European Journal of Pharmacology (v.392, #3)

The role of the G protein γ2 subunit in opioid antinociception in mice by Keiko Hosohata; Jennifer K Logan; Eva Varga; Thomas H Burkey; Todd W Vanderah; Frank Porreca; Victor J Hruby; William R Roeske; Henry I Yamamura (R9-R11).
We examined the role of the γ2 subunit of G proteins (Gγ2) in the antinociception produced by c[D-Pen2,D-Pen5]enkephalin (DPDPE) in mice. DPDPE produced 84.0±9.0% antinociception in vehicle-treated mice. After intracerebroventricular (i.c.v.) treatment with an antisense phosphorothioate oligodeoxynucleotide to the Gγ2 subunit, DPDPE-mediated antinociception decreased to 24.4±7.4%. The mismatch phosphorothioate oligodeoxynucleotide-treated mice showed 65.1±10.3% antinociception, while the missense phosphorothioate oligodeoxynucleotide-treated mice showed 76.4±23.6% antinociception by DPDPE. The reduction of analgesia in antisense phosphorothioate oligodeoxynucleotide-treated mice was significant in comparison with vehicle-treated (P<0.001), mismatch phosphorothioate oligodeoxynucleotide-treated (P<0.01) and missense phosphorothioate oligodeoxynucleotide-treated (P<0.05) mice. These results suggest that the G protein γ2 subunit is involved in the transduction pathway leading to antinociception by DPDPE.
Keywords: G protein γ2 subunit; DPDPE (c[D-Pen2,D-Pen5]enkephalin); Antinociception;

β-Amyloid peptide-induced death of PC 12 cells and cerebellar granule cell neurons is inhibited by long-term lithium treatment by Huafeng Wei; Peter R Leeds; Yanning Qian; Wenlin Wei; Ren-wu Chen; De-Maw Chuang (117-123).
Treatment of rat pheochromocytoma cells (PC 12) cells with β-amyloid peptide-(1–42) for 24 h induced a concentration-dependent decrease in cellular redox activity in the dose range of 1 to 20 μM. These effects were markedly attenuated by pretreatment with 2 mM LiCl for 7 days, whereas 1-day pretreatment was ineffective. Measurements of live and dead cells by double-staining with fluorescein diacetate and propidium iodide, respectively revealed that protracted lithium pretreatment attenuated PC 12 cell death induced by β-amyloid-(1–42) and cerebellar granule cell death induced by β-amyloid-(25–35). Preceding PC 12 cell death, β-amyloid peptide elicited a slight decrease in protein levels of Bcl-2. Conversely, 7-day pretreatment with lithium resulted in an approximate doubling of Bcl-2 protein levels in cells treated with or without β-amyloid peptide-(1–42). Lithium-induced Bcl-2 upregulation was temporally associated with the cytoprotective effects of this drug. Thus, lithium protection against β-amyloid peptide neurotoxicity might involve Bcl-2 overexpression, and lithium treatment for Alzheimer's disease should be reexamined.
Keywords: Lithium chloride; β-Amyloid; Bcl-2; Alzheimer's disease;

Functional consequences of receptor stimulation by quinpirole, a dopamine D2-like receptor agonist, were assessed using agonist-stimulated [35S]GTPγS binding in rat striatal membranes. Dopamine receptor antagonists inhibited quinpirole-stimulated [35S]GTPγS binding with the following rank order of potency: spiperone>haloperidol>clozapine>SCH 23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine), consistent with a dopamine D2-like profile. In contrast, the monoamine oxidase inhibitors Ro 41-1049 (N-(2-aminoethyl)-5-(3-fluorophenyl)-4-thiazolecarboxemide), and (+)- and (−)-deprenyl, which inhibit [3H]quinpirole binding, had no effect on agonist-independent or quinpirole-stimulated [35S]GTPγS binding. Clorgyline inhibited [35S]GTPγS binding by a non-dopamine D2 receptor-mediated mechanism. These findings demonstrate a notable discrepancy between the pharmacological profile of [3H]quinpirole binding and quinpirole-stimulated [35S]GTPγS binding.
Keywords: [35S]GTPγS binding; Dopamine D2-like receptor; Quinpirole; Monoamine oxidase inhibitor; Striatum; (Rat);

Effect of methylene methylimino linkage of antisense oligonucleotide to the platelet-derived growth factor A-chain on growth of vascular smooth muscle cells from spontaneously hypertensive rats by Hirobumi Kishioka; Noboru Fukuda; Mari Nakayama; Wen-Yang Hu; Chikara Satoh; Katsuo Kanmatsuse; Muthiah Manoharan (129-132).
Spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells show exaggerated growth and increased expression of platelet-derived growth factor (PDGF) A-chain mRNA. We examined the effect of methylene methylimino linkage of antisense oligodeoxynucleotide, a novel modification of antisense oligodeoxynucleotide designed to increase nuclease resistance, to PDGF A-chain on the exaggerated growth of vascular smooth muscle cells from SHR. Methylene methylimino-linked oligodeoxynucleotide provided complete resistance against S1 nuclease. Methylene methylimino linkage of antisense oligodeoxynucleotide to PDGF A-chain resulted in a rapid inhibition of basal DNA synthesis of vascular smooth muscle cells from SHR. This inhibition was much greater than that produced by phosphorothioate linkage of antisense oligodeoxynucleotide to PDGF A-chain. The methylene methylimino linkage of antisense oligodeoxynucleotide to PDGF A-chain may prove useful in the treatment of arterial proliferative diseases including hypertension.
Keywords: Methylene methylimino linkage; Antisense; Platelet-derived growth factor A-chain; Spontaneously hypertensive rat (SHR); Smooth muscle cell;

Biphasic effects of dithiocarbamates on the activity of nuclear factor-κB by Chul Hoon Kim; Joo Hee Kim; Seok Jun Moon; Chung Y Hsu; Jeong Taeg Seo; Young Soo Ahn (133-136).
Dithiocarbamates are well-known antioxidants and nuclear factor-κB (NF-κB) inhibitors. Recently, they have been characterized as zinc ionophores. Concentration-dependent biphasic effects of dithiocarbamates on NF-κB activity have been widely reported. We studied the mechanism of this phenomenon in relation to Zn2+ influx. Two dithiocarbamates, pyrrolidine dithiocarbamate and diethyldithiocarbamate, showed concentration-dependent biphasic effects in inhibiting NF-κB activation in cerebral endothelial cells. These unique effects of dithiocarbamates on NF-κB were tightly linked to their ability to elevate intracellular Zn2+ levels. At high concentrations (>500 μM), dithiocarbamates started to lose their ability to promote Zn2+ influx and to inhibit NF-κB activation. These results might provide insight into the appropriate use of dithiocarbamates in various disorders.
Keywords: Cerebral endothelial cell; (Bovine); Dithiocarbamate; NF-κB (nuclear factor-κB); Zn2+;

High affinity blockade of the HERG cardiac K+ channel by the neuroleptic pimozide by Jiesheng Kang; Lin Wang; Feng Cai; David Rampe (137-140).
Pimozide is an antipsychotic agent also used to treat facial tics. Pimozide can cause acquired long QT syndrome and ventricular arrhythmias. To elucidate the mechanism behind these clinical findings, we examined the effects of pimozide on the cloned human cardiac K+ channels HERG (human ether-a-go-go-related gene; rapid component of delayed rectifier), Kv1.5 (ultra-rapid delayed rectifier) and KvLQT1/minK (slow component of delayed rectifier). Using patch clamp electrophysiology, we found that pimozide was a potent inhibitor of HERG displaying an IC50 value of 18 nM. In contrast, pimozide (10 μM) was a weak inhibitor of KvLQT1/minK and Kv1.5. We conclude that pimozide is a specific, high affinity antagonist of HERG, and that this interaction leads to prolongation of cardiac repolarization.
Keywords: K+ channel; Pimozide; HERG (human ether-a-go-go-related gene); Arrhythmia; Antipsychotic agent;

The effect of a selective cyclic guanocine 3′,5′-monophosphate (cGMP)-dependent protein kinase Iα inhibitor, Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (Rp-8-p-CPT-CGMPS), on either N-methyl-d-aspartate (NMDA)- or N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)ethanamine (NOC-12, a nitric oxide (NO) donor)-produced thermal hyperalgesia was examined in the rat. Intrathecal administration of NMDA (15 pg/10 μl) or NOC-12 (10, 20 and 30 μg/10 μl) produced a marked curtailment of the tail-flick latency. Maximal NMDA- or NOC-12-produced facilitation of the tail-flick reflex was significantly and dose-dependently blocked by intrathecal pretreatment with Rp-8-p-CPT-CGMPS (7.5, 15 and 30 μg/10 μl). Rp-8-p-CPT-CGMPS given alone did not markedly alter baseline tail-flick latency. These results suggest that the activation of cGMP-dependent protein kinase Iα is required for NMDA- or NO-produced facilitation of thermal hyperalgesia at the spinal cord level.
Keywords: Protein kinase; N-methyl-d-aspartate (NMDA); Nitric oxide (NO); Nociception; Spinal cord; Hyperalgesia; Pain;

This investigation reports the possible role of the endocannabinoid anandamide on modulating the behavioral and neurochemical consequences of semi-starvation. We studied the effect of very low dose anandamide (0.001 mg/kg) administration on food intake, cognitive function and catecholaminergic and serotonergic pathways in two murine brain areas concerned with appetite (hypothalamus) and learning (hippocampus), and the peripheral corticosterone response to the stress of 40% diet restriction. Anandamide-treated mice consumed 44% more food (P<0.05) during 1 week of 2.5-h feeding each day. In the hypothalamus, there were significantly increased concentrations of norepinephrine (P<0.01), dopamine (P<0.05) and 5-hydroxytryptamine (5-HT) (P<0.001). In the hippocampus, anandamide increased significantly norepinephrine and dopamine, but decreased 5-HT (all at P<0.001). Diet restriction was accompanied in both areas by a significant decrease in all neurotransmitter concentrations that were partially restored by anandamide for dopamine and 5-HT, but not for norepinephrine. In animals on diet restriction, anandamide significantly improved impaired maze performance. Norepinephrine turnover and plasma corticosterone levels were also raised significantly by anandamide. The fact that low dose anandamide improved food intake, cognitive function and reversed some of the neurotransmitter changes caused by diet restriction, might have implications for the treatment of cachexia associated with acquired immunodeficiency syndrome (AIDS) and cancer, for mood changes sometimes associated with dieting, and in the extreme case, of patients with anorexia.
Keywords: Anandamide; Food intake; Diet restriction; Cognitive function; Hippocampus; Hypothalamus; Catecholamine, 5-hydroxytryptamine, serotonin (5-HT); Corticosterone;

Male rats exhibit significantly greater antinociception following central administration of morphine than female rats. The present study examined potential differences in β-endorphin (5.2–26 μg) antinociception elicited from the ventrolateral periaqueductal gray in adult sham-operated and gonadectomized male and female rats. Male rats displayed significantly greater peak (30 min) tail-flick latencies across the entire range of β-endorphin doses administered into the ventrolateral periaqueductal gray than female rats tested during the estrous phase of the estrous cycle. Adult gonadectomy failed to appreciably change the pattern of this effect in either males of females. Thus, antinociception elicited from the ventrolateral periaqueductal gray by β-endorphin, like morphine, is sensitive to sex differences.
Keywords: Pain; Antinociception; Sex difference; Gonadectomy, adult; β-Endorphin;

In the present study, the role of reactive oxygen species and the contribution of antioxidant defence in the time course of changes in acetylcholine-stimulated endothelium-dependent and sodium nitroprusside-stimulated endothelium-independent relaxation were investigated in aortic rings isolated from 6-month streptozotocin-diabetic and age-matched control rats. Although there were no significant differences in the degree of the peak relaxations produced by a single administration of acetylcholine (1 μM) or sodium nitroprusside (0.01 μM) between control and diabetic rings, the endothelium-dependent and -independent relaxant responses were more transient and the time required to reach a peak relaxation after addition of acetylcholine was shorter in diabetic vessels. Pretreatment of diabetic vessels with superoxide dismutase (100 U/ml) normalized the recovery phases of endothelium-dependent and -independent relaxations, but had no effect on the peak responses to acetylcholine and sodium nitroprusside. In the presence of diethyldithiocarbamate (5 mM), an inhibitor of superoxide dismutase, the transient nature of the relaxant response to acetylcholine or sodium nitroprusside was more marked and the peak relaxations were inhibited; these effects of diethyldithiocarbamate were more pronounced in diabetic than in control rings. Catalase, 160 U/ml, decreased the peak relaxant response to acetylcholine and accelerated fading of the relaxation in diabetic aorta. Similar results were obtained for control aorta with a higher concentration of catalase (550 U/ml). Pretreatment with 3-amino-1,2,4 triazole (5 mM), a catalase inhibitor, inhibited the peak relaxant response to acetylcholine in diabetic rings. The combination of superoxide dismutase (100 U/ml) plus 3-amino-1,2,4 triazole (5 mM) produced an increase of the transient nature of endothelium-dependent relaxation of diabetic rings greater than that with 3-amino-1,2,4 triazole alone. Neither catalase nor 3-amino-1,2,4 triazole affected the characteristics of sodium nitroprusside-induced relaxation. Desferrioxamine, an inhibitor of hydroxyl radical (·OH) production, or mannitol, a ·OH scavenger, had no effect on the characteristics of either acetylcholine- or sodium nitroprusside-induced relaxation in control and diabetic rings. Biochemical measurements revealed an inhibited superoxide dismutase activity in diabetic aorta together with activated catalase. Our findings suggest that, during the chronic phase of streptozotocin-diabetes, excess superoxide (O2 ·−) is responsible for the enhanced transient nature of endothelium-dependent and -independent relaxation of aorta via a reduction in bioavailable concentrations of nitric oxide (NO). However, the involvement of hydrogen peroxide (H2O2) in the establishment of acetylcholine-stimulated relaxation may be increased, which is likely to account for the maintenance of the relaxant effect of acetylcholine in chronically diabetic vessels.
Keywords: Aorta; Streptozotocin-diabetes; Endothelium-dependent relaxation; Endothelium-independent relaxation; Nitric oxide (NO); Superoxide; Hydrogen peroxide; Superoxide dismutase; Catalase;

Tumor necrosis factor-α augments contraction and cytosolic Ca2+ sensitivity through phospholipase A2 in bovine tracheal smooth muscle by Yuji Nakatani; Yoshihiro Nishimura; Teruaki Nishiuma; Hitoshi Maeda; Mitsuhiro Yokoyama (175-182).
To elucidate the effects of tumor necrosis factor-α (TNF-α) on tracheal smooth muscle contraction, we simultaneously measured isometric tension and intracellular Ca2+ ([Ca2+]i) in fura 2-loaded muscle strips. Smooth muscle force generation was evaluated in a high potassium (K+; 20.0–80.0 mM) solution and with acetylcholine (3 nM–10 μM ). TNF-α (1–100 ng/ml) did not directly contract muscle strips. The contractile response to acetylcholine was enhanced after application of 10 ng/ml of TNF-α for 30 min but not the response of [Ca2+]i. The contractile response and the response of [Ca2+]i to a high K+ solution were not altered after application of TNF-α. The [Ca2+]i–tension curve indicated that TNF-α enhanced the responsiveness of tracheal smooth muscle through the acetylcholine-mediated Ca2+ sensitivity of intracellular contractile elements. The augmentation of the acetylcholine concentration–response curves for muscle tension in the presence of TNF-α (10 ng/ml) was inhibited in part after application of manoalide, a phospholipase A2 inhibitor. We conclude that a low concentration of TNF-α enhances smooth muscle responsiveness to acetylcholine by agonist-mediated Ca2+ sensitivity facilitated by phospholipase A2.
Keywords: Cytokine; Bronchial hyperresponsiveness; Bronchial asthma; Arachidonic acid metabolite;

Evidence for the involvement of ATP, but not of VIP/PACAP or nitric oxide, in the excitatory effect of capsaicin in the small intestine by Loránd Barthó; Zsófia Lázár; László Lénárd; Rita Benkó; Gábor Tóth; Botond Penke; János Szolcsányi; Carlo Alberto Maggi (183-188).
The contractile effect of capsaicin in the guinea-pig small intestine involves an activation of enteric cholinergic neurons. Our present data show that the P2 purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 30 μM) significantly reduces the contractile response to capsaicin (2 μM) in the presence, but not in the absence, of the tachykinin receptor antagonists [O-Pro9, (Spiro-γ-lactam)Leu10, Trp11]physalaemin (1–11) (GR 82334; 3 μM) and (S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenylpiperidine-4-yl)-N-methylacetamide (SR 142801; 100 nM) (for blocking tachykinin NK1 and NK3 receptors, respectively). PPADS (30 μM) fails to influence submaximal cholinergic contractions evoked by cholecystokinin octapeptide (CCK-8; 2–3 nM) or senktide (1 nM), or the direct smooth muscle-contracting effect of histamine (100–200 nM). A higher concentration (300 μM) of PPADS is also without effect against the stimulatory action of cholecystokinin octapeptide. This means that PPADS can probably be safely used as a purinoceptor antagonist in intestinal preparations. The putative pituitary adenylate cyclase activating peptide (PACAP) receptor antagonist PACAP-(6–38) (3 μM) significantly reduces the contractile effect of PACAP-(1–38) (10 nM) and abolishes that of vasoactive intestinal polypeptide (VIP; 10 nM). PACAP-(6–38) (3 μM) fails to influence the effect of capsaicin (2 μM) both in the absence and in the presence of tachykinin receptor antagonists. The nitric oxide (NO) synthase inhibitor N G-nitro-l-arginine (l-NOARG; 100 μM) also fails to inhibit the capsaicin-induced motor response. We conclude that an endogenous ligand of PPADS-sensitive P2 purinoceptors (possibly ATP), but not a VIP/PACAP-like peptide or NO, is involved in the nontachykininergic activation of cholinergic neurons in the course of the capsaicin-induced contraction.
Keywords: Capsaicin; Myenteric neuron; PPADS (Pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid); VIP (Vasoactive intestinal polypeptide); PACAP (Pituitary adenylate cyclase activating peptide); NO (Nitric oxide); Small intestine;

The role of fractalkine in the recruitment of monocytes to the endothelium by Gayle A Chapman; Kitty E Moores; Jayneeta Gohil; Theo A Berkhout; Lisa Patel; Paula Green; Colin H Macphee; Brian R Stewart (189-195).
Recombinant fractalkine possesses both chemoattractive and adhesive properties in vitro. Previous studies have demonstrated an upregulation of this molecule on the membranes of activated human endothelial cells and hypothesised that fractalkine plays a role in the recruitment and adherence of monocytes to the activated endothelium. Here we present data analysing both the adhesive and chemoattractive properties of this chemokine expressed by activated human umbilical vein endothelial cells. We demonstrate that both recombinant fractalkine and endogenously produced fractalkine function as adhesion molecules, tethering monocytes to the endothelium. However, our data demonstrate that although recombinant fractalkine has the potential to function as a potent monocyte chemoattractant, the endogenous fractalkine cleaved from activated human umbilical vein endothelial cells is not responsible for the observed chemotaxis in this model. Instead, we show that monocyte chemoattractant protein-1 (MCP-1), secreted from the activated human umbilical vein endothelial cells, is responsible for the chemotaxis of these monocytes.
Keywords: Fractalkine; Chemokine; Chemotaxis; Adhesion; Umbilical vein; Endothelial cell;

Index (197-199).

Index (201-205).