Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid produced by cytochrome P450 epoxygenases responsible for important signaling that mediates a series of events, such as inflammation [1], pain [2], cancer [3], angiogenesis [4], and cardiovascular regulation [5].

Like EETs, nitric oxide (NO) has an intrinsic mechanism for regulating vascular tone [6]. Studies have shown that EETs can be considered an endothelium-derived hyperpolarizing factor (EDHF), as they cause hyperpolarization and subsequent relaxation of vascular smooth muscle cells by opening high-conductance calcium-activated potassium channels [7–10], whereas NO is an endothelium-derived relaxing factor (EDRF) that induces vasodilation through its pathway [11].

EETs and NO also have controversial roles in the nociceptive pathway and can be pro-nociceptive [2,12] or antinociceptive [13,14], depending on their concentration and route of administration. However, NO can also act as a mediator in the antinociceptive signaling of other antinociceptive molecules [15–18].

The description of the nitric oxide pathway in pain relief mechanisms was first outlined in the 1970s, with early findings showing that blocking mediators of this pathway prevents the pain-relieving effects of specific molecules, such as acetylcholine [19]. Later, Duarte et al. (1990) [13] demonstrated that nitroprusside, a non-enzymatic producer of NO, produced antinociception in a paw compression test in rats. They also found that L-arginine, a precursor of NO synthesis, induced antinociception, and that L-NMMA, an inhibitor of NO biosynthesis, blocked it. Ultimately, the researchers demonstrated that the pain-relieving effects of sodium nitroprusside and ACh were enhanced by the intraplantar injection of a cyclic GMP phosphodiesterase inhibitor (MY 5445). Therefore, it was only in the early 1990s that the link between this mediator and the NO pathway was established, providing evidence of the role of the NO pathway in peripheral antinociception.

Our group has described the influence of nitrergic pathway modulation as part of the endogenous control of inflammatory pain in the periphery [15] and as a key peripheral antinociceptive mechanism of drugs such as peptides from animal toxins [20], hydrogen peroxide [18], tramadol [16], and even cannabidiol [17].

Recently, our group described the antinociceptive potential of the four regioisomers of EETs in an acute pain model induced by PGE2. Now, in this study, our objective was to determine whether the nitrergic pathway mediates the analgesic effects of EETs.

Animals

Male Swiss mice weighing between 30 and 40 g, with free access to food and water, were kept on a 12-hour light/dark cycle (6:00-18:00 h) at a controlled temperature of 24 ± 2°C at the Bioterism Center of the Federal University of Minas Gerais (CEBIO-ICB/UFMG). After the tests, the animals were euthanized via high-dose intraperitoneal injection of an anesthetic mixture (300 mg/kg ketamine hydrochloride and 15 mg/kg xylazine hydrochloride, both from Sigma-Aldrich, USA). The Ethics Committee on Animal Experimentation approved the project under protocol number 75/2017.

Drugs

Prostaglandin E2 (PGE2; Sigma-Aldrich, USA) was diluted in 2% ethanol within an aqueous solution of sterile 0.9% sodium chloride (saline). 5,6-epoxieicosatrienoic acid (5,6-EET) (Cayman Chemical, USA) was dissolved in methyl acetate mixed with saline. 8,9-epoxieicosatrienoic acid (8,9-EET) (Cayman Chemical, USA) and 11,12-epoxieicosatrienoic acid (11,12-EET) (Cayman Chemical, USA) were dissolved in 6.4% ethanol in saline. 14,15-epoxieicosatrienoic acid (14,15-EET) (Cayman Chemical, USA) was dissolved in 25.6% ethanol in saline. Nonselective NO synthase (NOS) inhibitor L-NOarg [NG-nitro-L-arginine (RBI, USA)]; selective neuronal NO synthase inhibitor L-NPA [NW-propyl-L-arginine (Sigma-Aldrich)]; selective endothelial NO synthase inhibitor L-NIO [N5-(1-iminoethyl)-L-ornithine dihydrochloride (Sigma-Aldrich)]; and selective inducible NO synthase inhibitor L-NIL [L-N6-(1-iminoethyl) lysine hydrochloride (Sigma-Aldrich)] were dissolved in isotonic saline. Specific soluble guanylyl cyclase and phosphodiesterase 5 and 6 inhibitors ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; RBI) and Zaprinast (Cayman Chemical, USA) were dissolved in 10% DMSO in saline. All drugs were injected via intraplantar at a volume of 20 μg/paw.

Measurement of nociceptive threshold

Hyperalgesia was induced with prostaglandin E2 (PGE2; 2 µg) through a right hind paw intraplantar injection. The mechanical nociceptive threshold was measured in grams (g) using a paw pressure test (Ugo-Basile; SRL, Varese, Italy), as described in rats [21] and mice [22]. The test uses a cone-shaped paw-presser with a rounded tip that gradually increases force on the hind paw. The force required to elicit paw withdrawal was recorded as the nociceptive threshold. A cutoff value of 150 g was set to reduce the risk of paw damage.

The nociceptive threshold was measured in the right paw and calculated as the average of three consecutive trials recorded before and 180 minutes after PGE2 injection. Hyperalgesia was determined by the difference between these two averages (Δ of nociceptive threshold).

Experimental protocol

The animals were first randomly split into several groups.

The basal threshold was measured just before the experiments started, with no drugs present. Then, PGE2 was administered at zero minutes. Nociceptive threshold measurements were taken after 180 minutes, which is when hyperalgesia peaks, to evaluate the nitrergic pathway. EETs were given 5 minutes before measurement, representing the peak of the antinociceptive effect previously shown by our group [14]. The non-selective inhibitor of nitric oxide synthase isoforms, L-NAME, along with the selective isoform inhibitors L-NIL, L-NIO, and L-NPA, were injected at 135 minutes. The specific soluble guanylyl cyclase inhibitor ODQ was administered at 165 minutes. The specific phosphodiesterase 5 and 6 inhibitor Zaprinast was given at 115 minutes. The drug dosage and timing protocols in this study were based on previous research from our group and existing literature [15–18]. The timing of drug injections was coordinated with each inhibitor's peak activity to ensure that all inhibitors reached maximum effect when the nociceptive threshold was measured (180 minutes after the PGE2 injection in the hind paw).

The experiments were conducted in a double-blind manner, where the person who injected the solutions was not the same as the one who performed the nociceptive behavioral assessment. 

Data and statistical analysis

All results are shown in the figures as the mean ± standard error of the mean (S.E.M.). Statistical analysis was performed using GraphPad Prism 8.0.2, followed by one-way ANOVA and the Bonferroni post hoc test for multiple comparisons. Only p-values less than 0.05 (p<0.05) were considered statistically significant.

Participation of nitric oxide (NO) in EETs peripheral antinociceptive effect against PGE2-induced hyperalgesia

The peripheral antinociceptive effect of 5,6-, 8,9-, 11,12-, and 14,15-EET was blocked by L-NOArg (12, 18, and 24 µg/paw), a non-selective inhibitor of nitric oxide synthase (NOS) enzymes (Figure 1).

Selective inhibitors of inducible (iNOS; LNIL, 24 μg/paw) and endothelial (eNOS; LNIO, 24 μg/paw) isoforms of NOS partially reduced the antinociceptive effect of EETs. That is, there was a decrease in antinociception caused by EETs, but it did not return to the control threshold of hyperalgesia (Figures 2 and 3, respectively). Conversely, the selective inhibitor of neuronal NOS (nNOS; LNPA, 24 μg/paw) did not affect the peripheral antinociception induced by EETs (Figure 4). All these inhibitors, when administered alone, did not alter the response to PGE2 or the vehicle (Figure 7).

Role of cyclic guanosine monophosphate (cGMP) in the peripheral antinociceptive effects of EETs

The soluble guanylate cyclase (sGC) inhibitor, ODQ (50, 100, and 200 μg/paw), reversed the peripheral antinociceptive effect of EETs in a dose-dependent manner (Figure 5).

Additionally, a selective phosphodiesterase (PDE) inhibitor, Zaprinast (50 μg/paw), increased the peripheral antinociceptive effect of low doses of EETs, producing an effect similar to that of the maximum doses of these substances (Figure 6).ODQ and zaprinast, at the doses used, showed no effect when injected separately into either normal or hyperalgesic paws (Figure 7).

In Fonseca and Duarte (2024) [14], our group described the antinociceptive effects of the four regioisomeric forms of EETs and their receptor involvement in these effects in an acute pain model induced by PGE2. We used the paw pressure test to measure the mechanical nociceptive threshold, as outlined in rats by Randall and Selitto (1957) [21] and in mice by Kawabata et al. (1992) [22]. The maximum analgesic effects were observed at doses of 128 ng for 5,6-, 8,9-, and 11,12-EETs, and at 512 ng for 14,15-EET. The doses producing sub-maximal analgesic effects were 32 ng for 5,6-, 8,9-, and 11,12-EETs, and 256 ng for 14,15-EET. Therefore, both dose ranges were used in this study to examine the role of the nitric oxide pathway in the antinociceptive mechanism of EETs.

Besides their antinociceptive effects in the periphery, the review paper by Turnbull and Chapman (2024) [23] discusses how EETs also exert antinociceptive effects through mechanisms and pain control pathways in the central nervous system. 14,15-EET has been shown to reduce pain behavior after intrathalamic administration in a preclinical model of post-stroke pain. In this model, 14,15-EET increased allopregnanolone production, thereby enhancing γ-aminobutyric acid A receptor (GABA-AR) signaling [24]. The administration of 14,15-EET, but not 5,6-EET, 8,9-EET, or 11,12-EET, into the ventrolateral PAG also proved effective in inhibiting acute nociceptive responses via opioidergic mechanisms [25]. In addition to the literature describing how EETs can induce antinociception through central mechanisms, it is also important to explore other ways these molecules can cause peripheral antinociception.

Therefore, to determine whether the NO pathway is involved in the peripheral antinociception of EETs, we used a non-selective NOS inhibitor (L-NOArg). It was observed that blocking these enzymes reduced the peripheral antinociceptive effect of EETs, indicating that NOS enzymes play a role in this process. It is known that NOS has three different isoforms: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) [26]. To identify which NOS isoform is related to EET-induced antinociception, we used selective inhibitors for each isoform. The selective inhibitors of iNOS (L-NIL) and eNOS (L-NIO) partially reduced the antinociceptive effect of EETs. In contrast, the selective nNOS inhibitor (L-NPA) did not affect the peripheral antinociception caused by EETs. Thus, it is suggested that EETs are somehow associated with iNOS and eNOS, but not nNOS, leading to NO production.

Consistent with our findings, studies show that EETs can activate eNOS, leading to NO release and, consequently, vasodilation [27,28]. Tan et al. [29] concluded in their study that the vasodilation mechanism of 5,6-EET in the rabbit pulmonary circulation is mainly EDRF-NO dependent, as the use of LNNA (Nω-nitro-L-arginine, 10−4 M), an NO synthase inhibitor, blocked the vasodilation caused by EETs. Hercule et al. [27] reported that removing the endothelium or administering L-NAME, an NO synthase inhibitor, blocked 8,9-EET-dependent dilation and altered vascular function in mice. They showed that 8,9-EET could induce NO production in primary mouse aortic endothelial cells. These effects were also blocked by L-NAME, indicating that EETs can influence the availability and/or action of NO to produce vasodilation.

In antinociceptive signaling, NO promotes sGC activation, thereby increasing the conversion of GTP to cGMP and elevating intracellular cGMP levels [30]. This second messenger is also linked to antinociceptive effects in the periphery [13,31,32]. Therefore, we examine the role of the sGC enzyme in the peripheral antinociceptive effects of EETs using its inhibitor, ODQ. Inhibiting sGC reduces the antinociceptive effect of EETs, indicating that cGMP is involved in this process.

After the GTP-cGMP conversion, cGMP is quickly broken down by phosphodiesterases [33]. Therefore, inhibiting these enzymes would prolong the action of this second messenger. We used the selective cGMP-specific phosphodiesterase E (PDE) inhibitor, Zaprinast, to investigate cGMP's role in the peripheral antinociceptive effects of EETs. It was observed that PDE inhibition increased the antinociceptive effects of low doses of EETs, indicating that cGMP levels are key in this signaling.

Zhang et al. [34] showed that in vitro incubation of human platelets with 11,12-EET increases NOS activity, nitrite production, and cGMP levels in a concentration-dependent manner, indicating that some EETs can activate the NO pathway in different cells through various mechanisms. The findings of Hung et al. [35] reveal that 11,12-EET can also strongly promote neovasculogenesis, not only by increasing NO production but also through the endothelial nitric oxide synthase (eNOS) signaling pathway.

Our data, consistent with the literature, offers insight into the functions of EETs in nociception and how these substances interact with nitric oxide production and signaling, which seem to occur during their antinociceptive effects.

These results collectively indicate that 5,6-, 8,9-, 11,12-, and 14,15-EET, when administered peripherally, could be associated with iNOS and eNOS activation, but not nNOS. This process results in the production of nitric oxide, which stimulates sGC, increases cGMP, and ultimately produces the antinociceptive effect observed with these substances.

The authors declare that they have no conflicts of interest regarding this publication.

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; 303876/2023-9) and Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG).

FCSF: Data curation, Investigation, Methodology, Writing – original draft and Writing – review & editing. WCPB: Data curation, Methodology, Writing – original draft. DPDM: Data curation, Investigation and Methodology. DLA: Data curation, Investigation and Methodology. DDA: Data curation, Investigation and Methodology. BFGQ: Data curation, Investigation and Methodology. A.C.P.: Resources. T.R.L.R.: Resources. I.D.G.D.: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Validation, Visualization and Writing – review & editing.