Palmitoylethanolamide normalizes intestinal motility in a model of post-inflammatory accelerated transit: involvement of CB1 receptors and TRPV1 channels.

BACKGROUND AND PURPOSE: Palmitoylethanolamide (PEA), a naturally occurring acylethanolamide chemically related to the endocannabinoid anandamide, interacts with targets that have been identified in peripheral nerves controlling gastrointestinal motility, such as cannabinoid CB1 and CB2 receptors, TRPV1 channels and PPARα. Here, we investigated the effect of PEA in a mouse model of functional accelerated transit which persists after the resolution of colonic inflammation (post-inflammatory irritable bowel syndrome). EXPERIMENTAL APPROACH: Intestinal inflammation was induced by intracolonic administration of oil of mustard (OM). Mice were tested for motility and biochemical and molecular biology changes 4 weeks later. PEA, oleoylethanolamide and endocannabinoid levels were measured by liquid chromatography-mass spectrometry and receptor and enzyme mRNA expression by qRT-PCR. KEY RESULTS: OM induced transient colitis and a functional post-inflammatory increase in upper gastrointestinal transit, associated with increased intestinal anandamide (but not 2-arachidonoylglycerol, PEA or oleoylethanolamide) levels and down-regulation of mRNA for TRPV1 channels. Exogenous PEA inhibited the OM-induced increase in transit and tended to increase anandamide levels. Palmitic acid had a weaker effect on transit. Inhibition of transit by PEA was blocked by rimonabant (CB1 receptor antagonist), further increased by 5'-iodoresiniferatoxin (TRPV1 antagonist) and not significantly modified by the PPARα antagonist GW6471. CONCLUSIONS AND IMPLICATIONS: Intestinal endocannabinoids and TRPV1 channel were dysregulated in a functional model of accelerated transit exhibiting aspects of post-inflammatory irritable bowel syndrome. PEA counteracted the accelerated transit, the effect being mediated by CB1 receptors (possibly via increased anandamide levels) and modulated by TRPV1 channels.

CB1 and CB2 cannabinoid receptor agonists induce peripheral antinociception by activation of the endogenous noradrenergic system.

BACKGROUND: Cannabinoid agonists induce norepinephrine release in central, spinal, and peripheral sites. Previous studies suggest an interaction between the cannabinoid and adrenergic systems on antinociception. In this study, we sought to verify whether the CB1 and CB2 cannabinoid receptor agonists anandamide and N-palmitoyl-ethanolamine (PEA), respectively, are able to induce peripheral antinociception via an adrenergic mechanism. METHODS: All drugs were administered locally into the right hindpaw of male Wistar rats. The rat paw pressure test was used, with hyperalgesia induced by intraplantar injection of prostaglandin E2 (2 µg). RESULTS: Anandamide, 12.5 ng/paw, 25 ng/paw, and 50 ng/paw elicited a local peripheral antinociceptive effect that was antagonized by CB1 cannabinoid receptor antagonist AM251, 20 µg/paw, 40 µg/paw, and 80 µg/paw, but not by CB2 cannabinoid receptor antagonist AM630, 100 µg/paw. PEA, 5 µg/paw, 10 µg/paw, and 20 µg/paw, elicited a local peripheral antinociceptive effect that was antagonized by AM630, 25 µg/paw, 50 µg/paw, and 100 µg/paw, but not by AM251, 80 µg/paw. Antinociception induced by anandamide or PEA was antagonized by the nonselective α2 adrenoceptor antagonist yohimbine, 05 µg/paw, 10 µg/paw, and 20 µg/paw, and by the selective α2C adrenoceptor antagonist rauwolscine, 10 µg/paw, 15 µg/paw, and 20 µg/paw, but not by the selective antagonists for α2A, α2B, and α2D adrenoceptor subtypes, 20 µg/paw. The antinociceptive effect of the cannabinoids was also antagonized by the nonselective α1 adrenoceptor antagonist prazosin, 0.5 µg/paw, 1 µg/paw, and 2 µg/paw, and by the nonselective ß adrenoceptor antagonist propranolol, 150 ng/paw, 300 ng/paw, and 600 ng/paw. Guanethidine, which depletes peripheral sympathomimetic amines (30 mg/kg/animal, once a day for 3 days), restored approximately 70% the anandamide-induced and PEA-induced peripheral antinociception. Furthermore, acute injection of the norepinephrine reuptake inhibitor reboxetine, 30 µg/paw, intensified the antinociceptive effects of low-dose anandamide, 12.5 ng/paw, and PEA, 5 µg/paw. CONCLUSIONS: This study provides evidence that anandamide and PEA induce peripheral antinociception activating CB1 and CB2 cannabinoid receptors, respectively, stimulating an endogenous norepinephrine release that activates peripheral adrenoceptors inducing antinociception.

Activation and desensitization of TRPV1 channels in sensory neurons by the PPARα agonist palmitoylethanolamide.

BACKGROUND AND PURPOSE: Palmitoylethanolamide (PEA) is an endogenous fatty acid amide displaying anti-inflammatory and analgesic actions. To investigate the molecular mechanism responsible for these effects, the ability of PEA and of pain-inducing stimuli such as capsaicin (CAP) or bradykinin (BK) to influence intracellular calcium concentrations ([Ca²âº](i)) in peripheral sensory neurons, has been assessed in the present study. The potential involvement of the transcription factor PPARα and of TRPV1 channels in PEA-induced effects was also studied. EXPERIMENTAL APPROACH: [Ca²âº](i) was evaluated by single-cell microfluorimetry in differentiated F11 cells. Activation of TRPV1 channels was assessed by imaging and patch-clamp techniques in CHO cells transiently-transfected with rat TRPV1 cDNA. KEY RESULTS: In F11 cells, PEA (1-30 µM) dose-dependently increased [Ca²âº](i). The TRPV1 antagonists capsazepine (1 µM) and SB-366791 (1 µM), as well as the PPARα antagonist GW-6471 (10 µM), inhibited PEA-induced [Ca²âº](i) increase; blockers of cannabinoid receptors were ineffective. PEA activated TRPV1 channels heterologously expressed in CHO cells; this effect appeared to be mediated at least in part by PPARα. When compared with CAP, PEA showed similar potency and lower efficacy, and caused stronger TRPV1 currents desensitization. Sub-effective PEA concentrations, closer to those found in vivo, counteracted CAP- and BK-induced [Ca²âº](i) transients, as well as CAP-induced TRPV1 activation. CONCLUSIONS AND IMPLICATIONS: Activation of PPARα and TRPV1 channels, rather than of cannabinoid receptors, largely mediate PEA-induced [Ca²âº](i) transients in sensory neurons. Differential TRPV1 activation and desensitization by CAP and PEA might contribute to their distinct pharmacological profile, possibly translating into potentially relevant clinical differences.

Regulation of GPR119 receptor activity with endocannabinoid-like lipids.

The GPR119 receptor plays an important role in the secretion of incretin hormones in response to nutrient consumption. We have studied the ability of an array of naturally occurring endocannabinoid-like lipids to activate GPR119 and have identified several lipid receptor agonists. The most potent receptor agonists identified were three N-acylethanolamines: oleoylethanolamine (OEA), palmitoleoylethanolamine, and linoleylethanolamine (LEA), all of which displayed similar potency in activating GPR119. Another lipid, 2-oleoylglycerol (2-OG), also activated GPR119 receptor but with significantly lower potency. Endogenous levels of endocannabinoid-like lipids were measured in intestine in fasted and refed mice. Of the lipid GPR119 agonists studied, the intestinal levels of only OEA, LEA, and 2-OG increased significantly upon refeeding. Intestinal levels of OEA and LEA in the fasted mice were low. In the fed state, OEA levels only moderately increased, whereas LEA levels rose drastically. 2-OG was the most abundant of the three GPR119 agonists in intestine, and its levels were radically elevated in fed mice. Our data suggest that, in lean mice, 2-OG and LEA may serve as physiologically relevant endogenous GPR119 agonists that mediate receptor activation upon nutrient uptake.

Effects of palmitoylethanolamide on aqueous humor outflow.

PURPOSE: To study the effects of palmitoylethanolamide (PEA), a fatty acid ethanolamide, on aqueous humor outflow facility. METHODS: The effects of PEA on outflow facility were measured using a porcine anterior segment-perfused organ culture model. The involvements of different receptors in PEA-induced changes were investigated using receptor antagonists and adenovirus delivered small hairpin RNAs (shRNAs). PEA-induced activation of p42/44 mitogen-activated protein kinase (MAPK) was determined by Western blot analysis using an antiphospho p42/44 MAPK antibody. RESULTS: PEA caused a concentration-dependent enhancement of outflow facility, with the maximum effect (151.08 ± 11.12% of basal outflow facility) achieved at 30 nM of PEA. Pretreatment of anterior segments with 1 µM cannabinoid receptor 2 antagonist SR144528 and 1 µM PPARα antagonist GW6471, but not 1 µM cannabinoid receptor 1 antagonist SR141716A, produced a partial antagonism on the PEA-induced increase of outflow facility. Treatment of TM cells with PEA for 10 minutes activated phosphorylation of p42/44 MAPK, which was blocked by pretreatment with SR1444528 and GW6471, but not SR141716A. Knocking down the expression of either GPR55 or PPARα receptors with specific shRNAs for these receptors partially blocked PEA-induced increase in outflow facility and abolished PEA-induced phosphorylation of p42/44 MAPK. PD98059, an inhibitor of the p42/44 MAPK pathway, blocked both PEA-induced enhancement of aqueous humor outflow facility and PEA-induced phosphorylation of p42/44 MAPK. CONCLUSIONS: Our results demonstrate that PEA increases aqueous humor outflow through the TM pathway and these effects are mediated by GPR55 and PPARα receptors through activation of p42/44 MAPK.

Enhancement of the hypotensive effects of intrathecally injected endocannabinoids by the entourage compound palmitoylethanolamide.

The intrathecal (i.t.) injection of 50 and 100 nmol anandamide to urethane anesthetized rats induced a dose-dependent decrease in the mean blood pressure (-10.6+/-1.6 mmHg and -15.0+/-1.7 mmHg, respectively; n=6) whereas a lower dose of this endocannabinoid (25 nmol) was devoid of effect. Similar responses were obtained both with the non-metabolizable analog methanandamide and with the endocannabinoid N-arachidonoyldopamine. When the sub-effective dose (25 nmol) of each compound was co-injected with palmitoylethanolamide (100 nmol), significant decreases in the blood pressure were observed (-12.3+1.3 mmHg for anandamide; -12.1+/-0.8 mmHg for methanandamide; -12.1+/-0.8 mmHg for N-arachidonoyldopamine; n=4-6). Palmitoylethanolamide also enhanced the hypotensive responses to the 50 nmol-dose of both anandamide and methanandamide. The hypotensive response induced by co-administration of palmitoylethanolamide and 25 nmol anandamide was prevented both by the cannabinoid CB(1) receptor antagonist SR 144716A (20 nmol; i.t.) and by the vanilloid TRPV1 receptor antagonist capsazepine (20 nmol; i.t.) and enhanced by pretreatment with URB602 (3.5 nmol; i.t.), a putative inhibitor of palmitoylethanolamide degradation. These results suggest that in the spinal cord palmitoylethanolamide acts as an entourage compound for the hypotensive effects of i.t. administered endocannabinoids. The facilitative action of palmitoylethanolamide affects the vanilloid TRPV1 as well as the cannabinoid CB(1) receptor-mediated effects of endocannabinoids on the blood pressure control.

Mono-unsaturated fatty acids protect against beta-cell apoptosis induced by saturated fatty acids, serum withdrawal or cytokine exposure.

Long-chain saturated fatty acids are cytotoxic to pancreatic beta-cells while shorter-chain saturated and long-chain unsaturated molecules are better tolerated. Mono-unsaturated fatty acids are not, however, inert since they inhibit the pro-apoptotic effects of saturated molecules. In the present work we show that the mono-unsaturates palmitoleate (C16:1) or oleate (C18:1) also cause marked inhibition of apoptosis induced by exposure of clonal BRIN-BD11 beta-cells to serum withdrawal or a combination of interleukin-1beta plus interferon-gamma. This response was dose-dependent and not accompanied by changes in NO formation. Taken together, the results suggest that mono-unsaturated fatty acids regulate a distal step common to several apoptotic pathways in pancreatic beta-cells.

Endocannabinoids protect the rat isolated heart against ischaemia.

1 The purpose of this study was to determine whether endocannabinoids can protect the heart against ischaemia and reperfusion. 2 Rat isolated hearts were exposed to low-flow ischaemia (0.5-0.6 ml min(-1)) and reperfusion. Functional recovery as well as CK and LDH overflow into the coronary effluent were monitored. Infarct size was determined at the end of the experiments. Phosphorylation levels of p38, ERK1/2, and JNK/SAPK kinases were measured by Western blots. 3 None of the untreated hearts recovered from ischaemia during the reperfusion period. Perfusion with either 300 nM palmitoylethanolamide (PEA) or 300 nM 2-arachidonoylglycerol (2-AG), but not anandamide (up to 1 micro M), 15 min before and throughout the ischaemic period, improved myocardial recovery and decreased the levels of coronary CK and LDH. PEA and 2-AG also reduced infarct size. 4 The CB(2)-receptor antagonist, SR144528, blocked completely the cardioprotective effect of both PEA and 2-AG, whereas the CB(1)-receptor antagonist, SR141716A, blocked partially the effect of 2-AG only. In contrast, both ACEA and JWH015, two selective agonists for CB(1)- and CB(2)- receptors, respectively, reduced infarct size at a concentration of 50 nM. 5 PEA enhanced the phosphorylation level of p38 MAP kinase during ischaemia. PEA perfusion doubled the baseline phosphorylation level of ERK1/2, and enhanced its increase upon reperfusion. The cardioprotective effect of PEA was completely blocked by the p38 MAP kinase inhibitor, SB203580, and significantly reduced by the ERK1/2 inhibitor, PD98059, and the PKC inhibitor, chelerythrine. 6 In conclusion, endocannabinoids exert a strong cardioprotective effect in a rat model of ischaemia-reperfusion that is mediated mainly through CB(2)-receptors, and involves p38, ERK1/2, as well as PKC activation.

Antinociceptive activity of the endogenous fatty acid amide, palmitylethanolamide.

The endogenous fatty acid ethanolamide, palmitylethanolamide, alleviated, in a dose-dependent manner, pain behaviors elicited in mice by injections of formalin (5%, intraplantar), acetic acid (0.6%, 0.5 ml per animal, intraperitoneal, i.p.), kaolin (2.5 mg per animal, i.p.), and magnesium sulfate (120 mg per kg, i.p.). The antinociceptive effects of palmitylethanolamide were prevented by the cannabinoid CB2 receptor antagonist SR144528 [N-([1s]-endo-1.3.3-trimethylbicyclo[2.3.1]heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide], not by the cannabinoid CB1 receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide x HCl]. By contrast, palmitylethanolamide had no effect on capsaicin-evoked pain behavior or thermal nociception. The endogenous cannabinoid, anandamide (arachidonylethanolamide), alleviated nociception in all tests (formalin, acetic acid, kaolin, magnesium sulfate, capsaicin and hot plate). These effects were prevented by the cannabinoid CB1 receptor antagonist SR141716A, not the cannabinoid CB2 receptor antagonist SR141716A. Additional fatty acid ethanolamides (oleylethanolamide, myristylethanolamide, palmitoleylethanolamide, palmitelaidylethanolamide) had little or no effect on formalin-evoked pain behavior, and were not investigated in other pain models. These results support the hypothesis that endogenous palmitylethanolamide participates in the intrinsic control of pain initiation. They also suggest that the putative receptor site activated by palmitylethanolamide may provide a novel target for peripherally acting analgesic drugs.

Stimulation of myocyte insulin-responsive glucose transporters by the inhibition of fatty acid oxidation.

The effects of the fatty acid inhibitor 4-bromocrotonic acid (4-BCA) on glucose utilization was studied in isolated rat myocytes. In contrast to its potent inhibition of [1-14C]palmitate oxidation, 4-BCA strongly stimulated the oxidation of [1-14C]glucose and [2-14C]-pyruvate in a concentration-dependent manner. At a concentration of 300 microM, 4-BCA increased glucose oxidation threefold and that of pyruvate oxidation twofold. The rate of transport of [U-14C]-2-deoxyglucose was significantly stimulated by 4-BCA. The transport of 2-deoxyglucose was increased sevenfold with 200 microM 4-BCA, whereas insulin (10 microU)/ml enhanced 2-deoxyglucose transport twofold. The addition of insulin to myocytes preincubated with 4-BCA did not further increase glucose transport. Cytochalasin B and anti-GLUT 4 antibody decreased the 4-BCA-induced stimulation of glucose transport. These results suggest that the stimulation of 2-deoxy-glucose transport by 4-BCA occurs through an increase in the activity of insulin-responsive glucose transporters, GLUT 4, in the sarcolemmal membrane.

Injury induced by fatty acids or bile acid in isolated human colonocytes prevented by calcium.

Measurement of the modulation of the growth fraction of isolated normal colonocytes from adult subjects in primary monolayer culture was used as a sensitive quantitative assay to evaluate toxic effects of several endogenous compounds found within the colon. This assay was used to study the role of CaCl2 in blocking cell injury. When added simultaneously with the injurious agent, 5-10 mM CaCl2 blocked the toxicity of physiological concentrations of deoxycholic acid, oleic acid, palmitic acid and linoleic acid.

Heptakis(2,6-O-dimethyl)beta-cyclodextrin: a novel growth stimulant for Bordetella pertussis phase I.

The effect of cyclodextrins on the growth of Bordetella pertussis Tohama phase I in synthetic medium was evaluated. The addition of cyclodextrins, especially heptakis(2,6-O-dimethyl)beta-cyclodextrin (Me beta CD), to a complete synthetic medium such as Stainer-Scholte medium gave the same number of individual colonies and growth rates as those on Bordet-Gengou medium. Furthermore, with the addition of Me beta CD, growth inhibition by fatty acids such as oleic or palmitic acid was overcome and normal cell growth was observed. This modified Stainer-Scholte medium, designated as cyclodextrin solid medium (CSM), supported excellent growth of 20 lyophilized clinical isolates. Serotypes of the organisms after 10 passages on this CSM plate were not changed. These results suggest that Me beta CD is a significant growth stimulant and CSM is one of the most suitable synthetic media for culture of B. pertussis phase I.

Effects of palmitoyl CoA on citrate and malate transport by rat liver mitochondria.

Palmitoyl CoA inhibited citrate transport from isolated rat liver mitochondria. Under conditions described, 50% inhibition was observed at about 6-8 nmol/mg of mitochondrial protein per ml. The percentage inhibition was inversely proportional to the concentration of the counter-transporting anion in the medium. Although comparable levels of palmitoyl CoA had little effect on malate exit on the dicarboxylate carrier, higher concentrations inhibited both citrate and malate transport. The specificity of the inhibition by palmitoyl CoA was investigated by examination of the effects on the tricarboxylate transport system of fatty acids, CoASH, acetyl CoA, palmitoylcarnitine, deoxycholate, and other molecules with surface active properties. None of the above compounds, at sublytic concentrations, inhibited citrate transport appreciably. The inhibition of citrate transport by low concentrations of palmitoyl CoA was rapid and could be prevented or partially reversed by addition of albumin.