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Catecholamines and Ca2+ mediate an increase in reactive-thiol status of phosphofructokinase, associated with an increase in activation of the enzyme. A correlation was observed between the number of reactive thiols and activation (r = 0.878; P less than 0.001) for 14 different treatments, with approx. 1 group per 85,000-Mr subunit becoming available over the range from the lowest to the highest state of activation.

Epinephrine treatment of the perfused rat heart led to an increase in glucose uptake, detritiation of [5-3H] glucose, glycogenolysis, and the formation of lactate. The change in the rate of formation of 3H2O from [5-3H]glucose was slower to develop (commencing at approximately 30 s) than changes in cyclic AMP concentration, hexose-6-P concentration, and the phosphorylase a/(a + b) ratio which were maximal at 24 s. Epinephrine plus propranolol (alpha-adrenergic combination) treatment of the perfused heart also led to increases in glucose uptake, detritiation of [5-3H]glucose, and the formation of lactate, but these occurred without significant changes in cyclic AMP concentration, hexose-6-P concentration, or the phosphorylase a/(a + b) ratio. Half-maximal stimulation of glucose uptake occurred at 0.2 microM epinephrine, 1.5 microM methoxamine, and 1 microM isoproterenol. The increase in glucose uptake mediated by 1 microM epinephrine was blocked by 10 microM prazosin but unaffected by 10 microM propranolol. The increase in glucose uptake mediated by 10 microM epinephrine plus 10 microM propranolol was partly blocked by yohimbine and completely blocked by prazosin. A role for Ca2+ in the adrenergic regulation of glucose uptake was indicated by the sensitivity of the epinephrine dose curve to Ca2+ and the dependence of epinephrine on Ca2+. In addition the increases in glucose uptake mediated by 1 microM epinephrine, 1 microM epinephrine plus 10 microM propranolol, 1 microM isoproterenol, and by 10 mM CaCl2 were each blocked by the Ca2+ channel blocker nifedipine (1 microM). It is concluded that Ca2+-dependent alpha- and beta-adrenergic receptor mechanisms are present in rat heart for controlling glucose uptake. At submicromolar levels of epinephrine the predominant receptors utilized appear to be alpha 1.

Phosphofructokinase was determined at low substrate concentration using a new isotopic assay in extracts of perfused rat heart. Epinephrine treatment of the perfused heart resulted in an activation of the enzyme. Half-maximal activation of phosphofructokinase occurred at 5 X 10(-7) M epinephrine, which was approximately that required to produce half-maximal activation of phosphorylase. Time course studies indicated that epinephrine-mediated changes in beating rate, cyclic AMP concentration, and phosphorylase a activity were maximal at 1 to 2 min and preceded maximal activation of phosphofructokinase by approximately 3 min. the activated form of the enzyme as expressed in heart extracts was sensitized to the activators, cyclic AMP, AMP, glucose 1,6-bisphosphate, and fructose 1,6-bisphosphate. Passage of control extract that was untreated, activated by AMP, or inhibited by citrate through Sephadex G-25 columns gave eluate activities approaching control extract values. The epinephrine-activated form of the enzyme remained activated following similar treatment. The data suggest that epinephrine mediates a modification of phosphofructokinase that is independent of changes in intracellular effector concentration.

The activity ratio of phosphofructokinase in perfused rat heart and its activation by epinephrine was examined in non-obese, fat-fed obese, and genetically obese rats. For non-obese colony rats there was an age-dependent increase in the activity ratio of phosphofructokinase from 0.2 at 40 days to 0.4 at mature age (greater than 200 days). Epinephrine (10 microM) treatment of the heart for 5 min increased the ratio at all ages but the proportional increase diminished with age. For mature-age lean Zucker rats carrying the genetic determinant for obesity the results were similar to those obtained for comparable non-obese colony rats. For fat-fed obese rats the activity ratio of phosphofructokinase at 200 days of age was 0.2 and was increased to 0.6 by epinephrine treatment. For mature-age obese Zucker rats the activity ratio was 0.2 and no significant response to epinephrine occurred. The activity ratio of glycogen phosphorylase and its response to epinephrine (beta-adrenergic receptor mediated) in heart was unaffected by age, diet or the gene for obesity. The present findings indicate a specific defect in the adrenergic regulatory mechanism for phosphofructokinase in genetically obese rats.

The apparent insensitivity of phosphofructokinase to activation by adrenaline in hearts from genetically obese Zucker (fa/fa) rats [Patten, Filsell & Clark (1982) Metab. Clin. Exp. 31, 1137-1141] was examined in detail. Perfusion of hearts from obese (fa/fa) animals with medium containing 1nM-100 microM-adrenaline for 4 min did not significantly activate phosphofructokinase, but fully activated glycogen phosphorylase. Activation of phosphofructokinase occurred in hearts from lean (Fa/?) animals, with half-maximal activation at 10nM-adrenaline. Binding characteristics of cardiac alpha 1-adrenergic receptors were comparable between lean and obese animals. Properties of phosphofructokinase from obese and lean hearts were similar. High extracellular Ca2+ concentration, which activated phosphofructokinase in hearts from lean animals and phosphorylase in all hearts, failed to activate phosphofructokinase in hearts from obese rats. The data indicate that the failure of adrenaline to activate phosphofructokinase in hearts from obese (fa/fa) rats results from a post-receptor defect in the as-yet unidentified Ca2+-dependent phosphofructokinase-activating reactions.