The antitussive effects of endomorphin-1 and endomorphin-2 in mice

The antitussive effects of endomorphin-1 and endomorphin-2, endogenous mu-opioid receptor agonists, on capsaicin-induced coughs were examined in mice. Endomorphin-2, at doses of 3, 10 and 30 microg, i.c.v., dose-dependently inhibited the number of capsaicin-induced coughs. However, the same doses (3, 10 and 30 microg) of endomorphin-1 injected with i.c.v. had no significant effects on the number of capsaicin-induced coughs. The antitussive effect of endomorphin-2 was significantly reduced by beta-funaltrexamine, a mu(1)/mu(2)-opioid receptor antagonist, but not naloxonazine, a selective mu(1)-opioid receptor antagonist. Furthermore, the antitussive effect of endomorphin-2 was also partially but significantly reduced by nor-binaltorphimine, a selective kappa-opioid receptor antagonist. These results indicate that the administration of the endogenous mu-opioid ligand endomorphin-2, but not endomorphin-1, into the brain produces an antitussive effect via mainly naloxonazine-insensitive mu-opioid receptors, namely mu(2)-opioid receptors and partially kappa-opioid receptors.     

Central effects of the cannabinoid receptor agonist WIN55212-2 on respiratory and cardiovascular regulation in anaesthetised rats

1 The primary aim was to study the central respiratory effects of cannabinoids (CB). To this end, the cannabinoid receptor agonist WIN55212-2 was injected into the cisterna magna of urethane-anaesthetised rats and changes in respiratory parameters were observed. The secondary aim was to observe the centrally elicited cardiovascular actions of WIN55212-2. Involvement of opioid mechanisms in the central effects of WIN55212-2 was also studied. 2 Intracisternal (i.c.) application of WIN55212-2 (1, 3, 10 and 30 microg kg(-1)) dose-dependently decreased the respiratory rate and minute volume. Tidal volume was slightly increased, whereas peak inspiratory flow remained unchanged. In addition, WIN55212-2 increased mean arterial pressure and the plasma noradrenaline concentration and decreased heart rate. 3 I.c. injection of WIN55212-3 (1, 3, 10 and 30 microg kg(-1)), an enantiomer of WIN55212-2 lacking affinity for cannabinoid receptors, elicited no effects. All effects of WIN55212-2 were prevented by the CB1 receptor antagonist SR141716 (2 mg kg(-1) i.v.). I.c. administration of the opioid receptor agonist DAMGO (0.1, 0.3, 1 and 3 microg kg(-1)) markedly lowered the respiratory rate, tidal volume, minute volume and peak inspiratory flow. These effects were attenuated by the opioid receptor antagonist naloxone (0.2 mg kg(-1) i.v.). In contrast, naloxone did not affect the respiratory and cardiovascular effects of i.c. administered WIN55212-2. 4 Our results show that activation of CB1 cannabinoid receptors in the brain stem depresses respiration and enhances sympathetic tone and cardiac vagal tone. Opioid mechanisms are not involved in these central cannabinoid effects.     

Involvement of the opioid system in the anxiolytic-like effects induced by Delta(9)-tetrahydrocannabinol

RATIONALE: Recent studies have shown that several pharmacological actions induced by cannabinoids, including antinociception and reward, involve the participation of the endogenous opioid system. OBJECTIVES: The present study was designed to examine the possible involvement of the different opioid receptors in the anxiolytic-like responses induced by Delta(9)-tetrahydrocannabinol (THC). METHODS: The administration of a low dose of THC (0.3 mg/kg) produced clear anxiolytic-like responses in the light-dark box, as previously reported. The effects of the pretreatment with the CB(1) cannabinoid receptor antagonist, SR 141716A (0.5 mg/kg), or the micro -opioid receptor antagonist, beta-funaltrexamine (5 mg/kg), the delta-opioid receptor antagonist, naltrindole (2.5 mg/kg) and the kappa-opioid receptor antagonist, nor-binaltorphimine (2.5 mg/kg) were evaluated on anxiolytic-like responses induced by THC. RESULTS: SR 141716A completely blocked the anxiolytic-like response induced by THC, suggesting that this effect is mediated by CB(1) cannabinoid receptors. The micro -opioid receptor antagonist beta-funaltrexamine and the delta-opioid receptor antagonist naltrindole, but not the kappa-opioid receptor antagonist nor-binaltorphimine, abolished THC anxiolytic-like effects, suggesting an involvement of micro - and delta-opioid receptors in this behavioural response. CONCLUSIONS: These results demonstrate that the endogenous opioid system is involved in the regulation of anxiety-like behaviour by cannabinoids and provide new findings to clarify further the interaction between these two neuronal systems.     

Systemic effect of cannabinoids on the spontaneous firing rate of locus coeruleus neurons in rats

Previous reports have described modulation of noradrenergic activity by cannabinoid receptors. The aim of the present research was to examine the effect of two synthetic cannabinoid CB1/CB2 receptor agonists, R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)-methyl]pyrrolol-[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone (WIN 55212-2) and (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP 55940), on the spontaneous activity of locus coeruleus noradrenergic neurons by single-unit extracellular recordings in vivo and in vitro. In anaesthetized rats, intravenous administrations of WIN 55212-2 (31.3-500 microg/kg) or CP 55940 (31.3-500 microg/kg) increased the firing rate of locus coeruleus neurons in a dose-dependent manner. The stimulatory effect of WIN 55212-2 was blocked by pretreatment with the cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR 141716A; 2 mg/kg). Paradoxically, local administration of WIN 55212-2 (8.3-31.3 pmol) into the locus coeruleus and intracerebroventricular injections of WIN 55212-2 (10-20 microg) or CP 55940 (20-40 microg) failed to change the spontaneous firing rate of locus coeruleus neurons. Likewise, in rat brain slice preparations perfusion with WIN 55212-2 (10 microM) or CP 55940 (10-30 microM) did not specifically affect the spontaneous firing rate of locus coeruleus cells. Therefore, we conclude that synthetic cannabinoids increase the spontaneous firing activity of noradrenergic neurons in the rat locus coeruleus through cannabinoid CB1 receptors. This stimulation appears to be indirectly induced via a receptor mechanism probably located at the peripheral level.     

GABAA receptors modulate cannabinoid-evoked hypothermia

Cannabinoids evoke hypothermia by stimulating central CB(1) receptors. GABA induces hypothermia via GABA(A) or GABA(B) receptor activation. CB(1) receptor activation increases GABA release in the hypothalamus, a central locus for thermoregulation, suggesting that cannabinoid and GABA systems may be functionally linked in body temperature regulation. We investigated whether GABA receptors modulate the hypothermic actions of [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one] (WIN 55212-2), a selective cannabinoid agonist, in male Sprague-Dawley rats. WIN 55212-2 (2.5 mg/kg im) produced a rapid hypothermia that peaked 45-90 min postinjection. The hypothermia was attenuated by bicuculline (2 mg/kg ip), a GABA(A) antagonist. However, SCH 50911 (1-10 mg/kg ip), a GABA(B) blocker, did not antagonize the hypothermia. Neither bicuculline (2 mg/kg) nor SCH 50911 (10 mg/kg) by itself altered body temperature. We also investigated a possible role for CB(1) receptors in GABA-generated hypothermia. Muscimol (2.5 mg/kg ip), a GABA(A) agonist, or baclofen (5 mg/kg ip), a GABA(B) agonist, evoked a significant hypothermia. Blockade of CB(1) receptors with SR141716A (2.5 mg/kg im) did not antagonize muscimol- or baclofen-induced hypothermia, indicating that GABA-evoked hypothermia does not contain a CB(1)-sensitive component. Our results implicate GABA(A) receptors in the hypothermic actions of cannabinoids and provide further evidence of a functional link between cannabinoid and GABA systems.     

Differentiation of opioid receptor preference by [Dmt1]endomorphin-2-mediated antinociception in the mouse

The potent opioid [Dmt1]endomorphin-2 (Dmt-Pro-Phe-Phe-NH2) differentiated between the opioid receptor subtypes responsible for the antinociception elicited by endomorphin-2 in mice. Antinociception, induced by the intracerebroventricular administration of [Dmt1]endomorphin-2 and inhibited by various opioid receptor antagonists [naloxone, naltrindole, beta-funaltrexamine, naloxonazine], was determined by the tail-flick (spinal effect) and hot-plate (supraspinal effect) tests. The opioid receptor subtypes involved in [Dmt1]endomorphin-2-induced antinociception differed between these in vivo model paradigms: naloxone (non-specific opioid receptor antagonist) and beta-funaltrexamine (irreversible mu1/mu2-opioid receptor antagonist) blocked antinociception in both tests, although stronger inhibition occurred in the hot-plate than the tail-flick test suggesting involvement of other opioid receptors. Consequently, we applied naloxonazine (mu1-opioid receptor antagonist) that significantly blocked the effect in the hot-plate test and naltrindole (delta-opioid receptor antagonist), which was only effective in the tail-flick test. The data indicated that [Dmt1]endomorphin-2-induced spinal antinociception was primarily mediated by both mu2- and delta-opioid receptors, while a supraspinal mechanism involved only mu1/mu2-subtypes.     

Involvement of central opioid systems in human interferon-alpha induced immobility in the mouse forced swimming test

1. We investigated the mechanism by which human interferon-alpha (IFN-alpha) increases the immobility time in a forced swimming test, an animal model of depression. 2. Central administration of IFN-alpha (0.05 - 50 IU per mouse, i.cist.) increased the immobility time in the forced swimming test in mice in a dose-dependent manner. 3. Neither IFN-beta nor -gamma possessed any effect under the same experimental conditions. 4. Pre-treatment with an opioid receptor antagonist, naloxone (1 mg kg(-1), s.c.) inhibited the prolonged immobility time induced by IFN-alpha (60 KIU kg(-1), i.v. or 50 IU per mouse. i.cist. ). 5. Peripheral administration of naloxone methiodide (1 mg kg(-1), s. c.), which does not pass the blood - brain barrier, failed to block the effect of IFN-alpha, while intracisternal administration of naloxone methiodide (1 nmol per mouse) completely blocked. 6. The effect of IFN-alpha was inhibited by a mu(1)-specific opioid receptor antagonist, naloxonazine (35 mg kg(-1), s.c.) and a mu(1)/mu(2) receptor antagonist, beta-FNA (40 mg kg(-1), s.c.). A selective delta-opioid receptor antagonist, naltrindole (3 mg kg(-1), s.c.) and a kappa-opioid receptor antagonist, nor-binaltorphimine (20 mg kg(-1), s.c.), both failed to inhibit the increasing effect of IFN-alpha. 7. These results suggest that the activator of the central opioid receptors of the mu(1)-subtype might be related to the prolonged immobility time of IFN-alpha, but delta and kappa-opioid receptors most likely are not involved.     

The antinociceptive effect of amisulpride in mice is mediated through opioid mechanisms

Antinociceptive effects of various neuroleptics in animal acute pain-models have been described, mediated trough different pathways including the opioid system. In this study, we assessed the antinociceptive effects of the atypical neuroleptic drug amisulpride, which acts as a selective blocker of dopamine D2 and D3 receptors. Furthermore, at low doses amisulpride has a selective preference for presynaptic dopamine autoreceptors, while at high doses it manifests a preferential action at post-synaptic dopamine receptors. We found amisulpride to be a potent antinociceptor agent in the mouse tail-flick assay, with an ED50 of 36.6 mg/kg. This effect was antagonized by naloxone (P<0.05), indicating an involvement of opioid mechanisms as mediators of the antinociceptive effect of amisulpride. Beta-funaltrexamine (mu1- and mu2-opioid receptor antagonist), naloxonazine (selective mu1-opioid receptor antagonist), naltrindole (selective delta-opioid receptor antagonist), Nor-binaltorphamine (kappa1-opioid receptor antagonist) reversed amisulpride antinociception at the same dose that they antagonized morphine's antinociceptive effect (all P<0.005). We found that the sensitivity of amisulpride-induced antinociception is mediated through selective involvement of all three opioid receptor subtypes. Based on previous studies with risperidone, clozapine and olanzapine we tend to attribute this global interaction with the opioid system to amisulpride's action at the dopamine D2 receptor sites.     

The role of opioid receptor subtypes in the development of behavioral sensitization to ethanol.

Nonspecific blockade of opioid receptors has been found to prevent development of behavioral sensitization to ethanol. Whether this effect is achieved through a specific opioid receptor subtype, however, is not clear. The present study investigated, for the first time, the role of specific opioid receptor subtypes in the development of ethanol-(2.5 g/kg/day; six sessions) induced locomotor sensitization in mice. We confirmed previous results showing that the nonspecific antagonism of opioid receptors (naltrexone; 0-2 mg/kg) prevented the development of behavioral sensitization to ethanol, an effect attained at doses presumed to occupy only mu opioid receptors. This was confirmed by using the selective mu opioid receptor antagonist CTOP (0-1.5 mg/kg), which also blocked sensitization to ethanol. The selective delta receptor antagonist, naltrindole (0-10 mg/kg), however, did not alter sensitization. We further assessed the role of mu opioid receptors in sensitization to ethanol by exploring the involvement of mu(1), mu(1+2), and mu(3) opioid receptor subtypes. Results of these experiments revealed that the blockade of mu(1) (naloxonazine; 0-30 mg/kg) or mu(3) opioid receptors (3-methoxynaltrexone; 0-6 mg/kg) did not prevent locomotor sensitization to ethanol. Using naloxonazine under treatment conditions that block mu(1+2) opioid receptor subtypes we observed a retarded sensitization. The present data suggest that the concurrent inactivation of all mu opioid receptor subtypes may be required to prevent the neural adaptations underlying the development of behavioral sensitization to ethanol. In addition, these results support previous data suggesting a putative role for the mu opioid receptor endogenous ligand, beta-endorphin, and the hypothalamic arcuate nucleus in ethanol sensitization.     

Agmatine and a cannabinoid agonist, WIN 55212-2, interact to produce a hypothermic synergy

Agmatine blocks morphine withdrawal symptoms and enhances morphine analgesia in rats. Yet, the role of agmatine in the pharmacological effects of other abused drugs has not been investigated. The present study investigates the effect of agmatine administration on the hypothermic response to cannabinoids. Hypothermia is an effective endpoint because cannabinoid agonists produce a rapid, reproducible, and significant decrease in body temperature that is abolished by cannabinoid CB(1) receptor antagonists. WIN 55212-2, a cannabinoid agonist, was administered to rats by itself and with agmatine. WIN 55212-2 (1, 2.5, 5 and 10 mg/kg, i.m.) caused a significant hypothermia. Agmatine (10, 25 and 50 mg/kg, i.p.) was ineffective. For combined administration, agmatine (50 mg/kg, i.p.) enhanced the hypothermic effect of WIN 55212-2 (1, 2.5, 5 and 10 mg/kg, i.m.). The enhancement was strongly synergistic, indicated by a 2.7-fold increase in the relative potency of WIN 55212-2. The central administration of agmatine (25 and 50 mug/rat, i.c.v.) significantly increased the hypothermic effect of WIN 55212-2 (2.5 mg/kg, i.m.). This indicates that agmatine acts through a central mechanism to augment cannabinoid-evoked hypothermia. Idazoxan (2 mg/kg, i.p.), an imidazoline antagonist, blocked the enhancement by agmatine, thus suggesting that imidazoline receptor activation is required for agmatine to enhance cannabinoid-evoked hypothermia. The present data reveal that agmatine and a cannabinoid agonist interact to produce a hypothermic synergy in rats. These results show that agmatine acts in the brain and via imidazoline receptors to enhance cannabinoid-evoked hypothermia.     

Role of Opioidergic and Serotonergic Mechanisms in Cough and Antitussives

This paper provides an overview of our current understanding of the serotonergic and opioidergic mechanisms of cough and antitussives. Systemic administration of 8-OH-DPAT, at doses of 0.1 and 0.3 mg/kg, ip, markedly reduced the number of coughs in rats in a dose-dependent manner. The antitussive effects of 8-OH-DPAT, dihydrocodeine and dextromethorphan were significantly reduced by pretreatment with methysergide, but not with ketanserin. Therefore it is possible to speculate that 5-HT₁receptors, in particular the 5-HT_1Areceptors, may be more important than others with respect to the effect of antitussive drugs. DAMGO, a selective μ-opioid receptor agonist, and U-50,488H, a highly selective κ-opioid receptor agonist, have potent antitussive effects when administered either icv or ip. However, we did not observe a cough-depressant effect of DPDPE, a selective δ-opioid receptor agonist. These results indicate that the antitussive effects of opioids are mediated predominantly by μ- and κ-opioid receptors. On the other hand, naloxonazine, a selective μ₁-opioid receptor antagonist, had no effect on the antitussive effects associated with icv DAMGO. These results indicate that μ₂- rather than μ₁-opioid receptors are involved in μ-opioid receptor-induced antitussive effects. Antitussive effects of dextromethorphan and noscapine were significantly and dose-dependently reduced by pretreatment with rimcazole, a specific antagonist of sites. However, rimcazole did not have a significant effect on the antitussive effect of morphine. These results suggest that sites may be involved in the antitussive mechanism of non-narcotic antitussive drugs.     

Antitussive effect of dihydroetorphine in mice

The present study examined the opioid receptors involved in the antitussive effect of dihydroetorphine in mice. Dihydroetorphine suppressed coughs dose dependently at doses between 0.1–1 μg/kg i.p. Blockade of μ-opioid receptors by pretreatment with β-funaltrexamine significantly reduced the antitussive effect of dihydroetorphine. Furthermore, the antitussive effect of dihydroetorphine was also antagonized by nor-binaltorphimine, a κ-opioid receptor antagonist. However, pretreatment with naltrindole, a δ-opioid receptor antagonist, did not affect the antitussive effect of dihydroetorphine. These results indicate that the antitussive effect of dihydroetorphine is mediated by the activation of μ-opioid receptors and κ-opioid receptors, but not δ-opioid receptors.     

Agmatine enhances cannabinoid action in the hot-plate assay of thermal nociception

Agmatine–cannabinoid interactions are supported by the close association between cannabinoid CB₁ receptors and agmatine immunoreactive neurons and evidence that shared brain mechanisms underlie the pharmacological effects of agmatine and cannabinoid agonists. In the present study, we used the hot-plate assay of thermal nociception to determine if agmatine alters cannabinoid action through activation of imidazoline sites and/or alpha₂-adrenoceptors. WIN 55212-2 (1, 2 or 3 mg/kg, i.p.) or CP55,940 (1, 2 or 3 mg/kg, i.p.) administration increased hot-plate response latency. Agmatine (50 or 100 mg/kg, i.p.) was ineffective. Administration of agmatine (50 mg/kg, i.p.) with WIN 55212-2 (1, 2 or 3 mg/kg, i.p.) or CP55,940 (1, 2 or 3 mg/kg, i.p.) produced response-latency enhancement. Regression analysis indicated that agmatine increased the potency of WIN 55212-2 and CP55,940 by 3- and 4.4-fold, respectively, indicating synergy for both drug interactions. Idazoxan, a mixed imidazoline site/alpha₂-adrenoceptor antagonist, but not yohimbine (5 mg/kg, i.p.), a selective alphia₂-adrenoceptor antagonist, blocked response-latency enhancement produced by a combination of WIN 55212-2 (2 mg/kg) and agmatine. Response-latency enhancement produced by WIN 55212-2 (2 mg/kg) was blocked by SR 141716A (5 mg/kg, i.p.), a cannabinoid CB₁ receptor antagonist; attenuated by idazoxan (2 and 5 mg/kg); and not affected by yohimbine (5 mg/kg). These results demonstrate a synergistic interaction between agmatine and cannabinoid agonists and suggest that agmatine administration enhances cannabinoid action in vivo.     

Antagonism between the anti-inflammatory activity of the cannabinoid WIN 55212-2 and SR 141716A

The CB1/CB2 receptor agonist WIN 55212-2 (0.75 mg/kg, i.v.) caused a significant reduction in neurogenic plasma extravasation induced by electrical stimulation of the saphenous nerve in anesthetized rats; WIN 55212-2 at 2.5-10 mg/kg, s.c., also produced a significant reduction in the carrageenan-induced paw edema in conscious rats. The selective CB1 antagonist SR 141716A (0.075-0.75 mg/kg i.v.) antagonized the WIN 55212-2 effects in the plasma extravasation model and antagonized the WIN 55212-2 (2.5 mg/kg, s.c.)-induced decreases in rectal temperature and increases in tail-flick latencies. However, SR 141716A (10 mg/kg, p.o.) failed to antagonize the effects of Win 55212-2 (2.5 mg/kg, s.c.) in the carrageenan model, suggesting that cannabinoid receptors found in the periphery may be able to modulate inflammatory processes in rats.     

Effects of cannabinoids on adrenaline release from adrenal medullary cells.

1. The objective of the present study was to analyse the peripheral effects of cannabinoids on adrenaline release from adrenal chromaffin cells. 2. In pithed rabbits with electrically stimulated sympathetic outflow, intravenous injection of the cannabinoid receptor agonists WIN55212-2 and CP55940 (5, 50 and 500 microg x kg(-1)) markedly lowered the plasma adrenaline concentration. The effect of WIN55212-2 was attenuated by the selective CB1 cannabinoid receptor antagonist SR141716A (500 microg x kg(-1)). WIN55212-3 (same doses as WIN55212-2), the enantiomer of WIN55212-2 lacking affinity for cannabinoid receptors, had no effect on the plasma adrenaline concentration. 3. In rabbit isolated adrenal glands, the release of adrenaline elicited by electrical stimulation was measured by fast cyclic voltammetry. Electrically-evoked adrenaline release was inhibited by WIN55212-2 (0.3, 1, 3 and 10 microM) and this effect was antagonized by SR141716A (1 microM). The non-cholinergic component of adrenaline release observed after blockade of nicotinic (by hexamethonium 100 microM) and muscarinic (by atropine 0.5 microM) acetylcholine receptors was not depressed by WIN55212-2. WIN55212-3 (10 microM) had no effect on adrenaline release. 4. No detectable specific CB1 receptor binding and mRNA expression were found in rabbit adrenal glands with autoradiography and in situ hybridization. 5. The results show that cannabinoids inhibit adrenaline secretion in rabbit isolated adrenal glands; the likely mechanism is a presynaptic CB1 receptor-mediated inhibition of acetylcholine release from preganglionic sympathetic neurons. The inhibition of adrenaline secretion in adrenal glands most probably accounts for the decrease in the plasma adrenaline concentration observed after cannabinoid administration in pithed rabbits.     

Agmatine enhances cannabinoid action in the hot-plate assay of thermal nociception

Agmatine–cannabinoid interactions are supported by the close association between cannabinoid CB₁ receptors and agmatine immunoreactive neurons and evidence that shared brain mechanisms underlie the pharmacological effects of agmatine and cannabinoid agonists. In the present study, we used the hot-plate assay of thermal nociception to determine if agmatine alters cannabinoid action through activation of imidazoline sites and/or alpha₂-adrenoceptors. WIN 55212-2 (1, 2 or 3 mg/kg, i.p.) or CP55,940 (1, 2 or 3 mg/kg, i.p.) administration increased hot-plate response latency. Agmatine (50 or 100 mg/kg, i.p.) was ineffective. Administration of agmatine (50 mg/kg, i.p.) with WIN 55212-2 (1, 2 or 3 mg/kg, i.p.) or CP55,940 (1, 2 or 3 mg/kg, i.p.) produced response-latency enhancement. Regression analysis indicated that agmatine increased the potency of WIN 55212-2 and CP55,940 by 3- and 4.4-fold, respectively, indicating synergy for both drug interactions. Idazoxan, a mixed imidazoline site/alpha₂-adrenoceptor antagonist, but not yohimbine (5 mg/kg, i.p.), a selective alphia₂-adrenoceptor antagonist, blocked response-latency enhancement produced by a combination of WIN 55212-2 (2 mg/kg) and agmatine. Response-latency enhancement produced by WIN 55212-2 (2 mg/kg) was blocked by SR 141716A (5 mg/kg, i.p.), a cannabinoid CB₁ receptor antagonist; attenuated by idazoxan (2 and 5 mg/kg); and not affected by yohimbine (5 mg/kg). These results demonstrate a synergistic interaction between agmatine and cannabinoid agonists and suggest that agmatine administration enhances cannabinoid action in vivo.     

Cannabinoid CB1-mediated inhibition of stress-induced gastric ulcers in rats

The effect of cannabinoid drugs (i.p.) on cold/restraint stress-induced gastric ulcers was studied in rats. The cannabinoid receptor agonist (WIN 55,212-2, 0.1-1 mg/kg), but not the less active isomer WIN 55,212-3 (1 mg/kg), reduced gastric ulceration. The protective effect of WIN 55,212-2 (1 mg/kg) was counteracted by the cannabinoid CB1 receptor antagonist SR141716A, but not by the cannabinoid CB2 receptor antagonist SR144528. These results indicate that the antiulcer effect of the cannabinoid receptor agonist WIN 55,212-2 is mediated by cannabinoid CB1 receptors.     

The cannabinoid receptor agonists,anandamide and WIN 55,212-2, do not directly affect mu opioid receptors expressed in <Emphasis Type="Italic">Xenopus</Emphasis> oocytes

A functional link between the cannabinoid and opioid receptor pathways has been proposed based on data showing that cannabinoid effects can be blocked by opioid receptor antagonists and that cannabinoids can bind to opioid receptors. To explore this link in more detail at the receptor level, we tested the hypothesis that cannabinoids directly activate or modulate mu opioid receptor function. The G-protein coupled mu opioid receptor, MOR-1, and its effector, the G-protein activated potassium channel, GIRK2 (Kir3.2), were expressed together in Xenopus oocytes and potassium currents measured using the two-electrode voltage clamp technique. The specific mu receptor agonist DAMGO activated potassium currents in oocytes expressing the mu receptor that were fully inhibited by the mu receptor antagonist, naloxone. The endogenous cannabinoid, anandamide, and the synthetic cannabinoid, WIN 55,212-2, had no direct effects on potassium currents in the oocytes expressing the mu receptor. The cannabinoids also had no effect on the magnitude of the potassium currents activated by DAMGO or on the desensitization kinetics of MOR-1 in the continued presence of DAMGO. Both WIN 55,212-2 and anandamide activated cannabinoid CB1 receptors when co-expressed with GIRK2 in the oocytes. We conclude that neither anandamide nor WIN 55,212-2 directly activate or modulate mu opioid receptor function in oocytes and that interactions of cannabinoids with mu opioid receptors are likely to be indirect.     

Suppression by WIN55212-2, a cannabinoid receptor agonist, of inflammatory reactions in mouse ear: Interference with the actions of an endogenous ligand, 2-arachidonoylglycerol

The effect of WIN55212-2, a cannabinoid receptor agonist, on acute inflammation of mouse ear was investigated. We found that topical application of WIN55212-2 suppressed ear swelling induced by 12-O-tetradecanoylphorbol 13-acetate or 2-arachidonoylglycerol. Similar inhibition was observed with CP55940, another cannabinoid receptor agonist, and HU-308, a cannabinoid CB(2) receptor-selective agonist. WIN55212-2 also suppressed the infiltration of leukocytes induced by 12-O-tetradecanoylphorbol 13-acetate. On the other hand, WIN55212-3, an inactive enantiomer of WIN55212-2, exerted only small effects on inflammation. Notably, SR144528, a cannabinoid CB(2) receptor antagonist, also suppressed inflammatory reactions in mouse ear. Thus, both the cannabinoid CB(2) receptor agonist and antagonist are capable of reducing inflammatory reactions. We then investigated the mechanism underlying WIN55212-2-induced suppression of inflammation using cultured cells. We found that the addition of WIN55212-2 together with 2-arachidonoylglycerol blocked 2-arachidonoylglycerol-induced migration of human promyelocytic leukemia HL-60 cells that had been differentiated into macrophage-like cells. The restoration of 2-arachidonoylglycerol-desensitized cells and WIN55212-2-desensitized cells from an anergic condition was examined next. We found that 2-arachidonoylglycerol-treated cells rapidly recovered the capacity to respond to 2-arachidonoylglycerol. On the other hand, the anergic condition toward 2-arachidonoylglycerol continued for a longer period after pretreatment with WIN55212-2. These results suggest that the anti-inflammatory activity of WIN55212-2 is attributable, at least in part, to interference with the actions of the endogenous ligand, 2-arachidonoylglycerol.     

Effect of the cannabinoid receptor agonist WIN55212-2 on sympathetic cardiovascular regulation

1. The aim of the present study was to analyse the cardiovascular actions of the synthetic CB1/CB2 cannabinoid receptor agonist WIN55212-2, and specifically to determine its sites of action on sympathetic cardiovascular regulation. 2. Pithed rabbits in which the sympathetic outflow was continuously stimulated electrically or which received a pressor infusion of noradrenaline were used to study peripheral prejunctional and direct vascular effects, respectively. For studying effects on brain stem cardiovascular regulatory centres, drugs were administered into the cisterna cerebellomedullaris in conscious rabbits. Overall cardiovascular effects of the cannabinoid were studied in conscious rabbits with intravenous drug administration. 3. In pithed rabbits in which the sympathetic outflow was continuously electrically stimulated, intravenous injection of WIN55212-2 (5, 50 and 500 microg kg(-1)) markedly reduced blood pressure, the spillover of noradrenaline into plasma and the plasma noradrenaline concentration, and these effects were antagonized by the CB1 cannabinoid receptor-selective antagonist SR141716A. The hypotensive and the sympathoinhibitory effect of WIN55212-2 was shared by CP55940, another mixed CB1/CB2 cannabinoid receptor agonist, but not by WIN55212-3, the enantiomer of WIN55212-2, which lacks affinity for cannabinoid binding sites. WIN55212-2 had no effect on vascular tone established by infusion of noradrenaline in pithed rabbits. 4. Intracisternal application of WIN55212-2 (0.1, 1 and 10 microg kg(-1)) in conscious rabbits increased blood pressure and the plasma noradrenaline concentration and elicited bradycardia; this latter effect was antagonized by atropine. 5. In conscious animals, intravenous injection of WIN55212-2 (5 and 50 microg kg(-1)) caused bradycardia, slight hypotension, no change in the plasma noradrenaline concentration, and an increase in renal sympathetic nerve firing. The highest dose of WIN55212-2 (500 microg kg(-1)) elicited hypotension and tachycardia, and sympathetic nerve activity and the plasma noradrenaline concentration declined. 6. The results obtained in pithed rabbits indicate that activation of CB1 cannabinoid receptors leads to marked peripheral prejunctional inhibition of noradrenaline release from postganglionic sympathetic axons. Intracisternal application of WIN55212-2 uncovered two effects on brain stem cardiovascular centres: sympathoexcitation and activation of cardiac vagal fibres. The highest dose of systemically administered WIN55212-2 produced central sympathoinhibition; the primary site of this action is not known.