Bromoenol Lactone, an Inhibitor of Calcium-Independent Phospholipase A2, Suppresses Carrageenan-Induced Prostaglandin Production and Hyperalgesia in Rat Hind Paw

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 β, iPLA2 γ, 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.


Introduction
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 E 2 (PGE 2 ) and PGI 2 are responsible for the sensitization of nociceptors [1,2]. Recent studies further clarified the molecular mechanism of sensitization: PGE 2 and PGI 2 enhance or sensitize the activity of the capsaicin receptor transient potential receptor V1 (TRPV1) through the activation of PGE 2 EP1 and PGI 2 receptors, respectively [3]. Protein kinase C and protein kinase A are involved in the activation of TRPV1 by these prostaglandins. In contrast, cyclopentenone prostaglandins, including 15-d-PGJ 2 , PGA 2 , and PGA 1 , which are metabolites of PGD 2 , PGE 2 , and PGE 1 , respectively, directly activate the irritant receptor TRPA1 [4]. 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 [5], which involves three enzymatic steps: first phospholipase A 2 (PLA 2 ) cleaves membrane phospholipids and releases arachidonic acid; second, cyclooxygenase (COX) converts arachidonic acid into PGH 2 ; and, third, various types of prostaglandin isomerases convert PGH 2 to bioactive prostaglandins, including PGE 2 and PGI 2 . Each enzymatic reaction can involve multiple isozymes. Recent studies have shown that COX-2, an inducible isozyme of 2 Mediators of Inflammation COX, and microsomal prostaglandin synthase-1 (mPGES-1), an inducible isozyme of PGE synthase, are responsible for the generation of PGE 2 in inflamed tissues [6,7]. In these studies, administration of a COX-2 inhibitor to rats [7] or disruption of the mPGES-1 gene in mice lowered PGE 2 content in inflamed tissues and eased pain-related behaviors [6]. Thus, COX-2 and mPGES-1 could be pharmacological targets for the treatment of inflammatory pain.
However, it is not clear which isozyme of PLA 2 is responsible for the production of prostaglandins and development of hyperalgesia in inflamed tissues. PLA 2 has over ten isozymes which are classified into three categories based on their structural and functional similarities [8,9]. First, cytosolic PLA 2 s (cPLA 2 or group IV PLA 2 ) are located in the cytoplasm and are activated by low calcium ion concentrations ( M levels). Second, secretory PLA 2 s (sPLA 2 or group IB, II, V, or X PLA 2 ) are released into the extracellular space where they are activated by high concentrations (mM levels) of calcium ions. Third, calcium-independent PLA 2 s (iPLA 2 or group VI PLA 2 ) 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 PLA 2 s, a significant role for iPLA 2 in the production of prostaglandins in carrageenan-induced pleuritis in rats was demonstrated [10]. It is also reported that, in the spinal cord, cPLA 2 seems to be involved in inflammation-induced hyperalgesia [11]. In the present study, we examined if iPLA 2 and/or cPLA 2 are responsible for the production of prostaglandins and development of hyperalgesia in carrageenan-induced inflammation in the rat hind paw.

Animals.
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 8:00-20:00) with controlled temperature (25 ± 3 ∘ C) and humidity (55 ± 15%). Animals were allowed free access to food and water at all times.

Pharmacological Treatment.
The plantar surface of the left paw received a subcutaneous injection of either 3 mg type carrageenan (Sigma-Aldrich, St. Louis, MO) dissolved in 100 L saline or saline alone. Just after carrageenan injection, one of the two PLA 2 inhibitors (30 nmol) or vehicle (100 L of 0.1% dimethyl sulfoxide (DMSO) in saline) was injected into the same site. The inhibitor dose was determined according to the study by Gilroy et al. [10]. 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 −80 ∘ C until further processing. Rats were killed by decapitation. This time point was determined based on the following nociceptive behavior study, in which a PLA 2 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 PLA 2 inhibitors/vehicle in the manner described above. Thermally evoked paw-withdrawal response was assessed using a device developed in Yaksh's lab [12]. 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.

Biochemical Assay.
For analyses of PGE 2 and 6-keto-PGF 1 (a metabolite of PGI 2 ), the hind paws were coronally cut into 50 m thick frozen sections in a cryostat at −20 ∘ C. 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 4 ∘ C) and the supernatant was collected. Ethanol was evaporated from the supernatant using a vacuum centrifuge. Prostaglandins, which were retained in the tube, were dissolved in EIA buffer. PGE 2 and 6-keto-PGF 1 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.

Real-Time RT-PCR.
Frozen paw sections were prepared as described above. Twenty of these sections were placed into a vial containing RNA later (1 mL) and stored at −30 ∘ C 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, iPLA 2 , iPLA 2 , 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 , (1) Specificity of the PCR products was checked by the melting curve and by agarose gel electrophoresis in the Sybr Green protocol.
2.6. Statistics. The significance of differences between two groups was determined by t-test. The significance of differences among multiple groups was determined by multiple ttests with Bonferroni correction. Differences were considered significant at < 0.05. Data are presented as mean ± SEM.

Results
We examined the effects of PLA 2 inhibitors on PGE 2 and 6-keto-PGF 1 (a metabolite of PGI 2 ) levels in inflamed foot pad. Carrageenan and PLA 2 inhibitors/vehicle were injected into the right foot pad at the same time. Three hours after the injection, carrageenan significantly elevated PGE 2 and 6-keto-PGF 1 levels compared to injection of saline alone in vehicle-, BEL-and AACOCF 3 -coinjected groups ( = 4 in each group, = 0.0002-0.014) (Figure 1). BEL, an iPLA 2 inhibitor, significantly suppressed carrageenaninduced increases in PGE 2 by 57% ( = 0.009) and 6-keto-PGF 1 by 49% ( = 0.017) compared to vehicle. On the other hand, AACOCF 3 , a cPLA 2 inhibitor and less potent iPLA 2 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 salineinjected foot pad.
Real-time RT-PCR studies revealed that mRNAs of both iPLA 2 isozymes, that is, iPLA 2 and iPLA 2 , 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). Carrageenan significantly elevated the mRNA levels of IL1 and IL6. 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 iPLA 2 s resulting in suppression of prostaglandin levels in inflamed tissue.
Lastly, we studied the effects of PLA 2 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 ( = 5, < 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 ( = 6) and carrageenan + vehicle group ( = 5) was statistically significant ( = 0.026, 0.014, and 0.015, resp.). On the other hand, AACOCF 3 did not influence the thermal hyperalgesia ( Figure 5(a)). Injection of saline did not induce thermal hyperalgesia ( Figure 5(b)). Both BEL and AACOCF 3 did not show significant effect in saline-injected foot pad.

Discussion
PGE 2 and PGI 2 are known to sensitize nociceptors and develop hyperalgesia during inflammation [1,2]. However, which isoform of PLA 2 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 iPLA 2 , 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 iPLA 2 and iPLA 2 were constitutively expressed in the rat foot pad. This result is in line with the study by Gilroy et al. [10], who demonstrated that BEL reduced PGE 2 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. Grkovich et al.  Figure 2: Levels of mRNA of iPLA 2 and iPLA 2 relative to that of an internal control gene (GAPDH) in rat hind paw. Their relative levels were not influenced by carrageenan-induced inflammation. Open bars and filled bars represent results from saline (Sal)-injected group and carrageenan (Car)-injected group, respectively. = 4 in each group. [13] reported that BEL suppressed COX-2 expression in LPS-activated macrophages through the inhibition of phosphatidic acid phosphohydrolase-1. Therefore, we wondered if BEL might have reduced prostaglandin levels by suppressing COX-2 induction. 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 PGE 2 and PGI 2 synthesis, respectively.
In line with these results, BEL did not reduce carrageenaninduced 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 iPLA 2 enzyme activity to reduce carrageenan-induced PGE 2 and PGI 2 synthesis and, consequently, thermal hyperalgesia. Therefore, we conclude that iPLA 2 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. This may result in side effects of NSAIDs. On the other hand, since inhibitors of PLA 2 , such as BEL, reduce the supply of free arachidonic acid, PLA 2 inhibitors may be more ideal in the treatment of symptoms induced by eicosanoids than NSAIDs.
The inhibition of prostaglandin synthesis by BEL was partial-approximately 50% of the control (vehicle alone). This fact suggests that other PLA 2 isoforms, that is, cPLA 2 or sPLA 2 s, also participated in carrageenan-induced prostaglandin synthesis. Traditionally, cPLA 2 has been considered the major PLA 2 that cleaves arachidonic acid from the sn-2 position of membrane phospholipids during inflammation. In the present study injection of AACOCF 3 , a cPLA 2 inhibitor, into the foot pad neither reduced PGE 2 and PGI 2 nor suppressed thermal hyperalgesia, suggesting that cPLA 2 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. [10] showed no effect of AACOCF 3  on the PGE 2 content of cell-free inflammatory exudates of the pleural cavity 3 h after carrageenan injection. The second study by Lucas et al. [11] reported that intraplantar injection of AACOCF 3 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 cPLA 2 inhibitor reduced carrageenan-induced PGE 2 production in the rat air pouch [14]. The reason for the discrepancy is unclear at present, and we cannot completely exclude the possibility that cPLA 2 is partly involved in PGE 2 and PGI 2 production in inflammatory tissues. PGE 2 and possibly PGI 2 augment nociceptive responses by acting in multiple sites along the pain processing neural pathway. In each site, distinct isoforms of PLA 2 seem to be involved in prostaglandin synthesis. In the peripheral inflammatory site, these prostaglandins are produced by the action of iPLA 2 and sensitize nociceptors, as shown in this study. In the spinal cord, PGE 2 is produced in response to nociceptive neural inputs from the primary sensory neurons and possibly enhances the pain signal transmission [15]. In this site, cPLA 2 and sPLA 2 but not iPLA 2 are reported to play major roles in PGE 2 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 [17]. This is evidenced by an elevation of PGE 2 in the cerebrospinal fluid and induction of COX-2 in endothelial cells throughout the brain and spinal cord after intraplantar injection of carrageenan [18]. Antiserum to interleukin-6 suppressed these responses [17]. However, little is known about which isoform of PLA 2 is activated upstream of COX-2. Further studies are necessary to thoroughly determine which PLA 2 isoforms are involved in prostaglandin synthesis in the central nervous system under inflammatory conditions.

Conclusion
The present study has indicated the importance of iPLA 2 in prostaglandin synthesis at the inflammatory site and suggested that iPLA 2 is a possible target for the treatment of inflammatory hyperalgesia.