Glycogenolysis is catalysed by glycogen phosphorylactivation of phosphorylase. The NO effect was not addi-ase, an enzyme that exists in two interconvertible tive to maximal stimulation of glycogenolysis (7.7 { 0.2 forms, the active phosphorylated a-form and the inac- strictly depends on covalent activation of phosphoryl-The requirement for activation of phosphorylase was also evidenced by the ineffectiveness of NO in phosphor-ase, 1 by a specific phosphorylase kinase. The kinase is ylase-kinase-deficient livers of gsd/gsd rats. The NO activated by cyclic adenosine monophosphate (cAMP)-effect was blocked by co-administration of cyclooxy-dependent protein kinase, or by direct interaction of genase inhibitors (50 mmol/L ibuprofen, 50 mmol/L in-Ca 2/ with a calmodulin subunit. Ultimately, the catadomethacin, or 2 mmol/L aspirin), suggesting a media-lytic expression of phosphorylase a is under substrate tory role of prostanoids from nonparenchymal cells. control, 2 because cytosolic levels of inorganic phosphate This conclusion was confirmed by the fact that NO did (P i ) fail to saturate substrate demands of activated not activate phosphorylase in isolated hepatocytes. phosphorylase a. Moreover, NO was no longer glycogenolytic in liversControl of hepatic glycogenolysis involves intricate 3-7 Evidence has accumulated inhibited the basal glucose production. This coincided that prostanoids are engaged in the control of glycogenwith an increased elution of cyclic guanosine mono-olysis in liver. Although endotoxin, 8 platelet-activating phosphate (cGMP). Inhibition of glycogenolysis by NO factor, 9 and phorbol esters 3,8 stimulate glycogenolysis under these conditions was blocked by 1 mmol/L the-in intact liver, these compounds are ineffective when ophylline, suggestive for involvement of cGMP-stimu-tested directly on isolated hepatocytes. In the intact lated cAMP phosphodiesterase. However, we could not liver, cyclooxygenase inhibitors suppress stimulation confirm that an increase in cGMP resulted in a drop of prostanoid synthesis and activation of phosphorylase in cAMP. In conclusion, NO recruits opposing mechaby the former agents.10-12 Prostanoids may be directly involved in covalent activation of hepatocellular phosphorylase. 8,13 Indirectly, eicosanoids may trigger vaso- lytic response to platelet-activating factor is sup-

We perfused livers from fed rats with a balanced salt solution containing 1 mmol/L glucose. Under these conditions a low steady rate of glycogenolysis was observed (approximately 1.7 micromol glucose equivalents/g/min; 20% of the maximal glycogenolytic activity). Nitric oxide (NO) transiently stimulated hepatic glucose production. A maximal response (on average doubling basal glucose output) was observed with 34 micromol/L NO. The same concentration of nitrite (NO2-) was ineffective. Half-maximal effects were seen at 8 to 10 micromol/L NO, irrespective of the flow direction (portocaval or retrograde). This glycogenolytic response to NO corresponded to a partial activation of phosphorylase. The NO effect was not additive to maximal stimulation of glycogenolysis (7.7 +/- 0.2 micromol hexose equivalents/g/min; n = 4) by 100 micromol/L dibutyryl cyclic adenosine monophosphate (Bt2cAMP). The requirement for activation of phosphorylase was also evidenced by the ineffectiveness of NO in phosphorylase-kinase-deficient livers of gsd/gsd rats. The NO effect was blocked by co-administration of cyclooxygenase inhibitors (50 micromol/L ibuprofen, 50 micromol/L indomethacin, or 2 mmol/L aspirin), suggesting a mediatory role of prostanoids from nonparenchymal cells. This conclusion was confirmed by the fact that NO did not activate phosphorylase in isolated hepatocytes. Moreover, NO was no longer glycogenolytic in livers perfused with Ca2+-free medium, in agreement with the known mediatory role of Ca2+ in prostanoid-mediated responses. Surprisingly, in Ca2+-free medium NO inhibited the basal glucose production. This coincided with an increased elution of cyclic guanosine monophosphate (cGMP). Inhibition of glycogenolysis by NO under these conditions was blocked by 1 mmol/L theophylline, suggestive for involvement of cGMP-stimulated cAMP phosphodiesterase. However, we could not confirm that an increase in cGMP resulted in a drop in cAMP. In conclusion, NO recruits opposing mechanisms with respect to modulation of basal hepatic glycogenolysis. In the presence of Ca2+, activation of phosphorylase with stimulation of glycogenolysis dominates. Cyclooxygenase inhibitors abolish this effect. Activation by NO of the cyclooxygenase in nonparenchymal cells is a distinct possibility. In the absence of Ca2+, inhibition of basal glycogenolysis becomes observable. It remains to be established whether this results from cGMP-mediated stimulation of hydrolysis of cAMP.

We report on the 13C NMR visibility of the C-1 glycosidic carbon of alpha-particulate glycogen in perfused rat liver. We used rats fed ad libitum, animals refed after a 48 h fast with a sucrose supplement with or without glucocorticoid treatment, and gsd/gsd rats with a hepatic glycogen storage disease due to phosphorylase kinase deficiency. Thus we studied a wide range of glycogen levels (25-140 mg/g liver). All livers were perfused with 15 mM glucose, to maintain constant glycogen levels. Failure to activate glycogen phosphorylase ensures stable glycogen levels in gsd/gsd livers. Natural abundance 13C NMR signals were calibrated against a phantom containing a fixed amount of glycogen. Accumulated free induction decays were analysed after Fourier transformation by numerical integration, or by direct analysis of the signal in the time domain using a non-iterative method based on singular value decomposition. NMR quantification of the glycogen correlated well with the chemical determination over the whole concentration range. However, the precision (reproducibility) of glycogen determinations (be it by chemical methods or by NMR spectroscopy) may pose problems. Authors should be encouraged to report systematically on the precision of their methods.