European Journal of Pharmacology (v.506, #1)

Effects of U-37883A on intracellular Ca2+-activated large-conductance K+ channels in pig proximal urethral myocytes by Noriyoshi Teramoto; Manami Aishima; Hai-Lei Zhu; Toshihisa Tomoda; Takakazu Yunoki; Fumi Takahashi-Yanaga; Alison F. Brading; Yushi Ito (1-7).
Kinetic studies of U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N′-cyclohexyl-hydrochloride), a vascular ATP-sensitive K+ channel (KATP channel) blocker, were performed on pig urethral myocytes to investigate inhibitory effects on large-conductance intracellular Ca2+-sensitive K+ channels (i.e., BKCa channels; 225 pS K+ channels) by use of single-channel recordings (outside-out and inside-out configuration). BKCa channels in pig urethral smooth muscles showed extracellular iberiotoxin (300 nM) sensitivity and voltage dependency. The α subunit of BKCa channel proteins was detected in the membrane fraction by use of Western blot technique. Application of U-37883A (≥10 μM) reduced the activity of BKCa channels in a concentration-dependent manner, not only by decreasing mean openlife time but also by prolonging the mean closed time. These results shows that U-37883A affects channels other than the vascular KATP channel, and demonstrates how it inhibits the activities of BKCa channels in urethral smooth muscles.
Keywords: ATP-sensitive K+ channel; Channel kinetics; Intracellular Ca2+-activated large-conductance K+ channel; U-37883A;

Modulation of human GABAA and glycine receptor currents by menthol and related monoterpenoids by Adam C. Hall; Carolyn M. Turcotte; Brooke A. Betts; Wing-Yee Yeung; Abena S. Agyeman; Lâle A. Burk (9-16).
Effects of common monoterpenoid alcohols and ketones were investigated on recombinant human γ-aminobutyric acid A (GABAA; α1β2γ2s) and glycine (α1 homomers) receptors expressed in Xenopus oocytes. GABA currents were enhanced by coapplications of 10–300 μM: (+)-menthol>(−)-menthol>(−)-borneol≫(−)-menthone=camphor enantiomers>carvone enantiomers, with menthol acting stereoselectively. By contrast, thujone diastereomers inhibited GABAA receptor currents while glycine currents were only markedly potentiated by menthol. Positive modulation by (+)-menthol was explored given its pronounced effects (e.g., at 100 μM, GABA and glycine EC20 responses increased by 496±113% and 135±56%, respectively). (+)-Menthol, 100 μM, reduced EC50 values for GABA and glycine from 82.8±9.9 to 25.0±1.8 μM, and from 98.7±8.6 to 75.7±9.4 μM respectively, with negligible effects on maximal currents. This study reveals a novel neuroactive role for menthol as a stereoselective modulator of inhibitory ligand-gated channels.
Keywords: GABAA receptor; Glycine receptor; Monoterpenoid; Menthol;

Compound (+)-R,R-D-84 is an optically active trans-hydroxy-substituted derivative of 4-(2-benzhydryloxy-ethyl)-1-(4-fluorobenzyl)piperidine (D-164). As a hydroxypiperidine analog of GBR 12935, (+)-R,R-D-84 is a candidate dopamine transporter compound for the treatment of cocaine dependence. The present work addresses the functional activity of (+)-R,R-D-84 at monoamine transporters and its potential molecular mechanism involving acidic amino acids (D and E). The selectivity for the dopamine vs. serotonin transporter of (+)-R,R-D-84 was greater than that of (−)-S,S-D-83, its enantiomer, and the selectivity of both compounds was greater than that of GBR 12909 (diphenyl-fluorinated GBR 12935). Only (+)-R,R-D-84 displayed improved selectivity vs. the norepinephrine transporter. D313N or E215Q mutation did not alter the pattern of affinities (measured by membrane binding of the cocaine analog [3H]CFT) for the dopamine transporter of (+)-R,R-D-84, (−)-S,S-D-83, D-164 (non-hydroxylated analog), or GBR 12909. In contrast, D68N mutation specifically lowered the affinity of (+)-R,R-D-84, pointing to a role for D68 in the interaction with (+)-R,R-D-84, possibly through hydrogen bonding between the hydroxyl and the carboxyl group of D68 which is lacking in N68. The present results, combined with behavioral data, implicate D68 in the dopamine transporter in cocaine antagonist activity of (+)-R,R-D-84.
Keywords: GBR 12935 derivative; Dopamine transporter; Monoamine uptake; Hydrogen bonding; Cocaine antagonist; Striatum; (Rat);

KR-31378, a novel benzopyran analog, attenuates hypoxia-induced cell death via mitochondrial KATP channel and protein kinase C-ɛ in heart-derived H9c2 cells by Chang-Hyun Moon; Mi-Young Kim; Mi Jeong Kim; Min Hwa Kim; Sunkyung Lee; Kyu Yang Yi; Sung Eun Yoo; Dong-Ha Lee; Hong Lim; Ho Soon Kim; Soo Hwan Lee; Eun Joo Baik; Yi-Sook Jung (27-35).
A novel compound KR-31378 [(2S,3S,4R)-N″-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2-methly-2-dimethoxy-methly-2H-benzo-pyran-4-yl)-N-benzylguanidine] has been demonstrated as an anti-ischemic agent in rat heart and brain. Here, we report the effects of this compound on hypoxia-induced cell death and possible signaling pathways in heart-derived H9c2 cells. Treatment with KR-31378 (3–30 μM) 1 h before and during hypoxia significantly reduced hypoxia-induced cell death in a concentration-dependent manner. In addition, increase in hypoxia-induced transferase UTP nick end labeling (TUNEL)-positive cells was reduced by KR-31378, suggesting its antiapoptotic potential in H9c2 cells. The protective effect conferred by KR-31378 (10 μM) was abolished by cotreatment with 5-hydroxydecanoate (5HD), a specific blocker of the mitochondrial KATP (mtKATP) channel, but not by HMR-1883 (1-[[5-[2-(5-chloro-o-anisamido)ethyl]-methoxyphenyl]sulfonyl]-3-methylthiourea), a specific blocker of the sarcolemmal KATP channel. We observed that the treatment with KR-31378 could increase the expression of protein kinase C (PKC)-ɛ protein, but not other PKC isotypes (-α, -β, -δ, -ζ), in the particulate fraction. This increased level of PKC-ɛ was sustained during the hypoxic period up to 8 h. In addition, our results showed that treatment with KR-31378 induced the expression of PKC-ɛ mRNA as early as 15 min after the treatment. A specific inhibitor for PKC-ɛ isoform, ɛV1-2, completely blocked the protective effect of KR-31378 against hypoxia-induced cell death. In conclusion, our results suggest that KR-31378 can protect cultured H9c2 cells from hypoxia-induced death via the mtKATP channel and PKC-ɛ.
Keywords: KR-31378; Potassium channel; Protein kinase C-ɛ; Cardiac cell; Hypoxia;

Nicotine and epibatidine triggered prolonged rise in calcium and TH gene transcription in PC12 cells by Volodia D. Gueorguiev; Christopher M. Frenz; Kimberly M. Ronald; Esther L. Sabban (37-46).
The effect of epibatidine on regulation of [Ca2+]i and tyrosine hydroxylase (TH) transcription was examined. Epibatidine triggers a biphasic rise in [Ca2+]i in PC12 cells similar to that observed with nicotine. There was an immediate transient increase in [Ca2+]i and a subsequent sustained second elevation. In contrast to nicotine, the epibatidine-triggered increase in [Ca2+]i was independent of activation of α7 nicotinic acetylcholine receptors, as it was not altered by either methyllycaconitine or α-bungarotoxin. The second [Ca2+]i elevation involves calcium release from intracellular stores and is inhibited by dantrolene or xestospongin C. Epibatidine, like nicotine, elevated TH promoter driven reporter transcription, mostly mediated by the cyclic-AMP responsive motifs. Elevation in TH promoter activity requires Ca2+ and cAMP since it is inhibited by 1,2-bis(o-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic Acid Tetra (acetoxymethyl ester) (BAPTA-AM) or 2′,5′-dideoxyadenosine (DDA). The results reveal that epibatidine can elevate [Ca2+]i in an α7 independent manner and nevertheless induce TH transcription.
Keywords: Nicotine; Epibatidine; Tyrosine hydroxylase; Calcium; Transcription;

Quetiapine increases the firing rate of rat substantia nigra and ventral tegmental area dopamine neurons in vitro by Taco R. Werkman; Johanna E. Olijslagers; Benny Perlstein; Antonius H.J. Jansen; Andrew C. McCreary; Chris G. Kruse; Wytse J. Wadman (47-53).
The antipsychotic drug quetiapine increases the firing rate of dopamine neurons in the substantia nigra and the ventral tegmental area of the rat. In the present study we used an in vitro midbrain slice preparation and found that 3 μM quetiapine increases the firing rate of dopamine neuron in both structures by ∼30%. The magnitude of the increase was not correlated with the basal firing rate of the dopamine neurons. In addition, quetiapine was not able to antagonize the inhibition of the firing evoked by the dopamine D2 receptor agonist quinpirole. Only with a very high concentration (30 μM), quetiapine was able to counteract the amphetamine-induced inhibition of the firing of the ventral tegmental area neurons; this effect was less pronounced in substantia nigra neurons. These findings indicate that the increase in firing rate induced by quetiapine cannot solely be mediated through an interaction with the dopamine D2-like autoreceptor present on the dopamine neurons.
Keywords: Antipsychotic drug; Seroquel; Dopamine D2 autoreceptor; Extracellular recording; In vitro slice preparation;

Effects of baicalein on β-amyloid peptide-(25–35)-induced amnesia in mice by Sheng-Yun Wang; Hui-Hung Wang; Chin-Wen Chi; Chieh-Fu Chen; Jyh-Fei Liao (55-61).
Baicalein may act on the benzodiazepine binding sites to exert an anxiolytic-like effect in mice. Since many benzodiazepine drugs have amnesic side-effect and baicalein can protect cultured cortical neurons from β-amyloid peptide-(25–35)-induced toxicity, this study examined the amnesic effect of baicalein and its effects on β-amyloid peptide-(25–35) (3 nmol/mouse, i.c.v.)-induced amnesia in mice. Using the step-through passive avoidance test, the results showed that baicalein (10–100 mg/kg, i.p.), unlike the benzodiazepine drug chlordiazepoxide (10 mg/kg, i.p.), had no significant amnesic effect. Baicalein (10–50 mg/kg, i.p.) also had no facilitating effect on the learning and memory. However, one dosage pretreatment, but not post-treatment, of baicalein (5 or 10 mg/kg, i.p.) attenuated β-amyloid peptide-(25–35)-induced amnesia. Interestingly, post-treatment for 7 or 13 days of baicalein (10–15 mg/kg/day, i.p.), like melatonin (10 mg/kg/day, i.p.), also attenuated β-amyloid peptide-(25–35)-induced amnesia. Therefore, this study demonstrated that baicalein has protective effect on β-amyloid peptide-(25–35)-induced amnesia.
Keywords: Baicalein; Melatonin; Anti-amnesic effect; β-Amyloid peptide-(25–35); Passive avoidance test;

Δ9-Tetrahydrocannabinol-induced conditioned place preference and intracerebroventricular self-administration in rats by Daniela Braida; Stefania Iosuè; Simona Pegorini; Mariaelvina Sala (63-69).
On the basis of contradictory findings on the rewarding effects of Δ9-tetrahydrocannabinol (Δ9-THC) in laboratory animals, the effect of the compound on conditioned place preference and intracerebroventricular (i.c.v.) self-administration in a free-choice procedure, using a wide range of doses (0.015–6 mg/kg for conditioned place preference test and 0.01–1 μg/2 μl/infusion for i.c.v. self-administration), was studied in Wistar rats. The present results showed that Δ9-THC induced reward in both tests, but only at the lowest tested doses (0.075–0.75 mg/kg i.p. for conditioned place preference test and 0.01–0.02 μg/infusion for i.c.v. self-administration). This effect was fully antagonised by i.p. pretreatment with the cannabinoid CB1 receptor antagonist, SR 141716A [N-piperidino-5-(4-chlorophenyl)1-(2,4-dichlorophenyl)-4 methyl pyrazole 3-carboxamide] (0.25–1 mg/kg), and the opiate receptor antagonist, naloxone (0.5–2 mg/kg), suggesting the involvement of both endocannabinoid and opioid systems. In conclusion, these findings demonstrate, for the first time, that low doses of Δ9-THC can act as an effective reinforcer in Wistar rats providing a reliable animal model of human marijuana abuse.
Keywords: Δ9-THC; Conditioned place preference; i.c.v. self-administration;

Biphasic effects of oxotremorine-M on turning behavior induced by caffeine in 6-OHDA-lesioned rats by Juan Francisco Núñez-Taltavull; Gemma Prat; Antonia Rubio; Patricia Robledo; Miguel Casas (71-74).
This work studied the interactions between cholinergic and adenosine systems in the denervated striatum. For that purpose, we evaluated the effects of an intrastriatal administration of the muscarincic receptor agonist, oxotremorine-M on turning behavior induced by systemic caffeine in unilaterally 6-hydroxydopamine-lesioned rats. Low doses of oxotremorine-M (0.1 ng/μl) enhanced, whereas high doses (100 ng/μl) attenuated contralateral turning induced by caffeine. These results support a functional link between muscarinic and adenosinergic systems in the denervated striatum and suggest opposite actions of muscarinic M2 and M1 receptors on caffeine-induced turning behavior.
Keywords: Caffeine; Oxotremorine-M; Turning behavior; Striatum; Adenosine/acetylcholine interaction;

Interaction between 1,4-butanediol and ethanol on operant responding and the cardiovascular system by Lisa R. Gerak; Alissa R. Hicks; Peter J. Winsauer; Kurt J. Varner (75-82).
The current studies characterized the rate-decreasing and cardiovascular responses produced by 1,4-butanediol administered alone and in combination with ethanol to test the hypothesis that these effects resulted from the degradation of 1,4-butanediol to γ-hydroxybutyrate. One group of rats responded under a fixed-ratio 20 schedule of food presentation; ethanol and 1,4-butanediol dose-dependently decreased response rates. Ethanol administered in combination with 1,4-butanediol attenuated the rate-decreasing effects of 1,4-butanediol without altering the potency of ethanol. In separate groups of conscious rats, radio telemetry was used to record mean arterial pressure and heart rate. In contrast to its depressant effects on schedule-controlled responding, 1,4-butanediol increased mean arterial pressure and heart rate; these increases were attenuated by ethanol. Thus, the behavioral and cardiovascular actions of 1,4-butanediol are similar to those elicited by γ-hydroxybutyrate. The ability of ethanol to attenuate the behavioral and cardiovascular effects of 1,4-butanediol indicates that these effects require the conversion of 1,4-butanediol to γ-hydroxybutyrate.
Keywords: 1,4-Butanediol; Schedule-controlled behavior; Mean arterial pressure; Heart rate; Radio telemetry; Rat;

The role of endothelin peptides was evaluated on survival and organ injury in a model of polymicrobial sepsis, induced by caecal ligation and puncture with particular emphasis on the timing of the administration of its blocker bosentan in Swiss albino mice (20–40 g). The cardiovascular response pattern in this experimental model was characterized by an early, “hyperdynamic” phase starting at 5 h, followed by a late but “hypodynamic” phase that commence after 20 h, provided that the animals are “resuscitated” by injecting 1 ml of saline i.p. at the end of the surgery. However, if saline resuscitation is omitted, then only hypodynamic pattern is observed starting at 5 h without any hyperdynamic phase. Thus, mice were first allocated into saline-resuscitated or unresuscitated groups and endothelin receptor antagonist bosentan (30 mg kg−1, i.p., either 5 or 20 h after caecal ligation and puncture) was then administered. The control animals received the solvent of bosentan (i.e., saline: %0.9 NaCl, w/v). The survival rates in each group (n=14) were recorded over the following 144 h. In unresuscitated mice, the overall survival at 144 h was 14.3% in controls while bosentan treatment at 5 h (78.6%, P=0.0018) or 20 h (64.3%, P=0.0183) have both significantly improved the survival. However, in saline-resuscitated mice, bosentan administered at 20 h has significantly improved the survival (71.4%, P=0.0213) while its administration at 5 h has yielded exactly the same percent of survival (i.e., 21.4%) as observed in control animals. The beneficial effects of bosentan in preventing the tissue injury due to caecal ligation and puncture were also observed histopathologically in liver, spleen and kidney. Therefore, we concluded that the blockade of endothelin receptors by using bosentan during the later (hypodynamic) stages of septic shock is a promising therapeutic manoeuvre.
Keywords: Septic shock; Endothelin; Bosentan; Organ injury; Survival;