acute inflammation produces a condition known as hyperalgesia , which is characterized by enhanced pain sensation and reduced pain threshold .
this abnormal sensory state is brought about , at least in part , by sensitization of peripheral nociceptors .
accumulating evidence indicates that inflammatory mediators including prostaglandin e2 ( pge2 ) and pgi2 are responsible for the sensitization of nociceptors [ 1 , 2 ] .
recent studies further clarified the molecular mechanism of sensitization : pge2 and pgi2 enhance or sensitize the activity of the capsaicin receptor transient potential receptor v1 ( trpv1 ) through the activation of pge2 ep1 and pgi2 receptors , respectively .
protein kinase c and protein kinase a are involved in the activation of trpv1 by these prostaglandins .
in contrast , cyclopentenone prostaglandins , including 15-d - pgj2 , pga2 , and pga1 , which are metabolites of pgd2 , pge2 , and pge1 , respectively , directly activate the irritant receptor trpa1 .
for pharmacological control of inflammatory hyperalgesia , it is important to understand how these prostaglandins are produced in inflamed tissues .
prostaglandins are generated through the arachidonic acid cascade , which involves three enzymatic steps : first phospholipase a2 ( pla2 ) cleaves membrane phospholipids and releases arachidonic acid ; second , cyclooxygenase ( cox ) converts arachidonic acid into pgh2 ; and , third , various types of prostaglandin isomerases convert pgh2 to bioactive prostaglandins , including pge2 and pgi2 .
recent studies have shown that cox-2 , an inducible isozyme of cox , and microsomal prostaglandin synthase-1 ( mpges-1 ) , an inducible isozyme of pge synthase , are responsible for the generation of pge2 in inflamed tissues [ 6 , 7 ] . in these studies ,
administration of a cox-2 inhibitor to rats   or disruption of the mpges-1 gene in mice lowered pge2 content in inflamed tissues and eased pain - related behaviors .
thus , cox-2 and mpges-1 could be pharmacological targets for the treatment of inflammatory pain . however , it is not clear which isozyme of pla2 is responsible for the production of prostaglandins and development of hyperalgesia in inflamed tissues .
pla2 has over ten isozymes which are classified into three categories based on their structural and functional similarities [ 8 , 9 ] .
first , cytosolic pla2s ( cpla2 or group iv pla2 ) are located in the cytoplasm and are activated by low calcium ion concentrations ( m levels ) .
second , secretory pla2s ( spla2 or group ib , ii , v , or x pla2 ) are released into the extracellular space where they are activated by high concentrations ( mm levels ) of calcium ions .
third , calcium - independent pla2s ( ipla2 or group vi pla2 ) are present in the cytoplasm and do not require calcium ions for their enzymatic activity , although the precise mechanisms for activation are unclear . among these pla2s , a significant role for ipla2 in the production of prostaglandins in carrageenan - induced pleuritis in rats was demonstrated .
it is also reported that , in the spinal cord , cpla2 seems to be involved in inflammation - induced hyperalgesia . in the present study , we examined if ipla2 and/or cpla2 are responsible for the production of prostaglandins and development of hyperalgesia in carrageenan - induced inflammation in the rat hind paw .
male sprague - dawley rats ( nine weeks old , 300320  g ) were purchased from charles river laboratories ( yokohama , japan ) .
all chemicals were obtained from commercial suppliers : bromoenol lactone ( bel ; ipla2 inhibitor ) , arachidonyl trifluoromethyl ketone ( aacocf3 ; cpla2 inhibitor ) , pge2 enzyme immunoassay ( eia ) kit , and 6-keto - pgf1 eia kit from cayman chemical ( ann arbor , mi ) ; isogen ( rna extraction solution ) from nippon gene ( tokyo , japan ) ; reverse transcription kit from invitrogen ( carlsbad , ca ) ; pcr sybr green master mix , lightcycler taqman master , and taqman probes from roche diagnostics ( indianapolis , in ) ; and rnalater ( rna stabilization solution ) from ambion ( austin , tx ) .
all experiments were carried out according to protocols approved by the institutional animal care committee of kyoto prefectural university of medicine .
rats were housed four per cage and maintained on a 12  h light / dark cycle ( light on 8:0020:00 ) with controlled temperature ( 25  3c ) and humidity ( 55  15% ) .
the plantar surface of the left paw received a subcutaneous injection of either 3  mg type  carrageenan ( sigma - aldrich , st .
just after carrageenan injection , one of the two pla2 inhibitors ( 30  nmol ) or vehicle ( 100  l of 0.1% dimethyl sulfoxide ( dmso ) in saline ) was injected into the same site .
for biochemical analyses , each rat was anesthetized with pentobarbital ( 60  mg / kg , intraperitoneal injection ) 3  h after subcutaneous injection , and the left hind leg was cut at the knee , quickly frozen in dry ice powder , and kept at 80c until further processing .
this time point was determined based on the following nociceptive behavior study , in which a pla2 inhibitor was effective between 1  h and 3  h after injection . for the assessment of nociceptive behavior response
, rats were injected with the carrageenan / saline and pla2 inhibitors / vehicle in the manner described above .
thermally evoked paw - withdrawal response was assessed using a device developed in yaksh 's lab .
the paw - withdrawal latency was measured for the left hind paw before and every 60 minutes after the subcutaneous injection for a total of 360 minutes . for each time point , the latencies were measured 3 times in each rat and averaged .
paw - withdrawal latencies were expressed as ratios to the baseline value of each rat . for analyses of pge2 and 6-keto - pgf1 ( a metabolite of pgi2 )
, the hind paws were coronally cut into 50  m thick frozen sections in a cryostat at 20c .
twenty of these sections were collected in a plastic tube containing precooled ethanol ( 1  ml ) and indomethacin ( 10  g ) which prevents the synthesis of prostaglandins during tissue processing . after measuring wet tissue weight ,
the sections were homogenized with polytron for 30  s followed by sonication for 20  s. the homogenates were centrifuged ( 15000  rpm for 20  min at 4c ) and the supernatant was collected .
pge2 and 6-keto - pgf1 were measured using eia kits according to the manufacturer 's instructions .
tissue pellet remaining in the plastic tube was heated in a heat block to completely evaporate the ethanol .
the weight of dried pellet was considered to be the dry tissue weight of the paw from which the prostaglandins were extracted .
twenty of these sections were placed into a vial containing rna later ( 1  ml ) and stored at 30c until further processing . for rna extraction
, the samples were homogenized in 1  ml phenol - based rna extraction solution ( isogen ) with polytron for 30  s followed by sonication for 20  s. total rna was isolated according to the manufacturer 's instructions .
cdna was prepared from total rna using m - mlv reverse transcriptase and random hexamer as the primer .
the reverse - transcribed cdna was amplified using a light cycler ( roche diagnostics ) .
mrnas of cox-2 , mpges-1 , ipla2 , ipla2 , and gapdh were quantified with the sybr green protocol . for the quantification of mrnas of prostaglandin
i synthase ( pgis ) , interleukin-1 ( il1 ) , and interleukin-6 ( il6 ) , the taqman probe protocol was used .
primer pairs used for the pcr reaction were as follows :  cox-2 : 5-ctcactttgagtcattc-3 , 5-gattagtactgtagggttaatg-3 ,  mpges-1 : 5-aatgaacccacgcattcgct-3 , 5-cagccttcatggctccgtct-3 ,  ipla2 : 5-caaggaactgggcaagatgg-3 , 5-agagggcgttgaccagcact-3 ,  ipla2 : 5-gaataccacaacatacacga-3 , 5-acctaaaatacgtgtcagca-3 ,  gapdh : 5-tgaacgggaagctcactgg-3 , 5-tccaccaccctgttgctgta-3 ,  pgis : 5-atgccatcaacagcatcaaa-3 , 5-gctccaggtcgaaatgagtc-3 , taqman probe ( upl # 18 ) ,  il1 : 5-tgtgatgaaagacggcacac-3 , 5-cttcttctttgggtattgtttgg-3 , taqman probe ( upl # 78 ) ,  il6 : 5-cccttcaggaacagctatgaa-3 , 5-acaacatcagtcccaagaagg-3 , taqman probe ( upl # 20 ) ,  gapdh : 5-agctggtcatcaatgggaaa-3 , 5-atttgatgttagcgggatcg-3 , taqman probe ( upl # 9 ) .
cox-2 : 5-ctcactttgagtcattc-3 , 5-gattagtactgtagggttaatg-3 , mpges-1 : 5-aatgaacccacgcattcgct-3 , 5-cagccttcatggctccgtct-3 , ipla2 : 5-caaggaactgggcaagatgg-3 , 5-agagggcgttgaccagcact-3 , ipla2 : 5-gaataccacaacatacacga-3 , 5-acctaaaatacgtgtcagca-3 , gapdh : 5-tgaacgggaagctcactgg-3 , 5-tccaccaccctgttgctgta-3 , pgis : 5-atgccatcaacagcatcaaa-3 , 5-gctccaggtcgaaatgagtc-3 , taqman probe ( upl # 18 ) , il1 : 5-tgtgatgaaagacggcacac-3 , 5-cttcttctttgggtattgtttgg-3 , taqman probe ( upl # 78 ) , il6 : 5-cccttcaggaacagctatgaa-3 , 5-acaacatcagtcccaagaagg-3 , taqman probe ( upl # 20 ) , gapdh : 5-agctggtcatcaatgggaaa-3 , 5-atttgatgttagcgggatcg-3 , taqman probe ( upl # 9 ) .
relative expression levels of each gene were calculated with the following formula : ( 1)relative  expression  of  mrna=2cycle  number  of  gapdh2cycle  number  of  gene  of  interest .
specificity of the pcr products was checked by the melting curve and by agarose gel electrophoresis in the sybr green protocol .
the significance of differences among multiple groups was determined by multiple t - tests with bonferroni correction .
we examined the effects of pla2 inhibitors on pge2 and 6-keto - pgf1 ( a metabolite of pgi2 ) levels in inflamed foot pad .
carrageenan and pla2 inhibitors / vehicle were injected into the right foot pad at the same time .
three hours after the injection , carrageenan significantly elevated pge2 and 6-keto - pgf1 levels compared to injection of saline alone in vehicle- , bel- and aacocf3-coinjected groups ( n = 4 in each group , p = 0.00020.014 ) ( figure 1 ) .
bel , an ipla2 inhibitor , significantly suppressed carrageenan - induced increases in pge2 by 57% ( p = 0.009 ) and 6-keto - pgf1 by 49% ( p = 0.017 ) compared to vehicle .
on the other hand , aacocf3 , a cpla2 inhibitor and less potent ipla2 inhibitor , did not suppress the prostaglandin levels compared to the vehicle - treated rats .
the two inhibitors did not exert significant effects on the prostaglandin levels in the saline - injected foot pad .
real - time rt - pcr studies revealed that mrnas of both ipla2 isozymes , that is , ipla2 and ipla2 , were present in the noninflamed foot pad , and their levels did not change following carrageenan injection ( figure 2 ) .
we then asked if bel influences the induction of cox-2 , mpges-1 , and pgis , which are possibly involved in carrageenan - induced prostaglandin synthesis .
cox-2 mrna , mpges-1 mrna , and pgis mrna were increased by carrageenan injection though the increases did not reach the statistically significant level except for cox-2 mrna and pgis mrna in carrageenan + bel group ( figure 3 ) .
there was no significant effect of bel on the cox-2 mrna , mpges-1 mrna , or pgis mrna levels ( figure 3 ) .
we also examined the effects of bel on carrageenan - induced proinflammatory cytokine mrnas ( figure 4 ) .
bel slightly but significantly elevated carrageenan - induced il1 mrna and did not change carrageenan - induced il6 mrna .
therefore , bel seemed to act solely on the enzyme activity of ipla2s resulting in suppression of prostaglandin levels in inflamed tissue .
lastly , we studied the effects of pla2 inhibitors on carrageenan - induced thermal hyperalgesia . when carrageenan was injected with vehicle , foot withdrawal latency to radiant heat
was reduced by approximately 70% compared to the preinjection level ( at time point 0 ) , indicating the induction of thermal hyperalgesia ( figure 5(a ) ) .
the thermal hyperalgesia was statistically significant throughout the observation period of 6  h ( n = 5 , p < 0.005 ) .
carrageenan - induced thermal hyperalgesia was ameliorated by bel by 44% at 1  h , 61% at 2  h , and 46% at 3  h after injection compared to vehicle - treated group . at these 3 time points , the difference between carrageenan + bel group ( n = 6 ) and carrageenan + vehicle group (
n = 5 ) was statistically significant ( p = 0.026 , 0.014 , and 0.015 , resp . ) .
on the other hand , aacocf3 did not influence the thermal hyperalgesia ( figure 5(a ) ) .
both bel and aacocf3 did not show significant effect in saline - injected foot pad .
pge2 and pgi2 are known to sensitize nociceptors and develop hyperalgesia during inflammation [ 1 , 2 ] . however , which isoform of pla2 is involved in the synthesis of these prostaglandins in inflammatory tissues was unclear . in the present study , we showed that bel , a potent inhibitor of ipla2 , lowered these prostaglandins by approximately 50% in rat foot pads that were inflamed with carrageenan .
the inhibitor also halved carrageenan - induced thermal hyperalgesia during the first 3  h after injection .
we confirmed that mrnas of ipla2 and ipla2 were constitutively expressed in the rat foot pad .
this result is in line with the study by gilroy et al . , who demonstrated that bel reduced pge2 content in the cell - free inflammatory exudates of the pleural cavity 3  h after carrageenan injection .
it should be noted that bel is also a potent inhibitor of phosphatidic acid phosphohydrolase .
reported that bel suppressed cox-2 expression in lps - activated macrophages through the inhibition of phosphatidic acid phosphohydrolase-1 .
. however , in the present study , carrageenan - induced cox-2 mrna was not affected by bel .
this was also the case for mrnas of mpges-1 and pgis , the final enzymes in pge2 and pgi2 synthesis , respectively .
in line with these results , bel did not reduce carrageenan - induced proinflammatory cytokines , which are strong inducers of cox-2 and other enzymes in the arachidonic acid cascade .
these results strongly suggest that bel did not significantly suppress inflammation but inhibited ipla2 enzyme activity to reduce carrageenan - induced pge2 and pgi2 synthesis and , consequently , thermal hyperalgesia .
therefore , we conclude that ipla2 is responsible for the synthesis of prostaglandins in carrageenan - induced tissue inflammation .
nonsteroidal anti - inflammatory drugs ( nsaids ) are widely used for the treatment of inflammatory pain .
nsaids potently inhibit cox but do not suppress the supply of its substrate , that is , arachidonic acid .
thus , free arachidonic acid may enter lipoxygenase or epoxygenase pathways to be various potent eicosanoids such as leukotrienes .
since inhibitors of pla2 , such as bel , reduce the supply of free arachidonic acid , pla2 inhibitors may be more ideal in the treatment of symptoms induced by eicosanoids than nsaids .
this fact suggests that other pla2 isoforms , that is , cpla2 or spla2s , also participated in carrageenan - induced prostaglandin synthesis . traditionally , cpla2 has been considered the major pla2 that cleaves arachidonic acid from the sn-2 position of membrane phospholipids during inflammation . in the present study injection of aacocf3 , a cpla2 inhibitor , into the foot pad neither reduced pge2 and pgi2 nor suppressed thermal hyperalgesia , suggesting that cpla2 does not play a significant role in these responses
this result is consistent with those of two previous studies , the first of which by gilroy et al
.   showed no effect of aacocf3 on the pge2 content of cell - free inflammatory exudates of the pleural cavity 3  h after carrageenan injection .
the second study by lucas et al .   reported that intraplantar injection of aacocf3 did not suppress carrageenan - induced thermal hyperalgesia even with a dose approximately 20 times higher than that used in the present study .
on the other hand , it is reported that a new type of cpla2 inhibitor reduced carrageenan - induced pge2 production in the rat air pouch .
the reason for the discrepancy is unclear at present , and we can not completely exclude the possibility that cpla2 is partly involved in pge2 and pgi2 production in inflammatory tissues .
pge2 and possibly pgi2 augment nociceptive responses by acting in multiple sites along the pain processing neural pathway .
in each site , distinct isoforms of pla2 seem to be involved in prostaglandin synthesis . in the peripheral inflammatory site
, these prostaglandins are produced by the action of ipla2 and sensitize nociceptors , as shown in this study . in the spinal cord , pge2 is produced in response to nociceptive neural inputs from the primary sensory neurons and possibly enhances the pain signal transmission .
in this site , cpla2 and spla2 but not ipla2 are reported to play major roles in pge2 synthesis and hyperalgesic response [ 11 , 16 ] .
furthermore , in the spinal cord and brain , peripheral inflammation induces prostaglandin synthesis through the action of circulating inflammatory cytokines such as interleukin-6 .
this is evidenced by an elevation of pge2 in the cerebrospinal fluid and induction of cox-2 in endothelial cells throughout the brain and spinal cord after intraplantar injection of carrageenan .
. however , little is known about which isoform of pla2 is activated upstream of cox-2 .
further studies are necessary to thoroughly determine which pla2 isoforms are involved in prostaglandin synthesis in the central nervous system under inflammatory conditions .
the present study has indicated the importance of ipla2 in prostaglandin synthesis at the inflammatory site and suggested that ipla2 is a possible target for the treatment of inflammatory hyperalgesia .
 prostaglandin ( pg ) e2 and pgi2 are essential to hyperalgesia in inflammatory tissues . 
 these prostaglandins are produced from arachidonic acid , which is cleaved from membrane phospholipids by the action of phospholipase a2 ( pla2 ) . 
 which isozyme of pla2 is responsible for the cleavage of arachidonic acid and the production of prostaglandins essential to inflammation - induced hyperalgesia is not clear . in this study , we examined the effects of two pla2 isozyme - specific inhibitors on carrageenan - induced production of pge2 and pgi2 in rat hind paw and behavioral nociceptive response to radiant heat . 
 local administration of bromoenol lactone ( bel ) , an inhibitor of calcium - independent pla2 ( ipla2 ) , significantly reduced carrageenan - induced elevation of prostaglandins in the inflamed foot pad 3  h after injection . 
 it also ameliorated the hyperalgesic response between 1  h and 3  h after carrageenan injection . on the other hand , aacocf3 , an inhibitor of cytosolic pla2 , suppressed neither prostaglandin production nor the hyperalgesic response . 
 bel did not suppress the mrna levels of ipla2 , ipla2 , cyclooxygenase-2 , microsomal prostaglandin e synthase , prostaglandin i synthase , or proinflammatory cytokines in the inflamed foot pad , indicating that bel did not suppress inflammation itself . 
 these results suggest that ipla2 is involved in the production of prostaglandins and hyperalgesia at the inflammatory loci . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion