European Journal of Pharmacology (v.450, #1)

Lipoteichoic acid-induced cyclooxygenase-2 expression requires activations of p44/42 and p38 mitogen-activated protein kinase signal pathways by Chien-Huang Lin; I-Hui Kuan; Chun-Hua Wang; Horng-Mo Lee; Wen-Sen Lee; Joen-Rong Sheu; George Hsiao; Chih-Hsiung Wu; Han-Pin Kuo (1-9).
This study investigated the role of p44/42 and p38 mitogen-activated protein kinase (MAPK) in cyclooxygenase-2 expression caused by lipoteichoic acid in human pulmonary epithelial cell line (A549). Lipoteichoic acid-induced increases in cyclooxygenase activity and cyclooxygenase-2 expression were attenuated by tyrosine kinase inhibitors (genistein and tyrphostin AG126), a MAPK/extracellular signal-regulated protein kinase (MEK) inhibitor [2′-amino-3′-methoxyflavone] (PD 98059) and a p38 MAPK inhibitor [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] (SB 203580). Lipoteichoic acid-induced p44/42 MAPK activation was inhibited by protein kinase C (PKC) inhibitors [12-(2-cyanoethyl)6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] (Go 6976) and {3-[1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide]} (Ro 31-8220), genistein and PD 98059. Lipoteichoic acid-induced increase in p38 MAPK activity was inhibited by Go 6976, Ro 31-8220, genistein and SB 203580. Lipoteichoic acid-mediated formation of nuclear factor-κB (NF-κB)-specific DNA–protein complex was inhibited by genistein, tyrphostin AG126, PD 98059 and SB 203580. These results suggest that the activations of both p44/42 and p38 MAPK by lipoteichoic acid result in stimulation of NF-κB-specific DNA–protein binding and subsequent cyclooxygenase-2 expression in A549 cells. Both events required activation of upstream tyrosine kinase and PKC.
Keywords: Lipoteichoic acid; Cyclooxygenase-2; p44/42 mitogen-activated protein kinase (MAPK); p38 MAPK; A549 cell;

In order to determine whether 5-[bis(carboxymethyl) amino]-2-carboxy4-cyano-3-thiopheneacetic acid distrontium salt (S12911-2) inhibits bone resorption by acting on the differentiation and/or function of osteoclasts, its effects were assessed on the 1,25-dihydroxyvitamin D3-induced expression of carbonic anhydrase II and vitronectin receptor in chicken bone marrow cells, and on the resorbing activity of authentic rat osteoclasts cultured on bone slices. S12911-2 dose-dependently inhibited, after a 6-day exposure, the expression of carbonic anhydrase II and vitronectin receptor in stimulated osteoclasts (46% and 40%, respectively, at 10−3 M Sr2+, P<0.05). A pre-incubation of bone slices with S12911-2 induced a dose-dependent inhibition of bone resorbing activity from 32% at 10−4 M Sr2+ to 66% at 10−3 M Sr2+ (P<0.05 in each case). A continuous incubation (10−3 M Sr2+) induced a greater inhibition of bone resorbing activity (73%, P<0.05). The inhibition of bone resorption obtained specifically with S12911-2 is related to an inhibition of the differentiation and resorbing activity of the osteoclasts.
Keywords: S12911-2; Osteoclast; Bone resorption; Differentiation;

A nonpeptide oxytocin receptor antagonist radioligand highly selective for human receptors by Wei Lemaire; Julie A O'Brien; Maryann Burno; Ashok G Chaudhary; Dennis C Dean; Peter D Williams; Roger M Freidinger; Douglas J Pettibone; David L Williams (19-28).
A novel, potent nonpeptide oxytocin receptor antagonist (1-(1-(2-(2,2,2-trifluoroethoxy)-4-(1-methylsulfonyl-4-piperidinyloxy) phenylacetyl)-4-piperidinyl)-3,4-dihydro-2(1H)-quinolinone) has been identified that can be labeled to high specific activity with [35S]. In binding studies, this compound exhibits sub-nanomolar affinity and a high degree of selectivity (900–1800-fold) for human oxytocin receptors compared to human vasopressin receptors. This compound appears suitable for studying the pharmacology of oxytocin receptors in human and nonhuman primate tissues, for which there is currently a paucity of highly selective tools. It may also be useful as a nonlabeled competitor or as a radioligand in autoradiographic studies of oxytocin receptor localization in these tissues.
Keywords: Oxytocin receptor antagonist; Oxytocin; Radioligand; Oxytocin receptor; Vasopressin;

High-threshold Ca2+ channels and tetrodotoxin-resistant Na+ channels are highly expressed in small dorsal root ganglion neurons. In acutely isolated rat dorsal root ganglion neurons, the effects of neomycin, one of the aminoglycoside antibiotics, on high-threshold Ca2+ currents and tetrodotoxin-resistant Na+ currents were examined using whole-cell patch recording. We showed for the first time that neomycin dose-dependently inhibited peak high-threshold Ca2+ currents and peak tetrodotoxin-resistant Na+ currents with half-maximal inhibitory concentrations at 3.69 μM (n=20) and 1213.44 μM (n=25), respectively. Inactivation properties of high-threshold Ca2+ currents and activation properties of tetrodotoxin-resistant Na+ currents were also affected by neomycin with reduction of excitability of small dorsal root ganglion neurons. Half-maximal inactivation voltage of high-threshold Ca2+ currents was −45.56 mV before and −50.46 mV after application of neomycin (n=10). Half-maximal activation voltage of tetrodotoxin-resistant Na+ currents was −19.93 mV before and −11.19 mV after administration of neomycin (n=15). These results suggest that neomycin can inhibit high-threshold Ca2+ currents and tetrodotoxin-resistant Na+ currents in small dorsal root ganglion neurons, which may contribute to neomycin-induced peripheral and central analgesia.
Keywords: Ca2+ channel, high-threshold; Neomycin; Whole-cell patch recording; Na+ channel, tetrodotoxin-resistant; Pain;

A comparison of the receptor binding and HERG channel affinities for a series of antipsychotic drugs by Sathapana Kongsamut; Jiesheng Kang; Xiao-Liang Chen; Joachim Roehr; David Rampe (37-41).
Many antipsychotic drugs produce QT interval prolongation on the electrocardiogram (ECG). Blockade of the human cardiac K+ channel known as human ether-a-go-go-related gene (HERG) often underlies such clinical findings. In fact, HERG channel inhibition is now commonly used as a screen to predict the ability of a drug to prolong QT interval. However, the exact relationship between HERG channel blockade, target receptor binding affinity and clinical QT prolongation is not known. Using patch-clamp electrophysiology, we examined a series of seven antipsychotic drugs for their ability to block HERG, and determined their IC50 values. We then compared these results to their binding affinities (K i values) for the dopamine D2 receptor, the 5-HT2A receptor and, where available, to clinical QT prolongation data. We found that sertindole, pimozide and thioridazine displayed little (<10-fold) or no selectivity for dopamine D2 or 5-HT2A receptors relative to their HERG channel affinities. This lack of selectivity likely underlies the significant QT interval prolongation observed with administration of these drugs. Of the other drugs tested (ziprasidone, quetiapine, risperidone and olanzapine), olanzapine displayed the greatest selectivity for dopamine D2 and 5-HT2A receptor binding (100–1000-fold) compared to its HERG channel IC50. We also compared these HERG channel IC50 values to QT interval prolongation and plasma drug levels obtained in a recent clinical study. We found that the ratio of total plasma drug concentration to HERG IC50 value was indicative of the degree of QT prolongation observed. Target receptor affinity and expected clinical plasma levels are important parameters to consider for the interpretation of HERG channel data.
Keywords: HERG channel; Antipsychotic; Arrhythmia; QT prolongation; K+ channel;

Heme oxygenase catalyzes the formation of CO, Fe2+ and biliverdin from the substrate heme. In these studies, we attempted to define the roles heme oxygenase play in pain-related behaviors induced by intrathecal injection of the spinal neurotransmitter glutamate. The intrathecal injection of glutamate or the more selective agonists N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in C57Bl/6 mice lead to caudally directed pain behaviors which were sensitive to the heme oxygenase inhibitors tin protoporphyrin (Sn-protoporphyrin) and chromium mesoporphyrin (Cr-mesoporphyrin). Intrathecal injections of glutamate in heme oxygenase type 2 (HO-2) null-mutant animals resulted in reduced pain-related behaviors when compared with wild type animals. Glutamate, NMDA and AMPA stimulated cGMP accumulation in mouse spinal cord slices, which was blocked by heme oxygenase inhibitors. Glutamate did not stimulate cGMP production in HO-2 null-mutant animals. Our data are consistent with the hypothesis that pain-related behaviors induced by spinal glutamate rely on the activation of HO-2 and subsequent production of cGMP.
Keywords: Heme oxygenase; Guanylate cyclase; cGMP; Glutamate; Null-mutant; Metalloporphyrin;

We have studied and compared the antinociceptive and anti-hyperalgesic effect of the partial opioid receptor agonist buprenorphine in normal and neuropathic rats. In normal rats, systemic buprenorphine produced dose-dependent antinociception on the hot plate test. In rats with peripheral nerve or spinal cord injury, buprenorphine markedly alleviated neuropathic pain-related behaviors, including mechanical and cold allodynia/hyperalgesia at doses comparable to that producing antinociception. The results suggest that buprenorphine may be a useful analgesic for treating neuropathic pain and thus is an atypical opioid since morphine tends to be less potent after nerve injury.
Keywords: Morphine; Opioid; Sciatic nerve; Spinal cord;

Distribution of [3H]BU224, a selective imidazoline I2 binding site ligand, in rat brain by Emma S.J Robinson; Robin J Tyacke; David J Nutt; Alan L Hudson (55-60).
BU224 (2-(4,5-dihydroimidaz-2-yl)-quinoline) is a selective imidazoline I2 binding site ligand characterised in both competition binding assays and functional studies. However, in some studies, BU224 has been reported to have a different functional effect from that seen with another selective imidazoline I2 binding site ligand 2-BFI (2-(2-benzofuranyl)-2-imidazoline). This effect may reflect differing efficacies of the ligands or a difference in their brain distribution. The present study has investigated the distribution of the tritiated form of BU224 in rat brain and correlated this distribution with other imidazoline I2 binding site ligands, [3H]idazoxan and [3H]2-BFI. Saturation studies revealed binding of [3H]BU224 was of high affinity and saturable. The central distribution of [3H]BU224 was similar to that previous reported for imidazoline I2 binding site in rat brain. Autoradiography revealed that the highest levels of binding were in the arcuate nucleus, interpeduncular nucleus, area postrema, pineal gland and ependymal cell layer lining the ventricles. Correlation analysis of the binding distribution with our previous published studies revealed a highly significant correlation between [3H]BU224 and both [3H]idazoxan (r=0.94) and [3H]2-BFI (r=0.96). These data indicate [3H]BU224 labels the same population of imidazoline I2 binding site in rat brain as seen with [3H]idazoxan and [3H]2-BFI. Therefore, the differences in functional effects observed with these compounds may reflect agonist and antagonist properties.
Keywords: Imidazoline binding site; Autoradiography; BU224 (2-(4,5-dihydroimidaz-2-yl)-quinoline; Brain;

Enalapril and quinapril improve endothelial vasodilator function and aortic eNOS gene expression in l-NAME-treated rats by Vito De Gennaro Colonna; Giuseppe Rossoni; Antonello E Rigamonti; Sara Bonomo; Barbara Manfredi; Ferruccio Berti; Eugenio E Muller (61-66).
Endothelial dysfunction ensuing inhibition of nitric oxide synthase (NOS) was investigated in male Sprague–Dawley rats given N ω-nitro-l-arginine methyl ester (l-NAME) in drinking water for 8 weeks. Age-matched rats served as controls. l-NAME-treated rats, as compared to control animals, showed: (1) a clear-cut increase in systolic blood pressure; (2) a consistent decrease of endothelial-cell NOS (eNOS) gene expression in aortic tissue; (3) a reduction of the relaxant activity of acetylcholine (ACh, from 10−10 to 10−4 M) on norepinephrine-precontracted aortic rings (reduction by 52±5%); (4) a marked decrease (−50%) of the basal release of 6-keto-prostaglandin F (6-keto-PGF) from aortic rings. In l-NAME-treated rats, administration in the last 2 weeks of either the angiotensin-converting enzyme inhibitor enalapril (1 mg/kg/day) or the cognate drug quinapril (1 mg/kg/day) decreased systolic blood pressure levels, completely restored eNOS mRNA levels in aortic tissue, and allowed a consistent recovery of both the relaxant activity of ACh and the generation of 6-keto-PGF. No difference was present in the ability of the two angiotensin-converting enzyme inhibitors to reverse NAME-induced endothelial dysfunction. These findings indicate that l-NAME-induced hypertension in the rat relies on the marked impairment of the endothelial vasodilator function, with an ensuing contribution by a decreased production of prostacyclin by the endothelial cells. Angiotensin-converting enzyme inhibition by enalapril or quinapril was equally effective in improving endothelial vasodilator function, prostacyclin endothelial production and restoring aortic eNOS mRNA.
Keywords: Endothelial dysfunction; NO (Nitric oxide); Hypertension; Angiotensin-converting enzyme inhibitor;

A role for superoxide in gentamicin-mediated nephropathy in rats by Salvatore Cuzzocrea; Emanuela Mazzon; Laura Dugo; Ivana Serraino; Rosanna Di Paola; Domenico Britti; Angela De Sarro; Simone Pierpaoli; Achille P Caputi; Emanuela Masini; Daniela Salvemini (67-76).
Gentamicin is an antibiotic effective against Gram-negative infection, whose clinical use is limited by its nephrotoxicity. Oxygen free radicals are considered to be important mediators of gentamicin-mediated nephrotoxicity, but the exact nature of the radical in question is not known with certainty. We have investigated the potential role of superoxide in gentamicin-induced renal toxicity by using M40403, a low molecular weight synthetic manganese containing superoxide dismutase mimetic, which selectively removes superoxide. Administration of gentamicin at 100 mg/kg, s.c. for 5 days to rats induced a marked renal failure, characterised by a significant decrease in creatinine clearance and increased plasma creatinine levels, fractional excretion of sodium, lithium, urine γ glutamyl transferase (γGT) and daily urine volume. A significant increase in kidney myeloperoxidase activity and lipid peroxidation was also observed in gentamicin-treated rats. M40403 (10 mg/kg, i.p. for 5 days) attenuated all these parameters of damage. Immunohistochemical localisation demonstrated nitrotyrosine formation and poly(ADP-ribose) synthetase (PARS) activation in the proximal tubule of gentamicin-treated rats. Renal histology examination confirmed tubular necrosis. M40403 significantly prevented gentamicin-induced nitrotyrosine formation, poly(ADP-ribose) synthetase activation and tubular necrosis. These results confirm our hypothesis that superoxide anions play an important role in gentamicin-mediated nephropathy and support the possible clinical use of low molecular weight synthetic superoxide dismutase mimetics in those conditions that are associated with over production of superoxide.
Keywords: Gentamicin; Superoxide anion; Superoxide dismutase mimetic; Compound M40403;

Modulation of gastric emptying and gastrointestinal transit in rats through intestinal cannabinoid CB1 receptors by Marco Landi; Tiziano Croci; Murielle Rinaldi-Carmona; Jean-Pierre Maffrand; Gérard Le Fur; Luciano Manara (77-83).
We studied the delay in gastric emptying and gastrointestinal transit induced by the cannabinoid receptor agonists (+)-WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate) and CP 55,940 ((−)-cis-3[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol), as prevented by the selective cannabinoid CB1-receptor antagonist SR141716 ((N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide)) in rats after systemic or central drug administration. Oral SR141716 showed comparable potency (ID50 range 1.0–3.9 mg/kg) in antagonizing gastric emptying and gastrointestinal transit delay by (+)-WIN 55,212-2 or CP 55,940. Gastric emptying and gastrointestinal transit delay after intracerebroventricular (i.c.v.) (+)-WIN 55,212-2 was prevented by oral or i.c.v. SR141716, but i.c.v. SR141716 did not significantly reduce the effect of i.p. (+)-WIN 55,212-2. Pertussis toxin prevented the delaying action of i.c.v. (+)-WIN 55,212-2 on both gastric emptying and gastrointestinal transit, but had no effect on (+)-WIN 55,212-2 i.p. These findings are consistent with a primary role of peripheral cannabinoid CB1 receptor mechanisms in gastrointestinal transit delay by specific agonists.
Keywords: Gastric emptying; Gastrointestinal transit; Pertussis toxin; Cannabinoid CB1 receptor;

Our previous results showed that L-RNA, extracted from lipopolysaccharide-stimulated macrophages, induces interleukin-1, interleukin-8 and tumor necrosis factor-α (TNF-α) in resident macrophages. It was demonstrated the promoter of these cytokine genes contain nuclear factor-kB (NF-κB) binding sites. We hypothesized that this effect of L-RNA is mediated by RNA-dependent protein kinase (PKR) through NF-κB activation. We found that L-RNA activates PKR and induces NF-κB activation through degradation of its inhibitor I-κBα. These data support the idea that L-RNA acts as a regulatory RNA. A model for the mechanism of action of L-RNA is proposed.
Keywords: RNA; PKR (RNA-dependent protein kinase); NF-κB (nuclear factor-kB); Lipopolysaccharide; Macrophage;

Corrigendum (91).

“The role of melanocortins in body weight regulation: opportunities for the treatment of obesity” by Douglas J. MacNeil; Andrew D. Howard; Xiaoming Guan; Tung M. Fong; Ravi P. Nargund; Maria A. Bednarek; Mark T. Goulet; David H. Weinberg; Alison M. Strack; Donald J. Marsh; Howard Y. Chen; Chun-Pyn Shen; Airu S. Chen; Charles I. Rosenblum; Tanya MacNeil; Michael Tota; Euan D. MacIntyre; Lex H.T. Van der Ploeg (93-109).
Five G-protein-coupled melanocortin receptors (MC1–MC5) are expressed in mammalian tissues. The melanocortin receptors support diverse physiological functions, including the regulation of hair color, adrenal function, energy homeostasis, feed efficiency, sebaceous gland lipid production and immune and sexual function. The melanocortins (adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hormone (α-MSH), β-MSH and γ-MSH) are agonist peptide ligands for the melanocortin receptors and these peptides are processed from the pre-prohormone proopiomelanocortin (POMC). Peptide antagonists for the melanocortin MC1, MC3 and MC4 receptors include agouti-related protein (AgRP) and agouti. Diverse lines of evidence, including genetic and pharmacological data obtained in rodents and humans, support a role for the melanocortin MC3 and MC4 receptors in the regulation of energy homeostasis. Recent advances in the development of potent and selective peptide and non-peptide melanocortin receptor ligands are anticipated to help unravel the roles for the melanocortin receptors in humans and to accelerate the clinical use of small molecule melanocortin mimetics.
Keywords: Melanocortins; Melanocortin receptor; Neuropeptide; ACTH (adrenocorticotropic hormone); AgRP (agouti-related protein); POMC (proopiomelanocortin); Energy homeostasis; Obesity; Sexual function;