European Journal of Pharmacology (v.577, #1-3)

Angiotensin-converting enzyme (ACE) inhibitors have different selectivity for bradykinin binding sites of human somatic ACE by Claudio Ceconi; Gloria Francolini; Adriana Olivares; Laura Comini; Tiziana Bachetti; Roberto Ferrari (1-6).
The angiotensin-converting enzyme (ACE) has two natural substrates and two catalytic domains: one cleaving angiotensin I and one inactivating bradykinin. The aim of this study was to investigate the comparative binding affinity of ACE inhibitors for the two binding sites of human endothelial ACE. In vitro binding assays were performed to test the ability of bradykinin, angiotensin I, or various ACE inhibitors (enalaprilat, perindoprilat, quinaprilat, ramiprilat, and trandolaprilat) to displace a saturating concentration of [125I]351A, a radiolabeled lisinopril analogue, from ACE binding sites. The calculated IC50 values for the ACE inhibitors were in the nanomolar range, while those for the natural substrates were in the micromolar range. The bradykinin/angiotensin I selectivity ratios calculated from double displacement experiments were: perindoprilat, 1.44; ramiprilat, 1.16; quinaprilat, 1.09; trandolaprilat, 1.08; enalaprilat, 1.00. The ACE inhibitors generally had higher affinity for the bradykinin than the angiotensin I binding sites, supporting the idea that these agents are primarily inhibitors of bradykinin degradation, and secondarily inhibitors of angiotensin II production. Perindoprilat had the highest selectivity for bradykinin versus angiotensin I binding sites, and enalaprilat has the lowest. These results indicate that there are differences in the affinity of ACE inhibitors toward sites for bradykinin degradation, which could lead to differences in efficacy in cardiovascular disease.
Keywords: ACE; ACE inhibitor; Angiotensin I; Binding affinity; Bradykinin; Cardiovascular disease; HUVEC; Perindopril; Perindoprilat; Selectivity;

Calcitonin gene-related peptide (CGRP) is a highly potent vasodilator known to be involved in many physiological functions within the cardiovascular, gastrointestinal, immune, and nervous systems. This study assessed the desensitization of CGRP receptors by measuring agonist-mediated activation of adenylate cyclase in a model system employing human neuroblastoma-derived SK-N-MC cells. In these cells, we demonstrated that pre-incubation with CGRP (20 nM) induces a rapid desensitization of CGRP signaling (t 1/2  ≤ 3 min) by causing a decrease in potency and efficacy. CGRP's desensitization potency (DC50  = 0.29 nM) is similar to its activation potency on non-desensitized cells (EC50  = 0.20 nM). The desensitized receptors exhibited slow and incomplete re-sensitization upon removal of the pre-incubated ligand, resulting in 52–65% functional recovery after 3–5 h while CGRP binding sites were completely restored. Additional agonists within the calcitonin/CGRP family of peptides (calcitonin, amylin, adrenomedullin, and adrenomedullin 2) were compared to CGRP with regard to their ability to activate and desensitize CGRP receptors. Calcitonin and amylin did not cause receptor activation nor did they produce desensitization. Adrenomedullin and adrenomedullin 2 activated the receptors and produced desensitization, but at a slower rate and with a weaker desensitization potency than CGRP-induced desensitization. Adrenomedullin exhibited similar potency for receptor activation and desensitization, whereas adrenomedullin 2 has a 4-fold higher preference for receptor desensitization than for receptor activation. Activation and desensitization induced by CGRP, adrenomedullin and adrenomedullin 2 were blocked by the CGRP receptor antagonist CGRP8-37. These data indicate that CGRP receptors are desensitized by select peptides in the calcitonin/CGRP family. Slow recovery from the desensitized state may provide a strategy for timed modulation of the CGRP signaling pathway.
Keywords: Calcitonin gene-related peptide; Desensitization; Cyclic AMP; SK-N-MC cell;

Induction of Nrf2-regulated genes by 3H-1, 2-dithiole-3-thione through the ERK signaling pathway in murine keratinocytes by Sarala Manandhar; Jeong-Min Cho; Jung-Ae Kim; Thomas W. Kensler; Mi-Kyoung Kwak (17-27).
Electrophile and free radical detoxifying enzymes including NAD(P)H:quinine oxidoreductase 1 (Nqo1) play an important role in the defense system by enhancing cellular antioxidant capacity. Chemopreventive efficacy of 3H-1,2-dithiole-3-thione (D3T) is mediated through activation of the transcription factor Nrf2 and subsequent elevation of detoxifying enzymes. In the present study, we have investigated the potential role of extracellular signal-regulated kinase (ERK) in regulation of D3T-induced and Nrf2-dependent gene expression in murine keratinocytes. Expression levels of Nqo1 were highly inducible by D3T treatment and increased nuclear levels of Nrf2 were observed in these cells. Treatment with pharmacological inhibitor of ERK1/2 largely blocked nuclear accumulation of Nrf2, ARE-driven reporter gene expression, and induction of Nqo1, as well as other phase 2 genes. Activation of ERK1/2 has been demonstrated following treatment with D3T. While, inhibitors of p38, PKC and PI3K did not affect ARE-driven gene expression. Involvement of the ERK1/2 cascade in inducible ARE-transcription activities was also observed in cells treated with other types of inducers oltipraz, sulforaphane and hydrogen peroxide. Collectively, current study suggests that phosphorylation cascade via ERK1/2 is associated with the activation process of Nrf2 and subsequent transactivation of its target gene Nqo1 following treatment with dithiolethione in murine keratinocyte.
Keywords: Chemopreventive agent; Dithiolethiones; Nrf2; ERK;

Acetylbritannilactone suppresses lipopolysaccharide-induced vascular smooth muscle cell inflammatory response by Yue-Ping Liu; Jin-Kun Wen; Bin Zheng; Di-Qun Zhang; Mei Han (28-34).
To investigate the mechanism of action by which a new anti-inflammatory active compound, 1-O-acetylbritannilactone (ABL) isolated from Inula britannica-F., inhibits inflammatory responses in vascular smooth muscle cells (VSMCs). Enzyme immunoassay was used to measure the levels of prostandin E2 (PGE2) production. Immunocytochemistry staining and Western blot analysis were performed to detect the nuclear translocation of nuclear factor-κB (NF-κB) p65 and the expression of IκB-α, pIκB-α and cyclooxygenase-2 (COX-2). Electrophoretic mobility shift assays (EMSA) were used to detect DNA-binding activity of NF-κB in VSMCs. ABL (5, 10, 20 μmmol/l) had several concentration-dependent effects, including inhibition of lipopolysaccharide (LPS)-induced PGE2 production and COX-2 expression, and blockade of NF-κB activation and translocation. These effects were owing to reductions in IκB-α phosphorylation and degradation induced by LPS. In addition, ABL directly inhibited the binding of active NF-κB to specific DNA cis-element. These results indicate that ABL is a potent inhibitor of LPS-stimulated VSMC inflammatory responses through blockade of NF-κB activity and inhibition of inflammatory gene COX-2 expression.
Keywords: 1-O-acetylbritannilactone; Nuclear factor-κB; Vascular smooth muscle cell; Cyclooxygenase-2;

Adenosine uptake-dependent C6 cell growth inhibition by Satoko Ohkubo; Koichi Nagata; Norimichi Nakahata (35-43).
In C6 glioma cells, adenine nucleotides, especially AMP, and adenosine inhibited cell proliferation in time- and concentration-dependent manners. α,β-methylene-ADP, an ecto-5′-nucleotidase inhibitor, suppressed the hydrolysis of AMP and reversed the inhibition of cell growth induced by AMP but not by adenosine. Adenosine deaminase eliminated both AMP- and adenosine-mediated growth inhibitions. 5′-N-ethylcarboxamidoadenosine, an adenosine receptor agonist, had little effect on the cell growth. Equilibrative nucleoside transporters, ENT-1 and ENT-2, were expressed in C6 cells by determining their mRNAs. ENT inhibitors, nitrobenzylthioinosine and dipyridamole, suppressed the uptake of [3H]adenosine into C6 cells, and attenuated AMP- or adenosine-mediated growth inhibition. Furthermore, an adenosine kinase inhibitor 5-iodotubercidin reversed the growth inhibition induced by AMP and adenosine. When uridine was added in the extracellular space, AMP- or adenosine-induced cell growth inhibition was completely reversed, suggesting that intracellular pyrimidine starvation would be involved in their cytostatic effects. These results indicate that extracellular adenine nucleotides inhibit C6 cell growth via adenosine, which is produced by ecto-nucleotidases including CD73 at the extracellular space and then incorporated into cells by ENT2. Intracellular AMP accumulation by adenosine kinase after adenosine uptake would induce C6 cell growth inhibition through pyrimidine starvation.
Keywords: AMP; Adenosine; Nucleoside transporter; Adenosine kinase; Pyrimidine starvation; C6 glioma cells; Cell growth;

Differential desensitization of dopamine D2 receptor isoforms by protein kinase C: The importance of receptor phosphorylation and pseudosubstrate sites by Stephen J. Morris; Irit Itzhaki Van-Ham; Mireille Daigle; Liliane Robillard; Naghmeh Sajedi; Paul R. Albert (44-53).
Altered regulation of dopamine D2 receptors is implicated in addiction, schizophrenia and movement disorders, as well as lactotroph growth and regulation. Dopamine D2S and dopamine D2L receptors are alternately-spliced variants that differ by 29 amino acids in the third intracellular (i3) domain and display different sensitivity to desensitization by protein kinase C (PKC). In the present studies we determined the specific phosphorylation sites on the dopamine D2S receptor that confer PKC-mediated desensitization. In dopamine D2L receptors, we identified a PKC pseudosubstrate site responsible for the relative insensitivity of the receptor to PKC-induced uncoupling. In transiently transfected Ltk fibroblast cells, 2-min preactivation of PKC with 12-O-tetradecanoyl 4β-phorbol 13α-acetate (TPA) completely inhibited calcium mobilization induced by the dopamine D2S receptor, but not the dopamine D2L variant. Point mutation of i3 PKC sites Ser228/229Gly rendered the dopamine D2S receptor resistant to PKC action, with lesser effects of other Ser and Thr mutations. Inactivation of the PKC pseudosubstrate motif in the dopamine D2L receptor sensitized the receptor to PKC, and this was reversed by mutation of i3 PKC sites Ser228/229. A phospho-specific antibody generated against phospho-Ser228/229 demonstrated PKC-induced phosphorylation at these sites of dopamine D2S, but not D2L receptors, in Ltk cells. Conversely, the pseudosubstrate dopamine D2L receptor mutant displayed PKC-induced phosphorylation at Ser228/229, which was abolished when these sites were mutated. Similar phosphorylation results were observed using GH4 cells stably transfected with dopamine D2 receptors and mutants. Thus the relative location of phosphorylation and pseudosubstrate sites provides an important determinant substrate sensitivity to PKC.
Keywords: Dopamine; Protein kinase C; Calcium; Desensitization; Uncoupling; Phosphorylation;

ICAM-1 and p38 MAPK mediate fibrinogen-induced migration of human vascular smooth muscle cells by Bernhard H. Rauch; Birgit Müschenborn; Marina Braun; Artur-Aron Weber; Karsten Schrör (54-57).
Fibrinogen deposition in the vessel wall represents an independent atherogenic risk factor. In Boyden-chamber assays, fibrinogen concentration-dependently (1–100 μM) induced migration of human vascular smooth muscle cells (SMC). This was inhibited by antibodies to intercellular adhesion molecule-1 (ICAM-1, 10 μg/ml), and by inhibitors of PI3-kinase (LY294002, 10 μM) and MAPK (mitogen-activated protein kinase) p38 (SB203580, 10 μM). The MEK (MAP kinase kinase) inhibitor PD98059 (10 μM) and the GPIIb/IIIa antagonist abciximab (10 μg/ml) had no effect. ICAM-1 antibodies inhibited fibrinogen-induced Akt and p38 phosphorylation. Thus fibrinogen stimulates human SMC migration through binding to ICAM-1 and activation of Akt and p38.
Keywords: Fibrinogen; Migration; Human smooth muscle cell; ICAM-1; p38 MAPK;

Neuroprotective effects of emodin-8-O-β-d-glucoside in vivo and in vitro by Chaoyun Wang; Dalei Zhang; Hongmei Ma; Juntian Liu (58-63).
Emodin-8-O-β-d-glucoside extracted from the traditional Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc is widely used to treat acute hepatitis possibly by antioxidative mechanisms. The present study was designed to investigate whether emodin-8-O-β-d-glucoside exerted neuroprotective effects on the focal cerebral injury induced by ischemia and reperfusion in vivo and on the neuronal damage induced by glutamate in vitro, and to study the possible mechanisms. Male Wistar rats were used to establish the model of ischemia and reperfusion. The behavioral test was performed and the cerebral infarction area was assessed in the brain slices stained with 2% 2,3,5-triphenyl tetrazolium chloride to evaluate the neuroprotective effects of emodin-8-O-β-d-glucoside. Superoxide dismutase (SOD) activity, total antioxidative capability and malondialdehyde (MDA) level in the brain tissue were determined with spectrophotometrical methods to probe the primary mechanisms of emodin-8-O-β-d-glucoside. In vitro, the neuroprotective effects of emodin-8-O-β-d-glucoside were tested in the cultured cortical cells of fetal rats exposed to glutamate. Emodin-8-O-β-d-glucoside concentration in plasma and brain tissue was also measured to examine distribution of emodin-8-O-β-d-glucoside in the brain. The results showed that the treatment of rats with emodin-8-O-β-d-glucoside reduced the neurological deficit score and the cerebral infarction area, increased SOD activity and total antioxidative capability, and decreased MDA level in the brain tissue in dose-dependent way. Emodin-8-O-β-d-glucoside also inhibited the neuronal damage induced by glutamate. Besides, emodin-8-O-β-d-glucoside was able to penetrate blood-brain barrier and distribute in the brain tissue. These findings demonstrate that emodin-8-O-β-d-glucoside is able to provide neuroprotection against cerebral ischemia-reperfused injury and glutamate induced neuronal damage through exerting antioxidative effects and inhibiting glutamate neurotoxicity.
Keywords: Polygonum cuspidatum Sieb. et Zucc; Emodin-8-O-β-d-glucoside; Ischemia and reperfusion; Neuroprotection; Lipid peroxidation;

The goal of the present study was to analyse to what extent variables such as (1) injected volume, (2) nature of the solvent of drugs (saline versus distilled water) and (3) placement of an additional cannula to inject the solvent of the drugs at the opposite side of the brain, influenced the behavioural effects of the combined administration of the dopamine D1 receptor agonist (±)-1-phenyl-2,3,4,5-tetrahydro-[1H]-3-benzazepine-7,8-diol (SKF 38393, 5.0 μg) and the dopamine D2/D3 receptor agonist quinpirole (10.0 μg) into the shell or core of the nucleus accumbens of freely moving rats. First, we found that increasing the injected volume from 0.2 μl to 0.5 μl significantly increased the amount of contralateral turning after injection of the drugs into the shell and, especially, the core of rats equipped with one cannula. More importantly, the type of turning behaviour changed: instead of a predominance of pivoting, both pivoting and circling appeared. Second, replacing the solvent saline by distilled water resulted in a minor, but significant, decrease of the amount of contralateral turning elicited from either the shell or the core of the nucleus accumbens of rats equipped with one cannula. The type of turning was not changed by this new solvent. Third, and most importantly, this study showed that the vehicle injection into the right core exerted a potentiating effect on the number of contralateral rotations elicited by injections of SKF 38393 + quinpirole into the left core, whereas such a vehicle injection into the right shell did not affect the number of contralateral rotations elicited by injections of SKF 38393 + quinpirole into the left shell. The type of turning in these rats was not changed when compared to rats equipped with one cannula. It is hypothesized that the fluid injected into the core, directly or indirectly, enhanced the dopaminergic asymmetry between the left and the right brain, implying that this manipulation anyhow reduced the dopaminergic activity in the region under discussion. In conclusion, subtle changes in the methodology used to study both the behaviour-specificity and the region-specificity of drug injections into the brain significantly directs the outcome of such studies.
Keywords: Behaviour; Microinjection; Striatum; Cannula; Catecholamine; (Rat);

Performance- and task-dependent effects of the dopamine D1/D5 receptor agonist SKF 38393 on learning and memory in the rat by Francesco Amico; Laura Spowart-Manning; Roger Anwyl; Michael J. Rowan (71-77).
Dopamine D1/D5 receptor agonists may enhance cognition by mimicking dopamine's neurophysiological actions on the processes underlying learning and memory. The present study examined the task- and performance- dependence of the cognitive effects of a partial agonist at dopamine D1/D5 receptors, SKF 38393 [(±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide], in rats. Spatial working memory was assessed in a T-maze, spatial reference memory in a water maze and habituation learning in a novel environment, a hole board. The muscarinic acetylcholine receptor antagonist scopolamine (1.5 mg/kg, i.p.) was used to cause an impairment of performance of these learning tasks. Administration of SKF 38393 (6 mg/kg, i.p.) alone had no significant effect on spontaneous alternation in the T-maze, latency to escape to a hidden platform in the water maze or the habituation of spontaneous behaviour in the hole board. In contrast, in scopolamine-treated rats, whereas SKF 38393 prevented the scopolamine-induced deficit in the T-maze, it exacerbated the impairment in the water maze and did not significantly alter the disruption of habituation. These results suggest that dopamine D1/D5 receptor activation has performance- and task-dependent effects on cognitive function.
Keywords: Morris water maze; T-maze; Hole board; Scopolamine; Reference memory; Working memory; Novelty habituation;

Senktide-induced gerbil foot tapping behaviour is blocked by selective tachykinin NK1 and NK3 receptor antagonists by Monika Sundqvist; Elin Kristensson; Rebecka Adolfsson; Agnes Leffler; Ingela Ahlstedt; Susanna Engberg; Tomas Drmota; Kalle Sigfridsson; Rainer Jussila; Jennie de Verdier; Anna Novén; Anders Johansson; Ingrid Påhlman; Bengt von Mentzer; Erik Lindström (78-86).
Intracerebroventricular (i.c.v.) administration of tachykinin NK1 receptor agonists induces tapping of the hind legs in gerbils, so-called gerbil foot tapping, which is thought to reflect a fear-related response. The aim of the present study was to examine how ligands selective for NK1, NK2 and NK3 receptors affect the gerbil foot tap response. Agonists selective for NK receptor subtypes were administered i.c.v. and the gerbil foot tap response was monitored. The effect of systemically administered antagonists was also studied. The interaction of ligands with gerbil NK1 receptors was evaluated using autoradiography on gerbil brain slices with [3H]-Sar,Met(O2)-substance P or [3H]GR205171 as radioligand. The effects of ligands on NK1 and NK3 receptor-mediated increases in intracellular calcium in vitro were studied in Chinese hamster ovary cells expressing the cloned gerbil receptors. The selective NK1 receptor agonist ASMSP and the selective NK3 receptor agonist senktide induced dose-dependent increases in gerbil foot tapping with similar potency. The maximal effect of senktide was approximately 40% of the maximal response evoked by ASMSP. The effects of ASMSP and senktide were blocked by administration of the selective NK1 receptor antagonist CP99,994 (10 μmol/kg s.c.). The effects of senktide, but not ASMSP, were blocked by administration of the selective NK3 receptor antagonist SB223412 (50 μmol/kg i.p.). Senktide did not displace NK1 receptor radioligand binding and was > 1000-fold less potent than ASMSP at activating gerbil NK1 receptors. The selective NK3 receptor agonist senktide evokes fear-related gerbil foot tapping, an effect which probably involves indirect enhancement of NK1 receptor signalling.
Keywords: Gerbil foot tap; Neurokinin; Senktide; Tachykinin receptor; Tachykinin receptor antagonist;

The N-methyl-d-aspartate (NMDA) receptor system is thought to be underactive in schizophrenia which may contribute to attentional dysfunction in this disease. In a visual signal detection task that required discrimination of signaled-trials from trials with no signal, the NMDA receptor antagonist, dizocilpine (0.05 mg/kg), increased errors on non-signal trials. Co-administration of dizocilpine and 10.0 mg/kg d-cycloserine, a co-agonist at the glycine site on the NMDA receptor, significantly decreased the error rate on non-signal trials compared to dizocilpine alone. These results suggest that drugs targeting the glycine site may be beneficial for attenuating attentional deficits associated with an underactive NMDA receptor system.
Keywords: Glutamate; MK-801; Schizophrenia; Vigilance;

Effects of trans- and cis-resveratrol on Ca2+ handling in A7r5 vascular myocytes by Manuel Campos-Toimil; Jacobo Elíes; Ezequiel Álvarez; Ignacio Verde; Francisco Orallo (91-99).
Although the natural polyphenol resveratrol posses a direct vasorelaxant effect, its effects on cytoplasmic Ca2+ concentration ([Ca2+]i) in vascular cells remain still unclear. Here, we have investigated the effects of the isomers trans- and cis-resveratrol on agonist- and high-K+-induced [Ca2+]i increases and on voltage-activated transmembrane Ca2+ fluxes using imaging and patch-clamp techniques in vascular A7r5 myocytes. Arginine vasopressin (AVP) or angiotensin II caused a biphasic increase in [Ca2+]i that was reduced by preincubation with trans-resveratrol and cis-resveratrol. Both isomers also reduced the agonist-induced increase in [Ca2+]i in absence of extracellular Ca2+. In high-K+ Ca2+-free solution, reintroduction of Ca2+ caused a sustained rise in [Ca2+]i that was reduced by preincubation with trans-resveratrol or cis-resveratrol. When the isomers were applied during the plateau phase of the agonist- or the high-K+-induced response, a biphasic change in [Ca2+]i was observed: a transient reduction of the plateau (< 5 min) followed by an increase (> 10 min). Finally, trans-resveratrol and cis-resveratrol inhibited voltage-dependent L-type Ca2+ currents (I Ca(L)). In conclusion, resveratrol isomers exert a dual effect on [Ca2+]i handling in A7r5 myocytes: 1) a blockade of I Ca(L) and 2) an increase in [Ca2+]i by depletion of intracellular Ca2+ stores (which interferes with the agonist-induced release of intracellular Ca2+) and influx of Ca2+, mainly due to activation of capacitative Ca2+ entry, although other Ca2+-permeable channels are also involved. Taken together, these effects may explain, in part, the endothelium-independent vasorelaxant effects of resveratrol in rat aorta.
Keywords: A7r5; Calcium; Fura-2; Patch-clamp; Resveratrol;

WB1106, a novel nitric oxide-releasing derivative of telmisartan, inhibits hypertension and improves glucose metabolism in rats by Yong-Qi Li; Hui Ji; Yi-Hua Zhang; Wei-Bing Shi; Zhao-Ke Meng; Xiu-Ying Chen; Guan-Tao Du; Jide Tian (100-108).
Angiotensin converting enzyme (ACE) inhibitors usually cause severe coughing and intolerance while antagonists for angiotensin AT1 receptor do not stimulate the production of nitric oxide (NO). NO has been shown to regulate arterial hypertension and insulin resistance. Hence, new hybrids of antagonist for angiotensin AT1 receptor and a NO donor may have potent anti-hypertensive effect and regulate glucose metabolism and insulin resistance. Herein, the effects of [6-(nitrooxymethyl)pyridin-2-yl] methyl 4′-[1-(1,7′-dimethyl-2′-propyl-1H,3′H-2,5′-bibenzo[d]imidazol-3′-yl)ethyl] biphenyl-2-carboxylate (WB1106), a novel NO-releasing derivative of telmisartan newly synthesized, on the vasocontraction, hypertension and diet-induced insulin resistance were examined in vitro using rat aortic strips and in normotensive and spontaneous hypertension rats (SHR rats). Apparently, WB1106 induced the vasorelaxation of contracted rat aortic strips in a dose- and time-dependent manner, which depended on the activity of guanylate cyclase, a characteristic of NO-related function. Furthermore, WB1106 reduced the contractile and blood pressure responses to angiotensin II, which relied on the release of telmisartan. Moreover, treatment with WB1106 significantly reduced the blood pressure with similar potency to telmitarsan and increased the contents of cGMP in SHR rats. Therefore, WB1106 possesses both the angiotensin AT1 receptor antagonist activity of telmisartan and the NO-releasing property of a ‘slow NO donor’. Importantly, in contrast to equimolar telmisartan, treatment with WB1106 significantly attenuated body weight gains and improved glucose tolerance in high-fat and carbohydrate-fed rats, reflecting a synergistic effect of NO and telmisartan. Potentially, WB1106 may be a potent anti-hypertensive drug for treatment of hypertension and diabetes-related cardiovascular diseases in the clinic.
Keywords: WB1106; Nitric oxide; Angiotensin II; Hypertension; Glucose tolerance;

Pharmacological nature of nicotine-induced contraction in the rat basilar artery: Involvement of arachidonic acid metabolites by Xu Ji; Tsuyoshi Nishihashi; Cristina C. Trandafir; Aimin Wang; Yoshiharu Shimizu; Kazuyoshi Kurahashi (109-114).
The pharmacological nature of nicotine-induced contraction in the rat basilar artery is poorly understood. The purpose of this study was to investigate the endothelium dependency and involvement of arachidonic acid metabolites in nicotine-induced contraction in the rat basilar artery. The rat basilar artery was removed from the brain and cut into a spiral preparation. Nicotine (3 × 10− 5 to 10− 2 M) induced the concentration-dependent contraction in the rat basilar artery, and the maximal contraction was obtained at 3 × 10− 3 M. The contraction induced by nicotine (3 × 10− 3 M) was significantly attenuated by the presence of saponin (0.05 mg/ml, 15 min). Phospholipase C (PLC) inhibitors (NCDC and U-73122), calcium-independent phospholipase A2 (iPLA2) inhibitor (BEL), cyclooxygenase-2 (COX-2) inhibitors (nimesulide, L-745,337 and celecoxib), and a 5-lipoxygenase (5-LOX) inhibitor (ZM-230487) concentration-dependently attenuated the nicotine-induced contraction. A cytosolic phospholipase A2 (cPLA2) inhibitor (AACOCF3), secretory phospholipase A2 (sPLA2) inhibitor (indoxam), and cyclooxygenase-1 (COX-1) inhibitors (flurbiprofen and ketoprofen) did not affect the nicotine-induced contraction. From these results, it was suggested that nicotine-induced contraction in the rat basilar artery is endothelium-dependent and is due to arachidonic acid metabolites.
Keywords: Rat basilar artery; Nicotine; Contraction; Endothelium; Cyclooxygenase-2 (COX-2); Arachidonic acid metabolite;

ATP-dependent potassium channels (KATP) have been implicated in cardioprotection both during myocardial ischemia and reperfusion. We compared the effect of a non-selective KATP inhibitor glibenclamide, a selective mitochondrial KATP inhibitor 5-hydroxy-decanoate (5-HD) and a selective sarcolemmal KATP blocker HMR 1883, on survival and incidence of arrhythmias during myocardial ischemia in conscious, and during ischemia-reperfusion in pentobarbitone anesthetized rats. Glibenclamide (5 mg/kg i.p.) or HMR 1883 (3 mg/kg i.v.) reduced ischemia-induced irreversible ventricular fibrillation and improved survival during myocardial ischemia (64% and 61% vs. 23% in controls, respectively). 5-HD (5 mg/kg i.v.) did not influence survival and the incidence of ventricular arrhythmias. The incidence of reperfusion-induced arrhythmias was reduced by both glibenclamide and HMR 1883 (3 or 10 mg/kg) resulting in improved survival during reperfusion (81%, 82% and 96% vs. 24% in controls, respectively) in anesthetized rats. 5-HD did not reduce the incidence of lethal reperfusion arrhythmias. Glibenclamide and HMR 1883 prolonged (89 ± 4.6 and 89 ± 4.9 ms vs. 60 ± 2.4 ms in controls), while 5-HD did not change the QT interval. In conclusion, inhibition of sarcolemmal KATP reduces the incidence of lethal ventricular arrhythmias and improves survival both during acute myocardial ischemia and reperfusion in rats. This beneficial effect correlates with the prolongation of repolarization. Inhibition of mitochondrial KATP does not improve survival or reduce the occurrence of ischemia and/or reperfusion-induced arrhythmias and does not prolong the QT interval. The present results also suggest that the antiarrhythmic effect of KATP inhibitors is not influenced by pentobarbitone anesthesia.
Keywords: Sarcolemmal KATP channels; Glibenclamide; HMR 1883; 5-hydroxy-decanoate; Ventricular arrhythmias;

Sulphonylureas induced vasorelaxation of mouse arteries by Bo Jiang; Lingyun Wu; Rui Wang (124-128).
Sulphonylureas, such as glybenclamide and gliclazide, are classical blockers of ATP-dependent potassium channels (KATP). Closure of KATP channels in vascular smooth muscles by sulphonylureas would lead to membrane depolarization and vasoconstriction but this hypothesis has not been consistently proved in different types of vascular tissues. Our present study examined mouse vascular contractility changes induced by sulphonylureas using a wire myograph. The phenylephrine-precontracted aorta (n  = 6), super mesenteric arteries (n  = 10) and third-order mesenteric artery (n  = 10) from C57BL mice were relaxed by glybenclamide with IC50 of 116 ± 13.0, 61.8 ± 5.5, and 41.4 ± 1.3 μM, respectively. Gliclazide or U37883A (a vascular KATP blocker structurally different from that of sulphonylureas) had similar vasorelaxant effects on mesenteric arteries with IC50 of 14.8 ± 1.5 μM (n  = 5) or 15.6 ± 1.3 μM (n  = 9), respectively. The presence of Nomega-nitro-L-arginine methyl ester hydrochloride (l-NAME, 300 μM, n  = 8), apamin + charybdotoxin (n  = 4), or 1H-[1, 2, 4]-oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ) (10 μM, n  = 10) partially reduced vasorelaxant effect of glybenclamide on mesenteric arteries. Removal of endothelium (n  = 7) or precontraction with 100 mM [K+]o (n  = 10) also significantly reduced vasorelaxant effect of glybenclamide on mesenteric arteries. The relaxation of mouse resistance arteries by KATP channel blockers calls for further investigation into the selectivity and suitability of these drugs in treatment of different vascular disease.
Keywords: Glybenclamide; Mesenteric artery; Vasorelaxation; Sulphonylureas; KATP channels; Endothelium; (Mouse);

Cardiovascular effects of CDP-choline and its metabolites: Involvement of peripheral autonomic nervous system by Mehmet Cansev; Mustafa Sertac Yilmaz; Yesim Ozarda Ilcol; Emre Hamurtekin; Ismail Hakki Ulus (129-142).
Intraperitoneal administration of CDP-choline (200–900 μmol/kg) increased blood pressure and decreased heart rate of rats in a dose- and time-dependent manner. These responses were accompanied by elevated serum concentrations of CDP-choline and its metabolites phosphocholine, choline, cytidine monophosphate and cytidine. Blood pressure increased by intraperitoneal phosphocholine (200–900 μmol/kg), while it decreased by choline (200–600 μmol/kg) administration; phosphocholine or choline administration (up to 600 μmol/kg) decreased heart rate. Intraperitoneal cytidine monophosphate (200–600 μmol/kg) or cytidine (200–600 μmol/kg) increased blood pressure without affecting heart rate. Pressor responses to CDP-choline, phosphocholine, cytidine monophosphate or cytidine were not altered by pretreatment with atropine methyl nitrate or hexamethonium while hypotensive effect of choline was reversed to pressor effect by these pretreatments. Pretreatment with atropine plus hexamethonium attenuated or blocked pressor response to CDP-choline or phosphocholine, respectively. Heart rate responses to CDP-choline, phosphocholine and choline were blocked by atropine and reversed by hexamethonium. Cardiovascular responses to CDP-choline, phosphocholine and choline, but not cytidine monophosphate or cytidine, were associated with elevated plasma catecholamines concentrations. Blockade of α-adrenoceptors by prazosin or yohimbine attenuated pressor response to CDP-choline while these antagonists blocked pressor responses to phosphocholine or choline. Neither bilateral adrenalectomy nor chemical sympathectomy altered cardiovascular responses to CDP-choline, choline, cytidine monophosphate or cytidine. Sympathectomy attenuated pressor response to phosphocholine. Results show that intraperitoneal administration of CDP-choline and its metabolites alter cardiovascular parameters and suggest that peripheral cholinergic and adrenergic receptors are involved in these responses.
Keywords: CDP-choline; Cardiovascular; Phosphocholine; Choline; Cytidine;

Effects of antimuscarinics on voiding function after cerebral infarction in a rat model of overactive bladder by Anwar Yusup; Hironobu Akino; Yoshiji Miwa; Nobuyuki Oyama; Yoshitaka Aoki; Hideaki Ito; Kazuya Tanase; Yosuke Matsuta; Masaharu Nakai; Osamu Yokoyama (143-149).
Muscarinic receptor antagonists are used clinically for their therapeutic peripheral effects on bladder function. However, these agents may also act on central muscarinic receptors, especially in individuals with compromised blood–brain barrier function. We compared the effects of atropine and tolterodine, agents that do and do not readily cross the blood–brain barrier, respectively, administered peripherally (intravenous [i.v.]) and centrally (intracerebroventricular [i.c.v.]) on cystometrography in conscious rats after cerebral infarction induced by middle cerebral artery occlusion or sham surgery. We hypothesized that tolterodine would produce greater improvement in bladder capacity and less impairment in bladder contractility and that the effects of both agents would be greater in rats with cerebral infarction and sham-operated rats after peripheral administration, but that tolterodine and atropine would exert similar effects after central administration. Bladder capacity was markedly reduced following cerebral infarction. Low-dose i.v. tolterodine (≤ 20 nmol/kg) significantly reversed this effect without altering residual volume or bladder contraction pressure. Low-dose i.v. atropine (2 nmol/kg) had no effect on bladder capacity but significantly decreased bladder contraction pressure. Higher doses of i.v. atropine (≥ 20 nmol/kg) significantly increased bladder capacity but also significantly increased residual volume and decreased bladder contraction pressure. Tolterodine was significantly more efficacious than atropine in increasing bladder capacity, whereas atropine produced significantly greater increases in residual volume and reductions in bladder contraction pressure; these treatment group differences were also observed in sham-operated animals. Tolterodine and atropine administered i.c.v. significantly increased bladder capacity following cerebral infarction or sham surgery; however, this was accompanied by significantly increased residual volume and decreased bladder contraction time. The decrease in bladder contraction time was significantly smaller after tolterodine vs atropine. Peripherally acting muscarinic receptor antagonists may be preferable to centrally acting agents for minimizing adverse events, such as incomplete bladder emptying, even in individuals with compromised blood–brain barrier function.
Keywords: Cerebral infarction; Muscarinic; Micturition; Overactive bladder; Tolterodine; (Rat);

Human epididymal and prostatic tracts of vas deferens: Different contraction response to noradrenaline stimulation in isolated organ bath assay by Nicola A. Colabufo; Vincenzo Pagliarulo; Francesco Berardi; Marialessandra Contino; Roberto Perrone; Mauro Niso; Giancarlo Albo; Gaetano de Rienzo; Arcangelo Pagliarulo (150-155).
In the present study epididymal and prostatic portions of human vas deferens were separately isolated and stimulated with exogenous noradrenaline to study their contractile properties. The results displayed that the epididymal tract produced a phasic–tonic response, while the prostatic strip produced only a phasic response suggesting a different functional role of each vas deferens segment. Moreover, it has been verified if α1-adrenoceptor antagonists doxazosin, alphuzosin and terazosin could differently block the noradrenaline response in each segment.Doxazosin, the most potent antagonist, displayed similar potency in epididymal and prostatic tract (pA2  = 8.51 and 8.42, respectively). Analogously, alphuzosin, although less potent than doxazosin, displayed in the same tracts a superimposed potency (pA2  = 7.25 and 7.30, respectively). In contrast with doxazosin and alphuzosin, terazosin displayed higher potency in blocking the contractile response in prostatic tract (pA2  = 7.67) than in epididymal segment (pA2  = 6.43).These results showed that α1-adrenoceptor antagonists doxazosin and alphuzosin, although with a different potency, did not discriminate between epididymal and prostatic segment while terazosin showed high potency in prostatic tract and only a moderate activity in epididymal section. Moreover, the biological model employed in our experiments could be a valid screening method to test the potential interferences of drugs indicated for bladder outlet obstruction with the peristaltic activity or the global tone of the human vas deferens.
Keywords: Human vas deferens; α1-Adrenoceptor antagonist; Doxazosin; Alphuzosin; Terazosin;

Haemostatic imbalance following carrageenan-induced rat paw oedema by Carla Cicala; Silvana Morello; Alessio Alfieri; Valentina Vellecco; Stefania Marzocco; Giuseppina Autore (156-161).
Carrageenan-induced rat paw oedema is a widely used model to investigate the physiopathology of an acute local inflammation. Recently, much attention has been focused on the link between haemostasis and inflammation, and on the impact that inflammation might have on thrombotic events. It is known that the systemic response to inflammation is the “acute phase reaction” that represents a highly complex reaction of the organism to a variety of injuries, aimed to restore homeostasis; one important feature of the acute phase reaction is the hepatic synthesis of proteins involved in the coagulation cascade. Much attention has been focused on the role that systemic inflammation might have on thrombotic events, while there is not much information on the role played by an acute local inflammation on haemostasis, that can lead toward a pro-thrombotic state. The present study was conducted to evaluate the haemostatic balance in the early and the late phase of carrageenan-induced rat paw oedema; i.e. at 3 h, when paw inflammation is maximally expressed, and 24 h following carrageenan injection, when there is an almost complete absence of local inflammatory symptoms. We found that in inflamed animals, 24 h following oedema induction, there was an increase in plasma fibrinogen levels, antithrombin III activity and serum interleukin-6 levels, concomitant to a shortened prothrombin time and to an increased platelet responsiveness to ADP. Furthermore, in inflamed tissues at 3 h there was an increase in antithrombin III proteic expression. Our results demonstrate that a haemostatic imbalance occurs following carrageenan-induced rat paw oedema.
Keywords: Carrageenan; Haemostasis; Fibrinogen; Platelet aggregation; Antithrombin III;

Topical levamisole hydrochloride therapy attenuates experimental murine allergic rhinitis by Heyao Wang; Jiali Zhang; Chunsheng Gao; Ying Zhu; Chen Wang; Wenjie Zheng (162-169).
Allergic rhinitis is one of the most common chronic diseases. There are a number of effective therapeutic options for allergic rhinitis patients, such as intranasal corticosteroids. How to avoid the adverse effects of these traditional medicines has come to public attention and started the search for effective and safe medicine. We used BALB/c mice with experimental allergic rhinitis, and determined that levamisole delivered locally (intranasal, i.n.) could attenuate early-phase inflammatory response, decrease histamine, suppress edema and eosinophil infiltration, and diminish the ovalbumin-specific serum IgE level. Detailed analysis of cytokine gene expression showed that levamisole can decrease IL-4, IL-5 and IL-13 mRNA and increase IL-12, IL-18 and IFN-γ mRNA. Levamisole showed analogous effects of down-regulating Th2 cytokines with budesonide and distinct up-regulating effects on Th1 cytokines gene expression. Our findings offer potential options for allergic rhinitis therapy.
Keywords: Levamisole hydrochloride; Allergic rhinitis; Eosinophil; (Mouse);

β-adrenoceptor blockade ameliorates the clinical course of experimental allergic encephalomyelitis and diminishes its aggravation in adrenalectomized rats by M. Dimitrijević; A. Rauški; K. Radojević; D. Kosec; S. Stanojević; I. Pilipović; G. Leposavić (170-182).
As glucocorticoids influence both catecholamine synthesis and adrenoceptor expression by immune cells, the current study was undertaken to distinguish their direct effects on the development of experimental allergic encephalomyelitis from those induced by alteration of catecholamine signaling. We examined the influence of 16-day-long β-adrenoceptor blockade with propranolol (0.40 mg/100 g body weight/day, s.c.) beginning 3 days before immunization on the development of experimental allergic encephalomyelitis in adrenalectomized (7 days before immunization) and in non-operated male Dark Agouti rats. Adrenalectomy aggravated the clinical course of experimental allergic encephalomyelitis. In contrast, propranolol attenuated both the clinical signs of the disease and decreased the number of lesions in the spinal cord. Furthermore, propranolol prevented adrenalectomy-induced aggravation of the disease course without affecting mortality. We also found that the percentage of CD4+CD25+ T lymphocytes (recently activated or regulatory cells) was increased in peripheral blood of experimental allergic encephalomyelitis rats over that in the corresponding non-immunized and bovine serum albumin immunized rats. However, the percentage of these cells was reduced in adrenalectomized and/or propranolol-treated experimental allergic encephalomyelitis rats compared to control experimental allergic encephalomyelitis rats. Our findings, coupled with the clinical course of the disease and the underlying pathomorphological changes, clearly suggest that differential mechanisms were responsible for the changes in the percentage of CD4+CD25+ T lymphocytes in propranolol-treated adrenalectomized rats and only propranolol-treated rats with experimental allergic encephalomyelitis. Our results, when viewed globally, indicate that: i) β-adrenoceptor-dependent mechanisms are involved in the immunopathogenesis of experimental allergic encephalomyelitis, ii) experimental allergic encephalomyelitis has a more severe course in adrenalectomized rats and iii) β-adrenoceptor-mediated mechanisms operate in adrenalectomy-induced aggravation of the disease.
Keywords: Adrenalectomy; β-Adrenoceptor blockade; Experimental allergic encephalomyelitis; CD4+CD25+ peripheral blood lymphocytes;

Pancreatic islet cell death is the cause of deficient insulin production in diabetes mellitus. Approaches towards prevention of cell death are of prophylactic importance in control and management of hyperglycemia. Generation of oxidative stress is implicated in streptozotocin, a beta cell specific toxin-induced islet cell death. In this context, antioxidants raise an interest for therapeutic purposes. Curcumin, a common dietary spice is a well known antioxidant and hence we investigated its effect on streptozotocin-induced islet damage in vitro. Isolated islets from C57/BL6J mice were incubated with curcumin for 24 h and later exposed to streptozotocin for 8 h. The effect of streptozotocin exposure to islets was determined with respect to islet viability and functionality, cellular reactive oxygen species concentrations and levels of activated poly (ADP-ribose) polymerase-1. Cellular antioxidant potential (Cu/Zn superoxide dismutase) and advanced glycation end-product related damage was assessed to determine the metabolic status of treated and untreated islets. Islet viability and secreted insulin in curcumin pretreated islets were significantly higher than islets exposed to streptozotocin alone. Curcumin retarded generation of islet reactive oxygen species along with inhibition of Poly ADP-ribose polymerase-1 activation. Although curcumin did not cause overexpression of Cu/Zn superoxide dismutase, it prevented reduction in levels of cellular free radical scavenging enzymes. Our data shows that curcumin protects islets against streptozotocin-induced oxidative stress by scavenging free radicals. We show here for the first time, that prophylactic use of curcumin may effectively rescue islets from damage without affecting the normal function of these cellular structures.
Keywords: Prevention; Islet death; Streptozotocin; Reactive oxygen species; Curcumin;

C-type natriuretic peptide stimulates pancreatic exocrine secretion in the rat: Role of vagal afferent and efferent pathways by María E. Sabbatini; Myrian R. Rodríguez; Paula Dabas; Marcelo S. Vatta; Liliana G. Bianciotti (192-202).
We previously reported that C-type natriuretic peptide (CNP) increases amylase release in isolated pancreatic acini through natriuretic peptide receptor C activation and enhances pancreatic exocrine secretion via vagal pathways when applied to the brain. In the present study we sought to establish whether CNP was involved in the peripheral regulation of pancreatic secretion. Anesthetized rats were prepared with pancreatic duct cannulation, pyloric ligation and bile diversion into the duodenum. CNP dose-dependently enhanced pancreatic flow, chloride and protein excretion but did not modify bicarbonate output. A selective natriuretic peptide receptor C agonist enhanced pancreatic flow and mimicked CNP-evoked protein output but failed to modify chloride secretion. Truncal vagotomy, perivagal application of capsaicin and hexamethonium reduced CNP-evoked pancreatic flow and abolished chloride excretion but did not affect protein output. Furthermore, pre-treatment with atropine reduced both CNP-stimulated pancreatic flow and chloride excretion but failed to modify protein excretion. Partial muscarinic blockade of CNP-evoked chloride output suggested that mediators other than acetylcholine were involved. However, CNP response was unaltered by cholecystokinin and vasoactive intestinal peptide receptor blockade or by nitric oxide synthase inhibition. In conclusion, CNP-stimulated pancreatic flow through the activation of the natriuretic peptide receptor C and the vago-vagal reflex but it increased protein output only by natriuretic peptide receptor C activation and chloride excretion by vago-vagal reflexes. Present results suggest that CNP may play a role as a local regulator of the exocrine pancreas.
Keywords: Pancreatic exocrine secretion; Vago-vagal reflex; Natriuretic peptide; Natriuretic peptide receptor C;

Melatonin reduces formalin-induced nociception and tactile allodynia in diabetic rats by Rosaura Arreola-Espino; Héctor Urquiza-Marín; Mónica Ambriz-Tututi; Claudia Ivonne Araiza-Saldaña; Nadia L. Caram-Salas; Héctor I. Rocha-González; Teresa Mixcoatl-Zecuatl; Vinicio Granados-Soto (203-210).
The purpose of this study was to assess the antinociceptive and antiallodynic effect of melatonin as well as its possible mechanism of action in diabetic rats. Streptozotocin (50 mg/kg) injection caused hyperglycemia within 1 week. Formalin-evoked flinching was increased in diabetic rats as compared to non-diabetic rats. Oral administration of melatonin (10–300 mg/kg) dose-dependently reduced flinching behavior in diabetic rats. In addition, K-185 (a melatonin MT2 receptor antagonist, 0.2–2 mg/kg, s.c.) completely blocked the melatonin-induced antinociception in diabetic rats, whereas that naltrexone (a non-selective opioid receptor antagonist, 1 mg/kg, s.c.) and naltrindole (a selective δ opioid receptor antagonist, 0.5 mg/kg, s.c.), but not 5′-guanidinonaltrindole (a selective κ opioid receptor antagonist, 1 mg/kg, s.c.), partially reduced the antinociceptive effect of melatonin. Given alone K-185, naltrexone, naltrindole or 5′-guanidinonaltrindole did not modify formalin-induced nociception in diabetic rats. Four to 8 weeks after diabetes induction, tactile allodynia was observed in the streptozotocin-injected rats. On this condition, oral administration of melatonin (75–300 mg/kg) dose-dependently reduced tactile allodynia in diabetic rats. Both antinociceptive and antiallodynic effects were not related to motor changes as melatonin did not modify number of falls in the rotarod test. Results indicate that melatonin is able to reduce formalin-induced nociception and tactile allodynia in streptozotocin-injected rats. In addition, data suggest that melatonin MT2 and δ opioid receptors may play an important role in these effects.
Keywords: Melatonin; Melatonin MT2 receptor; Naltrexone; Naltrindole; Diabetes; Tactile allodynia;

(R)-ACX, a pancreatic β-cell selective sulfonylurea, does not aggravate myocardial ischemia-reperfusion damage by Iyuki Namekata; Yusuke Yamaguchi; Shuhei Moriguchi; Satoshi Yamazaki; Akihiko Terasawa; Reiko Yamagishi; Tokiko Aikawa; Tomoaki Saito; Katsumi Kurashima; Kenji Seri; Yorishige Imamura; Hiroyuki Akita; Koki Shigenobu; Hikaru Tanaka (211-218).
The organ selectivity and the effect on myocardial ischemia-reperfusion injury of (R)-acetoxyhexamide ((R)-ACX), a novel sulfonylurea, were examined. (R)-ACX, as well as glibenclamide, concentration-dependently stimulated insulin release from INS-1 cell, a cell line derived from pancreatic β-cells. The potency of (R)-ACX was about 1/10 of that of glibenclamide. In isolated guinea pig ventricular myocardial tissue, glibenclamide concentration-dependently inhibited the action potential shortening by NIP-121, an ATP-sensitive potassium channel opener, but (R)-ACX showed only slight inhibition. In isolated rat aortic rings contracted with norepinephrine, glibenclamide concentration-dependently inhibited the relaxation by NIP-121, while (R)-ACX showed only slight inhibition. In coronary-perfused guinea pig ventricular preparations, glibenclamide reduced the recovery of contractile force after ischemia-reperfusion, while (R)-ACX did not. In conclusion, (R)-ACX is a β-cell selective sulfonylurea which, unlike glibenclamide, does not aggravate cardiac ischemia-reperfusion damage.
Keywords: Heart; Ischemia-reperfusion; (R)-ACX;

Impairment of recognition memory in interleukin-6 knock-out mice by Anna Hryniewicz; Izabela Bialuk; Karol A. Kamiński; Maria M. Winnicka (219-220).
We have used transgenic male mice not expressing interleukin-6 (IL-6) [C57BL/6JIL-6/-tm Kopf] in object recognition test to assess the role of endogenous IL-6 in recognition memory. Wild-type controls showed better memory than IL-6 knock-out mice. Results of our experiment suggest that endogenous IL-6 may play an important role in the process of recognition memory in mice.
Keywords: Interleukin-6 (IL-6) knock-out mice; Recognition memory;

Corrigendum to “In-vitro experimental models for the risk assessment of antibiotic-induced QT prolongation” by Hua Rong Lu; Eddy Vlaminckx; Andre Van de Water; Jutta Rohrbacher; An Hermans; David J. Gallacher (222-232).
The prolongation of the ventricular repolarization and proarrhythmic effects (Torsade de Pointes: TdP) of five reference antibiotics were compared in four in-vitro models. 1. Using the patch clamp technique on the human ether-a-gogo-related gene (HERG) current, the rank order for blockade of the HERG-current (IC50) was: sparfloxacin (44 μM) > telithromycin = moxifloxacin = erythromycin (± 100 μM). 2. Assessing their effects on action potential duration (APD90) and incidence of early afterdepolarizations in isolated rabbit Purkinje fibers, the rank order was: sparfloxacin >moxifloxacin > telithromycin > erythromycin (prolongation of APD90 at 100 μM: 83%, 48%, 33% and 17% from baseline, respectively, compared to + 5% with solvent, P  < 0.05). 3. Assessing the drug effects on the APD60, triangulation, reverse use-dependency, and instability in isolated Langendorff-perfused rabbit hearts, the rank order was: moxifloxacin > sparfloxacin > telithromycin > erythromycin. 4. Assessing their torsadogenic potentials (scores of effects on QT-interval, peak of the T wave to end of T wave: T p–e, T p–e/QT ratio, R wave on T wave (R on T) and TdP in isolated rabbit left ventricular wedge preparations, the rank order for their TdP risk score was: sparfloxacin > erythromycin > moxifloxacin > telithromycin. Additional experiments with grepafloxacin indicate that the rank order to detect grepafloxacin-induced long QT was the wedge preparation > the Purkinje fiber > HERG > the isolated heart, where the isolated heart was unable to detect grepafloxacin-induced APD prolongation. The present study demonstrates that the first three in vitro models can be used to assess the ability of antibiotic compounds to delay ventricular repolarization. However, with respect to the perceived clinical arrhythmogenic potential (QT prolongation/TdP) of these fluoroquinolones, the wedge preparation appears to be more predictive and suitable for detecting torsadogenic action of antibiotics.
Keywords: Action potential duration; Antibiotics; Drug-induced long QT; Early afterdepolarizations; TdP (Torsade de Pointes); Long QT syndrome;