European Journal of Pharmacology (v.415, #2-3)

In the present study, we tested the hypothesis that the activation of imidazoline I1-receptor, which is coupled to phosphatidylcholine-specific phospholipase C, results in downstream activation of mitogen-activated protein kinase (p42mapk and p44mapk isoforms) in PC12 cells. PC12 cells pretreated with nerve growth factor (50 ng/ml, 48 h) to initiate neuronal differentiation were incubated with [methyl-3H]choline and [3H]myristate. Activation of imidazoline I1 receptor by rilmenidine (10 μM) caused time-dependent increases in diacylglycerol accumulation and phosphocholine release. The Western blotting analysis showed that rilmenidine (10 μM) produced a time-dependent activation of p42mapk and p44mapk that reached its maximum at 15 min and returned to control levels after 30 min. This finding was confirmed by immunofluorescence labeling of activated mitogen-activated protein kinase in the same model system. Efaroxan (imidazoline I1-receptor antagonist) or tricyclodecan-9-yl-xanthogenate (D609, phosphatidylcholine-specific phospholipase C inhibitor) attenuated the phosphorylation of p42mapk and p44mapk induced by rilmenidine. Nerve growth factor-induced phosphorylation of both mitogen-activated protein kinase isoforms was not affected by D609. These results support the hypothesis that the activation of the imidazoline I1 receptor coupled phosphatidylcholine-specific phospholipase C results in the downstream activation of mitogen-activated protein kinase.
Keywords: MAP kinase; Phosphatidylcholine-specific phospholipase C; Imidazoline I1 receptor; PC12 Cell; Diacylglycerol; Rilmenidine;

Pharmacological characterisation of pyrimidinoceptor responses in NG108-15 cells by Katrin Sak; Külli Samuel; Merike Kelve; Tania E Webb (127-133).
In the present study, the P2Y receptor(s) mediating the effects of the pyrimidines UTP and UDP on phospholipase C activation in the mouse neuroblastoma×rat glioma hybrid cell line NG108-15 was investigated. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis detected transcripts for the P2Y6 and P2Y2 receptors, but not for P2Y1 and P2Y4. UTP and UDP were equipotent agonists and their effects were partially additive. Suramin, reactive blue 2 and pyridoxal phosphate-6-azophenyl-2′,4′disulfonic acid (PPADS) antagonised the phospholipase C response to both UTP and UDP. High micromolar concentrations of adenosine, 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS-21680), 2′,3′-O-isopropylideneadenosine (iPAdo) and adenosine 3′:5′-cyclic monophosphate (3′,5′-cAMP) were able to antagonise the effect of UTP on phospholipase C but not that of UDP. The additivity of the UTP and UDP responses, novel P2 receptor antagonist profile and the distinguishing action of adenosine may indicate the expression of a pyrimidine selective P2Y receptor in addition to the P2Y6 type in these cells.
Keywords: NG108-15 cell line; Pyrimidinoceptor; UTP-preferring receptor; Inositol phospholipid; Adenosine;

Interaction of the neuroprotective drug riluzole with GABAA and glycine receptor channels by Bahram Mohammadi; Klaus Krampfl; Hafis Moschref; Reinhard Dengler; Johannes Bufler (135-140).
Riluzole is used as therapeutic agent in amyotrophic lateral slerosis. We investigated the interaction of riluzole with recombinant GABA (γ-aminobutyric acid)A receptor channels (α1β2γ2-subunits) and glycine receptor channels (α1β-subunits) transiently expressed in HEK293 cells. For electrophysiological experiments, the patch–clamp technique in combination with tools for ultrafast solution exchange was used. Saturating concentrations of GABA or glycine were applied with different concentrations of riluzole to outside-out patches containing α1β2γ2 GABAA receptor channels or α1β-glycine receptor channels on their surface, respectively. The current declined after application of GABA or glycine with three time constants of desensitization to a steady-state current amplitude. Application of riluzole resulted in a shift to fast desensitized states at both receptors. The proportion of the time constants of fast desensitization increased and the time constants of slow desensitization and the steady-state current decreased whereas the maximal current amplitudes were not affected by riluzole. The data of the study demonstrate for the first time interaction of GABAergic and glycinergic currents with riluzole under physiological conditions.
Keywords: GABAA receptor channel; Glycine receptor channel; Patch–clamp technique; Rapid application; Riluzole;

Plasticity in transmission and modulatory systems are implicated in mechanisms of neuropathic pain. Studies demonstrate the importance of high voltage-activated Ca2+ channels in pain transmission, but the role of low voltage-activated, T-type Ca2+ channels in nociception has not been investigated. The Kim and Chung rodent model of neuropathy [Pain 50 (1992) 355] was used to induce mechanical and cold allodynia in the ipsilateral hindpaw. In vivo electrophysiological techniques were used to record the response of dorsal horn neurones to innocuous and noxious electrical and natural (mechanical and thermal) stimuli after spinal nerve ligation. Spinal ethosuximide (5–1055 μg) exerted dose-related inhibitions of both the electrically and low- and high-intensity mechanical and thermal evoked neuronal responses and its profile remained unaltered after neuropathy. Measures of spinal cord hyperexcitability were most susceptible to ethosuximide. This study, for the first time, indicates a possible role for low voltage-activated Ca2+ channels in sensory transmission.
Keywords: Ca2+ channels; Neuropathic pain; T-type channel; Analgesia; Nociception; Dorsal horn;

Characterisation of P2Y1-like receptor in cultured rat pineal glands by Zulma S Ferreira; Regina P Markus (151-156).
The rat pineal gland possesses P2 receptors which potentiate the effect of noradrenaline-induced N′-acetyl-5-hydroxytryptamine (N′-acetyl-5-HT) production. In the current study, this receptor was characterised according to agonist selectivity and signal transduction mechanisms. 2-MethylthioATP (2MeSATP), 2-chloroATP (2-ClATP), adenosine 5′-O-2-thiodiphosphate, (ADPβS), ATP and ADP, but not UTP, potentiated noradrenaline-induced N′-acetyl-5-HT production in a concentration-dependent manner. 2MeSATP neither induced the production of adenosine 3′:5′-cyclic monophosphate (cyclic AMP), nor inhibited its formation when the glands were stimulated by forskolin. The phospholipase C inhibitor 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), but not the inactive analogue, 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione (U73343), blocked the 2MeSATP effect. The P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-dissulphonic acid (PPADS), which inhibits phospholipase C-coupled P2Y1 receptors, blocked the 2MeSATP effect. In conclusion, our data strongly suggest that the P2-like receptor that is present in rat pinealocytes and which is responsible for the potentiation of noradrenaline-induced N′-acetyl-5-HT production is a P2Y1-like receptor, coupled to a G protein which stimulates phospholipase C.
Keywords: P2Y1 receptor; Pineal gland; N′-acetyl-5-hydroxytryptamine production; Melatonin; Phospholipase C;

Central and peripheral activity of cholinesterase inhibitors as revealed by yawning and fasciculation in rats by Hiroo Ogura; Takashi Kosasa; Yuka Kuriya; Yoshiharu Yamanishi (157-164).
This study was designed to investigate the central and peripheral activity profile of cholinesterase inhibitors in rats. Intravenous injection of cholinesterase inhibitors caused fasciculation, a fine involuntary muscular movement. This peripheral cholinergic sign was tightly correlated with in vitro anti-acetylcholinesterase activity by cholinesterase inhibitors, suggesting that fasciculation is a valid index of peripheral cholinergic activation. Yawning, used as a marker of central cholinergic activation, was also monitored. E2030 (3-(2-(1-(1,3-dioxolan-2-ylmethyl)-4-piperidyl)ethyl)-2H-3,4-dihydro-1,3-benzoxazin-2,4-dione hydrochloride) elicited yawning at more than 4 mg/kg, while fasciculation was significantly intensified only at a dose of 16 mg/kg. Donepezil and tacrine induced both yawning and fasciculation at doses greater than 4 mg/kg, whereas physostigmine induced both behaviors at a dose of 8 mg/kg and above. Finally, ipidacrine elicited yawning at a dose of 16 mg/kg and fasciculation at doses greater than 8 mg/kg. Thus, all putative centrally acting cholinesterase inhibitors elicited yawning. TAK-147 (3-[1-(phenylmethyl)-4-piperidinyl]-1-(2,3,4,5-tetrahydro-1H-benzazepin-8-yl)-1-propanone fumarate) did not significantly elicit yawning at doses under 16 mg/kg, but elicited fasciculation at a dose of more than 4 mg/kg. Distigmine, a peripherally acting cholinesterase inhibitor, evoked fasciculations, but not yawning. When mild to moderate fasciculation was evoked, donepezil and E2030 elicited more than nine yawns over 30 min, while the other cholinesterase inhibitors elicited approximately five yawns at most during this period. These results indicated that E2030 and donepezil exhibited the most marked preferential central cholinergic activity, relative to peripheral activity, among cholinesterase inhibitors tested. Scopolamine, a centrally acting antimuscarinic drug, completely inhibited E2030-induced yawning, while peripherally acting methylscopolamine did not. Haloperidol, a dopamine receptor antagonist, partially blocked E2030-induced yawning, but did not block donepezil-induced yawning. These results suggest that central cholinergic and, in part, dopaminergic mechanisms are involved in E2030-induced yawning.
Keywords: Cholinesterase inhibitor; Donepezil; E2030; Yawning; Fasciculation; (Rat);

Ultrasonic vocalizations of preweanling rats: involvement of both α2-adrenoceptor and κ-opioid receptor systems by Arbi Nazarian; Catherine M Krall; James R Osburn; Sanders A McDougall (165-171).
Stimulation of α2-adrenoceptors and κ-opioid receptors increases the ultrasonic vocalizations of preweanling rats. The purpose of the present study was to determine whether α2-adrenoceptors and κ-opioid receptors modulate ultrasonic vocalization production via a common mechanism. To that end, 11-day-old rats were injected with the α2-adrenoceptor antagonist yohimbine (0, 0.5, or 1.0 mg/kg, i.p.) or the κ-opioid receptor antagonist nor-binaltorphimine (0, 5, or 10 mg/kg, i.p.). After 15 min, the same rats were injected with saline, the α2-adrenoceptor agonist clonidine (0.25 mg/kg, i.p.), or the κ-opioid receptor agonist trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate (U-50,488; 2.5 mg/kg, i.p.). Results showed that both clonidine and U-50,488 increased the ultrasonic vocalizations of preweanling rats. Not surprisingly, clonidine-induced ultrasonic vocalizations were blocked by yohimbine, while U-50,488-induced vocalizations were blocked by nor-binaltorphimine. Importantly, yohimbine also attenuated the vocalizations produced by U-50,488, whereas nor-binaltorphimine did not alter clonidine-induced ultrasonic vocalizations. Thus, it appears that α2-adrenoceptor and κ-opioid receptor stimulation increases ultrasonic vocalization production via a common mechanism. It is likely that the κ-opioid receptors responsible for modulating ultrasonic vocalizations are located “upstream” from the α2-adrenoceptors.
Keywords: Ultrasonic vocalization; α2-Adrenoceptor; κ-Opioid receptor; Clonidine; U-50,488;

Failure of GPI compounds to display neurotrophic activity in vitro and in vivo by Arnaud Bocquet; Geneviève Lorent; Bruno Fuks; Renée Grimée; Patrice Talaga; Jean Daliers; Henrik Klitgaard (173-180).
The aim of this study was to evaluate the neurotrophic and neuroprotective properties of a series of immunophilin ligands and to assess the potential involvement of FK506 Binding Protein 12 kDa (FKBP12) rotamase inhibition in this activity. Both FK506 and rapamycin induced a potent inhibition of the FKBP12 rotamase activity (pIC50 values of 7.3 and 7.4, respectively) but only a modest inhibition was observed with 1-(3,3-dimethyl-2-oxo-pentanoyl)-pyrrolidine-2-carboxylic acid S-3-pyridin-3-yl-propyl ester (GPI 1046) (5.8), its N-oxide (5.4) and thioester (6.3) analogues. Compared to nerve growth factor, all these immunophilin ligands only induced marginal increases in neurite outgrowth of rat dissociated newborn dorsal root ganglia cells. Furthermore, systemic administration of GPI 1046 and its N-oxide and thioester analogues failed to prevent striatal dopamine depletion induced by acute or chronic i.p. treatment with 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP). These results suggest that inhibition of FKBP12 rotamase activity is not predictive for neurotrophic and neuroprotective properties of immunophilin ligands and question their therapeutic utility in neurodegenerative diseases like Parkinson's disease.
Keywords: Immunophilin; Rotamase; Dorsal root ganglia; Neuroprotection; MPTP (1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine); (Mouse);

Sedative and anxiolytic effects of zopiclone's enantiomers and metabolite by Jeffrey N Carlson; Rene Haskew; Jennifer Wacker; Isabelle M Maisonneuve; Stanley D Glick; Thomas P Jerussi (181-189).
We evaluated racemic zopiclone, its (S)- and (R)-enantiomers and a metabolite, (S)-desmethylzopiclone, for their actions on locomotor activity, rotarod performance, the elevated plus maze and the Vogel conflict test of anxiety, and electroconvulsive shock-induced seizures duration. Zopiclone and its (R)- and (S)-enantiomers reduced locomotor activity, and zopiclone and its (S)-enantiomer disrupted rotarod performance at 10 mg/kg. (S)-desmethylzopiclone did not alter these measures at doses of less than 200 mg/kg. (S)-desmethylzopiclone altered plus maze performance at the lowest dose of all the zopiclone derivatives tested, caused a dose-related effect on the Vogel conflict test and caused a dose-related reduction of electroconvulsive shock-induced seizure durations. The data indicate that (S)-desmethylzopiclone can bring about an anxiolyic effect without a substantial degree of central nervous system depression, and suggest that the agent may be particularly useful clinically in the treatment of anxiety.
Keywords: Zopiclone; (S)-Desmethylzopiclone; Anxiolytic effect;

CGP 36216 is a selective antagonist at GABAB presynaptic receptors in rat brain by Jennifer Ong; Sotiria Bexis; Victor Marino; David A.S Parker; David I.B Kerr; Wolfgang Froestl (191-195).
In rat neocortical preparations maintained in Mg2+-free Krebs medium, baclofen depressed the frequency of spontaneous discharges in a concentration-dependent manner (EC50=6 μM), sensitive to (3-aminopropyl)ethylphosphinic acid (CGP 36216) (100, 300 and 500 μM) (pA 2=3.9±0.1). By contrast, CGP 36216, up to 1 mM, was ineffective in antagonising baclofen-induced hyperpolarisations, mediated through γ-aminobutyric acidB (GABAB) postsynaptic receptors. In electrically stimulated brain slices preloaded with [3H]GABA, CGP 36216 increased [3H]GABA release (IC50=43 μM), which was reversed by baclofen (20 μM). While CGP 36216 is ineffective at GABAB postsynaptic receptors, it is appreciably more active at presynaptic receptors.
Keywords: GABAB receptor antagonist; Neocortex; Baclofen; CGP 36216;

The forced swim test and tail suspension test are often used in laboratory practice to identify compounds that possess antidepressant-like activity. This experiment was conducted to determine whether housing conditions per se influence the response of mice in these antidepressant screening procedures. Male NIH Swiss mice were housed individually or in groups (five per cage) for 8 weeks prior to testing. After 8 weeks, the animals were exposed to the forced swim and tail-suspension tests. Group housed mice displayed high levels of immobility in the forced swim and tail suspension tests. Desipramine injection 60 min prior testing, in doses 7.5 and 15 mg/kg, produced significant reductions in the immobility time in forced swimming and tail suspension tests. Individually housed mice, when exposed to these tests, displayed lower levels of immobility with a magnitude comparable to the effect of desipramine in group housed mice. Desipramine given to individually housed mice did not reduce the duration of immobility either in the forced swim test or in the tail suspension test. These results indicate that both tests are sensitive to housing conditions. This observation suggests that long lasting group housing may be critical to the behavioral response in these preclinical screening procedures in mice.
Keywords: Forced swim test; Tail suspension test; Group housing; Single housing; Desipramine; (Mouse);

Corticotropin-releasing factor (CRF) has been suggested to play an important role in the development of drug dependence and withdrawal. Based on the recent finding that CRF receptor antagonists inhibit the stress-induced relapse to opiate dependence and attenuate anxiety-like responses related to cocaine withdrawal, the present experiment was performed to examine the possible effect of different CRF receptor antagonists on reactivation of cocaine-conditioned place preference induced by cocaine and stress in rats. The results show that a single injection of cocaine (10 mg/kg, i.p.) could reactivate cocaine-conditioned place preference following a 28-day extinction, and pretreatment with i.c.v. 10 μg α-helical CRF, a nonspecific CRF receptor antagonist, significantly attenuated this reactivation of conditioned place preference. However, pretreatment with i.p. 1 or 10 mg/kg CP-154,526 (butyl-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-ethylamine), a specific CRF receptor subtype 1 antagonist, and i.c.v. 1 or 10 μg AS-30 ([d-Phe11,His12]Svg-(11-40)), a specific CRF receptor subtype 2 antagonist, failed to show the same effects. In addition, a single footshock stress also elicited the reactivation of cocaine-conditioned place preference following a 28-day extinction and pretreatment with α-helical CRF (10 μg, i.c.v.) and CP-154,526 (1 or 10 mg/kg, i.p.) significantly blocked this effect. In contrast, pretreatment with AS-30 at a dose of 1 or 10 μg (i.c.v.) did not affect the stress-induced reactivation of cocaine-conditioned place preference. The present study demonstrated that CRF receptor type 1, but not CRF receptor type 2, mediates the stress-induced reactivation of cocaine-conditioned place preference. These findings suggest that CRF receptor subtype 1 antagonists might be of some value in the treatment and prevention of stress-induced relapse to drug dependence long after detoxification.
Keywords: CRF (corticotropin-releasing factor); CRF receptor; Stress; Cocaine; Conditioned place preference; Relapse;

Release of endothelial nitric oxide in coronary arteries by celiprolol, a β1-adrenoceptor antagonist: possible clinical relevance by Kumiko Noda; Michiko Oka; Fu-H Ma; Satoru Kitazawa; Yojiro Ukai; Noboru Toda (209-216).
Mechanisms underlying celiprolol-induced vasodilatation were analyzed in isolated porcine coronary arteries. Celiprolol induced dose-related relaxation of the artery rings with endothelium, an effect which was suppressed by N G-nitro-l-arginine methylester (l-NAME), nitric oxide (NO) scavenger, guanylate cyclase inhibitor, endothelium denudation, and removal of Ca2+. l-NAME contracted, and superoxide dismutase relaxed, the arteries only when the endothelium was preserved. Neither superoxide dismutase nor β-adrenoceptor antagonists changed celiprolol-induced relaxations. Celiprolol increased the cyclic GMP content in the tissue. The release of NO from endothelium, estimated by the extracellular production of cyclic GMP in arteries incubated in medium containing guanylate cyclase and GTP, was augmented by celiprolol, and l-NAME abolished this action of celiprolol. It is concluded that celiprolol elicits relaxation by acting on sites other than β-adrenoceptors in the endothelium and by releasing NO, which activates soluble guanylate cyclase in smooth muscle and produces cyclic GMP. Scavenging of superoxide anions from the endothelium does not seem to account for the induced relaxation.
Keywords: Celiprolol; β-Adrenoceptor antagonist; Nitric oxide (NO); Endothelium; Coronary artery;

Effect of simvastatin on vascular smooth muscle responsiveness: involvement of Ca2+ homeostasis by Marı́a Álvarez de Sotomayor; Concepción Pérez-Guerrero; Marı́a Dolores Herrera; Elisa Marhuenda (217-224).
This report is focused on the study of simvastatin-induced relaxation of rat aorta through its effects on vascular smooth muscle and Ca2+ signalling. The presence of endothelium affected only the simvastatin-induced relaxation of aortic rings precontracted with noradrenaline, but not by depolarization with KCl 80 mM. Blockade of Ca2+ entry through voltage-operated Ca2+ channels (VOCCs) by diltiazem abolished the endothelium-dependent and direct relaxation, whereas Ca2+-ATPase inhibition by cyclopiazonic acid (3×10−5 M) only affected the endothelium-dependent relaxation. In KCl-depolarised arteries concentration–response curves for CaCl2 were shifted to the right in the presence of simvastatin (3×10−6 and 3×10−5 M) or diltiazem (10−6 and 10−7 M). The transient contraction caused by noradrenaline in Ca2+-free medium, which is mainly due to intracellular Ca2+ release, was inhibited by simvastatin (3×10−5 M) or cyclopiazonic acid (3×10−5 M) and the contraction induced by CaCl2 (2×10−3 M) added after noradrenaline was inhibited by diltiazem and simvastatin. All the reported effects of simvastatin were inhibited by the product of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, mevalonate (10−3 M). These findings demonstrate that the vascular effects of simvastatin may involve both Ca2+ release from intracellular stores, which could promote activation of endothelial factors, and blockade of extracellular Ca2+ entry, which promote relaxations independent of the presence of endothelium. This action on Ca2+ could be related to the inhibition of isoprenoid synthesis, which subsequently affects the function of G-proteins involved in communication among intracellular Ca2+ pools and capacitative Ca2+ entry.
Keywords: HMG-CoA reductase; Simvastatin; Ca2+; Ca2+-ATPase; Diltiazem;

Diabetes potentiates acetylcholine-induced relaxation in rabbit renal arteries by José A Alabadı́; Francisco J Miranda; Silvia Lloréns; Rosa F Ruiz de Apodaca; José M Centeno; Enrique Alborch (225-232).
The response of rabbit renal arteries to acetylcholine and its endothelial modulation in diabetes were investigated. Acetylcholine induced concentration-related endothelium-dependent relaxation of renal arteries that was significantly more potent in diabetic rabbits than in control rabbits. Pretreatment with N G-nitro-l-arginine (l-NOArg), indomethacin, or l-NOArg plus indomethacin induced partial inhibition of acetylcholine-induced relaxation. Inhibition induced by l-NOArg plus indomethacin was significantly higher in arteries from diabetic rabbits than in arteries from control rabbits. In renal arteries depolarised with KCl 30 mM and incubated with l-NOArg plus indomethacin, acetylcholine-induced relaxation was almost abolished in both groups of rabbits and this response was not different from that obtained in arteries without endothelium. Sodium nitroprusside induced concentration-dependent relaxation of renal arteries from control and diabetic rabbits without significant differences between the two groups of animals. These results suggest that diabetes potentiates the acetylcholine-induced relaxation in rabbit renal arteries. Increased release of nitric oxide and prostacyclin could be responsible for the enhanced relaxant potency of acetylcholine in diabetes.
Keywords: Diabetes; Renal artery; Acetylcholine; Endothelium;

2-Arachidonoylglycerol, a candidate of endothelium-derived hyperpolarizing factor by Satomi Kagota; Yu Yamaguchi; Kazuki Nakamura; Takayuki Sugiura; Keizo Waku; Masaru Kunitomo (233-238).
We investigated whether 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, is involved in acetylcholine- and calcium ionophore A23187-induced relaxations in the presence of N G-nitro-l-arginine methyl ester (l-NAME) and indomethacin, which is considered to be mediated by endothelium-derived hyperpolarizing factor (EDHF). In rabbit mesenteric arterial rings pre-constricted with noradrenaline, 2-arachidonoylglycerol caused concentration-dependent relaxation. The 2-arachidonoylglycerol-induced relaxations were not affected by endothelium removal. N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morholinyl-1H-pyrazole-3-carboxamide (AM281), cannabinoid CB1 receptor antagonists, significantly attenuated 2-arachidonoylglycerol-induced relaxation and the acethycholine-induced relaxation only slightly, but not the calcium ionophore A23187-induced relaxation. On the other hand, charybdotoxin plus apamin, K+ channel blockers, significantly attenuated acetylcholine and calcium ionohore A23187-induced relaxations but not 2-arachidonoylglycerol-induced relaxations. These results suggest that 2-arachidonoylglycerol can cause relaxations via cannabinoid CB1 receptors, but is not involved in EDHF-mediated relaxations.
Keywords: 2-Arachidonoylglycerol; Cannabinoid; EDHF (endothelium-derived hyperpolarizing factor);

Contribution of peroxynitrite to the beneficial effects of preconditioning on ischaemia-induced arrhythmias in rat isolated hearts by Sedat Altup; A.Tuncay Demiryürek; Dilek Ak; Muzaffer Tungel; ⋖lker Kanzık (239-246).
We studied the effects of urate, a peroxynitrite scavenger, on ischaemia- and peroxynitrite-induced preconditioning in rat isolated hearts. Isolated hearts perfused with Krebs–Henseleit solution were preconditioned either by 3 min of coronary artery occlusion or by peroxynitrite administration (1 μM) for 3 min, followed by 10 min of reperfusion and 30 min of coronary artery occlusion. Both ischaemia and peroxynitrite produced a marked reduction in arrhythmias. Urate (1 mM) added to the perfusate 10 min prior to ischaemic preconditioning or peroxynitrite infusion and maintained until coronary artery occlusion, markedly reversed the beneficial effects in the ischaemic and peroxynitrite-treated groups. Urate administration in the peroxynitrite-treated group increased the incidence of ventricular tachycardia from 57% (n=11) to 100% (n=6) and total ventricular fibrillation from 0% (n=0) to 44% (n=4). Similarly, urate augmented the incidence of ventricular tachycardia from 47% (n=8) to 85% (n=6) in the ischaemic preconditioning group. On its own, urate did not affect the severity of cardiac arrhythmias. Peroxynitrite infusion caused a marked increase in the effluent nitrate levels, from 0.05±0.1 μM (n=5) to 0.4±0.2 μM (n=6), and urate significantly decreased these levels to 0.08±0.03 μM (n=9). These results suggest that peroxynitrite at low concentrations contributes to the beneficial effects of preconditioning on ischaemia-induced arrhythmias in rat isolated hearts.
Keywords: Preconditioning; Peroxynitrite; Ischaemia-induced arrhythmia; Urate; Nitrate level;

The action of melatonin to alter calcitonin gene-related peptide (CGRP)-mediated vasodilation and stimulation of adenylate cyclase activity in middle cerebral arteries of rats was investigated. Concentration-dependent dilation of the rat middle cerebral artery produced by CGRP (EC50 of 9.4×10−10 M) was significantly inhibited in the presence of 10−8 M melatonin (EC50 of 3.4×10−9 M). In addition, CGRP (10−7 M)-mediated increase in adenylate cyclase activity was also significantly attenuated by the receptor mediated action of melatonin. These results indicate that melatonin may interact with CGRP to regulate cerebral arterial tone.
Keywords: CGRP (calcitonin gene-related peptide); Melatonin; Cerebral artery; Adenylate cyclase; Vascular tone; Cerebral circulation;

Relaxations to adenosine and analogues were investigated in the mouse aorta in the presence of the adenosine A1 receptor-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 30 nM), which did not affect relaxations to adenosine or its analogue N 6-R-phenylisopropyladenosine (R-PIA) but abolished contractile adenosine A1 receptor-mediated responses to these agonists. Relaxations to adenosine, 5′-N-ethylcarboxamidoadenosine, R-PIA, 2-[p-(2-carbonylethyl)-phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680), and N 6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) were unaffected by the adenosine A1/A2 receptor antagonist 8-sulphophenyltheophylline (100 μM). IB-MECA relaxations were unaffected by the adenosine A3 receptor-selective antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1191, 30 μM) and R-PIA relaxations were unaffected by N G-nitro-l-arginine methyl ester (100 μM) and endothelium removal. In conclusion, relaxant responses to adenosine and analogues do not involve adenosine A1, A2 or A3 receptors and are endothelium- and nitric oxide-independent.
Keywords: Adenosine; Aorta; Xanthine; Purinoceptor; Endothelium;

The effect of hypothyroidism on gastrointestinal β1- and β3-adrenoceptor function and expression was examined in rat ileal smooth muscle preparations. (−)-Isoprenaline and the selective β3 agonist disodium (R,R)-5-[2-[[2-3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316234) relaxed both control and hypothyroid tissues in a dose-dependent manner. Responses to isoprenaline were reduced in tissues from hypothyroid rats, as was the shift produced with the β3-adrenoceptor antagonist, 3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol oxalate (SR 59230A). No change was seen in responses to CL 316243. Experiments with a selective β1-adrenoceptor antagonist produced results suggesting that isoprenaline did not act at this receptor. Messenger RNA levels for both β1- and β2-adrenoceptors were not affected by hypothyroidism. These results show that, unlike in adipose tissues, ileal β1- and β3-adrenoceptors are not directly regulated by thyroid hormone and that β3-adrenoceptor coupling to the relaxation response is reduced in a rat model of hypothyroidism.
Keywords: β-Adrenoceptors; Smooth muscle; Hypothyroidism; Messenger RNA; relaxation;

The potency of the putatively α1B-adrenoceptor selective drug, 1-[biphenyl-2-yloxy]-4-imino-4-piperidin-1-yl-butan-2-ol (AH11110A), to antagonize contraction upon stimulation of α1A-adrenoceptors in rat vas deferens and rat perfused kidney, α1B-adrenoceptors in guinea-pig spleen, mouse spleen and rabbit aorta, and α1D-adrenoceptors in rat aorta and pulmonary artery was evaluated and compared to that of a number of subtype-discriminating antagonists. N-[3-[4-(2-Methoxyphenyl)-1-piperazinyl]propyl]-3-methyl-4-oxo-2-phenyl-4H-1-benzopyran-8-carboxamide (Rec 15/2739) and (±)-1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-benzodioxin-5-yl)-1-piperazinyl]propyl]amino]-2,4(1H,3H)-pyrimidinedione (B8805-033) were confirmed as selective for α1A-adrenoceptors, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY 7378), 8-[2-(1,4-benzodioxan-2-ylmethylamino)ethyl]-8-azaspiro[4.5]decane-7,9-dione (MDL 73005EF), and cystazosin were found to be selective for α1D-adrenoceptors, whereas spiperone was weakly selective for α1B-over α1A-adrenoceptors. However, from the functional affinity profile obtained for AH11110A at α1A-adrenoceptors (pA 2=6.41 in rat vas deferens), α1B-adrenoceptors (pA 2=5.40–6.54) and α1D-adrenoceptors (pA 2=5.47–5.48), the affinity and presumed selectivity previously obtained for AH11110A in radioligand binding studies at native α1B- and cloned α1b-adrenoceptors (pK i=7.10–7.73) could not be confirmed. Additionally, AH11110A enhanced the general contractility of rat vas deferens, produced a bell-shaped dose–response curve of vasodilation in perfused rat kidney, and its antagonism in most other tissues was not simply competitive. The affinity of AH11110A for prejunctional α2-adrenoceptors in rabbit vas deferens (pA 2=5.44) was not much lower than that displayed for α1-adrenoceptor subtypes, revealing that AH11110A, besides α1-adrenoceptors, also interacts with α2-adrenoceptors, and thus may be unsuitable for α-adrenoceptor subtype characterization, at least in smooth muscle containing functional studies.
Keywords: α1-Adrenoceptor; Subtype A, B and D; α2-Adrenoceptor; AH11110A; Selectivity;

Tachykinin NK2 receptor mediates contraction and ion transport in rat colon by different mechanisms by Riccardo Patacchini; Helen M Cox; Sandra Ståhl; Iain R Tough; Carlo Alberto Maggi (277-283).
We have characterized the tachykinin NK2 receptor-mediated contraction and vectorial ion transport responses in the muscularis mucosae and mucosa of the rat isolated distal colon, respectively. The tachykinin NK2 receptor-selective antagonist nepadutant (c{[(β-d-GlcNAc)Asn-Asp-Trp-Phe-Dpr-Leu]c(2β-5β)}) produced competitive antagonism of [βAla8]neurokinin A-(4-10)-induced contraction (pK B=9.3) in the muscularis mucosae, and insurmountable blockade of increases in short-circuit current (I sc) responses (pK B=8.6) in the mucosa. However, this latter effect was completely reversed by washout of the antagonist. [βAla8]Neurokinin A-(4-10)-induced contractions were unaffected by indomethacin (3 μM). In sharp contrast, I sc responses induced by [βAla8]neurokinin A-(4-10) (100 nM) were inhibited (>70%) by indomethacin (3 μM), while I sc responses to substance P (3 μM) were unchanged. Our study provides the first evidence that in the same organ stimulation of tachykinin NK2 receptors leads to two independent responses mediated by different effector mechanisms both of which are blocked (albeit with different kinetics) by the potent and selective tachykinin NK2 receptor antagonist, nepadutant.
Keywords: Nepadutant; Tachykinin NK2 receptor; Ion transport; Colon mucosa (rat);

Dysidotronic acid, a new sesquiterpenoid, inhibits cytokine production and the expression of nitric oxide synthase by Inmaculada Posadas; M.Carmen Terencio; Clelia Giannini; Maria Valeria D'Auria; Miguel Payá (285-292).
In a previous study, we reported a new bioactive sesquiterpenoid, named dysidotronic acid, to be a potent, selective human synovial phospholipase A2 inhibitor. Dysidotronic acid is a novel, non-complex manoalide analogue lacking the pyranofuranone ring. We now investigate the effect of this compound on cytokine, nitric oxide and prostanoid generation on the mouse macrophage cell line RAW 264.7, where it showed a dose-dependent inhibition with inhibitory concentration 50% values in the micromolar range. This effect was also confirmed in the mouse air pouch injected with zymosan. Dysidotronic acid inhibited the production of tumor necrosis factor alpha and interleukin-1 beta as well as the production of nitric oxide, prostaglandin E2 and leukotriene B4. Decreased nitric oxide generation was the consequence of inhibition of the expression of nitric oxide synthase, whereas PGE2 and LTB4 reduction was due to inhibition of arachidonic acid bioavailability through a direct inhibitory effect of dysodotronic acid on secretory phospholipase A2.
Keywords: Dysidotronic acid; Cytokine; Nitric oxide (NO); RAW 264.7; Zymosan; Air pouch;

Effects of F2833 on cholesterol metabolism in the genetically hyperlipidemic rat by Khadija Ouguerram; Claude Lutton; André Delhon; Thierry Magot (293-299).
The effects of the new hypolipidemic agent, F2833 or (chloro 2′ (1-1′) biphenyl-4)-2 propanol-2, on cholesterol metabolism were studied in genetically hyperlipidemic rats (RICO). Cholesterolemia decreased after 2 days of treatment to 60% of its initial value (1.20±0.10 g/l vs. 1.99±0.08, P<0.001) and then stabilised within 10 days. This hypocholesterolemic action was effective for as long as 3 months. Concerning the different classes of lipoproteins, a significant drop was observed in HDL (high density lipoproteins) (25%, 0.49±0.02 g/l vs. 0.66±0.007, P<0.01) and particularly in LDL (low density lipoproteins) (70%, 0.30±0.04 g/l vs. 0.92±0.05, P<0.001). Whole body cholesterol showed a higher fractional catabolic rate (0.25±0.02 vs. 0.17±0.005 day−1, P<0.01) together with an increased cholesterol synthesis (60±5 vs. 36±4 mg/day, P<0.01). LDL kinetics showed that the decrease in these lipoproteins is essentially caused by an increase in the fractional catabolic rate (10.6±0.1%/h vs. 5.2±0.1%/h, P<0.001) and by a lesser decrease in the LDL production rate. This cholesterol metabolic profile created by treatment suggests an effect through stimulation of cholesterol output (biliary cholesterol elimination or cholesterol transformation into bile acids).
Keywords: Hypocholesterolemic drug; Cholesterol turnover; Low density lipoprotein kinetics;

Author index (303-305).

Keyword index (307-312).