European Journal of Pharmacology (v.424, #1)

Inhibitors of arachidonic acid metabolism potentiate tumour necrosis factor-α-induced apoptosis in HL-60 cells by Jan Vondráček; Jiřı́ Štika; Karel Souček; Kateřina Minksová; Luděk Bláha; Jiřina Hofmanová; Alois Kozubı́k (1-11).
We investigated whether and how could various modulators of arachidonic acid metabolism affect apoptosis induced by tumour necrosis factor-α (TNF-α) in human myeloid leukaemia HL-60 cells. These included arachinonyltrifluoromethyl ketone (AACOCF3; cytosolic phospholipase A2 inhibitor), indomethacin (cyclooxygenase inhibitor), MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethyl propanoic acid; 5-lipoxygenase-activating protein inhibitor), nordihydroguaiaretic acid (general lipoxygenase inhibitor), and arachidonic acid itself. Incubation of HL-60 cells with nordihydroguaiaretic acid resulted in apoptosis and it was characterised by mitochondria membrane depolarisation, release of cytochrome c from mitochondria into cytosol and activation of caspase-3. Indomethacin and nordihydroguaiaretic acid synergistically potentiated TNF-α-induced apoptosis, while arachidonic acid, AACOCF3 and MK-886 did not modulate its effects. Furthermore, indomethacin potentiated apoptosis in cells treated with a differentiating agent, all-trans retinoic acid, which induces resistance to TNF-α. However, the observed effects were probably not associated either with the cyclooxygenase- or lipoxygenase-dependent activities of indomethacin and nordihydroguaiaretic acid, respectively. Since indomethacin may reportedly activate peroxisome proliferator-activated receptors (PPARs), the effects of specific ligands of PPARs on apoptosis were studied as well. It was found that selective PPARs ligands had no effects on TNF-α-induced apoptosis. The findings suggest that arachidonic acid metabolism does not play a key role in regulation of apoptosis induced by TNF-α in the present model. Nevertheless, our data raise the possibility that indomethacin could potentially be used to improve the treatment of human myeloid leukaemia.
Keywords: Retinoic acid; TNF-α (tumor necrosis factor-α); Fas; Arachidonic acid; Lipoxygenase; Cyclooxygenase; PPAR (Peroxisome proliferator-activated receptor);

Agonist properties of pindolol at h5-HT1A receptors coupled to mitogen-activated protein kinase by Mark J Millan; Adrian Newman-Tancredi; Delphine Duqueyroix; Didier Cussac (13-17).
At h5-HT1A receptors, stably transfected into Chinese Hamster Ovary Cells (CHO-h5-HT1A), the selective 5-HT1A receptor agonist, (+)8-hydroxy-dipropyl-amino-tetralin, ((+)8-OH-DPAT), transiently activated mitogen-activated protein kinase (MAPK) with a pEC50 of 8.5. The arylalkylamine, (−)-pindolol, also behaved as an agonist with a maximal effect of 57% relative to (+)8-OH-DPAT (100%), and with a pEC50 of 7.2. The selective 5-HT1A receptor antagonist, N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclo-hexane carboxamide (WAY100,635), blocked (+)8-OH-DPAT- and (−)-pindolol-induced MAPK activation with pK Bs of 9.7 and 9.9, respectively, whereas the selective 5-HT1B receptor antagonist, 1′-Methyl-5-[2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-ylcarbonyl]-2,3,6,7-tetrahydro-5H-spiro[furo[2,3-f]indole-3,4′-piperidine] (SB224,289) was inactive. Pertussis toxin blocked the actions of (+)8-OH-DPAT and (−)-pindolol demonstrating implication of Gi/Go proteins. Thus, stimulation of MAPK provides an intracellular marker and signal for expression of the agonist actions of (−)-pindolol at h5-HT1A receptors.
Keywords: MAPK (Mitogen-activated protein kinase); h5-HT1A receptor; (−)-Pindolol; (+)8-OH-DPAT ((+)8-Hydroxy-dipropyl-amino-tetralin); G protein;

Modulation by taurine of the pacemaking activity and the underlying ionic currents in rabbit sino-atrial nodal cells was investigated at low and high cellular Ca2+ concentrations ([Ca2+]i) using a patch-clamp technique. At both pCa 8 and 6, taurine depressed the spontaneous activity, more strongly at pCa 6 than at pCa 8. Taurine, 20 mM, markedly inhibited the L-type Ca2+ current: by 56.9±2.8% (n=6, P<0.001) at pCa 8, and by 97.6±3.8% (n=7, P<0.001) at pCa 6. Also at 20 mM, taurine decreased the delayed rectifier K+ current by 26.8±2.6% (n=6, P<0.01) at pCa 6, whereas taurine had less or no effect at pCa 8. The hyperpolarization-activated inward current also decreased at both pCa 8 and 6, by 18.3±1.3% (n=8, P<0.05) and by 20.8±3.3% (n=8, P<0.05) in 20 mM taurine, respectively. Taurine caused a more potent inhibitory effect at pCa 6. Taurine often elicited dysrhythmias, at 20 mM, in 3 of 17 cells at pCa 8 and in 12 of 16 cells at pCa 6. During washout, the incidence of dysrhythmias or arrest increased further. These results indicate that taurine exerts more potent inhibitory actions on ionic currents under Ca2+ overload conditions in rabbit sino-atrial nodal cells. However, taurine would possibly elicit a cellular Ca2+ overload, when taurine application was discontinued.
Keywords: Taurine; Automaticity; Spontaneous action potential; Ionic current; Ca2+ overload; Sino-atrial nodal cell;

Comparison of apomorphine, amphetamine and dizocilpine disruptions of prepulse inhibition in inbred and outbred mice strains by Geoffrey B Varty; Nafesa Walters; Mary Cohen-Williams; Galen J Carey (27-36).
The dopamine agonist apomorphine robustly disrupts prepulse inhibition of the acoustic startle response in the rat, yet published studies have not demonstrated a robust disruption of prepulse inhibition with apomorphine in the mouse. The aim of these studies was to establish the optimal prepulse conditions (using manipulations to prepulse intensity and inter-stimulus interval) and mouse strain(s) for testing apomorphine, and also the prepulse inhibition disrupting drugs amphetamine, and dizocilpine (MK-801). The effects of these drugs on startle response and prepulse inhibition were tested in outbred CD-1 and Swiss Webster (CFW) strains, and the inbred C57BL/6, 129X1/SvJ, and A/J strains. There were strain differences with baseline startle and prepulse inhibition in that the CD-1, CFW, and C57BL/6 strains exhibited high levels of startle and prepulse inhibition, the 129X1/SvJ strain exhibited low levels of startle but high levels of prepulse inhibition, while the A/J strain exhibited low startle and no prepulse inhibition. Apomorphine disrupted prepulse inhibition in the CFW and C57BL/6 strains and the effect was only evident when using a short 30 ms inter-stimulus interval. Amphetamine disrupted prepulse inhibition in the CFW, C57BL/6, and 129X1/SvJ strains, and dizocilpine disrupted prepulse inhibition in the CD-1, CFW, C57BL/6, and 129X1/SvJ strains. The effects of amphetamine and dizocilpine were independent of the inter-stimulus interval. These studies demonstrated clear strain differences in the startle response and prepulse inhibition, and the pharmacological disruptions of prepulse inhibition, and suggest that inter-stimulus intervals less than 100 ms may be optimal for detecting the effects of apomorphine in mice.
Keywords: Acoustic startle response; Prepulse inhibition; Schizophrenia; (Mouse strain); Apomorphine; Amphetamine; Dizocilpine;

Anti-ischemic and cognition-enhancing properties of NNC-711, a γ-aminobutyric acid reuptake inhibitor by Alan W O'Connell; Gerard B Fox; Connie Kjøller; Helen C Gallagher; Keith J Murphy; John Kelly; Ciaran M Regan (37-44).
NNC-711 [1-(2-((diphenylmethylene)amino)oxy)ethyl)-1,2,4,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride], a γ-aminobutyric acid (GABA) reuptake inhibitor with anticonvulsant activity, was investigated with respect to its cognition-enhancing and neuroprotective potency. In the rat, administration of NNC-711 immediately prior to training prevented amnesia for a passive avoidance task induced by the acetylcholine receptor antagonist scopolamine. NNC-711 was also effective in protecting against ischemia-induced death of CA1 pyramidal neurons in a model of bilateral common carotid artery occlusion in the gerbil. In addition to a neuroprotective activity, NNC-711 exhibited significant cognition-enhancing actions. Daily administration of NNC-711, immediately prior to a spatial learning task, significantly reduced escape latencies in the water maze paradigm in both mature (postnatal day 80) and aged (28 months) rats. All of the above actions exhibited a bell-shaped response with an optimal dose of 0.5–1.0 mg/kg. These investigations with NNC-711 and previous clinical observations on the structurally related anticonvulsant tiagabine confirm the potential of GABA reuptake inhibitors as anti-amnesia and cognition-enhancing agents.
Keywords: GAT1 inhibitor; Tiagabine; Avoidance conditioning; Spatial learning; Carotid occlusion; CA1 cell death;

We used the formalin test to clarify the 5-hydroxytryptamine (5-HT) receptor subtypes involved in the modulation of spinal nociceptive transmission in rats. Intrathecal administration of a 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)-tetraline (8-OH-DPAT; 1, 10, and 30 μg), or a 5-HT1B receptor agonist, 1, 4-dihydro-3-(1, 2, 3, 6-tetrahydro-4-pyridinyl)-5H-pyrrol (3, 2-b) pyridin-5-one (CP 93129; 1 and 10 μg), produced no significant change in the number of flinches. A 5-HT2 receptor agonist, (±)-2, 5-dimethoxy-4-iodoamphetamine (DOI ; 10, 30, and 100 μg), and a 5-HT3 receptor agonist, 2-methyl-5-HT (100 and 300 μg), produced dose-dependent decreases in the number of flinches in phases 1 (1 to 6 min) and 2 (10 to 61 min) of the test. The antinociceptive effects of DOI and 2-methyl-5-HT were antagonized by intrathecal pretreatment with a 5-HT2 receptor antagonist, ketanserin, and a 5-HT3 receptor antagonist, 3-tropanyl-3, 5-dichlorobenzoate (MDL-72222), respectively. These results suggest that 5-HT2 and 5-HT3 receptors in the spinal cord mediate antinociception to chemical stimuli.
Keywords: 5-HT (5-hydroxytryptamine, serotonin); 5-HT receptor subtype; Antinociception; Spinal cord; Formalin test; (Rat);

The effect of adrenergic compounds on neurogenic dural vasodilatation by Simon Akerman; David J Williamson; Raymond G Hill; Peter J Goadsby (53-58).
The pharmacology of neurogenic trigeminovascular vasodilator responses in the dura mater is of interest for understanding the pathophysiology of migraine and to develop new therapies for this disabling common condition. Aminergic mechanisms have been implicated in migraine through direct study of amines in patients, and by inference from the pharmacology of many effective anti-migraine compounds, particularly preventative agents. This study used intravital microscopy to assess the role of aminergic transmission in neurogenic dural vasodilatation (NDV) by measuring directly the diameter of dural arteries in sodium pentobarbitone anaesthetised rats. Electrical stimulation of a closed cranial window produces, by local depolarisation of nerves, dural vessel dilation that is monitored continuously on-line using video-microscopy and a video dimension analyser. This dural vasodilatation was not affected by pre-treatment with an α1-adrenoceptor agonist (phenylephrine, 1 and 5 μg/kg), or antagonist (corynanthine, 1 and 2 mg/kg), nor by an α2-adrenoceptor agonist (UK14,304, 5 μg/kg) or antagonist (yohimbine, 1 and 3 mg/kg). Similarly, we saw no effect of β-adrenoceptor blockade (propranolol, 1 and 3 mg/kg). The lack of an inhibitory effect of UK14,304 the model of neurogenic dural vasodilation contrasts with its effect in neurogenic dural plasma protein extravasation model. The lack of inhibition of β-adrenoceptor antagonists in the neurogenic vasodilatation model contrasts with their usefulness as migraine prophylactics, and suggests that their mechanism of action in migraine is unlikely to be through sensory trigeminal fibre terminals at the neurovascular junction. Moreover, the data indicate that the adrenergic system does not play a significant role in neurogenic dural vasodilation.
Keywords: Trigeminovascular; Migraine; Headache; Adrenergic compound;

Effects of the endothelin receptor antagonist Bosentan on ischaemia/reperfusion injury in rat skeletal muscle by Kevin J Herbert; Michael J Hickey; Diana A Lepore; Kenneth R Knight; Wayne A Morrison; Alastair G Stewart (59-67).
We examined the role of endothelin in ischaemia/reperfusion injury in skeletal muscle, using the endothelin receptor antagonist Bosentan. In the rat hindlimb tourniquet ischaemia model, one hindlimb was rendered ischaemic for 2 h at 36 °C, then blood flow was re-established for either 24 h to assess muscle survival or 1.5 h for a study of capillary perfusion. In the first set of rats, the gastrocnemius muscle was removed from the postischaemic limb and assessed for viability histochemically using the nitro blue tetrazolium stain. Tissue water content (a measure of oedema) and myeloperoxidase activity (a measure of neutrophil accumulation) were also assessed in the ischaemic muscle, the contralateral non-ischaemic muscle and the lungs. In the second set of rats, the hind limb was infused with India ink after 2-h ischaemia and 1.5-h reperfusion and the muscle was harvested, fixed and cleared. In control rats, muscle viability was 17±2% (S.E.M.). In rats treated with Bosentan (10 mg/kg, i.p.) 30 min before release of the tourniquet, muscle viability (48±7%) was significantly increased compared to the control group (P<0.01). Bosentan treatment had no significant effect on tissue water content or myeloperoxidase activity in the ischaemic muscle, the contralateral non-ischaemic muscle or the lung. Immunoreactive endothelin levels in serum increased to a peak at 90 min of reperfusion and returned to control levels by 24-h reperfusion. India ink studies demonstrated a significantly increased functional capillary density in postischaemic Bosentan-treated muscles compared with postischaemic control muscles (P<0.05). These results suggest that endothelin plays an important role in the necrosis which results from a period of ischaemia and reperfusion in skeletal muscle, by mediating a decrease in postischaemic microvascular perfusion.
Keywords: Endothelin; Ischaemia/reperfusion; Skeletal muscle; (Rat); Bosentan; No-reflow; Neutrophil; Oedema;

The role of nitric oxide in the gastric acid secretion induced by ischemia–reperfusion in the pylorus-ligated rat by Junko Tanaka; Yasukatu Yuda; Shigeharu Inouye; Toshio Yamakawa (69-74).
In a rat model of the ischemia–reperfusion with pylorus ligation, gastric ulcer was formed, although gastric acid secretion was reduced. When the polymorphonuclear leukocytes were inactivated in advance, gastric ulcer was not formed, but acid secretion was increased, indicating that gastric acid is not a cause of the ulcer formation in this model. The mechanism of gastric acid suppression accompanied by ischemia–reperfusion was examined in relation to the role of oxygen-free radicals in this rat model. Prior administration of superoxide dismutase did not modulate acid secretion, but N-nitro-l-arginine methyl ester (l-NAME) increased acid secretion. The action of l-NAME was antagonized specifically by l-arginine, but not by d-arginine. S-nitroso-N-acetylpenicillamine did not inhibit basal acid secretion but antagonized the action of l-NAME. Aminoguanidine increased significantly the gastric acid output that was suppressed by ischemia–reperfusion. When polymorphonuclear leukocytes were inactivated by treatment with their antibody, the gastric acid output recovered to the level in the pylorus-ligated rat without ischemia–reperfusion. These results suggested that nitric oxide (NO) produced by the infiltrated polymorphonuclear leukocytes plays an important role in the suppression of acid secretion induced by ischemia–reperfusion.
Keywords: Nitric oxide (NO); Ischemia–reperfusion; Gastric acid; Polymorphonuclear leukocyte;

Effect of N-acetylcysteine on gentamicin-mediated nephropathy in rats by Emanuela Mazzon; Domenico Britti; Angela De Sarro; Achille P Caputi; Salvatore Cuzzocrea (75-83).
Studies were performed on the mechanisms of the protective effects of free-radical scavengers against gentamicin-mediated nephropathy. Administration of gentamicin, 100 mg/kg s.c., for 5 days to rats induced marked renal failure, characterised by a significantly decreased creatinine clearance and increased blood creatinine levels, fractional excretion of sodium Na+, lithium Li+, urine gamma glutamyl transferase and daily urine volume. A significant increase in kidney myeloperoxidase activity and lipid peroxidation was observed in gentamicin-treated rats. Immunohistochemical localisation demonstrated nitrotyrosine formation and poly(ADP-ribose)synthase activation in the proximal tubule from gentamicin-treated rats. Renal histology examination confirmed the tubular necrosis. N-acetylcysteine (10 mg/kg i.p. for 5 days) caused normalisation of the above biochemical parameters. In addition, N-acetylcysteine treatment significantly prevents the gentamicin-induced tubular necrosis. These results suggest that (1) N-acetylcysteine has protective effects on gentamicin-mediated nephropathy, and (2) the mechanisms of the protective effects can be, at least in part, related to interference with peroxynitrite-related pathways.
Keywords: Nitric oxide (NO); Peroxynitrite; Poly(ADP ribose)synthetase; Gentamicin; Renal injury;