.     

Biphasic effects of the beta-adrenoceptor agonist, BRL 37344, on glucose utilization in rat isolated skeletal muscle.

1. The effects of the selective beta 3-adrenoceptor agonist, BRL 37344 (BRL) on glucose uptake and phosphorylation (i.e. glucose utilization; GU) and glycogen synthesis in rat isolated soleus and extensor digitorium longus (EDL) muscle preparations in vitro were investigated by use of 2-deoxy-[3H]-glucose (GU) and [U-14C]-glucose (glycogen synthesis). 2. Low concentrations of BRL (10(-11)-10(-9) M) significantly increased GU, with maximal increases of 30% in soleus and 24% in EDL at 10(-11) M. Neither the selective beta 1-adrenoceptor antagonist, atenolol (10(-8)-10(-6) M), nor the selective beta 2-adrenoceptor antagonist, ICI 118551 (10(-8)-10(-6) M) had any effect on the stimulation of GU induced by 10(-11) M BRL. 3. High concentrations of BRL (10(-6)-10(-5) M) caused significant inhibition (up to 30%) of GU in both soleus and EDL muscles. The inhibition of 10(-6) M BRL was blocked completely by 10(-6) and 10(-7) M ICI 118551 in soleus, and by 10(-6)-10(-8) M ICI 118551 in EDL; atenolol (10(-8)-10(6) M) had no effect. 4. Another selective beta 3-adrenoceptor agonist, CL 316,243, also caused a significant stimulation of muscle GU, with maximal increases of 43% at 10(-9) M in soleus and 45% at 10(-10) M in EDL. The stimulation of GU declined with further increases in the concentration of CL 316,243, but no inhibition of GU was seen, even at the highest concentration (10(-5) M) tested. 5. BRL at 10(-5) M inhibited completely insulin-stimulated glycogen synthesis in both soleus and EDL, but this inhibitory effect of BRL was abolished by 10(-6) M ICI 118551. BRL at 10(-11) M (with or without 10(-6) M ICI 118551) had no effect on insulin-stimulated glycogen synthesis. 6. It is concluded that: (i) low (< nM) concentrations of BRL stimulate GU via an atypical beta-adrenoceptor that is resistant to conventional beta 1-adrenoceptor and beta 2-adrenoceptor antagonists; (ii) the stimulation of GU is negated by the activation of beta 2-adrenoceptors that occurs at higher (> nM) concentrations of BRL; (iii) inhibition of GU via beta 2-adrenoceptor activation is associated with inhibition of glycogen synthesis, possibly due to activation of glycogenolysis; (iv) the opposing effects of beta 2-adrenoceptor and atypical beta-adrenoceptor activation on GU suggest that in skeletal muscle these adrenoceptors are linked to different post-receptor pathways.     

Effect of hindlimb suspension and clenbuterol treatment on polyamine levels in skeletal muscle

Polyamines are unbiquitous, naturally occurring small aliphatic, polycationic, endogenous compounds. They are involved in many cellular processes and may serve as secondary or tertiary messengers to hormonal regulation. The relationship of polyamines and skeletal muscle mass of adductor longus, extensor digitorum longus, and gastrocnemius under unloading (hindlimb suspension) conditions was investigated. Unloading significantly affected skeletal muscle polyamine levels in a fiber-type-specific fashion. Under loading conditions, clenbuterol treatment increased all polyamine levels, whereas under unloading conditions, only the spermidine levels were consistently increased. Unloading attenuated the anabolic effects of clenbuterol in predominately slow-twitch muscles (adductor longus), but had little impact on clenbuterol's action as a countermeasure in fast- twitch muscles such as the extensor digitorum longus. Spermidine appeared to be the primary polyamine involved in skeletal muscle atrophy/hypertrophy.     

Hemodynamic effects on hepatic blood flow of a selective beta 2-adrenoceptor agonist, clenbuterol, in rat.

Acute clenbuterol administration (50 micrograms/kg, i.v.) to anesthetized normotensive rats, produce a marked reduction in the mean blood pressure, (MBP), about 58 mm Hg. Indocyanine Green clearance analysis (control, 1.83 +/- 0.15: clenbuterol, 1.10 +/- 0.20 ml/min/100 g, P < 0.05) showed that the action in the hepatic vascular bed is opposite to its systemic vasodilator effects. The hepatic blood flow (HBF) appears significantly reduced (control, 8.24 +/- 0.35: clenbuterol, 3.83 +/- 0.71 ml/min/100 g, P < 0.05) whereas the hepatic uptake and excretion proceedings were apparently not affected (control hepatic extraction coefficient, 0.225 +/- 0.024: clenbuterol, 0.300 +/- 0.04, NS). These findings show that a marked reduction in HBF follows systemic vasodilator effects produced by clenbuterol.     

Effect of sodium fluoride on protein and DNA synthesis, ornithine decarboxylase activity, and polyamine content in LS cells.

Sodium fluoride exhibited a dose dependent inhibitory effect on protein and DNA synthesis at concentrations from 1.3 mM in growing LS cells. The activity of ornithine decarboxylase (ODC) was slightly stimulated by 0.5 mM-NAF, but inhibited at 1.3 mM and above. The reduced enzyme activity seemed to be due to a reduced de novo formation of the enzyme caused by an inhibition of the protein synthesis. In spite of a reduction of ODC-activity, fluoride had no effect on the cellular polyamine content during the experimental period (10 hours).     

Transforming growth factor betas are upregulated in the rat masseter muscle hypertrophied by clenbuterol, a beta2 adrenergic agonist

1. The regulatory mechanism for the hypertrophy of skeletal muscles induced by clenbuterol is unclear. The purpose of the present study was to determine the extent to which transforming growth factor betas (TGFbetas), fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), and platelet-derived growth factors (PDGFs) are involved in the hypertrophy of rat masseter muscle induced by clenbuterol. 2. We measured the mRNA expression levels for TGFbetas, FGFs, HGF, and PDGFs in rat masseter muscle hypertrophied by oral administration of clenbuterol for 3 weeks and determined correlations between the weight of masseter muscle and mRNA expression levels by regression analysis. We determined immunolocalizations of TGFbetas and their receptors (TGFbetaRs). 3. The mRNA expression levels for TGFbeta1, 2, and 3, and for PDGF-B demonstrated clenbuterol-induced elevations and positive correlations with the weight of masseter muscle. In particular, TGFbeta1, 2, and 3 showed strong positive correlations (correlation coefficients >0.6). The mRNA expression levels for PDGF-A, FGF-1 and 2, and HGF showed no significant differences between the control and clenbuterol groups, and no significant correlations. TGFbeta1, 2, and 3 were principally localized in the connective tissues interspaced among myofibers, and TGFbetaRI and II were localized in the periphery and sarcoplasm of the myofibers. 4. These results suggest that paracrine actions of TGFbeta1, 2, and 3 via TGFbetaRI and II could be involved in the hypertrophy of rat masseter muscle induced by clenbuterol. This is the first study to document the involvement of TGFbetas in the hypertrophy of skeletal muscles induced by clenbuterol.     

Effect of praziquantel and oxamniquine on prostacyclin synthesis by the rat arterial and myometrial tissues.

1. The influence of the two antischistosomal drugs (+/-) praziquantel and (+/-) oxamniquine on PGI2 synthesis by the male rat thoracic aorta and day-20 pregnant rat myometrium in vitro was investigated using a rat platelet antiaggregatory bioassay method. 2. Pretreatment of the tissues with praziquantel (64-512 microM) or oxamniquine (36-288 microM) for 30 min at 37 degrees C significantly inhibited basal PGI2 synthesis in a concentration-dependent manner (P less than 0.005, n = 5-6). 3. Both drugs failed to inhibit PGI2 synthesis in presence of exogenous arachidonic acid (AA) (16.6 microM). 4. Furthermore, they did not antagonize AA (4 nmol kg-1)-induced hypotension in urethane-anaesthetized rats. Thus, the drugs seemed to act via inhibition of phospholipase A2 enzyme (PLA2). 5. The highly lipophilic drugs may interact with membrane phospholipids resulting in prevention of interaction between the substrates and the enzyme's active site.     

Prolonged, intravenous paraquat infusion in the rat. II. Paraquat-induced alterations in lung polyamine metabolism

The effects of paraquat (PQ) on lung putrescine, spermidine, and spermine levels, and ornithine decarboxylase (ODC) activity were assessed in rats after 7 days of iv infusion of the herbicide via osmotic minipump. Paraquat administration at a rate of 250 nmol/hr [673 +/- 40 nmol/kg/hr (n = 15)] had no effect on these parameters. In contrast, significant (p less than 0.05) elevations in lung putrescine (407% of control), spermidine (202% of control), and ODC activity (174% of control were measured in lungs of rats given 500 nmol PQ/hr [1.31 +/- 0.53 mumol/kg/hr (n = 14)]. Since evidence of lung damage was, likewise, observed only in the high-dose PQ rats, these changes in polyamine metabolism could have been a nonspecific response to PQ-induced lung injury rather than a direct biochemical effect of PQ. The results suggest that stimulation of polyamine biosynthesis may play an important role in PQ-induced lung injury. This role may involve regulation of repair mechanisms or, conversely, the polyamines may actually mediate PQ-induced fibrotic changes in the lung.     

Involvement of FAD-dependent polyamine oxidase in the metabolism of milacemide in the rat.

1. It has previously been established that monoamine oxidase (MAO)-B participates in the metabolism of milacemide [2-(pentylamino)acetamide]. Furthermore, in rats, inhibition of FAD-dependent polyamine oxidase (PAO) was found to decrease the urinary excretion of two milacemide metabolites, termed UK1 and UK2. 2. Using gas chromatography-mass spectrometry, UK1 was identified as oxamic acid and UK2 as 2-hydroxyacetamide, confirming that PAO is involved in the metabolism of milacemide. 3. Thus, two FAD-dependent amine-oxidizing enzymes, MAO and PAO, contribute to the metabolism of milacemide. Milacemide appears to be the first non-polyamine xenobiotic in the metabolism of which PAO participates.     

Effect of enoxacin, felbinac, and sparfloxacin on fatty acid metabolism and glucose concentrations in rat tissues

Multiple changes in metabolic levels could be useful for understanding physiological toxicity. To explore further risk factors for the convulsions induced by the interaction of nonsteroidal anti-inflammatory and new quinolone antimicrobial drugs, the effect of sparfloxacin, enoxacin, and felbinac on fatty acid metabolism and glucose concentrations in the liver, brain, and blood of rats was investigated. The levels of long-chain acyl-CoAs (C(18:1) and C(20:4)) in the liver and brain were decreased at the onset of convulsions induced by the coadministration of enoxacin with felbinac. Then, glucose concentrations in the liver and blood were decreased, whereas they were increased in a dose-dependant manner in the brain. However, the formation of acyl-CoAs and glucose levels in the liver, brain, and blood was not significantly influenced by enoxacin, felbinac, and sparfloxacin alone, respectively. The disturbance of both fatty acid metabolism and glucose levels might be associated with the increased susceptibility to convulsions, which may contribute to further understanding of the toxic effects associated with these drugs.     

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

The beta 2-adrenoceptor agonist, clenbuterol (initially 5 mg/kg), was found to significantly reduce plasma tyrosine and raise brain tryptophan levels (P less than 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 ED50 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 beta 2-antagonist, ICI 118,551, but not by the beta 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 beta 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.     

Evaluation of a new beta-adrenoceptor agonist, procaterol, based on metabolic responses in rats.

Intravenous injection of 5-(1-hydroxy-2-isopropylaminobutyl)-8-hydroxycarbostyril hydrochioride hemihydrate (procaterol) or isoproterenol into fasted rats caused increases in blood levels of glucose, lactate, free fatty acids (FFA), glycerol, immunoreactive insulin and cyclic AMP. Procaterolinduced alterations of these metabolic parameters, other than FFA, were durable; the increases were observable over a period longer than 2 hr, in contrast to a much shorter duration of isoproterenol-induced metabolic changes. These actions of procaterol were antagonized by propranolol, were observed in adrenodemedullated rats, and were enhanced by theophylline. It is thought therefore, that metabolic changes induced by procaterol are actually mediated via β-adrenoceptors, as are its pharmacological actions. Procaterol caused hyperlactacidemia at molar doses ten to one hundred times lower than those required for isoproterenol, trimetoquinol or salbutamol. A much higher dose of procaterol was required to increase blood levels of FFA, glycerol and insulin. In view of our findings that butoxamine, a selective β₂-adrenoceptor antagonist, antagonized the isoproterenol-induced blood lactate, while practolol, a selective ₁-antagonist, effectively inhibited the stimulatory actions of isoproterenol on FFA, glycerol and insulin, it is concluded that procaterol is a more selective ₂-adrenoceptor agonist than isoproterenol or trimetoquinol, and is more potent than salbutamol.     

Characterization of the rat parotid beta-adrenoceptor.

1 The effects of various beta-adrenoceptor agonists on amylase secretion from the rat parotid gland were studied by means of two different in vitro techniques. 2 The dose-response relation for each agonist was established, as also were the ED50 values. 3 All drugs appeared to act directly on the acinar cells, as reserpine-treatment did not abolish their secretagogic effects. 4 Two groups of agonists could be distinguished: one group consisting of adrenaline, noradrenaline and the B1-selective agonist prenalterol (H133/22) with a high enzyme discharge potency and a second group consisting of the beta 2-agonists, terbutaline and salbutamol, with a markedly lower effect. 5 The present data further support the theory that rat parotid acinar cells are supplied mainly with beta-adrenoceptors of the beta 1-subtype, similar to those present in heart and adipose tissue.