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Biomedical subjects

M R Munday

Publications and source records attributed to M R Munday.

At least 37 records · Page 2Linked to original sources
PubMed1993

Regulation of human lung mast cell function by phosphatase inhibitors.

Okadaic acid, a cell-permeant inhibitor of serine/threonine phosphatases (PP) was found to attenuate the IgE-mediated release of both performed (histamine) and newly formed (leukotriene C4) mediators from human lung mast cells (HLMC). Optimal inhibition (82 +/- 6%) of the IgE-triggered release of histamine was observed after a 2-h incubation of HLMC with okadaic acid (1 microM). Shorter (< 2 h) incubation times and lower (< 1 microM) concentrations of okadaic acid were less effective at inhibiting histamine release from HLMC. The extent to which okadaic acid prevented histamine release was highly dependent upon the strength of the stimulus with lower levels of stimulation more readily modulated. The efficacy of okadaic acid as an inhibitor was not dependent upon the nature of the stimulus because matched levels of histamine release (approximately 30%) induced by either IgE-mediated processes or the calcium ionophore A23187 were attenuated to comparable extents. A series of analogues of okadaic acid and a structurally-distinct PP inhibitor, calyculin, were also found to inhibit the IgE-induced release of mediators from HLMC with the following rank order of potency; calyculin (approximate IC50; 0.015 microM) > okadaic acid (0.2 microM) > okadaol (3.3 microM) > nor-okadaone (> 10 microM). These same PP inhibitors displayed a similar rank order of activity for the inhibition of mediator release induced by the ionophore A23187. Extracts of purified HLMC were found to liberate 32P from radiolabeled glycogen phosphorylase and this PP activity was inhibited by both low (2 nM) and high (5 microM) concentrations of okadaic acid suggesting that HLMC contain both PP type 2A (PP2A) and PP type 1 (PP1). The okadaic acid, analogues of okadaic acid, and calyculin attenuated mediator release at concentrations and with a rank order of activity that closely parallels their activities as inhibitors of PP suggests that PP are important in the regulation of HLMC function. This contention is further supported by the finding that PP activities are constitutively associated with HLMC and that these activities could be inhibited by okadaic acid.

Ethers, Cyclic↗
PubMed1993

Mechanisms involved in the coordinate regulation of strategic enzymes of glucose metabolism.

In this review, we evaluate the relative regulatory importance of specific strategic enzymes (in particular glycogen synthase, acetyl-CoA carboxylase [ACC] and the pyruvate dehydrogenase complex [PDH]) for carbohydrate utilization as an anabolic precursor and as an energy substrate during the nutritional transitions between the fed and fasted states. The involvement of the specific protein kinases contributing to the inactivation of these enzymes by phosphorylation [cyclic AMP-dependent protein kinase, AMP-activated protein kinase and PDH kinase] in achieving each regulatory response is also assessed. We demonstrate a striking temporal correlation between hepatic glycogen mobilization and PDH and ACC inactivation by phosphorylation during the immediate postabsorptive period; in contrast, rates of hepatic glycogen synthesis and PDH and ACC expressed activities do not change in parallel during refeeding. The results are consistent with shifting of the primary sites of control for overall hepatic carbon flux during the fed-to-starved and starved-to-fed nutritional transitions achieved, at least in part, by a complex pattern of regulation by protein phosphorylation and metabolites which is critically dependent on the precise nutritional status. Data are also presented that demonstrate asynchronous suppression of glucose uptake/phosphorylation and pyruvate oxidation in cardiac and skeletal muscle during progressive starvation. Analogous asynchrony is observed in the reactivation of these processes in cardiac and skeletal muscle during refeeding after starvation. We provide evidence in support of the concept that selective suppression of pyruvate oxidation in oxidative muscles during early starvation and during the initial phase of refeeding is achieved because of differential sensitivity of glucose uptake/phosphorylation and pyruvate oxidation to lipid-fuel utilization. We discuss the relative importance of regulatory events governing local fatty acid production and utilization (via lipoprotein lipase and carnitine palmitoyltransferase 1, respectively) or overall fatty acid supply (dictated by events at the adipocyte) for fuel utilization by muscle during nutritional transitions. Finally, we assess the regulatory importance of glycogen synthesis in determining overall rates of glucose clearance by skeletal muscle during alimentary hyperglycemia and hyperinsulinemia.

Acetyl-CoA Carboxylase↗
PubMed1992

Diurnal rhythm of phosphorylation of rat liver acetyl-CoA carboxylase by the AMP-activated protein kinase, demonstrated using freeze-clamping. Effects of high fat diets.

1. Acetyl-CoA carboxylase was purified to homogeneity, in the presence of protein phosphatase inhibitors, from rat liver sampled without freeze-clamping. The enzyme was in a highly phosphorylated state (4.8 mol/subunit) of low specific activity, and could be dramatically reactivated by treatment with protein phosphatase-2A. Amino acid sequencing and fast-atom-bombardment mass spectrometry showed that the enzyme was phosphorylated in Ser79, Ser1200 and Ser1215, the three sites known to be phosphorylated in cell-free assays by the AMP-activated protein kinase. 2. The inactive enzyme could also be completely reactivated using a limited treatment with trypsin, which removes the N-terminal segment containing Ser79 and reduces the phosphate content to 3.5 mol/subunit. These results strengthen previous findings that it is phosphorylation at Ser79 by the AMP-activated protein kinase that is responsible for the inactivation, and not the phosphorylation of the 220-kDa core fragment (which contains Ser1200 and Ser1215). 3. Analysis of the phosphorylation state of Ser79 in acetyl-CoA carboxylase from rat liver showed that phosphorylation occurs post mortem if freeze-clamping is not used. The higher phosphorylation observed in extracts made without freeze-clamping correlates with a large increase in AMP and decrease in ATP (presumably caused by hypoxia during removal of the liver), and with increased activity of the AMP-activated protein kinase. These results provide a rational explanation for the post mortem phosphorylation events, and re-emphasize the point that rapid cooling of cells and tissues is essential when measuring the expressed activity of acetyl-CoA carboxylase (as well as 3-hydroxy-3-methylglutaryl-CoA reductase). 4. Using the freeze-clamping procedure, the ratio of 'expressed' activity (measured in the presence of protein phosphatase inhibitors) to 'total' activity (measured after complete dephosphorylation) of rat liver acetyl-CoA carboxylase showed a marked diurnal rhythm, changing from 50% in the active form in the middle of the dark period to less than 10% active in the middle of the light period. The very low activity in the light period was associated with a high level of phosphorylation in Ser79. This diurnal rhythm is very similar to that previously described for the phosphorylation of 3-hydroxy-3-methylglutaryl-CoA reductase, another substrate for the AMP-activated protein kinase. Neither the activity of the AMP-activated protein kinase nor the content of AMP, ADP or ATP changed between the dark or light periods.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetyl-CoA Carboxylase↗
PubMed1991

The short-term regulation of hepatic acetyl-CoA carboxylase during starvation and re-feeding in the rat.

Rapid inhibition of acetyl-CoA carboxylase (ACC) activity in rat liver in response to 6 h starvation and rapid re-activation in response to 2-6 h of re-feeding chow were shown to be due to changes in the expressed activity of existing enzyme. Decreases and increases in ACC concentration occurred at later stages of the transitions, i.e. 6-48 h starvation and 8-24 h re-feeding respectively. The decrease in expressed activity of ACC was due primarily to changes in its phosphorylation state, demonstrated by a significantly decreased Vmax. and significantly increased Ka for citrate of enzyme purified by avidin-Sepharose chromatography from 6 h- or 48 h-starved rats. These effects were totally reversed within 2-4 h of chow re-feeding. Changes in the activity of purified ACC closely correlated with reciprocal changes in the activity of AMP-activated protein kinase (AMP-PK) over the fed to starved to re-fed transition. Increases in the activity ratio of cyclic-AMP-dependent protein kinase in response to starvation lagged behind the increase in AMP-PK and the decrease in ACC activity. Changes in AMP-PK and ACC activities of rat liver closely correlated with changes in plasma insulin concentration in response to time courses of starvation and re-feeding.

AMP-Activated Protein Kinases↗
PubMed1991

Regulation of fatty acid synthesis in lactating rat mammary gland in the fed to starved transition: asynchronous control of pyruvate dehydrogenase, phosphofructokinase and acetyl-CoA carboxylase.

1. Withdrawal of food from lactating rats produced a rapid and dramatic decrease in the uptake of glucose by the mammary gland and an inhibition of the rate of fatty acid synthesis that could not be explained alone by decreased substrate supply to the tissue. 2. Within the first 6 hr starvation, fatty acid synthesis and pyruvate dehydrogenase activity were inhibited by 87 and 80%, respectively, but acetyl-CoA carboxylase activity did not change significantly. 3. Between 6 and 24 hr starvation, total and expressed activities of acetyl-CoA carboxylase decreased by 62 and 55%, respectively. 4. The ratio of fructose-6-phosphate/fructose-1,6-bisphosphate concentration in mammary tissue increased 9-fold during the first 6 hr starvation, indicating an inhibition of 6-phosphofructo-1-kinase. However, the major inhibition of this enzyme occurred between 6 and 24 hr starvation when this metabolite ratio increased a further 160-fold in parallel with increased tissue citrate concentration. 5. The increase in citrate concentration between 6 and 24 hr starvation correlated with acetyl-CoA carboxylase inactivation and ketone body accumulation in the mammary gland. 6. This study confirms the asynchronous control of three important regulatory steps in the pathway of glucose utilization and fatty acid synthesis in the lactating rat mammary gland.

Acetyl-CoA Carboxylase↗
PubMed1988

Negative interactions between phosphorylation of acetyl-CoA carboxylase by the cyclic AMP-dependent and AMP-activated protein kinases.

We have reported previously that cyclic AMP-dependent protein kinase phosphorylates two sites on acetyl-CoA carboxylase (site 1: Arg-Met-Ser(P)-Phe, and site 2: Ser-Ser(P)-Met-Ser-Gly-Leu), while the AMP-activated protein kinase also phosphorylates site 1, plus site 3 (Ser-Ser-Met-Ser(P)-Gly-Leu), the latter being two residues C-terminal to site 2. We now report that prior phosphorylation of site 2 by cyclic AMP-dependent protein kinase prevents the subsequent phosphorylation of site 3 and the consequent large decrease in Vmax produced by the AMP-activated protein kinase. Similarly, prior phosphorylation of site 3 by the AMP-activated protein kinase prevents subsequent phosphorylation of site 2 by cyclic AMP-dependent protein kinase.

Acetyl-CoA Carboxylase↗
PubMed1988

Identification by amino acid sequencing of three major regulatory phosphorylation sites on rat acetyl-CoA carboxylase.

We have examined the sites phosphorylated on acetyl-CoA carboxylase by three protein kinases which have been shown to inactivate the enzyme, i.e. cyclic-AMP-dependent protein kinase, acetyl-CoA carboxylase kinase-2 (ACK2, purified from rat mammary gland) and the AMP-activated protein kinase (formerly called acetyl-CoA carboxylase kinase-3, purified from rat liver). Each protein kinase phosphorylates two out of three sites (termed 1-3) which have been established by amino acid sequencing. The two sites phosphorylated by each kinase can be recovered on separate peptides, TC1 and TC2, derived by combined digestion of the native enzyme by trypsin and chymotrypsin: TC1 = Ser-2Ser(P)-Met-3Ser(P)-Gly-Leu; TC2 = Arg-Met-1Ser(P)-Phe- Cyclic-AMP-dependent protein kinase phosphorylates sites 1 and 2 exclusively, whereas the AMP-activated protein kinase phosphorylates sites 1 and 3, plus at least one other minor site. ACK2 phosphorylates site 1 and, more slowly, an unidentified site(s) within TC1. We have also established the structures of the single major phosphopeptides (T1 and C1 respectively) which are recovered by HPLC after acetyl-CoA carboxylase phosphorylated by cyclic-AMP-dependent protein kinase is digested with trypsin or chymotrypsin alone. T1 is related to TC1, and has the structure: Ser-Ser(P)-Met-Ser-Gly-Leu-His-Leu-Val-Lys. C1 is identical with TC2. We have carried out studies on the correlation of the activity of acetyl-CoA carboxylase with the occupancy of sites 1, 2 and 3 during phosphorylation by each of the three protein kinases. The results suggest that phosphorylation of site 3 is primarily responsible for the large decrease in Vmax produced by the AMP-activated protein kinase, while phosphorylation of site 1 may be primarily responsible for the increase in A0.5 for citrate and more modest depression of Vmax produced by cyclic-AMP-dependent protein kinase and ACK2. Our results emphasize that amino acid sequence information is essential in the unequivocal interpretation of data from phosphopeptide mapping experiments and allow a more complete interpretation of previous data on phosphorylation of acetyl-CoA carboxylase in intact cells. They also open the way to experiments which could establish the physiological roles of these protein kinases in the control of fatty acid synthesis.

Acetyl-CoA Carboxylase↗
PubMed1987

Effects of angiotensin I converting enzyme inhibition and calcium channel blockade on plasma levels of active and inactive renin in conscious rabbits.

The interplay of juxtaglomerular (jg) calcium fluxes and exposure to AII in the regulation of jg renin secretion, was examined in vivo. An inhibitor of angiotensin I converting enzyme (captopril), a blocker of calcium channels (verapamil) and AII amide were infused, singly or in combination, into the ear vein of conscious rabbits. The effects on arterial pressure, and on levels of active and inactive plasma renin were monitored. Captopril (50 micrograms X min-1 X kg-1) produced a greater percentage increase in renin secretion than did verapamil (20 micrograms X min-1 X kg-1), whilst the percentage fall in arterial pressure was similar. AII amide counteracted more effectively the actions of captopril than those of verapamil. When captopril was infused first, addition of verapamil did not enhance renin secretion (P greater than 0.2). When verapamil was infused first, addition of captopril greatly enhance renin secretion (P less than 0.01). However, when captopril was infused first, and its actions then suppressed by AII amide, addition of verapamil led to extremely high rates of renin secretion. The findings suggest the following: the short loop negative feedback plays in vivo an important role in the rapid modulation of jg renin secretion and the action of AII may involve up- and down-regulation at the receptor and/or post-receptor level; infused agents have rapid access to the critical sites of jg cells; exposure to raised concentrations to AII not only reduces the effectiveness of AII, but also enhances jg secretory responses to lack of AII, as well as to calcium channel blockade. Thus, at least some of the jg calcium channels appear to respond both to AII and to blockers; extreme changes in the levels of active renin are possible without changes of inactive renin levels. Secretion of the latter may be under separate control, or its secretion rate parallelled by the rate of its activation.

Angiotensin Amide↗
PubMed1987

Insulin activation of lipogenesis in isolated mammary acini from lactating rats fed on a high-fat diet. Evidence that acetyl-CoA carboxylase is a site of action.

Feeding lactating rats on high-fat cheese crackers in addition to laboratory chow increased the dietary intake of fat from 2 to 20% of the total weight of food eaten and decreased mammary-gland lipogenesis in vivo by approx. 50%. This lipogenic inhibition was also observed in isolated mammary acini, where it was accompanied by decreased glucose uptake. These inhibitions were completely reversed by incubation with insulin. Insulin had no effect on the rate of glucose transport into acini, nor on pyruvate dehydrogenase activity as estimated by the accumulation of pyruvate and lactate, suggesting that these are not the sites of lipogenic inhibition. Insulin stimulated the incorporation of [1-14C]acetate into lipid in acini from high-fat-fed rats. In the presence of alpha-cyanohydroxycinnamate, a potent inhibitor of mitochondrial pyruvate transport, and with glucose as the sole substrate, neither [1-14C]glucose incorporation into lipid nor glucose uptake were stimulated by insulin. Insulin did stimulate the incorporation of [1-14C]acetate into lipid in the presence of alpha-cyanohydroxycinnamate, and this was accompanied by an increase in glucose uptake by the acini. This indicated that increased glucose uptake was secondary to the stimulation of lipogenesis by insulin, which therefore must occur via activation of a step in the pathway distal to mitochondrial pyruvate transport. Insulin stimulated acetyl-CoA carboxylase activity measured in crude extracts of acini from high-fat-fed rats, restoring it to values close to those of chow-fed controls. The effects of insulin on acetyl-CoA carboxylase activity and lipogenesis were not antagonized by adrenaline or dibutyryl cyclic AMP.

Acetates↗
PubMed1986

The role of acetyl-CoA carboxylase phosphorylation in the control of mammary gland fatty acid synthesis during the starvation and re-feeding of lactating rats.

Activation of acetyl-CoA carboxylase during incubation of crude extracts of lactating rat mammary gland with Mg2+ and citrate can be blocked by NaF, suggesting that it represents a dephosphorylation of the enzyme. The greater extent of activation in extracts from 24 h-starved rats (200%) compared with fed controls (70%) implies that the decrease in acetyl-CoA carboxylase activity in response to 24 h starvation may involve increased phosphorylation of the enzyme. Acetyl-CoA carboxylase was purified from the mammary glands of lactating rats in the presence of protein phosphatase inhibitors by avidin-Sepharose chromatography. Starvation of the rats for 24 h increased the concentration of citrate giving half-maximal activation by 75%, and decreased the Vmax. of the purified enzyme by 73%. This was associated with an increase in the alkali-labile phosphate content from 3.3 +/- 0.2 to 4.5 +/- 0.4 mol/mol of enzyme subunit. Starvation of lactating rats for 6 h, or short-term insulin deficiency induced by streptozotocin injection, did not effect the kinetic parameters or the phosphate content of acetyl-CoA carboxylase purified from mammary glands. The effects of 24 h starvation on the kinetic parameters and phosphate content of the purified enzyme were completely reversed by re-feeding for only 2.5 h. This effect was blocked if the animals were injected with streptozotocin before re-feeding, suggesting that the increase in plasma insulin that occurs on re-feeding was responsible for the activation of the enzyme. The effects of re-feeding 24 h-starved rats on the kinetic parameters and phosphate content of acetyl-CoA carboxylase could be mimicked by treating enzyme purified from 24 h-starved rats with protein phosphatase-2A in vitro. Our results suggest that, in mammary glands of 24 h-starved lactating rats, insulin brings about a dephosphorylation of acetyl-CoA carboxylase in vivo, which may be at least partly responsible for the reactivation of mammary lipogenesis in response to re-feeding.

Acetyl-CoA Carboxylase↗
PubMed1984

Isolation of three cyclic-AMP-independent acetyl-CoA carboxylase kinases from lactating rat mammary gland and characterization of their effects on enzyme activity.

Three cyclic AMP-independent acetyl-CoA carboxylase kinases (A, B1 and B2) have been isolated from lactating rat mammary gland, using phosphocellulose chromatography, high performance gel filtration, and affinity chromatography on casein-Sepharose and phosvitin-Sepharose. These protein kinases have been identified with previously described kinases by the following criteria. Kinase A phosphorylates the same sites on rabbit mammary acetyl-CoA carboxylase as acetyl-CoA carboxylase kinase 2, which was originally described as a contaminant of rabbit mammary acetyl-CoA carboxylase purified by the poly(ethylene glycol)procedure. Kinase A will henceforth be referred to as acetyl-CoA carboxylase kinase-2. Kinase B1 has been identified with casein kinase II by its heparin sensitivity, elution behaviour on phosphocellulose, molecular mass, substrate specificity and subunit composition. Kinase B2 has been identified with casein kinase I by its elution behaviour on phosphocellulose, molecular mass, substrate specificity and subunit composition. The three kinases phosphorylate distinct sites on acetyl-CoA carboxylase. Phosphorylation by either casein kinase I or II does not affect enzyme activity. However, acetyl-CoA carboxylase kinase 2 inactivates acetyl-CoA carboxylase reversibly, in an identical manner to cyclic-AMP-dependent protein kinase, and phosphorylates sites located on identical peptides. Acetyl-CoA carboxylase kinase-2 can, however, be distinguished from the free catalytic subunit of cyclic-AMP-dependent protein kinase by its molecular mass, its substrate specificity, its elution behaviour on phosphocellulose, and its complete lack of sensitivity to the protein inhibitor of cyclic-AMP-dependent protein kinase. We also present evidence that phosphorylation of acetyl-CoA carboxylase by cyclic-AMP-dependent protein kinase occurs directly and not via a bicyclic cascade system as proposed by other laboratories.

Acetyl-CoA Carboxylase↗
PubMed1984

Biochemical basis of dietary influences on the synthesis of the macronutrients of rat milk.

In this contribution the regulation of the synthesis of the macronutrients (lactose, lipid, protein) of rat milk are briefly reviewed. Lactose synthesis and lipid synthesis in mammary gland have glucose as the common precursor, and the rates of both processes show diurnal changes that correlate with the variations in food intake. Short-term (6-h) removal of food results in large decreases in the rates of synthesis of lactose and lipid, despite any change in plasma glucose or insulin. Similarly, an intragastric load of fat rapidly decreases de novo lipogenesis in the mammary gland. The existence of a factor in the circulation that signals the dietary intake of carbohydrate is postulated. This putative factor may originate from the intestine. Synthesis of protein appears less sensitive to dietary intake and is regulated mainly by prolactin.

Amino Acids↗
PubMed1983

Diurnal variations in food intake and in lipogenesis in mammary gland and liver of lactating rats.

Despite the hyperphagia, the food intake of the lactating rat showed marked diurnal changes which paralleled those of virgin rats. The major difference was that lactating rats consumed a higher proportion (35%) of their diet during the light period than did virgin rats (14%). The peak rate of lipogenesis in the lactating mammary gland occurred around midnight, and this decreased by 67% to reach a nadir around mid-afternoon; this corresponded with the period of lowest food intake. The diurnal variations in hepatic lipogenesis in lactating rats were much less marked. The changes in hepatic glycogen over 24 h suggest that it acts to supply carbon for lipogenesis during the period of decreased food intake. The activation state of acetyl-CoA carboxylase in mammary gland altered during 24 h, but the changes did not always correlate with alterations in the rate of lipogenesis. The changes in plasma insulin concentration tended to parallel the food intake in the lactating rats, but they did not appear to be sufficient to explain the large alterations in lipogenic rate in the mammary gland.

Acetyl-CoA Carboxylase↗