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The objective of the study was to evaluate the neuroprotective effects of bacoside A and bromelain against dichlorvos-incited toxicity. Healthy 6–8-week old, male Swiss mice were administered subacute doses of dichlorvos (40 mg/kg bw), bacoside A (5 mg/kg bw) and bromelain (70 mg/kg bw). AChE, BChE, GABA, serotonin and total protein content and their expressions were used for determination of toxic action of dichlorvos. Protective effects of bacoside A and bromelain were evaluated on the same parameters. Exposure to dichlorvos leads to significant decline in activities of AChE (p

The history of the research on the glucose receptor represents the main matter of the present chapter. A short introduction mentions two reviews published in 1972 and introduces the concept of a pancreatic islet B-cell glucoreceptor. Attention is then drawn to the anomeric specificity of glucose-stimulated insulin secretion and its possible metabolic determinants. The verbal care required for the use of the glucoreceptor word is duly underlined. Examples of the modulation of key enzyme activity in insulin-producing pancreatic islet cells by D-glucose itself or one of its metabolites are then provided in the framework of the interaction of phosphorylase a with D-glucose anomers, the activation of glucokinase by a fructose-1-phosphate-sensitive regulatory protein, and the activation of phosphofructokinase by fructose-2,6-bisphosphate. The riddle of L-glucose pentaacetate insulinotropic action, the effects of artificial sweeteners on insulin release, and last but not least, the presence of the sweet taste TIR3 receptor in pancreatic insulin-producing islet cells are eventually and duly also considered.

A series of linear glucan saccharides (GS) with defined quantity and degree of polymerization (DP) were synthesized from α-d-glucose 1-phosphate (α- d -Glc 1-P) by phosphorylase-a. The GS product fractions with average DP 11, 22, 38, 52, 60, 70, and 79 were measured by HPSEC-ELSD system. Then the same seven fractions were resolved into individual peaks with DP: 6–14, 10–32, 27–55, 37–67, 44–75, 49–83 and 53–89 by HPAEC-PAD system. Results showed that measurement of α- d -Glc 1-P amount consuming during GS synthesis by both systems enable calculation of reaction yield. The reaction yield for the 24 h biosynthesis of the GS product was 25.3% (measured by HPSEC-ELSD) or 29.1% (measured by HPAEC-PAD). The HPSEC-ELSD and HPAEC-PAD systems were also successfully used for phosphorylase-a activity measurement in order to perform its kinetic characterization. This study established feasible systems for preparation of various sizes of the GS with defined DP and quantity as well as characterization of phosphorylase-a kinetics.

survived freezing exposure at 21.5 to 22.5uC for 6-h periods (80% survival), but uniformly died when exposed to these same temperatures for 24-h freezing bouts. Hepatic glucose levels and phosphorylase a activities increased significantly during freezing, but hepatic glucose levels during freezing remained low, only reaching levels approximating those prior to freezing in freeze-tolerant species. Moreover, muscle glucose and hepatic glycogen levels did not vary with freezing, suggesting little mobilization of glucose from hepatic glycogen stores during freezing, contrasting with patterns in freeze-tolerant frogs. Temperatures in soil cracks and burrows potentially used for hibernacula were variable, with some sites remaining above the freezing point of the body fluids throughout the winter, some sites dropping below the freezing point for only short periods, and some sites dropping below the freezing point for extended periods. These data suggest that cricket frogs in South Dakota, as in other portions of their range, survive overwinter by locating hibernacula that prevent freezing, although their toleration of short freezing bouts may expand the range of suitable hibernacula. These data also suggest that overwinter mortality may be high at the northern range boundary and might limit cricket frogs from expanding their range northward.

Zusammenfassung Es wurde die Fasertypenentwicklung der Ferkelmuskulatur in den ersten 12 Lebenstagen mit histochemischen Methoden untersucht. Dabei wurden folgende Ergebnisse erzielt: Am 1. Lebenstag sind mittels der ATP-ase-Reaktion und der α-GDH-Reaktion jeweils 2 Fasertypen (Typ I und Typ II) sicher unterscheidbar. Nach saurer Prainkubation last sich zu diesem Zeitpunkt bei der ATP-ase auch der Subtyp II C nachweisen. Mit dem 6. Lebenstag last auch die Phosphorylasereaktion die Differenzierung von 2 Fasertypen zu. Am 9. Tag konnen anhand der ATP-ase-Reaktion (incl. saurer Prainkubation) 4 verschiedene Fasertypen dargestellt werden (Typ I; Typ II A; II B und II C). Wahrend am 1. Lebenstag samtliche Fasern Fett in feintropfiger Verteilung aufweisen, zeigt sich mit zunehmendem Alter eine Konzentrierung der Fetttropfchen auf die Typ-I- und Typ-II-A-Fasern. Summary Histological studies on the development of skeletal muscle of piglets in the neonatal period The fibre type development of piglet musculature in the first 12 days was studied by histochemical methods. The following results were obtained: on the first day after birth two fibre types (Type 1 and Type 2) were detected with certainty by the use of the ATP-ase reaction and the α-GDH reaction. After acid pre-incubation sub-type 11 C was also detected at this stage by ATP-ase. On the sixth day of life the phosphorylase reaction also allowed differentiation of the two types. On the ninth day the ATP-ase reaction (including acid pre-incubation) showed 4 different fibre types (Type I; Type II A, II B and II C). Whereas at one day old all the fibres showed the presence of fat in fine droplet distribution, with increasing age a concentration of the fat droplets in Type I and Type II A fibres was found. Resume Recherches histochimiques sur le developpement de la maculature du squelette du porcelet dans la periode neonatale On a examine avec des methodes histochimiques le developpement des fibres musculaires du porcelet pendant les douze premiers jours de vie. Les resultats suivants ont ete obtenus: 2 types de fibres (type I et II) ont ete differencies avec certitude le premier jour avec la reaction de l'ATP-ase et la reaction α-GDH. Un sous-type II c a egalement ete mis en evidence dans cette periode avec l'ATP-ase apres une preincubation acide. La reaction a la phosphorylase a permis la differenciation de deux types de fibres le sixieme jour. Quatre types de fibres differents sont apparus au neuvieme jour avec la reaction de l'ATP-ase (y compris une preincubation acide): Type I, II A, IIB et II C. Alors que toutes les fibres presentaient le premier jour une repartition de graisse en fines gouttelettes, il est apparu une concentration des gouttelettes graisseuses dans les types I et II avec l'âge. Resumen Estudios histoquimicos sobre el desarrollo de la musculatura del esqueleto del lechon en el periodo de recien nacido Se examino con metodos histoquimicos el desarrollo de tipos de fibras musculares de los lechones en los 12 primeros dias de vida. Los resultados obtenidos fueron los siguientes: El primer dia de vida se pueden distinguir con seguridad mediante la reaccion de ATFasa y de la α-GDH 2 tipos cada vez de fibras (tipos I y II). Tras preincubacion acida tambien se puede identificar en este tiempo en la reaccion de ATFasa el subtipo II C. El dia 6° de vida tambien permite la reaccion de fosforilasa la diferenciacion de 2 tipos de fibras. El dia 9° se pueden revelar con ayuda de la reaccion de ATFasa (incl. preincubacion acida) 4 tipos diferentes de fibras (tipos I; II A, II B y II C). Mientras que el primer dia de vida presentan todas las fibras una distribucion de grasa en gotas finisimas, al aumentar la edad aparece una concentracion de gotitas adiposas en las fibras de tipos I y II A.

Glycogen phosphorylase a activity, the contents of adenine nucleotides and isometric tension were measured in rabbit colon smooth muscle after exposure to 2,4-dinitrophenol (DNP). DNP caused a dose-dependent increase in phosphorylase a activity, with an ED50 value of 1 X 10(-4)M. Since adenine nucleotides, especially 5-AMP by inhibiting the phosphorylase a to b conversion, might increase the phosphorylase a activity, a time-response study was undertaken in order to analyse the time course of changes in the adenine nucleotide content and phosphorylase a activity after addition of DNP. The latter activity was increased after only 1 min. of incubation. At this time no changes were found in the contents of ATP, ADP, 5-AMP or CP. Not until the phosphorylase a activity had reached its maximum after 5-7.5 min. did the 5-AMP content increase. The phosphorylase a activity then started to decline, but the 5-AMP content continued to rise. The effect of DNP on isometric tension was also studied to test whether the cytoplasmic Ca2+ concentration was increased. An increase in tension was observed 5 min. after administration of the drug and was maximal at 15 min. The results seem to dispute against 5-AMP as being a mediator for the DNP-stimulated increased in phosphorylase a activity in smooth muscle. The finding that DNP elicited a contraction of rabbit colon supports the earlier suggestion that the cytoplasmic Ca2+ concentration was increased (Pettersson 1983).

It was observed in earlier studies that when the phosphorylase a activity of rabbit colon smooth muscle was increased by anoxia or 2,4-dinitrophenol (DNP), the calcium content of the mitochondrial fraction decreased. Despite this, under basal conditions there was no significant increase in the Ca2+ content in the fraction consisting of microsomes and cytoplasm. In the present study it was therefore investigated whether mitochondrial Ca2+ released by anoxia and DNP is translocated from the smooth muscle cells into the extracellular fluid. The Ca2+ efflux from rabbit colon into a Ca2+-free Krebs-Ringer bicarbonate buffer was measured by using a Ca2+-selective electrode. Both anoxia and DNP (6.6 × 10-5M) increased the Ca2+ efflux from the smooth muscle cells. The local anaesthetic D-mepivacaine, at a concentration of 1×10 3M, reduced the increase in Ca2+ efflux and simultaneously enhanced the anoxic or DNP-induced rise in phosphorylase a activity. The replacement of external Na+ by choline was found to reduce the basal Ca2+ efflux and to moderately increase the phosphorylase a activity. On addition of DNP to the Na+-free and Ca2+-free buffer there was no significant change in the Ca2+ efflux, but the increase in phosphorylase a activity was greater than in the physiological buffer containing 137 mM Na+. These observations support the suggestion that anoxia and DNP, by releasing Ca2+ from the mitochondria, increase the phosphorylase a activity of smooth muscle.

The action of glycogen phosphorylase (GP) is essentially reversible, although GP is generally classified as a glycogen-degrading enzyme. In this study, we developed a highly sensitive and convenient assay for GP activity by analysing its chain-lengthening action on a fluorogenic maltooligosaccharide derivative in a glucose-1- phosphate-rich medium. Characterization of the substrate specificity of GP using pyridylaminated (PA-) maltooligosaccharides of various sizes revealed that a maltotetraosyl (Glc 4 ) residue comprising the non-reducing-end of a PA-maltooligosaccharide is indispensable for the chain-lengthening action of GP, and PA-maltohexaose is the most suitable substrate for the purpose of this study. By using a high-performance liquid chromatograph equipped with a fluorescence spectrophotometer, PA-maltoheptaose produced by the chain elongation of PA-maltohexaose could be isolated and quantified at 10 fmol. This method was used to measure the GP activities of crude and purified GP preparations, and was demonstrated to have about 1,000 times greater sensitivity than the spectrophotometric orthophosphate assay.

Activation of either coexisting beta1- or beta2 -adrenoceptors with noradrenaline or adrenaline, respectively, causes maximum increases of contractility of human atrial myocardium. Previous biochemical work with the beta2 -selective agonist zinterol is consistent with activation of the cascade beta2 -adrenoceptors-->Gsalpha-protein-->adenylyl cyclase-->cAMP-->protein kinase (PKA)-->phosphorylation of phospholamban, troponin I, and C-protein-->hastened relaxation of human atria from nonfailing hearts. However, in feline and rodent myocardium, catecholamines and zinterol usually do not hasten relaxation through activation of beta2 -adrenoceptors, presumably because of coupling of the receptors to Gi protein. It is unknown whether the endogenously occurring beta2 -adrenoceptor agonist adrenaline acts through the above cascade in human atrium and whether its mode of action could be changed in heart failure. We assessed the effects of (-)-adrenaline, mediated through beta2 -adrenoceptors (in the presence of CGP 20712A 300 nM to block beta1 -adrenoceptors), on contractility and relaxation of right atrial trabecula obtained from nonfailing and failing human hearts. Cyclic AMP levels were measured as well as phosphorylation of phospholamban, troponin I, and protein C with Western blots and the back-phosphorylation procedure. For comparison, beta1 -adrenoceptor-mediated effects of (-)-noradrenaline were investigated in the presence of ICI 118,551 (50 nM to block beta2 -adrenoceptors). The positive inotropic effects of both (-)-noradrenaline and (-)-adrenaline were accompanied by reductions in time to peak force and time to reach 50% relaxation. (-)-Adrenaline caused similar positive inotropic and lusitropic effects in atrial trabeculae from failing hearts. However, the inotropic potency, but not the lusitropic potency, of (-)-noradrenaline was reduced fourfold in atrial trabeculae from heart failure patients. Both (-)-adrenaline and (-)-noradrenaline enhanced cyclic AMP levels and produced phosphorylation of phospholamban, troponin I, and C-protein to a similar extent in atrial trabeculae from nonfailing hearts. The hastening of relaxation caused by (-)-adrenaline together with the PKA-catalyzed phosphorylation of the three proteins involved in relaxation, indicate coupling of beta2 -adrenoceptors to Gs protein. The phosphorylation of phospholamban at serine16 and threonine17 evoked by (-)-adrenaline through beta2 -adrenoceptors and by (-)-noradrenaline through beta1 -adrenoceptors was not different in atria from nonfailing and failing hearts. Activation of beta2 -adrenoceptors caused an increase in phosphorylase a activity in atrium from failing hearts further emphasizing the presence of the beta2 -adrenoceptor-Gsalpha-protein pathway in human heart. The positive inotropic and lusitropic potencies of (-)-adrenaline were conserved across Arg16Gly- and Gln27Glu-beta2 -adrenoceptor polymorphisms in the right atrium from patients undergoing coronary artery bypass surgery, chronically treated with beta1 -selective blockers. The persistent relaxant and biochemical effects of (-)-adrenaline through beta2 -adrenoceptors and of (-)-noradrenaline through beta1 -adrenoceptors in heart failure are inconsistent with an important role of coupling of beta2 -adrenoceptors with Gialpha-protein in human atrial myocardium.

(1) Changes in the activity of hepatic glycogen phosphorylase a + b and a (GPh-ase a + b and a ), liver glycogen content and blood glucose level during acclimation to moderate high environmental temperature (35±1 °C) were studied. (2) Experiments were carried out on adult fed Wistar rats of both sexes, previously given either short-term (1, 4 and 7 days) or long-term (14, 21, 30 and 60 days) exposure to high environmental temperature. The controls were continuously kept at room temperature (20±2 °C). (3) The results obtained showed that in the period of short-term exposure the liver glycogen content was decreased significantly (after the first and fourth days in male rats and after first day in female rats) and the GPh-ase a activity increased (after first day in male rats and after first, fourth and seventh day in female rats). Long-term exposure caused significant increased liver glycogen content (beginning from the 14th day in male rats and the 21st day in female rats) until the end of the acclimation period (60 days). The elevated activity of GPh-ase a persists after 14th day of exposure only in female rats while there are no significant changes over the rest of the acclimation period in both sexes. There were no significant changes in total GPh-ase activity during the whole period of exposure. Blood glucose level was significantly decreased throughout the whole period of acclimation to high environmental temperature, in both sexes (except in the 1 day exposed groups). (4) The increased activity of hepatic GPh-ase a and decreased glycogen content suggested that the short-term exposure to heat stimulates the glycogenolytical processes. Decreased blood glucose level, and elevated liver glycogen content ( r = - 0.7467 in male and r = - 0.6548 in female rats) suggested that prolonged exposure to high environmental temperature stimulated glycogenogenesis, without changes in the GPh-ase activity.

Glucokinase and phosphorylase both have a high control strength over hepatocyte glycogen metabolism and are potential therapeutic targets for type 2 diabetes. We tested whether combined phosphorylase inactivation and glucokinase activation is a more effective strategy for controlling hepatic glycogen metabolism than single-site targeting. Activation of glucokinase by enzyme overexpression combined with selective dephosphorylation of phosphorylase-a by an indole carboxamide that favors the T conformation of phosphorylase caused a greater stimulation of glycogen synthesis than the sum of either treatment alone. This result is explained by the complementary roles of elevated glucose-6-phosphate (G6P; a positive modulator) and depleted phosphorylase-a (a negative modulator) in activating glycogen synthase and also by synergistic inactivation of phosphorylase-a by glucokinase activation and the indole carboxamide. Inactivation of phosphorylase-a by the indole carboxamide was counteracted by 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside, which is metabolized to an AMP analog; this effect was reversed by G6P. Our findings provide further evidence for the inverse roles of G6P and AMP in regulating the activation state of hepatic phosphorylase. It is proposed that dual targeting of glucokinase and phosphorylase-a enables improved potency and efficacy in controlling hepatic glucose metabolism.

Glycogen phosphorylase (GP(a)) is a specific target for the design of inhibitors and may prevent glycogenolysis under high glucose conditions in type II diabetes. The carboxamides first reported by Hoover D. J. et al. (J. Med. Chem. 1998, 41, 2934-2938) are one of the major classes of GP(a) inhibitors other than glucose derivatives. The recent, X-ray crystallographic analyses (Oikonomakos et al. Biochim. Biophys. Acta 2003, 1647, 325-332) have revealed a distinct mechanism of action for these inhibitors, which bind at a new allosteric site away from the inhibitory and catalytic sites. To elucidate the essential structural and physicochemical requirements responsible for binding to the GP(a) enzyme and to develop predictive models, CoMFA and docking studies have been carried out on a series of indole-2-carboxamide derivates. The CoMFA model developed using pharmacophoric alignments and hydrogen-bonding fields demonstrated high predictive ability against the training (r2 = 0.98, q2 = 0.68) and the test set (r2pred = 0.85). Further the superimposition of PLS coefficient contour maps from CoMFA with the GP(a) active site (PDB: 1lwo) has shown a high level of compatibility.

The protein serine/threonine phosphatase (PP) type 2A family consists of three members: PP2A, PP4, and PP6. Specific rabbit and sheep antibodies corresponding to each catalytic subunit, as well as a rabbit antibody recognizing all three subunits, were utilized to examine the expression of these enzymes in select rat tissue extracts. PP2A, PP4, and PP6 catalytic subunits (PP2A(C), PP4(C), and PP6(C), respectively) were detected in all rat tissue extracts examined and exhibited some differences in their levels of expression. The expression of alpha4, an interacting protein for PP2A family members that may function downstream of the target of rapamycin (Tor), was also examined using specific alpha4 sheep antibodies. Like the phosphatase catalytic subunits, alpha4 was ubiquitously expressed with particularly high levels in the brain and thymus. All three PP2A family members, but not alpha4, bound to the phosphatase affinity resin microcystin-Sepharose. The phosphatase catalytic subunits were purified to apparent homogeneity (PP2A(C) and PP4(C)) or near homogeneity (PP6(C)) from bovine testes soluble extracts following ethanol precipitation and protein extraction. In contrast to PP2A(C), PP4(C) and PP6(C) exhibited relatively low phosphatase activity towards several substrates. Purified PP2A(C) and native PP2A in cellular extracts bound to GST-alpha4, and co-immunoprecipitated with endogenous alpha4 and ectopically expressed myc-tagged alpha4. The interaction of PP2A(C) with alpha4 was unaffected by rapamycin treatment of mammalian cells; however, protein serine/threonine phosphatase inhibitors such as okadaic acid and microcystin-LR disrupted the alpha4/PP2A complex. Together, these findings increase our understanding of the biochemistry of alpha4/phosphatase complexes and suggest that the alpha4 binding site within PP2A may include the phosphatase catalytic domain.

High glucose concentration suppresses hepatic glycogenolysis by allosteric inhibition and dephosphorylation (inactivation) of phosphorylase-a. The latter effect is attributed to a direct effect of glucose on the conformation of phosphorylase-a. Although glucose-6-phosphate (G6P), like glucose, stimulates dephosphorylation of phosphorylase-a by phosphorylase phosphatase, its physiological role in regulating glycogenolysis in intact hepatocytes has not been tested. We show in this study that metabolic conditions associated with an increase in G6P, including glucokinase overexpression and incubation with octanoate or dihydroxyacetone, cause inactivation of phosphorylase. The latter conditions also inhibit glycogenolysis. The activity of phosphorylase-a correlated inversely with the G6P concentration within the physiological range. The inhibition of glycogenolysis and inactivation of phosphorylase-a caused by 10 mmol/l glucose can be at least in part counteracted by inhibition of glucokinase with 5-thioglucose, which lowers G6P. In conclusion, metabolic conditions that alter the hepatic G6P content affect glycogen metabolism not only through regulation of glycogen synthase but also through regulation of the activation state of phosphorylase. Dysregulation of G6P in diabetes by changes in activity of glucokinase or glucose 6-phosphatase may be a contributing factor to impaired suppression of glycogenolysis by hyperglycemia.

The role of cAMP in beta(2)-adrenoceptor signaling and its functional relevance in adult rat heart has been the subject of considerable controversy. Therefore, we investigated the beta(2)-adrenoceptor pathways in both adult cardiomyocytes and in the intact hearts of Wistar rats with respect to protein kinase A (at Ser16)-, the key event in shortening of relaxation time, and CaM kinase II (at Thr17)-dependent phospholamban phosphorylation.Contractile and cellular beta(1)/beta(2)-adrenergic responses were studied in parallel on the same perfused rat heart. (-)Isoproterenol and the beta(2)-adrenergic agonists zinterol and procaterol were used to discriminate the beta-adrenoceptor subtype-related actions.Beta(2)-adrenoceptor stimulation induces protein kinase A-dependent phospholamban phosphorylation in both adult cardiomyocytes and in adult hearts of rats. The beta(2)-adrenoceptor-mediated shortening of relaxation time in the heart correlates with Ser16 phosphorylation. Adenosine elicited antiadrenergic action on both beta(1)- and beta(2)-adrenergic signaling cascades by reducing the phosphorylation status of phospholamban. Only beta(1)-adrenoceptor stimulation produced significant CaM kinase II-related Thr17 phosphorylation, troponin I phosphorylation and activation of phosphorylase a.Our findings clearly show that beta(2)-adrenoceptor signaling is coupled to phospholamban phosphorylation and shortening of relaxation time in the adult rat heart.

To develop our knowledge of specificity determinants for protein phosphatase-1, mutants of phosphorylase b have been converted to phosphorylase a and examined for their efficacy as substrates for protein phosphatase-1. Mutants focused on the N-terminal primary sequence surrounding the phosphoserine (R16A, R16E, and I13G) and at a site that interacts with the phosphoserine in phosphorylase a, (R69K and R69E). The success achieved studying protein kinase substrate specificity with peptide substrates has not extended to protein phosphatases. Protein phosphatases are believed to recognize higher order structure in substrates in addition to the primary sequence surrounding the phosphoserine or threonine. Peptide studies with protein phosphatase-1 have revealed a preference for basic residues N-terminal to the phosphoserine. Arginine 16 in phosphorylase a may be a positive determinant. In this work, protein phosphatase-1 preferred the positive charge on arginine 16. R16A exhibited a similar K(m) but reduced V(max), and R16E had an increased K(m) and a decreased V(max) when compared to phosphorylase. I13G had a similar K(m) but an increased V(max). The R69 mutants were also dephosphorylated preferentially over phosphorylase a. The K(m) for R69K was unchanged but had a higher V(max). R69E exhibited the most changes, with a 4-fold increase in K(m) and a 10-fold increase in V(max). These results suggest that proper presentation of the phosphoserine can greatly affect the rate of dephosphorylation.

We have demonstrated previously that 24 h of ethanol treatment potentiates angiotensin II (ANG II)–stimulated p42/p44 mitogen-activated protein kinase (MAPK) activity in hepatocytes. This potentiation of p42/p44 MAPK by ethanol exhibited agonist selectivity. To compare the effects of acute (24 h) versus chronic (6 weeks) ethanol treatment, ANG II-induced intracellular signaling was examined in (1) rat hepatocytes treated with ethanol for 24 h and (2) hepatocytes obtained from rats fed ethanol for 6 weeks. In hepatocytes obtained from rats fed ethanol for 6 weeks, ANG II-stimulated phosphorylase a was reduced, and this activity was calcium dependent and p42/p44 MAPK independent. Surprisingly, ANG II-stimulated p42/p44 MAPK activation was not affected in hepatocytes obtained from rats fed ethanol chronically (6 weeks). However, chronic (6 weeks) ethanol treatment decreased ethanol potentiation of p42/p44 MAPK by about 56.3% ± 3.6% for p42 MAPK and 61.3% ± 11.7% for p44 MAPK. Furthermore, ethanol had no effect on the expression of angiotensinogen and c- myc mRNA in hepatocytes. A decrease in ANG II-activated phosphorylase a , but not in p42/p44 MAPK activation, after chronic (6 weeks) ethanol treatment leads to the conclusion that they may not be dependent on each other.

This study aimed to investigate the effect of long-term oral nicotine administration on insulin resistance in an animal model of obesity. Eight-week-old male Zucker fatty rats (ZFRs) were administered nicotine tartrate dihydrate (4.6 mg/kg/day) in the drinking water. The control group was pair-fed. The body weights and food intake over 8 weeks were similar in both groups. Plasma glucose levels at 3, 6, 9, 12, and 15 min after insulin administration (0.5 U/kg) in the nicotine group were significantly lower than those in the control group. The calculated KITT value for the nicotine group was significantly higher than that for the control group. Wet weight of the liver in the nicotine group was significantly lower than that in the control group. Transaminases and histological examination of the liver revealed no alteration by nicotine administration. Glycogen, glycogen synthetase activity and gluconeogenesis in the liver in the nicotine group were significantly lower than those in the control group. Phosphorylase-a activity of the liver in the nicotine group was significantly higher than that in the control group. Glycogen, glycogen synthetase, and phosphorylase-a activity of skeletal muscle were similar in both groups. These results suggest that long-term oral nicotine administration may reduce insulin resistance in obese diabetic rats through a reduced hepatic glucose release and, in part, contribute to lowering blood glucose levels. D 2002 Elsevier Science B.V. All rights reserved.

Two distinct allosteric inhibitors of glycogen phosphorylase, 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) and CP-91149 (an indole-2-carboxamide), were investigated for their effects on the phosphorylation state of the enzyme in hepatocytes in vitro. CP-91149 induced inactivation (dephosphorylation) of phosphorylase in the absence of hormones and partially counteracted the phosphorylation caused by glucagon. Inhibition of glycogenolysis by CP-91149 can be explained by dephosphorylation of phosphorylase a. This was associated with activation of glycogen synthase and stimulation of glycogen synthesis. DAB, in contrast, induced a small degree of phosphorylation of phosphorylase. This was associated with inactivation of glycogen synthase and inhibition of glycogen synthesis. Despite causing phosphorylation (activation) of phosphorylase, DAB is a very potent inhibitor of glycogenolysis in both the absence and presence of glucagon. This is explained by allosteric inhibition of phosphorylase a, which overrides the increase in activation state. In conclusion, two potent phosphorylase inhibitors exert different effects on glycogen metabolism in intact hepatocytes as a result of opposite effects on the phosphorylation state of both phosphorylase and glycogen synthase.

Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue, 270 proteins were identified, and 11 different crystallins were found to contain a total of 73 sites of modification. Modifications identified in the crystallin proteins included Ser, Thr, and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, and Met, Tyr, and Trp oxidations. The method presented will be useful in discovering co- and posttranslational modifications of proteins.

Dietary creatine supplementation has been used to improve skeletal muscle performance. However, dietary creatine manipulation also affects glucose homeostasis. The aim of this study was to investigate the effect of dietary creatine supplementation on insulin secretion, glucose tolerance, and quadriceps glycogen metabolism in chow-fed rats. Forty-eight rats in total were divided into 2 groups of 24 and were then subdivided into 6 groups of 8. Rats were fed a diet supplemented with 0% (CON) or 2% (CREAT) creatine for 2, 4, or 8 weeks. At these 3 time points an oral glucose tolerance test was performed. Two days later, rats were euthanized and the pancreas and quadriceps muscles were collected. The peak insulin response to a glucose challenge was significantly elevated after both 4 (CON 327 +/- 72 v CREAT 735 +/- 140 pmol/L, P =.01) and 8 (CON 248 +/- 48 v CREAT 588 +/- 136 pmol/L, P =.02) weeks. Fasting insulin levels were also increased by creatine supplementation for 8 weeks (CON 78 +/- 14 v CREAT 139 +/- 14 pmol/L, P =.01). Glucose tolerance was not affected until 8 weeks at which point the peak plasma glucose was elevated in the creatine supplemented group (CON 10.1 +/- 0.6 v CREAT 13.5 +/- 1.5 mmol/L, P =.05). A significant increase in pancreatic total creatine content was seen in supplemented animals at 2 (CON 1.2 +/- 0.1 v CREAT 2.7 +/- 0.1 micromol/g wet wt, P =.005), 4 (CON 1.5 +/- 0.2 v CREAT 2.7 +/- 0.3 micromol/g wet wt, P =.02) and 8 (CON 1.5 +/- 0.1 v CREAT 2.6 +/- 0.1 micromol/g wet wt, P =.005) weeks, whereas no change in quadriceps total creatine or glycogen content was observed at any individual time point. This study shows that prolonged creatine supplementation induces abnormalities in pancreatic insulin secretion and changes in glucose homeostasis.

Host cell invasion by Toxoplasma gondii tachyzoites relies on many coordinated processes. The tachyzoite participates in invasion by providing an actomyosin-dependent force driving it into the nascent parasitophorous vacuole as well as by releasing molecules which contribute to the vacuole membrane. Exposure to type 1/2A protein phosphatase inhibitors, okadaic acid (OA) or tautomycin significantly impairs tachyzoite invasiveness. Furthermore, the tachyzoite extract contains a biochemically active type 1, but not a type 2A, serine-threonine protein phosphatase, which is immunologically related to eukaryotic phosphatase type 1 catalytic subunit. When tachyzoite extracts are incubated with a monoclonal antibody reactive to human type 1 catalytic subunit, other T. gondii molecules are coprecipitated among which one competes with the inhibitory toxin OA. Finally, in vitro phosphate labelling assays indicate that the biochemically characterized PP1 activity controls the phosphorylation of several proteins. Taken together, these data strongly suggest that the type 1 phosphatase activity detected in invasive tachyzoites is implicated in the control of the host cell invasion process.

The aim of this study was to understand by which intrahepatic mechanism metformin (Met) may inhibit basal hepatic glucose production (HGP) in type 2 diabetes. We studied rats that were fed for 6 weeks a high-fat (HF) diet, supplemented (HF-Met) or not (HF) with Met (50 mg · kg−1 · day−1). Basal HGP, assessed by 3-[3H]glucose tracer dilution, was lower by 20% in HF-Met rats compared with HF-rats: 41.6 ± 0.7 vs. 52 ± 1.5 μmol · kg−1 · min−1 (means ± SE, n = 5; P < 0.01). Glucose-6 phosphatase (Glc6Pase) activity, assayed in a liver lobe freeze-clamped in situ, was lower by 25% in HF-Met rats compared with HF-rats (7.9 ± 0.4 vs. 10.3 ± 0.9 μmol · min−1 · g−1 wet liver; P < 0.05). Glucose-6 phosphate and glycogen contents, e.g., 42 ± 5 nmol/g and 3.9 ± 2.4 mg/g, respectively, in HF-rats were dramatically increased by three to five times in HF-Met rats, e.g., 118 ± 12 nmol/g and 19.6 ± 4.6 mg/g (P < 0.05 and P < 0.01, respectively). Glucose-6 phosphate dehydrogenase activity was increased in HF-Met compared with HF rats (1.51 ± 0.1 vs. 1.06 ± 0.08 μmol · min−1 · g−1; P < 0.01). Intrahepatic lactate concentration tended to be lower in the Met-group (−30%; NS), whereas plasma lactate concentration was higher in HF-Met rats (1.59 ± 0.15 mmol/l) than in HF rats (1.06 ± 0.06 mmol/l; P < 0.05). We concluded that Met decreases HGP in insulin-resistant HF-fed rats mainly by an inhibition of hepatic Glc6Pase activity, promoting glycogen sparing. Additional mechanisms might involve the diversion of glucose-6 phosphate into the pentose phosphate pathway and an inhibition of hepatic lactate uptake.

This study aimed to determine physiologically relevant kinetic and allosteric effects of Pi, AMP, ADP, and caffeine on isolated skeletal muscle glycogen phosphorylase a (Phos a). In the absence of effectors, Phos a had Vmax= 221 ± 2 U/mg and Km= 5.6 ± 0.3 mM Piat 30°C. AMP and ADP each increased Phos a Vmaxand decreased Kmin a dose-dependent manner. AMP was more effective than ADP (e.g., 1 μM AMP vs. ADP: Vmax= 354 ± 2 vs. 209 ± 8 U/mg, and Km= 2.3 ± 0.1 vs. 4.1 ± 0.3 mM). Both nucleotides were relatively more effective at lower Pilevels. Experiments simulating a range of contraction (exercise) conditions in which Pi, AMP, and ADP were used at appropriate physiological concentrations demonstrated that each agent singly and in combination influences Phos a activity. Caffeine (50–100 μM) inhibited Phos a( Km∼8–14 mM, ∼40–50% reduction in activity at 2–10 mM Pi). The present in vitro data support a possible contribution of substrate (Pi) and allosteric effects to Phos a regulation in many physiological states, independent of covalent modulation of the percentage of total Phos in the Phos a form and suggest that caffeine inhibition of Phos a activity may contribute to the glycogen-sparing effect of caffeine.

D-Gluco- and D-xylopyranosylidene-spiro-hydantoins and -thiohydantoins were prepared from the parent sugars in a six-step, highly chemo-, regio-, and stereoselective procedure. In the key step of the syntheses C-(1-bromo-1-deoxy-beta-D-glycopyranosyl)formamides were reacted with cyanate ion to give spiro-hydantoins with a retained configuration at the anomeric center as the major products. On the other hand, thiocyanate ions gave spiro-thiohydantoins with an inverted anomeric carbon as the only products. On the basis of radical inhibition studies, a mechanistic rationale was proposed to explain this unique stereoselectivity and the formation of C-(1-hydroxy-beta-D-glycopyranosyl)formamides as byproducts. Enzyme assays with a and b forms of muscle and liver glycogen phosphorylases showed spiro-hydantoin 12 and spiro-thiohydantoin 14 to be the best and equipotent inhibitors with K(i) values in the low micromolar range. The study of epimeric pairs of D-gluco and D-xylo configurated spiro-hydantoins and N-(D-glucopyranosyl)amides corroborated the role of specific hydrogen bridges in binding the inhibitors to the enzyme.

The present study examines the reactivity of the glial metabolic enzyme, glycogen phosphorylase, within the amygdala and bed nucleus of the stria terminalis. Reactivity for phosphorylase a, the active form of glycogen phosphorylase, was higher in all parts of the medial amygdaloid nucleus, in the medial division of the central amygdaloid nucelus, in the anterior amygdaloid area and in the bed nucleus of stria terminalis than in all parts of the lateral amygdaloid nucleus, the anterior cortical amygdaloid nucleus, the posteromedial and posterolateral cortical amygdaloid nuclei, the intercalated nucleus of the amygdala, main part and the intercalated nuclei. A greater degree of phosphorylase a reactivity was also observed in the basolateral amygdaloid nucleus, anterior and posterior parts, and in the basomedial amygdaloid nucleus, anterior part, while other parts of these nuclei were less reactive. Reactivity attributed to total glycogen phosphorylase enzyme, phosphorylase a+phosphorylase b activated by AMP, was higher and homogeneous across the amygdala. Phosphorylase a patterns are likely to reflect differences in the contribution of glycogenolysis to the metabolic support of cells in the amygdala and bed nucleus of the stria terminalis. Possible relationships to local neuronal activity and to differences in glycogenolytic neuromodulatory input are discussed.

Fenugreek (Trigonella foenum-graecumL. seed) is a food with traditional medicinal use in diabetes. Beneficial effects have been demonstrated in diabetic animals and both insulin-dependent and non-insulin-dependent diabetic subjects. Effects of a lipid extract A, crude ethanolic extract B, further sub-fractions of B (saponin-free C, saponin D and sapogenin E) and a gum fibre fraction F on intestinal sodium-dependent glucose uptake were investigatedin vitrousing rabbit intestinal brush border membrane vesicles. All fractions except A inhibited glucose-uptake at 0.33 and/or 3.3mg/mL (p< 0.001). Greatest inhibition was observed with fractions D and E. Diosgenin and trigonelline (compounds reported in fenugreek)also inhibited glucose-uptake (IC50values approximately 3mg/ml, equivalent to 8mM and 19mM respectively) but did not account for the activity of the crude extracts. Fenugreek extracts had no effect on basal levels of glycogen phosphorylase a (HGPa) activity in rat hepatocyte suspensions. However fractions C and E caused a marginal but statistically significant inhibition (18.9 and 15.1% respectively,p< 0.05) of glucagon induction of this enzyme suggesting a glucagon-antagonist effect. Diosgenin (1.65mg/ml; 4mM) inhibited glucagon-induced HGPa activity by 20% (p< 0.05), and was more effective than trigonelline (non significant inhibition of 9.4% at 1.65mg/ml, 10mM).

Caffeine, an allosteric inhibitor of glycogen phosphorylase a (GPa), has been shown to act synergistically with the potential antidiabetic drug (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarboxylate (W1807). The structure of GPa complexed with caffeine and W1807 has been determined at 100K to 2.3 A resolution, and refined to a crystallographic R value of 0.210 (Rfree = 0.257). The complex structure provides a rationale to understand the structural basis of the synergistic inhibition between W1807 and caffeine. W1807 binds tightly at the allosteric site, and induces substantial conformational changes both in the vicinity of the allosteric site and the subunit interface which transform GPa to the T'-like state conformation already observed with GPa-glucose-W1807 complex. A disordering of the N-terminal tail occurs, while the loop of polypeptide chain containing residues 192-196 and residues 43'-49', from the symmetry related subunit, shift to accommodate W1807. Caffeine binds at the purine inhibitor site by intercalating between the two aromatic rings of Phe285 and Tyr613 and stabilises the location of the 280s loop in the T state conformation.

pRB, a negative-growth regulatory protein, is a demonstrated substrate for type 1 serine/threonine protein phosphatases (PP1). In a recent report from this laboratory, we demonstrated that select forms of phosphorylated as well as hypophosphorylated pRB can be found complexed with the alpha-isotype of PP1 (PP1alpha). This complex can also be observed when PP1 is rendered catalytically dead by toxin inhibition. These data suggested to us that pRB may bind to PP1 at one or more sites other than the catalytically active one on the enzyme and that such binding may play a role other than bringing the substrate into contact with the enzyme to facilitate catalysis. To address this possibility we utilized a series of pRB deletion mutants and coprecipitation studies to map the pRB domain involved in binding to PP1. Together with competition assays using in vivo expression of SV40 T-antigen, we show here that the carboxyl-terminal region of pRB is both necessary and sufficient for physical interaction with PP1. Subsequent biochemical analyses demonstrated inhibition of PP1 catalytic activity toward the standard substrate phosphorylase a when this enzyme is bound to pRB containing this region. K(m) and V(max) calculations revealed that pRB binds to PP1 in a non-competitive manner. These data support the notion that pRB, in addition to being a substrate for PP1, also functions as a PP1 inhibitor. The significance of this finding with respect to the functional importance of this interaction is discussed.

Injection of excitotoxins, such as quinolinic acid (QA), into the striatum has been extensively used as an experimental model of Huntington's disease, while injection of 6-hydroxydopamine (6-OHDA) into the dopaminergic nigrostriatal pathway provides a well established model of Parkinson's disease. In the present study, we have examined the metabolic changes induced by an intrastriatal injection of QA or 6-OHDA using histochemical staining for the metabolic markers cytochrome oxidase (COx) and active glycogene phosphorylase (GPa). Intrastriatal injection of QA produced major changes in COx (decrease of staining) and GPa (increase of staining, except in the core of the lesion where the staining was virtually absent) histochemistry at the level of the striatum and of most of the other basal ganglia nuclei. Although attenuated over time, these changes persisted up to one year after the lesion. On the contrary, after the intrastriatal injection of 6-OHDA (which induces only a partial lesion of the nigrostriatal pathway), we did not observe any remarkable changes in COx or GPa staining. This study illustrates the discrepancies between the morphological changes and metabolic changes that are induced when using these experimental models of neurodegenerative disorders.

There is increasing evidence that endogenous glycogen depletion may affect excitation–contraction (E–C) coupling events in vertebrate skeletal muscle. One approach employed in physiological investigations of E–C coupling involves the use of mechanically skinned, single fibre preparations obtained from tissues stored under paraffin oil, at room temperature (RT: 20–24°C) and 4°C for several hours. In the present study, we examined the effect of these storage conditions on the glycogen content in three muscles frequently used in research on E–C coupling: rat extensor digitorum longus (EDL) and soleus (SOL) and toad iliofibularis (IF). Glycogen content was determined fluorometrically in homogenates prepared from whole muscles, stored under paraffin oil for up to 6 h at RT or 4°C. Control muscles and muscles stored for 0.5 and 6 h were also analysed for total phosphorylase (Phostotal) and phosphorylase a (Phos a) activities. No significant change was observed in the glycogen content of EDL and SOL muscles stored at RT for 0.5 h. In rat muscles stored at RT for longer than 0.5 h, the glycogen content decreased to 67.6% (EDL) and 78.7% (SOL) of controls after 3 h and 25.3% (EDL) and 37.4% (SOL) after 6 h. Rat muscles stored at 4°C retained 79.0% (EDL) and 92.5% (SOL) of glycogen after 3 h and 75.2% (EDL) and 61.1% (SOL) after 6 h. The glycogen content of IF muscles stored at RT or 4°C for 6 h was not significantly different from controls. Phostotal was unchanged in all muscles over the 6 h period, at both temperatures. Phos a was also unchanged in the toad IF muscles, but in rat muscles it decreased rapidly, particularly in EDL (4.1-fold after 0.5 h at RT). Taken together these results indicate that storage under paraffin oil for up to 6 h at RT or 4°C is accompanied by minimal glycogen loss in toad IF muscles and by a time- and temperature-dependent glycogen loss in EDL and SOL muscles of the rat.

Background —Recent studies of β-adrenergic receptor (β-AR) subtype signaling in in vitro preparations have raised doubts as to whether the cAMP/protein kinase A (PKA) signaling is activated in the same manner in response to β 2 -AR versus β 1 -AR stimulation. Methods and Results —The present study compared, in the intact dog, the magnitude and characteristics of chronotropic, inotropic, and lusitropic effects of cAMP accumulation, PKA activation, and PKA-dependent phosphorylation of key effector proteins in response to β-AR subtype stimulation. In addition, many of these parameters and L-type Ca 2+ current ( I Ca ) were also measured in single canine ventricular myocytes. The results indicate that although the cAMP/PKA-dependent phosphorylation cascade activated by β 1 -AR stimulation could explain the resultant modulation of cardiac function, substantial β 2 -AR–mediated chronotropic, inotropic, and lusitropic responses occurred in the absence of PKA activation and phosphorylation of nonsarcolemmal proteins, including phospholamban, troponin I, C protein, and glycogen phosphorylase kinase. However, in single canine myocytes, we found that β 2 -AR–stimulated increases in both I Ca and contraction were abolished by PKA inhibition. Thus, the β 2 -AR–directed cAMP/PKA signaling modulates sarcolemmal L-type Ca 2+ channels but does not regulate PKA-dependent phosphorylation of cytoplasmic proteins. Conclusions —These results indicate that the dissociation of β 2 -AR signaling from cAMP regulatory systems is only apparent and that β 2 -AR–stimulated cAMP/PKA signaling is uncoupled from phosphorylation of nonsarcolemmal regulatory proteins involved in excitation-contraction coupling.

Deletion and mutational analyses of the rat liver glycogen-targeting subunit (GL) of protein phosphatase 1 (PP1) have identified three separate domains that are responsible for binding of PP1, glycogen and phosphorylase a. The glycogen-binding domain spans the centre of GL between residues 144 and 231 and appears to be distinct from the glycogen-binding (storage) site of phosphorylase. The regulatory high-affinity binding site for phosphorylase a lies in the 16 amino acids at the C-terminus of GL. The PP1-binding domain is deduced to comprise the -RVXF- motif [Egloff, Johnson, Moorhead, Cohen and Barford (1997) EMBO J. 16, 1876–1887] located at residues 61–64 of GL and preceding lysine residues at positions 56, 57 and 59. A possible approach for increasing glycogen synthesis in certain disorders is discussed.

This study investigated the transformational and posttransformational control of skeletal muscle glycogen phosphorylase and pyruvate dehydrogenase (PDH) at three exercise power outputs [35, 65, and 90% of maximal oxygen uptake (V˙o2 max)]. Seven untrained subjects cycled at one power output for 10 min on three separate occasions, with muscle biopsies at rest and 1 and 10 min of exercise. Glycogen phosphorylase in the more active ( a) form was not significantly different at any time across power outputs (21.4–29.6%), with the exception of 90%, where it fell significantly to 15.3% at 10 min. PDH transformation increased significantly from rest (average 0.53 mmol ⋅ kg wet muscle−1⋅ min−1) to 1 min of exercise as a function of power output (1.60 ± 0.26, 2.77 ± 0.29, and 3.33 ± 0.31 mmol ⋅ kg wet muscle−1⋅ min−1at 35, 65, and 90%, respectively) with a further significant increase at 10 min (4.45 ± 0.35) at 90%V˙o2 max. Muscle lactate, acetyl-CoA, acetylcarnitine, and free ADP, AMP, and Piwere unchanged from rest at 35% V˙o2 maxbut rose significantly at 65 and 90%, with accumulations at 90% being significantly higher than 65%. The results of this study indicate that glycogen phosphorylase transformation is independent of increasing power outputs, despite increasing glycogenolytic flux, suggesting that flux through glycogen phosphorylase is matched to the demand for energy by posttransformational factors, such as free Piand AMP. Conversely, PDH transformation is directly related to the increasing power output and the calculated flux through the enzyme. The rise in PDH transformation is likely due to increased Ca2+concentration and/or increased pyruvate. These results demonstrate that metabolic signals related to contraction and the energy state of the cell are sensitive to the exercise intensity and coordinate the increase in carbohydrate use with increasing power output.

Glucagon-like peptide 1(7-36)amide (GLP-1) is currently under investigation as a possible tool in the treatment of non-insulin-dependent diabetes mellitus. In addition to enhancing nutrient-stimulated insulin release, the peptide also favors glycogen synthesis and glucose use in liver, muscle, and adipose tissue. GLP-1 also activates glycogen synthase a in hepatocytes from both normal and diabetic rats. In the present study, the kinetic aspects of such an activation were investigated in hepatocytes from normal rats and from animals rendered diabetic induced by injection of streptozotocin, either in the adult age (insulin-dependent diabetes mellitus model) or in days 1 or 5 after birth (non-insulin-dependent diabetes mellitus models). GLP-1 increased, in a dose-dependent manner, glycogen synthase a activity in the hepatocytes from all groups studied. The activation of the enzyme reached a steady state within 1 min exposure to GLP-1, which, at 10(-12) M, caused a half-maximal activation. When comparing fed vs. overnight-starved normal rats, a somewhat lower basal activity of glycogen synthase a in fasted animals (P < 0.05) coincided with a greater relative increment in reaction velocity in response to GLP-1. The basal activity of glycogen synthase a and the relative extent of its inhibition by glucagon or activation by insulin and GLP-1 were modulated by the extracellular concentration of D-glucose. The activation of glycogen synthase a by either insulin or GLP-1 resulted not solely in an increase in maximal velocity but also in a decrease in affinity of the enzyme for uridine diphosphate-glucose; in diabetic animals, the capacity of insulin or GLP-1 to increase the maximal velocity and Michaelis-Menten constant were less marked than in normal rats. In conclusion, this study indicates that the GLP-1-induced activation of glycogen synthase a displays attributes of rapidity, sensitivity, and nutritional dependency that are well suited for both participation in the physiological regulation of enzyme activity and therapeutic purpose.

Inhibition of aggregation by Ro 44-9883, a potent and selective non-peptide GPIIb/IIIa antagonist, resulted in inhibition of serotonin secretion induced by weak agonists such as ADP or low doses of either thrombin receptor agonist peptide (TRAP) or collagen. In contrast, alpha granule secretion was inhibited to different extents dependent on donor, averaging 60% inhibition. Inhibition of serotonin secretion correlated with an inhibition of thromboxane A2 (TxA2) formation, both of which were overcome by higher doses of TRAP or collagen. Ro 44-9883 had no effect on the already reduced serotonin secretion and TxA2 formation in Glanzmann's thrombasthenic platelets. Restoration of serotonin secretion in the absence of aggregation requires both TxA2 and lysophosphatidic acid. In addition, Ro 44-9883 inhibition of TxA2 formation was not due to a lack of phospholipase A2 (PLA2) phosphorylation and activation as assayed in vitro. These results suggest that aggregation is required for weak or low dose agonist induced in vivo activity of PLA2, possibly by either regulating phospholipid substrate availability or interaction of PLA2 with platelet membranes.

Acetaminophen (N-acetyl-p-aminophenol [APAP]) hepatotoxicity is a process characterized by Ca2+ deregulation. Cellular functions utilizing Ca2+ as a second messenger molecule affect both cytosolic and nuclear signal transduction. Many studies have independently shown Ca2+-related effects on target molecules in response to toxic doses of APAP; however, the primary Ca2+ target resulting in liver necrosis has not been determined. We hypothesize that Ca2+-dependent DNA damage is a critical event in liver necrosis caused by alkylating hepatotoxins. In this study, Ca2+-dependent endonuclease activity was determined from DNA single-strand lesions measured by fluorometric analysis of DNA unwinding. The status of cytosolic Ca2+ was determined by measuring Ca2+-dependent activation of glycogen phosphorylase a. Primary cultures of mouse hepatocytes exposed to a toxic concentration of APAP showed twofold and greater increases in glycogen phosphorylase a stimulation at 6 hours, which was reversible with Ca2+-chelating agents. Cell death was preceded by a large decline in intact, double-stranded DNA. Following toxic administration of APAP, the percentage of total double-stranded DNA was significantly reduced by 2 hours. At 6 and 24 hours, genomic integrity was compromised by 26% and 37%, respectively, compared with untreated controls. Hepatotoxic effects of APAP-mediated Ca2+ deregulation were confirmed in both primary mouse hepatocytes and the human hepatoblastoma HepG2 cell line by lactate dehydrogenase (LDH) release and tetrazolium reduction using the 3-4,5-dimethylthiazole-2-yl-2,5-diphenyltetrazolium bromide thiazol blue(MTT) assay. The Ca2+ chelator, ethylene glycol-bis (beta-aminoethyl ether) N',N',N', N'-tetraacetic acid (EGTA), blocked APAP-induced phosphorylase a activation and necrotic cell death, but failed to inhibit phosphorylase a activation by the adenosine 3',5'-cyclic monophosphate (cAMP) analogue, dibutyryl cAMP, indicating little or no contribution of the cAMP pathway to phosphorylase a stimulation during APAP-induced necrotic death. Results with these in vitro models of liver injury are interpreted as supporting the hypothesis that increased Ca2+ availability plays a major role in the progression of APAP-dependent cellular necrosis, and that the nucleus is a critical target for APAP hepatotoxicity.

We have previously reported on the M-phase specific dephosphorylation of pRb and identified a type 1 serine/threonine protein phosphatase (PP1) as the enzyme mediating pRb dephosphorylation. In this report, we have characterized the pRb-directed phosphatase activity found in mitotic cells with respect to dose dependence and demonstrate that the pRb isoform conversion detected in vitro mirrors the pRb isoform conversion which occurs during mitosis of intact cells. Cell fractionation and PP1 catalytic subunit isolation studies support the notion that the pRb-directed phosphatase activity involves subpopulations of PP1 catalytic subunits. Coprecipitation studies revealed that PP1 can form a complex with hypophosphorylated pRb which was converted from the hyperphosphorylated form in mitotic cell extracts. Taken together with data from previous reports in the literature, a model for the regulation of PP1 activity towards pRb during mitotic exit is proposed.

The mechanism of activation of glycogen phosphorylase is incompletely understood, although adenosine and inosine nucleotides are known to be important allosteric activators. In this study the activation of glycogen phosphorylases a and b from bovine liver by adenosine 5′-monophosphate (AMP) and inosine 5′-monophosphate (IMP) has been investigated and the results compared with the activation of the muscle isozyme by the same nucleotides. Enzyme activity was determined by spectrophotometric measurement of inorganic phosphate produced in the phosphorylase-catalysed reaction of glycogen synthesis. Liver phosphorylase b binds both nucleotides non-co-operatively (Hill coefficients of 1.0 ± 0.1), with changes in the maximum velocity to 75 or 80 μmol min −1 mg −1 in the presence of adenosine 5′-monophosphate or inosine 5′-monophosphate, respectively, but no change in the enzyme affinity towards the substrate, glucose-1-phosphate. Binding of glucose-1-phosphate is co-operative and the kinetic data have been fitted with the Monod-Wyman-Changeux model. Liver phosphorylase a has a maximum velocity similar to that of the b form in the presence of nucleotides. Binding of glucose-1-phosphate to the enzyme is non-co-operative (Hill coefficient of 1.0 ± 0.1) and the affinities in the presence of the nucleotides (Michaelis constants of 28 ± 0.2 mM or 27 ± 0.2 mM for adenosine 5′-monophosphate or inosine 5′-monophosphate) are stronger than those of the b form. It is concluded that the activity of bovine liver phosphorylase a and b is similarly influenced by adenosine 5′-monophosphate and inosine 5′-monophosphate. The b form seems to behave like muscle phosphorylase b in response to inosine 5′-phosphate; however, the binding of adenosine 5′-phosphate does not induce the conformational change necessary to activate the liver enzyme, as occurs with the muscle isozyme.

The hormonal responsiveness of freshly isolated rat hepatocytes was compared to that of a) cold-preserved isolated hepatocytes and b) hepatocytes isolated from cold-preserved whole liver. Cold-preserved hepatocytes and cells isolated from cold-preserved whole liver increased phosphorylase a activity in response to norepinephrine (plus propranolol), vasopressin, angiotensin II and glucagon. However, the maximal response to these agents was smaller than that of freshly isolated hepatocytes. Basal phosphorylase a activity was increased in cold-preserved hepatocytes. Similarly, cold preservation decreased the accumulation of cyclic AMP induced by glucagon and the effects of norepinephrine (plus propranolol), vasopressin and angiotensin II on the production of inositol phosphates. Basal levels of cyclic AMP were similar in the three conditions studied but basal production of [ 3 H]IP 2 plus [ 3 H]IP 3 was increased in cold-preserved hepatocytes. There was a very small effect of β-adrenergic activation on phosphorylase activity and a small accumulation of cyclic AMP in response to isoproterenol in the conditions studied.

The GLP-1 structurally related peptides exendin-4 and exendin(9-39)amide were found to act, in rat liver and skeletal muscle, as agonist and antagonist, respectively, of the GLP-1(7-36)amide effects on glucose metabolism. Thus, like GLP-1(7-36)amide, exendin-4 increased glycogen synthase a activity and glucose incorporation into glycogen in both tissues and also stimulated exogenous D-glucose utilization and oxidation in muscle. These effects of GLP-1(7-36)amide and exendin-4 were inhibited by exendin(9-39)amide. Our findings provide further support to the proposed use of GLP-1, or exendin-4, as a tool in the treatment of diabetes mellitus. Thus, in addition to the well-known insulinotropic action of the peptides, they act both in liver and in muscle in a manner most suitable for restoration of glucose homeostasis, with emphasis on their positive effects upon glycogen synthesis in the two tissues and on the stimulation of exogenous glucose catabolism in muscle.

In striatal membrane preparation used for receptor binding experiments high levels of protein phosphatase 1 and 2A activities were detected using [ 32 P]phosphorylase a as substrate. Sodium chloride decreased the activity of protein phosphatase 2A and increased the activity of protein phosphatase 1 in a concentration-dependent manner. Sodium chloride facilitated the saturation binding of naloxone and naltrindole in rat striatal membrane preparation preincubated with ATP (50 μM) and MgCl 2 (5 mM). Preincubation with calyculin A (1 nM) further increased the binding of naloxone. Addition of okadaic acid in a concentration of 2 nM, which is specific for the inhibition of protein phosphatase 2A, augmented the number of binding sites of naloxone or naltrindole. The results suggest a protein phosphatase-dependent regulation of the binding of opiate ligands in the striatum.

Exposure of rat kidney cortical slices to cisplatin (2 mM) significantly increased the activity of cytosolic phosphorylase a, indicating that the concentration of cytosolic Ca2+ was increased. Dithiothreitol (DTT), a sulphydryl reducing agent, markedly reversed this effect but N,N′-diphenylphenylenediamine (DPPD), an antioxidant, did not. Cisplatin inhibited protein kinase C (PKC) activity in both mitochondrial and cytosolic fractions after the slices were exposed to cisplatin. Both DTT and DPPD prevented these inhibitory effects of cisplatin on PKC but diethylmaleate, a glutathione depletor, potentiated this inhibitory effect. These results suggest that the increase in cytosolic Ca2+ is related to depletion of SH-groups, but independent of lipid peroxidation, whereas inhibition of PKC may be associated with cisplatin-induced depletion of thiols and with lipid peroxidation.

Alignment of 23 branching enzyme (BE) amino acid sequences from various species showed conservation of two arginine residues. Phenylglyoxal (PGO) was used to investigate the involvement of arginine residues of maize BEI and BEII in catalysis. BE was significantly inactivated by PGO in triethanolamine buffer at pH 8.5. The inactivation followed a time- and concentration-dependent manner and showed pseudo first-order kinetics. Slopes of 0.73 (BEI) and 1.05 (BEII) were obtained from double log plots of the observed rates of inactivation against the concentrations of PGO, suggesting that loss of BE activity results from as few as one arginine residue modified by PGO. BE inactivation was positively correlated with [14C]PGO incorporation into BE protein and was considerably protected by amylose and/or amylopectin, suggesting that the modified arginine residue may be involved in substrate binding or located near the substrate-binding sites of maize branching enzymes I and II.

Analysis of the primary structure of mBEII, with those of other branching and amylolytic enzymes as reference, identifies four highly conserved regions which may be involved in substrate binding and in catalysis. When one of the amino acid residues corresponding to the putative catalytic sites of mBEII, i.e., Asp-386, Glu-441, and Asp-509, was replaced, activity disappeared. These putative catalytic residues are located in three different regions (regions 2-4) of the four highly conserved regions (regions 1-4) which exist in the primary structure of most starch hydrolases and related enzymes, including branching enzymes. Region 3, which contains Glu-441 as one of the putative catalytic residues, was located downstream of the carboxyl-terminal position previously reported. The importance of the carboxyl amino acid residues was also demonstrated by chemical modification of the branching enzyme protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.