Role of nitration in control of phosphorylase and glycogenolysis in mouse skeletal muscle.

Phosphorylase is one of the most carefully studied proteins in history, but knowledge of its regulation during intense muscle contraction is incomplete. Tyrosine nitration of purified preparations of skeletal muscle phosphorylase results in inactivation of the enzyme and this is prevented by antioxidants. Whether an altered redox state affects phosphorylase activity and glycogenolysis in contracting muscle is not known. Here, we investigate the role of the redox state in control of phosphorylase and glycogenolysis in isolated mouse fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) muscle preparations during repeated contractions. Exposure of crude muscle extracts to H2O2 had little effect on phosphorylase activity. However, exposure of extracts to peroxynitrite (ONOO-), a nitrating/oxidizing agent, resulted in complete inactivation of phosphorylase (half-maximal inhibition at ∼200 µM ONOO-), which was fully reversed by the presence of an ONOO- scavanger, dithiothreitol (DTT). Incubation of isolated muscles with ONOO- resulted in nitration of phosphorylase and marked inhibition of glycogenolysis during repeated contractions. ONOO- also resulted in large decreases in high-energy phosphates (ATP and phosphocreatine) in the rested state and following repeated contractions. These metabolic changes were associated with decreased force production during repeated contractions (to ∼60% of control). In contrast, repeated contractions did not result in nitration of phosphorylase, nor did DTT or the general antioxidant N-acetylcysteine alter glycogenolysis during repeated contractions. These findings demonstrate that ONOO- inhibits phosphorylase and glycogenolysis in living muscle under extreme conditions. However, nitration does not play a significant role in control of phosphorylase and glycogenolysis during repeated contractions.NEW & NOTEWORTHY Here we show that exogenous peroxynitrite results in nitration of phosphorylase as well as inhibition of glycogenolysis in isolated intact mouse skeletal muscle during short-term repeated contractions. However, repeated contractions in the absence of exogenous peroxynitrite do not result in nitration of phosphorylase or affect glycogenolysis, nor does the addition of antioxidants alter glycogenolysis during repeated contractions. Thus phosphorylase is not subject to redox control during repeated contractions.

Kinetics of denaturation of rabbit skeletal muscle glycogen phosphorylase b by guanidine hydrochloride.

The kinetics of denaturation and aggregation of rabbit muscle glycogen phosphorylase b in the presence of guanidine hydrochloride (GuHCl) have been studied. The curve of inactivation of phosphorylase b in time includes a region of the fast decline in the enzymatic activity, an intermediate plateau, and a part with subsequent decrease in the enzymatic activity. The fact that the shape of the inactivation curves is dependent on the enzyme concentration testifies to the dissociative mechanism of inactivation. The dissociation of phosphorylase b dimers into monomers in the presence of GuHCl is supported by sedimentation data. The rate of phosphorylase b aggregation in the presence of GuHCl rises as the denaturant concentration increases to 1.12 M; at higher concentration of GuHCl, suppression of aggregation occurs. At rather low concentration of the protein (0.25 mg/ml), the terminal phase of aggregation follows the kinetics of a monomolecular reaction (the reaction rate constant is equal to 0.082 min(-1); 1 M GuHCl, 25 degrees C). At higher concentration of phosphorylase b (0.75 mg/ml), aggregation proceeds as a trimolecular reaction.

Insulin increased cAMP phosphodiesterase activity antagonizing metabolic actions of glucagon in rat hepatocytes cultured with herbimycin A.

The baseline activity of cyclic nucleotide phosphodiesterase 4 was markedly lowered by primary culture of rat hepatocytes with herbimycin A for 4 h [Eur. J. Biochem. 260 (1999) 398-408.]. We now report that insulin added to this preparation of hepatocytes, which had been completely freed of herbimycin, increased the thus lowered phosphodiesterase activity, consequently antagonizing glucagon-induced production of cAMP and activation of glycogen phosphorylase. The insulin receptor beta-subunits and alpha-tubulin were tyrosine-phosphorylated upon the addition of insulin. The phosphorylation of alpha-tubulin afforded conditions unfavorable for microtubule assembly that is responsible for phosphodiesterase inhibition. These effects of insulin observed in herbimycin-pretreated hepatocytes were not inhibited by wortmannin that actually abolished insulin-induced activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase) under the same conditions. The physiological significance of the insulin action not mediated by PtdIns 3-kinase in herbimycin-pretreated hepatocytes is discussed.

Effect of an Asp905Tyr mutation of the glycogen-associated regulatory subunit of protein phosphatase-1 on the regulation of glycogen synthesis by insulin and cyclic adenosine 3',5'-monophosphate agonists.

The glycogen-associated regulatory subunit of protein phosphatase-1 (PP-1G) plays a major role in insulin-stimulated glycogen synthesis and thus the regulation of nonoxidative glucose disposal in skeletal muscle. In a general population of Caucasians a polymorphism at codon 905 of PP-1G from an aspartate to tyrosine has been reported to be associated with insulin resistance and hypersecretion. In this report functional studies were performed on rat skeletal muscle L6 cells stably transfected with an Asp905Tyr mutant PP-1G to evaluate the impact of this mutation on cellular responsiveness to insulin and cAMP. Although transfection resulted in a 3-fold increase in mutant PP-1G subunit expression, basal and insulin-stimulated PP-1 catalytic activities were decreased when compared with L6 cells transfected with wild-type PP-1G. The Asp905Tyr mutation resulted in an increase in cellular sensitivity to cAMP agonist, resulting in an inhibition of insulin's stimulatory effect on glycogen synthesis. More importantly, low concentrations of (Bu)2cAMP completely reversed insulin's stimulatory effects on glycogen synthesis when added to insulin-treated cells expressing mutant PP-1G. This was due to a rapid activation of glycogen phosphorylase a and a simultaneous inactivation of glycogen synthase via cAMP-mediated reductions in insulin-stimulated PP-1 catalytic activities. We conclude that an Asp905Tyr mutation of PP-1G is accompanied by a relative increase in sensitivity to cAMP agonists as well as a diminished capacity of the mutant PP-1G to effectively mediate the inhibitory effects of insulin on glycogen breakdown via PP-1 activation.

Nerve-dependent factors regulating transcript levels of glycogen phosphorylase in skeletal muscle.

1. Muscle glycogen phosphorylase (MGP), the rate-limiting enzyme for glycogen metabolism in skeletal muscle, is neurally regulated. Steady-state transcript levels of the skeletal muscle isozyme of MGP decrease significantly following muscle denervation and after prolonged muscle inactivity with an intact motor nerve. These data suggest that muscle activity has an important influence on MGP gene expression. The evidence to this point, however, does not preclude the possibility that MGP is also regulated by motor neuron-derived trophic factors. This study attempts to distinguish between regulation provided by nerve-evoked muscle contractile activity and that provided by the delivery of neurotrophic factors. 2. Steady-state MGP transcript levels were determined in rat tibialis anterior (TA) muscles following controlled interventions aimed at separating the contributions of contractile activity from axonally transported trophic factors. The innervated TA was rendered inactive by daily epineural injections of tetrodotoxin (TTX) into the sciatic nerve. Sustained inhibition of axonal transport was accomplished by applying one of three different concentrations of the antimicrotubule agent, vinblastine (VIN), to the proximal sciatic nerve for 1 hr. The axonal transport of acetylcholinesterase (AChE) was assessed 7, 14, and 28 days after the single application of VIN. 3. MGP transcript levels normalized to total RNA were reduced by 67% in rat TA, 7 days after nerve section. Daily injection of 2 microg TTX into the sciatic nerve for 7 days eliminated muscle contractile activity and reduced MGP transcript levels by 60%. 4. A single, 1-hr application of 0.10% (w/v) VIN to the sciatic nerve reduced axonal transport but did not alter MGP transcript levels in the associated TA, 7 days after treatment. Application of 0.10% VIN to the sciatic nerve also did not affect IA sensory or motor nerve conduction velocities or TA contractile function. 5. Treatment of the sciatic nerve with 0.40% (w/v) VIN for 1 hr reduced axonal transport and decreased MGP transcript levels by 50% within 7 days, but also reduced sensory and motor nerve conduction velocities and depressed TA contractile function. 6. Myogenin, a member of a family of regulatory factors shown to influence the transcription of many muscle genes, including MGP, was used as a molecular marker for muscle inactivity. Myogenin transcript levels were increased following denervation and after treatment with TTX or 0.40% VIN but not after treatment with 0.10% VIN. 7. The results suggest that MGP transcript levels in TA are regulated predominantly by muscle activity, rather than by the delivery of neurotrophic factors. Intrinsic myogenic factors, however, also play a role in MGP expression, since denervation did not reduce MGP transcript levels below 30% of control TA. The dominant influence of activity in the regulation of MGP contrasts with the proposed regulation of oxidative enzyme expression, which appears to depend on both activity and trophic factor influences.

Preserved GLP-I effects on glycogen synthase a activity and glucose metabolism in isolated hepatocytes and skeletal muscle from diabetic rats.

To search if biological effects of GLP-I on glucose metabolism in extrapancreatic tissue are present in diabetic states, we have studied the action of GLP-I and insulin on glycogen-enzyme activity, glycogen synthesis, and glucose metabolism in isolated hepatocytes and soleus muscle from adult streptozotocin (STZ)- and neonatal STZ-treated diabetic rats. This work confirms the previously reported insulin-like effects of GLP-I on glucose metabolism in both muscle and liver tissue from normal rats (control). The present study extends those observations to the muscle and liver tissue of diabetic animals. In both muscle and liver tissue, the metabolism of D-glucose, in the absence of added peptides, was more severely affected in adult STZ (IDDM model) than in neonatal STZ (nSTZ; NIDDM model) rats, and the magnitude of hormonal effect on metabolic variables was lower in diabetic rats than in control rats, as a rule. Nevertheless, in liver and muscle tissue of diabetic rats, GLP-I was able to increase glycogen synthase activity, augment the net rate of D-[U-14C]glucose incorporation into glycogen, and increase D-[5-3H]glucose utilization, D-[U-14C]glucose oxidation, and lactate production. In conclusion, GLP-I exerts insulin-like effects on D-glucose metabolism in both muscle and liver tissue in IDDM or NIDDM animal models, and present observations reinforce the view that GLP-I may represent a most promising tool in the treatment of diabetic patients.

Dexfenfluramine modulates hepatic glycogen metabolism by a calcium-dependent pathway.

In this study, the mechanism of action of dexfenfluramine (DEXF) at the hepatic level was investigated. The drug is shown to bind to the alpha 1-adrenergic receptor and to increase intracellular calcium in isolated rat hepatocytes, thereby activating phosphorylase via a calcium-dependent mechanism. Moreover, phosphorylase activation by DEXF was inhibited by different agents that interfere with the alpha 1-adrenergic signalling system: prazosin, phorbol 12 alpha-myristate 13 beta-acetate (PMA), and DEXF itself. We also show that phosphorylase activation induced by catecholamines and analogues (epinephrine, phenylephrine), whose actions are mediated by a calcium-dependent mechanism, was counteracted by the drug in the submillimolar range (0.1-1 mM). The activation of glycogenolysis by the drug is accompanied by a stimulation of the glycolytic flux (54% increase in lactate plus pyruvate accumulation), consistent with an increase in fructose-2,6-bisphosphate (F-2,6-BP) levels (36%). These results indicate that the interaction of DEXF with the alpha 1-adrenergic receptor channels glucose 6-phosphate derived from glycogen away from glucose production into the glycolytic pathway.

Bombyxin, an insulin-related peptide of insects, reduces the major storage carbohydrates in the silkworm Bombyx mori.

The effects of an insect insulin-related peptide, bombyxin, on carbohydrate metabolism were investigated in the silkworm Bombyx mori. Bombyxin lowered the concentration of the major hemolymph sugar, trehalose, in a dose-dependent manner when injected into neck-ligated larvae. Bombyxin also caused elevated trehalase activity in the midgut and muscle, suggesting that bombyxin induces hypotrehalosemia by promoting the hydrolysis of hemolymph trehalose to glucose and thereby facilitating its transport into tissues. In addition, bombyxin reduced the glycogen content in the fat body and concurrently raised the percentage of active glycogen phosphorylase in this tissue. Because hemolymph trehalose is also a major storage form of carbohydrate in insects, our results indicate that bombyxin reduces the amount of both principal storage carbohydrates in B. mori larvae. It is therefore suggested that although bombyxin is involved in the control of carbohydrate metabolism like insulin, the physiological role of bombyxin in insects is different from that of insulin in mammals.

Effects of diadenosine triphosphate and diadenosine tetraphosphate on rat liver cells. Differences and similarities with ADP and ATP.

Liver cells possess multiple types of purinoceptors that mediate the effects of extracellular nucleotides. Like ADP and ATP, the dinucleotides diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) fully activated glycogen phosphorylase, with ED50 values of 0.31 microM and 1.3 microM, respectively. At variance with ATP, neither the dinucleotides nor ADP significantly increased the levels of IP3.Ap4A (and also ADP) moderately increased IP3 (+/- 72%) whereas Ap3A was completely ineffective. Like ATP, Ap3A, Ap4A, and ADP inhibited the cAMP increase after glucagon. Phorbol-12-myristate-13-acetate (PMA) pretreatment of the hepatocytes clearly inhibited the glycogenolytic potency of Ap3A and ADP, but had only a minor effect on the potency of Ap4A or ATP. It is concluded that, depending upon the effect studied (glycogenolytic effect with or without PMA, increasing IP3 potency, or inhibition of cAMP increase), different analogies between the agonists studied emerged, indicating the complexity of the interaction of ATP and its analogues with liver purinoceptors and/or of the transduction mechanism(s) initiated by the different nucleotides.

Cloning and expression of cystolic phospholipase A2 (cPLA2) and a naturally occurring variant. Phosphorylation of Ser505 of recombinant cPLA2 by p42 mitogen-activated protein kinase results in an increase in specific activity.

Full-length cytosolic phospholipase A2 (cPLA2) was cloned from U937 cells and polymorphonuclear leukocytes (PMNLs) while a naturally occurring variant of cPLA2, which lacks residues Val473-Ala749 but has a C-terminal extension of ILMNLSEYMLWMSKVKRFM (DcPLA2) was cloned from PMNLs and mononuclear leukocytes. We were unable to clone DcPLA2 from U937 cells. When cPLA2 and DcPLA2 were expressed in insect cells, both proteins were detected in cell lysates by SDS/PAGE as single bands of apparent molecular masses 100 kDa and 57 kDa, respectively. Full-length cPLA2 was phosphorylated stoichiometrically by p42 mitogen-activated protein (MAP) kinase in vitro at a similar rate to other physiological substrates of this protein kinase and the major site of phosphorylation was identified by amino acid sequencing as Ser505. [32P]Ser(P)505 in cPLA2 was only dephosphorylated at a slow rate by mammalian tissue homogenates. Protein phosphatases 2A, 2B and 2C all contributed significantly to the overall dephosphorylation of cPLA2. The phosphorylation of cPLA2 by p42 MAP kinase correlated with an approximately 1.5-fold increase in specific enzyme activity which was reversed by dephosphorylation.

Signal transduction of adipokinetic hormones involves Ca2+ fluxes and depends on extracellular Ca2+ to potentiate cAMP-induced activation of glycogen phosphorylase.

Adipokinetic hormone (AKH)-induced mobilization of insect fat body glycogen occurs through activation of glycogen phosphorylase. In the migratory locust, signal transduction of AKH-I, -II and -III has been shown to involve the formation of cAMP. In the present study, we show that both the elevation of fat body cAMP levels and the activation of phosphorylase by the three AKHs in vitro depend on the presence of extracellular Ca2+; in the absence of Ca2+ in the medium, no phosphorylase activation occurs, whereas a concentration of at least 1.5 mM Ca2+ in the medium is required for maximal activation by each of the hormones. Furthermore, we show that AKH-I, -II and -III increase the influx of extracellular calcium into the fat body, as well as the efflux of cytosolic calcium from the fat body into the medium within 1 min of incubation. Although the time courses of their effects and the maximal responses to massive doses (40 nM) of the three hormones do not differ, AKH-III induces the highest increase in Ca2+ efflux when applied in a physiological dose (4 nM). No difference in the levels of Ca2+ influx induced by 4 nM of the hormones was observed. Quantitative analysis of the data suggests that the AKH-induced influx is larger than the efflux, implying a net rise in the fat body Ca2+ concentration.

Identification of the molecular basis for phosphorylase hypersensitivity in cultured diabetic cardiomyocytes.

The focus of this study was to identify the molecular basis for the hypersensitive response of glycogen phosphorylase activation to epinephrine stimulation in alloxan diabetic-derived cardiomyocytes. Cyclic AMP levels were found not to be significantly different between normal and diabetic-derived cells while cGMP concentrations were found consistently to be significantly lower in diabetic-derived cells than in normal cells. Treatment with cyclic GMP analogues did not affect phosphorylase activation by epinephrine in normal cardiomyocytes whereas, IBMX, a nonselective phosphodiesterase inhibitor, had a significant effect on basal and agonist-stimulated phosphorylase activity in both normal and diabetic-derived cardiomyocytes. Differences in the time course for the rate of decay of phosphorylase a from agonist-stimulated to basal levels were observed between normal and diabetic cells. After 3 h in primary culture, phosphorylase a activity returned to basal levels more quickly in normal than in diabetic-derived cells while after 24 h in culture, the time for phosphorylase a decay was not significantly different between normal and diabetic myocytes and was longer than the 3 h response. After 3 h response. After 3 h in primary culture, no significant difference in phosphorylase kinase activity was observed between normal and diabetic-derived cells exposed to epinephrine whereas, after 24 h in culture, phosphorylase kinase activity was significantly decreased in diabetic cells under basal and agonist-stimulation conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of vanadium treatment on the alterations of cardiac glycogen phosphorylase and phosphorylase kinase in streptozotocin-induced chronic diabetic rats.

Supersensitivity to isoproterenol (ISO) induced activation of cardiac phosphorylase in diabetic rat heart has been previously demonstrated and was also reproduced in this study. To explore further the nature of this supersensitivity, we examined the activity of phosphorylase kinase and the level of cyclic AMP (cAMP) in this tissue. We observed a significantly enhanced activation of phosphorylase kinase but no increase in cAMP levels in response to ISO stimulation in diabetic rat heart, suggesting that the supersensitivity of phosphorylase activation in diabetic heart may result from an enhanced activation of phosphorylase kinase that does not involve the cAMP pathway. On the other hand, perfusion of diabetic rat heart with verapamil (5 x 10(-8) M) prior to ISO stimulation abolished the enhanced cardiac phosphorylase activation, suggesting a role for calcium in the supersensitivity of phosphorylase activation. Furthermore, treatment of the diabetic rats with an insulin-like compound, vanadyl sulphate, completely abolished the enhanced cardiac phosphorylase activation and restored the increase in ISO-induced cAMP elevation in diabetic heart. The present study has provided further information on the changes of phosphorylase activation in the diabetic rat heart and demonstrated beneficial effects of vanadyl sulphate on the pathway leading to phosphorylase activation in diabetic rat heart.

Glycogen synthase, glycogen phosphorylase and alpha-amylase activity in homogenates of islets of GK rats: comparison with hepatic and pancreatic extracts.

Glycogen accumulation in pancreatic islet cells in situations of sustained hyperglycaemia may participate in the phenomenon of so-called B-cell glucotoxicity. Unexpectedly, however, previously little if any glycogen was found in islet cells of non-insulin-dependent diabetic Goto-Kakizaki rats (GK rats). Therefore, the activities of glycogen synthase, glycogen phosphorylase and alpha-amylase were measured in islets of control and GK rats. No significant difference in enzymatic activity was observed between the control and diabetic animals. In the liver, the activity of glycogen synthase appeared even somewhat higher in GK rats than in control animals. It is concluded that the diabetic syndrome in the GK rats does not involve any major anomaly of glycogen synthase and glycogen phosphorylase activity in the liver of these animals, as well as alpha-amylase, in pancreatic islets.

[The role of capsaicin-sensitive afferent nerves in regulating carbohydrate metabolism in the liver]. / Rol' kapsaitsin-chuvstvitel'nykh afferentnykh nervov v reguliatsii uglevodnogo obmena v pecheni.

Stimulation of afferent neurons with capsaicin enhanced the glycogen phosphorylase activity and decreased the glycogen contents in the rat liver. The glucose concentration in the blood serum was risen. Neurotoxic doses of capsaicin also enhanced the glycogen phosphorylase activity, more obviously so in adult rats. Preliminary administration of the toxic doses of capsaicin somewhat diminished the stimulating effect of capsaicin.

Evidence that the Ca2+ inflow pathway in hepatocytes stimulated by thapsigargin is similar to that activated by vasopressin.

Experiments were conducted to characterize the thapsigargin-stimulated plasma membrane Ca2+ inflow pathway in hepatocytes. Ca2+ inflow was estimated by measurement of the initial rate of activation of glycogen phosphorylase a following the addition of Ca2+ to cells previously incubated in the absence of added Ca2+. Pretreatment of hepatocytes with thapsigargin caused a substantial stimulation of the rate of Ca2+ activation of glycogen phosphorylase a. This was interpreted to reflect a stimulation of plasma membrane Ca2+ inflow. The effect of thapsigargin on plasma membrane Ca2+ inflow was approximately 65% of the magnitude of the effect caused by vasopressin. When thapsigargin and vasopressin were combined as a stimulus, the degree of stimulation was similar to that caused by vasopressin alone. The thapsigargin-induced stimulation of the rate of Ca2+ activation of glycogen phosphorylase a was inhibited in a concentration-dependent manner by both Zn2+ and 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365). The concentration of each agent required for half-maximal inhibition was approximately 20 microM. It is concluded from: (i) the apparent lack of additivity in the responses of thapsigargin and vasopressin, and (ii) the sensitivity to inhibitors, that the Ca2+ inflow pathway in hepatocytes stimulated by thapsigargin is likely to be similar to that which is activated by vasopressin.

[Action of dopamine on glucose level and glycogen phosphorylase activity of blood plasma in experimental coronary occlusion]. / Deistvie dofamina na uroven' gliukozy i glikogenfosforilaznuiu aktivnost' plazmy krovi pri éksperimental'noi koronarookkliuzii.

The effects of dopamine (5 micrograms/kg.min) on the blood plasma glucose level and glycogen-phosphorylase activity in experimental coronary occlusion were studied. The absence of the raising of the plasma glucose level and glycogen-phosphorylase "a" and "b" activities has been observed in 25 min after the coronary occlusion. It is supposed that the functional improvement of the ischemized myocardium by dopamine (5 micrograms/kg.min) is the result of the stimulation of presynaptic D-2-dopaminergic and postsynaptic D-1-dopaminergic receptors in coronary arteries.

Inhibition of glucose-6-phosphate phosphohydrolase by 3-mercaptopicolinate and two analogs is metabolically directive.

3-Mercaptopicolinae (3-MP) blocks gluconeogenesis from lactate, pyruvate, alanine, and other substrates through its inhibition of phosphoenolpyruvate carboxykinase. Nevertheless, we observed increased glycogenesis, net glucose uptake, and glucose-6-P levels in livers perfused with glucose in the presence of 3-MP. In perfusions with 20 mM dihydroxyacetone, increased glycogenesis and decreased glucose production were observed with 3-MP. These metabolic effects suggested additional site(s) of action of 3-MP. Further studies showed that 3-MP inhibits glucose-6-P phosphohydrolase activity of intact liver microsomes. Several compounds with structural similarities to 3-MP (2-mercaptonicotinic acid, picolinic acid, cysteine, reduced glutathione, nicotinic acid, quinolinic acid, tryptophan, and pyridine) were tested for their effect on glucose-6-P phosphohydrolase activity. Two of these compounds, 2-mercaptonicotinic acid and picolinic acid, were found to inhibit. In perfusions including 7.5 mM fructose, the addition of 3-MP, 2-mercaptonicotinic acid, or picolinic acid increased glycogenesis, decreased glucose production, and increased hepatic glucose-6-P concentrations. These observations indicate that the inhibition of glucose-6-P phosphohydrolase may play a role in enhanced glycogenesis from glucose, dihydroxyacetone, and fructose in isolated livers from 48-h fasted rats perfused with 3-MP or certain sulfhydryl-containing and sulfhydryl-devoid analogs.

Purification and properties of calmodulin from Phymatotrichum omnivorum.

Mycelia of Phymatotrichum omnivorum obtained at 10 day intervals during 10 to 50 days of growth were used for isolating calmodulin, and studying its effect on glycogen synthase, phosphorylase, phosphorylase kinase, cyclic AMP phosphodiesterase and Ca++ATPase. Glycogen synthase was inhibited until the 30th day by calmodulin, whereas calmodulin obtained from the 40th day stimulated glycogen synthase activity and the 50th day sample had no effect. cAMP phosphodiesterase and Ca++ATPase of P. omnivorum were stimulated by the respective calmodulin. Molecular weight of the purified fungal calmodulin was approximately 18 kD as revealed by sodium dodecyl sulphate gel electrophoresis. Trifluoperazine, dibucaine and lidocaine inhibited calmodulin activity and calmodulin activation of PDE, respectively.

Hexachlorophene induced alterations in brain carbohydrate metabolism of Wistar rat.

Effect of repeated oral administration of hexachlorophene (HCP) on glycolytic and oxidative pathways was studied in the rat brain. The rats were divided into three batches of six in each batch. The first batch was treated with paralytic dose (60 mg.kg-1.day-1) of HCP for 7 days. The second batch of animals was treated with sublethal dose (18 mg.kg-1.day-1) for 7 days. The third batch of animals was served as the age matched controls which received vehicle (corn oil) only. The glycolytic and oxidative metabolism of carbohydrates was significantly inhibited in the brain of rat during HCP treatment and the inhibition was more pronounced in paralytic dose treatment as compared to sublethal dose treatment. The inhibition of NADP-isocitrate dehydrogenase coupled with glucose 6-phosphate dehydrogenase indicates reduced generation of NADPH2 and pentoses for the synthesis of fatty acids and nucleotides.