Antidepressant like effect of selective serotonin reuptake inhibitors involve modulation of imidazoline receptors by agmatine

Recent findings demonstrated the dysregulation of imidazoline receptor binding sites in major depression and their normalization by chronic treatment with antidepressants including selective serotonin reuptake inhibitors (SSRIs). Present study investigated the role of agmatine and imidazoline receptors in antidepressant like effect of SSRIs and imipramine in mouse forced swimming test (FST) paradigm. The antidepressant like effect of fluoxetine or paroxetine was potentiated by imidazoline I₁/I₂ receptor agonist agmatine (5-10 mg/kg, ip), imidazoline I₁ receptor agonists, moxonidine (0.25-0.5 mg/kg, ip) and clonidine (0.015-0.03 mg/kg, ip), imidazoline I₂ receptor agonist, 2-(2-benzofuranyl)-2-imidazoline (5-10 mg/kg, ip) as well as by the drugs known to increase endogenous agmatine levels in brain viz., l-arginine, an agmatine biosynthetic precursor (40 μg/mouse, icv), ornithine decarboxylase inhibitor, difluoromethyl ornithine (12.5 μg/mouse, icv), diamine oxidase inhibitor, aminoguanidine (6.5 μg/mouse, icv) and agmatinase inhibitor, arcaine (50 μg/mouse, icv). Conversely, prior administration of I₁ receptor antagonist, efaroxan (1 mg/kg, ip), I₂ receptor antagonist, idazoxan (0.25 mg/kg, ip) and arginine decarboxylase inhibitor, d-arginine (100 mg/kg, ip) blocked the antidepressant like effect of paroxetine (10 mg/kg, ip) and fluoxetine (20 mg/kg, ip). On the other hand, antidepressant like effect of imipramine was neither augmented nor attenuated by any of the above drugs. Mice pretreated with SSRIs but not imipramine and exposed to FST showed higher concentration of agmatine in brain as compared to saline control. This effect of SSRIs on agmatine levels was completely blocked by arginine decarboxylase inhibitor d-arginine but not by imidazoline receptor antagonists, efaroxan or idazoxan. These results demonstrate that modulation of imidazoline receptors by agmatine are implicated in the antidepressant like effect of SSRIs and may be projected as a potential therapeutic target for the treatment of depressive disorders.     

Mechanisms involved in the antinociception caused by ethanolic extract obtained from the leaves of Melissa officinalis (lemon balm) in mice

The present study examined the antinociceptive effect of the ethanolic extract from Melissa officinalis L. and of the rosmarinic acid in chemical behavioral models of nociception and investigates some of the mechanisms underlying this effect. The extract (3-1000 mg/kg), given orally (p.o.) 1 h prior to testing, produced dose-dependent inhibition of acetic acid-induced visceral pain, with ID50 value of 241.9 mg/kg. In the formalin test, the extract (30-1000 mg/kg, p.o.) also caused significant inhibition of both, the early (neurogenic pain) and the late (inflammatory pain), phases of formalin-induced licking. The extract (10-1000 mg/kg, p.o.) also caused significant and dose-dependent inhibition of glutamate-induced pain, with ID50 value of 198.5 mg/kg. Furthermore, the rosmarinic acid (0.3-3 mg/kg), given p.o. 1 h prior, produced dose-related inhibition of glutamate-induced pain, with ID50 value of 2.64 mg/kg. The antinociception caused by the extract (100 mg/kg, p.o.) in the glutamate test was significantly attenuated by intraperitoneal (i.p.) treatment of mice with atropine (1 mg/kg), mecamylamine (2 mg/kg) or l-arginine (40 mg/kg). In contrast, the extract (100 mg/kg, p.o.) antinociception was not affected by i.p. treatment with naloxone (1 mg/kg) or d-arginine (40 mg/kg). It was also not associated with non-specific effects, such as muscle relaxation or sedation. Collectively, the present results suggest that the extract produced dose-related antinociception in several models of chemical pain through mechanisms that involved cholinergic systems (i.e. through muscarinic and nicotinic acetylcholine receptors) and the l-arginine-nitric oxide pathway. In addition, the rosmarinic acid contained in this plant appears to contribute for the antinociceptive property of the extract. Moreover, the antinociceptive action demonstrated in the present study supports, at least partly, the ethnomedical uses of this plant.     

Evidence for the involvement of glutamatergic receptors in the antinociception caused in mice by the sesquiterpene drimanial

Drimanial, a new sesquiterpene isolated from the barks of the plant Drimys winteri (Winteraceae), given systemically, intraplantarly, or by spinal or supraspinal routes, produced pronounced antinociception against both phases of formalin-induced licking. The systemic injection of drimanial also inhibited, in a graded manner, the pain-related behaviours induced by intraplantar or intrathecal (i.t.) administration of glutamate. Moreover, drimanial also caused marked inhibition of the nociception induced by i.t. administration of a metabotropic glutamate agonist (1S,3R)-ACPD, without affecting nociceptive responses induced by ionotropic agonists (NMDA, kainate, AMPA) or by substance P. The antinociception caused by drimanial was not influenced by naloxone, nor did it interfere with the motor coordination of animals in the rota-rod test. Furthermore, drimanial caused graded inhibition of [(3)H]glutamate binding in cerebral cortical membranes from mice, with an IC(50) value of 4.39 micro M. Together, these results provide strong evidence indicating that the sesquiterpene drimanial produces antinociception in mice at peripheral, spinal and supraspinal sites. An interaction with metabotropic glutamate receptors seems to contribute to the mechanisms underlying its antinociceptive action.     

The alpha2A-adrenoceptor subtype is not involved in inflammatory hyperalgesia or morphine-induced antinociception

The purpose of the present study was to investigate the role of the alpha(2A)-adrenoceptor subtype in inflammatory hyperalgesia, and in adrenergic-mu-opioid interactions in acute pain and inflammatory hyperalgesia. Behavioral responses to mechanical and thermal stimuli were studied in alpha(2A)-adrenoceptor knockout mice and their wild-type controls. Thermal nociception was evaluated as paw withdrawal latencies to radiant heat applied to the hindpaws. Mechanical nociception was measured using von Frey monofilament applications to the hindpaws. Mechanical and thermal hyperalgesia, induced with intraplantar carrageenan (1 mg/40 microl) were compared in alpha(2A)-adrenoceptor knockout and wild-type mice. The effects of the systemically administered mu-opioid receptor agonist morphine (1-10 mg/kg) were evaluated on mechanical withdrawal responses under normal and inflammatory conditions in knockout and wild-type mice. Withdrawal responses to radiant heat and von Frey monofilaments were similar in alpha(2A)-adrenoceptor knockout and wild-type mice before and after the carrageenan-induced hindpaw inflammation. Also, the antinociceptive effects of morphine in mechanical nociceptive tests were similar before and after carrageenan-induced hindpaw inflammation. Our observations indicate that alpha(2A)-adrenoceptors are not tonically involved in the modulation of inflammation-induced mechanical and thermal hyperalgesia. In addition, alpha(2A)-adrenoceptors do not appear to play an important role in mu-opioid receptor-mediated antinociception or antihyperalgesia.     

Roles of serotonin receptor subtypes for the antinociception of 5-HT in the spinal cord of rats

The contribution of 5-HT (5-hydroxytryptamine) receptor subtypes to the antinociception produced by intrathecal 5-HT in the formalin test was investigated in rats. Intrathecal 5-HT suppressed both phases of behaviors produced by 5% formalin, and this was blocked by antagonists for 5-HT(1B) (3-[3-(Dimethylamino)propyl]-4-hy-droxy-N-[4-(4-pyridinyl)phenyl]benzamide dihydrochloride, GR 55562), 5-HT(2C) (N-ormethylclozapine/8-Chloro-11-(1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine, D-MC), 5-HT3 (1-Methyl-N-(8-methyl-8-azabicyclo[3.2.1]-oct-3-yl)-1H-indazole-3-carboxamide maleate, LY-278,584) and 5-HT4 receptors (4-Amino-5-chloro-2-metho-xy-benzoic acid 2-(diethylamino)ethyl ester hydrochloride, SDZ-205,557), but not the 5-HT(1D) receptor antagonist 3-[4-(4-Chlorophenyl)piperazin-1-yl]-1,1-diphenyl-2-propanol hydrochloride (BRL 15572). The 5-HT(1A) receptor antagonist N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]-N-2-pyridinyl-cyclohexanecarboxamide maleate (WAY-100635) decreased only the second phase antinociception of 5-HT. Intrathecal administration of agonists for 5-HT(1A) (3-(N,N-Dipropylaminoethyl)-1H-indole-5-carboxamide maleate, Dipropyl-5CT), 5-HT(1B) (7-Trifluoromethyl-4(4-met-hyl-1-piperazinyl)-pyrrolo[1,2-a]quinoxaline maleate, CGS-12066A), 5-HT(2C) (6-Ch-loro-2-(1-piperazinyl)pyrazine hydrochloride, MK 212), 5-HT3 (N-(3-Chlorophenyl)imidodicarbonimidic diamide hydrochloride, m-CPBG) and 5-HT4 receptors (2-[1-(4-Piperonyl)piperazinyl]benzothiazole, BZTZ) suppressed both phases of the formalin response. The results of the present study indicate that spinal 5-HT(1B,) 5-HT(2C,) 5-HT3 and 5-HT4 receptors, but not the 5-HT(1D) receptor, mediate antinociception produced by 5-HT in the formalin test. The relevance of the 5-HT(1A) receptor is less clear because of the different effects of antagonist and agonist.     

Involvement of a NO-cyclic GMP-PKG signaling pathway in nitrous oxide-induced antinociception in mice

The antinociceptive effect of nitrous oxide (N₂O) is dependent on nitric oxide (NO); however, the next step in the pathway activated by NO is undetermined. The present study was conducted to test the hypothesis that a N₂O action involves sequential activation of NO synthase, soluble guanylyl cyclase and protein kinase G to induce an antinociceptive effect in mice. The antinociceptive responsiveness of male NIH Swiss mice to N₂O was assessed using the acetic acid abdominal constriction test. Different groups of mice were pretreated with either saline, the NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (carboxy-PTIO), the guanylyl cyclase-inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), the protein kinase G-inhibitor Rp-isomer of 8-(4-chlorophenylthio)-guanosine-3′,5′-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS) or the selective phosphodiesterase V-inhibitor 1,2-dihydro-2-[(2-methyl-4-pyridinyl)methyl]-1-oxo-8-(2-pyrimidinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride (T 0156). Vehicle (saline)-pretreated mice responded to N₂O in a concentration-dependent manner. This antinociceptive effect was antagonized by systemic pretreatment with carboxy-PTIO and ODQ and central pretreatment with Rp-8-pCPT-cGMPS. In each case, the dose–response curve for N₂O was progressively shifted to the right by increasing the dose of each pretreatment drug. On the other hand, N₂O-induced antinociception was enhanced by systemic pretreatment with T 0156; the dose–response curve for N₂O was shifted to the left. The ATP-sensitive potassium channel blocker glibenclamide was without influence on the antinociceptive effect of N₂O. These results support the hypothesis that N₂O-induced antinociception in mice is mediated by a NO–cyclic GMP–PKG pathway.     

Nitric oxide discriminates the sites and mechanisms of action of centrally acting anti-hypertensive drugs in rabbits

The aim of the present study was to further investigate the mechanisms of the central hypotensive action of catecholamines and imidazolines, in particular the role of nitric oxide (NO). Microinjections into the nucleus reticularis lateralis (NRL/RVLM) located in the rostroventrolateral part of the medulla (RVLM) and/or into the nucleus tractus solitarii (NTS) were performed in pentobarbital-anesthetized rabbits. Microinjections of brimonidine (1 ng/kg), which binds both alpha(2)-adrenergic receptors (alpha(2)-ARs) and I(1) imidazoline receptors (I(1)Rs), into the NRL/RVLM induced hypotension (69+/-2 vs. 88+/-2 mm Hg) (p<0.05). Microinjections of S23757 (1 microg/kg), an antagonist highly selective for I(1)Rs, into the same site, prevented the hypotensive effect of brimonidine. These data show that the hypotensive effects of low doses of brimonidine involve the I(1)Rs of the NRL/RVLM. Alpha-methylnoradrenaline (alpha-MNA) (0.5 microg/kg) microinjected into the NTS induced hypotension (76+/-4 vs. 91+/-4 mm Hg) (p<0.05). Microinjections of a low dose of brimonidine (1 ng/kg) into the NTS had no blood pressure (BP) effect at all. In contrast, a higher dose (10 ng/kg) acting on alpha(2)-ARs induced hypotension (72+/-3 vs. 96+/-2 mm Hg) (p<0.05). Nomega-Nitro-L-arginine (L-NNA) (1.5 microg/kg) injected into the NRL/RVLM prevented the hypotensive effect of both alpha-MNA and the higher dose of brimonidine injected into the NTS. Bicuculline (1.5 microg/kg) injected into the NRL/RVLM prevented the hypotensive effect of alpha-MNA injected into the NTS. It is demonstrated that (i) the activation of alpha(2)-ARs of NTS triggers a neuronal GABAergic pathway projecting to the NRL/RVLM region which is NO dependent (ii) both alpha(2)-adrenergic (NTS) and non-adrenergic I(1)R (NRL/RVLM) mechanisms account for the very powerful hypotensive effect of brimonidine, a compound with high affinities at both types of receptors.     

Investigation of the SSRI augmentation properties of 5-HT(2) receptor antagonists using in vivo microdialysis

Recent evidence that 5-HT₂ receptors exert a negative influence on central 5-hydroxytryptamine (5-HT) neurones suggests that 5-HT₂ receptor antagonists may augment the effects of serotonin selective reuptake inhibitors (SSRIs). The present study investigated whether pre-treatment with 5-HT₂ receptor antagonists enhances the effect of SSRI administration on hippocampal extracellular 5-HT of freely moving rats. Administration of the SSRI citalopram at a low (2 mg kg⁻¹) and higher (4 mg kg⁻¹) dose, increased dialysate 5-HT by 5- and 8-fold, respectively. Pre-treatment with the 5-HT₂ receptor antagonist ketanserin (4 mg kg⁻¹) augmented the effect of 4 mg kg⁻¹ but not 2 mg kg⁻¹ citalopram. The effect of 4 mg kg⁻¹ citalopram was also augmented by pre-treatment with either the 5-HT_2C receptor antagonist SB 242084 (0.5 mg kg⁻¹) or the 5-HT_2A receptor antagonist MDL 100907 (0.5 mg kg⁻¹). As with citalopram, fluoxetine elevated dialysate 5-HT at both a low (5 mg kg⁻¹) and higher (20 mg kg⁻¹) dose. However, neither dose of fluoxetine was augmented by ketanserin (4 mg kg⁻¹). These results confirm recent findings that 5-HT₂ receptor antagonists augment the effect of citalopram on extracellular 5-HT, and indicate the involvement of 5-HT_2C and possibly 5-HT_2A receptors. The lack of augmentation of fluoxetine might reflect the intrinsic 5-HT₂ receptor antagonist properties of this drug.     

Pregabalin, S-(+)-3-isobutylgaba, activates the descending noradrenergic system to alleviate neuropathic pain in the mouse partial sciatic nerve ligation model

We have previously demonstrated that gabapentin supraspinally activates the descending noradrenergic system to alleviate neuropathic pain. In this study, we investigated whether pregabalin, an antiepileptic and analgesic drug that is also designed as a structural analogue of gamma-aminobutyric acid (GABA), exhibits supraspinal analgesic effects similar to those of gabapentin involving the descending noradrenergic system. Both systemically (intraperitoneally; i.p.) and locally (intracerebroventricularly or intrathecally; i.c.v. or i.t.) injected pregabalin reduced thermal and mechanical hypersensitivity in a murine chronic pain model that was prepared by partial ligation of the sciatic nerve (the Seltzer model), suggesting that pregabalin acts at both supraspinal and spinal loci. The supraspinal analgesic action of pregabalin was observed only after peripheral nerve injury, and pregabalin (i.p. and i.c.v.) did not affect acute thermal and mechanical nociception. Depletion of spinal noradrenaline (NA) or pharmacological blockade of spinal alpha(2)-adrenoceptors with yohimbine (i.p. or i.t.), but not alpha(1)-adrenoceptors with prazosin (i.p.), reduced the analgesic effects of pregabalin (i.p. or i.c.v.) on thermal and mechanical hypersensitivity. Moreover, i.c.v.-administered pregabalin dose-dependently increased the spinal 4-hydroxy-3-methoxyphenylglycol (MHPG) content and the MHPG/NA ratio only in mice with neuropathic pain, whereas the concentrations of NA, serotonin, 5-hydroxyindoleacetic acid and dopamine were unchanged, demonstrating that supraspinal pregabalin accelerated the spinal turnover of NA. Together, these results indicate that pregabalin supraspinally activates the descending noradrenergic pain inhibitory system coupled with spinal alpha(2)-adrenoceptors to ameliorate neuropathic pain.     

Evidence that 5-HT2C receptor antagonists are anxiolytic in the rat Geller-Seifter model of anxiety

Four non-selective 5-HT_2C/5-HT_2A receptor antagonists, mianserin (2–8 mg/kg), 1-naphthyl piperazine (1-NP) (0.5–1 mg/kg), ICI 169,369 (20 mg/kg) and LY 53857 (5 mg/kg), increased punished responding for a food reward in the rat Geller-Seifter test 30 min after subcutaneous (SC) administration. This property was shared by the benzodiazepine anxiolytic chlordiazepoxide (5 mg/kg SC). However, the selective 5-HT_2A receptor antagonists ketanserin (0.2–1 mg/kg SC) and altanserin (0.5, 1 mg/kg SC) had little effect. The 5-HT_1A, 5-HT_1B and-adrenergic receptor antagonists pindolol and cyanopindolol (6 mg/kg SC) did not affect punished responding either, nor did the 5-HT_1D receptor partial agonist and ₂ adrenergic receptor antagonist yohimbine (2.5 mg/kg SC) or the histamine H₁ receptor antagonist mepyramine (1 mg/kg SC). Unpunished responding was also modestly increased after some doses of the 5-HT_2C/5-HT_2A receptor antagonists. However, this effect was inconsistent and was also seen after chlordiazepoxide. Furthermore, it was not associated with the increase in punished responding observed in rats orally treated with mianserin (10, 20 mg/kg), 1-NP (10, 20 mg/kg) or ICI 169,369 (50 mg/kg). The action of the 5-HT_2C/5-HT_2A receptor antagonists tested is therefore consistent with anxiolysis. The results also strongly suggest that this effect is mediated by blockade of the 5-HT_2C receptor, although the possibility of 5-HT_2B receptor mediation is discussed.     

Anxiolytic-like action of MTEP expressed in the conflict drinking Vogel test in rats is serotonin dependent

The purpose of the present study was to investigate whether the anxiolytic-like action of a selective and brain penetrable group I metabotropic glutamate (mGlu5) receptor antagonist 3-[(2-methyl-1,3-tiazol-4-yl)ethynyl]-pyridine (MTEP) is dependent upon the serotonergic system. Experiments were performed on male Wistar rats. The Vogel conflict drinking test was used to detect anxiolytic-like activity. MTEP administered intraperitoneally at doses of 1, 3 and 6 mg/kg induced anxiolytic-like effect. The potential anxiolytic effect of MTEP (1 mg/kg) was inhibited by a nonselective 5-HT receptor antagonist metergoline (2 mg/kg i.p.) and 5-HT2A/2C receptor antagonist ritanserin (0.5 mg/kg i.p.), but not by a 5-HT1A receptor antagonist N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridynyl)cyclohexane-carboxamide (WAY 100635) (0.1 mg/kg i.p). The anxiolytic effect of MTEP (6 mg/kg) was attenuated by ritanserin (1 mg/kg i.p.). Moreover, MTEP-induced a dose-dependent release of serotonin in the frontal cortex. The obtained results suggest that the potential anxiolytic effect of the mGlu5 receptor antagonist MTEP is due to the increased serotonin release with subsequent activation of 5-HT2A/2C receptors, most probably located postsynaptically, but not by the 5-HT1A receptors.     

5-Hydroxytryptamine receptors with a 5-HT6 receptor-like profile stimulating adenylyl cyclase activity in pig caudate membranes

This study deals with the characterization of 5-hydroxytryptamine (5-HT, serotonin) receptors positively linked to adenylyl cyclase in membranes from pig brain caudate. 5-HT and related agonists induced a concentration-dependent stimulation of adenylyl cyclase activity in pig caudate membranes, with the following rank order of potency (mean pEC₅₀ values): 5-HT (7.1) 5-methoxytryptamine (6.9) > 5-carboxamidotryptamine (5.6) > sumatriptan (<5). Maximal stimulation by 5-HT averaged 35 pmol cyclic AMP/min/mg protein over a basal activity of 159 pmol cyclic AMP/min/mg protein. 5-Methoxytryptamine and 5-carboxamidotryptamine had similar efficacies to that of 5-HT, whereas sumatriptan was about half efficacious. Other compounds known as agonists at some 5-HT receptors were weakly potent (mean pEC₅₀ values <5). They include the 5-HT_1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), the 5-HT₄ receptor agonist, renzapride and the 5-HT₂ receptor agonist, (1-(2,5-dimethoxy-4-iodophenyl)-2 aminopropane) (DOI). In antagonist studies, methiothepin (0.1 and 1 mol/l) shifted the 5-HT curve to the right with no depression of the E_max, yielding pK_B values of 7.4–8.0. Clozapine (1 mol/l) also produced surmountable antagonism of 5-HT-induced effects (pK_B 6.9). Ketanserin (10 mol/l) weakly antagonized 5-HT (pK_B 5.0). The 5-HT₄ receptor antagonists, tropisetron (ICS 205–930) and SDZ 205–557 (2-methoxy-4-amino-5-chloro-benzoic acid 2-(diethylamino) ethyl ester), each at 1 mol/l, did not significantly alter the concentration-response curve of 5-HT. The present receptor shares some characteristics of the recently cloned 5-HT₆ receptor (Monsma et al. (1993) Mol Pharmacol 43:320–327): similar pharmacological profile, location (striatum) and ability to stimulate adenylyl cyclase. It may thus represent the functional 5-HT₆ receptor in its natural environment. Correspondence to: P. Schoeffter at the above address     

The rostroventromedial medulla is not involved in α2-adrenoceptor-mediated antinociception in the rat

The aim of the current study was to investigate the role of the rostroventromedial medulla (RVM) in α₂-adrenoceptor-mediated antinociception. Medetomidine or clonidine, selective α₂-adrenoceptor agonists were microinjected into the RVM in unanesthetized rats with a chronic guide cannula. The antinociceptive effects were evaluated using the tail-flick and hot-plate tests. For comparison, medetomidine was microinjected into the cerebellum or the periaqueductal gray (PAG). To study the role of medullospinal pathways, the tail-flick latencies were also measured in spinalized rats. The reversal of the antinociception induced by intracerebral microinjections of medetomidine was attempted by s.c. atipamezole, a selective α₂-adrenoceptor antagonist. The reversal of the antinociception induced by systemic administration of medetomidine was attempted by microinjections of 5% lidocaine or atipamezole into the RVM. When administered into the RVM, medetomidine produced a dose-dependent (1–30 μg) antinociception in the tail-flick and hot-plate tests, which antinociceptive effect was completely reversed by atipamezole (1 mg/kg, s.c.). Also clonidine produced a dose-dependent (3–30 μg) antinociception following microinjection into the RVM. Microinjections of medetomidine into the cerebellum or the PAG produced an identical dose-response curve in the tail-flick test as that obtained following microinjection into the RVM. In spinalized rats the antinociceptive effect (tail-flick test) induced by medetomidine microinjected into the RVM was not less effective than in intact rats. Lidocaine (5%) or atipamezole (5 μg) microinjected into the RVM did not attenuate the antinociception induced by systemically administered medetomidine (100 μg/kg, s.c.). The adapting skin temperature of the tail was increased in a nonmonotonic fashion following medetomidine. The results indicate that the RVM is not a site which is critical for the α₂-adrenergic antinociception. The antinociception following intracerebral microinjections of medetomidine into the RVM, PAG or the cerebellum in the current study can be explained by a spread of the α₂-adrenoceptor agonist into the spinal level to activate directly spinal α₂-adrenoceptors. Also, the antinociception following systemic administration of medetomidine can be explained by spinal α₂-adrenergic mechanisms. The medetomidine-induced increase of the adapting skin temperature may have attenuated the medetomidine-induced increases in the response latencies to noxious heat.     

Mechanisms involved in the antinociception induced by systemic administration of guanosine in mice

Background and purpose:

It is well known that adenine-based purines exert multiple effects on pain transmission. However, less attention has been given to the potential effects of guanine-based purines on pain transmission. The aim of this study was to investigate the effects of intraperitoneal (i.p.) and oral (p.o.) administration of guanosine on mice pain models. Additionally, investigation into the mechanisms of action of guanosine, its potential toxicity and cerebrospinal fluid (CSF) purine levels were also assessed.

Experimental approach:

Mice received an i.p. or p.o. administration of vehicle (0.1 mM NaOH) or guanosine (up to 240 mg·kg⁻¹) and were evaluated in several pain models.

Key results:

Guanosine produced dose-dependent antinociceptive effects in the hot-plate, glutamate, capsaicin, formalin and acetic acid models, but it was ineffective in the tail-flick test. Additionally, guanosine produced a significant inhibition of biting behaviour induced by i.t. injection of glutamate, AMPA, kainate and trans-ACPD, but not against NMDA, substance P or capsaicin. The antinociceptive effects of guanosine were prevented by selective and non-selective adenosine receptor antagonists. Systemic administration of guanosine (120 mg·kg⁻¹) induced an approximately sevenfold increase on CSF guanosine levels. Guanosine prevented the increase on spinal cord glutamate uptake induced by intraplantar capsaicin.

Conclusions and implications:

This study provides new evidence on the mechanism of action of the antinociceptive effects after systemic administration of guanosine. These effects seem to be related to the modulation of adenosine A₁ and A_2A receptors and non-NMDA glutamate receptors.     

Neuropharmacological reassessment of the discriminative stimulus properties ofd-lysergic acid diethylamide (LSD)

The neuropharmacological mechanisms underlying the behavioral effects ofd-lysergic acid diethylamide (LSD) were assessed by comparing the discriminative stimulus properties of LSD with those of agonists and antagonists that act selectively at putative serotonin (5-hydroxytryptamine; 5-HT) receptor subtypes (5-HT₁ and 5-HT₂). Male Sprague-Dawley rats (N=23) were trained to discriminate LSD (0.08 mg/kg) from saline and given substitution tests with the following agents: 8-hydroxy-2(di-n-propylamino) tetralin (8-OHDPAT; 0.02–0.64 mg/kg), Ru 24969 (0.2–3.2 mg/kg),m-chlorophenylpiperazine (MCPP; 0.1–1.6 mg/kg), 1-(m-trifluoromethylphenyl)piperazine (TFMPP; 0.1–1.6 mg/kg), and quipazine (0.2–3.2 mg/kg). Only quipazine mimicked LSD. In combination tests, BC 105 (0.2–3.2 mg/kg), 2-bromolysergic acid diethylamide (BOL; 0.1–1.6 mg/kg), Ly 53857 (0.4–3.2 mg/kg), metergoline (0.05–0.8 mg/kg), ketanserin (0.2–3.2 mg/kg), and pipenperone (0.0025–0.08 mg/kg), all of which act as 5-HT₂ antagonists, blocked the LSD cue; only spiperone (0.02–0.32 mg/kg) was without effect. Although commonalities may exist among 5-HT agonists, the present results demonstrate that such agonists are not identical. Since putative 5-HT₁ agonists do not mimic LSD and the LSD cue is potently blocked by 5-HT₂ antagonists, it appears that 5-HT₂ neuronal systems are of greater importance than 5-HT₁ systems in mediating the discriminative stimulus and, perhaps, other effects of LSD.     

Characterization of putative 5-HT7 receptors mediating tachycardia in the cat

1. It has been suggested that the tachycardic response to 5-hydroxytryptamine (5-HT) in the spinal-transected cat is mediated by ‘5-HT₁-like'' receptors since this effect, being mimicked by 5-carboxamidotryptamine (5-CT), is not modified by ketanserin or MDL 72222, but it is blocked by methiothepin, methysergide or mesulergine. The present study was set out to reanalyse this suggestion in terms of the IUPHAR 5-HT receptor classification schemes proposed in 1994 and 1996.
2. Intravenous (i.v.) bolus injections of the tryptamine derivatives, 5-CT (0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 μg kg⁻¹), 5-HT (3, 10 and 30 μg kg⁻¹) and 5-methoxytryptamine (3, 10 and 30 μg kg⁻¹) as well as the atypical antipsychotic drug, clozapine (1000 and 3000 μg kg⁻¹) resulted in dose-dependent increases in heart rate, with a rank order of agonist potency of 5-CT >> 5-HT > 5-methoxytryptamine >> clozapine.
3. The tachycardic effects of 5-HT and 5-methoxytryptamine were dose-dependently antagonized by i.v. administration of lisuride (30 and 100 μg kg⁻¹), ergotamine (100 and 300 μg kg⁻¹) or mesulergine (100, 300 and 1000 μg kg⁻¹); the highest doses of these antagonists used also blocked the tachycardic effects of 5-CT. Clozapine (1000 and 3000 μg kg⁻¹) did not affect the 5-HT-induced tachycardia, but attenuated, with its highest dose, the responses to 5-methoxytryptamine and 5-CT. However, these doses of clozapine as well as the high doses of ergotamine (300 μg kg⁻¹) and mesulergine (300 and 1000 μg kg⁻¹) also attenuated the tachycardic effects of isoprenaline. In contrast, 5-HT-, 5-methoxytryptamine- and 5-CT-induced tachycardia were not significantly modified after i.v. administration of physiological saline (0.1 and 0.3 ml kg⁻¹), the 5-HT_1B/1D receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (500 μg kg⁻¹) or the 5-HT_3/4 receptor antagonist, tropisetron (3000 μg kg⁻¹).
4. Intravenous injections of the 5-HT₁ receptor agonists, sumatriptan (30, 100 and 300 μg kg⁻¹) and indorenate (300 and 1000 μg kg⁻¹) or the 5-HT₄ receptor (partial) agonist cisapride (300 and 1000 μg kg⁻¹) were devoid of effects on feline heart rate per se and failed to modify significantly 5-HT-induced tachycardic responses.
5. Based upon the above rank order of agonist potency, the failure of sumatriptan, indorenate or cisapride to produce cardioacceleration and the blockade by a series of drugs showing high affinity for the cloned 5-ht₇ receptor, the present results indicate that the 5-HT receptor mediating tachycardia in the cat is operationally similar to other putative 5-HT₇ receptors mediating vascular and non-vascular responses (e.g. relaxation of the rabbit femoral vein, canine external carotid and coronary arteries, rat systemic vasculature and guinea-pig ileum). Since these responses represent functional correlates of the 5-ht₇ gene product, the 5-HT₇ receptor appellation is reinforced. Therefore, the present experimental model, which is not complicated by the presence of other 5-HT receptors, can be utilized to characterize and develop new drugs with potential agonist and antagonist properties at functional 5-HT₇ 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.     

Characterization of the contraction to 5-HT in the canine colon longitudinal muscle

1. Although conscious dogs have often been used for colonic motility studies with 5-hydroxytryptamine (5-HT), the effects of 5-HT on the isolated colon have not been thoroughly characterized yet. The current study was undertaken to characterize the response to 5-HT of the canine isolated colon longitudinal muscle.
2. Longitudinal strips of canine midcolon deprived of (sub)mucosa were prepared for isotonic measurement. 5-HT induced contractions from 3 nM onwards, which were not affected by selective inhibition of 5-HT re-uptake, monoamine oxidase or blockade of α-adrenoceptors. Tetrodotoxin (0.3 μM) did not affect the responses to 5-HT, suggesting that smooth muscle 5-HT receptors are involved. The selective 5-HT₄ receptor antagonist SB 204070 (10 nM) slightly enhanced contractions to 5-HT and therefore it was included in the organ bath solution in all further experiments. The 5-HT₁ and 5-HT₂ receptor antagonist methysergide (0.1 μM) depressed the curve to 5-HT, but the selective 5-HT₃ receptor antagonist granisetron (0.3 μM) had no effect.
3. Besides 5-HT, α-methyl-5-HT (α-Me-5-HT), 5-methoxytryptamine (5-MeOT), 2-methyl-5-HT (2-Me-5-HT) and 5-carboxamidotryptamine (5-CT) also induced contractions, with the following rank order of potency (pEC₅₀ values in parentheses): 5-HT (6.9)=α-methyl-5-HT (6.9)>2-Me-5-HT (5.8)=5-MeOT (5.7)=5-CT (5.6), indicative of 5-HT₂ receptor involvement. α-Me-5-HT produced a bell-shaped curve, which was not affected by α-adrenoceptor blockade. 5-HT, 5-MeOT, 2-Me-5-HT and 5-CT produced a monophasic concentration-response curve, consistent with an interaction with a single receptor site. 8-Hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and tryptamine only induced contractions at a concentration exceeding 1 μM.
4. The selective 5-HT_2B receptor antagonist SB 204741 (0.3 μM) did not affect the curve to 5-HT. Ketanserin, cisapride and spiroxatrine behaved as competitive antagonists with pK_b values of, respectively, 8.4, 8.1 and 6.7. Spiroxatrine (1 μM) shifted the curve to 5-MeOT rightward yielding an apparent pA₂ of 7.1. Other antagonists at 5-HT_2A receptors also surmountably inhibited the contractions to 5-HT (apparent pA₂ value in parentheses): mesulergine (8.2), cinanserin (8.2), yohimbine (6.2) and mianserin (8.6). However, as well as a rightward shift, methiothepin (8.3), pizotifen (8.6) and spiperone (8.8) also caused a depression of the curve, indicative of ‘pseudo-irreversible'' antagonism. Taken together, the above mentioned affinity estimates most closely corresponded to literature affinity values for 5-HT_2A receptors.
5. It was concluded that 5-HT induces contractions of the canine midcolon longitudinal muscle primarily by stimulation of smooth muscle 5-HT_2A receptors. The presence of inhibitory 5-HT₄ receptors cannot be ruled out.

     

5-HT1-like and 5-HT2A receptors mediate 5-hydroxytryptamine-induced contraction of rabbit isolated mesenteric artery

The contractions induced by 5-hydroxytryptamine (5-HT) and the 5-HT₁-like receptor agonist, sumatriptan, were investigated in the open ring preparations of rabbit mesenteric artery in order to characterize the 5-HT receptors. 5-HT induced concentration-dependent contractions. Sumatriptan did not induce any contraction of unstimulated rings, whereas it elicited concentration-dependent contractions in preparations given a moderate tone by a threshold concentration of prostaglandin F₂ (PGF₂). Pargyline, cocaine or normetanephrine were without significant effect on the contractions induced by 5-HT and sumatripan. The 5-HT concentration-effect curve was clearly biphasic. Methiothepin (0.01 M) shifted the both phases of the concentration-effect curve to the right. Ketanserin (0.1 M) shifted the second, low affinity, phase and prazosin did not alter concentration-effect curve to 5-HT. The sumatriptan concentration-effect curve was shifted by methiothepin (0.01 M) to the right (pK_B = 9.19) but not by ketanserin (1 M). Concentration-effect curves to 5-HT and sumatriptan were not affected by the 5-HT₃ receptor antagonist tropisetron (1 M). These results suggest that 5-HT₁-like type receptors are responsible for the first phase of 5-HT-induced contraction and 5-HT_2A receptor for the second phase, in rabbit mesenteric artery. Sumatriptan-induced contractions appear to be mediated by 5-HT₁-like type receptors in this artery. These results also suggest that this kind of amplification may be a common feature of vascular 5-HT₁-like type receptor as has been shown in other vascular segments such as rabbit femoral, iliac and renal arteries, and guinea-pig iliac artery.     

Blockade of phencyclidine-induced hyperlocomotion by olanzapine, clozapine and serotonin receptor subtype selective antagonists in mice

In humans, phencyclidine (PCP) is known to produce a syndrome of behavioral effects which have many characteristics in common with schizophrenia. Therefore, antagonism of PCP effects might be evidence for antipsychotic efficacy of a compound. In the present studies, the effects of the D₂-like antagonist haloperidol, the mixed D₂-like/5-HT₂ antagonists olanzapine and clozapine, and a series of 5-HT receptor subtype selective antagonists on the hyperlocomotion produced by PCP were evaluated in mice. PCP (0.3–10 mg/kg) produced a dose-related increase in locomotor activity, with a peak effect at 3.0 mg/kg. The D₂-like antagonist haloperidol produced a dose-related decrease in locomotor activity when administered alone, and blocked the hyperactivity effects of PCP over the same dose-range (minimal effective dose, MED = 0.3 mg/kg for both effects). In contrast, olanzapine and clozapine reversed the hyperlocomotion effects of PCP at doses (MED = 0.03 and 0.3 mg/kg, respectively) approximately 30-and 10-fold, respectively, below those that decreased activity when administered alone (MED = 1.0 and 3.0 mg/kg, respectively). The selective 5-HT₂ antagonist LY53857 (0.3–3.0 mg/kg) administered alone had no effect on locomotor activity but reversed (MED = 0.1 mg/kg) the effects of PCP. Similarly, the selective 5-HT_2A/2C antagonist ritanserin (0.001–1.0 mg/kg) alone had no effect on locomotor activity, but reversed (MED = 0.01 mg/kg) the effects of PCP. The selective 5-HT_2A antagonists ketanserin (MED = 3.0 mg/kg) and MDL 100,907 (MED = 0.3 mg/kg) produced dose-related decreases in locomotor activity and ketanserin (MED = 0.1 mg/kg) and MDL 100,907 (MED = 0.003 mg/kg) reversed the effects of PCP. The selective 5-HT₃ antagonist zatosetron (0.01–10 mg/kg) and the selective 5-HT_1A antagonist WAY 100,635 (0.001–3 mg/kg) were without effects on spontaneous locomotor activity. Zatosetron reversed the effects of 3.0 mg/kg PCP at the nonselective dose of 10 mg/kg whereas WAY 100,635 (0.001–1 mg/kg) did not affect PCP-induced hyperlocomotion. The present results indicate that PCP increases locomotor activity, at least in part, due to actions at 5-HT_2A, but not 5-HT₃ or 5-HT_1A, receptors. Further, the present findings support the hypothesis that antagonism at 5-HT_2A receptors contributes to the in vivo actions of atypical antipsychotics such as olanzapine and clozapine. Received: 27 June 1996/Final version: 20 August 1996