European Journal of Pharmacology (v.438, #1-2)

Polymorphisms of the renin–angiotensin system are associated with cardiovascular disorders, possibly as a consequence of increased brain angiotensin II activity. Within the brain, angiotensin controls blood pressure, fluid balance and hormone secretion; it also influences behaviour: reduction of central angiotensin function has both antidepressant-like and axiolytic-like actions. Evidence concerning the role of the renin–angiotensin system in learning and memory is contradictory, although more studies support the proposal that angiotensin reduces cognitive function. Studies of renin–angiotensin system genotype and psychological status have suggested an association between the angiotensin-converting enzyme deletion allele and age related cognitive decline, but a greater prevalence of the insertion allele in Alzheimer's disease. The deletion allele has also been associated with depressive illness, as has the M allele of the angiotensinogen gene although other studies have failed to replicate these findings. The role of the brain renin–angiotensin system in human psychopathology remains to be fully explored.
Keywords: Renin–angiotensin system; Affective disorder; Cognition; Memory;

Effect of glutathione S-transferases on the survival of patients with acute myeloid leukaemia by Judith L Autrup; Peter Hokland; Lars Pedersen; Herman Autrup (15-18).
The objective of the study was to investigate the effect of genetic polymorphisms in glutathione S-transferases (GST) on the survival of acute myeloid leukaemia patients receiving adriamycin induction therapy. A total of 89 patients were included in the study. Patients who carried at least one GSTM1 allele had trend towards a better survival (mortality rate ratio (RR) 0.588; 95% CI 0.334–1.036) than GSTM1*0/0 patients. However, at low accumulated adriamycin dose, GSTM1*0/0 cases had a better survival than people expressing the gene (RR=6.1; 95% CI=1.2–11.0). The GSTT1 and GSTP1 genotype did not influence the survival in any of the groups.
Keywords: Leukaemia; Chemotherapy; Survival; GST (glutathione S-transferase);

Methotrexate-induced apoptosis in hepatocytes after partial hepatectomy by Kaoru Kobayashi; Chieko Terada; Ikuyo Tsukamoto (19-24).
To investigate apoptosis induced by methotrexate in hepatocytes in vivo, rats received a single injection of methotrexate immediately after partial hepatectomy and apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) and gel electrophoresis of DNA. Characteristic DNA fragmentation was obvious at 2 h and peaked at 4 h after partial hepatectomy with methotrexate injection. TUNEL-positive staining was observed in nuclei and nuclear fragments of hepatocytes in the methotrexate-injected liver (partial hepatectomy with methotrexate), with negligible background staining in the control (partial hepatectomy only) and in the methotrexate-injected normal (normal with methotrexate) rat liver. The involvement of the c-Jun N-terminal kinase (JNK) activator protein 1 (AP-1) pathway and p53 in apoptosis was also examined. The activity of JNK increased at 15 min and peaked at 1 h after partial hepatectomy. This increase was repressed by methotrexate injection. Western blot analysis showed that the levels of c-Fos and c-Jun protein expression, which increased at 1 h after partial hepatectomy, were also reduced by methotrexate. The levels of p53 protein were markedly increased after partial hepatectomy with methotrexate injection. The increase in p53 protein was followed by an up-regulation of p21 WAF1/CIP1 protein at 2 h after partial hepatectomy. These results suggested that the inhibition of the JNK-AP-1 pathway and concurrent up-regulation of p53 and p21 WAF1/CIP1 were involved in hepatocyte apoptosis induced by partial hepatectomy with methotrexate.
Keywords: Apoptosis; JNK (c-Jun N-terminal kinase); c-Fos; c-Jun; p53; p21 WAF1/CIP1 ;

Identification and characterisation of functional bombesin receptors in human astrocytes by Sarah Mason; Darren Smart; Ian C.B Marshall; Alexander McKnight; Jeremy N Skepper; Shaun McNulty (25-34).
Reverse transcription polymerase chain reaction (RT-PCR) demonstrated the presence of bombesin BB2 receptor mRNA but not bombesin BB1 receptor or bombesin BB3 receptor mRNA in cultured human astrocytes. Neuromedin C hyperpolarised human astrocytes in whole-cell current and voltage clamp recordings and increased the intracellular free Ca2+ ion concentration ([Ca2+]i) in single astrocytes. Treatment with neuromedin C caused larger and more frequent increases in [Ca2+]i than those triggered by neuromedin B, with 96% and 78% of cells responding, respectively. The stimulatory effects of neuromedin C were inhibited significantly by treatment with U73122 or the bombesin BB2 receptor antagonist [d-Phe6, des-Met14]bombesin-(6–14) ethylester. A Fluorometric Imaging Plate Reader (FLIPR) was used to measure [Ca2+]i in cell populations. Neuromedin C was approximately 50-fold more potent than neuromedin B in elevating [Ca2+]i in astrocytes and Chinese hamster ovary (CHO) cells expressing human bombesin BB2 receptors (hBB2-CHO). However, in CHO cells expressing the bombesin BB1 receptor hBB1-CHO, neuromedin B was 32-fold more potent than neuromedin C. [d-Phe6, des-Met14]bombesin-(6–14) ethylester was a partial agonist in hBB1-CHO cells (E max=55%) but was a noncompetitive antagonist in both hBB2-CHO cells and astrocytes. These studies report the first identification of functional bombesin receptors on cultured human astrocytes and have demonstrated that the bombesin BB2 receptor contributes significantly to astrocyte physiology.
Keywords: Astrocyte; CHO cell; FLIPR; Ca2+; Bombesin; [d-Phe6, des-Met14]bombesin-(6–14) ethylester;

Isobolographic analysis of non-depolarising muscle relaxant interactions at their receptor site by Matthias Paul; Christoph H Kindler; Ralf M Fokt; Natalie C.J Dipp; C Spencer Yost (35-43).
Administration of certain combinations of non-depolarising muscle relaxants produces greater than expected neuromuscular blockade. Synergistic effects may be explained by drug interactions with the postsynaptic muscle nicotinic acetylcholine receptor. To investigate this hypothesis, the adult mouse muscle nicotinic acetylcholine receptor (α2βδε) was heterologously expressed in Xenopus laevis oocytes and activated by the application of acetylcholine (10 μM). The effects of five individually applied muscle relaxants and six combinations of structurally similar and dissimilar compounds were studied. Drug combinations containing equipotent concentrations of two agents were tested and dose–response curves were determined. All compounds tested alone and in combination produced rapid and readily reversible, concentration-dependent inhibition. Isobolographic and fractional analyses indicated additive interactions for all six tested combinations. These findings suggest that synergistic neuromuscular blocking effects, observed for the administration of certain combinations of muscle relaxants, do not result from purely postsynaptic binding events at the muscle nicotinic acetylcholine receptor, but rather from differential actions on pre- and postsynaptic sites.
Keywords: Acetylcholine receptor; Muscle relaxant; (Antagonist); Interaction; Isobologram; Potency; Xenopus oocyte;

Intracellular cAMP increases during the positive inotropism induced by androgens in isolated left atrium of rat by Lucı́a Velasco; Manuel Sánchez; José Manuel Rubı́n; Agustı́n Hidalgo; Carmen Bordallo; Begoña Cantabrana (45-52).
Molecular interactions of androgens with the plasma membrane may produce rapid cardiovascular effects that cannot be explained by the classic genomic mechanisms. In this sense, 5α- and 5β-dihydrotestosterone-induced an acute positive inotropic effect in isolated left atrium of rat, an effect which may be due to cAMP-dependent mechanisms. To prove this, intracellular levels of cAMP, after exposure to androgens in the organ bath, and binding to β1-adrenoceptors were evaluated. After a 4-min exposure, 5α- and 5β-dihydrotestosterone increased cAMP levels from 3.83±0.61 to 6.15±1.1 and 11.18±2.4 pmol cAMP/mg of protein, respectively. These increases were inhibited by atenolol and not modified by treatment of the rats with reserpine. The androgen-induced cAMP increase seems to be produced via an extracellular interaction, because positive inotropism and raised levels of cAMP were produced by 5α-dihydrotestosterone conjugated with bovine serum albumin (BSA). In addition, it is independent of β1-adrenoceptor activation, because neither androgen displaced [3H]dihydroalprenolol binding. Therefore, the androgens induced a positive inotropic effect via a postsynaptic effect that increases intracellular levels of cAMP. This effect is modulated by transcriptional mechanisms or by a protein with a short half-life.
Keywords: Androgen; Atrium; cAMP;

AMPA-induced Ca2+ influx in cultured rat cortical nonpyramidal neurones: pharmacological characterization using fura-2 microfluorimetry by Wolfgang Fischer; Heike Franke; Peter Scheibler; Clemens Allgaier; Peter Illes (53-62).
Immunocytochemical and Co2+ uptake studies revealed that in primary cultures of rat cortical neurones, the majority of neurones are γ-aminobutyric acid (GABA) immunopositive and can express Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors. By fura-2 microfluorimetry, it was shown that the stimulation with the selective agonist (S)-AMPA (0.3–300 μM) induced a concentration-dependent but cell-variable increase in intracellular Ca2+ concentration ([Ca2+]i) (EC50 value 7.4 μM) in more than 80% of the medium-sized multipolar neurones studied. The AMPA-induced rise in [Ca2+]i seems to be due to Ca2+ entry through AMPA receptor channels, because the response was abolished in Ca2+-free solution and by AMPA receptor selective antagonists, but was not significantly influenced by cyclopiazonic acid, an inhibitor of the endoplasmatic Ca2+-ATPase, by selective N-methyl-d-aspartic acid (NMDA) receptor antagonists, as well as the Na+ channel blocker tetrodotoxin and the majority of tested Ca2+ channel blockers. In conclusion, the results indicate that the cerebral cortical neurones in culture represent mostly GABAergic interneurone-like cells and the majority of them possess Ca2+-permeable AMPA receptors, important for intracellular signal transduction and neuronal plasticity.
Keywords: AMPA receptor; Cortical neurone; GABA (γ-aminobutyric acid) immunocytochemistry; Co2+ uptake; Fura-2 microfluorimetry; Ca2+ concentration;

Na+ channel effects of remacemide and desglycinyl-remacemide in rat cortical synaptosomes by Sarah Santangeli; Graeme J Sills; George G Thompson; Martin J Brodie (63-68).
The effects of the novel anticonvulsant, remacemide hydrochloride and its active metabolite, desglycinyl-remacemide, on veratridine-induced Na+ influx in rat cortical synaptosomes were investigated and compared to established Na+ channel blocking antiepileptic drugs. Remacemide and desglycinyl-remacemide reduced veratridine-stimulated Na+ influx to 30.7% (IC50=160.6 μM) and 13.2% (IC50=85.1 μM) of control, respectively. Carbamazepine, phenytoin and lamotrigine similarly reduced Na+ influx to 20.1% (IC50=325.9 μM), 79.8% and 27.9% (IC50=23.0 μM) of control, respectively. Resting internal Na+ concentrations were significantly increased by desglycinyl-remacemide (1 and 10 μM) and, conversely, decreased by desglycinyl-remacemide and carbamazepine (both 1000 μM). These studies support previous electrophysiological investigations, which suggest that remacemide and desglycinyl-remacemide exert their antiepileptic effects, at least in part, by an inhibitory action on voltage-gated Na+ channels. Desglycinyl-remacemide may have an additional action on Na+ homeostasis that merits further exploration.
Keywords: Remacemide; Desglycinyl-remacemide; Antiepileptic drug; Na+ channel; Synaptosome;

Inhibition of nitric oxide-dependent activation of soluble guanylyl cyclase by the antimalarial drug, artemisinin by Irina S Severina; Natalya V Pyatakova; Olga G Bussygina; Felix S Mikhailitsyn; Yuri V Khropov (69-73).
The influence of artemisinin on the activity of human platelet soluble guanylyl cyclase was investigated. Artemisinin (0.1–100 μM) had no effect on the basal activity of the enzyme. Artemisinin inhibited in a concentration-dependent manner the sodium nitroprusside-induced activation of human platelet guanylyl cyclase with an IC50 value 5.6 μM. Artemisinin (10 μM) also inhibited (by 71±4.0%) the activation of the enzyme by the thiol-dependent nitric oxide (NO) donor, the derivative of furoxan, 3,4-dicyano-1,2,5-oxadiazole 2-oxide (10 μM), but did not influence the stimulation of soluble guanylyl cyclase by protoporphyrin 1X. Inhibition of guanylyl cyclase activation by NO donors but not by protoporphyrin 1X represents a possible additional mechanism of the pharmacological action of this drug.
Keywords: Guanylyl cyclase; Nitric oxide (NO); Artemisinin;

Phencyclidine increases vesicular dopamine uptake by Michael J Crosby; Jarom E Hanson; Annette E Fleckenstein; Glen R Hanson (75-78).
Phencyclidine (PCP) rapidly (within 1 h) increased vesicular dopamine uptake and binding of the vesicular monoamine transporter-2 (VMAT-2) ligand, dihydrotetrabenazine. Uptake returned to basal values 3 h in the striatum after a high-dose administration of this drug (15 mg/kg i.p.). In contrast, a similar pretreatment with another non-competitive NMDA receptor antagonist, dizocilpine;([5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine; MK-801; 1 mg/kg, i.p.), was without effect on vesicular dopamine uptake. Pretreatment with the dopamine D2 receptor antagonist, eticlopride, blocked the increase in vesicular dopamine uptake caused by PCP administration. These data demonstrate a heretofore unreported mechanism that may contribute to the ability of PCP to influence dopamine neuronal function and exert its pharmacological effects.
Keywords: Vesicular monoamine transporter; Dopamine D2 receptor; NMDA receptor;

The effects of the nitric oxide (NO) donor, sodium nitroprusside, on l-DOPA and dopamine release from striatal tissue were evaluated using a static incubation system in which the striatal tissue released between three and six times more l-DOPA than DA, although the DA content was four times higher than that of l-DOPA. Sodium nitroprusside stimulated l-DOPA release in a time- and concentration-dependent (25, 50 and 100 μM) manner. This effect was not due to an increase in l-DOPA synthesis because sodium nitroprusside did not modify the tyrosine hydroxylase activity of striatal tissue. DA release was also stimulated by sodium nitroprusside but it required a higher concentration (500 μM) and longer incubation (60 min). Neither basal nor sodium nitroprusside-stimulated l-DOPA release was influenced by Ca2+ deprivation (EGTA 5 mM) and/or the presence of nitrendipine (1 μM), a blocker Ca2+ channel, in the incubation medium. However, cGMP (1 mM) increased l-DOPA release, and the soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ) (5 μM), partially blunted the stimulatory effect of sodium nitroprusside 100 μM. In addition, the presence of certain scavengers of free radicals, such as uric acid (300 μM) or melatonin (300 μM) but not of superoxide dismutase (1000 UI/ml) or salicylic acid (300 μM), completely blocked sodium nitroprusside (100 μM)-induced l-DOPA release. These results show that NO stimulates l-DOPA release from striatal tissue by an apparently Ca2+-independent mechanism, mediated by cGMP but also by peroxynitrite.
Keywords: c-GMP; Dopamine; l-DOPA (l-3,4-dihydroxyphenylalanine); Nitric oxide (NO); Peroxynitrite; Striatum;

Pharmacological evidence for the activation of K+ channels by diclofenac by Mario I Ortiz; Jorge E Torres-López; Gilberto Castañeda-Hernández; Rodolfo Rosas; Guadalupe C Vidal-Cantú; Vinicio Granados-Soto (85-91).
The involvement of K+ channels in the antinociceptive action of diclofenac was assessed in the formalin test. Local administration of diclofenac produced a dose-dependent antinociceptive effect due to a local action because drug administration in the contralateral paw was ineffective. Pretreatment of the injured paw with glibenclamide and tolbutamide (ATP-sensitive K+ channel inhibitors), charybdotoxin and apamin (large- and small-conductance Ca2+-activated K+ channel blockers, respectively), 4-aminopyridine or tetraethylammonium (voltage-dependent K+ channel inhibitors) prevented diclofenac-induced antinociception. Given alone, K+ channel inhibitors did not modify formalin-induced nociceptive behavior. Pinacidil (an ATP-sensitive K+ channel opener) also produced antinociception which was blocked by glibenclamide. The peripheral antinociceptive effect of morphine (positive control) was blocked by glibenclamide and 4-aminopyridine but not by charybdotoxin or apamin. The results suggest that the peripheral antinociceptive effect of diclofenac may result from the activation of several types of K+ channels, which may cause hyperpolarization of peripheral terminals of primary afferents.
Keywords: Diclofenac; Apamin; Charybdotoxin; Glibenclamide; Pinacidil; Morphine;

Tacrolimus, a specific inhibitor of calcineurin, modifies the locomotor activity of quinpirole, but not that of SKF82958, in male rats by Masatsuna Sakanoue; Norio Mori; Nori Takei; Masayoshi Kawai; Kunihiko Tani; Katsuaki Suzuki; Yasuhide Iwata; Yoshimoto Sekine; Charles R Ashby; Yoshio Minabe (93-97).
In the present study, we examined the effect of tacrolimus, a specific inhibitor of calcineurin, on the locomotor activity elicited by the selective dopamine D1 receptor agonist (±) 6-chloro-7,8-dyhydroxy-3allyl-1-phenyl-2,3,4,5-tetra-hydro-1H-benzazepine (SKF82958) and the dopamine D2/D3 receptor agonist quinpirole, in male Wistar rats. Tacrolimus (0.5, 1, 2 or 5 mg/kg, i.p.) alone had no significant effect on basal locomotor activity. The dose-related increase in locomotor activity produced by the administration of SKF82958 (0.1, 1 or 5 mg/kg, i.p.) was not significantly altered by 2 mg/kg of tacrolimus. In addition, the increase in locomotor activity produced by 1 mg/kg of SKF82958 was not significantly altered by tacrolimus (0.5, 1, 2 or 5 mg/kg, i.p.). The administration of quinpirole (0.1, 0.25, 0.5, 1 or 3 mg/kg, i.p.) produced a biphasic response, with the minimum and maximal increase in locomotor activity occurring at 0.1 and 1 mg/kg, respectively. The pretreatment of 2 mg/kg of tacrolimus, compared to vehicle-treated animals, significantly lowered the dose of quinpirole that produce a maximal effect on locomotor activity from 1 to 0.5 mg/kg but did not significantly alter the minimum response. The increase in locomotor activity produced by 0.5 mg/kg of quinpirole was significantly potentiated by 0.5, 1, 2 or 5 mg/kg of tacrolimus compared to vehicle-treated animals. Our results suggest that calcineurin may play a role in the alteration of locomotor activity produced by dopamine D2/D3 receptors, but not dopamine D1 receptors.
Keywords: Tacrolimus; Calcineurin; Dopamine D1 receptor; Dopamine D2 receptor; Dopamine D3 receptor; Locomotor activity;

Antagonism of α3β4 nicotinic receptors as a strategy to reduce opioid and stimulant self-administration by Stanley D Glick; Isabelle M Maisonneuve; Barbara A Kitchen; Mark W Fleck (99-105).
The iboga alkaloid ibogaine and the novel iboga alkaloid congener 18-methoxycoronaridine are putative anti-addictive agents. Using patch–clamp methodology, the actions of ibogaine and 18-methoxycoronaridine at various neurotransmitter receptor ion-channel subtypes were determined. Both ibogaine and 18-methoxycoronaridine were antagonists at α3β4 nicotinic receptors and both agents were more potent at this site than at α4β2 nicotinic receptors or at NMDA or 5-HT3 receptors; 18-methoxycoronaridine was more selective in this regard than ibogaine. In studies of morphine and methamphetamine self-administration, the effects of low dose combinations of 18-methoxycoronaridine with mecamylamine or dextromethorphan and of mecamylamine with dextromethorphan were assessed. Mecamylamine and dextromethorphan have also been shown to be antagonists at α3β4 nicotinic receptors. All three drug combinations decreased both morphine and methamphetamine self-administration at doses that were ineffective if administered alone. The data are consistent with the hypothesis that antagonism at α3β4 receptors is a potential mechanism to modulate drug seeking behavior. 18-Methoxycoronaridine apparently has greater selectivity for this site than other agents and may be the first of a new class of synthetic agents acting via this novel mechanism to produce a broad spectrum of anti-addictive activity.
Keywords: 18-Methoxycoronaridine; Dextromethorphan; Mecamylamine; Morphine; Methamphetamine; Nicotine receptor; Drug addiction;

We examined the effects of γ-aminobutyric acid (GABA) on the blood pressure in spontaneously hypertensive rats and normotensive Wistar–Kyoto rats. A single oral administration (0.5 mg/kg) significantly lowered the systolic blood pressure in spontaneously hypertensive rats, but not in normotensive rats. In the mesenteric arterial bed, the perivascular nerve stimulation-induced increase in perfusion pressure and noradrenaline release were significantly inhibited by GABA in spontaneously hypertensive rats, but not in normotensive rats, and attenuated by the selective GABAB receptor agonist, baclofen, but not by the selective GABAA receptor agonist muscimol. The inhibitory effects of GABA on the perivascular nerve stimulation-induced increase in perfusion pressure and noradrenaline release were completely antagonized by the selective GABAB receptor antagonist, saclofen, but not by the selective GABAA receptor antagonist, bicuculline. These results suggest that, in spontaneously hypertensive rats, GABA has an antihypertensive effect due to its inhibition of noradrenaline release from sympathetic nerves in the mesenteric arterial bed via presynaptic GABAB receptors.
Keywords: GABA (γ-Aminobutyric acid); Noradrenaline; Blood pressure; Mesenteric arterial bed; Perivascular nerve stimulation; Spontaneously hypertensive rat (SHR);

We have previously shown that coupling bath application of dopamine with 50 Hz tetani induces long-term depression in rat prefrontal slices [Neuroscience 85 (1998) 669]. Here, we report a reliable protocol for inducing long-term potentiation in the same preparation. Long-term potentiation was induced by the same dopamine–tetani coupling protocol when the coupling was preceded (∼30 min) by a single bath application of dopamine. We suggest that metaplastic processes triggered by the first application of dopamine might underlie the LTP induction.
Keywords: Long-term potentiation; Dopamine; Cortex;