Rat liver 6-phosphofructokinase (ATP-D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was partially purified free of interfering enzymes by a rapid procedure. Fructose 2,6-bisphosphate, at micromolar concentrations, greatly stimulated the enzyme by increasing its affinity for fructose 6-phosphate and relieving the inhibition by ATP. Its action was synergistic with that of AMP. As a stimulator of liver phosphofructokinase, fructose 2,6-bisphosphate was approximately 1000-and 2500-fold more efficient than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate, respectively. The concentration at which a half-maximal effect was obtained with the hexose bisphosphates was dependent upon the experimental conditions. It was relatively high at physiological concentrations of substrates, AMP, and Pi, and under these conditions the positive effect of fructose 1,6-bisphosphate was no longer detectable. This was probably due to the negative effect of fructose 1,6-bisphosphate as a reaction product inhibitor. It is concluded that fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate controls, in association with other effectors, the activity of phosphofructoldnase in the liver.There is a general agreement in the literature that 6-phosphofructokinase (ATP:D-fructose-6-phosphate l-phosphotransferase, EC 2.7.1.11) plays a major role in the control ofglycolysis in nearly all types of cells (1-6). The activity of this enzyme is controlled by several metabolites, most notably its two substrates, fructose 6-phosphate and ATP. The rate of the reaction is a sigmoidal function of fructose 6-phosphate concentration, and the saturation curve is shifted to the left in the presence of positive effectors, among which fructose 1,6-bisphosphate (7,8), the product of the reaction, and AMP are currently believed to play a major role. Negative effectors such as ATP and citrate have the opposite effect. The rate ofthe reaction plotted as a function of ATP concentration exhibits a typical inhibition by excess substrate. This inhibition is relieved by the positive effectors and by large concentrations of fructose 6-phosphate, whereas it is intensified by the negative effectors.Fructose 2,6-bisphosphate is a newly recognized, extremely effective, positive effector of liver and muscle phosphofructokinase (9). Its concentration in the liver is greatly increased under conditions in which glycolysis is active and is decreased by glucagon (10). It has also been found to inhibit, at micromolar concentrations, liver and muscle fructose-1,6-bisphosphatase (11). It therefore appears to be a major regulator of both glycolysis and gluconeogenesis in the liver. The purpose of the present work was to investigate the action of fructose 2,6-bisphosphate on the kinetics of liver phosphofructokinase and to compare it to that of its isomers fructose 1,6-bisphosphate and glucose 1,6-bisphosphate and that of AMP. When the effect of fructose 1,6-bisphosphate was examined, pyruvate kinase (20 ,g/ml) and lactate dehydrogenase (25 Mg/ ml) were u...

1 Prostacyclin (PGI 2 ) possesses various physiological functions, including modulation of nociception, inflammation and cardiovascular activity. Elucidation of these functions has been hampered by the absence of selective IP receptor antagonists. 2 Two structurally distinct series of IP receptor antagonists have been developed: 4,5-dihydro-1H-imidazol-2-yl)-[4-(4-isopropoxy-benzyl)-phenyl]-amine (RO1138452) and R-3-(4-fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid (RO3244794). 3 RO1138452 and RO3244794 display high affinity for IP receptors. In human platelets, the receptor affinities (pK i ) were 9.370.1 and 7.770.03, respectively; in a recombinant IP receptor system, pK i values were 8.770.06 and 6.970.1, respectively. 4 Functional antagonism of RO1138452 and RO3244794 was studied by measuring inhibition of carbaprostacyclin-induced cAMP accumulation in CHO-K1 cells stably expressing the human IP receptor. The antagonist affinities (pK i ) of RO1138452 and RO3244794 were 9.070.06 and 8.570.11, respectively. 5 Selectivity profiles for RO1138452 and RO3244794 were determined via a panel of receptor binding and enzyme assays. RO1138452 displayed affinity at I 2 (8.3) and PAF (7.9) receptors, while RO3244794 was highly selective for the IP receptor: pK i values for EP 1 (o5), EP 3 (5.38), EP 4 (5.74) and TP (5.09). 6 RO1138452 (1-10 mg kg , p.o.) significantly reduced carrageenan-induced mechanical hyperalgesia and edema formation. RO3244794 (1 and 10 mg kg À1 , p.o.) also significantly reduced chronic joint discomfort induced by monoiodoacetate. 7 These data suggest that RO1138452 and RO3244794 are potent and selective antagonists for both human and rat IP receptors and that they possess analgesic and anti-inflammatory potential.

Drugs that are clinically effective (mexiletine and desipramine) or ineffective (fluoxetine) in the treatment of human neuropathic pain were evaluated for efficacy in rat models involving central sensitization (i.e., formalin model and the L5/L6 spinal nerve ligation model of neuropathic pain) using tests that differ in stimulus modality: noxious chemical stimulus (formalin model) as well as noxious (pin prick) and innocuous mechanical stimuli (application of von Frey filaments). Mexiletine (10-100 mg/kg, s.c.) significantly (P < 0.05) attenuated hyperalgesia in formalin-treated (60 mg/kg and 100 mg/kg) and neuropathic rats (100 mg/kg) as well as tactile allodynia in neuropathic rats (100 mg/kg). Desipramine (1-100 mg/kg, s.c.), on the other hand, reduced hyperalgesia significantly (P < 0.05) in formalin-treated (3, 10, 30 and 100 mg/kg) and neuropathic rats (10 mg/kg and 100 mg/kg), but did not reduce tactile allodynia in the neuropathic rats. Fluoxetine (3-30 mg/kg, s.c.) did not inhibit either hyperalgesia or allodynia in any of the tests employed. Fluoxetine, which is relatively ineffective in reducing neuropathic pain in humans, was also ineffective in reducing hyperalgesia and allodynia associated with central sensitization in rats. Thus, drugs which are effective in reducing human neuropathic pain consistently attenuated hyperalgesia in formalin-treated or neuropathic rats. Desipramine also distinguished mechanical hyperalgesia from tactile allodynia in rats rendered neuropathic by spinal nerve ligation. These data are consistent with the hypothesis that the neuronal mechanisms underlying these two manifestations of neuropathic pain are different.

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