European Journal of Pharmacology (v.396, #2-3)

Effect of norepinephrine release on adrenoceptors in severe seizure genetically epilepsy-prone rats by Dong Ook Seo; Chan Young Shin; Jae Ryun Ryu; Jae Hoon Cheong; Chang-Rak Choi; John W Dailey; Maarten E.A Reith; Phillip C Jobe; Kwang Ho Ko (53-58).
The genetically epilepsy-prone rat (GEPR) seizure model is characterized by extensive abnormalities in brain noradrenergic function. Earlier studies had suggested that GEPRs might not regulate adrenoceptors in a normal fashion. The purpose of the present study was to determine if GEPR-9s are capable of up and down regulation of α1- and β-adrenoceptors in response to increments or decrements in extracellular norepinephrine. Seizure induction has been shown to increase extracellular norepinephrine. Chronic sound or electroshock-induced seizures caused down regulation of β-adrenoceptors in frontal cortex and in hippocampus from GEPR-9s. Similarly, chronic daily treatment with the norepinephrine reuptake inhibitor desmethylimipramine produced down regulation of β-adrenoceptors in frontal cortex and in hippocampus from GEPR-9s. As is the case in neurologically normal animals, chronic electroshock-induced seizure did not cause down regulation of β-adrenoceptors in 6-hydroxydopamine pretreated GEPR-9s. Chronic electroshock treatment also caused up-regulation of α1-adrenoceptors in frontal cortex but not in hippocampus. In 6-hydroxydopamine pretreated GEPR-9s, chronic electroshock treatment caused a further up-regulation of α1-adrenoceptors in frontal cortex but not in hippocampus. Taken together, these results indicate that GEPR-9s are capable of up and down regulation of α1- and β-adrenoceptors in a manner that is qualitatively similar to the regulation of these receptors in normal animals. Whether the regulation of brain adrenoceptors is quantitatively different in GEPRs from normal animals remains to be established.
Keywords: GEPR (genetically epilepsy-prone rat); Electroshock; Convulsion; α1-Adrenoceptor; β-Adrenoceptor; Receptor regulation;

The pharmacology of a dopamine receptor in the locust nervous tissue by Joern Degen; Michael Gewecke; Thomas Roeder (59-65).
A dopamine receptor in the nervous tissue of the desert locust (Schistocerca gregaria Forskål) was studied using [3H]lysergic acid diethylamide (LSD) as the radioligand. Its expression is almost entirely restricted to the mushroom bodies, centres for learning and memory in the insect brain. This G-protein coupled receptor is present in relatively low concentrations in the locust brain (35 fmol/mg protein). The pharmacological characterisation reveals high affinity for the putative natural agonist dopamine (K i=28 nM). Substances with high subtype specificity for vertebrate dopamine receptors such as SCH 23390 (K i=639 nM) and sulpiride (K i=21,200 nM) have low affinity for the locust neuronal dopamine receptor. In opposite, substances with a broad pharmacological profile such as LSD, spiperone (K i=7.26 nM), and chlorpromazine (K i=9.52 nM) have high affinity properties. Comparison of the pharmacological data reveals no significant homology to any vertebrate dopamine receptor class characterised so far. This uncertainty about the pharmacological relatedness of insect dopamine receptors mirrors the available molecular data. It is almost impossible to classify cloned insect dopamine receptors into vertebrate dopamine receptor schemes. This lack of pharmacological relatedness opens the opportunity to develop highly specific insecticides against insect dopamine receptors.
Keywords: Dopamine receptor; [3H]Lysergic acid diethylamide (LSD); (Locust); (Insect); Mushroom body;

Activation of G protein by opioid receptors: role of receptor number and G-protein concentration by Ann E Remmers; Mary J Clark; Andrew Alt; Fedor Medzihradsky; James H Woods; John R Traynor (67-75).
The collision-coupling model for receptor-G-protein interaction predicts that the rate of G-protein activation is dependent on receptor density, but not G-protein levels. C6 cells expressing μ- or δ-opioid receptors, or SH-SY5Y cells, were treated with β-funaltrexamine (μ) or naltrindole-5′-isothiocyanate (δ) to decrease receptor number. The time course of full or partial agonist-stimulated [35S]GTPγS binding did not vary in C6 cell membranes containing <1–25 pmol/mg μ-opioid receptor, or 1.4–4.3 pmol/mg δ-opioid receptor, or in SHSY5Y cells containing 0.16–0.39 pmol/mg receptor. The association of [35S]GTPγS binding was faster in membranes from C6μ cells than from C6δ cells. A 10-fold reduction in functional G-protein, following pertussis toxin treatment, lowered the maximal level of [35S]GTPγS binding but not the association rate. These data indicate a compartmentalization of opioid receptors and G protein at the cell membrane.
Keywords: μ-Opioid receptor; δ-Opioid receptor; G-protein; [35S]GTPγS binding; Collision-coupling model; Compartmentalization;

Antinociceptive activity of combination of morphine and NMDA receptor antagonists depends on the inter-injection interval by Irina V Belozertseva; Olga A Dravolina; Olga N Neznanova; Wojciech Danysz; Anton Y Bespalov (77-83).
The actual time-course of morphine antinociception is shorter than what would be predicted from its elimination kinetics, suggesting the presence of an acute tolerance phenomenon. Since antagonists acting at NMDA subtype of glutamate receptors were repeatedly shown to prolong acute morphine antinociception, acute tolerance may be attributed to hyperactivity of NMDA receptors. The ability of various site-selective NMDA receptor antagonists to affect morphine antinociception (tail-flick test) was assessed in mice 30 and 120 min after acute morphine challenge. Competitive NMDA receptor antagonist 3-(2-carboxypiperazin-4-yl)-1-propenyl-1-phosphonic acid (d-CPPene) (SDZ EAA 494; 0.1–1 mg/kg), low-affinity channel blockers 1-amino-3,5-dimethyl adamantane (memantine) (1–10 mg/kg) and 1-amino-1,3,3,5,5-pentamethyl-cyclohexan hydrochloride (MRZ 2/579) (1–10 mg/kg), glycine site antagonists 5-nitro-6,7-dichloro-1,4-dihydro-2,3-quinoxalinedione (ACEA-1021) (5 or 10 mg/kg) and 8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridaliono(4,5-b)quinoline-5-oxide choline salt (MRZ 2/576) (1–10 mg/kg) were administered intraperitoneally (i.p.) 15 or 30 min prior to the tail-flick test (i.e., interval between injections of morphine and NMDA receptor antagonist was either 0–15 or 90–105 min). ACEA-1021, MRZ 2/576 and to the lesser extent, memantine and MRZ 2/579 enhanced morphine antinociception when tests were conducted 120 but not 30 min post-morphine. d-CPPene potentiated morphine antinociception irrespective of the interval between morphine administration and the tail-flick test. The results suggest that NMDA receptor antagonists may restore analgesic activity of morphine in acutely tolerant mice.
Keywords: Morphine; Antinociception; NMDA receptor antagonist; d-CPPene (SDZ EAA 494); Memantine; MRZ 2/579; MRZ 2/576; ACEA-1021;

Role of the endogenous cannabinoid system in the formalin test of persistent pain in the rat by Pierre Beaulieu; Tiziana Bisogno; Shahid Punwar; W.Paul Farquhar-Smith; Gerolmina Ambrosino; Vincenzo Di Marzo; Andrew S.C Rice (85-92).
It has been suggested that administration of a cannabinoid CB1 (SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1-H-pyrazole-3-carboxamide]) and CB2 (SR144528 {N-[(1S)-endo-1, 3, 3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide}) receptor antagonists to mice potentiates inflammatory hyperalgesia by removing an endogenous cannabinoid tone. We examined whether the behavioural response to s.c. formalin injection in rats is similarly enhanced. A total of 30 animals received SR141716A (0.5 or 5 mg/kg) or SR144528 (0.3 or 3 mg/kg) 30 min before 1% formalin. Pain behaviour was quantified using the composite weighted pain score technique (CPS-WST0,1,2). An overall CPS-WST0,1,2 was calculated for each phase and groups were compared (analysis of variance). The results obtained in the control group confirmed the characteristic biphasic behavioural response to formalin injection. None of antagonist groups had a significant increase in overall CPS-WST0,1,2 compared to the control. Indeed, a significant decrease in CPS-WST0,1,2 scores for both phases was detected in most of all of the groups, except SR141716A at 5 mg/kg. Levels of endogenous cannabinoids (anandamide, palmitoylethanolamide, 2-arachidonylglycerol) were measured from rats hind-paw skin 1 h after s.c. injection of 0.9% saline (100 μl), 1% (50 μl), 2.5% (50 μl) and 5% (100 μl) formalin. The concentration of endocannabinoids did not differ between control and formalin-induced inflammation groups. The activity of anandamide amidohydrolase in hind-paw skin also did not change after treatment with formalin. In conclusion, cannabinoid antagonists do not enhance formalin-evoked pain behaviour. These results suggest that, in this model, endogenous cannabinoids do not tonically attenuate inflammatory hyperalgesia.
Keywords: Cannabinoid CB1 receptor; Cannabinoid CB2 receptor; Cannabinoid receptor antagonist; Hyperalgesia; Anandamide; Palmitoylethanolamide; 2-Arachidonylglycerol; Nociception; Inflammation;

Lack of synergism between caffeine and SKF 38393 on rotational behavior in 6-hydroxydopamine-denervated rats by Miquel Casas; Gemma Prat; Antonia Rubio; Manel Barbanoj; Francesc Jané (93-99).
We have recently shown a synergistic effect between caffeine and the dopamine D2 receptor agonist, bromocriptine, on contralateral rotational behavior in unilaterally 6-hydroxydopamine-denervated rats. In addition, we found that bromocriptine prevented caffeine-induced tolerance to this behavior following repeated treatment. In the present study, we investigated whether or not the dopamine D1 receptor agonist, (±)-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol (SKF 38393), presented similar characteristics. Different groups of rats received simultaneous injections of either vehicle plus vehicle, caffeine (40 mg/kg) plus vehicle, SKF 38393 (0.5, 1, 2, and 4 mg/kg) plus vehicle, or caffeine plus SKF 38393 (0.5, 1, 2, and 4 mg/kg) for 5 consecutive days, and both ipsilateral and contralateral rotational behavior was measured. Results showed that, on the first day of treatment, caffeine produced significantly more rotational behavior than did a low dose of SKF 38393 (0.5 mg/kg), and significantly less turning than at higher doses (2 and 4 mg/kg). Combined treatment with caffeine and a high dose of SKF 38393 (4 mg/kg) produced significantly more rotational behavior than did caffeine plus vehicle. With repeated administration, caffeine produced sustained tolerance to its effects on rotational behavior, whereas SKF 38393 did not. In the groups treated with low doses of SKF 38393 (0.5, and 1 mg/kg) plus caffeine, tolerance was observed while in the groups that received high doses of SKF 38393 (2 and 4 mg/kg) plus caffeine, no tolerance was observed to rotational behavior. These results suggest that maximal stimulation of dopamine D1 receptors may be needed to prevent the tolerance effects of caffeine in this animal model. This finding may have clinical relevance to the therapeutic treatment of Parkinson's disease.
Keywords: Dopamine D1 receptor; 6-Hydroxydopamine; Turning behavior; Behavioral tolerance;

Antiparkinson potential of δ-opioid receptor agonists by Thomas J Hudzik; Andrew Howell; Kemal Payza; Alan J Cross (101-107).
δ-Opioid receptors, present in very high concentrations in striatum and overlying cortex, are thought to be involved in a number of processes, including analgesia, mood, reward, modulation of neuronal excitability, and alterations in neurotransmitter release. Given the localization of the receptors in motor circuits in brain, we thought it of interest to study the antiparkinson potential of δ-opioid receptor agonists. Rats were given unilateral 6-hydroxydopamine lesions of the nigrostriatal tract, and following recovery, were tested for rotational activity. Tonazocine mesylate is a nonpeptide, partial δ-opioid receptor agonist with μ-receptor antagonist properties. Tonazocine (0.1–10 mg/kg) evoked a dose-related, ipsilateral rotation, consistent with augmentation of dopaminergic function on the unlesioned side. The rotation evoked by tonazocine was blocked by the selective δ-opioid receptor antagonist naltrindole, suggesting that the effect was mediated by δ-opioid receptors. The full δ-opioid receptor agonist (+)-4-[9-α-R)-α-(2S,5RO-4-allyl-2,5-dimethyl-1-piperaziny l)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80) produced both contralateral and ipsilateral rotation. Tonazocine additionally augmented the effects of l-3,4 dihydroxyphenylalanine (l-DOPA) on reserpine-induced suppression of motor activity. Binding affinities and efficacies of tonazocine and SNC-80 against μ-, κ-, and δ-opioid receptors were also confirmed and compared to standards. These data suggest therapeutic potential of agents interacting with δ-opioid receptors, and indicate some differences in the activities of tonazocine and SNC-80.
Keywords: δ-Opioid receptor agonist; Tonazocine; SNC-80; Rotation; Parkinson's disease;

Negative inotropic effect of endothelin-1 in the mouse right ventricle by Masanori Izumi; Shigeki Miyamoto; Masatoshi Hori; Hiroshi Ozaki; Hideaki Karaki (109-117).
Effects of endothelin-1 on the contraction and cytosolic Ca2+ concentrations ([Ca2+]i) of the mouse right ventricle were investigated. Endothelin-1 (1–300 nM) elicited a negative inotropic effect in a concentration-dependent manner. The endothelin-1-induced negative inotropy was antagonized by a selective endothelin ETA receptor antagonist, BQ-123 (cyclo [Asp-Pro-Val-Leu-Trp-]; 3, 10 μM). Endothelin-1 reduced the peak amplitudes of both the [Ca2+]i transient and contraction without changing inward Ca2+ current. The relationship between peak amplitude of [Ca2+]i and peak force generated by changing the extracellular Ca2+ concentration ([Ca2+]o) was not affected by endothelin-1. In addition, the trajectory of the [Ca2+]i-contraction phase plane diagram obtained at 2 mM [Ca2+]o in the absence of endothelin-1 was superimposable on that obtained at 4 mM [Ca2+]o in the presence of endothelin-1 (300 nM). Endothelin-1 (300 nM) translocated protein kinase C from cytosol to membrane, suggesting activation of protein kinase C. Further, a selective protein kinase C inhibitor, bisindolylmaleimide I (10 μM), inhibited the endothelin-1-induced negative inotropy. These results suggest that endothelin-1 elicits negative inotropy by reducing the amplitude of the [Ca2+]i transient without changing inward Ca2+ current through the activation of the endothelin ETA receptor followed by protein kinase C activation in the mouse right ventricle.
Keywords: Endothelin-1; Cardiac muscle; [Ca2+]i transient; Protein kinase C;

The sodium–hydrogen exchanger-isotype 1 (NHE-1) plays a critical role in myocardial ischemia–reperfusion injury. While studies employing less selective sodium–hydrogen inhibitors have demonstrated antiarrhythmic activity, only one study has examined the in vivo efficacy of selective NHE-1 inhibition in a canine model of ischemia–reperfusion-induced arrhythmia. In the present study, the antiarrhythmic activity of Benzamide,N-(aminoiminomethyl)-4-[4-(2-furanylcarbonyl)-1-piperazinyl]-3-(methylsulfonyl), methanesulfonate (BIIB 513), a novel NHE-1 inhibitor, was examined. An in vivo canine model of myocardial ischemia–reperfusion injury in which 60 min of left anterior descending coronary artery (LAD) occlusion followed by 3 h of reperfusion was employed. BIIB 513 was infused either prior to ischemia or prior to reperfusion. Arrhythmias were quantified by single lead electrocardiogram. Infarct size, determined by triphenyltetrazolium staining, was expressed as a percent of the area-at-risk. In vivo, NHE-1 inhibition did not affect phase 1a arrhythmias, which occur within the first 10 min of occlusion, however, BIIB 513 significantly reduced the incidence of ischemia-induced phase 1b arrhythmias which occur between 10 and 30 min following occlusion and the incidence of reperfusion-induced ventricular fibrillation. Furthermore, NHE-1 inhibition significantly reduced infarct size, when the drug was administered either prior to ischemia or prior to reperfusion. NHE-1 inhibition selectively reduces both ischemia-induced phase 1b arrhythmias and reperfusion-induced ventricular fibrillation, and also markedly reduces myocardial infarct size when the drug is administered prior to ischemia or prior to reperfusion.
Keywords: Sodium–hydrogen exchanger; Arrhythmias; Myocardial infarct size;

Aminoguanidine prevents concanavalin A-induced hepatitis in mice by Toshihiro Okamoto; Yoriko Masuda; Toru Kawasaki; Motoki Shinohara; Katsuhiro Matsuzaki (125-130).
Aminoguanidine is an inhibitor of the inducible form of nitric oxide synthase (iNOS). In the present study, the effect of aminoguanidine on concanavalin A-induced hepatitis was examined. Treatment of mice with concanavalin A (10 mg/kg, i.v.) induced interferon-γ and iNOS mRNA expression in the liver before the elevation of plasma alanine aminotransferase activity. Immunohistochemical study showed the induction of iNOS protein expression in the area of necrosis. Aminoguanidine (1, 3 and 10 mg/kg, i.p.) inhibited the concanavalin A-induced elevation of alanine aminotransferase activity. Aminoguanidine (10 mg/kg, i.p.) did not inhibit concanavalin A-induced interleukin-2, interferon-γ, tumor necrosis factor-α or iNOS mRNA expression in the liver. The plasma nitrite/nitrate level was elevated at 6 and 24 h after concanavalin A treatment. The elevation of nitrite/nitrate was inhibited by aminoguanidine (10 mg/kg, i.p.). From these results, we conclude that nitric oxide formed by iNOS may be involved in the development of concanavalin A-induced hepatitis.
Keywords: Concanavalin A; Aminoguanidine; Hepatitis;

Clonidine injected intracerebroventricularly (i.c.v.) (0.47 nmol/rat) exerted gastric mucosal protective effect against acidified ethanol. Evidence was obtained that the gastroprotective effect of clonidine was blocked by i.c.v. injected α2-adrenoceptor antagonists yohimbine (non-subtype selective antagonist), prazosin and 2-[2-(4-(O-methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione (ARC-239) (representative α2B/2C-adrenoceptor blocking agents) and opioid receptor antagonists naloxone (a non-selective, moderately μ-opioid receptor preferring antagonist), naltrindole and naltriben δ-opioid receptor antagonists). The centrally injected naltrindole (0.5 nmol/rat) antagonised also the gastroprotective effect of clonidine — but not that of the δ-agonist [d-Ala2, d-Leu5]enkephalin — administered peripherally. The results suggest that central α2B/2C-adrenoceptor subtypes and opioid — particularly δ — receptors are likely to be involved in the gastric mucosal protective effect of clonidine.
Keywords: Gastroprotection; Clonidine; α2B-Adrenoceptor; Opioid receptor;

Specific uptake of 5-hydroxytryptamine is reduced in lungs from hypoxic pulmonary hypertensive rats by Trina K Jeffery; Lesley J Bryan-Lluka; Janet C Wanstall (137-140).
In this study, the aim was to determine whether 5-hydroxytryptamine (5-HT) removal by the pulmonary endothelium is reduced in 1-week hypoxic, pulmonary hypertensive rats by directly measuring [3H]5-HT uptake in isolated lungs. In lungs from hypoxic rats, specific 5-HT uptake was reduced. This was due to a 50% decrease in the maximal initial rate of uptake rather than a decrease in affinity of 5-HT for its transporter. It is possible that reduced removal of 5-HT may contribute to the elevation in plasma levels of this vasoactive amine in pulmonary hypertension.
Keywords: Chronic hypoxia; 5-HT (5-hydroxytryptamine, serotonin) uptake; Pulmonary hypertension; Lung;

Immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB2 receptor by Nancy E Buckley; Kathleen L McCoy; Éva Mezey; Tom Bonner; Anne Zimmer; Christian C Felder; Michelle Glass; Andreas Zimmer (141-149).
Cannabinoids have immunomodulatory as well as psychoactive effects. Because the central cannabinoid receptor (cannabinoid CB1 receptor) is highly expressed in many neuronal tissues and the peripheral cannabinoid receptor (cannabinoid CB2 receptor) is highly expressed in immune cells, it has been suggested that the central nervous system effects of cannabinoids are mediated by cannabinoid CB1 receptors and that the immune effects are mediated by cannabinoid CB2 receptors. To test this hypothesis, we have generated the first mouse strain with a targeted mutation in the cannabinoid CB2 receptor gene. Binding studies using the highly specific synthetic cannabinoid receptor agonist (−)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol ([3H]CP 55,940) revealed no residual cannabinoid binding sites in the spleen of the cannabinoid CB2 receptor knockout mice, while binding in the central nervous system was unchanged. Cannabinoid CB2 receptor knockout mice, which appear healthy, are fertile and care for their offspring. Fluorescence activated cell sorting (FACS) analysis showed no differences in immune cell populations between cannabinoid CB2 receptor knockout and wildtype mice. We investigated the immunomodulatory effects of cannabinoids in cannabinoid CB2 receptor deficient mice using a T cell co-stimulation assay. Δ9Tetrahydrocannabinol inhibits helper T cell activation through macrophages derived from wild type, but not from knockout mice, thus indicating that this effect is mediated by the cannabinoid CB2 receptor. In contrast, central nervous system effects of cannabinoids were not altered in these mice. Our results suggest that cannabinoid CB2 receptor-specific ligands may be clinically useful in the modulation of macrophage immune function while exhibiting no central nervous system activity. Furthermore, we conclude that the cannabinoid CB2 receptor knockout mouse is a useful animal model in which to study the role of the cannabinoid system in immunoregulation.
Keywords: Knockout mouse; Cannabinoid receptor; Δ9Tetrahydrocannabinol; Macrophage;

Index (151-152).

Index (153-156).