European Journal of Pharmacology (v.419, #1)

Functional pharmacology of GABAA receptors containing the chicken brain γ4 subunit by Ian C. Forster; Robert J. Harvey; Mark G. Darlison; Jack A. Benson (1-7).
The functional pharmacology of receptors composed of the chicken brain GABAA receptor γ4 subunit and the mammalian GABAA receptor α3 and β2 subunits was studied by heterologous expression in Xenopus laevis oocytes using the two electrode voltage-clamp technique. GABA-evoked currents had an EC50 of 180±30 μM. Responses were blocked by the competitive and non-competitive GABAA receptor antagonists, bicuculline methochloride and picrotoxin. Sodium pentobarbital reversibly potentiated the current several-fold, and Zn2+ ions blocked the current with high potency (IC50=20 μM). GABA-evoked currents were potentiated by the benzodiazepine site full agonists flunitrazepam and triazolam and less by the partial agonists abecarnil and bretazenil. The inverse agonists methyl-β-carboline-3-carboxylate (β-CCM) and methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) reduced the current. However, the imidazobenzodiazepine Ro 15-4513, which acts as an inverse agonist at mammalian αxβyγ2 GABAA receptors (where x=1, 2, 3 or 5, and y=1, 2 or 3), acted as a positive agonist at the γ4 subunit-containing receptors.
Keywords: Benzodiazepine pharmacology; Electrophysiology; GABAA receptor; Xenopus oocyte; Zn2+ inhibition;

Protein kinase C-α1b-adrenoceptor coimmunoprecipitation: effect of hormones and phorbol myristate acetate by Rocı́o Alcántara-Hernández; Dinorah Leyva-Illades; J.Adolfo Garcı́a-Sáinz (9-13).
α1b-Adrenoceptors immunoprecipitated with protein kinase C α, δ, and ε isoforms under basal conditions and such coimmunoprecipitations were increased in cells treated with phorbol myristate acetate. The increased coimmunoprecipitations induced by phorbol myristate acetate were concentration-dependent and reached their maxima 1 to 2 min after the addition of the tumor promoter. No coimmunoprecipitation of protein kinase C ζ and α1b-adrenoceptors was detected. Norepinephrine, endothelin-1, lysophosphatidic acid and epidermal growth factor were also able to increase the coimmunoprecipitation of protein kinase C isoenzymes and α1b-adrenoceptors. These data support the idea that protein kinase-receptor complexes might form and could be relevant in receptor desensitization.
Keywords: α1-Adrenoceptor; Protein kinase C; Desensitization;

Pharmacological characterization of the dermorphin analog [Dmt1]DALDA, a highly potent and selective μ-opioid peptide by Claire L Neilan; Thi M.-D Nguyen; Peter W Schiller; Gavril W Pasternak (15-23).
The dermorphin-derived peptide [Dmt1]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2), labels μ-opioid receptors with high affinity and selectivity in receptor binding assays. In mouse, radiant heat tail-flick assay [Dmt1]DALDA produced profound spinal and supraspinal analgesia, being approximately 5000- and 100-fold more potent than morphine on a molar basis, respectively. When administered systemically, [Dmt1]DALDA was over 200-fold more potent than morphine. Pharmacologically, [Dmt1]DALDA was distinct from morphine. [Dmt1]DALDA displayed no cross-tolerance to morphine in the model used and it retained supraspinal analgesic activity in morphine-insensitive CXBK mice. Supraspinally, it also differed from morphine in its lack of sensitivity towards naloxonazine. Finally, in antisense mapping studies, [Dmt1]DALDA was insensitive to MOR-1 exon probes that reduced morphine analgesia, implying a distinct receptor mechanism of action. Thus, [Dmt1]DALDA is an interesting and extraordinarily potent, systemically active peptide analgesic, raising the possibility of novel approaches in the design of clinically useful drugs.
Keywords: μ-Opioid peptide; Analgesia; Tail-flick assay; Radioligand binding; Splice variant; Enkephalin; Opioid; μ-Opioid receptor;

Antinociceptive effects of intracerebroventricularly administered P2 purinoceptor agonists in the rat by Masato Fukui; Takayuki Nakagawa; Masabumi Minami; Masamichi Satoh (25-31).
We examined the effects of adenosine 5′-triphosphate (ATP) and its analogues administered intracerebroventricularly on nociceptive thresholds in rats. Intracerebroventricular (i.c.v.) administration of ATP (10 and 100 nmol/rat), α,β-methylene-ATP (1–30 nmol/rat) and 2′, 3′-O-(4-benzoylbenzoyl)-ATP (1–30 nmol/rat) dose-dependently elevated the mechanical nociceptive threshold in the paw pressure test. These antinociceptive effects were rapid and short-lasting, peaking at 5 min and disappearing by 20 min after the administration. However, i.c.v. administration of β,γ-methylene-ATP (1–30 nmol/rat) and UTP (10 and 100 nmol/rat) had no significant effects on the mechanical nociceptive threshold. In other tests, i.c.v. administration of α,β-methylene-ATP (10 and 30 nmol/rat) prolonged the thermal nociceptive latency in the hot plate test, but only a higher dose (30 nmol/rat) of α,β-methylene-ATP prolonged the latency in the tail flick test. α,β-Methylene-ATP produced no motor deficit in the inclined plane test. These results suggest that P2X purinoceptors play an inhibitory role in nociception at the supraspinal level.
Keywords: ATP; α,β-Methylene-ATP; Antinociception; Purinoceptor;

C-Terminal glycine is crucial for hyperalgesic activity of nociceptin/orphanin FQ-(1–6) by Jolanta Kotlińska; Piotr Suder; Agnieszka Sciubisz; Anna Łęgowska; Julita Eilmes; Krzysztof Rolka; Jerzy Silberring (33-37).
A C-terminal analog of the hexapeptide orphanin FQ/nociceptin-(1–6), [Ala6]-orphanin FQ/nociceptin-(1–6), and a pentapeptide orphanin FQ/nociceptin-(1–5) were tested in vivo for their analgesic/hyperalgesic activity in the hot-plate test with rats. Replacement of the C-terminal glycine by l-alanine (Phe-Gly-Gly-Phe-Thr-Ala) in orphanin FQ/nociceptin-(1–6) abolished the hyperalgesic potency of native orphanin FQ/nociceptin-(1–6) (Phe-Gly-Gly-Phe-Thr-Gly), but analgesic activity was retained and was diminished by naloxone. Removal of the C-terminal amino acid (glycine or alanine) from orphanin FQ/nociceptin-(1–6) caused a significant loss of analgesic activity. It is anticipated that glycine plays a crucial role in the biphasic activity of orphanin FQ/nociceptin-(1–6). This may suggest the existence of a mechanism for terminating the biological action of orphanin FQ/nociceptin.
Keywords: Orphanin FQ/nociceptin; Metabolism; Glycine; Opioid receptor; Pain;

Inhibition of morphine tolerance and dependence by MS-153, a glutamate transporter activator by Takayuki Nakagawa; Tohru Ozawa; Kaori Shige; Rie Yamamoto; Masabumi Minami; Masamichi Satoh (39-45).
We investigated the effects of (R)-(−)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), which is reported to accelerate glutamate uptake, on the development of morphine tolerance and physical dependence in mice. For the induction of morphine tolerance and dependence, mice were twice daily treated with morphine (10–45 mg/kg, s.c.) for 5 days. First, co-administration of MS-153 (12.5 mg/kg, s.c.) did not affect the morphine's potency for its acute antinociceptive effect (1 and 3 mg/kg, s.c.). Next, co-administrations of MS-153 (1, 3 and 12.5 mg/kg, s.c.) during repeated morphine treatments significantly attenuated the development of tolerance to the antinociceptive effect of morphine (3 mg/kg, s.c.) and suppressed the naloxone (10 mg/kg, i.p.)-precipitated withdrawal signs (jumps and body weight loss). The inhibitory effect of MS-153 on the withdrawal signs was due to the attenuation of the development of dependence rather than that of expression of withdrawal signs. These results suggest that MS-153, a glutamate transporter activator, has an inhibitory effect on the development of morphine tolerance and physical dependence.
Keywords: MS-153; Morphine tolerance; Morphine dependence; Glutamate; Glutamate transporter;

Effect of tibolone and raloxifene on the tail temperature of oestrogen-deficient rats by Hemmie H.G. Berendsen; Albert H.J. Weekers; Helenius J. Kloosterboer (47-54).
Oestradiol, clonidine, tibolone and raloxifene were tested for their effects on the tail temperature of oestrogen deficient rats, a potential new model that can be used to test compounds that may be of use in the treatment of hot flushes in humans. Rats underwent ovariectomies or sham operations and their tail temperature and physical activity were measured telemetrically. Oestrogen depletion affected tail temperature in the rats' active, but not their resting phase. During the transition from the resting to the active phase, tail temperature in normal rats dropped by about 6°C, but only by approximately 1°C after ovariectomy. Treatment of the ovariectomised rats with oestrogen, clonidine or tibolone dose-dependently restored the drop in tail temperature. However, raloxifene did not change the tail temperature of ovariectomised rats. Thus, tibolone and raloxifene have different effects on the temperature regulation in the tail. This method of measuring tail temperature free of stress in ovariectomised rats may serve as a useful procedure for selecting compounds that are of potential use in the treatment of hot flushes.
Keywords: Menopause; Hot flush; Tail temperature; Telemetry; Tibolone; Raloxifene;

Histamine H3 receptor-mediated inhibition of sympathetically evoked mydriasis in rats by Yongxin Yu; Masahiro Kawarai; Michael C Koss (55-59).
This study was designed to determine if the histamine H3 receptor agonist R-α-methylhistamine would play a role in modulation of sympathetically evoked mydriasis in anesthetized rats, and if so, to ascertain the specific receptor subtype(s) involved. Reproducible frequency–response curves of pupillary dilation were generated by stimulation of the cervical preganglionic sympathetic nerve (1–32 Hz). Systemic administration of R-α-methylhistamine (0.3–3.0 mg kg−1) produced a dose-related inhibition of the evoked mydriasis. The greatest inhibition was seen at lower frequency levels, with about 43% depression observed at 2 Hz. The specific histamine H3 receptor antagonist, clobenpropit (3.0 mg kg−1, i.v.), blocked the inhibitory effect of R-α-methylhistamine, whereas neither the histamine H2 receptor antagonist, cimetidine (5.0 mg kg−1, i.v.), nor the histamine H1 receptor antagonist, chlorpheniramine (0.5 mg kg−1, i.v.), was effective. The histamine H2 receptor agonist, dimaprit (10 mg kg−1, i.v.), was also without effect on the evoked mydriasis. R-α-methylhistamine (3.0 mg kg−1) did not inhibit phenylephrine-induced mydriasis. These results support the conclusion that R-α-methylhistamine produces inhibition of sympathetically evoked mydriasis via histamine H3 receptor stimulation, presumably by an action on presynaptic histamine H3 receptors.
Keywords: Histamine H3 receptor; R-α-methylhistamine; Clobenpropit; Mydriasis; Iris; Sympathetic nerve stimulation;

Anorectic effect of dehydroepiandrosterone combined with dexfenfluramine or thionisoxetine by Neil E Rowland; Misty Marshall; Kimberly Robertson (61-64).
Free feeding rats given supplementary 1 h access per day to a palatable dessert test meal were tested for the anorectic effect of dehydroepiandrosterone alone or in combination with either the serotonin releasing agent dexfenfluramine or the norepinephrine uptake inhibitor thionisoxetine (LY 368975). Isobolographic analysis showed that the effect of dehydroepiandrosterone combined with either dexfenfluramine or thionisoxetine was within the range predicted for additivity of action.
Keywords: Dessert test; 5-HT (5-hydroxytryptamine, serotonin); Norepinephrine; Isobologram; Additivity; Synergy;

Novel selective hypotensive vasopressin peptides: cardiovascular and structure–activity-relationship studies by W.Y Chan; Roberto Levi; Nga Ching Wo; Motohiro Koyama; Stoytcho Stoev; Ling Ling Cheng; Maurice Manning (65-72).
Recently, we discovered a series of peripheral acting selective hypotensive vasopressin peptides. Whether these peptides may interact with receptors outside the vasopressin receptor family and affect cardiac function could not be excluded. Accordingly, we tested the effects of these hypotensive vasopressin peptides on blood pressure and heart rate in intact rats and on the heart rate, ventricular contractile force and coronary flow of isolated perfused rat hearts. We found that the hypotensive vasopressin peptides did not modify cardiac function, either in vivo or in vitro. The vasodepressor potency was reduced when assayed in rats with vasopressin-maintained baseline blood pressure, suggesting that vasopressin and the hypotensive peptide compete for a common vasodilating vasopressin receptor in the vasculature. We have now synthesized more potent and radioiodinatable hypotensive peptides that could serve as lead compounds for the development of a radiomarker for the putative vasodilating vasopressin receptor.
Keywords: Vasopressin peptide; Vasopressin receptor; Vasopressin receptor subtype; Structure–activity–relationship;

Low concentrations of certain KATP channel openers have been reported to exert a moderate inhibitory effect on arrhythmias during post-ischaemic early myocardial reperfusion, but the accompanying effects on the time course of changes in action potentials in intact hearts have not yet been studied. We report that in rat isolated hearts, reperfusion following 10 min of regional no-flow ischaemia was associated with both an acute, marked, but transient, shortening of ventricular repolarisation (by 63%) during reperfusion, and a high incidence (90%) of ventricular tachyarrhythmias. The KATP channel opener Ro 31-6930 [2-(6-cyano-2,2-dimethyl-2H-1-benzopyran-4-yl)-pyridine 1-oxide], delivered prior to ischaemia at a relatively low concentration (0.5 μM), significantly reduced the incidence and duration of reperfusion arrhythmias, and prevented the associated acute action potential shortening during reperfusion, each in a glibenclamide (1 μM)-sensitive manner (P<0.05, n=10–15 hearts). This was associated with a moderate and non-arrhythmogenic action potential shortening during ischaemia (a potentially “cardioprotective” effect). However, these data highlight the potential harm these drugs may cause, since a higher concentration of Ro 31-6930 caused marked shortening of action potentials and significant pro-arrhythmia during ischaemia.
Keywords: K+ channel; Reperfusion; Heart; Action potential; Ventricular arrhythmia;

Effects of sulfonylurea derivatives on ischemia-induced loss of function in the isolated rat heart by Roger J Legtenberg; Ralph J.F Houston; Berend Oeseburg; Paul Smits (85-92).
This study determined whether sulfonylurea derivatives affect cardiac function prior to and after a mild ischemic incident (stunning). This was investigated using an isolated, erythrocyte-perfused, working rat heart model. In total, 11 groups were studied: five increasing (clinically relevant) concentrations of the classical glibenclamide (range 0.005–4 μmol l−1), five increasing concentrations of the newly developed glimepiride (range 0.005–0.8 μmol l−1), and one control group. Pre-ischemically, glibenclamide and glimepiride reduced coronary blood flow concentration dependently to 55.2±4.5% and 58.5±5.5%, respectively (P<0.001). Twenty minutes after a 12-min ischemic incident, these reductions of flow were even more pronounced (to 38.3±6.7% and 45.8±5.8%, P<0.001). This shows that both sulfonylureas reduce coronary blood flow at concentrations slightly higher than therapeutic ones. In the control group, the ischemic incident significantly lowered cardiac function by 22.2±2.9%. In the therapeutic range, glimepiride, but not glibenclamide, significantly reduced this ischemia-induced cardiac functional loss to 4.9±1.2% (P<0.01). Therefore, we suggest that both sulfonylureas and in particular glimepiride can be used safely in patients with type 2 diabetes mellitus, as long as the coronary vascular system is not compromised. Because of the obvious vasocontrictor response to sulfonylurea derivatives, these drugs must be used with caution in patients with a reduced coronary reserve.
Keywords: Diabetes type 2; KATP channel; Cardiovascular; Heart; Ventricular;

TY-12533, a novel Na+/H+ exchange inhibitor, prevents myocardial stunning in dogs by Kazuyuki Aihara; Hiroaki Hisa; Jun Sasamori; Fumiya Yoneyama; Fuminari Yamaguchi; Isamu Satoh; Susumu Satoh (93-97).
Anesthetized open-chest dogs were subjected to 15-min myocardial ischemia followed by 2-h reperfusion to induce myocardial stunning. A novel Na+/H+ exchange inhibitor 6,7,8,9-tetrahydro-2-methyl-5H-cyclohepta[b]pyridine-3-carbonylguanidine maleate (TY-12533), administered 10 min before or 10 min after start of ischemia (3 mg/kg/10 min, i.v.), did not affect reductions in regional myocardial wall thickening, blood flow and pH during ischemia, but it significantly improved recovery of the wall thickening and blood flow after reperfusion. These results indicate that TY-12533, even when administered during ischemia, could prevent myocardial stunning without affecting myocardial dysfunction or acidosis induced by brief ischemia.
Keywords: TY-12533; Na+/H+ exchange inhibitor; Myocardial wall thickening; Myocardial blood flow; Myocardial pH;

Induction of choleresis by immunosuppressant FK506 through stimulation of insulin-like growth factor-I production in the liver of rats by Ikuo Kawamura; Shigeru Takeshita; Mariko Fushimi; Miyuki Mabuchi; Jiro Seki; Toshio Goto (99-105).
FK506 (Tacrolimus) is an effective immunosuppressant currently used worldwide in organ transplantation. Based on our recent findings that insulin-like growth factor-I (IGF-I) is important for the stimulation of choleresis in vivo, in this study we investigated the effect of FK506 on bile flow and the plasma and hepatic levels of IGF-I in rats. Intravenous treatment of rats with FK506 resulted in a significant increase in bile flow, whereas cyclosporin A induced a significant decrease. A significant increase in plasma levels of IGF-I was observed in rats 30 min after a single intravenous administration of FK506. Oral treatment of rats with FK506 for 1 week also resulted in an increase in both plasma and hepatic levels of IGF-I. Overall, this study showed that FK506 treatment increased bile flow and also induced an increase in the plasma and hepatic levels of IGF-I in rats, suggesting that a stimulation of hepatic IGF-I production by FK506 may contribute to its choleretic profile.
Keywords: FK506 (Tacrolimus); IGF-I (Insulin-like growth factor-I); Bile flow; Choleresis; Cyclosporin A; Cholestasis;