British Journal of Pharmacology

In endothelium‐intact rings precontracted to the thromboxane A₂ mimetic, U46619, anandamide (0.01 – 30 μM) induced slowly developing concentration‐dependent relaxations (pEC₅₀ [negative log of EC₅₀]=6.1±0.1; R_max [maximum response]=81±4%). Endothelium denudation caused a 10 fold rightward shift of the anandamide concentration‐relaxation curve without modifying R_max. Methanandamide was without effect on U46619‐induced tone.

The anandamide‐induced relaxation was unaffected by the cannabinoid receptor antagonist, SR 141716A (3 μM), the vanilloid receptor antagonist, capsazepine (3 and 10 μM) or the nitric oxide synthase inhibitor, L‐NAME (100 μM).

The cyclo‐oxygenase inhibitor, indomethacin (3 and 10 μM) and the anandamide amidohydrolase inhibitor, PMSF (70 and 200 μM), markedly attenuated the anandamide response. The anandamide transport inhibitor, AM 404 (10 and 30 μM), shifted the anandamide concentration‐response curve to the right.

Precontraction of endothelium‐intact rings with 25 mM KCl attenuated the anandamide‐induced relaxations (R_max=7±7%), as did K⁺ channel blockade with tetraethylammonium (TEA; 3 μM) or iberiotoxin (100 nM). Blockade of small conductance, Ca²⁺‐activated K⁺ channels, delayed rectifier K⁺ channels, K_ATP channels or inward rectifier K⁺ channels was without effect.

These data suggest that the relaxant effects of anandamide in sheep coronary arteries are mediated in part via the endothelium and result from the cellular uptake and conversion of anandamide to a vasodilatory prostanoid. This, in turn, causes vasorelaxation, in part, by opening potassium channels.

The vanilloid receptor antagonist, capsazepine (10 μM), significantly attenuated the contractile effect of anandamide, the response to 100 μM anandamide being 40.53±7.04% in the presence of vehicle and 1.57±8.93% in the presence of 10 μM capsazepine. The contractile actions of anandamide and AM404 were markedly enhanced by the peptidase inhibitor thiorphan.

The log concentration‐response curve of anandamide was unaltered by the CB₁ receptor antagonist, SR141716A. The pEC₅₀ values for anandamide were 4.88±0.08 and 5.17±0.19 in the presence of vehicle and SR141716A (1 μM) respectively.

The lipoxygenase inhibitors 5,8,11,14‐eicosatetraynoic acid (ETYA) and 5,8,11 eicosatriynoic acid (ETI) reduced the effect of 100 μM anandamide from 34.7±1.9% (vehicle) to 7.7±5% (ETYA, 10 μM) and from 41.85±4.25% (n=6) (vehicle) to 10.31±3.54 (n=6) (ETI, 20 μM). Neither inhibitor significantly affected contraction of the tissue by substance P.

This study provides evidence that anandamide acts on vanilloid receptors in the guinea‐pig isolated bronchus. These data raise the possibility that the contractile action of anandamide may be due, at least in part, to lipoxygenase metabolites of this fatty acid amide that are vanilloid receptor agonists.

For short‐chain alcohols from methanol to 1‐hexanol, potency for inhibition of GluR1 and GluR3 receptor‐mediated current increased in proportion to the chain length or hydrophobicity of the alcohol.

The IC₅₀ values of these alcohols for GluR1 were: methanol, 702 mM; ethanol, 170 mM; 1‐propanol, 69 mM; 1‐butanol, 20 mM; 1‐pentanol, 17 mM; and 1‐hexanol, 10 mM. For GluR3, IC₅₀ values were: methanol, 712 mM; ethanol, 238 mM; 1‐propanol, 50 mM; 1‐butanol, 32 mM; 1‐pentanol, 13 mM; and 1‐hexanol, 7 mM.

For long‐chain alcohols, 1‐heptanol was less potent than 1‐hexanol (estimated IC₅₀: 19 mM for GluR1 and 18 mM for GluR3), 1‐octanol had little effect only on GluR3, and 1‐nonanol and 1‐decanol did not significantly inhibit both GluR1 and GluR3 responses.

The observations indicate that straight‐chain n‐alcohols exhibit a cutoff in their potency for inhibition of the function of non‐NMDA glutamate receptor subunits, GluR1 and GluR3. The cutoff in potency of n‐alcohols for inhibition of non‐NMDA glutamate receptor function is consistent with the interpretation that alcohols affect the function of these receptor‐channels by interacting with an alcohol binding site of specific dimensions on the receptor protein.

Changing pH_o from 7.4 to 6.4 or 8.4 produced a depolarisation or hyperpolarisation, respectively, in mesenteric and pulmonary arteries. Anandamide (10 μM) or bupivacaine (100 μM) reversed the hyperpolarisation associated with alkaline pH_o, shifting the E_M of both vessels to levels comparable to that at pH 6.4. In pulmonary arteries, clofilium (100 μM) caused a significant reversal of hyperpolarisation seen at pH 8.4 but was without effect at pH 7.4.

K⁺ channel blockade by 4‐aminopyridine (4‐AP) (5 mM), tetraethylammonium (TEA) (10 mM), Ba²⁺ (30 μM) and glibenclamide (10 μM) depolarised the pulmonary artery. However, shifts in E_M with changes in pH_o remained and were sensitive to anandamide (10 μM), bupivacaine (100 μM) or Zn²⁺ (200 μM).

Anandamide (0.3–60 μM) or bupivacaine (0.3–300 μM) caused a concentration‐dependent increase in basal tone in pulmonary arteries.

RT–PCR demonstrated the expression of TASK‐1, TASK‐2, THIK‐1, TRAAK, TREK‐1, TWIK‐1 and TWIK‐2 in mesenteric arteries and TASK‐1, TASK‐2, THIK‐1, TREK‐2 and TWIK‐2 in pulmonary arteries. TASK‐1, TASK‐2, TREK‐1 and TWIK‐2 protein was demonstrated in both arteries by immunostaining.

These experiments provide evidence for the presence of two‐pore domain K⁺ channels in rat mesenteric and pulmonary arteries. Collectively, they strongly suggest that modulation of TASK‐1 channels is most likely to have mediated the pH‐induced changes in membrane potential observed in these vessels, and that blockade of these channels by anandamide or bupivacaine generates a small increase in pulmonary artery tone.

Effects of glycine on release of catecholamines in the striatum were examined by microdialysis in freely‐moving rats. Transcription of the genes encoding strychnine‐sensitive glycine receptors was assessed in the striatum and substantia nigra, by use of reverse transcription followed by the polymerase chain reaction.

Glycine administered via the microdialysis probe dose‐dependently increased concentrations of dopamine and its metabolites, dihydroxyphenylacetic acid and homovanillic acid, in the perfusate, indicating increased local release and metabolism of dopamine. Strychnine markedly attenuated these responses. Whereas striatal tissue did not contain mRNA for either the adult or neonatal form of strychnine‐sensitive glycine receptor, nigral tissue contained a message for the adult form.

The results suggest that dopaminergic cells in the substantia nigra synthesize strychnine‐sensitive glycine receptors and transport the receptors to terminals in the striatum. Occupation of the glycine receptors then exerts a net stimulatory effect on striatal dopamine release in vivo.

Twenty μM cis‐oleamide (but not trans) reversibly enhanced GABA_A currents and depressed the frequency of spontaneous excitatory and inhibitory synaptic activity in cultured networks.

cis‐oleamide stereoselectively blocked veratridine‐induced (but not K⁺‐induced) depolarisation of mouse synaptoneurosomes (IC₅₀, 13.9 μM).

The cis isomer stereoselectively blocked veratridine‐induced (but not K⁺‐induced) [³H]‐GABA release from mouse synaptosomes (IC₅₀, 4.6 μM).

At 20 μM cis‐oleamide, but not trans, produced a marked inhibition of Na⁺ channel‐dependent rises in intrasynaptosomal Ca²⁺.

The physiological significance of these observations was examined by isolating Na⁺ spikes in cultured pyramidal neurones. Sixty‐four μM cis‐oleamide did not significantly alter the amplitude, rate of rise or duration of unitary action potentials (1 Hz).

cis‐Oleamide stereoselectively suppressed sustained repetitive firing (SRF) in these cells with an EC₅₀ of 4.1 μM suggesting a frequency‐ or state‐dependent block of voltage‐gated Na⁺ channels.

Oleamide is a stereoselective modulator of both postsynaptic GABA_A receptors and presynaptic or somatic voltage‐gated Na⁺ channels which are crucial for synaptic inhibition and conduction. The modulatory actions are strikingly similar to those displayed by sedative or anticonvulsant barbiturates and a variety of general anaesthetics.

Oleamide may represent an endogenous modulator for drug receptors and an important regulator of arousal.

The 5‐HT‐induced current was inhibited by the following cannabinoid receptor agonists (at decreasing order of potency): Δ⁹‐THC, WIN55,212‐2, anandamide, JWH‐015 and CP55940. The WIN55,212‐2‐induced inhibition was not altered by SR141716A, a CB₁ receptor antagonist. WIN55,212‐3, an enantiomer of WIN55,212‐2, did not affect the 5‐HT‐induced current.

WIN55,212‐2 did not change the EC₅₀ value of 5‐HT in stimulating current, but reduced the maximum effect.

The CB₁ receptor ligand [³H]‐SR141716A and the CB₁/CB₂ receptor ligand [³H]‐CP55940 did not specifically bind to parental HEK293 cells. In competition experiments on membranes of HEK293 cells transfected with the h5‐HT_3A receptor cDNA, WIN55,212‐2, CP55940, anandamide and SR141716A did not affect [³H]‐GR65630 binding, but 5‐HT caused a concentration dependent‐inhibition.

In conclusion, cannabinoids stereoselectively inhibit currents through recombinant h5‐HT_3A receptors independently of cannabinoid receptors. Probably the cannabinoids act allosterically at a modulatory site of the h5‐HT_3A receptor. Thus the functional state of the receptor can be controlled by the endogenous ligand anandamide. This site is a potential target for new analgesic and antiemetic drugs.