European Journal of Pharmacology (v.572, #2-3)

Recent research in cell signaling has shown that the assembly of G protein coupled receptors into signaling complexes or signalplexes represents the primary mechanism by which receptor-mediated signaling is established and maintained. In this review, we summarize the current state of knowledge regarding protein interactions that comprise the dopamine D2 receptor signalplex within the brain. Studies based on conventional and advanced two-hybrid methodologies, as well as bioinformatic and computational analysis of sequence information from completed genomes have demonstrated interactions between dopamine D2 receptors and a cohort of dopamine receptor interacting proteins (DRIPs). DRIP interactions appear to regulate key aspects of receptor function including the signaling and membrane trafficking of dopamine D2 receptors. Disruptions or modifications of the signalplex, using membrane permeant competing peptide or dominant negative approaches, may represent promising new strategies for the selective targeting of the dopamine D2 receptor in cells and in native tissue. DRIP interactions provide a novel platform for understanding the mechanisms of dopamine receptor signaling, and for the potential development of novel treatments for brain disease.
Keywords: Dopamine D2 receptor; Signalplex; Interacting protein; Proteomics; G protein coupled receptor;

Antagonism of neurosteroid modulation of native γ-aminobutyric acid receptors by (3α,5α)-17-phenylandrost-16-en-3-ol by Stephen P. Kelley; Jamie K. Alan; Todd K. O'Buckley; Steven Mennerick; Kathiresan Krishnan; Douglas F. Covey; A. Leslie Morrow (94-101).
Endogenous pregnane neurosteroids are allosteric modulators at γ-aminobutyric acid type-A (GABAA) receptors at nanomolar concentrations. There is direct evidence for multiple distinct neurosteroid binding sites on GABAA receptors, dependent upon subunit composition and stoichiometry. This view is supported by the biphasic kinetics of various neuroactive steroids, enantioselectivity of some neurosteroids, selective mutation studies of recombinantly expressed receptors and the selectivity of the neurosteroid antagonist (3α,5α)-17-phenylandrost-16-en-3-ol (17PA) on 5α-pregnane steroid effects on recombinant GABAA receptors expressed in Xenopus oocytes and native receptors in dissociated neurons. However, it is unclear whether this antagonist action is present in a mature mammalian system. The present study evaluated the antagonist activity of 17PA on neurosteroid agonists both in vivo and in vitro by examining the effects of 17PA on 5α-pregnane-induced sedation in rats, native mature GABAA receptor ion channels utilizing the chloride flux assay and further studies in recombinant α1β2γ2 receptors. The data show that 17PA preferentially inhibits 3α,5α-THP vs. alphaxalone in vivo, preferentially inhibits 3α,5α-THDOC vs. alphaxalone potentiation of GABA-mediated Cl uptake in adult cerebral cortical synaptoneurosomes, but shows no specificity for 3α,5α-THDOC vs. alphaxalone in recombinant α1β2γ2 receptors. These data provide further evidence of the specificity of 17PA and the heterogeneity of neurosteroid recognition sites on GABAA receptors in the CNS.
Keywords: Neuroactive steroid; Native GABA receptor; Recombinant α1β2γ2 receptor; Sedation;

Inhibition of cyclooxygenase-2 down-regulates osteoclast and osteoblast differentiation and favours adipocyte formation in vitro by Maarit Kellinsalmi; Vilhelmiina Parikka; Juha Risteli; Teuvo Hentunen; Hannu-Ville Leskelä; Siri Lehtonen; Katri Selander; Kalervo Väänänen; Petri Lehenkari (102-110).
Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenases (COX) and are widely used for post-trauma musculoskeletal analgesia. In animal models, NSAIDs have been reported to delay fracture healing and cause non-union, possibly due to the drug-induced inhibition of osteoblast recruitment and differentiation. To further investigate the cellular effects of these drugs in the context of bone healing, we examined the effects of COX-1 inhibitor indomethacin and COX-2 inhibitors, parecoxib and NS398 on osteoclast and osteoblast differentiation and activity in vitro. We discovered that all tested COX-inhibitors significantly inhibited osteoclast differentiation, by 93%, 94% and 74% of control for 100 μM indomethacin, 100 μM parecoxib and 3 μM NS398, respectively. Furthermore, inhibition of COX-2 reduced also the resorption activity of mature osteoclasts. All tested COX-inhibitors also significantly inhibited osteoblast differentiation from human mesenchymal stem cells. Simultaneously, the number of adipocytes was significantly increased. The adipocyte covered areas in the cultures with 1 μM indomethacin, 1 μM parecoxib and 3 μM NS398 were 9%, 29% and 24%, respectively, as compared with 6% in the control group. This data suggests that COX-2 inhibition disturbs bone remodelling by inhibiting osteoclast differentiation and diverting stem cell differentiation towards adipocyte lineage instead of osteoblast lineage. In conclusion, our results further suggest cautious use of COX-2 inhibitors after osseous trauma.
Keywords: Cyclooxygenase; Human mesenchymal stem cell; Adipocyte; Parecoxib;

Mechanisms of prodigiosin cytotoxicity in human neuroblastoma cell lines by Roser Francisco; Ricardo Pérez-Tomás; Pepita Gimènez-Bonafé; Vanessa Soto-Cerrato; Pol Giménez-Xavier; Santiago Ambrosio (111-119).
Prodigiosin is a bacterial red pigment with cytotoxic properties and potential antitumor activity that has been tested against different cancerous cells. In this study we report the effect and mechanisms of action of prodigiosin against different human neuroblastoma cell lines: SH-SY5Y, LAN-1, IMR-32 (N-type) and SK-N-AS (S-type). We compare the anticancerous effect of prodigiosin with that of cisplatin at different concentrations during 24 h of exposure. Prodigiosin is more potent, with IC50 values lower than 1.5 μM in N-type neuroblastoma cells and around 7 μM in the S-type neuroblastoma cell line. We describe prodigiosin as a proton sequestering agent that destroys the intracellular pH gradient, and propose that its main cytotoxic effect could be related to its action on mitochondria, where it exerts an uncoupling effect on the electronic chain transport of protons to mitochondrial ATP synthase. As a result of this action, ATP production is reduced but without decreasing in oxygen consumption. This mechanism of action differs from those induced by conventional chemotherapeutic drugs, suggesting a possible role for prodigiosin to enhance the effect of antitumor agents in the treatment of neuroblastoma.
Keywords: Neuroblastoma; Prodigiosin; Cisplatin; Apoptosis;

Methamphetamine-induced hyperthermia and lethal toxicity: Role of the dopamine and serotonin transporters by Yohtaro Numachi; Arihisa Ohara; Motoyasu Yamashita; Setsu Fukushima; Hideaki Kobayashi; Harumi Hata; Hidekazu Watanabe; F. Scott Hall; Klaus-Peter Lesch; Dennis L. Murphy; George R. Uhl; Ichiro Sora (120-128).
We examined the hyperthermic and lethal toxic effects of methamphetamine in dopamine transporter (DAT) and/or serotonin transporter (SERT) knockout (KO) mice. Methamphetamine (45 mg/kg) caused significant hyperthermia even in the mice with a single DAT gene copy and no SERT copies (DAT+/− SERT−/− mice). Mice with no DAT copies and a single SERT gene copy (DAT−/− SERT+/− mice) showed significant but reduced hyperthermia when compared to wild-type mice after methamphetamine. Surprisingly, DAT/SERT double KO mice exhibited a paradoxical hypothermia after methamphetamine. These results demonstrate that methamphetamine exerts a hyperthermic effect via DAT, or via SERT, in the absence of DAT. The selective norepinephrine transporter blocker (20 mg/kg nisoxetine) caused hyperthermia in DAT/SERT double KO mice, suggesting that the norepinephrine system is not responsible for methamphetamine-induced paradoxical hypothermia in the double KO mice. DAT gene deletion in mice strikingly increased LD50 of methamphetamine by 1.7–1.8 times that of wild-type mice, suggesting that the lethal toxic effect of methamphetamine is mainly dependent on DAT. Moreover, dissociation between hyperthermic and lethal toxic effects of methamphetamine in DAT single KO mice and DAT/SERT double KO mice suggest that hyperthermia is not a prerequisite for methamphetamine-induced lethality. Methamphetamine (45 mg/kg) significantly increased mRNA of interleukin-1β, which is the major endogenous pyrogen, in the hypothalamus of wild-type mice but not in DAT/SERT double KO mice, which provides a partial mechanism of methamphetamine-induced paradoxical hypothermia. These results suggest that DAT and SERT are key molecules for hyperthermic and lethal toxic effects of methamphetamine.
Keywords: Monoamine; Norepinephrine; Paradoxical hypothermia; Interleukin-1β; Transgenic knockout mouse;

Both muscarinic and nicotinic receptors are implicated in cognition. We have previously suggested that stimulation of the muscarinic M1 receptor has a beneficial effect on cognition, based upon evidence that the muscarinic M1 receptor agonist of N-desmethylclozapine, the major metabolite of clozapine, may contribute to the ability of clozapine to improve some domains of cognition in schizophrenia. Present study examined the effectiveness of a new muscarinic M1 receptor agonist, 4-[3-(4-butylpiperidin-1-yl)-propyl]-7-fluoro-4H-benzo[1,4]oxazin-3-one (AC260584), to increase the release of acetylcholine and dopamine in the rat medial prefrontal cortex and hippocampus. Using microdialysis in awake, freely moving rats, AC260584, 3 and 10, but not 1 mg/kg (s.c.), significantly increased dopamine release in the medial prefrontal cortex and hippocampus. However, only the high dose of AC260584, 10 mg/kg (s.c.), significantly increased acetylcholine release in these regions. Moreover, the increases in acetylcholine release produced by AC260584, 10 mg/kg, were attenuated by the muscarinic M1 receptor antagonist telenzepine (3 mg/kg, s.c.) but not by the 5-HT1A receptor antagonist N-[2-(4-2-methoxyphenyl)-1-piperazinyl]-N-(2-pyridyl) cyclohexanecarboxamide (WAY100635, 0.2 mg/kg, s.c.). However, the increase in dopamine release produced by 10 mg/kg AC260584 was blocked by both telenzepine and WAY100635. In addition, pretreatment with the atypical antipsychotic drug risperidone (0.1 mg/kg, s.c.) potentiated AC260584 (1.0 mg/kg, s.c.)-induced acetylcholine and dopamine release in the medial prefrontal cortex. These findings suggest that the muscarinic M1 receptor agonist property of AC260584 contributes to its enhancement of cortical acetylcholine and dopamine efflux. Therefore, AC260584, as well as other muscarinic M1 receptor agonists, may be a valuable target for the development of drugs which can improve the cognitive deficits in schizophrenia and perhaps other neuropsychiatric disorders, as well.
Keywords: AC260584; Muscarinic; Acetylcholine; Dopamine; Cognition; Microdialysis;

Role of 4-aminopyridine-sensitive potassium channels in peripheral antinociception by Tufan Mert; Yasemin Gunes; Ismail Gunay (138-141).
Previous studies has report the modulation of K+ channels play key roles in the induction of peripheral antinociception induced by many types of drugs. However, the possible participation of 4-aminopyridine-sensitive K+ channels to local antinociception induced by tramadol, a μ opioid receptor agonist, and lidocaine, a local anaesthetic, has been less studied. In this study, we therefore investigated this by using thermal plantar test. Tramadol or lidocaine administered intraplantarly into the hind paw elicited an antinociceptive effect. 4-aminopyridine caused an increase in the antinociception produced by lidocaine. However, tramadol induced antinociception remained unaffected by intraplantar administration of 4-aminopyridine. These results suggest that 4-aminopyridine-sensitive K+ channels may play an important role in the thermal peripheral antinociception produced by lidocaine, but not tramadol.
Keywords: 4-Aminopyridine; Tramadol; Lidocaine; Antinociception; Plantar test;

The effects of (−)-N1phenethyl-norcymserine (PEC, 5 mk/kg, i.p.) on acetylcholine release and cholinesterase activity in the rat cerebral cortex were compared with those of donepezil (1 mg/kg, i.p.), a selective acetylcholinesterase inhibitor, and rivastigmine (0.6 mg/kg, i.p.), an inhibitor of acetylcholinesterase and butyrylcholinesterase. Acetylcholine extracellular levels were measured by microdialysis coupled with HPLC; acetylcholinesterase and butyrylcholinesterase activity were measured with colorimetric and radiometric methods. It was found that comparable 2–3 fold increases in cortical extracellular acetylcholine level, calculated as areas under the curve, followed the administration of the three drugs at the doses used. At the peak of acetylcholine increase, a 27% acetylcholinesterase inhibition and no butyrylcholinesterase inhibition was found after donepezil (1 mg/kg, i.p) administration. At the same time point, rivastigmine (0.6 mg/kg, i.p.) inhibited acetylcholinesterase by 40% and butyrylcholinesterase by 25%. After PEC (5 mg/kg, i.p.) administration, there was a 39% butyrylcholinesterase inhibition and no effect on acetylcholinesterase. Since in the present study it was also confirmed that in the brain butyrylcholinesterase activity is only about 10% of acetylcholinesterase activity, it is surprising that its partial inhibition is sufficient to increase extracellular acetylcholine levels. The importance of butyrylcholinesterase as a “co-regulator” of synaptic acetylcholine levels should thus be reconsidered.
Keywords: Acetylcholine; Acetylcholinesterase; Butyrylcholinesterase; Cholinesterase inhibitors; Microdialysis;

Cholinergic drugs affect novel object recognition in rats: Relation with hippocampal EEG? by Anke Sambeth; Wim J. Riedel; Laura Th. Smits; Arjan Blokland (151-159).
This study examined the role of cognitively enhancing cholinergic drugs on both object memory and brain activity in rats, as well as the possible relation between the two measures. A group of twenty-four animals was used for assessing object recognition. In another group of eight rats, an electrode was implanted into the dorsal hippocampus to record an electroencephalogram (EEG) and auditory evoked potentials (AEP). In both groups, animals were treated with saline, 0.1 mg/kg scopolamine, 0.1 mg/kg methylscopolamine, 3 mg/kg donepezil, donepezil combined with scopolamine, 0.1 mg/kg nicotine, and nicotine combined with scopolamine. Scopolamine, but not methylscopolamine, impaired object recognition. Both donepezil and nicotine reversed this impairment. The N1 and N2 components of the AEP became closer to baseline after scopolamine, which was not reversed by donepezil or nicotine. Scopolamine increased the theta frequency in the EEG. When combined with donepezil, theta increased even more. Conversely, nicotine reversed the theta increment to control level. It is suggested that scopolamine caused a decrement in arousal in this study. Furthermore, the current results suggest a relation between EEG and object memory after cholinergic drug treatment. However, there was a clear dissociation between memory performance and EEG after combined treatment with drugs, which makes additional research where EEG and performance measures are co-registered imperative.
Keywords: Acetylcholine; Animal model; Auditory evoked potential (AEP); Donepezil; Electroencephalogram (EEG); Nicotine; Scopolamine;

Lurasidone (SM-13496), a novel atypical antipsychotic drug, reverses MK-801-induced impairment of learning and memory in the rat passive-avoidance test by Takeo Ishiyama; Kumiko Tokuda; Tadashi Ishibashi; Akira Ito; Satoko Toma; Yukihiro Ohno (160-170).
Lurasidone (SM-13496) is a novel atypical antipsychotic with high affinities to dopamine D2, serotonin 5-HT7, 5-HT2A, 5-HT1A receptors and α2C adrenoceptor. In this study, the effects of lurasidone on the rat passive-avoidance response and its impairment by the N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 (dizocilpine) were evaluated and compared with those of other antipsychotics. The passive-avoidance response was examined by measuring the step-through latency, 1 day after the animals received foot-shock training. When given before the training session, lurasidone did not affect the passive-avoidance response at any dose tested (1–30 mg/kg, p.o.). All the other atypical antipsychotics examined (i.e., risperidone, olanzapine, quetiapine, clozapine and aripiprazole), however, significantly reduced the step-through latency at relatively high doses. A pre-training administration of lurasidone significantly and dose-dependently reversed the MK-801-induced impairment of the passive-avoidance response. At doses lower than those that affected the passive-avoidance response, risperidone, quetiapine, and clozapine partially reduced the MK-801-induced impairment, whereas haloperidol, olanzapine, and aripiprazole were inactive. In addition, the post-training administration of lurasidone was as effective in countering the MK-801 effect as the pre-training administration, suggesting that lurasidone worked, at least in part, by restoring the memory consolidation process disrupted by MK-801. These results suggest that lurasidone is superior to other antipsychotics in improving the MK-801-induced memory impairment and may be clinically useful for treating cognitive impairments in schizophrenia.
Keywords: Learning; Memory; NMDA receptor antagonist; Serotonin-dopamine antagonist; Schizophrenia; Passive avoidance;

SB 277011-A (trans-N-[4-[2-(6-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolinecarboxamide) and S 33084 [(3aR,9bS)-N[4-(8-cyano-1,3a,4,9b-tetrahydro-3H-benzopyrano[3,4-c]pyrrole-2-yl)-butyl](4-phenyl)benzamide] were already shown to lack cataleptogenic actions. Further to that, we report that SB 277011 exerted a dose-dependent dampening on the development of haloperidol-induced catalepsy in the dose-range of 13.5–30 mg/kg p.o. while S 33084, at the dose of 0.625 mg/kg sc. significantly inhibited catalepsy induced by haloperidol; had no effect at 1.25 mg/kg, and further augmented the effect of haloperidol after 2.5 mg/kg. The compounds also produced effective inhibition when administered 2 hours after haloperidol. The results underline that dopamine D3 receptor antagonist action may have therapeutic value in the treatment of schizophrenia.
Keywords: Haloperidol-induced catalepsy; Dopamine D3 receptor antagonist; SB 277011; S 33084;

Rat/mouse hemokinin-1 is a mammalian tachykinin peptide whose biological functions have not been well characterized. In the present study, an attempt has been made to investigate the effect and mechanism of action of rat/mouse hemokinin-1 on systemic arterial pressure after intravenous (i.v.) injections in anesthetized rats by comparing it with that of substance P. Our data showed that injection of rat/mouse hemokinin-1 (0.1, 0.3, 1, 3 and 10 nmol/kg) lowered systemic arterial pressure dose-dependently. This effect was significantly blocked by pretreatment with SR140333 (a selective tachykinin NK1 receptor antagonist) and the NO synthase inhibitor L-NAME (N w-nitro-l-arginine methyl ester hydrochloride), respectively, but was not affected by bilateral vagotomy or the muscarinic receptor blocker atropine. Compared to rat/mouse hemokinin-1, a dose of 3 nmol/kg of substance P caused biphasic changes in systemic arterial pressure (depressor and pressor responses). The results suggest that the mechanism of the depressor response caused by substance P was similar to rat/mouse hemokinin-1 in that it was inhibited by SR140333 and L-NAME, respectively, but that there was a component of the cardiovascular change induced by rat/mouse hemokinin-1 (but not substance P) that was attenuated by SR48968 (a selective tachykinin NK2 receptor antagonist). The depressor response induced by rat/mouse hemokinin-1 (i.v.) might be explained primarily by the action on endothelial tachykinin NK1 receptors to release endothelium-derived relaxing factor (NO) and this effect was not affected by vagal components. In addition, rat/mouse hemokinin-1 could not induce the pressor response through stimulation of sympathetic ganglion like substance P in anesthetized rats.
Keywords: Rat/mouse hemokinin-1; Substance P; SR140333 and SR48968; Systemic arterial pressure;

Aldosterone causes vasoconstriction in coronary arterioles of rats via angiotensin II type-1 receptor: Influence of hypertension by Motoi Kushibiki; Masahiro Yamada; Koichi Oikawa; Hirofumi Tomita; Tomohiro Osanai; Ken Okumura (182-188).
Aldosterone is involved in many cardiovascular diseases with increased oxidative stress. Aldosterone-induced cardiac fibrosis is abolished by blockade of angiotensin II Type-1 (AT1) receptor. Recently, non-genomic vasoconstrictor effects of aldosterone were reported in various vascular beds. We tested the hypothesis that aldosterone stimulates angiotensin AT1 receptor, and causes vasoconstriction by increasing oxidative stress in coronary microcirculation. Coronary arterioles (60–120 μm) were isolated from spontaneously hypertensive rats (SHR) and control Wistar Kyoto (WKY) rats, aged 23–26 weeks. They were cannulated, and pressurized at 60 cm H2O. Effect of aldosterone (10− 15 to 10− 6 M) on coronary arteriolar diameter was examined. Aldosterone rapidly and dose-dependently decreased coronary arteriolar diameter in WKY rats and SHR (diameter changes, 8.4 ± 0.7% vs 13.9 ± 0.8%, P  < 0.05). Aldosterone-induced vasoconstriction was enhanced by 1.6-folds in SHR compared to WKY rats (P  < 0.05). Mineralocorticoid receptor antagonist spironolactone (10− 6 M) did not influence aldosterone-induced vasoconstriction. Selective angiotensin AT1 receptor blocker valsartan (10− 4 M) or candesartan (10− 7 M) abolished aldosterone-induced vasoconstriction. Similarly, superoxide dismutase (SOD, 300 U/ml), and NADPH oxidase inhibitor apocynin (10− 4 M) abolished it. Moreover, the vasoconstrictor effect of aldosterone disappeared in denuded vessels. Real-time quantitative RT-PCR revealed that angiotensin AT1 receptor mRNA level in coronary arterioles of SHR was upregulated by 1.5-folds compared to that in WKY rats (P  < 0.05). Aldosterone causes vasoconstriction in coronary arterioles, and this vasoconstrictor effect is enhanced by genetically defined hypertension. Aldosterone-induced vasoconstriction is mediated by angiotensin AT1 receptor presumably via oxidative stress.
Keywords: Aldosterone; Coronary microcirculation; Vasoconstriction; Hypertension; Angiotensin receptor;

The ability of agmatine, formed from l-arginine by the enzyme arginine decarboxylase (ADC), to modulate vasomotor function in rat aorta was investigated in the present study. Agmatine-mediated modulation of vasomotor tone was studied in organ chambers, protein expression quantified by Western blot analysis and cyclic guanosine 5′-monophosphate (cGMP) levels measured by radioimmunoassay. Agmatine (10− 10 to 10− 3 M) produced concentration-dependent relaxations (82 ± 5%) in phenylephrine-contracted endothelium intact rat aorta. Relaxations to agmatine were diminished on denudation of endothelium and nitric oxide synthase (NOS) inhibition by l-N ω-nitro arginine or soluble guanylate cyclase inhibition by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (P  < 0.001) abolished agmatine-mediated relaxations, while relaxations were insensitive to inducible NOS inhibition by 1400W. Agmatine-treated aorta demonstrated increased protein expression of phosphorylated S473-Akt and phosphorylated S1177-endothelial nitric oxide synthase (eNOS), and elevated the levels of cyclic GMP (P  < 0.01). Agmatine-mediated potentiation of relaxations and elevation of cGMP levels was sensitive to phosphatidylinositol 3′-kinase inhibitor, wortmannin. Relaxations to agmatine were also affected by pre-treatment with tetraethylammonium (P  < 0.01) or apamin (P  < 0.05), and were not affected by charybdotoxin. Relaxations to agmatine were partially affected by pre-treatment of aortic rings with barium chloride (P  < 0.05), and glybenclamide (P  < 0.05). Results obtained suggest that agmatine activates protein kinase B/Akt to phosphorylate eNOS and elevate cyclic GMP levels to produce vasodilatation of aorta. Agmatine-mediated relaxations in rat aorta seems to be mediated mainly by endothelial NO-mediated activation of small conductance Ca2+-activated K+ channels, and partly by ATP-sensitive and inward rectifying K+ channels.
Keywords: Rat aorta; Nitric oxide synthase; Potassium channels; Soluble guanylate cyclase; Akt; Vasorelaxation;

This study investigated the effects and mechanisms of action of carbon monoxide-releasing molecule-2 (CORM-2), compared to those of carbon monoxide (CO), in murine gastric fundus and jejunal circular smooth muscle. Functional in vitro experiments and cGMP measurements were conducted. In both tissues, CO and CORM-2 induced concentration-dependent relaxations. CO-induced relaxations were abolished by the soluble guanylyl cyclase (sGC) inhibitor ODQ, while CORM-2-evoked inhibitory responses were only partly prevented by ODQ. Relaxations elicited by CO (300 μM) were associated with a significant increase in cGMP levels, whereas for CORM-2 (300 μM) no significant increase in cGMP levels could be measured. The sGC sensitizer YC-1 was able to accelerate and potentiate both CO- and CORM-2-induced relaxations. Furthermore, the intermediate- and large-conductance Ca2+-activated K+ (IKCa-BKCa) channel blocker charybdotoxin significantly reduced CO- and CORM-2-induced relaxations in jejunal tissue; this same effect was observed with the BKCa channel blocker iberiotoxin. The combination of apamin plus charybdotoxin significantly reduced relaxations in gastric fundus and had synergistic inhibitory effects in jejunum. The NOS inhibitor l-NAME had no effect on the induced relaxations in gastric fundus, but significantly reduced CO- and CORM-2-evoked relaxations in jejunum. In conclusion, these results demonstrate that CO and CORM-2 produce relaxation in gastric fundus and jejunum via sGC and activation of KCa channels, and a nitric oxide (NO)-mediated amplification of CO signaling in jejunum is suggested.
Keywords: Carbon monoxide; Carbon monoxide-releasing molecule-2; Relaxation; Soluble guanylyl cyclase; KCa channels; Gastric fundus; Jejunum; Interstitial cells of Cajal; (Mouse);

Comparative in vivo uroselectivity profiles of anticholinergics, tested in a novel anesthetized rabbit model by Krishna S. Naruganahalli; Sandeep Sinha; Laxminarayan G. Hegde; Ashwinkumar V. Meru; Anita Chugh; Naresh Kumar; Jung B Gupta; Abhijit Ray (207-212).
The aim of this study was to describe a new experimental animal model for simultaneous measurement of carbachol-induced increase in intravesical pressure and salivary secretion in rabbits. Further, we also compared the in vivo potency and urinary bladder versus salivary gland selectivity profiles of Oxybutynin, Tolterodine, Solifenacin and Darifenacin. The intravesical pressure and salivary secretion were evoked by intra-arterial injection of carbachol (1.5 μg/kg). The carbachol-induced increase in intravesical pressure and salivation was simultaneously recorded before and after increasing doses of test drugs administered intravenously. The basal mean changes in intravesical pressure and salivation subsequent to carbachol administration were in the range of 6.7–7.5 mm Hg and 0.5–0.7 g respectively. Repeated administration of vehicle did not elicit any appreciable changes in intravesical pressure and salivary secretion to carbachol administration from the basal values till 3 h. All the test drugs exhibited a dose-dependent inhibition of carbachol-induced increase in intravesical pressure and salivary secretion. Darifenacin demonstrated a greater potency compared to other muscarinic receptor antagonists for inhibiting carbachol-induced increase in intravesical pressure. It also exhibited functional selectivity for the urinary bladder versus salivary gland. In contrast, Oxybutynin was functionally more selective in inhibiting carbachol-induced increase in salivary secretion. The observed urinary bladder versus salivary selectivity values were 0.6 ± 0.2, 1.1 ± 0.2, 1.7 ± 0.5, and 2.3 ± 0.5 for Oxybutynin, Tolterodine, Solifenacin and Darifenacin respectively. These results suggest that the functional selectivity of muscarinic receptor antagonists between urinary bladder and salivary glands can be readily detected in this model. Thus rabbits may represent a useful animal model for evaluating putative bladder selective muscarinic receptor antagonists for the treatment of overactive bladder.
Keywords: Anticholinergics; Dry mouth; Muscarinic receptor; Overactive bladder; Salivary gland;

Effects of bis(α-furancarboxylato)oxovanadium(IV) on glucose metabolism in fat-fed/streptozotocin-diabetic rats by Yanfen Niu; Weiping Liu; Changfu Tian; Mingjin Xie; Lihui Gao; Zhihe Chen; Xizhu Chen; Ling Li (213-219).
Bis(α-furancarboxylato)oxovanadium(IV) (BFOV) is a new orally active anti-diabetic organic vanadium complex. In the previous studies, we found that BFOV exhibited a glucose-lowering activity following oral administration to type 1-like diabetic mice induced by alloxan and rats induced by streptozotocin, and the mechanism was not related to enhancing the insulin synthesis and secretion. Since the enhancement of insulin sensitivity is involved in one of the mechanisms by which vanadium exerts its anti-diabetic effects, BFOV has been further tested on fat-fed/streptozotocin-treated rats, a type 2-like diabetic animal model, in the present study. The results showed that 4 weeks of BFOV treatment significantly improved hyperglycemia, glucose intolerance and hyperinsulinemia, as well as increased insulin sensitivity index in the fat-fed/streptozotocin-diabetic rats. Furthermore, BFOV efficiently activated glucokinase, increased hepatic glycogen content and suppressed phosphoenolpyruvate carboxykinase gene expression in the liver and kidney of the diabetic rats, which contributed to augmentation of hepatic glucose disposal and maintenance of blood glucose homeostasis. These findings suggested that BEOV had anti-diabetic and insulin-sensitizing effects in the diabetic rats, exhibiting the potential to be developed as a new therapeutic agent for the treatment of type 2 diabetes.
Keywords: Bis(α-furancarboxylato)oxovanadium(IV); Fat-fed/streptozotocin rats; Blood glucose; Insulin; Glucokinase; Phosphoenolpyruvate carboxykinase;

We have previously shown that monocyte conditioned medium (MCM) from patients with liver fibrosis stimulated proliferation of hepatic stellate cells (HSCs), the major cell involved in hepatic fibrosis. To investigate the potential role of fetuin and pentoxifylline in fibrosis we used MCM samples obtained from patients with biopsy proven hepatic fibrosis related to Hepatitis C (HCV). Our results indicate that the MCM obtained from patients with HCV-related liver fibrosis significantly stimulated collagen synthesis in HSCs as assessed by tritiated proline incorporation into a collagenase sensitive trichloroacetic acid (TCA) precipitate. Collagen synthesis was also stimulated in HSCs using transforming growth factor beta (TGFβ) and this effect was neutralized using TGFβ antibody. Incubation of HSCs with fetuin (but not TGFβ antibody) significantly inhibited collagen synthesis in HSCs that were stimulated by HCV MCM samples. Patient MCM samples would also stimulate proliferation of HSCs as assessed by tritiated thymidine uptake but this effect was not attenuated by fetuin. Likewise the significant stimulatory effect of platelet derived growth factor (PDGF) on HSC proliferation and collagen synthesis was not inhibited by fetuin but could be significantly reduced by 70% and 40% respectively, when treated with pentoxifylline. We also investigated the ability of samples obtained from patients with hepatic fibrosis to inhibit HSC apoptosis, as determined by okadaic acid-induced 4-hydroxynonenal immunocytochemistry in HSCs. We have previously reported that okadaic acid induces apoptosis in HSCs as assessed by Hoescht and TUNEL. Okadaic acid treatment produced a positive 4-hydroxynonenal (4-HNE) immunoreactivity in HSCs and treatment with HCV patient MCM or TGFβ decreased the 4-HNE positive immunoreactivity in HSCs treated with okadaic acid. Our results suggest that fetuin may be beneficial in hepatic fibrosis and suggest that combination of fetuin and pentoxifylline may target the two key events in hepatic fibrosis by modifying the effects of TGFβ and PDGF, the two major growth factors in fibrosis.
Keywords: Liver fibrosis; Okadaic acid; Apoptosis; Collagen; Pentoxifylline; Fetuin;

Here we demonstrate that the widely abused drug methylenedioxymethamphetamine (MDMA; “Ecstasy”) suppresses innate interferon (IFN)-γ production in mice following an in vivo lipopolysaccharide (LPS) challenge. IFN-γ signalling was also impaired by MDMA, as indicated by reduced phosphorylation of signal transducer and activator of transcription-1 (STAT1) and reduced expression of interferon-γ inducible protein 10 (IP-10/CXCL10); a chemokine induced by IFN-γ. MDMA also suppressed production of interleukin (IL)-12 and IL-15; two cytokines that induce IFN-γ production. Our results demonstrate that in vitro exposure to MDMA does not mimic the suppression of innate IFN-γ observed in vivo, indicating that observed suppression is most likely due to the release of endogenous immunomodulatory substances following drug administration. In this regard, we previously demonstrated that MDMA increases production of the anti-inflammatory cytokine IL-10 in vivo, an event that is mediated by β-adrenoceptor activation on immune cells. Considering that increased IL-10 production precedes suppression of IFN-γ induced by MDMA, and also considering that IL-10 can inhibit IL-12 and IFN-γ production, we examined the possibility that IL-10 was an essential mediator of the suppressive effect of MDMA on the IFN-γ response. By pre-treating mice with an anti-IL-10 receptor antibody we demonstrate that IL-10 is a critical mediator of MDMA-induced suppression of IFN- γ production and signalling. Consistent with a role for β-adrenoceptor activation in the immunosuppressive actions of MDMA, pre-treatment with the β-adrenoceptor antagonist nadolol blocked the MDMA-induced increase in IL-10, and also inhibited the suppressive action of MDMA on the innate IFN-γ response. The potential clinical significance of these findings for MDMA users is discussed.
Keywords: MDMA; IFN-γ; Immunosuppression; IL-10; Ecstasy; Drug abuse;

Anti-inflammatory activity of 21(α, β)-methylmelianodiols, novel compounds from Poncirus trifoliata Rafinesque by Hong Yu Zhou; Eun Myung Shin; Lian Yu Guo; Li Bo Zou; Guang Hua Xu; Seung-Ho Lee; Keum Ryon Ze; Eun-Kyung Kim; Sam Sik Kang; Yeong Shik Kim (239-248).
The fruits of Poncirus trifoliata (L.) are widely used in Oriental medicine as a remedy for allergic inflammation. As a part of our program to screen medicinal plants for potential anti-inflammatory compounds, 21α-methylmelianodiol (21α-MMD) and 21β-methylmelianodiol (21β-MMD), which are two isomers of 21-methylmelianodiol isolated from the fruits of P. trifoliata for the first time, were found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. 21α-MMD and 21β-MMD attenuated LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 protein expressions as well as the mRNA levels of iNOS, COX-2, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). To investigate the mechanism involved, we examined the effect of 21α-MMD and 21β-MMD on LPS-induced nuclear factor-κB (NF-κB) activation. Both 21α-MMD and 21β-MMD significantly inhibited LPS-induced NF-κB transcriptional activity in RAW 264.7 macrophages. Moreover, the in vivo anti-inflammatory effect of 21α-MMD was examined in two mouse models of acute inflammation. In the carrageenan-induced paw edema model, administration of 21α-MMD (20 and 100 mg/kg, i.p.) dose-dependently reduced paw swelling. In addition, 21α-MMD significantly inhibited the dye leakage in an acetic acid-induced vascular permeability assay. Taken together, our data indicate that 21-methylmelianodiol is an important constituent of the fruit of P. trifoliata, and that the inhibition of iNOS and COX-2 expression by 21α-MMD and 21β-MMD might be one of the mechanisms responsible for their anti-inflammatory effects.
Keywords: 21(α; Anti-inflammation; Inducible nitric oxide synthase; Cyclooxygenase-2; Nuclear factor-κB; In vivo activity;