European Journal of Pharmacology (v.451, #1)

The role of neutrophil elastase in acute lung injury by Kazuhito Kawabata; Tetsuya Hagio; Shozo Matsuoka (1-10).
Beside its physiological function as a powerful host defense, neutrophil elastase is also known as one of the most destructive enzymes in the body. Current notion holds that neutrophil elastase is able to escape from regulation by multiple protease inhibitors at inflammatory sites. Once unregulated, this enzyme disturbs the function of the lung permeability barrier and induces the release of pro-inflammatory cytokines. These actions then cause symptoms that are typical in the pathophysiology of acute lung injury. In this article, we review recent progress in the understanding of the physiological activity of neutrophil elastase and its role in acute lung injury. Evidence in this review that supports the involvement of neutrophil elastase in the pathophysiology of acute lung injury includes: (1) neutrophil elastase levels are increased in both clinical and animal models of acute lung injury; (2) topical or systemic administration of neutrophil elastase produces typical symptoms of acute lung injury both in vitro and in vivo; and (3) inhibition of increased neutrophil elastase activity reduces symptoms of acute lung injury in animal models. A greater understanding of the role of this enzyme in the pathophysiology of acute lung injury will lead to better treatments for this complicated disease.
Keywords: Neutrophil elastase; Lung injury; Respiratory distress syndrome; Permeability; Edema;

Avasimibe and atorvastatin synergistically reduce cholesteryl ester content in THP-1 macrophages by Gemma Llaverı́as; Mireia Jové; Manuel Vázquez-Carrera; Rosa M Sánchez; Cristina Dı́az; Gonzalo Hernández; Juan C Laguna; Marta Alegret (11-17).
Evidence suggests that the inhibition of both acyl-CoA:cholesterol acyltransferase and hydroxymethyl glutaryl-CoA reductase causes a synergistic direct antiatherosclerotic effect on the vessel wall. To investigate this synergism in a single cell type and to avoid the confounding effect of plasma cholesterol lowering by these drugs, we have used an in vitro model of human macrophages (phorbol ester-treated THP-1 cells). In macrophages incubated simultaneously with acetyl low-density lipoproteins, the novel acyl-CoA:cholesterol acyltransferase inhibitor avasimibe (0.01–0.5 μM) caused a concentration-dependent reduction in cell cholesteryl ester content that was not accompanied by an increase in intracellular free cholesterol. A 5 μM concentration of atorvastatin enhanced by approximately twofold the ability of 0.5 μM avasimibe to reduce the mass of esterified cholesterol, and this was reversed by co-incubation with 200 μM mevalonate or 10 μM geranyl-geraniol. Based on these data, we propose that the synergism between acyl-CoA:cholesterol acyltransferase and hydroxymethyl glutaryl-CoA reductase inhibitors found in several in vivo studies may be explained by a direct additive effect of both agents reducing the lipid content of the macrophages present in the lesion area.
Keywords: Avasimibe; Atorvastatin; Acyl-CoA cholesterol acyltransferase; Hydroxymethyl glutaryl-CoA reductase; Macrophage; Cholesteryl ester;

TAK-778 [(2R,4S)-(−)-N-(4-Diethoxyphosphorylmethylphenyl)-1,2,4,5-tetrahydro-4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide: mw 505.52], a novel compound promoting osteoblast differentiation, promotes osteogenesis in vitro and enhances bone formation during skeletal repair in vivo. In this study, we further evaluated the effects of TAK-778 on the differentiation of cultured bone marrow stromal cells into osteoblasts in the presence of dexamethasone, paying particular attention to the expression of transcription factors involved in regulating osteoblast differentiation. Treatment of TAK-778 (10−7–10−5 M) for 4 h resulted in an increase in the mRNA expression of Msx2, but not Cbfa1 or Dlx5. This transcriptional alteration preceded the changes in other markers related to the osteoblast phenotype, such as alkaline phosphatase and osteocalcin mRNA. The transfection of Msx2-antisense in the cells caused a significant reduction in the levels of alkaline phosphatase mRNA expression induced by TAK-778. These results suggest that TAK-778 promotes osteoblast differentiation partly through the expression of Msx2, a homeobox-related gene.
Keywords: Osteoblast differentiation; Alkaline phosphatase; Growth factor; Msx2; TAK-778;

Enhanced amphetamine-mediated dopamine release develops in PC12 cells after repeated amphetamine treatment by Lana Kantor; Yang Hae Park; Kevin K.W Wang; Margaret E Gnegy (27-35).
We previously demonstrated that rats treated with repeated, intermittent amphetamine displayed enhanced amphetamine-mediated dopamine release in the striatum. In this study, we examined whether amphetamine pretreatment would elicit enhanced amphetamine-mediated dopamine release in a cultured cell line in the absence of intact synaptic connections. PC12 cells pretreated with 1 μM amphetamine produced over twofold increase in amphetamine-mediated dopamine release upon challenge with 1 μM amphetamine as compared with vehicle-treated cells. No change in norepinephrine transporter density or [3H]dopamine uptake was detected. A withdrawal time of 6 days was required to observe the enhanced amphetamine-mediated dopamine release. Differentiation of the cells with nerve growth factor did not alter the amphetamine-mediated dopamine release in control cells or the development of enhanced release in amphetamine-treated cells. Our results demonstrate that repeated, intermittent amphetamine leads to a neuroadaptation resulting in enhanced amphetamine-induced dopamine release in catecholaminergic cells without the need of an intact neuroanatomy.
Keywords: Nisoxetine; Norepinephrine transporter; Withdrawal time; Intermittent; Nerve growth factor; Dopamine uptake;

Zoniporide: a potent and highly selective inhibitor of human Na+/H+ exchanger-1 by Ravi B Marala; Janice A Brown; Jimmy X Kong; W.Ross Tracey; Delvin R Knight; Ronald T Wester; Dexue Sun; Scott P Kennedy; Ernest S Hamanaka; Roger B Ruggeri; Roger J Hill (37-41).
We evaluated the in vitro pharmacological profile of a novel, potent and highly selective Na+/H+ exchanger-1 (NHE-1) inhibitor, [1-(Quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine hydrochloride monohydrate (zoniporide or CP-597,396). The potency and selectivity of zoniporide were determined via inhibition of 22Na+ uptake by PS-120 fibroblast cell lines overexpressing human NHE-1, -2 or rat NHE-3. Additionally, potency for endogenous NHE-1 was confirmed via ex vivo human platelet swelling assay (PSA), in which platelet swelling was induced by exposure to sodium propionate. The pharmacological profile of zoniporide was compared with that of eniporide and cariporide. Zoniporide inhibited 22Na+ uptake in fibroblasts expressing human NHE-1 in a concentration-dependent manner (IC50=14 nM) and was highly selective (157-fold and 15,700-fold vs. human NHE-2 and rat NHE-3, respectively). Zoniporide was 1.64- to 2.6-fold more potent at human NHE-1 than either eniporide or cariporide (IC50=23 and 36 nM, respectively). Zoniporide was also more selective at inhibiting human NHE-1 vs. human NHE-2 than either eniporide or cariporide (157-fold selective compared with 27- and 49-fold, respectively). All three compounds inhibited human platelet swelling with IC50 values in low nanomolar range. From these results, we conclude that zoniporide represents a novel, potent and highly selective NHE-1 inhibitor.
Keywords: Na+/H+ exchanger; Platelet swelling assay;

GABAA receptor activation and open-channel block by volatile anaesthetics: a new principle of receptor modulation? by Rainer Haseneder; Gerhard Rammes; Walter Zieglgänsberger; Eberhard Kochs; Gerhard Hapfelmeier (43-50).
The rapid application of solutions containing the volatile anaesthetics isoflurane or sevoflurane induced inward currents in human embryonic kidney (HEK293) cells carrying rat recombinant α1β2γ2L GABAA receptor assemblies. The responses evoked by the anaesthetics applied via a fast delivery system were recorded using the patch-clamp technique in the whole-cell mode. The anaesthetics induced a fast inward current which was followed by a prominent tail current upon the rapid withdrawal of the agent. These currents were simulated using a kinetic scheme embodying two agonist-like binding steps required for receptor activation, and one binding step by which the anaesthetic induces an open-channel block. According to this model of a biphasic receptor modulation, the open-channel block delays the ion flux through the ligand-gated receptors and, thus, prolongs the overall duration of the current response. Open-channel blocks might also be operative in other ligand-gated ion channels to modulate synaptic strength.
Keywords: GABAA receptor; Isoflurane; Patch-clamp; Kinetic modelling; Open-channel block;

In the present study, we demonstrate the involvement of GABAA receptors in the ethanol-mediated modulation of extracellular signal-regulated kinases (ERK). Intraperitoneal (i.p.) administration of ethanol (3.5 g), flurazepam (75 mg) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cycloheptane-5,10-iminemaleate (MK-801) (0.4 mg/kg body weight) decreased, while picrotoxin (10 mg/kg body weight) increased, the phosphorylation of ERK following 10 min of their injection. However, the picrotoxin-induced phosphorylation of ERK was inhibited by ethanol, but was not affected by MK-801. These results indicate that ethanol's inhibitory effect on ERK phosphorylation may involve the modulation of GABAA receptor function.
Keywords: Ethanol; GABA (γ-amino butyric acid); MAP (mitogen-activated protein) kinase;

Group III mGlu receptor agonists potentiate the anticonvulsant effect of AMPA and NMDA receptor block by Giovambattista De Sarro; Alba Chimirri; Brian S Meldrum (55-61).
We report the anticonvulsant action in DBA/2 mice of two mGlu Group III receptor agonists: (R,S)-4-phosphonophenylglycine, (R,S)-PPG, a compound with moderate mGlu8 selectivity, and of (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid, ACPT-1, a selective agonist for mGlu4α receptors. Both compounds, given intracerebroventricularly at doses which did not show marked anticonvulsant activity, produced a consistent shift to the left of the dose–response curves (i.e. enhanced the anticonvulsant properties) of 1-(4′-aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-one hydrochloride, CFM-2, a noncompetitive AMPA receptor antagonist, and 3-((±)-2-carboxypiperazin-4-yl)-1-phosphonic acid, CPPene, a competitive NMDA receptor antagonist, in DBA/2 mice. In addition, (R,S)-PPG and ACPT-1 administered intracerebroventricularly prolonged the time course of the anticonvulsant properties of CFM-2 (33 μmol/kg, i.p.) and CPPene (3.3 μmol/kg, i.p.) administered intraperitoneally. We conclude that modest reduction of synaptic glutamate release by activation of Group III metabotropic receptors potentiates the anticonvulsant effect of AMPA and NMDA receptor blockade.
Keywords: Epilepsy; mGlu receptor; NMDA receptor antagonist; AMPA receptor antagonist; Antiepileptic drug;

Role of nitric oxide in systemic effect of theophylline on mouse body temperature by Mohammad-Reza Zarrindast; Mohammad Adl; Mohammad Sharifzadeh; Taraneh Bahreini (63-68).
In the present study, the interaction of nitric oxide synthase (NOS) inhibitors, l-NAME (N G-nitro-l-arginine methyl ester HCl) and l-NA (N ω-nitro-l-arginine), and its precursor, l-arginine (2-(S)-2-amino-5-[(aminoiminomethyl)amino] pentatonic acid), with theophylline on mouse body temperature was studied. Intraperitoneal (i.p.) injection of different doses of theophylline altered body temperature. Lower doses of theophylline (12.5 and 25 mg/kg) increased, but a higher dose (100 mg/kg) reduced, the animals' body temperature. The combination of l-arginine (20 and 40 mg/kg) with the highest dose of theophylline potentiated the hypothermic effect induced by the latter drug, while l-arginine by itself did not alter body temperature. l-NAME (10-80 mg/kg) or l-NA (10 mg/kg) plus a lower dose of theophylline (12.5 mg/kg) reduced the theophylline-induced hyperthermic response. l-NA (1, 5, and 10 mg/kg) in combination with the high dose of theophylline (100 mg/kg) also induced greater hypothermia. Both l-NAME and l-NA by themselves reduced body temperature. It is concluded that nitric oxide (NO) may be involved in the effects of theophylline on body temperature in mice.
Keywords: Theophylline; Nitric oxide (NO); l-Arginine; N G-nitro-l-arginine methylester (l-NAME); N ω-nitro-l-arginine (l-NA); Temperature (mouse);

Arylalkylamines are a novel class of positive allosteric modulators at GABAB receptors in rat neocortex by David I.B Kerr; Jennifer Ong; Ni Made Puspawati; Rolf H Prager (69-77).
Using grease-gap recording from rat neocortical slices, the γ-aminobutyric acidB (GABAB) receptor agonists baclofen (3–100 μM) and SKF 97541 (3-aminopropyl-methylphosphinic acid) (1–30 μM) elicited reversible and concentration-dependent hyperpolarizing responses, with EC50 values of 10 and 3 μM, respectively. The hyperpolarizations were antagonised by the GABAB receptor antagonist Sch 50911 ((+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid) (1, 5 and 10 μM). Fendiline (N-[3,3-diphenylpropyl)-α-methylbenzylamine) (5–50 μM) and its congeners, prenylamine (N-[3,3-diphenylpropyl)-α-methylphenylethylamine) (10–100 μM) and F551 (N-[3,3-diphenylpropyl)-α-methyl-3-methoxybenzylamine) (1–30 μM) reversibly enhanced hyperpolarizing responses to the agonists; such effects were reduced by Sch 50911. These arylalkylamines produced leftward shifts of the concentration–response curves, with a marked increase in the maximal hyperpolarization obtained, compared with the agonists alone, F551 being the most potent. These findings suggest that these arylalkylamines represent a new class of positive modulators of GABAB receptor-mediated function.
Keywords: Neocortex; Baclofen; SKF 97541; Positive modulator; Fendiline; Prenylamine;

Endomorphins suppress nociception-induced c-Fos and Zif/268 expression in the rat spinal dorsal horn by Shingo Tateyama; Tetsuya Ikeda; Kazuko Kosai; Tadashi Nakamura; Toshiharu Kasaba; Mayumi Takasaki; Toshikazu Nishimori (79-87).
We evaluated the potency of endomorphin-1 and -2 as endogenous ligands on c-Fos and Zif/268 expression in the spinal dorsal horn by formalin injection to the rat hind paw. Endomorphin-1, -2, or morphine was administered intrathecally or intracerebroventricularly 5 min before formalin injection (5%, 100 μl). All drugs produced marked reductions of formalin-induced c-Fos and Zif/268 immunoreactivity in laminae I and II, and laminae V and VI in the rat lumbar spinal cord. The reductions of Zif/268 expression by endomorphins were greater than those by morphine, while the reductions of c-Fos expression by endomorphins were smaller than those by morphine. These effects of endomorphins were attenuated by pretreatment with naloxone. These results indicate that endomorphin-1 and -2 act as endogenous ligands of μ-opioid receptor in neurons of the spinal dorsal horn and suppress the processing of nociceptive information in the central nervous system.
Keywords: Endomorphin-1; Endomorphin-2; c-Fos; Zif/268; Formalin; Spinal dorsal horn;

The mediators of bronchoconstriction of isolated lungs and trachea from ovalbumen sensitized guinea-pigs to adenosine and ovalbumen were examined using relevant antagonists. Changes in perfusion pressure and tension of paired lung halves and tracheal spiral strips, respectively, were recorded in response to adenosine (1 mM lung, 300 μM trachea), histamine (10 μM), methacholine (10 μM) and ovalbumen (10 μg). One half was perfused with antagonist while the other received vehicle. Tracheal strips were superfused throughout with the P1 receptor antagonist 8-phenyltheophylline, to examine 8-phenyltheophylline-resistant responses. The histamine H1 receptor antagonist, mepyramine (1.5 mM), the cyclooxygenase inhibitors, indomethacin (5 mM) and diclofenac (5 mM), the leukotriene receptor antagonist, zafirlukast (1 mM), and the lipoxygenase inhibitor, zileuton (20 mM), alone failed to inhibit bronchoconstriction by adenosine and ovalbumen of the lung and trachea. When two antagonists were combined, only mepyramine and zafirlukast significantly reduced the lung responses to adenosine and ovalbumen. The tracheal adenosine response was substantially reduced, although not significantly, while ovalbumen was significantly reduced. When mepyramine, indomethacin and zafirlukast were combined, the lung constriction by adenosine and ovalbumen were virtually abolished. Similarly, the combination of mepyramine, diclofenac and zafirlukast significantly attenuated the lung responses to adenosine and ovalbumen. Thus, histamine, cyclooxygenase products and leukotrienes alone are not responsible for the bronchoconstriction of isolated sensitized lung tissues to adenosine or ovalbumen, which appears to be due to the release of all three mediators.
Keywords: Adenosine; Bronchconstriction; Guinea-pig; Histamine; Leukotriene; Cyclooxygenase product;

Involvement of Raf-1 in chronic δ-opioid receptor agonist-mediated adenylyl cyclase superactivation by Eva V Varga; Marc Rubenzik; Vanessa Grife; Masano Sugiyama; Dagmar Stropova; William R Roeske; Henry I Yamamura (101-102).
Chronic δ-opioid receptor agonist treatment of Chinese hamster ovary (CHO) cells stably expressing the human δ-opioid receptor (hDOR/CHO) leads to increased cAMP formation after the removal of the agonist (adenylyl cyclase superactivation). We have previously found that at the same time, chronic δ-opioid receptor agonist treatment augments phosphorylation of the adenylyl cyclase VI isoenzyme. Since phosphorylation of adenylyl cyclase VI by Raf-1 protein kinase was recently shown, we tested the role of Raf-1 in adenylyl cyclase superactivation in hDOR/CHO cells. We found that pretreatment of the cells with the selective Raf-1 inhibitor GW5074 (3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one) (10 μM, 30 min) attenuates chronic deltorphin II-mediated increase in forskolin-stimulated cAMP formation by 40% (n=6, P<0.05). Better understanding of the molecular mechanism of adenylyl cyclase superactivation should aid in the development of analgesics that act longer and have fewer side effects.
Keywords: Adenylyl cyclase superactivation; δ-Opioid receptor; Raf-1;