Biochemical SocietyTransactions ( 1 992) 20 Clenbuterol induces a propranolol-sensitive accumulation of actin message in rat skeletal muscle PETER A. MACLENNAN and RICHARD H.T. EDWARDS Department of Medicine University of Liverpool, P.O. Box 147, Liverpool L69 3BX. Certain beta-adrenergic agonists exert dramatic muscle protein anabolic effects when administered to a variety of species (1). It has been suggested that these effects may be mediated, not via the betaadrenergic receptor, but through a novel signalling mechanism (2). This hypothesis was based on the observation that administration of propranolol in combination with the beta agonist clenbuterol did not abolish the muscle growth effects of the agonist. Our subsequent studies demonstrated, however, that the experimental protocol employed by the previous investigators did not block fully the beta effects of clenbuterol on muscle. Moreover, administration of blocker in quantities sufficient to abolish the effect of the agonist on intramuscular CAMP concentrations also abolished the effects of clenbuterol on muscle growth (3). We have explored the question of how betaadrenergic stimulation might influence muscle growth in our system by examining the effects of clenbuterol and propranolol on muscle messenger RNA (mRNA) concentrations. Groups of female Wistar rats were treated twice daily either with vehicle only, clenbuterol only, propranolol only or with both clenbuterol and propranolol. The doses and routes of administration were as described previously (3). After treatment for 7 days the animals were anaesthetised with sodium pentobarbital and gastrocnemius muscle samples were rapidly frozen in liquid nitrogen. RNA was isolated by an adaption of the method described in (4) with a recovery of 82-107%. Recoveries were calculated from the A260 of the isolated material and the total RNA/wet weight ratio (evaluated by standard methods eg 3). The quality of the isolated RNA was routinely assessed by electrophoresis and from the A260/A280 ratio. Northern blots were prepared by formaldehyde gel electrophoresis and transfer to nylon membranes ( 5 ) , dot blots by applying aliquots of RNA directly to the membrane. The blots were hybridised at 37°C for 72 h with a "P labelled probe prepared from mouse skeletal muscle actin cDNA. Autoradiography of the Northern blots and comparison with RNA standards demonstrated that more than 90% of radioactivity was hybridised to a single RNA species of an appropriate base length thereby demonstrating the specificity of our hybridisation protocol. The quantity of probe bound to dot blots was measured by scintillation counting. Highly linear relationships between the quantity of RNA applied to each dot and the binding of j r P were obtained. Figura 1: Tha affects

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clenbuterol and proprinolol on actin mRNA T

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