Palmitoylethanolamide treatment reduces blood pressure in spontaneously hypertensive rats: involvement of cytochrome p450-derived eicosanoids and renin angiotensin system

Abstract

Palmitoylethanolamide (PEA), a peroxisome proliferator-activated receptor-α agonist, has been demonstrated to reduce blood pressure and kidney damage secondary to hypertension in spontaneously hypertensive rat (SHR). Currently, no information is available concerning the putative effect of PEA on modulating vascular tone. Here, we investigate the mechanisms underpinning PEA blood pressure lowering effect, exploring the contribution of epoxyeicosatrienoic acids, CYP-dependent arachidonic acid metabolites, as endothelium-derived hyperpolarizing factors (EDHF), and renin angiotensin system (RAS) modulation. To achieve this aim SHR and Wistar-Kyoto rats were treated with PEA (30 mg/kg/day) for five weeks. Functional evaluations on mesenteric bed were performed to analyze EDHF-mediated vasodilation. Moreover, mesenteric bed and carotid were harvested to measure CYP2C23 and CYP2J2, the key isoenzymes in the formation of epoxyeicosatrienoic acids, and the soluble epoxide hydrolase, which is responsible for their degradation in the corresponding diols. Effect of PEA on RAS modulation was investigated by analyzing angiotensin converting enzyme and angiotensin receptor 1 expression. Here, we showed that EDHF-mediated dilation in response to acetylcholine was increased in mesenteric beds of PEA-treated SHR. Western blot analysis revealed that the increase in CYP2C23 and CYP2J2 observed in SHR was significantly attenuated in mesenteric beds of PEA-treated SHR, but unchanged in the carotids. Interestingly, in both vascular tissues, PEA significantly decreased the soluble epoxide hydrolase protein level, accompanied by a reduced serum concentration of its metabolite 14-15 dihydroxyeicosatrienoic acid, implying a reduction in epoxyeicosatrienoic acid hydrolisis. Moreover, PEA treatment down-regulated angiotensin receptor 1 and angiotensin converting enzyme expression, indicating a reduction in angiotensin II-mediated effects. Consistently, a damping of the activation of angiotensin receptor 1 underlying pathways in mesenteric beds was shown in basal conditions in PEA-treated SHR. In conclusion, our data demonstrate the involvement of epoxyeicosatrienoic acids and renin angiotensin system in the blood pressure lowering effect of PEA.

Fig 1. Effect of PEA on EDHF-mediated relaxation on mesenteric arterial bed on stable tone of MTX.

In order to visualize the contribution of EDHF in mesenteric bed, a curve concentration-response to Ach (1–1000 pmoles) in Krebs solution medicated with INDO (10 μM) and L-NAME (100 μM) was performed on MTX stable tone. (A) The EDHF-mediated relaxation resulted significantly reduced in SHR compared with WKY (*P<0.05). The treatment with PEA significantly increased EDHF-mediated relaxation in SHR compared with SHR group (###P<0.001). (B) The increase in perfusion pressure (mmHg) achieved by the adding of INDO (10 μM) plus L-NAME (100 μM) was significantly higher in PEA-treated SHR compared to SHR (##P<0.01). Data are expressed as means ± SEM for 4–5 different animals.

Fig 2. Effect of PEA on CYP2C23 and CYP2J2 protein expression in mesenteric bed and carotid.

Representative Western blots show bands in mesenteric bed (A and B) and carotid (C and D) of SHR and WKY rats. Densitometric evaluations of protein levels were obtained from 5 different animals. Data are expressed as means ± SEM. *P<0.05 and ***P<0.001 vs WKY; #P<0.05 and ###P<0.001 vs SHR.

Fig 3. sEH protein expression in mesenteric bed and carotid and its modulation by PEA treatment.

Immunoblot of sEH and densitometric analysis of protein band from mesenteric bed (A) and carotid (B) of SHR and WKY rats are shown. Serum 14-15DHET (ng/ml) is reported in panel C. Data are expressed as means ± SEM. *P<0.05 and ***P<0.001 vs WKY; #P<0.05, ##P<0.01, and and ###P<0.001 vs SHR.

Fig 4. Effect of PEA on AT1 and ACE protein expression in mesenteric bed and carotid.

Representative Western blots show bands in mesenteric bed (A and B) and carotid (C and D) of SHR and WKY rats. Densitometric evaluations of protein levels were obtained from 5 different animals. Data are expressed as means ± SEM. *P<0.05, **P<0.01 and ***P<0.001 vs WKY; ##P<0.01 and ###P<0.001 vs SHR.

Fig 5. Activation of transcription factors in mesenteric bed and its modulation by PEA treatment.

IκBα (A), pSTAT3/STAT (B), and pERK1/2 (C) immunoblots are shown. Densitometric evaluations of protein levels were obtained from 5 different animals. Data are expressed as means ± SEM. *P<0.05 and **P<0.01 vs WKY; #P<0.05, ##P<0.01 and ###P<0.001 vs SHR.