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Stimulation of RBL-2H3 m1 mast cells through the IgE receptor with antigen, or through a G protein-coupled receptor with carbachol, leads to the rapid appearance of phosphothreonine in nonmuscle myosin heavy chain II-A (NMHC-IIA). We demonstrate that this results from phosphorylation of Thr-1940 by calcium/calmodulin-dependent protein kinase II (CaM kinase II), activated by increased intracellular calcium. The phosphorylation site in rodent NMHC-IIA was localized to the carboxyl terminus of NMHC-IIA distal to the coiled-coil region, and identified as Thr-1940 by site-directed mutagenesis. A fusion protein containing the NMHC-IIA carboxyl terminus was phosphorylated by CaM kinase II in vitro, while mutation of Thr-1940 to Ala eliminated phosphorylation. In contrast to rodents, in humans Thr-1940 is replaced by Ala, and human NMHC-IIA fusion protein was not phosphorylated by CaM kinase II unless Ala-1940 was mutated to Thr. Similarly, co-transfected Ala --Thr-1940 human NMHC-IIA was phosphorylated by activated CaM kinase II in HeLa cells, while wild type was not. In RBL-2H3 m1 cells, inhibition of CaM kinase II decreased Thr-1940 phosphorylation, and inhibited release of the secretory granule marker hexosaminidase in response to carbachol but not to antigen. These data indicate a role for CaM kinase stimulation and resultant threonine phosphorylation of NMHC-IIA in RBL-2H3 m1 cell activation.

Phosphorylation of the alpha-subunits of the branched chain alpha-keto acid and the pyruvate dehydrogenases was measured in mitochondria isolated from rat hearts perfused in the presence of [32P]phosphate and various substrates. Mitochondrial isolations were accomplished rapidly under conditions which would preclude the interconversion of these two enzyme complexes between their active (dephosphorylated) and inactive (phosphorylated) forms. Inactivation of the branched chain complex and the concomitant incorporation of [32P]phosphate into the alpha-chain of the dehydrogenase subcomponent were observed in hearts perfused with glucose (10 mM) or pyruvate (10 mM). alpha-Ketoisocaproate infusion caused a 15-fold activation and dephosphorylation of the branched chain dehydrogenase, while pyruvate dehydrogenase remained inactive and phosphorylated under these conditions. Both dehydrogenase components were partially activated, and most of the [32P]phosphate was removed from their respective alpha-subunits during substrate-free perfusion. Both enzyme complexes were activated and dephosphorylated completely during infusion of dichloroacetate (1 mM) and glucose (10 mM).

1-O-Alkyl-2-acetyl-sn-glyceryl 3-phosphorylcholine or acetylglyceryl ether phosphorylcholine (AGEPC) stimulated glycogenolysis in perfused livers from fed rats at concentrations as low as 10(-11) M. At the lower AGEPC concentrations, e.g. 2 X 10(-10) M, a single transient phase of enhanced hepatic glucose output was elicited upon infusion of this agonist. At higher concentrations, e.g. 2 X 10(-8) M, a sharp transient spike of glucose output was observed, followed by a stable elevated steady state rate of glucose output until the AGEPC infusion was terminated. Increased rates of lactate and acetoacetate output and a diminished hepatic oxygen consumption were characteristic of the response of the livers to AGEPC at 2 X 10(-10) M. Neither alpha- nor beta-adrenergic antagonists blocked the glycogenolytic response of AGEPC. Repeated infusion of AGEPC led to homologous desensitization of the response, but the response of the liver to the alpha-adrenergic agonist, phenylephrine, or to glucagon, subsequent to AGEPC stimulation, was unaffected. Increasing the period of perfusion between successive additions of AGEPC, from 7 to 30 min, resulted in an increased glycogenolytic response to this agonist. When the perfusate calcium concentration was reduced from 1.25 to 0.05 mM, the glycogenolytic response to AGEPC was markedly diminished; calcium efflux from the liver following stimulation with AGEPC was not observed. The data presented in this study illustrate a potent agonist effect of AGEPC on the glycogenolytic system in the rat liver.

Infusion of heat-aggregated immunoglobulin G (IgG) into perfused livers from fed rats resulted in a dose-dependent but transient stimulation of hepatic glucose output. Oxygen consumption and lactate production by the liver also were stimulated, while the lactate/pyruvate ratio of the effluent perfusate was increased. Upon return of hepatic glucose output to the basal rate following immune aggregate stimulation, a second infusion of immune aggregate caused little or no increase in glucose output, indicating desensitization of the response. Infusion of immune aggregate into rat livers also resulted in the appearance of platelet-activating factor activity in the effluent perfusate. Synthetic platelet-activating factor or acetylglyceryl ether phosphorylcholine (AGEPC) has been demonstrated to be a potent glycogenolytic agonist in the perfused liver (Buxton, D.B., Shukla, S. D., Hanahan, D. J., and Olson, M.S. (1984) J. Biol. Chem. 259, 1468-1471). The glycogenolytic response of the liver to immune aggregate infusion was not desensitized by prior infusion of AGEPC. Co-infusion of the cyclooxygenase inhibitor indomethacin prevented the immune aggregate-induced glycogenolytic response but had no effect on AGEPC-stimulated hepatic glucose output. It is suggested that the mechanism by which aggregated IgG activates hepatic glycogenolysis requires the synthesis of AGEPC by the liver by a pathway which involves the metabolism of arachadonic acid.

The regulation of the pyruvate dehydrogenase multienzyme complex was investigated during alpha-adrenergic stimulation with phenylephrine in the isolated perfused rat liver. The metabolic flux through the pyruvate dehydrogenase reaction was monitored by measuring the production of 14CO2 from infused [1-14C] pyruvate. In livers from fed animals perfused with a low concentration of pyruvate (0.05 mM), phenylephrine infusion significantly inhibited the rate of pyruvate decarboxylation without affecting the amount of pyruvate dehydrogenase in its active form. Also, phenylephrine caused no significant effect on tissue NADH/NAD+ and acetyl-CoA/CoASH ratios or on the kinetics of pyruvate decarboxylation in 14CO2 washout experiments. Phenylephrine inhibition of [1-14C]pyruvate decarboxylation was, however, closely associated with a decrease in the specific radioactivity of perfusate lactate, suggesting that the pyruvate decarboxylation response simply reflected dilution of the labeled pyruvate pool due to phenylephrine-stimulated glycogenolysis. This suggestion was confirmed in additional experiments which showed that the alpha-adrenergic-mediated inhibitory effect on pyruvate decarboxylation was reduced in livers perfused with a high concentration of pyruvate (1 mM) and was absent in livers from starved rats. Thus, alpha-adrenergic agonists do not exert short term regulatory effects on pyruvate dehydrogenase in the liver. Furthermore, the results suggest either that the rat liver pyruvate dehydrogenase complex is insensitive to changes in mitochondrial calcium or that changes in intramitochondrial calcium levels as a result of alpha-adrenergic stimulation are considerably less than suggested by others.

Infusion of platelet-activating factor (alkyl acetylglycerophosphocholine (AGEPC] into isolated perfused rat livers caused a dose-dependent, transient increase in portal vein pressure, indicative of constriction of the hepatic vasculature. A close correlation was observed between the changes in portal pressure and concomitant transient increases in hepatic glucose output. The two processes displayed similar dose dependence and were attenuated to a similar extent by reducing the perfusate calcium concentration. Reducing the perfusate free calcium concentration to 1 nM by co-infusion of EGTA did not abolish completely the hepatic responses to AGEPC. Verapamil inhibited both the hemodynamic and glycogenolytic responses to AGEPC in a dose-dependent fashion; the IC50 was approximately 10 microM at an AGEPC concentration of 6.6 X 10(-11) M. Also, both responses displayed similar degrees of tachyphylaxis in response to repeated short infusions of AGEPC. Measurement of glycogen phosphorylase a in extracts from freeze-clamped livers demonstrated a rapid increase in phosphorylase a in response to infusion of AGEPC. A small but significant increase in whole tissue ADP was found in response to AGEPC (2 X 10(-8) M); cAMP levels were not changed by AGEPC infusion. It is concluded that glycogenolysis in the perfused liver in response to AGEPC may be a result of the hemodynamic effects of AGEPC, rather than a direct effect of the phospholipid mediator on the hepatocyte.

Acetylglyceryl ether phosphorylcholine (AGEPC), or 1-O-Alkyl-2-acetyl-sn-glyceryl 3-phosphorylcholine, has been shown to have a dramatic influence on phosphoinositide metabolism in isolated rat hepatocytes and upon glycogenolysis in the intact perfused rat liver. Addition of 5 X 10(-10) M AGEPC to 32Pi-labeled rat hepatocytes resulted in up to a 30 to 40% decrease in the [32Pi]phosphatidylinositol 4,5-bisphosphate within 10 s. The 32P content of phosphatidylinositol 4-phosphate decreased approximately 25% within 60 s, while a 5 to 8% decrease in [32P]phosphatidylinositol was observed only after 2 to 5 min of incubation of hepatocytes with AGEPC. Infusion of AGEPC (2 X 10(-10) M) into perfused livers resulted in a 3-fold increase in the glucose output in the effluent perfusate within 2 min. Interestingly, when a 500-fold higher concentration, i.e. 1 X 10(-7) M, of 1-O-alkyl-sn-glyceryl 3-phosphorylcholine or the stereoisomer 3-O-alkyl-2-acetyl-sn-glyceryl 1-phosphorylcholine was infused, no increase in the hepatic glucose output was seen. These observations lead to the conclusion that AGEPC exerts a potent influence on the polyphosphoinositide metabolism and glycogenolysis in rat liver and establishes the liver as an ideal system in which to conduct a detailed inquiry into the biochemical mechanism(s) responsible for the biological action of this unusual phospholipid.