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M R Munday

Publications and source records attributed to M R Munday.

At least 19 recordsLinked to original sources
PubMed2007

Role of calcineurin in the regulation of human lung mast cell and basophil function by cyclosporine and FK506.

BACKGROUND AND PURPOSE: Cyclosporine and FK506 are thought to act by targeting the Ca2+-dependent protein phosphatase, calcineurin. The aim of the present study was to determine whether cyclosporine and FK506 stabilize mast cells and basophils by interacting with calcineurin. EXPERIMENTAL APPROACH: The effects of cyclosporine and FK506 on the IgE-mediated release of histamine from mast cells and basophils were evaluated. The presence of calcineurin in cells was determined by Western blotting. Ca2+-dependent protein phosphatase activities were assessed in cell extracts using a synthetic phosphorylated peptide that is known to serve as a substrate for calcineurin. KEY RESULTS: FK506 was about 100-fold more potent than cyclosporine as an inhibitor of IgE-dependent histamine release from mast cells and basophils. Immunoblotting of solubilized preparations of purified cells demonstrated the presence of calcineurin in mast cells and basophils. In enzyme assays, mast cells expressed approximately 7-fold higher Ca2+-dependent protein phosphatase activity than basophils. Whereas cyclosporine effectively inhibited Ca2+-dependent protein phosphatase activity in cell extracts, FK506 was considerably less effective. CONCLUSIONS AND IMPLICATIONS: FK506 and cyclosporine inhibit the stimulated release of histamine from mast cells and basophils. However, the ability of cyclosporine, but not FK506, to inhibit Ca2+-dependent protein phosphatase activity questions whether FK506 stabilizes mast cells and basophils by interacting with calcineurin.

Basophils↗
PubMed2003

Markers of experimental acute inflammation in the Wistar Han rat with particular reference to haptoglobin and C-reactive protein.

C-reactive protein (CRP), haptoglobin (Hp) and fibrinogen (Fbgn) are acute phase reactants (APRs), the blood levels of which increase during acute inflammation. However, although the levels of these APRs are used to monitor inflammation in man, their usefulness and sensitivity as markers of inflammation in rodents are less clear. We therefore wished to evaluate, in a comparative fashion, a prototype immunoassay for serum CRP, a commercial assay for serum Hp, and an automated assay for Fbgn, using a model of acute inflammation in the rat. Additionally, pro-inflammatory cytokines and serum protein fractions were also measured. The model of inflammation used was the intraperitoneal injection of Freund's complete adjuvant (FCA). In a concluding experiment, findings with Hp in the FCA rat model were validated in a toxicologically relevant study involving the induction of acute hepatic inflammation using the model hepatotoxicant carbon tetrachloride (CCl(4)). Female Wistar Han rats were treated with a single injection of FCA in a dose-response study (1.25-10.0 ml/kg, sampling at 36 h) and two time-course studies (over 40 h and 21 days). In a final experiment, rats were dosed with CCl(4) at 0.8 ml/kg and sampled over a 17-day period. In FCA and CCl(4) experiments, serum/plasma was prepared and tissues taken at autopsy for histological assessment (CCl(4) study only). In the dose-response study, serum CRP, Hp and plasma Fbgn were increased at all FCA dose levels at 36 h post-dosing. Serum alpha(2) and beta(1) globulin fractions were also increased, while albumin levels were decreased. In the 40-h time-course study, CRP levels peaked at 25-40 h post-dosing, to approximately 120% of control (as 100%). Hp levels increased to a maximum at 25 and 40 h post-dosing with values greater than 400% of control, and alpha(2) and beta(1) globulin fractions peaked at 30 and 40 h post-dosing to 221 and 187% of control, respectively. Increased serum interleukin-6 (IL-6) and interleukin-1beta (IL-1beta) levels peaked at 20 h (11-fold) and 25 h (19-fold), respectively. In a 21-day time-course study, no increased CRP levels were measured despite elevated levels of Hp, which peaked at 36 h (approximately 7-fold above control), and remained elevated up to 21 days. IL-6 and IL-1beta levels peaked at 12 h (19-fold) and 24 h (28-fold), respectively. Liver histopathology of animals treated with CCl(4) showed centrilobular hepatocellular degeneration and necrosis (most significant at 36 h) with an inflammatory response (most significant at 48 h). Resolution of the lesion was complete by 4 days post-dosing. Serum alanine aminotransferase, aspartate aminotransferase and glutamate dehydrogenase levels peaked at 36 h post-dosing. Hp levels increased maximally at 48 h (426% of control). We conclude that serum CRP is a poor marker of acute inflammation in the rat in comparison with serum Hp and plasma Fbgn. Between Hp and Fbgn, serum Hp is shown to be the most sensitive and useful marker of acute inflammation.

Acute Disease↗
PubMed2002

Regulation of mammalian acetyl-CoA carboxylase.

Acetyl-CoA carboxylase (ACC) plays a critical role in the regulation of fatty acid metabolism and its two isoforms, ACCalpha and ACCbeta, appear to have distinct functions in the control of fatty acid synthesis and fatty acid oxidation, respectively. They are regulated by similar short-term mechanisms of allosteric activation by citrate, and reversible phosphorylation and inactivation, and there is clearly interaction between these mechanisms. AMP-activated protein kinase is the important physiological ACC kinase for both isoforms and yet there is a potential physiological role for cAMP-dependent protein kinase in the hormonally mediated inactivation of ACCalpha, and phosphorylation of ACCbeta in its unique N-terminus.

Acetyl-CoA Carboxylase↗
PubMed1999

The regulation of acetyl-CoA carboxylase--a potential target for the action of hypolipidemic agents.

ACC exists as two major isoforms ACC1 or ACC alpha, and ACC2 or ACC beta, and there is evidence that they play separate roles in the production of malonyl-CoA for fatty acid synthesis and the control of mitochondrial beta-oxidation, respectively. ACC alpha can be regulated at the level of gene expression, allosteric regulation of the enzyme, and reversible phosphorylation by AMP-PK. Emerging lines of research suggest that similar mechanisms of regulation exist for ACC beta. Its inactivation in heart and skeletal muscle through phosphorylation by AMP-PK is becoming well-established. ACC is an important target of certain hypolipidemic drugs such as the fibrates. This is not simply because ACC alpha inhibition decreases the synthesis of a lipid component of VLDL because fatty acids synthesized de novo in liver are not always major contributors to VLDL lipid (158); it is also because ACC beta inhibition leads to a decrease in malonyl-CoA levels and the disinhibition of fatty acid oxidation. Partitioning fatty acids towards oxidation and away from esterification is an important aspect of the lipid-lowering effects of fibrates. Fibrates could use any of the mechanisms of ACC regulation to decrease activity. They could repress ACC gene expression through the activation of PPAR alpha, and fibroyl-CoA esters could inhibit ACC allosterically just as TOFyl-CoA does. However, we have demonstrated a rapid inactivation of ACC in cultured rat hepatocytes by gemfibrozil that is mediated by activation of AMP-PK and the subsequent phosphorylation of ACC. The end result is the inhibition of hepatic fatty acid synthesis and a possible activation of beta-oxidation as evidenced by the increased production of ketone bodies. The mechanism through which fibrates activate the AMP-PK cascade, the role of PPAR alpha, the physiological responses of biosynthesis and oxidation and the use of these mechanisms by other hypolipidemic agents are areas of ongoing investigation.

AMP-Activated Protein Kinases↗
PubMed1999

Role of protein phosphatases in the regulation of human mast cell and basophil function.

Many extracellular stimuli mediate physiological change in target cells by altering the phosphorylation state of proteins. These alterations result from the dynamic interplay of protein kinases, which mediate phosphorylations, and protein phosphatases, which catalyse dephosphorylations. The antigen-mediated aggregation of high-affinity receptors for IgE on mast cells and basophils triggers rapid changes in the phosphorylation of many proteins and culminates in the generation of inflammatory mediators involved in allergic inflammatory diseases such as asthma. Although protein kinases have an established role in this process, less is known about the involvement of protein phosphatases. This imbalance has been redressed in recent years by the availability of phosphatase inhibitors, such as okadaic acid, that facilitate investigations of the role of protein phosphatases in intact cells. Here we review a number of studies in which inhibitors of protein phosphatases have been used to shed light on the potential importance of these enzymes in the regulation of human mast cell and human basophil function.

Basophils↗
PubMed1998

Characterization of protein serine/threonine phosphatase activities in human lung mast cells and basophils.

1. The serine/threonine protein phosphatase (PP) inhibitors, okadaic acid and calyculin, attenuated the IgE-mediated release of histamine from human lung mast cells (HLMC) and basophils in a dose-dependent manner whereas an alternative PP inhibitor, microcystin, was ineffective. Calyculin was more potent than okadaic acid in both cell types. The concentration required to inhibit by 50% (IC50) the release of histamine was 15 (HLMC) and 50 nM (basophils) for calyculin and 200 (HLMC) and 300 nM (basophils) for okadaic acid. 2. Lysates of purified HLMC and basophils dephosphorylated radiolabelled glycogen phosphorylase, a substrate for both PP1 and PP2A. The PP activity in lysates of both cell types was inhibited in a dose-dependent fashion by the PP inhibitors with the following rank order of activity, calyculin (approximate IC50; 0.02-0.1 nM) > or = microcystin (0.1 nM) > okadaic acid (70 nM). 3. The PP1-selective inhibitor, inhibitor-2 (I-2), attenuated the dephosphorylation of glycogen phosphorylase in lysates of both HLMC and basophils. I-2 (20 nM) inhibited the glycogen phosphorylase PP activity by 71+/-3% and 49+/-13% in HLMC and basophil extracts, respectively. There were, approximately, 6 fold greater levels of I-2-sensitive activity in HLMC than in basophils. Qualitatively similar results were obtained with an alternative PP1-selective inhibitor, inhibitor-1 (I-1). 4. Lysates derived from HLMC and basophils dephosphorylated radiolabelled casein which is a PP2A-restricted substrate. HLMC lysates contained, approximately, 2.5 fold higher levels of casein PP activity than basophil lysates. 5. These data indicate that HLMC and basophils both contain PP1 and PP2A. The data suggest that, on a per cell basis, HLMC have higher levels of both PP1 and PP2A. Moreover, the ratio of PP1 to PP2A is higher in HLMC than in basophils.

Basophils↗
PubMed1997

The role of pyruvate dehydrogenase, phosphofructo-1-kinase and acetyl-CoA carboxylase in the regulation of fatty acid synthesis in the lactating rat mammary gland during the starved to re-fed transition.

Re-feeding 24-h-starved lactating rats resulted in a rapid (within 0.5 h) restoration of glucose uptake by the mammary gland and a slower (within 3 h) restoration of fatty acid synthesis. The rapid reactivation of glucose uptake (82% of fed value within 0.5 h of re-feeding) correlated with a rapid reactivation of 6-phosphofructo-1-kinase (6-PF-1-K) and glycolysis (as determined by a 97% decrease in the [fructose-6-phosphate]/[fructose-1,6-bisphosphate] ratio). This could not be fully explained by a fall (29%) in the tissue concentration of its allosteric inhibitor, citrate. The delayed reactivation of pyruvate dehydrogenase (PDH) correlated very closely with the delayed reactivation of fatty acid synthesis and explained the continued output of pyruvate and lactate within the first 0.5 h of re-feeding. PDH reactivation preceded the reactivation of acetyl-CoA carboxylase (ACC), which did not occur significantly until 1.5 h of re-feeding. ACC reactivation correlated with a decrease in the tissue concentration of citrate and a second late phase of 6-PF-1-K activation. It is clear that the important regulatory steps 6-PF-1-K, PDH and ACC, are reactivated asynchronously in the lactating mammary gland in response to re-feeding starved rats and that PDH is more important than ACC in the regulation of fatty acid synthesis.

Acetyl-CoA Carboxylase↗
PubMed1997

Preliminary characterization of the role of protein serine/threonine phosphatases in the regulation of human lung mast cell function.

1. Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE-dependent release of mediators from human lung mast cells (HLMC). The concentration of okadaic acid required to inhibit by 50% (IC50) the IgE-dependent release of histamine was 0.2 microM. Okadaic acid also inhibited the IgE-mediated generation of prostaglandin D2 (PGD2) and sulphopeptidoleukotrienes (sLT) with IC50 values of 0.2 microM and 0.6 microM respectively. 2. The IgE-mediated generation of histamine, PGD2 and sLT was inhibited by okadaic acid and two analogues of okadaic acid, okadaol and okadaone, with the following rank order of activity; okadaic acid > okadaol > okadaone. This order of activity for the inhibition of mediator release parallels the activity of these compounds as inhibitors of isolated PPs. 3. Extracts of HLMC liberated 32P from radiolabelled glycogen phosphorylase and this PP activity was inhibited by the PP inhibitors (all at 3 microM), okadaic acid (73 +/- 4% inhibition, P < 0.0005), okadaol (26 +/- 7% inhibition, P < 0.05) and okadaone (8 +/- 7% inhibition, P = 0.52). The rank order of activity of okadaic acid > okadaol > okadaone parallels the activity of these compounds as inhibitors of isolated PPs. 4. Dephosphorylation of radiolabelled glycogen phosphorylase by extracts of HLMC was inhibited by 15 +/- 3% (P < 0.001) by a low (2 nM) concentration of okadaic acid and by 88 +/- 4% (P < 0.0005) by a higher (5 microM) concentration of okadaic acid. Because 2 nM okadaic acid may act selectively to inhibit PP2A whereas 5 microM okadaic acid inhibits both PP1 and PP2A, these data suggest that both PP1 and PP2A are present in HLMC. 5. Inhibitor 2, a PP1-selective inhibitor, attenuated (71 +/- 3% inhibition, P < 0.05) PP activity in extracts of HLMC suggesting that HLMC contain PP1 and that it may constitute 71% of the phosphorylase PP activity in extracts of HLMC. 6. Radiolabelled casein, a PP2A-restricted substrate, was dephosphorylated by extracts of purified HLMC and this activity was inhibited (81 +/- 8% inhibition, P < 0.005) by 2 nM okadaic acid suggesting that PP2A is resident in HLMC. 7. Collectively, these data suggest that both PP1 and PP2A are resident in HLMC. However, although the data suggest that okadaic acid regulates responses in HLMC by interacting with PPs, it has not been possible to determine whether either PP1 or PP2A or both PPs are involved in the okadaic acid-induced inhibition of mediator release from HLMC.

Histamine Release↗
PubMed1996

Regulation of human basophil function by phosphatase inhibitors.

1. Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE-mediated release of the pre-formed mediator, histamine from human basophils in a time- and dose-dependent manner. Optimal inhibition (77 +/- 4%, P < 0.0001) of histamine release was observed following a 2 h incubation with 1 microM okadaic acid. 2. Okadaic acid and two analogues of okadaic acid were also studied and were found to inhibit the IgE-dependent release of histamine. Concentrations required to inhibit release by 50% (IC50) were 0.6 microM for okadaic acid and 7.5 microM for okadaol, whereas okadaone was inactive. 3. The structurally-unrelated PP inhibitor, calyculin A, also inhibited IgE-dependent histamine release from basophils dose-dependently and was approximately six fold more potent than okadaic acid. 4. The IgE-mediated generation of sulphopeptidoleukotrienes (sLT) from basophils was inhibited by okadaic acid and related analogues with the following rank order of potency; okadaic acid (approx. IC50 0.3 microM) > okadaol (3 microM) > okadaone (inactive). 5. Okadaic acid, okadaol and okadaone (all at 3 microM) inhibited the IgE-mediated generation of the cytokine interleukin 4 (IL4) from human basophils by 67 +/- 9% (P < 0.002), 48 +/- 14% (P < 0.05) and 8 +/- 7% (P = 0.31), respectively. 6. Extracts of purified human basophils liberated 32P from radiolabelled glycogen phosphorylase and this PP activity was inhibited by 17 +/- 3% (P < 0.0005) by a low (2 nM) concentration of okadaic acid and was inhibited by 96 +/- 1% (P < 0.0001) by a higher (5 microM) concentration of okadaic acid. Because a low (2 nM) concentration of okadaic acid inhibits PP2A selectively whereas a higher (5 microM) concentration inhibits both PP1 and PP2A, these findings suggest that both PP1 and PP2A are present in basophils. 7. In total these data suggest that PPs are resident in human basophils and that PPs may be important in the regulation of basophil function.

Basophils↗
PubMed1994

Yeast SNF1 is functionally related to mammalian AMP-activated protein kinase and regulates acetyl-CoA carboxylase in vivo.

The product of the SNF1 gene is a protein kinase whose activity is essential for transcriptional activation of glucose repressed genes in Saccharomyces cerevisiae. We have cloned a mammalian AMP-activated protein kinase (AMPK) that is 46% identical to the deduced amino acid sequence of SNF1 (Carling, D., Aguan, K., Woods, A., Verhoeven, A.J.M., Beri, R., Brennan, C.H., Sidebottom, C., Davison, M.D., and Scott, J. (1994) J. Biol. Chem. 269, 11442-11448). Mammalian AMPK plays a major role in the control of lipid metabolism and phosphorylating, thereby inactivating both acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase, key regulatory enzymes in the synthesis of fatty acids and cholesterol, respectively. We present evidence indicating that, in common with its mammalian homologue, SNF1 forms part of a protein kinase cascade. SNF1 is inactivated in vitro by treatment with protein phosphatase 2A and can be reactivated using a partially purified preparation of mammalian AMPK kinase. SNF1 undergoes a time-dependent increase in activity during growth in glucose-derepressing conditions, providing the first evidence that SNF1 activity is regulated by the level of available glucose. In wild-type yeast, but not in a snf1 deletion mutant, acetyl-CoA carboxylase shows a reciprocal change in activity compared with SNF1 under glucose derepressing conditions, indicating that SNF1 regulates acetyl-CoA carboxylase in vivo. These results suggest that, in addition to their structural similarity, the role of SNF1 and AMPK in the regulation of fatty acid synthesis has been highly conserved throughout evolution.

AMP-Activated Protein Kinases↗