The behavior of hepatic phosphorylase b kinase, phosphorylase a and b after administration of glucagon to patients with glycogen storage disease type VIa

In the patients with glycogen storage disease (GSD) type VIa and different serum glucose response to glucagon, the activities of hepatic phosphorylase b kinase, phosphorylase a and b were estimated before and after the intravenous administration of glucagon. 3 min after the administration of glucagon an increase in the activities of phosphorylase b kinase and phosphorylase a was found in liver tissue of all patients except one. These enzymatic activities, however, did not exceed the values of these enzymes in the control liver biopsies without glucagon loading. After the intravenous administration of glucagon an unsuspected increase of phosphorylase b activity was observed in the control liver tissues and in patients with GSD type VIa, except one. In vitro investigations revealed that an increase of hepatic phosphorylase b activity occurs during its conversion to phosphorylase a. We suppose that this phosphorylase b represents a partially phosphorylated form of this enzyme (an intermediate form) that is due to the action of the active phosphorylase b kinase. The correlations between the activities of phosphorylase b kinase, phosphorylase a and an intermediate form of phosphorylase b and hepatic glycogen degradation after administration of glucagon has been discussed.     

Effect of epinephrine infusions imitating plasma epinephrine levels in immobilization stress and of fasting on the activity of key enzymes of liver glycogen metabolism

In adult male rats anaesthetized with pentobarbital, intravenous infusion of 0.25 microgram kg-1 min-1 of epinephrine increased plasma epinephrine levels to more than 10 pmol ml-1 within 5 min, the values remaining constant during the next 20 min. On the other hand, a maximal increase of liver phosphorylase a was found after 5 min and this response was attenuated at 10 and 20 minutes in the fed rats. Fasting for 24 h extinguished the greatest part of this response. During infusion, a decrease of liver synthase a activity was observed in the fasted and of total synthase activity in both the fed and fasted animals. In fed rats an i.v. bolus of 0.50 microgram.kg-1 or doubling of the infusion rate for 5 min, both immediately after 20 min of infusion, led to maximal phosphorylase a activity again and to a decrease of synthase a activity. The levels of cAMP in the liver were increased by the infusion in one series and unchanged in another.     

Nicotinamide riboside phosphorylase from beef liver: purification and characterization

Nicotinamide riboside phosphorylase (NR phosphorylase) from beef liver has been purified to apparent homogeneity at 300-fold purification with a 35% yield. Kinetic constants for the enzyme-catalyzed phosphorolysis were as follows Knicotinamide riboside, 2.5 +/- 0.4 mM; Kinorganic phosphate, 0.50 +/- 0.12 mM; Vmax, 410 +/- 30 X 10(-6) mol min-1 mg protein-1, respectively. The molecular weights of the native enzyme and subunit structure were determined to be 131,000 and 32,000, respectively, suggesting the beef liver NR phosphorylase to be tetrameric in structure and consistent with the presence of identical subunits. The amino acid composition was shown to be very similar to that reported for human erythrocyte purine-nucleoside phosphorylase but differing considerably from that found for rat liver purine-nucleoside phosphorylase. In addition to catalytic activity with nicotinamide riboside, the beef liver enzyme catalyzed a phosphorolytic reaction with inosine and guanosine exhibiting activity ratios, nicotinamide riboside:inosine: guanosine of 1.00:0.35:0.29, respectively. These ratios of activity remained constant throughout purification of the beef liver enzyme and no separation of these activities was detected. Phosphorolysis of nicotinamide riboside was inhibited competitively by inosine (Ki = 75 microM) and guanosine (Ki = 75 microM). Identical rates of thermal denaturation of the beef liver enzyme were observed when determined for the phosphorolysis of either nicotinamide riboside or inosine. These observations coupled with studies of pH and specific buffer effects indicate the phosphorolysis of nicotinamide riboside, inosine, and guanosine to be catalyzed by the same enzyme.     

Decreased activity and impaired hormonal control of protein phosphatases in rat livers with a deficiency of phosphorylase kinase.

1. Livers from gsd/gsd rats, which do not express phosphorylase kinase activity, also contain much less particulate type-1 protein phosphatases. In comparison with normal Wistar rats, the glycogen/microsomal fraction contained 75% less glycogen-synthase phosphatase and 60% less phosphorylase phosphatase activity. This was largely due to a lower amount of the type-1 catalytic subunit in the particulate fraction. In the cytosol, the synthase phosphatase activity was also 50% lower, but the phosphorylase phosphatase activity was equal. 2. Both Wistar rats and gsd/gsd rats responded to an intravenous injection of insulin plus glucose with an acute increase (by 30-40%) in the phosphorylase phosphatase activity in the liver cytosol. In contrast, administration of glucagon or vasopressin provoked a rapid fall (by about 25%) in the cytosolic phosphorylase phosphatase activity in Wistar rats, but no change occurred in gsd/gsd rats. 3. Phosphorylase kinase was partially purified from liver and subsequently activated. Addition of a physiological amount of the activated enzyme to a liver cytosol from Wistar rats decreased the V of the phosphorylase phosphatase reaction by half, whereas the non-activated kinase had no effect. The kinase preparations did not change the activity of glycogen-synthase phosphatase, which does not respond to glucagon or vasopressin. Furthermore, the phosphorylase phosphatase activity was not affected by addition of physiological concentrations of homogeneous phosphorylase kinase from skeletal muscle (activated or non-activated). 4. It appears therefore that phosphorylase kinase plays an essential role in the transduction of the effect of glucagon and vasopressin to phosphorylase phosphatase. However, this inhibitory effect either is specific for the hepatic phosphorylase kinase, or is mediated by an unidentified protein that is a specific substrate of phosphorylase kinase.     

Progressive weakening of the response of key enzymes of liver glycogen metabolism to permanently increased plasma epinephrine levels

In adult male rats anaesthetized with pentobarbital the intravenous infusion of 0.5 micrograms.kg-1.min-1 of epinephrine increased liver phosphorylase a activity within 5 min, whereas later a weakening of the hormone effect was observed. After increasing the infusion rate to 1.0 micrograms.kg-1.min-1 and extending the study to more parameters, the diminishing effect on phosphorylase was confirmed and a similar response was established for liver cAMP. Concomitantly, a decrease and recovery of liver glycogen synthase a activity was observed. In rats with permanent catheters in one of their tail arteries for obtaining blood samples, the plasma epinephrine levels were shown to be permanently increased (from cca 1 pmol.ml-1 before infusion of 1.0 micrograms.kg-1.min-1 to more than 30 pmol.ml-1 during infusion) and remained at steady levels throughout the infusion. Therefore, the weakening of the epinephrine effect should be ascribed to changes at (or beyond) the catecholamine receptor level. A hitherto undescribed decrease of total glycogen synthase activity was observed during the infusions.     

Acute regulation of hepatic protein phosphatases by glucagon, insulin, and glucose

The intravenous administration of glucagon to anesthetized rats resulted within 5 min in a 20% drop in the hepatic phosphorylase phosphatase activity, as measured in a post-mitochondrial supernatant at low dilution, but it did not affect the activity of glycogensynthase phosphatase. On the other hand, the injection of insulin plus glucose caused increases by about 35% in both phosphatase activities. Upon subcellular fractionation these effects were recovered in the cytosol, but not in the glycogen/microsomal fraction. However, activity changes in the latter fraction were observed after recombination with the liver cytosol from a hormone-treated animal. Preincubation of the liver cytosol with modulator protein (a specific inhibitor of type-1 protein phosphatases) cancelled the activity changes induced by insulin plus glucose. No hormonal effects on hepatic protein phosphatase activities were observed when the fractions were either diluted an additional 10-fold or pretreated with trypsin. An acute hormonal regulation of protein phosphatases could also be demonstrated in the perfused liver. When added to the perfusion medium, glucose as well as insulin increased the cytosolic protein phosphatase activities by about 25%. Their effect was additive, irrespective of the order of addition. On the other hand, the addition of glucagon and/or vasopressin resulted in a 20% drop in the phosphorylase phosphatase activity. The presence of glucagon did not interfere with the effectiveness of insulin, and vice versa. The changes in the phosphorylase phosphatase activities induced by glucagon, insulin, and glucose represented changes in the Vmax only. We propose that the acute control of the hepatic glycogen synthase phosphatase and phosphorylase phosphatase activities is mediated by transferable, cytosolic effector(s).     

Effects of epinephrine and norepinephrine on glucose release from chinook salmon (Oncorhynchus tshawytscha) liver incubated in vitro

The effects of two catecholamines, epinephrine (EP) and norepinephrine (NE), on carbohydrate metabolism were studied by incubating chinook salmon liver in vitro. Basal release of glucose over the course of a 5-h incubation was 7.93 +/- 1.70 mumol/g dry weight. Both EP and NE (2 X 10(-7) M) stimulated glucose release rapidly during the first hour. After 5 h, EP and NE significantly increased glucose release over basal levels to 43.55 +/- 9.01 and 32.75 +/- 6.17 mumol/g dry weight, respectively. Epinephrine- and NE-stimulated glucose release was dose dependent, with a minimum effective dose of 10(-9) M. ED50 for both agents was approximately 2 X 10(-7) M; maximal stimulation occurred at 10(-5) M. No difference in potency between the two catecholamines was found. The effects of adrenergic agonists and antagonists were also studied. Alpha-agonists, methoxamine and phenylephrine, had no effect on glucose release. Isoproterenol, a beta-agonist, stimulated glucose release in a manner similar to EP. The beta-antagonist, propranolol, inhibited both catecholamine- and isoproterenol-stimulated glucose release. Alpha-antagonists (phentolamine, prazosin, and yohimbine) had no effect on either catecholamine- or isoproterenol-stimulated glucose release. Epinephrine and NE stimulate glycogen phosphorylase activity; propranolol inhibits catecholamine-stimulated phosphorylase activity. These results indicate that catecholamines stimulate glucose mobilization in salmon liver by promoting glycogenolysis mediated through beta-adrenergic receptors.     

The role played by hormonal factors in the rapid activation of liver glycogen phosphorylase in traumatized rats

In adult male SPF rats anaesthetized with pentobarbital and subjected to traumatization in revolving Noble-Collip drums for 2 min (= 120 revolutions) maximal increases of liver glycogen phosphorylase activity were observed. In experiments on rats with permanent arterial catheters for blood sampling no posttraumatic increase of plasma norepinephrine and an only slight increase of plasma epinephrine was observed if the animals were traumatized under anaesthesia, in contrast to the considerable increases in the plasma level of both hormones in rats subjected to the injury without anaesthesia. Time and extent of the phosphorylase response of anaesthetized rats after trauma were compared with changes in enzyme activity after i.v. administration of exogenous epinephrine or glucagon. A nearly maximal response after 1 microgram kg-1 epinephrine was present within 1 min, whereas after 0.1 micrograms kg-1 of glucagon there was comparable phosphorylase activation 2 min after administration of the hormone. The plasma renin-angiotensin activity was not increased after injury for 2 min under anaesthesia so that only the increase in plasma vasopressin fitted in with the criteria for possible activators of phosphorylase. An additional role of glucagon also cannot be excluded on the basis of data obtained by the present authors. The increase of phosphorylase activity in this type of stress is ensured by several mechanisms. Moreover, the high effectivity of these hormonal factors in evoking the phosphorylase response even without major activation of the sympathicoadrenal system is underlined.     

Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells.

Epinephrine and the alpha-adrenergic agonist phenylephrine activated phosphorylase, glycogenolysis, and gluconeogenesis from lactate in a dose-dependent manner in isolated rat liver parenchymal cells. The half-maximally active dose of epinephrine was 10-7 M and of phenylephrine was 10(-6) M. These effects were blocked by alpha-adrenergic antagonists including phenoxybenzamine, but were largely unaffected by beta-adrenergic antagonists including propranolol. Epinephrine caused a transient 2-fold elevation of adenosine 3':5'-monophosphate (cAMP) which was abolished by propranolol and other beta blockers, but was unaffected by phenoxybenzamine and other alpha blockers. Phenoxybenzamine and propranolol were shown to be specific for their respective adrenergic receptors and to not affect the actions of glucagon or exogenous cAMP. Neither epinephrine (10-7 M), phenylephrine (10-5 M), nor glucagon (10-7 M) inactivated glycogen synthase in liver cells from fed rats. When the glycogen synthase activity ratio (-glucose 6-phosphate/+ glucose 6-phosphate) was increased from 0.09 to 0.66 by preincubation of such cells with 40 mM glucose, these agents substantially inactivated the enzyme. Incubation of hepatocytes from fed rats resulted in glycogen depletion which was correlated with an increase in the glycogen synthase activity ratio and a decrease in phosphorylase alpha activity. In hepatocytes from fasted animals, the glycogen synthase activity ratio was 0.32 +/- 0.03, and epinephrine, glucagon, and phenylephrine were able to lower this significantly. The effects of epinephrine and phenylephrine on the enzyme were blocked by phenoxybenzamine, but were largely unaffected by propranolol. Maximal phosphorylase activation in hepatocytes from fasted rats incubated with 10(-5) M phenylephrine preceded the maximal inactivation of glycogen synthase. Addition of glucose rapidly reduced, in a dose-dependent manner, both basal and phenylephrine-elevated phosphorylase alpha activity in hepatocytes prepared from fasted rats. Glucose also increased the glycogen synthase activity ratio, but this effect lagged behind the change in phosphorylase. Phenylephrine (10-5 M) and glucagon (5 x 10(-10) M) decreased by one-half the fall in phosphoryalse alpha activity seen with 10 mM glucose and markedly suppressed the elevation of glycogen synthase activity. The following conclusions are drawn from these findings. (a) The effects of epinephrine and phenylephrine on carbohydrate metabolism in rat liver parenchymal cells are mediated predominantly by alpha-adrenergic receptors. (b) Stimulation of these receptors by epinephrine or phenylephrine results in activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase by mechanisms not involving an increase in cellular cAMP. (c) Activation of beta-adrenergic receptors by epinephrine leads to the accumulation of cAMP, but this is associated with minimal activation of phosphorylase or inactivation of glycogen synthase...     

Evidence for the non-identity of proteins having synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity in rat liver.

Synthase phosphatase, phosphorylase phosphatase and histone phosphatase in rat liver were measured using as substrates purified liver synthase D, phosphorylase alpha and 32P-labelled phosphorylated f1 histone, respectively. The three phosphatase enzymes had different sedimentation characteristics. Both synthase phosphatase and phosphorylase phosphatase were found to sediment with the microsomal fraction under our experimental conditions. Only 10% of histone phosphatase was in this fraction; the majority was in the cytosol. No change in histone phosphatase was observed in the adrenalectomized fasted rat whereas synthase phosphatase and phosphorylase phosphatase activities were decreased 5-10 fold. Fractionation of liver extract with ethanol produced a dissociation of the three phosphatase activities. When a partially purified fraction was put on a DEAE-cellulose column, synthase phosphatase and phosphorylase phosphatase both exhibited broad elution profiles but their activity peaks did not coincide. Histone phosphatase eluted as a single discrete peak. When the supernatant of CaCl2-treated microsomal fraction was put on a Sepharose 4B column, the majority of synthase phosphatase was found to elute with the larger molecular weight proteins whereas the majority of phosphorylase phosphatase eluted with the smaller species. Histone phosphatase migrated as a single peak and was of intermediate size. Synthase phosphorylase phosphatase by synthase D (Ki approximately 2 units/ml). The inhibition of synthase phosphatase by phosphorylase alpha was kinetically non-competitive with substrate. Histone phosphatase activity was not inhibited by synthase D or by phosphorylase alpha. The above results suggest that different proteins are involved in the dephosphorylation of synthase D, phosphorylase alpha and histone in the cell.     

Evidence against glycogen cycling of gluconeogenic substrates in various liver preparations

The effect of inhibition of glycogen phosphorylase by 1,4-dideoxy-1,4-imino-d-arabinitol on rates of gluconeogenesis, gluconeogenic deposition into glycogen, and glycogen recycling was investigated in primary cultured hepatocytes, in perfused rat liver, and in fed or fasted rats in vivo clamped at high physiological levels of plasma lactate. 1,4-Dideoxy-1,4-imino-d-arabinitol did not alter the synthesis of glycerol-derived glucose in hepatocytes or lactate-derived glucose in perfused liver or fed or fasted rats in vivo. Thus, 1,4-dideoxy-1,4-imino-d-arabinitol inhibited hepatic glucose output in the perfused rat liver (0.77 +/- 0.19 versus 0.33 +/- 0.09, p < 0.05), whereas the rate of lactate-derived gluconeogenesis was unaltered (0.22 +/- 0.09 versus 0.18 +/- 0.08, p = not significant) (1,4-dideoxy-1,4-imino-d-arabinitol versus vehicle, micromol/min * g). Overall, the data suggest that 1,4-dideoxy-1,4-imino-d-arabinitol inhibited glycogen breakdown with no direct or indirect effects on the rates of gluconeogenesis. Total end point glycogen content (micromol of glycosyl units/g of wet liver) were similar in fed (235 +/- 19 versus 217 +/- 22, p = not significant) or fasted rats (10 +/- 2 versus 7 +/- 2, p = not significant) with or without 1,4-dideoxy-1,4-imino-d-arabinitol, respectively. The data demonstrate no glycogen cycling under the investigated conditions and no effect of 1,4-dideoxy-1,4-imino-d-arabinitol on gluconeogenic deposition into glycogen. Taken together, these data also suggest that inhibition of glycogen phosphorylase may prove beneficial in the treatment of type 2 diabetes.     

Hormonal regulation of hepatic glycogen synthase phosphatase

Perfusion of livers from fed rats with medium containing glucagon (2 x 10(-10) or 1 x 10(-8) M) resulted in both time- and concentration-dependent inactivation of glycogen synthase phosphatase. Expected changes occurred in cAMP, cAMP-dependent protein kinase, glycogen synthase, and glycogen phosphorylase. The effect of glucagon on synthase phosphatase was partially reversed by simultaneous addition of insulin (4 x 10(-8) M), an effect paralleled by a decrease in cAMP. Addition of arginine vasopressin (10 milliunits/ml) resulted in a similar inactivation of synthase phosphatase and activation of phosphorylase, but independent of any changes in cAMP or its kinase. Phosphorylase phosphatase activity was unaffected by any of these hormones. Synthase phosphatase activity, measured as the ability of a crude homogenate to catalyze the conversion of purified rat liver synthase D to the I form, was no longer inhibited by glucagon or vasopressin when phosphorylase antiserum was added to the phosphatase assay mixture in sufficient quantity to inhibit 90-95% of the phosphorylase a activity. These data support the following conclusions: 1) hepatic glycogen synthase phosphatase activity is acutely modulated by hormones, 2) hepatic glycogen synthase phosphatase and phosphorylase phosphatase are regulated differently, 3) the hormone-mediated changes in synthase phosphatase cannot be explained by an alteration of the synthase D molecule affecting its behavior as a substrate, and 4) glycogen synthase phosphatase activity is at least partially controlled by the level of phosphorylase a.     

Thyrotropin-releasing hormone (TRH) and its analog (DN-1417): interaction with pentobarbital in choline uptake and acetylcholine synthesis of rat brain slices

TRH or its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-proliamide) given 15 min after intravenous (i.v.) administration of pentobarbital (30 mg/kg) markedly shortened the pentobarbital-induced sleeping time in rats. This effect was almost completely abolished by intracerebroventricular pretreatment with atropine methylbromide (20 micrograms/rat), thereby suggesting the involvement of cholinergic mechanism. The action mechanism was investigated using rat brain slices. TRH (10(-6)-10(-4)M) or DN-1417 (10(-7)-10(-5)M) caused significant increases in the uptake of [3H]-choline into striatal slices. TRH(10(-4)M) or DN-1417(10(-5)M) also stimulated the conversion of [3H]-choline to [3H]-acetylcholine in striatal slices. A 30% reduction of acetylcholine synthesis from [3H]-choline in hippocampal slices and a 40% reduction of [3H]-choline uptake in slices of cerebral cortex, hippocampus and hypothalamus were observed in rats pretreated with pentobarbital (60 mg/kg, i.v.). TRH or DN-1417 (20 mg/kg, i.v.) given 15 min after the administration of pentobarbital markedly reversed both of the pentobarbital effects. Direct application of pentobarbital (5 X 10(-4)M) to slices in vitro also caused a 20-40% reduction of [3H]-choline uptake of cerebral cortex, hippocampus and diencephalon. A concomitant application of TRH(10(-4)M) or DN-1417(10(-5)M) and pentobarbital abolished the pentobarbital effect. These results provide neurochemical evidence that the antagonistic effects of TRH and DN-1417 on pentobarbital-induced narcosis are closely related to alterations in the rat brain choline uptake and acetylcholine synthesis, which are considered to be measures of the activity of cholinergic neurons.     

Effect of free Mg2+ on liver phosphorylase kinase activity

When pig liver phosphorylase kinase was assayed at various concentrations of Mg2+, about 2-fold stimulation was observed around 2-3 mM Mg2+ (Mg2+/ATP ratio, 20-30) compared with the activity at 0.3 mM Mg2+ (Mg2+/ATP ratio, 3). This stimulation was specific for Mg2+ among the divalent cations tested and the process was reversible. Km values for ATP and phosphorylase b were decreased 3.6- and 9.5-fold, respectively, at 3 mM Mg2+ compared with those obtained at 0.3 mM Mg2+. These results indicate that the activity of liver phosphorylase kinase is influenced by free Mg2+.     

Regulation of glycogen metabolism in liver by the autonomic nervous system. VI. Possible mechanism of phosphorylase activation by the splanchnic nerve.

The effects of autonomic-nerve stimulation on the activities of phosphorylase (EC 2.4.1.1), dephospho-phosphorylase kinase (EC 2.7.1.38) and phosphorylase phosphatase (EC 3.1.3.17), and on the concentration of adenosine 3', 5'-monophosphate in rabbit liver were investiaged. Results were compared with the effects of epinephrine and glucagon on these enzymes. 1. The acitivity of liver phosphorylase increased rapidly and markedly on electrical stimulation of the splanchnic nerve, or after intraportal administration of epinephrine or glucagon. The activity was not affected by vagal stimulation. 2. The activity of dephospho-phosphorylase kinase increased about 2--3-fold 1 min after injections of epinephrine and glucagon, glucagon causing more activation than epinephrine. The enzyme activity was not altered by splanchnic-nerve, or vagal stimulation. 3. Injections of epinephrine and glucagon caused marked elevation of liver adenosine 3', 5'-monophosphate within a few minutes. With epinephrine, the nucleotide concentration rose to a maximum after 1 min and amounted to about 3-fold increase, while with glucagon the maximum increase of approximately 8-fold increase was observed after 2 min. Stimulation of the splanchnic nerve for 10 min did not affect the adenosine 3', 5'-monophosphate level in the liver. Vagal stimulation also had no effect on the level. 4. The activity of phosphorylase phosphatase decreased promptly (within 30 s) and markedly on splanchnic-nerve stimulation, but did not change significantly on administration of epinephrine of glucagon. A small but insignificant increase in phosphatase activity wasobserved upon vagal stimulation. 5. The effect of Ca-2+ on purified dephospho-phosphorylase kinase was studied. The activity was found to depend partially on free Ca-2+ at low Ca-2+ concentrations (1-10-minus 7--1-10-minus 5 M). 6. These results suggest that the rise in hepatic phosphorylase content upon splanchnic-nerve stimulation, unlike that induced by epinephrine and glucagon, is not mediated by adenosine 3', 5'-monophosphate and subsequent activation of dephospho-phosphorylase kinase, but rather by inactivation of phosphorylase phosphatase. The possible existence of a new factor in this mechanism is discussed.     

Fructose effect to suppress hepatic glycogen degradation

The effect of fructose on glycogen degradation was examined by measuring the flux of 14C from prelabeled glycogen in perfused rat livers. During 2-h refeeding of 24-h-fasted rats, newly synthesized hepatic glycogen was labeled by intraperitoneal injection of [U-14C] galactose (0.1 mg and 0.02 microCi/g of body weight). The livers of refed rats were then perfused in a nonrecirculating fashion for an initial 30 min with glucose alone (10 mM) for the following 60 min with glucose (10 mM) without (n = 5) or with fructose (1, 2, or 10 mM; n = 5 for each). When livers were exposed to fructose, release of label into the perfusate immediately declined and remained markedly suppressed through the end of perfusion (p less than 0.05). The suppression was dose-dependent; at steady state (50-70 min), label release was suppressed 45, 64, and 72% by 1, 2, and 10 mM fructose, respectively (p less than 0.0001). Suppression was not accompanied by significant changes in the activities of glycogen synthase or phosphorylase assessed in vitro. These results suggest the existence of allosteric inhibition of phosphorylase in the presence of fructose. Fructose 1-phosphate (Fru-1-P) accumulated in proportion to fructose (0.11 +/- 0.01 without fructose, 0.86 +/- 0.03, 1.81 +/- 0.18, and 8.23 +/- 0.60 mumol/g of liver with 1, 2, and 10 mM fructose, respectively; p less than 0.0001). Maximum inhibition of label release was 82%; the Fru-1-P concentration for half inhibition was 0.57 mumol/g of liver, well within the concentration of Fru-1-P attained during refeeding. We conclude that fructose enhances net glycogen accumulation in liver by suppressing glycogenolysis and that the suppression is presumably caused by allosteric inhibition of phosphorylase by Fru-1-P.     

Spin-labelled AMP - an activator of phosphorylase.

1. A spin-labelled AMP derivative and its diamagnetic analogue activate phosphorylase b in the same way, but do not activate phosphorylase a. 2. The electron-spin-resonance spectra of the spin-labelled AMP derivative bound to phosphorylase b and a have "powderlike" characteristics indicating that the spin label is immobilised on the protein. From changes in the electron-spin-resonance spectrum of spin-labelled AMP as phosphorylase b or a is added, the dissociation constants were calculated. 3. The interactions of spin-labelled AMP and the diamagnetic analogue with phosphorylase b and a have been monitored by observing changes in the spectral properties of fluorescent and spin-label probes covalently attached to the enzyme. 4. The dissociation constants of spin-labelled AMP and phosphorylase b or a are 175 +/- 25 muM and 15 +/- 5 muM respectively. Similar dissociation constants are obtained for the diamagnetic analogue. The effect of these AMP derivatives on the covalently attached probe groups and on phosphorylase activity is compared to the effect of AMP and IMP.     

Blood collection and processing for measurement of catecholamines in exercising rats

We have studied the time course of the decline in plasma catecholamines in the postexercise period in rats. Male Sprague-Dawley rats were run on the treadmill for 5 min at 31 m/min up a 15% grade. At the end of the exercise the rats were quickly anesthetized by intravenous injection of pentobarbital. Blood samples were collected as soon as possible (average of 43 s), at 2 and 7 min postexercise. Plasma epinephrine decreased from 0.79 +/- 0.09 ng/ml to 0.51 +/- 0.05 after 2 min and to 0.35 +/- 0.09 after 7 min. Plasma norepinephrine decreased from 0.89 +/- 0.16 ng/ml to 0.61 +/- 0.05 after 2 min and to 0.50 +/- 0.07 after 7 min. We also studied the effect of time of centrifugation with respect to time of blood collection on plasma catecholamines. If blood samples were kept on ice no significant change in plasma epinephrine occurred over a period of 1 h. A small (14%) but significant decrease in norepinephrine was observed after 15 and 60 min. These studies emphasize the importance of collecting rat blood samples as quickly as possible after the end of exercise. Catecholamines decline very quickly in the rat after intravenous pentobarbital anesthesia.     

An evaluation of three intravenous anesthetic regimens in New Zealand rabbits

Intravenous anesthetics can be readily administered to rabbits through the marginal ear vein. In this study, three intravenous anesthetic protocols were evaluated in New Zealand White rabbits. The three anesthetic regimens were: (a) pentobarbital (40 mg/kg); (b) ketamine-xylazine (25-5 mg/kg); (c) midazolam-xylazine-alfentanil (1-1-0.1 mg/kg). The anesthetics were injected slowly over defined time intervals. Reactions to noxious stimuli were determined before and after administration of the anesthetics. Additionally, the effects of the anesthetic agents on the rabbit's cardiopulmonary system were evaluated. Rabbits anesthetized with midazolam-xylazine-alfentanil did not have a pedal withdrawal or ear pinch reflex throughout the testing period. The ketamine-xylazine combination produced a shorter duration of non-responsiveness to noxious stimuli. Rabbits anesthetized with pentobarbital had the greatest variability in response to noxious stimuli. Apnea occurred in at least one rabbit in each group. A side effect unique to the midazolam-xylazine-alfentanil group was the occurrence of opisthotonus or seizure activity during or shortly after the administration of alfentanil. Hypotension, hypercapnia and respiratory acidosis were characteristic of the cardiopulmonary effects of the anesthetics. When choosing an anesthetic regimen for rabbits, intravenous infusion should be considered as an option. Advantages include ease of administration, possibility of redosing as required, and minimal requirements for equipment. Disadvantages of intravenous anesthetic infusion in rabbits include potential for lethal overdose and metabolic alterations after administration.     

Effect of pentobarbital dependence on adenylate cyclase activity and calmodulin levels in rat cerebral cortex

The effect of calcium (Ca2+) on the adenylate cyclase activity and calmodulin level of cerebral cortex was determined in pentobarbital dependent rats and age matched controls. Female Sprague-Dawley rats were made dependent and maintained on pentobarbital by eating a mixture of pentobarbital and rat chow (350 mg pentobarbital/30 g chow). Ca2+ activated then inhibited the adenylate cyclase activity associated with a 20,000 X g particulate fraction from pentobarbital dependent and age matched control rats. The values for one-half maximal stimulation and inhibition by Ca2+ did not differ significantly in either cortical preparation. However, the ability of Ca2+ to activate adenylate cyclase from pentobarbital dependent animals was significantly decreased (p less than 0.05) when compared to control animals. Pentobarbital (10(-4) - 10(-3) added to particulate fractions from naive control rats did not alter the ability of Ca2+ to activate adenylate cyclase. The calmodulin levels in the particulate fraction from pentobarbital dependent animals (30.2 +/- 6.7 ng calmodulin/mg protein) did not differ significantly when compared to control (33.0 +/- 4.7 ng/mg). By contrast, the calmodulin levels (37.9 +/- 5.9 ng/mg) in the 20,000 X g supernatant from cortex of pentobarbital dependent animals was significantly greater than the level in the supernatant from control animals (28.6 +/- 2.6 ng/mg). The ability of forskolin, dopamine, GTP or forskolin plus GTP (all at a concentration of 100 microM) to activate adenylate cyclase was significantly decreased in particulate preparations from pentobarbital dependent animals. In summary, our data show that alterations in calmodulin levels and a decreased responsivity of adenylate cyclase occur in animals physically dependent on pentobarbital.