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Characterization of a platelet activating factor using forskolin and methylcarbamyl PAF Wong, Sandra I. 1992

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CHARACTERIZATION OF PLATELET ACTIVATING FACTORUSING FORSKOLIN AND METHYLCARBAMYL PAFbySANDRA I. WONGB.Sc., Simon Fraser University, 1989A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Experimental Medicine)We accept this thesis as conformingto the reQuired staii dardTHE UNIVERSITY OF BRITISH COLUMBIASeptember 16, 1992© Sandra I. Wong, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of_____________The University of British ColumbiaVancouver, CanadaDate J99zDE-6 (2/88)AB STRACTPlatelet activating factor is an important mediator of septic shock andinflammation. The action of PAF on target cells is mediated throughspecific membrane bound receptors. PAF binding to receptors initiates acascade of biochemical signalling events leading to the aggregation ofplatelets or the activation of other target cells. In this work wecharacterized the PAF-receptor physiochemical properties in platelets andinvestigated its interaction with signalling systems. The effect of forskolinon PAF receptor binding was studied. Studies were also conducted towardselucidation of the nature of the PAF receptor. In this direction the bindingof the stable PAP analog l-0-hexadecyl-2-O-(methylcarbamyl)-sn-glycero-3-phosphocholine (MC-PAF), a compound with potential uses in PAFreceptor purification and antibody production was evaluated.Forskolin is commonly used in the investigation of the effects ofstimulated adenylyl cyclase activity in cells. In the first project the effectof forskolin on platelet activating factor receptor was explored. Rabbitplatelets treated with forskolin prior to PAP binding resulted in a 30-40%decrease in binding which translated to a change in receptor number on thecell surface. This decrease in PAF binding caused by forskolin wasconcomitant with a decrease in platelets’ physiological response to PAP.However, the forskolin induced decrease in PAF binding was not aconsequence of cAMP formation as the addition of a cAMP analog couldnot mimic the actions of forskolin. Additionally, the inactive analog offorskolin, dideoxyforskolin, which does not activate adenylyl cyclase, alsoreduced PAF binding to its receptor. Reduction of PAP binding byforskolin and dideoxyforskolin was also demonstrated in isolated plateletIImembranes. The action of forskolin was also found to be independent ofG-protein involvement. We therefore concluded the PAF receptor may beregulated by several factors that are triggered by forskolin. Theseelements are independent of adenylyl cyclase involvement.In the second project MC-PAF was evaluated using rabbit platelets.MC-PAF was found to be approximately 3-5 fold less active in causingplatelet aggregation and serotonin release. Aggregation caused by MC-PAFwas completely inhibited by WEB 2086, a PAF receptor antagonist. Ligandbinding studies revealed that MC-PAF binds with approximately two-foldlower affinity than PAF to the PAF receptor (Kd=l .47 and 0.62 nM forMC-PAF and PAF, respectively). Studies in anesthetised rats showed thatboth PAF and MC-PAP caused a similar reduction in blood pressurewithout changes in either heart rate or EKG parameters. The susceptibilityof PAF and MC-PAF to hydrolysis by serum acetyihydrolases wasinvestigated. PAP was found to be fully hydrolyzed after 5 mm in rabbitserum while MC-PAF was not degraded significantly after a 1 hourexposure. These studies suggested that MC-PAF is a full agonist of the PAFreceptor, both in vitro and in vivo, and is more stable than PAP againstserum acetylhydrolases, thus making MC-PAF a useful compound forpurifying the PAF receptor as well as raising PAF antibodies.II ITABLE OF CONTENTSPageTitle page.iAbstract iiTable of Contents ivList of Tables viiList of Figures viiiList of Abbreviations ixAcknowledgements XChapter 1. INTRODUCTION1.1 Introduction 11.2 PAF Structure 11.3 PAF Biosynthesis1.3.1 De novo pathway ofPAFbiosynthesis 21.3.2 Remodelling pathway of PAF biosynthesis 41.4 PAF Inactivation 61.4.1 Acetyihydrolase 61.5 Cellular Sourèes and Targets of PAF1.5.1 Neutrophils 81.5.2 Eosinophils 81.5.3 Platelets 91.5.4 Endothelial Cells 91.6 Involvement of PAF in Human Disease1.6.1 Asthma 101.6.2 Septic shock 111.6.3 Kidney disease 131.6 PAF Antagonists1.6.1 Structurally related PAF antagonist 141.6.2 Structurally unrelated PAF antagonists1.6.2.1 Naturally occuring antagonist-Gingkolides 151.6.2.2 Psychotropic agentsTriazolothienodiazepines 151.7 PAP and Signal Transduction1.7.1 PAF receptor binding 161.7.2 Receptor independent internalization of PAF 181.7.3 G-Protein 191.7.4 Phosphatidylinositol turnover 201.7.5 Protein kinase C 211.7.6 Adenylyl cyclase 21iv1.7.7 cAMP dependent protein kinase A 221.7.8 Ion channel 221.8 Receptor Purification 23Chapter 2 OBJECTIVES2.1 The effects of forskolin-an activator of adenylyl cyclaseon PAF binding in rabbit platelets 252.2 Evaluation of methyl-carbamyl PAF as an agonist ofPAF 26Chapter 3 MATERIALS AND METHODS3.1 Materials 283.2 Purification of platelets 283.3 Membrane preparation 293.3 Binding Assays 293.4 Platelet aggregation assay 303.5 cAMP assay 313.6[3H]Serotonin incorporation into platelets 313.7 Serotonin release assay 313.8 Serum acetylhydrolase assay 313.9 HPLC extraction of PAF 323.10 ilemodynamics and cardiovascular effects of PAF andMC-PAF 33Chapter 4 RESULTS4.1 Effects of Forskolin on PAF Binding4.1.1 Measurement of cAMP in treated cells 354.1.2 PAF binding to forskolin treated cells 374.1.3 CPT-cAMP, staurosporin and A3 treatedplatelets 394.1.4 PAF binding to dideoxyforskolin treatedplatelets 404.1.5 Binding to forskolin and dideoxyforskolin treatedmembranes 424.1.6 Effect of a GTP analog on PAF binding toforskolin and dideoxyforskolin treated platelets 444.1.7 Effect of forskolin and dideoxyforskolin on PAFinduced platelet aggregation 464.2 Evaluation of MC-PAF4.2.1 PAF and MC-PAF induced plateletaggregation 484.2.2 PAF and MC-PAF induced serotonin release 504.2.3 PAF and MC-PAF binding to rabbit platelets 524.2.4 Effect of PAF and MC-PAF on anesthetised rats 54V4.2.5 Serum acetyihydrolase susceptibility of PAF andMC-PAF 56CHAPTER 5 DISCUSSION5.1 Effects of forskolin on PAF binding 585.2 Evaluation of MC-PAF 62CHAPTER 6 BIBLIIOGRAPHV 6 6viLIST OF TABLESTable 1. Receptor number and binding affinity in platelets,neutrophils, HL6O cells and P388D1 cells 17viiLIST OF FIGURESFigure 1.1 PAF structure.1Figure 1.2 De novo pathway of PAF biosynthesis 3Figure 1.3 Remodelling pathway of PAF biosynthesis 5Figure 1.4 Inactivation of PAF 7Figure 3.1 Standard curve of HPLC extraction of PAF 33Figure 4.1 cAMP measurement in rabbit platelets 36Figure 4.2 Specific binding of PAF to rabbit platelets 38Figure 4.3 PAF binding to dideoxyforskolin treated platelets 41Figure 4.4 PAF binding to treated membranes 43Figure 4.5 GTP-g-S treatment 45Figure 4.6 PAF induced platelet aggregation of forskolin anddideoxyforskolin treated platelets 47Figure 4.7 PAF and MC-PAF induced platelet aggregation 49Figure 4.8 [3H]Serotonin release by PAF and MC-PAF in rabbitplatelets 51Figure 4.9 PAF and MO-PAF binding to rabbit platelets 53Figure 4.11 The cardiac and hemodynamic parameters of rats inresponse to PAF and MC-PAF 55Figure 4.12 PAF and MC-PAF hydrolysis by serumacetylhydrolases 57viiiABBREVIATIONSA3 N-(2-aminoethyl)-5-chloronaphthalene-1-sulfonamidehydrochlorideADP adenosine diphosphateA1F4 fluoroaluminateBSA bovine serum albumincAMP cyclic adenosine monophosphateCPT-cAMP 8-(4-chlorophenylthio)-adenosine 3 ‘-5’-cyclicmonophosphateDAG diacylglycerolGj inhibitory guanine nucleotide binding proteinG-protein guanine nucleotide binding proteinG phospholipase C linked guanine nucleotide bindingproteinG5 stimulatory guanine nucleotide binding proteinGTP guanosine triphosphate1P3 inositol- 1 ,4,5-triphosphateIBMX 3-isobutyl- 1 -methyl-xanthineLyso-PAF 1 -O-alkyl-sn-glycero-3-phosphocholineMC-PAF 1 -O-hexadecyl-2-O-(methylcarbamyl)-sn-glycero-3 -phosphocholinePAF platelet activating factor/i -O-alkyl-2-acetyl-sn-glycero-3-phosphocholinePIP2 phosphatidyl inositol-4,5-bisphosphatePKA cAMP dependent protein kinase APKC calcium dependent protein kinase CPLA phospholipase A2ixACKNOWLEDGEMENTSI would like to thank Dr. Hassan Salari for his supervision and help inobtaining my degree. I would also like to thank Dr. Vincent Duronio(Biomedical Research Center, U.B.C.), Peter Dryden (Resp. Med. U.B.C.),Sandra Howard (Resp. Med. U.B.C.) and Michael Pugsley (Dept.Pharmacology, U.B.C.) for their support and technical assistance.I also like to express my appreciation for the support my family andfriends have offered me during the past two years.xChapter 1Literature Review1.1 IntroductionPlatelet activating factor (PAF) was first described as a soluble factorthat caused the aggregation of platelets in IgE-sensitized rabbit basophils ina calcium and temperature dependent process (1). Since its discovery it hasbeen demonstrated that PAF is synthesized in a wide number of cell andtissues. Furthermore, studies have implicated PAF as an importantmediator in a number of diseases including asthma and septic shock.1.2 PAP StructureThe structure of PAF is defined as 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (2,3) (figure 1.1). The naturally occuring enantiomer ofPAF is primarily a mixture of C16 and C 18 ether chains at the first carbonand is in the (R) configuration at the second carbon. Both these structuralfeatures of PAF are essential forrn m-axirna1 stimulatory activity. Reducingchain length at the first carbon or changing isomerism at the second carboncan result in up to a thousand fold drop in activity (4,5).CH3-O-(CH2) CH3H3C—C —0—C-HII0 0C-0-P-0-(CH2)-N(CH3)H20Figure 1.1 PAF structure, n=14-17.11.3 PAF BiosynthesisIn cells PAF can be synthesized via two known pathways; the de novopathway and the remodelling pathway.1.3.1 De novo pathway of PAF biosynthesisIn the de novo pathway 1-O-alkyl-2-lyso-sn-glycero-3-phosphate isconverted to PAF in a stepwise sequence of reactions (figure 1.2). Thefirst step in the de novo pathway involves the acetylation of anintermediate at the second carbon by a PAF specific acetyltransferase. Aphosphohydrolase enzyme then dephosphorylates the compound at the thirdcarbon. This is followed by the transfer of a phosphocholine from CDPcholine to 1 -O-alkyl-2-acetyl-sn-glycerol by a CDP-cholinephosphotransferase.All the participating enzymes in the de novo pathway are located on theendoplasmic reticulum (6). Although the presence of CDP-cholinephosphotransferase- activity has been demonstrated in a number of tissues,thus far the physiological significance of the de novo pathway is unknownsince upon cell stimulation by different agonists it appears that it is theremodelling pathway that is activated (6,7,8,9)2r 0-CH2-R[-0-H1—0—alk!J 1—2—Ij so—sn—gIJ cero—3— PAcetj1CoAN IAcet!, ltransferaseCoASH0CH2R1o- C-CHIIL®o1—0— alkj l—2—acetj l—sn—glJ cero —3—PPhosphohjdro1ase—0-CH-RF-o- C-CH3[0-H 0I —O—a1kj1— 2—acetj1—sn—gl gcerolCDP_Choline I)CMPr 0CH2R[_o_ C-CH30CholinePlatelet—activating FactorFigure 1.2 De novo pathway of PAF biosynthesis, R= fattyacid (C14-C18).31.3.2 Remodelling pathway of PAF biosynthesisIn the remodelling pathway the formation of PAF is initiated by theaction of membrane bound phospholipase A2 (PLA2) (figure 1.3). Theactivation of PLA2 appears to be calcium dependent and it is generallyactivated by agonists that also stimulate calcium mobilization (10). Onceactivated, PLA2 deacylates membrane bound 1-alkyl-2-acyl-glycero-phosphocholine by cleavage at the 2(R) position thus releasing two productsinto the cytoplasm, 1 -0-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) anda free fatty acid. The free fatty acid most often released is arachidonic acidwhich serves as an important precursor for a number of inflammatorymediators via both the lipoxygenase and the cyclooxygenase pathway(11,12). The lyso-PAF that is formed is modified by a PAF specificacetyltransferase enzyme that attaches an acetyl group at the 2(R) position.Activation of acetyltransferase occurs within minutes of agonist stimulationand appears to be the rate limiting step in PAF formation (6,13,14,15). Asseen with PLA2, acetyltransferase activity correlates positively withcalciuminflux into the cell (16, 17).41°(E)—Choli ne1 —O—alkj1—2—acJl—sn—glJcero—3—phospbocholinePhospholipase A2Fatt!,j acidr° - C H2 -HH(g)—Choli ne1 —O—alkj1—sn—gbjcero—3—phospbocho1ineAcetglCoA xIAcet!, itransferaseCoASH‘—Choline1 —0—alkjl—2—acetJl—sn—gljcero—3—phosphocholine(Platelet Activating Factor)Figure 1.3 Remodelling pathway of PAF biosynthesis, R=fatty acid (C14-C18).051.4 PAF InactivationPAF that is released from cells is metabolized and cleared from theblood very rapidly, occurring in approximately 30 seconds (18).Inactivation of PAF occurs in a two step process (figure 1.4). The firststep in PAF degradation involves the cleavage of the acetyl group at the2(R) position by an acetyihydrolase enzyme that is highly specific forphospholipids with short acyl chains at the sn-2 position. This results inlyso-PAF which is cytotoxic to the cell, having both lytic and detergentproperties and therefore must be eliminated (19). Elimination isaccomplished by an acylation reaction in which an acyltransferase convertsthe lyso-PAF formed to alkylacylglycerophosphocholine by theintroduction of a long chain fatty acid onto lyso-PAF at the second carbon.It appears that phosphatidyicholine is the main source of arachidonic acidused to acylate lyso-PAF (20). The final step in the process of PAFinactivation is incorporation of the end product,alkylacylglycerophosphocholine into the cell membrane (21).1.4.1 AcetyihydrolaseAcetythydrolase is found in a variety of cells and tissues. It appearsthat both the intracellular and plasma forms of acetyihydrolase haveidentical substrate specificities but differ in molecular weight and do notcross react immunologically (18,22). In human neutrophils,acetylhydrolase appears to be a cytosolic enzyme (23). In human plasma70% of acetyihydrolase is found tightly associated with a low densitylipoprotein and the remainder is complexed with a high density lipoprotein.While both lipoproteins contain the same enzyme, it appears that theacetyihydrolase found with the low density form is more efficient athydrolyzing PAF (24).6)—Cho1i ne1 —0—alky i—2—acetg 1—sn—gig cero—3—phosphochoiine(PAF)Acetg lhg drolaseAcetgl groupr°- C H2 -0-HLcp...cho1Inc -1 —O—aikg i—sn—gig cero—3—phosphochoiine(Lg so—P Ar)Phosphatidgi—........Acgl groupcholine Acgitransferase10L®_cholj1 —O—alkgl—2—acgi—sn—gigcero—3—phosphocholineFigure 1.4 Inactivation of PAF, R= fatty acid (C 14-Cl 8).0- C H2 - R071.5 Cellular Sources and Targets of PAP1.5.1 NeutrophilsInflammatory responses are characterized by the activation andinfiltration of polymorphonuclear cells to the site of inflammation. Whenadministered in vivo by a variety of routes, PAF causes transientneutropenia in both animals and humans with apparent sequestration in thepulmonary vasculature (25,26,27,28,29,30). In vitro, PAF is capable ofcausing neutrophil chemotaxis and enhanced neutrophil adhesiveness,aggregation, degranulation and superoxide anion generation (31,32). PAFcan also prime neutrophils, thus resulting in a synergistic response whentreated with other neutrophil agonists. Many of the other mediators ofneutrophil activation can also prime neutrophils and not surprisinglysimultaneously stimulate PAF formation (33,34). The list of mediators thatcan illicit PAP formation in neutrophils include: thrombin, collagen, Ca2ionophore A23 187, f-methionyl-leucyl-phenylalanine and opsonizedzymosan (35,36,37,38). Inaddition to PAP, many of these mediators alsogenerate lyso-PAF. Therefore it is not unexpected to find that neutrophilscontain comparatively high levels of the acetyltransferase enzyme whichcan readily convert synthesized lyso-PAP into PAF (8).1.5.2 EosinophilsMany inflammatory mediators have been shown to cause neutrophilchemotaxis but very few are able to cause eosinophil chemotaxis. In vitroessentially three important chemotactic substances are able to attract andactivate eosinophils, leukotriene B4, eosinophil chemotactic factor andPAP (39). However, in vivo PAP appears to be the only mediator thatselectively attracts human eosinophils (40). PAP is very effective inactivating eosinophils to release basic proteins, leukotriene C4, oxygenradicals as well as more PAP (41,42,43,13). When eosinophils arestimulated with the Ca2 ionophore A23 187 they are capable of generatinglarge amounts of PAF of which 30-50% is release from the cell (15).81.5.3 PlateletsIn vivo, PAF causes thrombocytopenia when administeredintravenously and this is associated with the pulmonary sequestration ofplatelets (25,44,45). In vitro, PAF causes the aggregation of plateletsfrom a number of animal species including, human, rabbit, guinea pig anddog and is the most potent inducer of platelet activation known (46,47).Rabbit platelets are extremely sensitive to PAF and will aggregate inresponse to concentrations as low as 0.1 nM (3,48). In platelets, PAFinduces secretion of a number of endogenous mediators such asthromboxane A2, platelet factor 4, ADP, serotonin and histamine. Inaddition, human platelet aggregation induced by some of these endogenousmediators can be potentiated by prior treatment with PAF through theupregulation of platelet fibrinogen receptors (46). The formation of PAFby platelets seems to be both species and agonist dependent. Chignard etal. (49) first demonstrated the formation of PAF in human and rabbitplatelets in response to Ca2 ionophore A23 187 stimulation. Equivalentconcentrations of Ca2 ionophore A23 187 evokes 2.99 ± 3.30 pmol PAF/5X 108 rabbit platelets as compared to 1.75 ± 1.40 pmol PAF/5 X 1human platelets (50). While PAF formation in platelets can also be inducedby other platelet agonists such as thrombin and collagen (37, 38, 51), therealso are several agonists recognized that do not induce PAF formation inrabbit platelets and this includes ADP, arachidonic acid and PAF itself (22,37).1.5.4 Endothelial cellsEndothelial cells are able to respond to as well as synthesize PAF.When stimulated with PAF, endothelial cells mobilize calcium, releaseprostaglandin 12 and contract (52). When stimulated with other agonists,endothelial cells from a number of sources including bovine pulmonaryand systemic vessels, rabbit thoracic aorta, as well as human pulmonaryartery and umbilical vein have been shown to produce PAF (53,54,55).Human endothelial cell cultures are able to produce PAF when stimulatedby thrombin, leukotriene C4, leukotriene D4, histamine, bradykinin, ATP,angiotensin 11(54,56,57,58).9Essentially all of the PAF produced in endothelial cells remainsassociated with the cell (56,59,60). The PAP retained in endothelial cells isexpressed on the surface of the cells and serves as an intercellularmessenger (61). The interaction of neutrophils to the endothelial cells is anessential step in inflammation. Once neutrophils adhere to the endotheliumthey are free to migrate out of the vasculature towards the site ofinflammation. It was found that treating endothelial cells with agonistsknown to induce PAF synthesis results in changes to the cell surface ofthese cells that promote the binding of neutrophils (56,62). It wasdetermined that this interaction required the presence of an active PAFreceptor on the neutrophil. It was also demonstrated that treatment ofagonist stimulated endothelial cells with PAP acetyihydrolase reduced thebinding of neutrophils to the endothelial cells (63). The role of PAF as anintercellular messenger on endothelial has also been established withregards to other circulating inflammatory cells such as platelets (61).1.6 Thvolvement of PAF in Human Disease1.6.1 AsthmaAsthma is a respiratory disorder characterized by the narrowing of theairways, mucosal edema and the influx of inflammatory cells. The result isdypsnea, wheezing and cough. This disease affects approximately 3% ofthe North American population and despite efforts aimed at improveddiagnosis and treatment its prevalence and severity appear to be increasing(64). Theories put forth on its etiology include: airway epithelial damage,inflammatory cell infiltration, neurologic, cholinergic and/or mechanicalabnormalities.The hallmark of human asthma is airway hyperresponsiveness asdefined by the exaggerated narrowing of the airways in response tononspecific stimuli (65). In the airways of asthmatics there is acharacteristic infiltration of inflammatory cells which include, eosinophils,mast cells and neutrophils (66,67,68). There is a close relationshipbetween eosinophil count in the lungs and bronchial hyperresponsiveness(69). When stimulated, each of these cell types are a rich source of a10number of chemical mediators, each of which is capable of causinginflammation and bronchoconstriction. However, while many mediatorsare capable of causing epithelial damage and inflammation, PAF alone hasbeen demonstrated to induce bronchial hyperresponsiveness and eosinophilchemotaxis in vivo (70). PAF’s involvement in asthma is supported byboth in vitro and in vivo data.In vitro, PAF is able to induce contraction of isolated smooth muscletissue which includes lung strips from rabbits, rats and guinea pigs (71,72).In vivo, intravenous injection and aerosol administration of PAF results inbronchoconstriction and nonspecific bronchial reactivity in a number ofexperimental animals including guinea pigs (73), dogs (74), rabbits (75),sheep (76), monkeys (77,78) as well as in normal human subjects (26,27).In rabbits and baboons PAF will cause thrombocytopenia, neutropenia andbasopenia (79). In guinea pig airways PAF induced hyperreactivity isaccompanied by the pulmonary accumulation of neutrophils, eosinophilsand platelets and although a direct link to platelets has not been established,PAF induced airway hyperreactivity will not occur in some animal specieswhen pretreated with antiplatelet antiserum (73,80). In humans, it wasdemonstrated by Cuss (26,27) that when normal subjects inhaled PAF theyexperienced bronchoconstriction and increased reactivity to methacholinefor as long as 3 weeks after PAF challenge. Also in support of PAF’sinvolvement in asthma is that it has been detected in the lavage fluid ofasthmatic hyperreactives (81) and in the blood of allergic asthmaticsfollowing allergen-induced bronchoconstriction (82). But perhaps one ofthe strongest lines of evidence is the effectiveness of PAF antagonists inblocking PAF induced responses. Bronchial hyperreactivity induced byPAF in guinea pig model can be prevented by pretreatment with the PAFantagonists BN 52021 and WEB 2086 (83,84,85,86). These two agentswere also effective in inhibiting eosinophil and neutrophil infiltration inguinea pig lung induced both by PAF and antigen stimulation (87).1.6.2 Septic shockSeptic or endotoxic shock is the feature of blood contamination bybacterial toxin. It is defined by the inadequate perfusion of vital organs,11with associated systemic hypotension, decreased peripheral vascularresistence, and decreased cardiac output, all of which lead to totalcirculatory collapse (88,89). The morbidity and mortality rates for septicshock have not improved in the past 20 years and remains the mostcommon cause of death in intensive care units in the United States (88,90).To combat the assault on the body caused by the infection of gramnegative bacteria, various systems are activated in response to the infectionand include the complement, coagulation, kinin and endorphin systems(91,92). Circulating cells such as platelets and leukocytes are also activatedand release a number of inflammatory mediators including PAF. Althougha direct link to PAF is difficult to establish due to the complexities of thisdisorder, several lines of evidence show that PAF may play a strong role inthe events leading to the hemodynamic instability observed duringendotoxin induced shock. First, although PAF is not present in significantlevels in control animals increased levels of PAF have been demonstratedin experimental models of anaphylaxis and endotoxemia. PAF has beendetected in the serum from rabbits following antigen challenge (93), in theliver and spleen of sensitized mice following injection with IgG antibodies(94), in blood samples from rats following intravenous injection ofSalmonella eneritidis (95) and in the peritoneal exudate and spleen of ratsafter intraperitoneal infusion of Escherichia coli (96).Secondly, when administered systemically PAF is the single most potentendogenous agent known to produce a shock state (97). Strikingsimilarities have been noted between effects of administered PAF and thehemodynamics of endotoxemia and anaphylaxis, including pulmonaryhypertension, systemic hypotension, decreased cardiac output and enhancedvascular permeability. More importantly the reversal of shock states inexperimental animals has been achieved with PAF inhibitors (96). ThePAF receptor antagonist CV 3988 significantly reduced hypotensioninduced by both Escherichia coli and Salmonella abortus endotoxininfusion (98). Another PAF antagonist BN 52021 inhibited thehemodynamic effects of Salmonella enteritidis endotoxin on anesthetizedrats and guinea pigs and significantly reduced mortality (99,100)12It seems that PAFs main effects may be mediated by the PAF generatedrelease of secondary mediators. In the rat model, PAF is only able to causethe contraction of arterial strips in the presence of leukocytes or platelets.This action could be blocked with the thromboxane A2 antagonist ONO3708. It would appear in this situation that the contraction of the arterialstrips was a result of PAP-induced release of secondary mediator from thecirculating cells (101).1.6.3 KidneyIt is reported that the kidney is a sensitive target for the development ofinflammatory tissue injury. Various properties of PAF make it the idealcandidate for a role in various kidney disorders and PAF has beenimplicated in the pathogenesis of glomerular injury as well as in variousaspects of renal physiology. Both the mesangial and interstitial cells of therat kidney can produce PAF under different experimental conditions(102,103). Furthermore, the detection of PAF in human urine has raisedthe possibility that PAF may be generated by the kidney even under normalphysiological conditions (104). PAF possibly has a direct effect onmesangial cells of the kidney by inducing the production of variousintermediates or else PAF may act indirectly by causing the renaldeposition of circulating immune complexes. In vitro the application ofPAF onto mesangial cells isolated from rat glomeruli causes cellcontraction and prostaglandin E2, superoxide anion and hydrogen peroxidesynthesis (105,106). PAF can also cause the release of mediators fromhuman mesangial cells and from isolated rat and rabbit kidneys (107). Cellcontraction of the mesangial cells induced by PAF may favor the depositionof immune complexes in the kidney which serves to illicit the furtherrelease of inflammatory mediators. PAF’s enhancement of vascularpermeability may also facilitate the deposition of circulating immuneaggregates (108,109,110). In the rabbit, PAP was shown to be releasedduring kidney hyperacute allograph rejection (111). Also in the rabbitmodel, short term intrarenal administration of PAF resulted in platelet andneutrophil aggregation and degranulation in the glomerular capillaries.This resulted in the loss of glomerular negative charges due to the release13of cationic proteins and also resulted in mild proteinuria which lasted forseveral hours (40). Infusion of PAF in the renal arteries of anesthetizeddogs is associated with a profound decrease of renal blood flow,glomerular filtration rate, and urinary sodium excretion. All of thesereactions can be inhibited by prior infusion of BN 52021 (112). PAFantagonists can also reduce proteinuria and decrease the histopathologicalglomerular lesions in models of nephrotoxic serum nephritis in both ratsand rabbits (113).1.6 PAF AntagonistsThere are a large number of PAF antagonists commercially available.Generally they may be broadly classified into two groups, structurallyrelated compounds and structurally unrelated compounds.1.6.1 Stucturally related PAF antagonistThe first structurally related PAF antagonist reported was CV 3988(114). This antagonist is derived from the PAF framework with thefollowing structural features added; position 1 incorporates a octadecylcarbamate, a methylether occupies position 2 and a tiazaolium ethylphosphate is in position 3. At doses higher than those required forinhibition of PAF receptor binding, CV 3988 was also found to be effectiveagainst arachidonic acid, ADP (115), collagen (116) and calcium ionophoreA23 187 (117) induced platelet aggregation. In the rat, CV 3988 caninhibit PAF-induced hypotension (114), thrombocytopenia (118),endotoxin-induced disseminated intravascular coagulation (119) andcounteract blood pressure decrease in the hypertensive model (120). CV3988 can also prevent PAF-induced death in the mouse (121). In a double-blind, placebo controlled study, healthy volunteers received an IV infusionof CV 3988, this resulted in reduced platelet sensitivity to PAF (122). Atthe dose range of 750-2000 rig/kg it was found that blood pressure, pulserate and respiratory parameters were unaffected.Further modifications of CV 3988’s basic structure has given rise to CV6209, which has a octadecyl carbamate group in place of the phosphate14group at position 1 (2) and to SRI 63-119, which has a CH2unit inplace of the P03 group in position 3 (123). Both compounds are effectiveantagonists of PAF-induced aggregation of both rabbit and human platelets,with CV 6209 having the greater potency (124,125) . CV 6209 isapproximately 80 times more potent than CV 3988 both in vivo and invitro but is poorly absorbed per orally (126).1.6.2 Structurally unrelated PAF antagonists1.6.2.1 Naturally occuring antagonist-GingkolidesA family of naturally occuring PAF antagonists includes the ginkgolidesisolated from the Chinese tree Ginkgo biloba. The ginkgolides have beendesignated A, B, C, M and J, corresponding to the names BN 52020, BN52021, BN 52022, BN 52023 and BN 52024. Of the series BN 52021appears to be the most potent and is able to inhibit PAP binding to rabbit(127) and human washed platelets (128,129) as well as to human leukocytes(127>. Unlike CV 3988, BN 52021 inhibits aggregation of platelets only toPAP and not to ADP, collagen, arachidonic acid, thrombin or to calciumionophore A23187 (115,127,129,130). BN 52021 is also effective ininhibiting PAP-induced internal Ca2 increase in rabbit platelets(131,132), inhibiting PAF-induced aggregation and degranulation in humanneutrophils (127), counteracting PAP and opsonized zymosan-inducedleukotriene C4 release in eosinophils (133) and inhibiting the effects ofPAF on human endothelial cells (134).In vivo, BN 52021 inhibits PAP and IgG-induced hypotension,hemoconcentration and extravasation in the rat model (135). In the guineapig, BN 52021 antagonizes PAF and antigen-induced coronaryvasoconstriction in the isolated heart (136,137) and antagonizes PAPinduced airway vascular permeability (138) and the release of thromboxaneB2, prostaglandin E2 and leukotriene C4 in isolated lungs (139).1.6.2.2 Psychotropic agents-TriazolothienodiazepinesTriazolothieneodiazepines are drugs classically employed aspsychotropic agents. It was discovered that both alprazolam and triazolam15specifically inhibited PAF-induced human platelet aggregation (140)without affecting platelet response to ADP, thrombin, epinephrine,collagen, arachidonic acid or calcium ionophore A23 187 (85,86). Aseparation of CNS and PAF antagonistic properties in thetriazolothienodiazepine drugs was achieved with the synthesis of WEB2086 (141). WEB 2086 selectively inhibits PAF-induced aggregation ofhuman platelets and rabbit neutrophils (84,85,142). Studies indicate thatWEB 2086 inhibits PAF receptor binding by interacting with PAF at acommon site on the PAF receptor (142).WEB 2086 can also play a therapeutic role when administered in vivo.In the guinea pig WEB 2086 inhibits PAF-induced bronchoconstriction,hypotension, thrombocytopenia and neutropenia (84,85,143). In the rat,WEB 2086 also protects against PAF-induced hypotension as well asagainst gastric lesions (84,85,144).1.7 PAF and Signal Traitsduction1.7.1 PAF receptor bindingBinding of PAF to the cell surface receptor initiates a cascade ofinternal effector systems which results in cellular responses such aschemotaxis, secretion or shape change. The PAF receptor has beenidentified in a number of cell types from a wide variety of species.Included in the list of cells which have been shown to possess the PAFreceptor are rabbit platelets (145,146,147), human platelets (146,148,149)porcine platelets (150), human polymorphonuclear neutrophils (151, 152),human lung tissue (153), guinea pig smooth muscle (145) and rat liverplasma membranes (154). See table 1 for a comparison of bindingaffinities and receptor numbers in a variety of cell types.16CELL TYPE receptors/cell [ Binding REFERENCEBmax I AffinityL Kd (nM)PLATELETSHuman 1,399 37 148242 0.59 147438 0.15 1461983 1.58 155Rabbit 19,386 0.9 155689 0.68 150Porcine 281 0.29 150Rats 0 0 155NEUTROPHILSHuman 5.2 X 106- -0.11 1511,100 0.2 23GuineaPig 1.6X 1011 7.6 145CELL_LINESHL-60 Cells 5200 0.7 156(Differentiated)P388D1 Murine 7872 0.08 157MacrophagesTable 1 Receptor number and binding affinity inplatelets, neutrophils, HL6O cells and P388Dj. cells.17PAF binding is distinguished by a number of characteristics. One isthat a high degree of nonspecific binding to cell membranes is usually seen.This is likely due to PAFs hydrophobic nature which allows it to easilypenetrate biological membranes (158,159). Although the lipid nature ofPAF causes a high degree of nonspecific interaction with the cellmembrane, it is speculated that PAF’s lipid nature is necessary for themolecule to enter the lipid bilayer for interaction with its receptor (159).This was supported by studies by Duronio et al. (150) where no detectablePAF binding in whole cells was observed at 40 C and fewer receptors inmembranes at 4° C were detected than at room temperature. It wasproposed that increased membrane fluidity at higher temperatures facilitatethe interaction of PAF with its receptor.A second characteristic of PAP binding is its reversibility in some celltypes and not in others (147). In neutrophils PAF is readily metabolizedwithin minutes of contact and and therefore receptor binding can beconsidered irreversible (23). This makes estimates of binding affinity and- receptor number difficult and probably accounts for the wide range innumber reported by different research groups. In contrast, PAF bindingin platelets is stable and reversible. In platelets, one hour after[3H]PAFbinding more than 90% of PAF remained unmetabolized and can still becompeted off the receptor by excess unlabelled PAF (147,151,160).A third aspect of PAF receptor binding is its sensitivity to bothmonovalent and divalent cations. Sodium has an inhibitory effect on PAFbinding while potassium, cesium, rubidium, magnesium, calcium andmanganese have an enhancing effect (161).1.7.2 Receptor independent internalization of PAFReceptor independent internalization of PAP was first put forth byHomma (162) and then by Tokumura (163). Subsequently Bratton (164)proposed that cellular activation of neutrophils leads to physical changes inthe plasma membrane that in turn leads to the nonspecific internalization ofPAF by a mechanism of enhanced transmembrane flipping. It was shownthat internalization of PAP was nonspecific with regard to structuralfeatures of the molecule and did not require metabolism of the molecule.18The contribution and significance of this nonspecific uptake as compared tothe receptor mediated uptake of PAF by neutrophils is not known.1.7.3 G-ProteinGuanine nucleotide binding proteins (G-protein) are a family ofproteins which bind and hydrolyze GTP. These heterotrimeric proteinsare located on the cytoplasmic surface of the cell membrane and form linksbetween the extracellular receptor and their effector systems. Included inthis family of G-proteins are G which is reported to inhibit adenylylcyclase, G5 which stimulates adenylyl cyclase and G which stimulatesphospholipase C (165). Unlike Gi and Gs, G has not been biochemicallyidentified and its existence is based upon evidence that fluoroaluminate(A1F4-; a direct activator of G-proteins) and various GTP analogues areable to activate phospholipase C in intact cells and in membranepreparations (166,167,168,169,170). Also in support of a link betweenand phospholipase C is the loss of inositol and Ca2 signalling in somepertussis toxin treated cells (171 ,i72). There are several reports indicatingthat the PAF receptor is linked to G-proteins (23,161,173). PAFstimulation of GTPase activity was demonstrated by the breakdownproducts of [g-32P]GTPase in rabbit platelet membranes in a manner thatwas receptor dependent and required Na (161). Houslay et at. (174)demonstrate that activity of both G5 and G can be blocked by the NADdependent ADP-ribosylation of the active subunit with cholera toxin andpertussis toxin, respectively. In the case of it appears that at least twoforms exist, one that is sensitive to pertussis toxin and a form that is not,however both are insensitive to cholera toxin (165). Using these knowninhibitors of GTPase activity, it was shown that in platelets, PAP receptorsare coupled to a phosphoinositide-specific phospholipase C through ain a manner similar to thrombin. The G-protein is insensitive to choleratoxin and only slightly sensitive to pertussis toxin (175,176,177). Incontrast to platelets, PAF binding to neutrophils is inhibited by pertussistoxin implying a link to G or perhaps to the pertussis toxin sensitive formof G. Pertussis toxin was found to inhibit PAF-mediated chemotaxis,19superoxide generation, aggregation and the release of lysozyme fromhuman neutrophils. (178). Similarly to other G linked receptors, directaddition of GTP inhibits specific binding of PAF to neutrophils (179).1.7.4 Phosphatidylinositol turnoverReceptor mediated phospholipase C activation results in thephosphodiester cleavage of phosphatidyl inositol-4,5-bisphosphate (PIP2)which gives rise to inositol-1 ,4,5-triphosphate (1P3) and diacyiglycerol(DAG). This occurs within 5-10 sec of PAF challenge (180). It appearsthat in platelets, PAF may cause the hydrolysis of a common pool of PIP2that is shared by thrombin (181). Both products of PIP2 breakdown leadto the activation of calcium-activated, phospholipid dependent proteinkinase C (PKC). 1P3 induces intracellular calcium mobilization leadingindirectly to the activation of PKC (182). Recently, an 1P3 receptor hasbeen purified in rat cerebellar Purkinje cells and was determined to be aligand gated Ca2+ channel that exists in a multimeric form. The receptor-contains an 1P3 binding site and shares many common features with the-Ca2+ channel found on skeletal muscle sarcoplasmic reticulum(183,184,185). DAG is the second product of PIP2 breakdown andremains in the plasma membrane where it binds with and activates PKC byincreasing the affinity of PKC for phosphatidylserine and calcium,therefore activation can occur with lower calcium concentrations (186).Alternatively, evidence shows that intracellular Ca2 mobilized by 1P3may prime PKC to activation by translocating PKC to the membrane wheresmall amounts of DAG can then activate the enzyme. Siess and Lapetina(187) showed that increases in calcium concentration alone could promotethe translocation of PKC from the cytosol to the membrane. Whenexamined further, the role of DAG, 1P3-induced Ca2 release andactivation of PKC by PAF becomes even more complex. Pelech et al.(188) provide evidence that shows PKC activation by PAF appears tooperate independently of translocation and that PKC activation is alsoindependent of Ca2 and lipid stimulation.201.7.5 Protein kinase CPKC consists of a family of lipid and calcium dependent isozymes all ofwhich phosphorylate serine or threonine residues. The enzyme also servesas a receptor for phorbol esters, a class of tumour promoter (189). Theevidence for DAG and 1P3 activation of PKC following PAF receptoractivation includes the phosphorylation of a 40-47 kDa protein that servesas a marker protein substrate for PKC activity (190,191). This proteinundergoes phosphorylation when rabbit and human platelets are treatedwith PAF (187,192,193). Sphingosine, a PKC inhibitor is also able toblock the PAF-induced phosphorylation (194).Although it is difficult to assign a direct role to PKC following PAFactivation, several putative target substrates have been identified both at thelevel of PAF synthesis and at the level of signal transduction and cellularresponses. At the level of PAF synthesis, there are at least twophosphorylation/dephosphorylation events which regulate PAF production.Phosphorylation appears to both activate acetyltransferase and to inhibitPLA2. PLA2 inhibition occurs indirectly through the phosphorylation ofinhibitory proteins called lipocortins (195,196). Whether cAMP dependentprotein kinase A (PKA) or calcium dependent protein kinase C (PKC) isinvolved in these processes remains to be clarified since both have beenimplicated (21,195,197). At the level of cellular responses, PKCactivation leads to platelet activation and secretion (191,198). In variouscell types PKC activation leads to both the phosphorylation of responseelements as well as a dampening of some cellular functions. Theseopposing responses are poorly understood. It is found that direct activatorsof PKC either inhibit or enhance the degranulating actions of PAF inhuman neutrophils depending upon the concentrations used. It was foundthat treatment of neutrophils with the tumor promoter at concentrationsthat inhibited PAF-induced responses also caused a down regulation of highaffinity PAF receptors (199).1.7.6 Adenylyl cyclaseThe activation of biochemical pathways following PAF binding causesthe inhibition of aclenylyl cyclase. In human platelets PAF inhibited the21basal, prostaglandin El-stimulated and fluoride-stimulated adenylyl cyclaseactivities (200). In human peritoneal macrophages physiological doses ofPAF stimulated cAMP formation, likely through the formation ofarachidonic acid metabolites. But at higher PAF concentrations cAMPformation was inhibited in the macrophages as in platelets (201). Inneutrophils (178) and possibly in platelets (161,202) the inhibition is linkedto the GTPase activity of Gj but PAF may also decrease cAMPconcentration by increasing cAMP phosphodiesterase activity (200,203). Arole for PAF-induced reduction of cAMP may be to increase intracellularCa2. Decreased cAMP may impair the function of cAMP-dependentcalcium pumps which sequester intracellular calcium (204) and wouldotherwise inhibit phospholipase C-induced-PKC activation.1.7.7 cAMP dependent protein kinase AFormation of cAMP activates cAMP dependent protein kinase A (PKA)which like PKC also targets serine and threonine residues. It appears that---activation of the PKA can lead to a dampening of PAF-induced responses atmany levels. Undem et al (205) found that modest increases in cAMP-dependent protein kinase activity in mouse PT-18 mast cells leads to theinhibition of PLA2 cleavage of arachidonic acid resulting in an inhibitionof PAF synthesis. PKA may also play a role in inhibiting PAF responsesfollowing receptor activation. The 1P3 receptor isolated from the ratcerebellar Purkinje cells appears to be inhibited by PKA phosphorylation(206).1.7.8 Ion channelsThe application of PAF to target cells induces the mobilization ofcalcium from internal stores via the 1P3 receptor and from externalsources via calcium pumps. PAF strongly modifies cell Ca2+concentration in thymocytes, macrophages and vascular smooth muscle atconcentrations greater than 10-12 M (207). It is estimated that release ofCa2+ from internal stores accounts for less than 20% of the increase incalcium observed after PAF treatment (208, 209). In thymocytes,macrophages, platelets and vascular smooth muscle it is demonstrated that22PAF activates a Ca2+ pump. The effects of PAF on calcium influx wereantagonized by the PAF receptor antagonist BN 52021. Platelet activationby PAF causes the opening of a calcium channel which is maximal within 5sec and then closes with a half life of 45 sec (210). Evidence for the closeassociation of PAF receptor with a calcium channel comes fromexperiments using calcium channel blockers. Calcium channel blockers,diltiazem and verapamil are able to inhibit PAF-induced calcium-influx,platelet activation and PAF binding (211,212). However, it must be notedthat calcium channel blockers inhibit calcium influx by binding to channel-associated proteins rather than by directly blocking the channel. Theidentity of the protein remains unknown. The structural association of thisprotein with the receptor and the calcium channel also remains unsolved.Although PAP was shown not to directly affect Na transport systems,the increase in free cytosolic calcium did stimulate Na+IH+ exchange whichis one main regulator of cellular pH and opened Ca2+ dependent K+channels which are frequently activated during secretory and inflammatoryprocesses (213,214,215,216).1.8 Receptor PurificationUntil recently PAF receptor characterization has progressed at anextremely slow pace. Virtually no information existed as to the PAPreceptor’s structure. Past attempts at receptor characterization has beenhampered by the lack of a consistent procedure to solubilize the membraneprotein in an active form. The first reported isolation of the PAF receptorwas by Valone (217). Human platelet membranes were solubilized with5% sodium clodecyl sulphate and loaded on a sepharose column of PAFhuman serum albumin. Following sodium dodecyl sulphate polyacrylamidegel electrophoresis a single protein with an apparent molecular weight of180,000 was reported. However the identity of the isolated protein was inquestion. A second attempt to isolate the PAP receptor in human plateletmembranes was made. Triton solubilization was followed by DEAEcellulose, CM cellulose and Sephadex G-200 separation. A protein with amolecular weight of 160,000 was isolated (218). Chau et al. (219,220)then made two attempts at receptor purification. The first attempt used a23sucrose density gradient and sephacryl S-300. This isolated a protein of220,000 Da. The second attempt used a photoreactive radioiodinatedderivative of PAF and isolated a protein of 52,000 Da. However, thisgroup solubilized a[3HIPAF receptor complex, leaving the possibility thatthe procedure could not be used to solubilize a unbound receptor that is notprotected from denaturation. The authors also expressed difficulty indisplacing[3H]PAF following solubilization.Recently, a successful solubilization of the unbound PAF receptor wasaccomplished by our group (221). An active form of the receptor wassolubilized from rabbit platelet membranes and was shown to bind PAFwith a similar Kd to that in intact cells. The purification was likelyincomplete as reflected by the high molecular weight of 350 kDa reported.Important information obtained from this study included that the PAFreceptor was heat labile and sensitive to trypsin inactivation and it wastherefore confirmed to be a protein. Prior to this it was speculated thatsince PAF is a lipid it could in fact be binding tightly to lipid molecules inthemembrane (222).. The failure ofthesolubilized protein to bind tovarious lectin columns gave evidence that the PAF receptor is not acommon glycoprotein containing N-linked oligosacchrides.In a separate study using a gene expression approach, Honda (223)cloned the PAF receptor from guinea pig lung. This was then followed bycloning of the PAF receptor from leukocytes (224). This is the firstphospholipid agonist for which the receptor has been cloned. A cDNAlibrary was constructed from size fragmented guinea pig lung poly(A+)RNA that elicited an electrophysiological response in oocytes. The cDNAwas a 3020 nucleotide sequence with the longest open reading frametranslating to 342 amino acids. The predicted molecular mass of thereceptor was reported to be 39 kDa. From this work the PAF receptor isnow known to be related to a family of G-protein coupled receptors withseven putative transmembrane domains. The cytoplasmic tail of thereceptor contains four serine and five threonine residues that could bepossible sites for PKC phosphorylation. There are also 2 tyrosine residuesthat could serve as targets for regulation by tyrosine kinases. This studydid not further characterize the expressed protein.24Chapter 2ObjectivesA number of goals involving the elucidation of the PAF receptor andPAF receptor signalling systems were targeted. My first goal was to studythe effect of forskolin, an agent known to elevate cAMP levels in cells, onthe PAF receptor-effector systems. My second goal was to evaluate thespecificity of a PAF analog, 1 -O-hexadecyl-2-O-(methylcarbamyl)-sn-glycero-3-phosphocholine (MC-PAF), which has potential uses in receptorpurification and antibody production.2.1 The effects of forskolin-an activator of adenylyl cyclase onPAF binding in rabbit platelets.It has been shown that the PAF signal transduction system can be down-regulated by a widespread number of agonists. PAF binding and GTPaseactivity can be desensitized with prior exposure to PAF (146,J48). Thstermed homologous desensitization as opposed to heterologousdesensitization which occurs when a different agonist desensitizes thereceptor. The effects of cross signalling between transduction signallingmechanisms are very interesting. It has been shown that thrombin-treatedplatelets are desensitized to subsequent exposure to both PAF and thrombinhowever pretreatment with PAF has no effect on the thrombin signallingsystem. Also indicated in several reports is that agents that elevate cAMPlevels can also inhibit PAF-induced responses. It was shown in rat Kupffercells that forskolin and dibutyryl cAMP were able to down-regulate thePAF receptor. It was assumed that the effects were induced throughelevations of cAMP levels (225). These mechanisms of homologous andheterologous PAF receptor desensitization are poorly understood.We decided to explore the phenomenon of cAMP-induced PAF receptordesensitization seen by Chao et al in Kupffer cells (225). The first step wasto determine if cAMP-induced down-regulation of the PAP receptor canalso occur in rabbit platelet and if so by what mechanism. Chao et al.(225) and many other researchers have used forskolin, as a tool to elevate25cAMP levels in cell systems. Classically it has been thought that forskolinbinds directly to and activates the catalytic subunit of almost all mammalianadenylyl cyclase (226). As a result a number of the conclusions on the roleof cAMP in PAF receptor desensitization are based on results observedwith forskolin, however there are also non-cAMP mediated effects offorskolin which are often overlooked. The effects can be distinguishedusing dideoxyforskolin, an analogue of forskolin which does not activateadenylyl cyclase and will therefore serve as our negative control. Ourobjective was to determine if the effects seen with forskolin were a resultof cAMP-induced down regulation and if so by what mechanism. Usingdifferent inhibitors and effectors of the post signalling system we willexplore possible causes of PAF receptor desensitization.2.2 Evaluation of methyl-carbamyl PAF binding to the PAFreceptor.Our goal in this study was to evaluate a commercially availableanalogue of PAF modified at the second carbon by the addition of a methylcarbamyl group. There are two foreseeable problems with the use of PAFin antibody production and receptor purification. The first is the presenceof acetyihydrolases that rapidly inactivate PAF and the second is the lack ofa functional group for conjugation. The addition of a methyl-carbamylgroup to the sn-2 position on the PAF molecule theoretically makes MCPAF more resistant to degradation by acetyihydrolases which couldotherwise interfere with attempts at receptor purification and antibodyproduction. If functional, MC-PAF would also give a more stablestructure to which active groups could be added to the first or thirdcarbon. However, we have seen that past attempts at modifying PAFsnative structure can drastically decrease its binding affinity to its receptor.We needed to insure that this form of PAF retains its specificity and highaffinity binding to the PAF receptor. We chose to evaluate the potency ofMC-PAF in rabbit platelets because of the availability of proven bioassaysto test its activity. In addition, binding assays to rabbit platelets are muchmore reliable than those performed in other cell types such as neutrophilsin which there is rapid uptake and metabolism of PAF. The in vivo cardiac26effects of MC-PAF will be evaluated in anesthetised rats, a model whichhas already been used effectively to evaluate the actions of PAF (227).27Chapter 3Materials and Methods3.1 MaterialsPAF, forskolin, 1 ,9-dideoxyforskolin, 8 -(4-chlorophenylthio)-adenosine 3’-5’-cyclic monophosphate (CPT-cAMP), bovine serum albumin(B SA), protease inhibitors, Ficoll-Hypaque and 3-isobutyl- 1 -methylxanthine (lBMX) were all purchased from Sigma Chemical Company (St.Louis, MO). Staurosporin, and N-(2-aminoethyl)-5-chloronaphthalene- 1-sulfonamide hydrochloride (A3) were purchased from Biomol ResearchLaboratories, Inc (Philadelphia, PA). GF/C glass microfiber filters werepurchased from Whatman Inc. (Clifton NJ). Cellulose acetate filters(HAWP, 0.45 tM were obtained from Millipore Corporation (Bedford,MA). [3HJPAF (80 Cilmmol) and 5-hydroxy[G-3H]tryptamine creatininesulphate([H]serotonin) (11 Cilmmol) and cyclic AMP3H]radioassay kitwere obtained from Amersham radiochemicals (Arlington Height, IL).Ecolume and Cytoscint scintillation fluid was obtained from ICNB iomedicals, Inc. (Costa Mesa, CA). 1 -O-hexadecyl-2-O-(methylcarbamyl)-sn-glycero-3-phosphocholine (MC-PAF) was obtainedfrom Cayman Chemical Co. (Ann Arbor, MI). WEB 2086 was obtainedfrom Boeringer Ingelheim (Indianapolis, IN).3.2 Purification of platelets.Blood from healthy rabbits was collected in citrate dextrose. The bloodwas then mixed 3:1 with tyrodes buffer (0.25 % gelatin, 137 mM NaC1, 27nM KC1, 12 mM NaHCO3, 1 mM MgCl, 5.5 mM D-glucose), containing0.1 mM of EGTA, pH 6.5. The blood was spun at 1200 rpm for 10 mm ina Beckman GPR centrifuge (Fullerton, CA). The platelet rich plasma wasoverlayed on two mls of Ficoll-Hypaque and centrifuged at 3000 rpm for15 mm. The platelet bands located at the interface between the FicollHypaque layer and plasma was collected and pooled. The platelets wereresuspended back to its original volume in tyrodes with EGTA, pH 6.5 andcentrifuged at 3000 rpm for 15 mm. The platelet bands were collected andwashed twice in solution 1 (10 mM Tris-HC1 pH 7.5, 2 mM EDTA, 15028mM NaCl), pH 7.5, by centrifugation at 3000 rpm for 15 mm. Thesupernatant was discarded and the platelet pellets were resuspended in theappropriate assay buffer and counted by a Coulter Counter (VancouverGeneral Hospital, Dept. Haematology). Contamination by other cells types(white blood cells and red blood cells) was determined to be less than 1%.3.3 Membrane preparation.Isolated platelets were suspended in solution 2 (10 mM Tris pH 7.5, 2IIM EDTA, 5 mM MgCl2) containing 20j.tg/ml leupeptin, 10 Ig/ml soybeantrypsin inhibitor, 1 tg/ml pepstatin and 20tg/ml PMSF and then stored at -80° C until membranes were prepared. Cells were thawed and sonicated onice for 30 s at a 50% power setting on a Microson Ultrasonic CellDisrupter (Heat Systems-Ultrasonics, Model HS-MS5O, Farmingdale, NY).The membrane fraction was isolated on a cushion of 27% sucrose in 10mM Tris/HC1, pH 7.5, by centrifugation at 65,000 x g for 20 mm inBeckman TL- 100 ultracentrifuge cooled to 4° C. Membranes were pooledand washed oncein 10 mM Tris/HC1 p1-I 7.5 and then resuspended in 1 mlof Tris/HC1 pH 7.5. An 20 ii aliquot was taken for protein determinationby the method of Lowery et al. (228). BSA, MgC1 and KC1 were thenadded in concentrations required for binding assays.3.3 Binding AssaysThe conditions for PAF binding to rabbit platelets and membranes werepreviously established in our lab (150). Prepared platelets were washedonce with binding buffer (10 mM Tris-HC1, 0.25% BSA, 10 mM KC1 and5 mM MgC1) and then resuspended in binding buffer. 1 X 108 plateletsor 10 tg of platelet membranes were incubated in a total volume of 400 .tlof binding buffer for 2 mm at room temperature in the presence of IBMXand other agents. Subsequently,[3H]PAF (final conc. 0.05 nM for wholerabbit platelets and 0.5 nM for rabbit membranes) and variousconcentrations of unlabelled PAF (0-75 nM for rabbit platelets andmembranes) was added for a final volume of 500 p1. Following a 20 mmincubation at room temperature, the reaction was stopped by filtration on29Whatman GF/C glass fiber filters for the whole platelets and on celluloseacetate filters for platelet membranes. The filters were washed with 5 mlof binding buffer to remove excess unlabelled PAF, incubated in Cytoscintscintillation fluid overnight and then counted in a liquid scintillationcounter. The specific binding was obtained by subtracting the nonspecificbinding in the presence of 75 nM unlabeled PAF from the total bindingobtained.For the experiment involving GTP-g-S, platelets were first treated with10 tg/ml of saponin for 1 mm. The saponin permeabilized platelets werethen exposed to 200 tM of GTP-g-S and/or 50 jiM forskolin ordideoxyforskolin for 2 mm. [3H]PAF was then added and nonspecificbinding was obtained by adding 75 nM unlabelled PAF.3.4 Platelet aggregation assayPrepared platelets were place in tyrodes buffer with 1.3 mM calcium,pH 7.5. Aliquots of 4 X108 platelets per ml were assayed for PAF-inducedaggregation at-37° C using a Biodata aggregrometer (Biodata, Hatboro,PA) which correlates platelet aggregation to changes in light transmission.PAF and MC-PAF or the drugs to be assayed were added directly to theplatelets in a cuvette which contained a magnetic stir bar for rapid andhomogeneous mixing. All PAF and MC-PAF concentrations were made inthe above tyrodes buffer with 0.25% BSA. A blank of buffer alone wasnormalized as 100% transmission. A cuvette of platelet rich plasma wasthen calibrated as 0% transmission. Aggregation was measured as %increase in light transmission after the addition of PAF.303.5 cAMP assayPlatelets in calcium containing tyrodes, pH 7.2 at 2 X iü cells in 500tl were incubated with forskolin or various other agents for 5 mm at roomtemperature. All samples also included 0.5 mM IBMX to inhibitphosphodiesterase activity. The reactions were stopped by the addition of25 tl of concentrated trichioroacetic acid. The platelets were pelleted for5 mm in a Beckman microfuge and the supernatant was subsequentlyremoved for cAMP measurement. The level of cAMP in triplicate sampleswas measured by a cyclic AMP{3H]radioassay kit.3.6[H]Serotonin incorporation into plateletsTo incorporate{3H]serotonin, isolated rabbit platelets in tyrodes with0.1 mM EGTA, pH 7.0 were incubated at 37° C for two hours with 0.2j.tCi/ml{3Hjserotonin. After the incubation period, the unincorporated[3H]serotonin was removed by washing the platelets three times withtyrode’s buffer, with EGTA, pH 6.5. The platelets were reconstituted to 2X108.cells/mi in tyrode’s buffer containing 0.14 mg/mi CaC12, pH 7.2.3.7 Serotonin release assayAliquots of 0.5 ml of[3H]serotonin labelled platelets were incubatedwith various concentrations of either PAF or MC-PAF for 2 mm at 3 7°C.The reaction was stopped by centrifugation of the incubation mixture at15000 X g for 15 sec in a Beckman microfuge. The supematant (0.25 ml)containing the released[3H]serotonin was mixed with Ecolume scintillationfluid and counted in a liquid scintillation counter. For accuratecomparisons all samples were assayed in triplicate on the same day afterchallenge with the same batch of labelled rabbit platelets.3.8 Serum acetyihydrolase assayTo assess the susceptibility of PAF and MC-PAF to serumacetyihydrolases we incubated 20 jig samples of PAF and MC-PAF in seraobtained from rabbit blood. To obtain the sera, rabbits blood was allowedto coagulate for 1 hour on ice and then centrifuged at 2000 X g for 10 mm.Samples were then incubated at 37° C in 0.2 ml of serum for various time31periods. The reactions were stopped by the addition of 0.5 ml of ice coldmethanol. Proteins were allowed to precipitate overnight and then waterand chloroform were added for a final ratio of 1:1:1. The chloroformlayer was collected and PAF and MC-PAF were extracted from othercontaminants in the sera by HPLC.3.9 HPLC extraction of PAPEach sample was dried under nitrogen gas and then reconstituted in 100jil of chloroform!methanol (9:1). The samples were then injected into a4.1 X 150 mm, 10 jtM silica gel column (Altech Scientific Co., Deerfield,IL). A solvent system of isopropanol/toluene/acetic acid/H20(93:110:15:15) was used to extract PAF at a flow rate of 1 mi/mm. Thecolumn was calibrated daily using [3H]PAF to generate a standard curveand determine the fraction at which PAF was eluted (figure 3.1). Fivefractions on either side of the radioactive peak were pooled and evaporatedunder nitrogen. The dried residues were reconstituted in 100 j.il of tyrodesbuffer containing 0.25% BSA and assayed for it ability to induce serotoninrelease from labelled rabbit platelets.32300025002000Cl) 1500I—z1000C)5000Fraction NumberFigure 3.1 Standard curve of HPLC extraction of PAF..Fractions marked by * indicates those collected and pooled forbioassay of PAF activity.3.10 Hemodynamics and cardiovascular effects of PAF andMC-PAF.Male Sprague-Dawley rats (200-3 00 g) were used in accordance withthe guidelines of the University of British Columbia’s Animal CareCommittee. Rats (n=6 per group) were anesthetised with pentobarbitol (55mg/kg i.p.) and their right jugular vein and left carotid artery werecannulated for drug administration and blood pressure monitering,respectively. All animals were artificially ventilated via a tracheal cannulaat a stroke volume of 10 mi/kg and a rate of 60 strokes/mm to ensureadequate blood-gas levels (229). Body temperature was monitored byrectal thermometer and maintained between 36-37° C with a heating lamp.0 5 10 15 20 25 30 35 40 45 5033The EKG signals were recorded via needle electrodes placed along thesuspected anatomical axis (right atrium to apex) as determined bypalpitation. A superior electrode was placed at the level of the rightclavicle while the inferior electrode was placed on the left side of thethorax exemplifying Lead II configuration. EKG and blood pressuremeasurements were made directly from a Grass polygraph (model 7D) at abandwidth of 0.1-40 Hz. Using a random block experimental design,animals were either given PAF or MC-PAF over a dose range of 0.5-20jig/kg. Each dose was infused over 2 mm and all recordings were made 3mm later, just prior to the addition of the next dose. The exposure timewas chosen from preliminary studies at which pharmacological a steadystate response to both PAF and MC-PAF occurred.The in vivo data was analyzed for statistical significance by using theGeneral Linear Model Analysis of Variance (GLM-ANOVA) followed byDuncan’s multiple range test using the NCSS statistical package (230). Allvalues are presented as the mean ± s.d. A difference of p<O.05 wasconsidered significant. ----34Chapter 4Results4.1 Effects of Forskolin on PAF Binding4.1.1 Measurement of cAMP in treated cellscAMP formation in rabbit platelets plays a major role in signallingmechanisms in the cell. We wanted to insure that the forskolinconcentration of 50 IIM used by Chao et al. (225) in Kupffer cells wasadequate to stimulate cAMP formation in our cell system. When rabbitplatelets were incubated with forskolin in the presence of IBMX for 2 mm,approximately a 9-fold increase in cAMP was obtained. The level ofcAMP in control platelets was approximately 1.1 pmol/1 08 platelets andthis was increased to 9.2 pmol/108platelets when treated with forskolin(Figure 4.1). As expected PAF and dideoxyforskolin had no effect oncAMP production in rabbit platelets.35Figure 4.10•1-00CD000.0cAIvIP measurement in rabbit platelets.cAMP was measured in 108 platelets following 2 mmtreatments with PAF (2 nM). forskolin (50 tM) anddideoxyforskolin (5OiiM). The results are the mean oftriplicate determinations, and are representative of threeexperiments.10 -86-4.2-0-CONTROL PAF FORSKOUNTREATMENTDDFORSK364.1.2 PAF binding toforskolin treated cellsPlatelets treated with forskolin for 2 mm demonstrated a decrease ofapproximately 3 0-40% in PAF binding (figure 4.2). Scatchard plotanalysis of the data generated from binding of[3HIPAF to receptors treatedwith forskolin is also shown in the figure 4.2. Analysis of the datarevealed that the decrease in PAF receptor binding was a result of adecrease in PAF binding sites (Bmax) rather than a change in bindingaffinity (Kd). The Kds obtained for PAF binding in control and forskolintreated platelets was 0.84 ± 0.13 nM and 0.81 ± 0.18 nM (p>l.O, n=5)respectively. The number of binding sites obtained for PAF on plateletsfor control and forskolin treated cells were 1238 ± 199 and 747 ± 196receptors/platelet, respectively (p< 0.005, n=5). The results shown are themean ± standard deviations. All binding data was performed in triplicateand the results of the scatchard analysis was analyzed for statisticalsignificance using the Student t-test. -37(22-Lii0Li0Figure 4.23000Specific binding of PAF to rabbit plateletsPlatelets were exposed to 50 iM forskolin (I) for 2 mm priorto PAF binding. Control platelets (•) were treated withequivalent amounts of binding buffer. Each point is the meanof triplicate determinations with error bars indicating thestandard deviation. The data is representative of similarresults obtained in five separate experiments.Scatchard analysis of PAF binding to rabbit platelets. Thedata generated from PAF binding to treated platelets ispresented in the inset figure as a Scatchard plot. Kd forcontrol (•) and forskolin (I) was 0.84 ± 0.13 and 0.81 ± 0.18respectively. Bmax for control and forskolin was 1238 ± 199and 747 ± 196 receptors/platelet (n=5), respectively.250020001500100050000 1 2 3 4 5 6COLD PAF CONCENTRATION (nM)384.1.3 CPT-cAJvIP, staurosporin and A3 treated plateletsThe effect of the membrane permeable cAMP analog, CPT-cAMP onPAF binding to rabbit platelets was investigated. Incubation of rabbitplatelets with 10 nM and 100 nM of CPT-cAMP for 2 mm did not induceany apparent changes in PAF binding (data not shown). This implied thatcAMP was not the cause of the changes seen with forskolin treatment ofplatelets. We explored this further to determine if there was a possibleactivation of PKC or PKA in the action of forskolin. Treatment ofplatelets with the PKA inhibitor A3 (200 nM) (231) and the PKC-inhibitorstaurosporin (ijiM) prior to treatment with forskolin and dideoxyforskolindid not block the reduction in PAF binding (data not shown). Thissuggested that the action of forskolin was independent of PKA or PKCactivation.394.1.4 PAF binding to dideoxyforskolin treated plateletsThe ineffectiveness of CPT-cAMP, staurosporin and A3 in inhibitingthe effect of forskolin led us to believe that perhaps the effect of forskolinwas independent of cAMP formation. Dideoxyforskolin does not activateadenylyl cyclase activity in cells and therefore can be used to distinguishcAMP dependent and independent responses. Treatment of platelets withdideoxyforskolin (50 tM) also reduced PAF binding with the effect beingapproximately 10% greater than that seen with forskolin (figure 4.3).40Figure 4.3C,zz0-..-a.U,Ua.PAF binding to dideoxyforskolin treated plateletsPAF binding to dideoxyforskolin (50 tiM) () treated plateletsis compared to forskolin-treated platelets (I) and untreatedplatelets (•). Each point is the mean ± standard deviation oftriplicate determinations and is representative of 3 individualexperiments.30002000100000.0 1.0 2.0 3.0 4.0 5.0COLD PAF CONCENTRATION (nM)414.1.5 Binding to forskolin and dideoxyforskolin treated membranesExperiments were performed with isolated rabbit platelet membranes todetermine whether cellular activity was required for the actions offorskolin and dideoxyforskolin. Since previous experiments withstaurosporin and A3 failed to reverse the effects of forskolin it wasspeculated that perhaps the effect of forskolin was due to a direct effect ona membrane bound component rather than through an activation of a signaltransduction pathway. In these experiments, platelet membranes weretreated with forskolin or dideoxyforskolin 2 mm prior to the addition ofPAF. As shown in figure 4.4, addition of forskolin or dideoxyforskolin tothe purified platelet membranes reduced PAF binding. The changes seenwith forskolin treatment of isolate platelet membranes was similar to thoseseen with forskolin treatment of whole platelets, suggesting the action offorskolin and dideoxyforskolin is on a membrane bound component anddoes not require an intact cell to be activated.42Figure 4.4C!,z- -z0E-u-.05.—LUU)UPAF binding to treated membranesMembranes pretreated with forskolin (I) or dideoxyforskolin(Li) are compared with untreated membranes (•). Theexperiment was repeated 3 times and each point shown is themean ± standard deviations (n=6) obtained in a representativeexperiment.1000080006000400020000.0 0.5 1.0 1.5 2.0COLD PAF CONCENTRATION (nM)2.5434.1.6 Effect of GTP analog on forskolin and dideoxyforskolin treatedplateletsPAF receptor linkage to G-proteins has been established (see section1.7.3). To explore the possibility that the effects observed with forskolinand dideoxyforskolin was a result of G-proteinlreceptor interruption,studies were performed with platelets treated with GTP-g-S in the presenceor absence of forskolin or dideoxyforskolin. Binding of PAF to itsreceptors was found to be reduced by approximately 30% in forskolintreated cells (figure 4.5). When the two agents were added together PAFbinding was reduced by approximately 70%. The results fordideoxyforskolin and GTP-g-S were also similar. These observations of anadditive effect of GTP-g-S and forskolinldideoxyforskolin suggested thatforskolin and dideoxyforskolin were not acting on a G-protein to alter PAFbinding.44zzC-).-’TREATMENTGTP-g-S treatmentSaponin permeabilized rabbit platelets were treated for 2 mmwith 100 iM GTP-g-S prior to the addition of forskolin ordideoxyforskolin. The results were compared to untreatedplatelets and platelets treated with GTP- g-S, forskolin ordideoxyforskolin alone. The results are the mean ± standarddeviations of triplicate determinations.Figure 4.52000 0Iz0t >LI1z1000Li.1160012008004000z-Icn’iOL454.1.7. Effect offorskolin and dideoxyforskolin on PAF -induced plateletaggregationTo determine the physiological relevance of forskolin anddideoxyforskolin mediated changes in PAF receptor binding, plateletaggregation studies were perfonned. Washed rabbit platelets were exposedto either forskolin or dideoxyforskolin for 2 mm prior to the addition ofPAF. Aggregation in response to PAF was reduced significantly inplatelets pretreated with either forskolin or dideoxyforskolin for 2 mmprior to the addition of PAF (figure 4.6). Without forskolin ordideoxyforskolin treatment, platelets responded to PAF at less thannanomolar concentrations to induce aggregation, while platelets treatedwith forskolin or dideoxyforskolin resisted aggregation in response to PAFat much higher concentrations.46z00Ui0z0I0Ui00Figure 4.6 PAF-induced platelet aggregation offorskolin anddideoxyforskolin treated plateletsRabbit platelets were incubated for 2 mm with 0 pM (I), 0.5pM (D), 5 iM () and 50 iM () forskolin (A) ordideoxyforskolin (B) prior to the addition of variousconcentrations of PAF. Aggregation was measured during a 1mm incubation.806040200806040200-10 -9 -8-7log [PAF] (M)-10 -9 -8log [PAF] (M)-7474.2 Evaluation of MC-PAF4.2.1 PAF and MC-PAF-induced platelet aggregationPlatelet aggregation is independent of cyclooxygenase products or ADPrelease and still is a method widely used to assess the potency of new PAPanalogs (232,233). The popularity of platelet aggregation as a bioassay forPAF activity lies both in its simplicity and its relative sensitivity. Doseresponse curves were generated for both PAF and MC-PAF over the doserange of 0.01-100 nM. MC-PAF was able to mimic PAF in causingplatelet aggregation but had a 2-4 fold lower potency than PAF (Figure4.7). Full platelet aggregation was achieved with approximately 5 nM PAFas compared to approximately 10 nM for MC-PAF.Next, the specificity of MC-PAF for the PAF receptor was assessed. Weevaluated MC-PAF’s ability to cause platelet aggregation in the presence ofthe PAF antagonist WEB 2086 which interacts with PAF at a common siteon the receptor (142,84,85). Rabbit platelets were treated with variousconcentrations of WEB 2086 followed by 10-8 M MC-PAF. This test wasrepeated for the MC-PAF concentrations of M and 10-6 M. WEB2086 was able to competitively inhibit all concentrations of MC-PAFinduced platelet aggregation in a dose dependent manner.48Figure 4.7 A) PAF and MCPAF-induced platelet aggregation.The potency of MC-PAF (I) was compared to PAF (0) inits ability to stimulate the platelet response to aggregation.B) Inhibition ofMC-PAF-induced platelet aggregation byWEB 2086.Platelets were first treated with the PAF receptor antagonist,WEB 2086 at various concentrations and then three differentconcentrations of MC-PAF was added: 10-8 (), i0- M () and 106 M (0). In a dose dependent manner, WEB 2086was able to block MC-PAF-induced platelet aggregationz0C!,UiccC!,z0UiccC!,A0S5040302010•0—-1250403020100I I— I— I-11 -10 -9 -8Compound [log M]-7 -6-9 -8 -7 -6 -5WEB 2086 [log M]-4 -3494.2.2 PAF and MC-PAF-induced serotonin releaseSerotonin is present in platelets at relatively high amounts and is takenup from the external media against concentration gradients via a membranetransport system coupled to sodium transport (234,235). All plateletserotonin is able to exchange freely with serotonin in the external media(235) and when platelets are activated by PAF and other agonists theyrelease serotonin into the suspending media in dose dependent manner.This proven method of quantifying PAF activity was used to compare thein vitro physiological effects of MC-PAF with PAP (Figure 4.8). As withplatelet aggregation the potency of PAF was about 2-fold greater than MCPAF in causing serotonin release.50[3H]Serotonin release by PAF and MC-PAF in rabbit platelets.H]Serotonin labelled rabbit platelets were treated withincreasing concentrations of PAF (0) or MC-PAF (R)ranging from 10-11 M to 10-6 M. The results are the mean ±standard deviation of triplicate determinations and isrepresentative of that obtained in two separate experiments.Figure 4.80Vzz00LUCl)cv)10000800060004000 -2000 -0- —-12S-11 -10 -9 -8 -7 -6Compound [log M]-5514.2.3 PAF and MC-PAF binding to rabbit plateletsThe specificity of MC-PAF receptor binding was assessed in bindingstudies that were subsequently analyzed by Scatchard analysis to determinebinding affinities and receptor numbers (figure 4.9). The Kd for PAF andMC-PAF were determined to be 0.62 ± 0.07 and 1.47 ± 0.30 nM,respectively (n=3). This approximately two fold difference was alsoreflected in the apparent receptor density values (Bmax) of 750 ± 156 and1931 ± 515 receptors/cell for PAF and MC-PAF, respectively (n=3). ThePAF and MC-PAF binding studies were performed in triplicate in threeseparate experiments. The binding results confirmed that the difference inplatelet aggregation and serotonin release recorded with PAF and MC-PAFare due to differences in binding at a receptor level.52Figure 4.9 PAF and MC-PAF binding to rabbit platelets.Increasing concentrations of unlabelled PAF (0 ) and MC-PAP (R) were added to rabbit platelets in the presence of0.05 nM of[3H]PAF. The points shown are the mean ±standard deviations of triplicate determinations and representsimilar results obtained in three separate experiments. Thespecific binding data for PAF (0 ) and MC-PAF (R) shownin the inset.a•00zzC-)UC-)w0Cl)PAF CONCENTRATION (nM)80060040020000 5 10 15 20 25 30534.2.4 Effect of PAF and MC-PAF on anesthetised rats.In pentobarbitol anesthetised rats, the cardiac and hemodynamicresponses of MC-PAF were markedly similar to those of PAF. Neither ofthe two compounds significantly altered heart rate, even at a maximumdose of 20 jig/kg tested (Figure 4.1OA). Both PAF and MC-PAF were ableto reduce blood pressure in a dose-dependent manner (Figure 4.1OB). Theeffect of both of these agents was instantaneous and persisted for at least 5mm after treatment. PAF and MC-PAF did not induce tachyphylaxis, evenafter the higher doses were administered. The dose at which bloodpressure was reduced half maximally (EC5O) was approximately 0.5 jig/kgfor PAF and 1.0 jig/kg for MC-PAF. A cumulative dose of 20 jig/kg ofPAF reduced blood pressure to 18 ± 6 mm Hg while the same dose of MCPAF reduced blood pressure to 37 ± 10 mm Hg. Studies of the EKGvalues showed that the P-R interval was slightly prolonged by PAF athigher doses by not by MC-PAF, while neither of the compounds alteredthe QRS or Q-T interval (results not shown).54CEL.0004-0.0UiI—IUizE0000a.00Figure 4.11 The cardiac and hemodynamic parameters of rats in responseto PAF and MC-PAF.PAF () and MC-PAF ( ) from 1-20 jig/kg were administered toanesthetised rats. No significant changes in heart rate were observed (A).Both PAF and MC-PAF reduced blood pressure (B).5004003002001000- Y 0.5 1 4 10 20DOSE (igIkg)20010000 0.5 1 4 10DOSE (jig/kg)20554.2.5 Serum acetyihydrolase susceptibility of PAF and MC-PAFThe conversion of PAF to its inactive metabolite, lyso-PAF occurs viathe hydrolysis of the acetyl group at the sn-2 position by serumacetyihydrolases (see section 1.4.1). Since rabbits are often used as avehicle for generating antibodies, PAF and MC-PAF inactivation in rabbitsera was assessed. PAF and MC-PAF were first incubated in rabbit serafor various time periods and then purified by HPLC. The resulting eluateswere tested for their ability to induce[3H]serotonin release from rabbitplatelets (figure 4.12). There was approximately a 35% drop in PAFinduced serotonin release after only a two minute incubation in sera andthis rapidly decreased at 5 minutes to a 75% drop in PAF stimulatingactivity. In comparison, MC-PAF resisted degradation by acetyihydrolasesfor up to 60 mm and only experienced approximately 35% drop in activity.56Figure 4.120-oz0I0UiU)=PAF and MC-PAF hydrolysis by serum acetyihydrolases.The susceptibility of PAF (0 ) and MC-PAF (•) to serumacetyihydrolases. After only a 5 mm treatment PAF wasrapidly degraded while MC-PAF was resistant toacetyihydrolase for at least one hour. Each point is the mean ±standard deviation of triplicate determinations. The datarepresents results obtained in two experiments.4000TIME (mm)100008000600020000 10 20 30 40 50 60 7057CHAPTER 5DISCUSSION5.1 Effects of forskolin on PAF bindingThe results of the present study provide new insights into the action offorskolin on PAF binding. Forskolin was able to dramatically inhibit PAFbinding to its receptor and subsequently block PAF-induced physiologicalresponses. However, the results do not support the involvement ofadenylyl cyclase in this process and suggest that other possible mechanismsmust be explored when explaining forskolin’s mode of action.Although forskolin is best known as an activator of adenylyl cyclaseactivity, resulting in cAMP formation (226,236), it has been shown to havenon-adenylyl cyclase activity in cells. Several reports have indicated that anumber of forskolin-induced activities in various biological systems may beindependent of adenylyl cyclase activation. Many of these activities areshared by dideoxyforskolin, an analog of forskolin which does not activateadenylyl cyclase (237). Dideoxyforskolin is therefore a useful tool inseparating the cAMP dependent actions of forskolln from the cAMPindependent actions. McHugh and McGee (238) demonstrated that bothforskolin and dideoxyforskolin inhibited the nicotinic acetyicholinereceptor mediated Rb+ uptake in skeletal muscles that did not involve theactivation of adenylyl cyclase or a cAMP dependent phosphorylation.Further work by Wagoner and Pallota (239) confirmed that forskolininduced desensitization of the nicotinic acetylcholine receptor in skeletalmuscles was mediated by a mechanism that did not involve the activation of58adenylyl cyclase or a cAMP-dependent phosphorylation. Another adenylylcyclase independent action of forskolin includes the inhibition of glucosetransport in human platelets, erythrocytes and adipocytes (240,241,242).These novel activities of forskolin are poorly understood.Given the alternative actions of forskolin, we attempted to investigatewhether the action of forskolin on PAF receptor binding was dependent onadenylyl cyclase. We were unable to replicate the effects of forskolin bydirect addition of CPT-cAMP. Although Chao et al. (225) and others(243,244) were able to use dibutyryl cAMP (db-cAMP) to simulate thedecrease in PAF binding shown with forskolin, we chose instead to useCPT-cAMP, bearing in mind that db-cAMP and forskolin have actionsother than mimicking endogenous cAMP. It has been demonstrated thatfollowing uptake of db-cAMP, it is subjected to enzymatic degradation intomonobutyryl cAMP and butyrate (245,246). Although monobutyryl cAMPis regarded as the active component, some effects of db-cAMP can becontributed to the action of butyrate. In one study, the inhibition of N2Acell growth, histone acetylation, thyroid hormone receptor down regulationand elevation of concentrations of histone Hi protein were all in part dueto the presence of butyrate (247). To rule out the possible effects ofbutyrate in our system, we used CPT-cAMP which is known to be about100 times more potent than cAMP in mimicking the effects of vasopressinin the mammalian kidneys. This is probably due to its greater resistence tophosphodiesterase activity and its greater permeability across cellmembranes (248). Also Chao et al. (225) found that in order to achievethe effects seen with db-cAMP on PAF receptor binding, long term59treatment (24 hours) was necessary. This long incubation time injects someuncertainty into the results since others (249) have shown that even with 24hour storage of platelets, responses to PAF and thrombin decline severely.The changes observed with stored platelets included decrease in receptorbinding, aggregation and phosphoinositide turnover. We therefore choseto avoid long term incubations with any of the agents to prevent cellulardegradation of the compound and to prevent possible physiological changesin our model that might be seen with long term storage. Using treatmenttimes comparable to that of forskolin, we found CPT-cAMP to beineffective in reducing PAF binding. Our aggregation data showed that theeffect of forskolin was extremely rapid and thus we felt that treatmenttimes with CPT-cAMP should be adequate. It was reasoned that ifdesensitization is a result of cAMP formation, the concentration CPTcAMP used should also be adequate given that it is approximately 50, 000times greater than the in vivo cAMP concentration measured in forskolintreated platelets.After finding that CPT-cAMP was unable to mimic the results obtainedwith forskolin, we also found other lines of evidence that seemed to ruleout the involvement of adenylyl cyclase and cAMP formation in the processof forskolin-induced alterations of PAF binding. This includes the abilityof dideoxyforskolin to cause similar changes in PAF receptor binding. Theability of dideoxyforskolin to reduce PAF receptor binding conclusivelyeliminates the role of cAMP as the induced mediator. Chao et al. (225) didnot evaluate the effect of dideoxyforskolin in their studies of cAMPdesensitization of the PAF receptor pathway. Since forskolin was able to60affect the PAF biochemical pathways at the receptor level, the decreasedPAF-induced arachidonic acid release and eicosanoid production observedby Chao et al. may also be attributed to a mechanism other than cAMPproduction. In addition, the positive effect that forskolin anddideoxyforskolin had on isolated platelet membranes demonstrates thatthese agents act at the cell membrane level and do not require theparticipation of intact cellular enzymatic systems. The results obtainedwith PKA and PKC inhibitors also indicate that although the PAF receptorpossesses potential sites for protein phosphorylation (223), these twokinases do not participate in the reduction of PAP binding, unlike themechanism of desensitization seen with the B-adrenergic receptor (250).One possible explanation offered for the cAMP independent effects seenwith the nicotiniè receptor is that forskolin, an extremely lipophilicmolecule is able to enter the plasma membrane, disrupt the lipid structureand interfere with normal channel activities in a similar way as a generalanesthetic molecule. The observation of a slightly greater potency fordideoxyforskolin is consistent with the lipid perturbation theory, sincedideoxyforskolin is a more lipophilic molecule than forskolin due to thelack of two hydroxy groups on the molecule (238). Another possibility isthat perhaps forskolin and dideoxyforskolin were acting in a mannersimilar to that found for the glucose transporter where inhibition ofactivity was due to the direct binding of these agents to the transporter inrat adipocytes and human erythrocytes (251,252). It is possible that ahydrophobic interaction does exist and that binding of forskolin anddideoxyforskolin to specific sites may affect structures closely associated61with the PAF receptor. One possible site of action could be a guaninenucleotide binding protein (G-protein) to which the PAF receptor iscoupled. Previous work has linked the PAF receptor to the G which inturn is known to play an important role in the control of adenylyl cyclaseor to which is linked to phospholipase C (see section 1.7.3). The latterresults are supported by the work of Homma and Hanahan (244) whichshowed that db-cAMP pretreatment of rabbit platelets resulted in adecrease in the extent of PAF-induced GTPase activity. However in thepresent study, we propose that the action of forskolin and dideoxyforskolinis independent of G-protein involvement since GTP-g-S and forskolin hadan additive effect on PAF binding.In conclusion, the work presented in this study indicate that forskolinacts to reduce PAF binding to its receptor in a manner that does not seemto involve adenylyl cyclase, G-protein, PKA or PKC. Also we note thatgiven the direct effect that forskolin has on PAF binding, caution must beused when it is employed as an agent to study the effect of cAMP on thePAF receptor and signal transduction pathways.5.2 Evaluation of MC-PAFThe present work demonstrates that MC-PAF behaves in a mannerrepresentative of being a full PAF agonist. MC-PAF was evaluated both invitro and in vivo. First, in all the tests of in vitro bioactivity MC-PAFwas able to mimic the actions of PAF with only a two to five folddifference in activity. In receptor binding studies MC-PAF was shown tobind to the PAF receptor with only a two fold lower affinity than PAF.62Secondly, in vivo the cardiovascular changes induced by MC-PAF werevery similar to PAP. It is noted however that MC-PAF does not reduceblood pressure to the same degree as PAF, even at the maximum dose of 20jig/kg tested. The dose of MC-PAF that effectively reduces blood pressurehalf maximally, EC5O, is approximately twice that of PAF. Thus the invivo data also reflects the observations seen in vitro with rabbit platelets.As mentioned, tachycardia was not observed in our experimental model.This observation was first reported by Caillard et al. (253) however othersreport that tachycardia does not result from the severe systemichypotension produced by PAF (254). Our results support the latterobservations. The precise mechanism for PAF-mediated tachycardia undercertain experimental conditions is not known.Thirdly, MC-PAF was shown to be a relatively stable molecule asserum acetylhydrolases were not able to cleave the carbamyl moiety fromthe sn-2 position of the phospholipid molecule. The stability of MC-PAFshould prove to be a valuable asset for investigations involving PAP andthe PAP receptor. Potentially MC-PAF could be used for raisingantibodies against PAP as well as in receptor purification.Past attempts at PAF receptor solubilization have been hampered by thelack of a specific purification method for the PAF receptor. Although ourresearch group has successfully solubilized an active form of the PAPreceptor (221), the next step to be accomplished is a more precisepurification of the receptor based on the solubilization procedures we havedeveloped. Ideally this will be accomplished by the development of anaffinity column in which PAP is immobilized on a solid support.63Theoretically, the solubilized PAP receptor could be then be applied to thecolumn. Following multiple washes to rid the column of contaminatingproteins the PAP receptor could then be eluted in a highly purified form.There are two foreseeable problems with our aims. The first problem isthe presence of acetyihydrolases which can readily inactive the PAF boundon the affinity column into lyso-PAF a form unrecognizable by the PAFreceptor. The second problem is that PAF lacks a functional group whichcan be used to immobilize it to a solid support. In this study, we havedemonstrated that MC-PAF is resistant to acetyihydrolase activity.Therefore, by placing functional groups such as an amino or carboxylmoiety at the sn-i position or by variations at the sn-3 position such asethanolamine, monomethylethanolamine or dimethylethanolamine, it ispossible to effectively conjugate the methylcarbamyl analog to a carrier forantibody production or to a solid support such as cyanogen bromideactivated agarose for the purpose of receptor purification in an affinitycolumn. Potentially, large amounts of PAF receptors could be solubilizedand applied to the PAP affinity column. Elution of the column byappropriate buffers would result in large quantities of the purified receptorthat would help tremendously in studies involving receptorcharacterization. Although the receptor has been cloned, information onthe PAF receptor’s association with other membrane bound proteins has yetto be determined. Also lacking is a crystalline form of the receptor. 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