European Journal of Pharmacology (v.494, #1)

Triptolide (PG490) is a natural, biologically active compound extracted from the Chinese herb Tripterygium wilfordii. It has been shown to possess potent anti-inflammatory and immunosuppressive properties. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, triptolide inhibits nitric oxide (NO) production in a dose-dependent manner and abrogates inducible nitric oxide synthase (iNOS) gene expression. To investigate the mechanism by which triptolide inhibits murine iNOS gene expression, we examined activation of mitogen-activated protein kinases (MAP kinases) and nuclear factor-κB (NF-κB) in these cells. Addition of triptolide inhibited phosphorylation of c-Jun NH2-terminal kinase (JNK) but not that of extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein kinase. In addition, triptolide significantly inhibited the DNA binding activity of NF-κB. Taken together, these results suggest that triptolide acts to inhibit inflammation through inhibition of NO production and iNOS expression through blockade of NF-κB and JNK activation.
Keywords: Triptolide; NO (Nitric oxide); iNOS; NF-κB; JNK;

Role of extracellular cysteine residues in dimerization/oligomerization of the human prostacyclin receptor by Vincent Giguère; Maxime A Gallant; Artur J de Brum-Fernandes; Jean-Luc Parent (11-22).
Prostacyclin activation of prostanoid IP receptors may result in pain sensation, inflammatory responses, inhibition of platelet aggregation, and vasodilation in vascular tissue. The prostanoid IP receptor is a G-protein-coupled receptor. In the present study, we investigated the determinants responsible, at least in part, for the prostacyclin receptor (IP) dimerization/oligomerization. Using co-immunoprecipitation of differentially tagged IP expressed in COS-7 cells, we demonstrate that IP can form dimers and oligomers. Treatment of IP-expressing cells with the stable agonist carbaprostacyclin failed to alter the ratios of oligomeric/dimeric/monomeric forms of the receptor, suggesting that IP dimerization/oligomerization is an agonist-independent process. The reducing agents dithiothreitol and 2-mercaptoethanol were highly efficient in converting the receptor from its oligomeric form to the monomeric state, indicating the involvement of disulfide bonds in IP oligomerization. Immunoblotting of the osteoblastic MG-63 cell line lysates with an anti-IP specific antibody revealed the presence of endogenous IP oligomers which were converted to dimers and monomers upon treatment with dithiothreitol. Individual substitutions of the four extracellular IP Cys residues (Cys5, Cys92, Cys165 and Cys170) for Ser resulted in greatly decreased receptor protein expression in COS-7 cells. The C92–170S double mutant showed receptor protein expression level similar to the individual mutants. However, expression of the C92–165S and C165–170S mutants was drastically reduced, suggesting that there was formation of disulfide bonds between Cys5 and Cys165, and between Cys92 and Cys170. The Cys receptor mutants showed altered oligomer/dimer/monomer ratios. Dimerization/oligomerization likely occurs intracellularly since these Cys receptor mutants could still form dimers/oligomers despite their lack of expression at the cell surface.
Keywords: Prostacyclin; Prostanoid IP receptor; G-protein-coupled receptor; Dimerization; Oligomerization; Cysteine;

In the present study, we have evaluated the visceral analgesic property of buspirone, a 5HT1A receptor partial agonist, on colorectal distension-induced mean arterial pressure and behavioral changes in anesthetized and awake Wistar rats, respectively. The selection of the rat strain was based on the observation that anesthetized Wistar rats exhibited a more prominent mean arterial pressure change in response to colorectal distention when compared to other strains (Sprague–Dawley, Wistar–Kyoto and Spontaneously Hypertensive). Buspirone dose-dependently (0.1–1 mg/kg, i.v.) antagonized mean arterial pressure change over a range of distensions (10–90 mmHg). In parallel studies conducted in awake animals, buspirone (1–5 mg/kg, s.c.) attenuated the abdominal withdrawal response, a nociceptive behavior, in response to colorectal distension. This effect was antagonized by co-administration of the 5-HT1A receptor antagonist N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-2- pyridinylcyclohexanecarboxamide (WAY-100635) (5 mg/kg, s.c.). We conclude that buspirone exhibits significant visceral analgesic property in two models of abdominal nociception.
Keywords: Colorectal distension; Visceral pain; Anti-nociception; 5-HT1A receptor; Buspirone;

Effects of nicotine preexposure on sulpiride-induced dopamine release in the nucleus accumbens by Shafiqur Rahman; Jianhua Zhang; William A Corrigall (31-34).
We examined the effects of nicotine preexposure or saline on dopamine release to sulpiride in the rat nucleus accumbens. Microdialysis was used to locally perfuse the sulpiride into the ventral tegmental area while sampling dopamine levels in the nucleus accumbens. The increase (130% and 165% of basal) in extracellular accumbens dopamine levels observed during ventral tegmental area perfusion for 80 min with 10–100 μM sulpiride in saline-treated animals was reduced (128% and 105% of basal) in nicotine-preexposed animals. The reduction of sulpiride-induced nucleus accumbens dopamine release after nicotine treatment is likely the result of down-regulation of somatodendritic dopamine autoreceptors.
Keywords: Nicotine; Dopamine release; Dopamine autoreceptor; Ventral tegmental area; Nucleus accumbens; In vivo microdialysis;

Effects of repeated testing in two inbred strains on flesinoxan dose–response curves in three mouse models for anxiety by J.Adriaan Bouwknecht; Jan van der Gugten; Lucianne Groenink; Berend Olivier; Richard E Paylor (35-44).
Over the last decade, many genetically modified mice have been developed as models for psychiatric diseases such as anxiety. Limited availability of such mutant mice highlights the importance of studying the possibility of repeatedly testing the same individuals. We tested mice four times with 1-week intervals with the same dose of the 5-HT1A receptor agonist flesinoxan (0–0.3–1.0–3.0 mg/kg s.c.) in three anxiety-related paradigms: light–dark exploration, open-field activity and stress-induced hyperthermia. The two inbred strains studied were the highly anxious 129S6/SvEvTac (S6) and low-anxiety C57BL/6J (B6) mice. The results indicate that the effects of repeated testing were relatively mild. B6 mice showed some mild habituation in the open-field test when treated with vehicle, whereas S6 mice developed reduced initial activity in the light–dark box after drug treatment. In contrast, responses to flesinoxan treatment were strong and highly consistent for most parameters. In the open-field and light–dark tests, B6 mice showed reduced activity and anxiogenic-like behavioral responses, whereas S6 mice were minimally affected. Anxiolytic-like responses were found in both strains in the stress-induced hyperthermia paradigm. We conclude that B6 and S6 mice can be tested repeatedly with agents such as 5-HT1A receptor agonists with 1-week intervals in the three paradigms tested.
Keywords: Physiology; Behavior; Animal model; Flesinoxan; Repeated testing; 5-HT1A receptor;

Behavioral and physiological mouse models for anxiety: effects of flesinoxan in 129S6/SvEvTac and C57BL/6J mice by J Adriaan Bouwknecht; Jan van der Gugten; Lucianne Groenink; Berend Olivier; Richard E Paylor (45-53).
Serotonin1A (5-HT1A) receptors are involved in anxiety. This study focuses on the role of genetic factors on the anxiety-related effects of 5-HT1A receptor stimulation using both a within subject design. The effects of 5-HT1A receptor activation were studied in high- and low-anxiety mice (129S6/SvEvTac (S6) and C57BL/6J (B6), respectively) in behavioral and physiological anxiety-related assays. These two strains were also selected because they are frequently used in gene-targeting studies. Mice were treated with the selective 5-HT1A receptor agonist flesinoxan (0–0.3–1.0–3.0 mg/kg s.c.) and tested in either the open-field activity test, the light–dark exploration test, or the stress-induced hyperthermia paradigm. Flesinoxan unexpectedly increased anxiety, but also decreased activity on several behavioral measures in B6 mice. Flesinoxan produced only minimal effects in the behavioral tests in the high-anxiety S6 strain. In contrast, the physiological hyperthermia response showed anxiolytic-like effects of flesinoxan in both strains. Our data indicate that the role of 5-HT1A receptor activation on anxiety-related responses is dependent on genetic background and selected paradigm used to assess anxiety. These findings indicate that it is critical to use a multi-level approach to develop mouse models for human diseases. In addition, the implication of such findings for studies on genetically modified mice is discussed.
Keywords: Physiology; Behavior; Anxiety; Animal model; Flesinoxan; 5-HT1A receptor;

This study was undertaken to investigate the effect of some cannabinoid agonists on the bovine ciliary muscle. Both anandamide and CP 55,940 (cis-3-(2-hydroxy-4-(1,1-dimethyl heptyl) phenyl)-trans-4-(3-hydroxypropyl) cyclohexanol) produced a concentration-dependent contractile response in ciliary muscle. These responses were inhibited by SR 141716A (N-[piperidin-1-yl]-5-(4-cholophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) (0.1 and 1 μM) but not by SR 144528 (N-[1S)-endo-1,3,3-trimethyl bicyclo[2.2.1] heptan-2-yl] 5-(4-chloro-3-methylphenyl)-1-(4 methoxy benzyl)-pyrazole-3-carboxamide) (1 and 10 μM). A preincubation with Gi/o protein inhibitor pertussis toxin (500 ng/ml) for 20 min inhibited the contractile action of anandamide and CP 55,940. In addition, the phospholipase C inhibitor U73122 (1[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl] amino] hexyl]-1H-pyrrole-2,5-dione) blocked the anandamide- and CP 55,940-induced contractions, whereas the protein kinase C activator, phorbol 12,13 dibutyrate (PDBu) significantly potentiated the contractions evoked by cannabinoid receptor agonists. We evaluated the binding of [3H]CP 55,940, which specifically labelled a single class of cannabinoid sites with affinity in low subnanomolar range (K d=0.6 nM) and the maximal number of binding sites of 1243 fmol/mg protein. Binding of [3H]CP 55,940 was inhibited by ligands having a major selectivity for cannabinoid (CB1) receptors. These findings provide strong evidence of the involvement of cannabinoid CB1 receptors promoting contraction in the bovine ciliary muscle. Furthermore, the action of cannabinoid receptor agonists appears to be mediated via phospholipase C. These data also contribute to elucidate the cannabinoid CB1 receptor pivotal role in the modulation of intraocular pressure and to show that cannabinoid receptor agonists may be regarded as potential antiglaucoma agents.
Keywords: Cannabinoid CB1 receptor; Anandamide; Receptor binding assay; Glaucoma;

Role of endothelin ETB receptor in partial ablation-induced chronic renal failure in rats by Yuka Okada; Mariko Nakata; Hiromi Izumoto; Mai Takasu; Naoko Tazawa; Masanori Takaoka; Cheryl E Gariepy; Masashi Yanagisawa; Yasuo Matsumura (63-71).
We investigated the role of endothelin ETB receptor in the remnant kidney model of chronic renal failure, by using the spotting-lethal (sl) rat, which carries a naturally occurring deletion in the endothelin ETB receptor gene. After 5/6 nephrectomy, systolic blood pressure and renal functional parameters were measured for 12 weeks. At the end of the experimental period, arterial blood sample, remnant kidney, heart and aorta were collected and used for biochemical measurements and histopathological studies. The ETB-deficient sl/sl rats exhibited earlier and higher increases in systolic blood pressure, urinary protein excretion, blood urea nitrogen and plasma creatinine concentration, compared with cases in wild-type rats. Histopathologic examination of the kidney revealed glomerular and tubular lesions, alterations of which were more severe in sl/sl than in wild-type rats. While aortic endothelin-1 contents were increased similarly in both groups, the level of renal endothelin-1 content was significantly elevated in sl/sl rats, but not in the wild-type rats. These results suggest that enhanced endothelin-1 production is at least partly responsible for the increased susceptibility to partial ablation-induced chronic renal failure in ETB receptor-deficient rats and that ETB receptor-mediated actions are protective against vascular and renal injuries in this disease.
Keywords: Endothelin-1; ETB receptor; Chronic renal failure; Renal mass reduction; Hypertension;

Growth factor-induced contraction of human bronchial smooth muscle is Rho-kinase-dependent by Reinoud Gosens; Dedmer Schaafsma; Mechteld M Grootte Bromhaar; Bart Vrugt; Johan Zaagsma; Herman Meurs; S.Adriaan Nelemans (73-76).
Growth factors have been implicated in the pathophysiology of asthma. However, the putative effects of these growth factors on human airway smooth muscle tone are still largely unknown. We performed contraction experiments using human bronchial smooth muscle ring preparations. The growth factor insulin-like growth factor-1 (IGF-1) induced a slowly developing sustained contraction, which was dependent on Rho-kinase, since contraction was almost completely inhibited by (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632; 1 μM). Angiotensin II, a Gq-coupled receptor agonist which can act as a growth factor as well, induced a biphasic contraction, the sustained phase of which was also almost completely inhibited by Y-27632. We conclude that angiotensin II and IGF-1 induce a Rho-kinase-dependent sustained contraction of human bronchial smooth muscle. Since growth factors are associated with pathophysiologiocal conditions such as asthma, inhibition of Rho-kinase could be effective under these conditions.
Keywords: Growth factor; Bronchial smooth muscle; Rho-kinase;