Biosynthesis of N-Docosahexanoylethanolamine from Unesterified Docosahexaenoic Acid and Docosahexaenoyl-Lysophosphatidylcholine in Neuronal Cells.

We investigated the synthesis of N-docosahexaenoylethanolamine (synaptamide) in neuronal cells from unesterified docosahexaenoic acid (DHA) or DHA-lysophosphatidylcholine (DHA-lysoPC), the two major lipid forms that deliver DHA to the brain, in order to understand the formation of this neurotrophic and neuroprotective metabolite of DHA in the brain. Both substrates were taken up in Neuro2A cells and metabolized to N-docosahexaenoylphosphatidylethanolamine (NDoPE) and synaptamide in a time- and concentration-dependent manner, but unesterified DHA was 1.5 to 2.4 times more effective than DHA-lysoPC at equimolar concentrations. The plasmalogen NDoPE (pNDoPE) amounted more than 80% of NDoPE produced from DHA or DHA-lysoPC, with 16-carbon-pNDoPE being the most abundant species. Inhibition of N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) by hexachlorophene or bithionol significantly decreased the synaptamide production, indicating that synaptamide synthesis is mediated at least in part via NDoPE hydrolysis. NDoPE formation occurred much more rapidly than synaptamide production, indicating a precursor-product relationship. Although NDoPE is an intermediate for synaptamide biosynthesis, only about 1% of newly synthesized NDoPE was converted to synaptamide, possibly suggesting additional biological function of NDoPE, particularly for pNDoPE, which is the major form of NDoPE produced.

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.

Nebivolol-induced vasodilation of renal afferent arterioles involves ß3-adrenergic receptor and nitric oxide synthase activation.

Nebivolol is a ß(1)-adrenergic blocker that also elicits renal vasodilation and increases the glomerular filtration rate (GFR). However, its direct actions on the renal microvasculature and vasodilator mechanism have not been established. We used the in vitro blood-perfused juxtamedullary nephron technique to determine the vasodilator effects of nebivolol and to test the hypothesis that nebivolol induces vasodilation of renal afferent arterioles via an nitric oxide synthase (NOS)/nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP pathway and the afferent arteriolar vasodilation effect may be mediated through the release of NO by activation of NOS via a ß(3)-adrenoceptor-dependent mechanism. Juxtamedullary nephrons were superfused with nebivolol either alone or combined with the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or the NOS inhibitor N(ω)-nitro-l-arginine (l-NNA) or the ß-blockers metoprolol (ß(1)), butoxamine (ß(2)), and SR59230A (ß(3)). Nebivolol (100 µmol/l) markedly increased afferent and efferent arteriolar diameters by 18.9 ± 3.0 and 15.8 ± 1.8%. Pretreatment with l-NNA (1,000 µmol/l) or ODQ (10 µmol/l) decreased afferent vasodilator diameters and prevented the vasodilator effects of nebivolol (2.0 ± 0.2 and 2.4 ± 0.6%). Metoprolol did not elicit significant changes in afferent vasodilator diameters and did not prevent the effects of nebivolol to vasodilate afferent arterioles. However, treatment with SR59230A, but not butoxamine, markedly attenuated the vasodilation responses to nebivolol. Using a monoclonal antibody to ß(3)-receptors revealed predominant immunostaining on vascular and glomerular endothelial cells. These data indicate that nebivolol vasodilates both afferent and efferent arterioles and that the afferent vasodilator effect is via a mechanism that is independent of ß(1)-receptors but is predominantly mediated via a NOS/NO/sGC/cGMP-dependent mechanisms initiated by activation of endothelial ß(3)-receptors.

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.

Involvement of ß3-adrenoceptors in the inhibitory control of cholinergic activity in human bladder: Direct evidence by [(3)H]-acetylcholine release experiments in the isolated detrusor.

Bladder overactivity (OAB) is a multifactorial bladder disorder that requires therapeutics superior to the current pharmacological treatment with muscarinic antagonists. ß3-adrenoceptor (ß3-ADR) agonists represent a novel promising approach that differently addresses the parasympathetic pathway, but the clinical efficacy of these drugs has not been fully elucidated to date. Therefore, we aimed to study the pharmacological mechanisms activated by ß3-ADR agonists at muscular and neural sites in the isolated human bladder. Detrusor smooth muscle strips obtained from male patients undergoing total cystectomy were labelled with tritiated choline and stimulated with electrical field stimulation (EFS). EFS produced smooth muscle contraction and simultaneous acetylcholine ([(3)H]-ACh) release, which mostly reflects the neural origin of acetylcholine. Isoprenaline (INA), BRL37344 and mirabegron inhibited the EFS-evoked contraction and [(3)H]-ACh release in a concentration-dependent manner, yielding concentration-response curves (CRCs) that were shifted to the right by the selective ß3-ADR antagonists L-748,337 and SR59230A. Based on the agonist potency estimates (pEC50) and apparent affinities (pKb) of antagonists evaluated from the CRCs of agonists, our data confirm the occurrence of ß3-ADRs at muscle sites. Moreover, our data are consistent with the presence of inhibitory ß3-ADRs that are functionally expressed at the neural site. Taken together, these findings elucidate the mechanisms activated by ß3-ADR agonists because neural ß3-ADRs participate in the inhibition of detrusor motor drive by reducing the amount of acetylcholine involved in the cholinergic pathway.

Functional effects of ß3-adrenoceptor on pacemaker activity in interstitial cells of Cajal from the mouse colon.

We investigated the presence of ß3-adrenoceptor and its functional effects on pacemaker potentials in colonic interstitial cells of Cajal (ICCs) from mice. The whole-cell patch clamp technique was used to record pacemaker potentials in cultured ICCs and reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the mRNA transcript levels ß-adrenoceptors. The ß3-adrenoceptor agonist, BRL37344, reduced the frequency of pacemaker potentials in a concentration-dependent manner. The inhibitory effects of BRL37344 were blocked by the pretreatment of propranolol, a nonspecific ß-adrenoceptor antagonist, but not by the selective ß1-adrenoceptor antagonist atenolol and the selective ß2-adrenoceptor antagonist butoxamine. ß3-adrenoceptor antagonists SR59230A and L748337 blocked the inhibitory effects of BRL37344. RT-PCR revealed mRNA transcripts of ß1- and ß3-adrenoceptor, but not ß2-adrenoceptor, in c-kit- and Ano-1-positive colonic ICCs. The K(+) channel blockers tetraethylammonium, apamin, and glibenclamide did not block the effects of BRL37344. N(ω)-Nitro-l-arginine methyl ester hydrochloride (L-NAME), an NO synthase inhibitor, and chelerythrine, a protein kinase C inhibitor, also did not block the effects of BRL37344. Noradrenaline mimicked the effects of BRL37344 in colonic ICCs. However, the inhibitory effects of noradrenaline on pacemaker potentials were blocked only by pretreatment with atenolol but not by butoxamine, SR59230A, or L748337. In small intestinal ICCs, BRL37344 had no effect on pacemaker potentials and mRNA transcripts of ß1-and ß2-adrenoceptor, but not ß3-adrenoceptor were detected. These results suggest that ß3-adrenoceptors are present in colonic ICCs and may play a role in regulating gastrointestinal motility by the inhibition of pacemaker potentials.

Age-related subsensitivity of cerebellar Purkinje neurons to locally applied beta 1-selective adrenergic agonist.

Previous electrophysiological studies in aged rats have revealed a number of deficits in noradrenergic neurotransmission in the central nervous system. Such deficits include subsensitivity to the depressant effects of norepinephrine on cerebellar Purkinje neurons, which has been attributed specifically to altered beta adrenergic receptor-mediated processes. The objective of this study was to determine which beta adrenergic receptor subtype, beta 1 or beta 2, is responsible for this age-related subsensitivity. The effects of beta 1 and beta 2 agonists on spontaneous activity of Purkinje neurons was first examined in young rats and the selectivity of these agents was validated using selective beta 1 and beta 2 antagonists. The effects of the selective beta 1 and beta 2 agonists were then compared in young (3-month-old) and aged (18- and 26-month-old) Fischer 344 rats. These agents were applied to Purkinje neurons by pressure microejection from multibarreled micropipettes and the change in neuronal action potential discharge rate was recorded. Both dobutamine, a beta 1-selective agonist, and zinterol, a beta 2-selective agonist, induced dose-dependent inhibitions of Purkinje cell firing rate. Dobutamine-induced inhibitions were blocked by the selective beta 1 antagonist, ICI 89406 and not by the beta 2-selective antagonist, ICI 118551; conversely, zinterol-induced inhibitions were not blocked by ICI 89406 but were blocked by the presence of ICI 118551. Purkinje neurons of both groups of aged rats were significantly less sensitive to locally applied dobutamine than Purkinje cells of young rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The beta-adrenoceptors mediating relaxation of rat oesophageal muscularis mucosae are predominantly of the beta 3-, but also of the beta 2-subtype.

1. beta-Adrenoceptor-mediated relaxation of rat oesophageal smooth muscle was investigated by studying the effects of beta 1- and beta 2-selective antagonists on the relaxation induced by (-)-isoprenaline, the beta 2-selective agonists fenoterol and clenbuterol and the beta 3-agonist, BRL 37344. 2. The highly beta 1-selective antagonist CGP 20721A did not antagonize (-)-isoprenaline- or BRL 37344-induced relaxations in concentrations up to 10 microM. Only at 100 microM of CGP 20712A were clear rightward shifts of the agonist concentration-response curves (CRCs) observed, with pA2 values of 4.70 and 4.97 against (-)-isoprenaline and BRL 37344, respectively. 3. ICI 118,551, a potent and selective beta 2-antagonist, at 100 nM caused moderate rightward shifts of the CRCs of (-)-isoprenaline, fenoterol and clenbuterol; with fenoterol and clenbuterol, this was accompanied by a clear steepening of the curve. Only at the highest concentration (100 microM ICI 118,551) did the shifts to the right further increase substantially. Resulting Schild-plots were clearly biphasic. BRL 37344-induced relaxations were only antagonized at 100 microM ICI 118,551, yielding a pA2 value of 5.48. 4. These results clearly demonstrate that the BRL 37344-induced relaxation of rat oesophageal muscularis mucosae is mediated solely through beta 3-adrenoceptors, whereas (-)-isoprenaline-, fenoterol- and clenbuterol-induced relaxations were shown to involve both beta 2- and, predominantly, beta 3-adrenoceptors.

Choline supplementation inhibits diethanolamine-induced morphological transformation in syrian hamster embryo cells: evidence for a carcinogenic mechanism.

DEA, an amino alcohol, and its fatty acid condensates are widely used in commerce. DEA is hepatocarcinogenic in mice, but shows no evidence of mutagenicity or clastogenicity in a standard testing battery. However, it increased the number of morphologically transformed colonies in the Syrian hamster embryo (SHE) cell morphologic transformation assay. The goal of this work was to test the hypothesis that DEA treatment causes morphologic transformation by a mechanism involving altered cellular choline homeostasis. As a first step, the ability of DEA to disrupt the uptake and intracellular utilization of choline was characterized. SHE cells were cultured in medium containing DEA (500 microg/ml), and (33)P-phosphorus or (14)C-choline was used to label phospholipid pools. After 48 h, SHE cells were harvested, lipids were extracted, and radioactive phospholipids were quantified by autoradiography after thin layer chromatographic separation. In control cells, phosphatidylcholine (PC) was the major phospholipid, accounting for 43 +/- 1% of total phospholipid synthesis. However, with DEA treatment, PC was reduced to 14 +/- 2% of total radioactive phospholipids. DEA inhibited choline uptake into SHE cells at concentrations > or = 50 microg /ml, reaching a maximum 80% inhibition at 250-500 microg/ml. The concentration dependence of the inhibition of PC synthesis by DEA (0, 10, 50, 100, 250, and 500 microg/ml) was determined in SHE cells cultured over a 7-day period under the conditions of the transformation assay and in the presence or absence of excess choline (30 mM). DEA treatment decreased PC synthesis at concentrations > or = 100 microg/ml, reaching a maximum 60% reduction at 500 microg/ml. However, PC synthesis was unaffected when DEA-treated cells were cultured with excess choline. Under 7-day culture conditions, (14)C-DEA was incorporated into SHE lipids, and this perturbation was also inhibited by choline supplementation. Finally, DEA (10-500 microg/ml) transformed SHE cells in a concentration-dependent manner, whereas with choline supplementation, no morphologic transformation was observed. Thus, DEA disrupts intracellular choline homeostasis by inhibiting choline uptake and altering phospholipid synthesis. However, excess choline blocks these biochemical effects and inhibits cell transformation, suggesting a relationship between the two responses. Overall, the results provide a plausible mechanism to explain the morphologic transformation observed with DEA and suggest that the carcinogenic effects of DEA may be caused by intracellular choline deficiency.

Inhibition of Na(+)/Ca(2+) exchange by KB-R7943, a novel selective antagonist, attenuates phosphoethanolamine and free fatty acid efflux in rat cerebral cortex during ischemia-reperfusion injury.

Reversal of the Na(+)/Ca(2+) exchanger (NCX) occurs during ischemia-reperfusion injury as a result of changes in intracellular pH and sodium concentration. Inhibition of NCXs has been shown to be neuroprotective in vitro. In this study, we evaluated the effects of KB-R7943 (50 microM), a specific inhibitor of the reverse mode of NCX, applied topically onto rat cerebral cortex prior to and during ischemia. Amino acid and free fatty acid levels in cortical superfusates, withdrawn at 10-min intervals from bilateral cortical windows, were analyzed by high-performance liquid chromatography. During a 20-min period of ischemia in control animals, there were significant increases in all amino acids and in all FFAs. Following reperfusion, all FFAs remained significantly elevated. Application of KB-R7943 (50 microM) significantly inhibited effluxes of phosphoethanolamine, but had no effect on glutamate, aspartate, taurine or GABA levels. KB-R7943 also resulted in significant reductions in levels of myristic, docosahexaenoic and arachidonic acid during ischemia and in reperfusion levels of arachidonic and docosahexaenoic acids. These data indicate that inhibition of Na(+)/Ca(2+) exchange likely prevented the activation of phospholipases that usually occurs following an ischemic insult as evidenced by its attenuation of phosphoethanolamine and free fatty acid efflux. The inhibition of phospholipases may be an essential component of the neuroprotective benefits of Na(+)/Ca(2+) exchange inhibitors in ischemia-reperfusion injury and may provide a basis for their possible use in therapeutic strategies for stroke.

Positive chronotropic and inotropic responses to BRL 37344, a beta 3-adrenoceptor agonist in isolated, blood-perfused dog atria.

We investigated the chronotropic and inotropic responses to BRL 37344 (a beta 3-adrenoceptor agonist) and isoproterenol in isolated, blood-perfused dog atria. BRL 37344 (0.1-30 nmol) or isoproterenol (0.001-0.3 nmol) increased the sinus rate and contractile force dose dependently. BRL 37344 was 290 times less potent than isoproterenol to increase sinus rate and 140 times less potent to increase atrial force. Both propranolol and bisoprolol similarly inhibited the positive chronotropic and inotropic responses to BRL 37344 and isoproterenol dose dependently. ICI 118,551 (0.1 and 1 nmol) did not significantly affect the positive cardiac responses to BRL 37344 or isoproterenol. Neither imipramine nor tetrodotoxin significantly affected the positive cardiac responses to BRL 37344. These results suggest that the positive chronotropic and inotropic responses to BRL 37344 are mediated mainly by beta 1-adrenoceptors in the dog heart. It is unlikely that beta 3-adrenoceptors, as previously reported in adipose tissue or gastrointestinal smooth muscle, mediate chronotropic and inotropic responses in the normal dog heart.

Adrenoceptors involved in the contractile activity of islated pregnant rat uterus.

Cumulative log dose-response curves of soterenol, isoproterenol, phenylephrine and norepinephrine on isolated pregnant rat uterus at different days of gestation, were investigated. Soterenol produced a sustained inhibition of spontaneous motility during the whole pregnancy and this effect was blocked by butoxamine. The affinity of myometrium for the beta 2-adrenoceptor agonist was parallel to the concentration of progesterone in plasma during pregnancy. Isoproterenol, norepinephrine and phenylephrine caused dual, alpha- and beta-mediated responses, their relative dominance varied with the concentration and the days of pregnancy, alpha-Adrenoceptor effects of the amines coincided with increased plasma concentrations of estrogens, whereas beta ones were in parallel with the increment of plasma progesterone. It is concluded that: (a) there exist in the pregnant rat uterus beta 2-receptor-mediated responses influenced by the length of gestation; and (b) the concentration-pregnancy-dependent biphasic actions of isoproterenol, norepinephrine or phenylephrine suggest that their variable hormone-modulated ability interacts with both alpha- and beta-adrenoceptive uterine sites.

Regulation of liver base-exchange activity by acidic phospholipids.

Liver microsomes were enriched in liposomal acidic lipids by Ca(2+)-dependent fusion of liposomes at pH 7.0. The extent of fusion was monitored by the transfer of radioactive cholesteryl oleate. The enrichment of membranes in phosphatidylserine inhibited ethanolamine base-exchange, whereas the fusion with phosphatidylinositol inhibited both ethanolamine and serine base-exchange reactions. In contrast, these two phospholipids had scarce effects on choline base-exchange. Phosphatidic acid did not suppress any of the three base-exchange activities. Possible functional implications are discussed.

Relaxant and beta 2-adrenoceptor blocking activities of labetalol, dilevalol, amosulalol and KF-4317 on the rat isolated aorta.

The KCl-contracted rat aorta is relaxed by labetalol, dilevalol, amosulalol and KF-4317. These relaxations are not reversed by ICI 118,551 at 10(-6) M and, therefore, are not due to beta-adrenoceptor agonism. At 10(-7) M, labetalol, dilevalol, amosulalol and KF-4317 were beta 2-adrenoceptor antagonists as they inhibited the relaxant responses of rat aorta to procaterol.

The effects of beta agonists on muscle cells in culture.

Increases in protein synthesis of 12% were found with two myogenic cell lines (L6 and G8-1) on treatment for 6 hr with the beta-adrenergic agonist cimaterol. In L6 cells, propranolol blocked the effect. Protein breakdown measured over 18-24 hr was unchanged. The Kd for cimaterol binding to the L6 beta-receptor was 26 nM which was compatible with its EC50 for the stimulation of protein synthesis (approx 5 nM). Evidence provided with muscle cell lines indicates a direct effect of cimaterol on protein synthesis, which may contribute to muscle accretion in cimaterol-fed animals.

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.

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.

Yohimbine antagonises α1A- and α1D-adrenoceptor mediated components in addition to the α2A-adrenoceptor component to pressor responses in the pithed rat.

We have recently shown that responses to pressor nerve stimulation in the pithed rat are mediated by α(1A)- and α(1D)-adrenoceptors, with no evidence for α(2)-adrenoceptor involvement, and that responses previously identified as α(2)-adrenoceptor mediated are actually α(1D)-adrenoceptor mediated. We have now re-examined the subtypes of α-adrenoceptor involved in pressor responses produced by exogenous agonists in the pithed rat preparation to confirm whether α(2)-adrenoceptors are involved in these responses. The α(2)-adrenoceptor and α(1D)-adrenoceptor antagonist yohimbine (1mg/kg) and the α(2A)-adrenoceptor antagonist methoxy-idazoxan (5 mg/kg) significantly shifted, but the α(1D)-adrenoceptor antagonist BMY 7378 (8-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspir o[4.5]decane-7,9-dione dihydrochloride) (1 mg/kg) did not affect, the pressor potency of the α(2)-adrenoceptor agonist xylazine. α(1)-adrenoceptor antagonists showed low potency against pressor responses to xylazine. The pressor potency of the α(1)-adrenoceptor agonist amidephrine was not affected by BMY 3778 (1 mg/kg) but significantly shifted by prazosin (0.01 mg/kg) and by yohimbine (1 mg/kg). In contrast, the pressor potency of phenylephrine was significantly shifted by both yohimbine and BMY 7378 (1 mg/kg), but to a greater extent by the α(1A)-adrenoceptor antagonist RS 100329 (5-Methyl-3-[3-[3-[4-[2-(2,2,2,trifluroethoxy) phenyl]-1-piperazinyl]propyl]-2,4-(1H,3H)-pyrimidinedione] hydrochloride) (0.1 mg/kg). In conclusion, we have identified and separated α(1A)-, α(1D)- and α(2A)-adrenoceptor antagonist actions of yohimbine against pressor responses. Pressor responses to exogenous agonists in the pithed rat involve both α(1A)- and α(1D)-adrenoceptors and in addition, α(2A)-adrenoceptors.

Human atrial ß(1L)-adrenoceptor but not ß3-adrenoceptor activation increases force and Ca(2+) current at physiological temperature.

BACKGROUND AND PURPOSE: It has been proposed that BRL37344, SR58611 and CGP12177 activate ß3-adrenoceptors in human atrium to increase contractility and L-type Ca(2+) current (I(Ca-L)). ß3-adrenoceptor agonists are potentially beneficial for the treatment of a variety of diseases but concomitant cardiostimulation would be potentially harmful. It has also been proposed that (-)-CGP12177 activates the low affinity binding site of the ß1-adrenoceptor in human atrium. We therefore used BRL37344, SR58611 and (-)-CGP12177 with selective ß-adrenoceptor subtype antagonists to clarify cardiostimulant ß-adrenoceptor subtypes in human atrium. EXPERIMENTAL APPROACH: Human right atrium was obtained from patients without heart failure undergoing coronary artery bypass or valve surgery. Cardiomyocytes were prepared to test BRL37344, SR58611 and CGP12177 effects on I(Ca-L). Contractile effects were determined on right atrial trabeculae. KEY RESULTS: BRL37344 increased force which was antagonized by blockade of ß1- and ß2-adrenoceptors but not by blockade of ß3-adrenoceptors with ß3-adrenoceptor-selective L-748,337 (1 µM). The ß3-adrenoceptor agonist SR58611 (1 nM-10 µM) did not affect atrial force. BRL37344 and SR58611 did not increase I(Ca-L) at 37°C, but did at 24°C which was prevented by L-748,337. (-)-CGP12177 increased force and I(Ca-L) at both 24°C and 37°C which was prevented by (-)-bupranolol (1-10 µM), but not L-748,337. CONCLUSIONS AND IMPLICATIONS: We conclude that the inotropic responses to BRL37344 are mediated through ß1- and ß2-adrenoceptors. The inotropic and I(Ca-L) responses to (-)-CGP12177 are mediated through the low affinity site ß(1L)-adrenoceptor of the ß1-adrenoceptor. ß3-adrenoceptor-mediated increases in I(Ca-L) are restricted to low temperatures. Human atrial ß3-adrenoceptors do not change contractility and I(Ca-L) at physiological temperature.

Vasorelaxation to N-oleoylethanolamine in rat isolated arteries: mechanisms of action and modulation via cyclooxygenase activity.

BACKGROUND AND PURPOSE: The endocannabinoid-like molecule N-oleoylethanolamine (OEA) is found in the small intestine and regulates food intake and promotes weight loss. The principal aim of the present study was to evaluate the vascular effects of OEA. EXPERIMENTAL APPROACH: Perfused isolated mesenteric arterial beds were pre-contracted with methoxamine or high potassium buffers and concentration-response curves to OEA were constructed. Combinations of inhibitors to block nitric oxide production, sensory nerve activity, cyclooxygenase activity, potassium channels, chloride channels and gap junctions, and a cannabinoid CB(1) receptor antagonist, were used during these experiments. The effects of OEA on caffeine-induced contractions in calcium-free buffer were also assessed. Isolated thoracic aortic rings were used as a comparison. KEY RESULTS: OEA caused concentration-dependent vasorelaxation in rat isolated mesenteric arterial beds and thoracic aortic rings, with a greater maximal response in mesenteric vessels. This relaxation was sensitive to inhibition of sensory nerve activity and endothelial removal in both preparations. The cyclooxygenase inhibitor indomethacin reversed the effects of capsaicin pre-treatment in perfused mesenteric arterial beds and indomethacin alone enhanced vasorelaxation to OEA. The OEA-induced vasorelaxation was inhibited by a CB(1) receptor antagonist only in aortic rings. In mesenteric arteries, OEA suppressed caffeine-induced contractions in calcium-free buffer. CONCLUSIONS AND IMPLICATIONS: The vasorelaxant effects of OEA are partly dependent on sensory nerve activity and a functional endothelium in the vasculature. In addition, vasorelaxation to OEA is enhanced following cyclooxygenase inhibition. OEA may also interfere with the release of intracellular calcium in arterial preparations.