CALCIUM AND cAMP AS INTRACELLULAR MESSENGERS

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PIGMENT

Pyocyanine (Pyo)

Pyocyanine and its degradation product, 1-hydroxyphenazine (1-HP), are pigments produced by P aeruginosa. The molecular structures of these agents are shown in Figure 2. Both are virulence factors which contribute to the pathogenicity of P aeruginosa. Pyocyanine (5-methyl-1-hydroxyphenazium betaine) is a blue pigment responsible for the distinctive blue colouration observed in the pus of some patients infected with P. aeruginosa. Its presence led to the original designation of Paeruginosa as Bacillus pyocyaneus. The molecular weight of pyocyanine is 210.2. It forms dark blue needles in water and in the solid state it is stable for weeks in a dry and dark environment, but decomposes on long storage. Pyocyanine is zwitterionic in nature, conferring solubility in aqueous and organic solvents. It is also an electron acceptor, as well as a carrier. It is blue in its oxidized form and colourless in the reduced state. Solubility is achieved in hot water and hot ethanol.

(1-HP)

Although 1-hydroxyphenazine (1-HP) is synthesized directly by P. aeruginosa, it is also formed by the degradation of pyocyanine by demethylation of the 5-methyl substituent on the phenazine nucleus. 1-HP is also reffered to as hemipyocyanine or 1-phenaziol.It has a molecular weight of 196.2 and is soluble in aqueous alkaline solutions and slightly soluble in hot water.

PHENAZINE PIGMENT (Pyocyanine, 1-Hydroxyphenazine) PRODUCTION

The natural occurrence of phenazine pigments has been known since the nineteenth century (Ingram and Blackwood, 1970). The pigments contain the same basic structure as shown in Figure 3. Pyocyanine is produced by some strains of Pseudomonas aeruginosa under certain culture conditions and may be regarded as the prototype microbial phenazine pigment. It has attracted interest because of its antibiotic properties and because of its occurrence in pathogenic strains of the organism. The pigment was first isolated by Gessard (1890) following the growth of P. aeruginosa on complex media.The first documented evidence concerning the production of pyocyanine in a chemically defined media was that of Jordan (1899). He demonstrated the ability of some cultures to produce the pigment when grown on a mixture of salts plus asparagine or plus the ammonium salts of succinic, lactic, acetic, or citric acids. Since that time many media, usually modifications of that mentioned above, have been described in an effort to optimize the amount of pigment produced. The media usually contain glycerol as a carbon source, leucine and/or alanine as nitrogen sources, and Fe+2, Mg+2, SO/, and P04-3, although the level of phosphate is critical and must be kept low to ensure maximum production of pyocyanine.Studies by Valette and co-workers (1966) with the A-237 strain of P. aeruginosa showed that optimum pyocyanine production occurs at a concentration of O.08M phosphate when the organism is cultured on a synthetic medium of succinic acid and ammonium chloride. Halpern et al., (1962) studied the production of pyocyanine in resting cell suspensions. Growth as measured by optical density at 490nm occurred while the production of pyocyanine proceeded. When the effects of inhibitors of the production of pyocyanine were studied, it was found that inhibitors which produce inhibition of growth also inhibited the production of the pigment. The production of pyocyanine commences when the phosphate concentration reaches a critical minimum level and terminates as the phosphate level rises.Millican (1962) using a direct label incorporation, or an isotope dilution procedure,showed that quinic or shikimic acids serve as good sources of pyocyanine carbon.However, in each study, interpretation was complicated by bacterial growth indicating that the carbon source was metabolized prior to incorporation into pyocyanine. The problem of precursor metabolism prior to incorporation into pigment was circumvented by using a mutant, a-, which was unable to oxidize and hence, unable to metabolize shikimic and quinic acids (lngledew and Campbell, 1969). When cultures of the wild type, W, were added to a resuspension medium containing 2-ketogluconate-14C as the initial sole source of carbon, it was found by isotope dilution that 9% of the pyocyanine carbon was derived from shikimic acid. However, when the same experiment was repeated, with the Q- strain, 98% of the pyocyanine ring carbon was derived from shikimic acid, whereas 40% was derived from quinic acid. The authors also reported that quinic acid is not a direct precursor of pyocyanine carbon. Their data strongly suggested that quinic acid is converted to shikimic acid, which is then incorporated into the pyocyanine molecule.

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SUMMARy
SAMEVATTING
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF FIGURES
LIST OF TABLES
LIST OF ABBREViATIONS
CHAPTER 1: LITERATURE REViEW 
1.1 INTRODUCTION
1.2 CYSTIC FIBROSiS 
2 PHAGOCYTES
3 PROTEASES
3.1 Neutrophil elastase (NE)
3.2 Pseudomonas aeruginosa elastase (PE)
4 PiGMENTS
4.1 Pyocyanine (Pyo)
4.2 1-hydroxyphenazine (1-HP)
4.3 Phenazine pigment production (Pyo and 1-HP)
4.4 Effects of pyocyanine and 1-HP on neutrophil function
4.5 Effects of pyocyanine, 1-HP and PE on ciliated respiratory epithelium
4.6 Effects of pyocyanine and 1-HP on lymphocyte function
4.7 Effects of pyocyanine on other cell types
5 CALCIUM AND cAMP AS INTRACELLULAR MESSENGERS 
6 cAMP-BASED ANTI-INFLAMMATORY STRATEGIES 
7 cAMP-ELEVATING AGENTS 
7.1 Dibutyryl cAMP-vii7.1.1 Anti-inflammatory activities of dibutyryl cAMP
7.2 f32-adrenoreceptor agonists
7.2.1 Salmeterol and salbutamol
7.2.1.1 Anti-inflammatory effects of salbutamol
7.2.1.2 Anti-inflammatory effects of salmeterol
7.3 Phosphodiesterase inhibitors
7.3.1 Theophylline
7.3.1.1 Anti-inflammatory effects of theophylline
7.3.2 Rolipram
7.3.2.1 Anti-inflammatory effects of rolipram
7.3.4 Adenosine receptor agonists
7.3.4.1 Adenosine A 1 receptors
7.3.4.2 Adenosine A2 receptors
7.3.4.3 Adenosine A3 receptors
7.3.4.4 Anti-inflammatory effects of A2a receptor agonists
8 THE AIM OF THE STUDY 
CHAPTER 2: EXPOSURE OF FMLP-ACTIVATED HUMAN NEUTROPHILS TO THE Pseudomonas aeruginosa-DERIVED PIGMENT,1-HYDROXYPHENAZINE (1-HP), IS ASSOCIATED WITH IMPAIRED CALCIUM EFFLUX AND POTENTIATION OF PRIMARY GRANULE ENZYME RELEASE
2.1 INTRODUCTION
2.2 METHODS
2.2.1 Preparation of the pigment, 1-hydroxyphenazine
2.2.2 Chemicals and reagents
2.2.3 Preparation of neutrophils
2.2.4 Elastase and MPO release
2.2.5 Spectrofluorimetric measurement of Ca2+fluxes
2.2.6 Radiometric assessment of Ca2+ fluxes
2.2.6.1 45Ca2+-efflux out of FMLP-activated neutrophils
2.2.6.2 45Ca2+-influx into FMLP-activated neutrophils
-VIII2.2.7 Radiometric assessment of Na+ influx
2.2.8 Intracellular cAMP levels
2.2.9 cAMP-dependent Protein Kinase A (PKA) activity
2.2.10 Intracellular ATP levels
2.2.11 Statistical analysis
2.3 RESUL TS
2.3.1 The effects of 1-HP on elastase and MPO release
2.3.2 The effects of 1-HP on the fura-2 responses of FMLP-activated neutrophils
2.3.3 45Ca2+ fluxes in activated neutrophils
2.3.4 Efflux of 45Ca2+ from FMLP-activated human neutrophils
2.3.5 Influx of 45Ca2+ into FMLP-activated human neutrophils
2.3.6 22Na+ fluxes in neutrophils
2.3.7 Intracellular cAMP levels
2.3.8 Intracellular A TP levels
2.3.9 cAMP-dependent protein kinase A (PKA)
2.4 DiSCUSSiON
CHAPTER 3: THE EFFECTS OF CONVENTIONAL INTRACELLULAR cAMPELEVATING AGENTS ON 1-HYDROXYPHENAZINE-MEDIATED INTERFERENCE WITH THE CLEARANCE OF CYTOSOLIC CALCIUM AND ENHANCEMENT OF ELASTASE RELEASE FROM FMLPACTIVATED NEUTROPHILS
3.1 INTRODUCTION
3.2 MATERIALS AND METHODS 
3.3 RESUL TS 
3.3.1 Effects of the various cAMP-elevating agents on elastase release
3.3.2 Effects of the cAMP-elevating agents on 1-HP-mediated alterations in the fura-2 responses of FMLP-activated neutrophils
3.3.3 Effects of theophylline, salbutamol and salmeterol on neutrophil cAMP levels
3.4 DiSCUSSiON
CHAPTER 4: INVESTIGATION OF THE EFFECTS OF ADENOSINE RECEPTOR AGONISTS ON 1 HYDROXYPHENAZINE-MEDIATED ENHANCEMENT OF
RELEASE OF ELASTASE FROM ACTIVATED NEUTROPHILS AND ITS RELATIONSHIP TO ALTERATIONS IN INTRACELLULAR cAMP LEVELS
4.1 INTRODUCTION
4.2 MATERIALS AND METHODS 
4.3 RESUL TS 
4.3.1 Effects of the adenosine receptor agonists on elastase release from FMLPICB-activated neutrophils
4.3.2 Effects of the A2a adenosine receptor antagonist (ZM241385) on CGS21680 and IB-MECA-mediated interference with 1-HP-induced enhancement of elastase release
4.3.3 Effects of CPA, CGS21680 and IB-MECA on neutrophil cAMP levels
4.4 DiSCUSSiON
CHAPTER 5: CONCLUSIONS 
5.1 Conclusions
REFERENCES
APPENDiCES

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