European Journal of Pharmacology (v.569, #3)

Differential pharmacology of the cardiac anionic background current I AB by John J. Borg; Jules C. Hancox; Henggui Zhang; Christopher. Ian. Spencer; Hongyu Li; Roland Z. Kozlowski (163-170).
A novel anionic background conductance (I AB) in cardiac ventricular myocytes has recently been identified but at present there is comparatively little information on its pharmacological modulation. This study investigated the effects of on I AB of four pyrethroid agents tefluthrin (a selective activator of this current), tetramethrin, fenpropathrin and α-cypermethrin in addition to other well known chloride channel modulators (chlorotoxin, gadolinium and picrotoxin). Guinea-pig ventricular myocytes were isolated using an enzymatic and mechanical dispersion procedure and all electrophysiological measurements were made using the whole-cell patch-clamp technique. In contrast to other anion conductances (stretch- or volume-regulated chloride current (I Cl,vol), a cAMP-dependent Cl current (I Cl,cAMP)) I AB was augmented by tefluthrin, fenpropathrin, α-cypermethrin (but not tetramethrin). I AB was insensitive to chlorotoxin, gadolinium and picrotoxin. Thus, I AB exhibits a distinct pharmacological profile from other known cardiac anion conductances.
Keywords: Cardiac chloride channels; Pyrethroids; Anionic background current; Heart; Ion channels;

Advanced glycation end products and transforming growth factor-β (TGF-β) have been implicated in the development of diabetic complications such as cataract. The diverse metabolic effects of protocatechualdehyde (PCA, 3, 4-dihydroxybenzaldehyde) include the inhibition of aldose reductase and oxidation, two processes that are involved in the development of complications in diabetic patients. Here, the potential therapeutic effects of PCA in the treatment of diabetic complications were studied by determining this compound's ability to inhibit the formation of advanced glycation end products-bovine serum albumin (BSA) and the expression of receptor for advanced glycation end products and TGF-β1 in human lens epithelial cells cultured under diabetic conditions. In addition, the ability of PCA to suppress lens opacification in streptozotocin-diabetic rats was analyzed. PCA significantly reduced advanced glycation end products-BSA formation in vitro and was more effective than aminoguanidine. In human lens epithelial cells, PCA significantly inhibited the induction of receptor for advanced glycation end products protein and mRNA expression by the receptor for advanced glycation end products-specific ligand S100b. Moreover, PCA inhibited high glucose- or S100b-induced TGF-β1 protein and mRNA expression as well as nuclear accumulation of phosphorylated Smad2/3. In streptozotocin-induced diabetic cataract in rats, oral administration of PCA (25 mg/kg body weight) for 8 weeks significantly ameliorated the development of lens opacity (cataract) with effect on glycemic control. These results suggest that PCA is of therapeutic interest with respect to the prevention of diabetic complications such as diabetic cataract.
Keywords: Diabetic complication; Protocatechualdehyde; Advanced glycation end product; Receptor for advanced glycation end products; TGF-β; Lens opacity;

Melatonin attenuates metabolic disorders due to streptozotocin-induced diabetes in rats by Elena Ju. Sudnikovich; Yuri Z. Maksimchik; Svetlana V. Zabrodskaya; Valeri L. Kubyshin; Elena A. Lapshina; Maria Bryszewska; Russel J. Reiter; Ilya B. Zavodnik (180-187).
Enhanced oxidative stress and impairments in nitric oxide synthesis and bioavailability are of considerable importance in the pathogenesis of diabetic vascular diseases. The aim of the present work was to evaluate the metabolic effects of pharmacological doses of the melatonin, a known antioxidant, on streptozotocin-induced diabetic damage in rats. We investigated the indolamine's influence on the cellular redox-balance, nitric oxide (NO) level, and the activities of antioxidative defence enzymes, as well as the activities of enzymes involved in phase II detoxication and NADPH-generating pentose phosphate pathway. Blood glucose, glycated hemoglobin, bilirubin, as well as plasma alanine aminotransferase activities increased and body weight was reduced in rats with streptozotocin-induced (60 mg/kg, i.p.) diabetes (25 days). The NO level was markedly increased in diabetic plasma (by 50%) and aortic tissue (by 30%). The hyperglycemia resulted in reduced activities of glutathione peroxidase (by 25%), catalase (by 20%), glucose-6-phosphate dehydrogenase (by 55%) and transketolase (by 40%) in liver tissue of diabetic animals. Melatonin treatment (10 mg/kg, 18 days) did not influence the level of hyperglycemia or glycated hemoglobin and it had little effect on the activities of antioxidative enzymes. However, melatonin markedly reversed the activities of glucose-6-phosphate dehydrogenase and transketolase in liver tissue of diabetic rats. The most pronounced effect of the melatonin administration was the prevention of an increase in nitric oxide levels in blood plasma and aortic tissue during diabetes. In in vitro experiments, nitrosomelatonin formation in the presence of nitrosodonors was observed. This implies that melatonin might operate as an NO scavenger and carrier. Thus, melatonin treatment may have some beneficial effects in controlling diabetic vascular complications.
Keywords: Diabetes; Melatonin; Nitric oxide; Nitrosomelatonin;

The spinal anaesthetic effect of dextromethorphan, dextrorphan, and 3-methoxymorphinan by Yu-Wen Chen; Yen-Chia Chen; Ching-Nan Lin; Chin-Chen Chu; Mao-Tsun Lin; Jhi-Joung Wang; Cheng-Hsing Kao (188-193).
Dextromethorphan, an antitussive, has a complex pharmacologic profile and has not been well studied. Our aim was to evaluate whether dextromethorphan and its metabolites, dextrorphan and 3-methoxymorphinan, have a spinal anaesthetic effect. Using a method of spinal blockade in rats, we evaluated the potencies and durations of the effects of dextromethorphan and its metabolites on spinal blockades of motor function and nociception. Bupivacaine was the active control. We found that dextromethorphan and its metabolites produced a dose-related spinal blockade of motor function and nociception. On an ED50 basis, the ranks of potencies were bupivacaine > dextrorphan > 3-methoxymorphinan > dextromethorphan (p  < 0.05 for the differences). On an equipotent basis, dextrorphan and bupivacaine produced similarly longer nociceptive blockades than did dextromethorphan and 3-methoxymorphinan (p  < 0.05 for the differences). Co-administration of dextromethorphan or its metabolites with bupivacaine produced an additive effect. In conclusion, intrathecal injections of dextromethorphan or its metabolites, dextrorphan and 3-methoxymorphinan, produced dose-related spinal blockades of motor function and nociception. The suitability of these drugs as clinical spinal anaesthetics is worth further evaluation.
Keywords: 3-Methoxymorphinan; Dextromethorphan; Dextrorphan; Intrathecal; Local anaesthetic; Spinal blockade;

Pramipexole prevents neurotoxicity induced by oligomers of beta-amyloid by Daniela Uberti; Irene Bianchi; Luca Olivari; Giulia Ferrari-Toninelli; PierLuigi Canonico; Maurizio Memo (194-196).
Here we demonstrate that pramipexole, an antiparkinsonian dopamine receptor agonist drug, exerts neuroprotective effects against beta-amyloid neurotoxicity. Using a specific protocol to test individually oligomers, fibrils, or unaggregated amyloid beta-peptide, we found pramipexole able to protect cells against oligomers and fibrils. Unaggregated amyloid beta-peptide was found unable to cause cell death. Fibrils and oligomers were also found to produce elevated amount of free radicals, and this effect was prevented by pramipexole. We propose pramipexole may become in the future a coadjuvant in the treatment of neuropathologies, besides Parkinson's disease, where amyloid beta-peptide-mediated oxidative injury exerts a relevant role.
Keywords: Free radicals; Oxidative stress; Alzheimer's disease; Neurodegeneration;

This study investigated, in mice, the antidepressant like effect of hyperfoliatin, a prenylated phloroglucinol derivative isolated from the aerial parts of Hypericum perfoliatum, as well as its action on monoaminergic systems. In the forced-swimming test, hyperfoliatin dose-dependently reduced immobility time. Immobility was interpreted as an expression of “behavioural despair”, which could be a component of depression syndrome. The effect of hyperfoliatin did not result from the stimulation of animal motor activity. Hyperfoliatin inhibited, in a concentration-dependent manner, the [3H]-dopamine, [3H]-serotonin and [3H]-noradrenaline synaptosomal uptakes, but did not prevent the binding of specific ligands to the monoamine transporters. These data suggest that the antidepressant-like effect of hyperfoliatin on the forced-swimming test is probably associated to monoamine uptake inhibition, due to a mechanism of action different from that of known antidepressants.
Keywords: Antidepressant drug; Monoaminergic system; Forced-swimming test; Hypericum perfoliatum; Hyperfoliatin;

Low-dose treatment with atorvastatin leads to anti-oxidative and anti-inflammatory effects in diabetes mellitus by Alexander Riad; Jing Du; Sebastian Stiehl; Dirk Westermann; Zarah Mohr; Meike Sobirey; Wolfram Doehner; Volker Adams; Matthias Pauschinger; Heinz Peter Schultheiss; Carsten Tschöpe (204-211).
We investigated whether atorvastatin, given in a dose to low to influence the lipid profile, has any effect on oxidative stress, inflammation and endothelial function under streptozotocin-induced diabetic conditions. Diabetes mellitus was induced in male Sprague Dawley rats by a single injection of streptozotocin. Rats were treated chronically with atorvastatin (50 mg/kg/day; p.o.) or vehicle until day 48 and compared with controls. NAD(P)H activity, protein expression nuclear factor-κBp65 (NF-κBp65) and phosphorylation of the extracellular signal-regulated kinase (ERK1/2) were assessed in the quadriceps muscle. Protein and mRNA levels of intracellular and vascular adhesion molecules (ICAM-1, VCAM-1) and cytokines were measured by Taqman or immunohistochemistry staining, respectively. Endothelial function was investigated in vivo using the autoperfused hind limb model. Diabetic groups displayed similar severe hyperglycemia. Untreated diabetic rats showed enhanced NAD(P)H activity, activation of the ERK1/2/NF-κBp65-pathway, enhanced expression of cytokines and cellular adhesion molecules and impaired vascular function. Low-dose therapy by atorvastatin did not alter the lipid profile but led to a reduction of NAD(P)H activity (− 28%, P  < 0.05) associated with reduced protein expression of NF-κBp65 (− 53%, P  < 0.05) and phosphorylation of its regulator mitogen-activated protein kinase (MAPK) ERK1/2 in diabetic rats. Also inflammatory markers were reduced after atorvastatin treatment indexed by reduced mRNA expression of VCAM-1 (− 24%), tumor necrosis factor α (− 59%) and interleukin 1β (− 50%) and reduced ICAM-1 (− 81%) and VCAM-1 (− 74%) positive staining. These beneficial effects were associated with improved endothelium-dependent vasodilatation (maximal vasodilatation: + 101%; P  < 0.05). Lipid-independent anti-oxidative and anti-inflammatory effects of low-dose atorvastatin involving the ERK1/2/NF-κB-pathway are sufficient to improve endothelial function under experimental diabetic conditions.
Keywords: Diabetes mellitus; Endothelial dysfunction; Adhesion molecule; Inflammation; Atorvastatin; Oxidative stress; NF-κB; ERK;

The vasculature of the equine digit fulfils an important role in thermoregulation. In other species, it has been found that cooling may enhance the response of cutaneous vessels to 5-hydroxytryptamine (5-HT) and α2-adrenoceptor agonists. Translocation of α2-adrenoceptors to the smooth muscle cell membrane, mediated by Rho kinase, is thought to be involved in the cooling-enhanced response in mouse tail arteries. However, little is known about the effect of cooling on 5-HT receptor function. The present investigation compared the response of 5-bromo-6-(2-imidazolin-2-ylamino) quinoxaline (UK14304:1 nM to 30 μM), methoxamine (0.1 nM to 30 μM; in the presence of yohimbine 0.1 μM), 5-carboxamidotryptamine (5-CT; 0.1 nM to 10 μM) and α-methyl 5-HT (0.1 nM to 10 μM) in the isolated equine digital vein at 30 °C and 22 °C. The effect of the Rho kinase inhibitor, fasudil (1 μM), and the recovery of the response after the irreversible blockade of surface receptors with phenoxybenzamine (10 μM) or 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ;10 μM), was established. Moderate cooling significantly increased the maximum response to α-methyl 5-HT, 5-CT and UK14304 and shifted their response curves to the left. Cooling also augmented the phenoxybenzamine- and EEDQ-resistant response to UK14304 and 5-CT, respectively. Fasudil had no effect on the contractile response at 30 °C, but completely abrogated the effect of cooling on the response to 5-CT and UK14304. The response to methoxamine was not significantly affected by cooling. These results suggest that Rho kinase plays an important role in the cooling-enhanced response mediated by 5-HT1B/D receptors and α2-adrenoceptors. The exact mechanism by which Rho/Rho kinase enhances the functional responses mediated by these receptors in these vessels has yet to be determined.
Keywords: Cooling; Rho kinase; Fasudil; 5-HT; α-adrenoceptor; Laminitis; Equine digital vein;

Antipyrine clearance in comparison to conventional liver function tests in hepatitis C virus patients by Madiha Mahmoud; Rania Abdel-Kader; Moataz Hassanein; Samira Saleh; Sanaa Botros (222-227).
In this study, 15 healthy volunteers and 96 patients with hepatitis C virus, classified according to Child-Pugh into 36 Child-A, 31 Child-B and 29 Child-C, were examined. All subjects ingested 600 mg antipyrine in the form of hard gelatinous capsules after overnight fasting. One milliliter of saliva was collected at 4 and 24 h after ingestion of antipyrine and analyzed using high-performance liquid chromatography. Blood samples were collected from all subjects for examination, using conventional liver function tests. The pharmacokinetic variables for antipyrine were determined using the two concentration time points selected. A cut-off value of 0.34 ml/min/kg was used to distinguish between cirrhotic and non-cirrhotic patients. Alanine aminotransferase, aspartate aminotransferase and gamma-glutamyl transferase values were significantly higher with significantly lower antipyrine clearance in Child-A, B, and C patients than in normal volunteers. The total protein concentration was significantly lower in Child-B and C patients. Moreover, AST was significantly higher in Child-C patients and antipyrine clearance was lower in Child-B and C patients than in Child-A patients. Antipyrine clearance showed a significant negative correlation with Child-Pugh scores, total protein, the international normalization ratio of prothrombin time and globulin, and a positive correlation with albumin and albumin-to-globulin ratio. Unlike most of the conventional liver function tests, antipyrine clearance, which represents the intrinsic clearance capacity of the liver, measured using saliva, proved to be a sensitive marker of liver function. It was significantly impaired in the Child-Pugh group A patients with the least hepatic impairment. The international normalization ratio of prothrombin time was just as informative as antipyrine clearance in identifying minimal hepatic impairment.
Keywords: Antipyrine clearance; Liver function tests; Hepatitis C virus (HCV) patients; Child-Pugh classification;

Anti-inflammatory effects of compounds alpha-humulene and (−)-trans-caryophyllene isolated from the essential oil of Cordia verbenacea by Elizabeth S. Fernandes; Giselle F. Passos; Rodrigo Medeiros; Fernanda M. da Cunha; Juliano Ferreira; Maria M. Campos; Luiz F. Pianowski; João B. Calixto (228-236).
This study evaluated the anti-inflammatory properties of two sesquiterpenes isolated from Cordia verbenacea's essential oil, α-humulene and (−)-trans-caryophyllene. Our results revealed that oral treatment with both compounds displayed marked inhibitory effects in different inflammatory experimental models in mice and rats. α-humulene and (−)-trans-caryophyllene were effective in reducing platelet activating factor-, bradykinin- and ovoalbumin-induced mouse paw oedema, while only α-humulene was able to diminish the oedema formation caused by histamine injection. Also, both compounds had important inhibitory effects on the mouse and rat carrageenan-induced paw oedema. Systemic treatment with α-humulene largely prevented both tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) generation in carrageenan-injected rats, whereas (−)-trans-caryophyllene diminished only TNFα release. Furthermore, both compounds reduced the production of prostaglandin E2 (PGE2), as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) expression, induced by the intraplantar injection of carrageenan in rats. The anti-inflammatory effects of α-humulene and (−)-trans-caryophyllene were comparable to those observed in dexamethasone-treated animals, used as positive control drug. All these findings indicate that α-humulene and (−)-trans-caryophyllene, derived from the essential oil of C. verbenacea, might represent important tools for the management and/or treatment of inflammatory diseases.
Keywords: α-humulene; (−)-trans-caryophyllene; C. verbenacea's essential oil; Anti-inflammatory property; Oral effect;

Toll-like receptor 4 signal transduction inhibitor, M62812, suppresses endothelial cell and leukocyte activation and prevents lethal septic shock in mice by Masaki Nakamura; Yusuke Shimizu; Yasuko Sato; Yutaka Miyazaki; Tsutomu Satoh; Masashi Mizuno; Yutaka Kato; Yoshitaka Hosaka; Shoji Furusako (237-243).
Sepsis occurs when microbes activate toll-like receptors (TLRs) stimulating widespread inflammation and activating coagulation cascades. TLR4 signal transduction has been recognized as a key pathway for lipopolysaccharide (LPS)-induced activation of various cells and an attractive target for treatment of sepsis. We found a new benzisothiazole derivative, M62812 that inhibits TLR4 signal transduction. This compound suppressed LPS-induced upregulation of inflammatory cytokines, adhesion molecules and procoagulant activity in human vascular endothelial cells and peripheral mononuclear cells. The half maximal inhibitory concentrations in these assays ranged from 1 to 3 μg/ml. Single intravenous administration of M62812 (10–20 mg/kg) protected mice from lethality and reduced inflammatory and coagulatory parameters in a murine d-galactosamine-sensitized endotoxin shock model. M62812 (20 mg/kg) also prevented mice from lethality in a murine cecal ligation and puncture model. These results suggest that inhibition of TLR4 signal transduction can suppress coagulation as well as inflammation during sepsis and may be clinically beneficial in sepsis treatment.
Keywords: Sepsis; Toll-like receptor 4; Lipopolysaccharide;