European Journal of Pharmacology (v.431, #3)

The effects of a volatile anesthetic, isoflurane, on glycinergic miniature inhibitory postsynaptic currents (IPSCs) were investigated in mechanically dissociated rat trigeminal nucleus neurons with intact glycinergic interneuronal presynaptic nerve terminals. The nystatin-perforated patch recording configuration was used to record the miniature IPSCs under voltage-clamp conditions. Isoflurane shifted in a parallel fashion the glycine (Gly) concentration–response curve of enzymatically dissociated neurons to the left without changing the maximum response. Isoflurane reversibly increased the frequency of the miniature IPSCs and prolonged the decay time constant without affecting the mean amplitude. The increase in the frequency of miniature IPSCs in the presence of isoflurane was also observed in Ca2+-free external solution. Thapsigargin prohibited the facilitatory effect of isoflurane on the miniature IPSC frequency. It is concluded that isoflurane increases the Ca2+ concentration in the glycinergic presynaptic nerve terminal by enhancing the release and/or suppressing the uptake of Ca2+ into stores.
Keywords: Trigeminal nucleus neuron; Mechanical dissociation; Isoflurane; Glycinergic synaptic bouton preparation; Inhibitory postsynaptic current;

Cannabinoid CB1 receptor expression, activation and detection of endogenous ligand in trabecular meshwork and ciliary process tissues by W.D Stamer; S.F Golightly; Y Hosohata; E.P Ryan; A.C Porter; E Varga; R.J Noecker; C.C Felder; H.I Yamamura (277-286).
Elevated intraocular pressure is the primary risk factor for glaucoma. Cannabinoids interact with molecular targets in the eye and lower intraocular pressure by an unknown mechanism. The purpose of the present study was to examine eye tissues for functional cannabinoid receptors of the neuronal, CB1 class, and an endogenous ligand, anandamide. The trabecular meshwork and ciliary processes are the primary structures of the eye that contribute to intraocular pressure and thus were our focus. Total RNA, frozen sections, cellular membranes and primary cultures of cells were prepared from both bovine and cadaveric human tissues. Using cannabinoid CB1 receptor-specific oligodeoxynucleotide primers, cannabinoid CB1 receptor antiserum, and cannabinoid-specific compounds (CP-55,940, WIN55,212-2 and SR-141716A), the presence of cannabinoid CB1 receptors in ciliary processes and trabecular meshwork was determined. Using reverse transcription-polymerase chain reaction, we identified mRNA encoding cannabinoid CB1 receptor protein in ciliary process and trabecular meshwork cells. Specific binding of anti-CB1 immunoglobulin-G in tissue sections localized cannabinoid CB1 receptor protein to the non-pigmented epithelial cells of the ciliary process and cells of the trabecular meshwork. While CP-55,940 and WIN55,212-2 failed to stimulate [35S]GTPγS binding in membrane preparations from trabecular meshwork and ciliary process, CP-55,940 significantly stimulated whole cell [35S]GTPγS binding by 51% over basal in ciliary process epithelial cells and 69% over basal in trabecular meshwork cells permeabilized with 5 μM digitonin (p<0.001). Specificity of agonist stimulation was verified by complete blockade with the specific cannabinoid CB1 receptor antagonist, SR-141716A. Moreover, activation of cannabinoid CB1 receptors by CP-55,940 resulted in a 2.3±0.3 and 1.7±0.3-fold stimulation of cAMP accumulation in trabecular meshwork and ciliary process cells, respectively (p<0.01). Lastly, anandamide was detected in human trabecular meshwork (3.08 pmol/g), ciliary process (49.42 pmol/g) and neurosensory retinal (4.48 pmol/g) tissues. These data, for the first time, demonstrate in a single study the presence of both CB1 mRNA and protein in trabecular meshwork and ciliary processes from two different species. Activation of heterotrimeric G-proteins and stimulation of cAMP accumulation by cannabinoids in vitro suggest that their intraocular pressure-lowering effects in vivo result from activation of cannabinoid CB1 receptors in the trabecular meshwork and increase aqueous outflow.
Keywords: Anandamide; cAMP; G-protein; Glaucoma; GTPγS; Intraocular pressure;

Effects and mechanisms of aloe-emodin on cell death in human lung squamous cell carcinoma by Hong-Zin Lee; Shih-Lan Hsu; Mei-Chun Liu; Chun-Hsiung Wu (287-295).
Aloe-emodin (1,8-dihydroxy-3-(hydroxymethyl)-anthraquinone) is an active component from the root and rhizome of Rheum palmatum. The study investigated the effects and mechanisms of aloe-emodin-induced cell death in human lung squamous cell carcinoma cell line CH27. Aloe-emodin (40 μM)-induced CH27 cell apoptosis was confirmed by DNA fragmentation (DNA ladders and sub-G1 formation). Aloe-emodin-induced apoptosis of CH27 cells involved modulation of the expression of Bcl-2 family proteins, such as BclXL, Bag-1, and Bak, and was associated with the translocation of Bak and Bax from cytosolic to particulate fractions. Aloe-emodin-treated CH27 cells had an increased relative abundance of cytochrome c in the cytosolic fraction. Results demonstrated that the activation of caspase-3, caspase-8, and caspase-9 is an important determinant of apoptotic death induced by aloe-emodin. These results suggest that aloe-emodin induces CH27 cell death by the Bax and Fas death pathway.
Keywords: Aloe-emodin (1,8-dihydroxy-3-(hydroxymethyl)-anthraquinone); Human lung squamous cell carcinoma cell line CH27; Apoptosis; Bcl-2 family protein; Cytochrome c; Caspase;

Transport characteristics of grepafloxacin and levofloxacin in the human intestinal cell line Caco-2 by Hiroaki Yamaguchi; Ikuko Yano; Hideyuki Saito; Ken-ichi Inui (297-303).
Transport characteristics of grepafloxacin and levofloxacin across the apical membrane of Caco-2 cells were examined. Both grepafloxacin and levofloxacin uptakes increased rapidly, and were temperature-dependent. Grepafloxacin and levofloxacin uptakes showed concentration-dependent saturation with Michaelis constants of 3.9 and 9.3 mM, respectively. Uptake of grepafloxacin and levofloxacin increased in Cl-free and ATP depleted conditions, suggesting the involvement of an efflux transport system different from the uptake mechanism. However, cyclosporin A, a typical inhibitor of P-glycoprotein, did not affect the uptake of these drugs. Unlabeled grepafloxacin, unlabeled levofloxacin and quinidine inhibited the uptake of grepafloxacin and levofloxacin under Cl-free conditions. Tetraethylammonium, cimetidine, p-aminohippurate, probenecid, amino acids, β-lactam antibiotic or monocarboxylates did not inhibit the uptake of grepafloxacin and levofloxacin under the same conditions. In conclusion, our results suggested that grepafloxacin and levofloxacin uptakes were mediated by a specific transport system distinct from those for organic cations and anions, amino acids, dipeptides and monocarboxylates.
Keywords: Intestinal transport; Grepafloxacin; Levofloxacin; Caco-2 cell;

Regional mapping of low-affinity kainate receptors in mouse brain using [3H](2S,4R)-4-methylglutamate autoradiography by Alexis Bailey; Eve E Kelland; Angharrad Thomas; James Biggs; Duncan Crawford; Ian Kitchen; Nick J Toms (305-310).
Recent data indicate that (2S,4R)-4-methylglutamate is a selective agonist for low affinity (GluR5 and GluR6) kainate receptor subunits. In the present study, we have employed [3H](2S,4R)-4-methylglutamate to examine low affinity kainate receptor distribution in mouse brain. [3H](2S,4R)-4-Methylglutamate labelled a single site in murine cerebrocortical membranes (K d=9.9±2.7 nM, B max=296.3±27.1 fmol mg protein−1). The binding of 8 nM [3H](2S,4R)-4-methylglutamate was displaced by several non-NMDA receptor ligands (K i±S.E.M.): domoate (1.1±0.2 nM)>kainate (7.1±1.1 nM)≫l-glutamate (187.6±31.9 nM)≫(S)-α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) (>50 μM). [3H](2S,4R)-4-Methylglutamate autoradiography revealed a widespread regional distribution of low affinity kainate receptors. Highest binding densities occurred within deep layers of the cerebral cortex, olfactory bulb, basolateral amygdala and hippocampal CA3 subregion. Moderate labelling was also evident in the nucleus accumbens, dentate gyrus, caudate putamen, hypothalamus and cerebellar granule cell layer. These data show that [3H](2S,4R)-4-methylglutamate is a useful radioligand for selectively labelling low affinity kainate receptors.
Keywords: Kainate receptor; AMPA receptor; Radioligand binding; BALB/c;

Agonists of proteinase-activated receptor 2 excite guinea pig ileal myenteric neurons by David R Linden; Brian P Manning; Nigel W Bunnett; Gary M Mawe (311-314).
The effects of proteinase-activated receptor 2 (PAR2) agonists on the electrical properties of intact guinea pig ileal myenteric neurons were measured with intracellular microelectrodes. Approximately 52% of AH neurons and 41% of S neurons responded to pressure ejection of SLIGRL-NH2 or trypsin with a prolonged depolarization that was often accompanied by increased excitability. When added to the bathing solution, trypsin caused a concentration-dependent depolarization of responding neurons with an estimated EC50 value of 87 nM. Collectively, these novel observations indicate that PAR2 excites a proportion of myenteric neurons, which may contribute to dysmotility during intestinal inflammation.
Keywords: Euteric nervous system; Neuroimmune interaction; Electrophysiology; Thrombin receptor; Trypsin;

Effects of ketamine on different types of anxiety/fear and related memory in rats with lesions of the median raphe nucleus by Emine Babar; Tuncay Özgünen; Enver Melik; Sait Polat; Hasan Akman (315-320).
The aim of the present study was to determine the involvement of the median raphe serotonergic system in the effects of ketamine on anxiety behaviours and related memory. The effects of ketamine pretreatment (3 and 10 mg/kg, i.p.) on three types of fear-motivated behaviours, unconditioned one-way escape, conditioned avoidance and freezing were tested. Experiments were performed with the inhibitory avoidance apparatus in rats with ibotenic acid lesions of the median raphe nucleus. It was found that 10 mg/kg ketamine had an anxiogenic-like effect on one-way escape type of fear and anxiolytic-like effect on conditioned freezing-related fear; these effects were unaffected by median raphe lesions. Both ketamine doses impaired freezing-related fear memory. Ketamine (10 mg/kg) also produced an anxiolytic-like effect on avoidance type of fear and impaired avoidance memory. The median raphe lesions attenuated the anxiolytic action of the drug on the avoidance type of fear and prevented ketamine-induced avoidance memory impairment. These results suggest that the anxiolytic-like effect of ketamine on avoidance-type fear is mediated through the median raphe serotonergic system.
Keywords: Ketamine; Median raphe nucleus; 5-HT (5-hydroxytryptamine, serotonin); Anxiety; Fear; Memory;

Renal vascular reactivity to vasopressin in rats with diabetes mellitus by Cécile Loichot; Jérôme Anjuère; Dino Nisato; Wybren De Jong; Jean-Louis Imbs; Mariette Barthelmebs (321-329).
We evaluated how renal vascular reactivity to vasopressin changes when nitric oxide (NO) synthesis varies, as has been reported to occur in the course of insulin-dependent diabetes mellitus. Renal vasoconstrictor responses to vasopressin were obtained in young and older Sprague–Dawley control rats (3 and 10 months old) and in age-matched diabetic rats that had been treated with streptozotocin (60 mg/kg i.v.) at the age of 2 months. In young rats, vasopressin (3–1000 ng/kg/min i.v.) induced in vivo a dose-dependent decrease in renal blood flow, which was diminished in streptozotocin diabetic rats (P<0.05). Similarly, in in vitro perfused kidneys, the concentration–response curve for vasopressin (0.03–10 nM) was shifted 3-fold to the right in kidneys isolated from young diabetic rats (P<0.05). This shift was abolished in the presence of an inhibitor of nitric oxide synthesis, N G-nitro-l-arginine (100 μM), in the perfusate. In 10-month-old rats, the in vivo renal vasoconstrictor dose–response curve to vasopressin was shifted 10-fold to the left as compared to that for young rats (P<0.001). This shift was similar in both control and diabetic rats.In conclusion, the present study documented the existence of hyporesponsiveness to vasopressin in the early stage of diabetes, possibly related to nitric oxide overproduction. In contrast, renal vascular hyperreactivity to vasopressin occurs with aging, whether the rats are diabetic or not.
Keywords: Vasopressin; Renal vascular reactivity; Diabetes; Age; Nitric oxide (NO);

Absence of exacerbation of myocardial stunning in anesthetized dogs treated with KAD-1229, a novel hypoglycemic agent by Kiyoshi Ichikawa; Kazuyasu Maruyama; Makoto Murakami; Atsutoshi Tsuji; Tokuhisa Yamato; Hiroshi Kusama; Masami Kojima (331-338).
The effect of (+)-momocalcium bis[(2S,3a,7a-cis)-α-benzylhexahydro-γ-oxo-2-isoindolinebutyrate]dihydrate (KAD-1229), a novel hypoglycemic agent with a chemical structure different from that of the sulfonylureas, on myocardial stunning was assessed in anesthetized dogs by comparison with that of glibenclamide, a sulfonylurea. Even though their hypoglycemic effects were of similar magnitude, glibenclamide (1 mg/kg, i.v.), but not KAD-1229, exacerbated the myocardial stunning induced by occlusion/reperfusion of the descending coronary artery. In a receptor-binding experiment, unlabeled glibenclamide completely inhibited [3H]glibenclamide binding to the myocardium, but KAD-1229 did not. These results suggest that the difference in binding properties of KAD-1229 and glibenclamide toward cardiac sulfonylurea receptors is one of the causes of their different effects on myocardial stunning. It is likely that KAD-1229 is highly specific for pancreatic sulfonylurea receptors and is speculated to be a safer hypoglycemic agent than, at least, glibenclamide.
Keywords: KAD-1229; Glibenclamide; Myocardial stunning; Sulfonylurea receptor; Diabetes mellitus;

Effects of GABAB receptor antagonists on spontaneous and on GABA-induced mechanical activity of guinea-pig smooth muscle preparations by Atanas D Kristev; Damianka P Getova; Vassil A Spassov; Valentin I Turiiski (339-344).
The majority of GABAB receptor antagonists have been based on alterations of the acidic moiety of γ-aminobutyric acid (GABA) or baclofen, such as the first selective antagonist phaclofen. More recently, a new structural class of compounds derived by p-alkyl substitution in the phosphinic analog of GABA, such as CGP35348 (3-amino-propyl-(diethoxymethyl)-phosphinic acid), have been introduced as GABAB receptor antagonists. The present study examine the influence of a series of structurally related phosphinic acid analogues on mechanical activity and their effect on GABA-induced reactions in ileal smooth muscle. In our experiments, GABA exerted a biphasic contractile-relaxation effect with pronounced dose-dependent characteristics. 3-[[1-(S)-(3,4-Dihydrophenyl) ethyl]amino]-2-(S)-hydroxy-propyl]-(phenylmethyl)-phosphinic acid hydrochloride (CGP55845A) induced prolonged relaxation without changing the phasic activity of the ileum preparations. [3-[1-R-[[2-(S)-hydroxy-3-[hydroxy-4-methoxyphenyl]-methyl]-phosphinyl]-propyl]-aminoethyl]-benzoic acid (CGP62349) did not change the mechanical activity of smooth muscle preparation. Trans 3-[6-[[Cyclo hexylmethyl-hydroxy-phosphinyl]-methyl]-3-morpholinyl]-benzoic acid (CGP71982) itself induced smooth muscle contractions. GABAB receptor antagonists decreased concentration-dependently the relaxation phase of the action of GABA from 50% to 90%. Their effect on the contractile phase of the action of GABA was quite different—CGP55845A decreased it dose-dependently, whereas CGP62349 and CGP71982 did not change it significantly. These findings prompted us to assume that the GABAB receptor antagonists studied, being phosphinic analogues, probably act on GABAB receptors in guinea-pig ileum smooth muscles.
Keywords: GABAB receptor antagonist; GABA (γ-aminobutyric acid); Ileum; (Guinea pig);

Squalene synthase inhibitors reduce plasma triglyceride through a low-density lipoprotein receptor-independent mechanism by Hironobu Hiyoshi; Mamoru Yanagimachi; Masashi Ito; Takao Saeki; Ichiro Yoshida; Toshimi Okada; Hironori Ikuta; Daisuke Shinmyo; Keigo Tanaka; Nobuyuki Kurusu; Hiroshi Tanaka (345-352).
Inhibitors of squalene synthase are considered to be candidate drugs to reduce both plasma cholesterol and triglyceride. However, little is known about the mechanism of squalene synthase inhibitor-specific effect on plasma triglyceride. In this study, we confirmed the triglyceride-lowering effect of ER-27856, a potent squalene synthase inhibitor prodrug, in rhesus monkeys. To determine the role of low-density lipoprotein (LDL) receptor in the triglyceride-lowering effect of squalene synthase inhibitors, we intravenously administered ER-28448, the active form of ER-27856, to Watanabe heritable hyperlipidemic (WHHL) rabbits for 4 days. In heterozygotes, ER-28448 reduced plasma cholesterol and triglyceride by 52% and 37%, respectively. In homozygous rabbits, in contrast, ER-28448 lowered plasma triglyceride by 40% but did not lower plasma cholesterol. Orally administered ER-27856 reduced plasma triglyceride in homozygous animals but atorvastatin and bezafibrate did not. In hepatocytes isolated from homozygous WHHL rabbits, squalene synthase inhibitors but not atorvastatin reduced triglyceride biosynthesis. These data demonstrate that squalene synthase inhibitors reduced plasma triglyceride through an LDL receptor-independent mechanism, which was distinct from that of the triglyceride-lowering action of atorvastatin or bezafibrate. The reduction of hepatic triglyceride biosynthesis may play an important role in the hypotrigyceridemic action of squalene synthase inhibitors.
Keywords: Squalene synthase inhibitor; Triglyceride; Low-density lipoprotein (LDL) receptor; WHHL; Hypertriglyceridemia;

In the present study, we investigated the effect of fenoterol-induced constitutive β2-adrenoceptor activity on muscarinic receptor agonist- and histamine-induced bovine tracheal smooth muscle contractions. Bovine tracheal smooth muscle strips were incubated with 10 μM fenoterol or vehicle for various periods of time (5, 30 min, 18 h) at 37 °C. After extensive washout (3 h, 37 °C), isometric contractions were measured to the full muscarinic receptor agonist methacholine, the partial muscarinic receptor agonist 4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium (McN-A-343) and histamine. Fenoterol treatment significantly reduced the sensitivity (pEC50) to methacholine in a time-dependent manner, without affecting maximal contraction (E max). Fenoterol treatment similarly reduced the pEC50 of McN-A-343 and histamine; however, E max values were also reduced, to approximately 70% of control after 18-h treatment. The inverse agonist timolol, having no effect on control preparations, consistently restored the reduced pEC50 and E max values of the contractile agonists. Remarkably, in the presence of timolol the pEC50 values of McN-A-343 and histamine in fenoterol-treated airways were significantly enhanced compared to controls. In conclusion, fenoterol-induced constitutive β2-adrenoceptor activity reduces muscarinic receptor agonist- and histamine-induced contractions of bovine tracheal smooth muscle, which can be reversed by the inverse agonist timolol. Moreover, after β2-adrenoceptor agonist treatment, inverse agonism by β-adrenoceptor antagonists may cause enhanced airway reactivity to contractile mediators.
Keywords: β2-Adrenoceptor activity; Fenoterol; Smooth muscle; Airway reactivity;

The involvement of a central mechanism in the antiemetic effect of AS-8112 ((R)-5-bromo-N-(1-ethyl-4-methylhexahydro-1H-1,4-diazepin-6-yl)-2-methoxy-6-methylamino-3-pyridinecarboxamide·2 fumarate), a novel and potent dopamine D2, D3, and 5-HT3 receptor antagonist, was investigated in ferrets. Intracerebroventricularly administered AS-8112 dose dependently inhibited R(+)-7-OH-DPAT (R(+)-7-hydroxy-2-(N,N-di-n-propylamino) tetraline)-induced emesis (ID50; 0.11 μg/kg, i.c.v.). In addition, AS-8112 (10 μg/kg, i.c.v.) significantly inhibited emesis induced by cisplatin. Ondansetron (10 μg/kg, i.c.v.) also inhibited cisplatin-induced emesis, but did not inhibit R(+)-7-OH-DPAT-induced emesis. S(−)-eticlopride (10 μg/kg, i.c.v.) did not inhibit emesis induced by cisplatin. However, racemic CP-99,994 ((±)-(2S, 3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine) (10 μg/kg, i.c.v.) inhibited both cisplatin- and R(+)-7-OH-DPAT-induced emesis. These results suggest that the antiemetic effects of AS-8112 are centrally mediated via dopamine D3 and 5-HT3 receptors in ferrets.
Keywords: AS-8112 ((R)-5-bromo-N-(1-ethyl-4-methylhexahydro-1H-1,4-diazepin-6-yl)-2-methoxy-6-methylamino-3-pyridinecarboxamide·2 fumarate); R(+)-7-OH-DPAT (R(+)-7-hydroxy-2-(N,N-di-n-propylamino)tetraline); Cisplatin; Emesis; Area postrema; (Ferret);

Beclomethasone, budesonide and fluticasone propionate inhibit human neutrophil apoptosis by Xianzhi Zhang; Eeva Moilanen; Hannu Kankaanranta (365-371).
Inhaled glucocorticoids are widely used to treat chronic obstructive pulmonary disease without much evidence of efficiency in this disease where neutrophils may contribute to the pathophysiology. This prompted us to test the effects of several currently used inhaled and systemic glucocorticoids on human neutrophil apoptosis. Beclomethasone, budesonide, dexamethasone, fluticasone propionate, hydrocortisone and prednisolone inhibited apoptosis in a concentration-dependent manner as assessed by flow cytometric analysis, annexin-V binding and morphological analysis. The maximal inhibition of apoptosis was 50–60%. The order of potency was fluticasone propionate (EC50 0.6±0.2 nM)≈budesonide (EC50 0.8±0.2 nM)>dexamethasone≈prednisolone≈beclomethasone≈hydrocortisone. The inhibitory effects of glucocorticoids were reversed by mifepristone. Moreover, glucocorticoids slightly enhanced the inhibitory effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on neutrophil apoptosis. The present data suggests that budesonide and fluticasone propionate prolong human neutrophil survival by inhibiting apoptosis at clinically relevant drug concentrations via an effect on glucocorticoid receptor.
Keywords: Apoptosis; Neutrophil; Fluticasone propionate; Budesonide; Beclomethasone;

Author index (373-375).

Keyword index (377-384).