Brit. J. Pharmacol. (1964), 23, 184-200. THE EFFECTS OF ADRENALINE AND OTHER DRUGS AFFECTING CARBOHYDRATE METABOLISM ON CONTRACTIONS OF THE RAT DIAPHRAGM BY W. C. BOWMAN AND C. RAPER From the Department of Pharmacology, School of Pharmacy, University of London, Brunswick Square, London, W.C.1 (Received March 18, 1964) The ability of several drugs to restore directly elicited twitches of the rat diaphragm depressed by excess potassium chloride has been studied. The drugs found to be effective were sympathomimetic amines, insulin, glucagon, caffeine, theophylline, calcium chloride and hexosephosphates. The effects of the sympathomimetic amines and glucagon were blocked by P-receptor blocking agents. Phloridzin blocked the effect of insulin and depressed that of glucagon. The increase in twitch tension still occurred under anaerobic conditions and was not abolished by the glycolytic inhibitor, iodoacetate. All of the effective drugs are known to affect carbohydrate metabolism and the suggestion by Ellis (1955) that the effect on contractions may be a result of increased intracellular hexosephosphate levels is discussed. Adrenaline and some other sympathomimetic amines have long been known to cause an increase in the contractions of fast-contracting skeletal muscles, stimulated directly or through their motor nerves (see Bowman, Goldberg & Raper, 1962, for references). The mechanism underlying this potentiation is unknown, but Ellis, Davis & Anderson (1955) demonstrated that the relative potencies of a series of sympathomimetic amines in potentiating the contractions of the isolated diaphragm muscle of the rat and in stimulating glycogenolysis in this tissue were similar, suggesting that the two effects might be interrelated. Ellis & Beckett (1954) had previously shown on the same tissue that adrenaline retained its twitch-potentiating effect under anaerobic conditions and in the presence of the glycolytic inhibitor, iodoacetate. These results indicated that the action of adrenaline on the muscle contractions was dependent neither on oxidative metabolism, nor on the energy- yielding steps of the Embden-Myerhof pathway which, according to Bloom, Stetten & Stetten (1953), is the main pathway for carbohydrate metabolism in this tissue. Ellis (1955) therefore concluded that if the effect of adrenaline on contractions is related to stimulation of glycogenolysis, it must depend on the stage in which the intracellular content of hexosephosphates is increased. Ellis (1955) then showed that the addition of glucose-6-phosphate and fructose-1,6-diphosphate to the fluid bathing the isolated diaphragm muscle produced an increase in twitch tension like that produced by adrenaline, but glycero-p-phosphate did not. The suggestion was made by Ellis (1959) that the effect of adrenaline on the contractions might be the ADRENALINE ON THE RAT DIAPHRAGM 185 result of a change in the physicochemical environment of the contractile system brought about by a change in the cellular content of hexosephosphates. In the experiments described in this paper this suggestion has been further tested by comparing the effects of sympathomimetic amines with those of a number of other drugs known to influence carbohydrate metabolism. The experiments have been carried out on isolated diaphragm preparations of rats, in most cases depressed by excess potassium chloride, since in this condition the muscle is very sensitive to the potentiating action of adrenaline (Knox, McDowall & Montagu, 1951). METHODS Phrenic nerve-hemidiaphragm preparations from random-bred Wistar rats weighing 200 to 300 g were set up in a 50 ml. organ-bath containing Krebs-Henseleit solution at 32' C, according to the method of Bfilbring (1946). The composition of the Krebs-Henseleit solution was as follows (in g/l.): NaCl 6.95; KCl 0.34; CaCl2 0.28; KH2PO4 0.162; MgSO4 0.294; NaHCO3 2.1; and dextrose 2. In most experiments the mixture was continually gassed with 95% oxygen and 5% 'carbon dioxide, but when anaerobic conditions were required the oxygen was replaced by nitrogen. Each preparation was initially stimulated via the phrenic nerve with rectangular pulses of 100 /Asec duration and of three or four times the strength necessary to produce a maximal twitch. Tubocurarine, in a concentration sufficient to block neuro- muscular transmission completely (6 pgg/ml.) was then added to the reservoir of Krebs- Henseleit solution and the muscle was stimulated directly for the remainder of the experiment. Direct stimulation was applied between two silver pins embedded in the muscle near its origin in the ribs. The pins were also used to anchor the diaphragm to the electrode block. Twitches were elicited by stimulation at a frequency of 6 shocks/min with rectangular pulses of 1 msec duration and of a strength to produce contractions equal in amplitude to, or slightly greater than, maximal twitches produced by indirect stimulation. Stronger direct stimuli often produced greater contractions, probably because repetitive responses were elicited in an increasing number of muscle fibres. The electrodes on the phrenic nerve were left in position and indirect stimulation was occasionally applied throughout the experiment to check that curarization was complete. Contractions of the muscle were recorded on smoked paper by attaching the central tendon to a spring-loaded lever. Before studying the effects of drugs, the contractions of the diaphragm were usually partially depressed by the addition of 0.4 to 0.6 ml. of a 5% solution of potassium chloride. This produced an initial potentiation of the twitches followed by a slowly developing depres- sion (Hajdu & McDowall, 1949). The drug being tested was added when the twitches were depressed by 50 to 90%. After observing the effect of the drug, the bath fluid was changed two or three times. Normal contractions were then recorded for 10 to 15 min before a further depression was produced with potassium chloride solution and the effects of the same or of another drug were determined. In preparations used to study the effects of anoxia or of treat- ment with sodium iodoacetate, the optimal conditions outlined by Ellis & Beckett (1954) were used; the bath temperature was lowered to 270 C and the glucose concentration of the bath fluid was increased from 0.2 to 0.38%. Anaerobic conditions were established by stimulating the preparation at a frequency of 6 shocks/min while the bath fluid was gassed with 95% nitrogen and 5% carbon dioxide for at least 30 min before any drugs were added to the bath. The drugs used were: (-)-adrenaline (B.D.H.), (-)-noradrenaline bitartrate (Light & Co.), (-)-isoprenaline bitartrate (Wyeth), (±)-noradrenaline hydrochloride (Sterling Winthrop), (±)-isoprenaline sulphate (Bayer), (+)-adrenaline (Light & Co.), (+)-noradrenaline bitartrate (Sterling Winthrop), (±)-N-ethylnoradrenaline hydrochloride (Sterling Winthrop), dopamine hydrochloride (Light & Co.), N-isopropyldopamine hydrochloride (Sterling Winthrop), tyra- mine hydrochloride (Light & Co.), phenylethylamine (Winthrop), (-)-ephedrine hydrochloride (Light & Co.), (±)-amphetamine sulphate (Light & Co.), dichloroisoprenaline hydrochloride (Lilly), phentolamine (Ciba), pronethalol (I.C.I.), isopropylmethoxamine (Burroughs Wellcome, G 186 W. C. BOWMAN and C. RAPER 61-43), methoxamine hydrochloride (B.W. & Co.), phloridzin (Light & Co.), caffeine (B.D.H.), theophylline (B.D.H.), glucagon (Lilly), insulin (Burroughs Wellcome), disodium glucose-6- phosphate (Light & Co.), disodium glucose-l-phosphate (Light & Co.), monosodium fructose-l, 6-diphosphate (Light & Co.), L-thyroxine sodium (Light & Co.), 5-hydroxytryptamine creatinine sulphate (May & Baker), hydroxy-L-proline (Light & Co.) and L-lysine (Light & Co.). The doses of the sympathomimetic amines and of 5-hydroxytryptamine refer to the base. Doses of all other compounds refer either to the base or to the salt as indicated in the above list. Caffeine and theophylline were dissolved in 0.1 N-hydrochloric acid, and phloridzin was made up as a suspension. The phloridzin dissolved completely when added to the bath fluid. In all experiments control injections of the diluent alone were completely without effect. RESULTS Adrenaline is known to exert two independent effects on skeletal muscle con- tractions, one on the neuromuscular junction and one on the muscle fibres them- selves (Bowman et al., 1962). These experiments were concerned with the latter action and, to avoid the complications of simultaneous actions at the neuromuscular junction, all drugs were studied during direct stimulation of the fully curarized preparation. Both potassium chloride and adrenaline exert anticurare actions (Wilson & Wright, 1936) and, since the effects of the drugs were studied in the presence of excess potassium chloride, it was necessary to ascertain that neuro- muscular transmission remained blocked throughout. Fig. 1 illustrates an initial 10 min dTC KCI Adr W W Fig. 1. Twitches of the diaphragm elicited once every 10 sec. by alternate periods of indirect (I) and direct (D) stimulation. Contractions downwards. At dTC, tubocurarine (6 ,tg/ml.), at KC1, potassium chloride (0 5 mg/ml.) and at Adr, (-)-adrenaline (20 ng/ml.) were added. At W, the bath was washed three times with normal Krebs-Henseleit solution. Tubocurarine com- pletely abolished the responses to indirect stimulation and these were not restored by potassium chloride or by adr naline. Twitches in response to indirect stimulation reappeared only after the second washing period. Time calibration