The Ca²⁺ sensitizer levosimendan (LEV) improves myocardial contractility by enhancing the sensitivity of the contractile apparatus to Ca²⁺. In addition, LEV promotes Ca²⁺ entry through L-type channels in human cardiac myocytes. In this study, which was performed using microdialysis, infusion of LEV at 0.25 μM for 160 min increased dopamine (DA) concentrations (up to fivefold baseline) in dialysates from the striatum of freely moving rats. Ca²⁺ omission from the perfusion fluid abolished baseline DA release and greatly decreased LEV-induced DA release. Reintroduction of Ca²⁺ in the perfusion fluid restored LEV-induced DA release. Chelation of intracellular Ca²⁺ by co-infusing 1,2-bis (o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM, 0.2 mM) did not affect basal DA release and scarcely affected LEV-induced increases in dialysate DA. In addition, co-infusion of the L-type (Ca_v 1.1 – 1.3) voltage-sensitive Ca²⁺-channel inhibitor nifedipine failed to inhibit LEV-induced increases in dialysate DA, which, in contrast, was inhibited by co-infusion of the N-type (Ca_v 2.2) voltage-sensitive Ca²⁺-channel inhibitor ω-conotoxin GVIA. We conclude that LEV promotes striatal extracellular Ca²⁺ entry through N-type Ca²⁺ channels with a consequent increase in DA release.

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We investigated the effect of doxazosin on cholinergic and adrenergic agonists responses in detrusor smooth muscle preparations from sham-operated and 2-week partially obstructed rat bladders. Male Wistar albino rats, 200 – 250 g, were randomly allocated to 4 experimental groups consisting of 12 animals each: sham-operated bladder, sham-operated bladder treated with doxazosin, partially obstructed bladder, and partially obstructed bladder treated with doxazosin. Partial outlet obstruction of the rat was surgically induced. The response to carbachol (10⁻⁷ – 10⁻⁴ M), isoproterenol (10⁻⁶ – 10⁻³ M), and 80 mM KCl were recorded. Carbachol caused concentration-dependent contractile responses in the detrusor smooth muscles from sham-operated and partially obstructed bladder. Isoproterenol produced concentration-dependent relaxation responses in the detrusor strips from all groups. Dose-response curves for carbachol and isoproterenol showed a shift to the left in rat detrusor smooth muscles from partially obstructed bladder when compared with the results obtained in detrusor muscles from sham-operated bladder. These responses were reversed to normal by doxazosin treatment in rat detrusor smooth muscles from partially obstructed bladder. KCl produced contractile responses in rat detrusor smooth muscles from all groups. The contractile responses to KCl were not significantly changed in all groups. We have shown that carbachol and isoproterenol responses were shifted to the left in rat detrusor smooth muscles from partially obstructed bladder and these responses were reversed by doxazosin treatment.

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A radioligand binding assay for the HERG (human ether-a-go-go-related gene) K⁺ channel was developed to identify compounds which may have inhibitory activity and potential cardiotoxicity. Pharmacological characterization of the [³H]astemizole binding assay for HERG K⁺ channels was performed using HERG-expressing HEK293 cells. The assay conditions employed yielded 90% specific binding using 10 μg/well of membrane protein with 1.5 nM of [³H]astemizole at 25°C. The K_d and B_max values were 5.91 ± 0.81 nM and 6.36 ± 0.26 pmol/mg, respectively. The intraassay and interassay variations were 11.4% and 14.9%, respectively. Binding affinities for 32 reference compounds (including dofetilide, cisapride, and terfenadine) with diverse structures demonstrated a similar potency rank order for HERG inhibition to that reported in the literature. Moreover, the [³H]astemizole binding data demonstrated a rank order of affinity that was highly correlated to that of inhibitory potency in the electrophysiological studies for HERG in HEK293 (r_SP = 0.91, P<0.05). In conclusion, the [³H]astemizole binding assay is rapid and capable of detecting HERG inhibitors.

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The effects of selective inhibitors of phosphodiesterase type IV (PDE4) on ischemia-reperfusion-induced gastric injuries were investigated in rats. Gastric ischemia was induced by applying a small clamp to the celiac artery, and reoxygenation was performed by removal of the clamp. Ischemia-reperfusion produced gastric hemorrhagic injuries and increased the content of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) and myeloperoxidase (MPO) activity in gastric mucosa. Rolipram (0.03 – 0.3 mg/kg, s.c.) and Ro-20-1724 (0.3 – 3 mg/kg, s.c.) prevented the development of gastric injury in a dose-dependent manner, and it also inhibited the increase in mucosal TNF-α content and MPO activity induced by ischemia-reperfusion. The anti-ulcer drug irsogladine (1 – 10 mg/kg, p.o.), which is known to possess a PDE4 inhibitory action, also inhibited the gastric injury produced by ischemia-reperfusion, as well as the increase in TNF-α levels and MPO activity. It is concluded that the ability of PDE4 inhibitors to inhibit cytokine TNF-α synthesis and the infiltration of polymorphonuclear leukocytes underlies their gastroprotective effects in ischemia-reperfusion-induced gastric injury. Our experiments suggest that drugs that inhibit PDE4 isoenzyme, such as the anti-ulcer drug irsogladine, may be a useful adjunct therapy for the treatment of the gastric damage that follows ischemia-reperfusion.

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We previously demonstrated that repeated electroconvulsive shock (ECS) treatment enhanced serotonin (5-HT)_1A- and 5-HT₃-receptor-mediated responses in hippocampal CA1 pyramidal neurons. The electrophysiological studies were performed to elucidate the effects of ECS treatment on depolarization, which was an additional response induced by 5-HT, and the second messenger system involved in this depolarization of hippocampal CA1 neurons. Bath application of 5-HT (100 μM) induced depolarization of the membrane potential in the presence of 5-HT_1A-receptor antagonists. This depolarization was mimicked by 5-HT₄-receptor agonists, RS 67506 (1 – 30 μM) and RS 67333 (0.1 – 30 μM), in a concentration-dependent manner. 5-HT- and RS 67333-induced depolarization was attenuated by concomitant application of RS 39604, a 5-HT₄-receptor antagonist. H-89, a protein kinase A (PKA) inhibitor, inhibited 5-HT-, RS 67506-, and RS 67333-induced depolarizations, while forskolin (10 μM), an activator of adenylate cyclase, induced depolarization. Furthermore, RS 67333-induced depolarization was not significantly different between hippocampal slices prepared from rats administered ECS once a day for 14 days and those from sham-treated rats. These findings suggest that 5-HT₄-receptor-mediated depolarization is caused via the cAMP-PKA system. In addition, repeated ECS-treatment did not modify 5-HT₄-receptor functions in contrast to 5-HT_1A- and 5-HT₃-receptor functions.

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The present study is part of a project on phospholipase D (PLD) in cardiac hypertrophy and analyzed effects on PLD activity of two growth stimuli, norepinephrine (NE) and cardiotrophin-1 (CT-1), in incubated rat heart. Phosphatidylcholine (PC) was labeled by ³H-myristic acid. PLD produced ³H-phosphatidylethanol (³H-PEth) from ³H-PC in the presence of ethanol and maintained a basal formation of ³H-PEth. Short-term and long-term exposure to NE for 2 or 13 h, respectively, enhanced the formation of ³H-PEth, which was blocked by prazosin. Long-term pretreatment with NE or CT-1 increased the incorporation of ³H-myristic acid into PC, which was blocked by atenolol. When the ³H-PEth formation was expressed as a fraction of ³H-PC, PLD activity seemingly was unchanged (NE) or markedly reduced (CT-1); the true effects, namely, stimulation by NE and nonresponsiveness towards CT-1, were unraveled by atenolol (NE) or when PLD activity was expressed as ³H-PEth per ng protein. In conclusion, α-adrenoceptor activation increased PLD activity. Long-term treatment with NE (via β-receptors) or CT-1 enhanced the ³H-myristic acid incorporation into a PC compartment, that was not available for the α-receptor-mediated PLD activation. These results were discussed in regard to cellular mechanisms of cardiac hypertrophy and to the transphosphatidylation assay of PLD.

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Although mannitol has been used as an osmotherapeutic drug on brain injury, the clinical efficiency of the drug are still controversial. In the present study, we examined the effects of mannitol on the edema in a hippocampal slice due to brief ischemia. To evaluate the effects, we employed an image analysis system that consists of an infrared-differential interference contrast (IR-DIC) microscope, an infrared CCD camera, and a computer with custom-made software. By this system, severity of the edema can be quantified as the coefficient of variation (CV) of digitalized slice images. The dose-dependent improvement on the deteriorated hippocampal slices could be obtained by administration of mannitol (10, 50, and 100 mM) after 10-min ischemia. However, field excitatory postsynaptic potentials (fEPSP) in CA1 stratum radiatum, which disappeared during 10-min ischemia, were never recovered by mannitol after more than 20-min treatment. fEPSP were blocked by the effective dose of mannitol for morphological recovery, but the effects found to be reversible. Although we failed to find positive rescuing effects of mannitol on the synaptic activities after ischemia, the protective effects of the drug on ischemic edema may rescue the secondary damages around the infarct area.

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Our purpose was to investigate whether the local or systemic factors of pregnancy are associated with inducible nitric oxide synthase (iNOS) mRNA expression and to determine the inhibitory effects of pharmacological agents that increase cGMP levels in rat myometrium. iNOS mRNA expression was determined in uterine tissues from nonpregnant rats and on day 17 of gestation in the pregnant and non-pregnant uterine horns by RT-PCR. In addition, uterine rings from the pregnant and non-pregnant uterine horns were placed in Krebs-Henseleit solution for isometric recordings of spontaneous contractions. Concentration-inhibition relationships to diethylamine/nitric oxide complex, 8-bromo-cGMP, and the selective phosphodiesterase V inhibitor were obtained. Compared to nonpregnant rats, expression of iNOS mRNA in myometrium increased during pregnancy, which was maximal on day 17, followed by a decrease on day 21 of gestation. Expression of iNOS mRNA at day 17 of gestation was greater in pregnant uterine horns than in nonpregnant ones. Maximal inhibition of phosphodiesterase V and increasing cGMP induced similar inhibition of spontaneous contractions in nonpregnant and pregnant uterine horns, while NO induced less inhibition in the former. The results suggest that the local pregnancy factor is needed for signal transduction from NO to soluble guanylate cyclase at a time when maximal expression of iNOS mRNA is evident.

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We investigated the effects of single and repeated administrations of S-312-d (methyl-4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitrophenyl)-thieno-[2,3-b]pyridine-5-carboxylate), a newly synthesized L-type Ca²⁺-channel blocker, on tonic convulsions and absence-like seizures in the spontaneously epileptic rat (SER: zi/zi, tm/tm), a genetically based animal model of human epilepsy. Single oral administrations of S-312-d dose-dependently inhibited tonic convulsions and the effects lasted for more than 2 h, although they did not attenuate the absence-like seizures. We also examined the effects of repeated administrations of S-312-d at 1 mg/kg once a day for 4 days on SER. A significant decrease in the number and total duration of tonic convulsions was observed 45 and 75 min after the first administration of the drug, respectively. The effects lasted for 24 h without changes in the background EEG or blood pressure. This inhibitory effect on the tonic convulsions was gradually strengthened by subsequent daily administrations of S-312-d and lasted for 3 days after the cessation of drug treatment. In contrast, the repeated treatment with S-312-d did not influence absence-like seizures of SER. These results suggest that S-312-d is a candidate drug that has antiepileptic effects against the convulsive seizures in human epilepsy.

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Single ventricular cells were enzymatically isolated from guinea pig hearts and the effects of sevoflurane on the delayed rectifier K⁺ current were investigated by the patch clamp method. The rapidly (I_Kr) and slowly activating delayed rectifier K⁺ current (I_Ks) were isolated using chromanol 293B, a selective blocker for I_Ks or E4031 (N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl]methanesulfonamide dihydrochloride), a blocker for I_Kr. Sevoflurane and halothane decreased I_Ks in a concentration-dependent manner with an IC₅₀ value of 0.38 mM for sevoflurane and 1.05 mM for halothane. I_Ks inhibition was characterized by suppression of maximum conductance with little effect on activation kinetics. Inhibition occurred immediately after anesthetic application and recovered upon wash-out. In contrast to the marked inhibition of I_Ks, I_Kr was hardly affected by sevoflurane. Under the current clamp, sevoflurane prolonged the action potential duration in a reversible manner and this effect was more marked when I_Kr was inhibited by E4031. The results suggest that sevoflurane inhibits I_Ks, and not I_Kr, in a concentration-dependent manner at clinically relevant concentrations. The resulting prolongation of ventricular repolarization may partly account for the clinical observation of excessive QT prolongation by these anesthetics.

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In the present study, we investigated the antidepressant- and anxiolytic-like effects of (+)-4-[(aR)-a-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80), a non-peptidic selective δ-opioid receptor agonist, in various animal models in rodents. SNC80 significantly reduced the duration of immobility in the forced swimming test. Furthermore, in the elevated plus-maze test, SNC80 dose-dependently and significantly increased the time spent in the open arms of the plus-maze. These effects were completely antagonized by a selective δ-opioid-receptor antagonist, naltrindole. In the conditioned fear stress test, which examines psychological stress-induced motor suppression, desipramine did not produce any significant effect on the conditioned suppression of locomotor activity. However, SNC80 completely attenuated the conditioned suppression of locomotor activity in the conditioned fear stress test. In conclusion, our results suggest that δ-opioid receptors may play an important role in the regulation of emotional responses. Furthermore, it is possible that δ-opioid-receptor agonists might be novel and potent antidepressants that also have anxiolytic-like effects.

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Aims were to observe pharmacokinetics, pharmacodynamics, and toxicity for constructing a Sino-pegylated liposomal platform. Human hepatocarcinoma cells (Bel7402) and murine hepatocarcinoma cells (H₂₂) were used for the cytotoxicity assay and the in vivo solid xenograft tumor model in mice, respectively. Pharmacokinetic results in mice showed that the pegylated liposomal doxorubicin markedly prolonged the blood circulation of doxorubicin. Elimination half-time (T_1/2,γ) of pegylated, regular liposomal doxorubicin and free doxorubicin were 46.09 ± 14.44, 26.04 ± 3.34, and 23.72 ± 5.13 h, respectively. The area under the concentration-time curves (AUC_0–∞) (h · μg/g) of the pegylated and regular liposomal doxorubicin were 6.8- and 2.6-fold higher than that of free doxorubicin, respectively. Cytotoxicity and antitumor activity in vivo indicated that activity of the pegylated liposomal doxorubicin was higher than that of the regular or the free one, respectively. After two weeks of tail intravenous injection of the pegylated liposomal doxorubicin at a single dose of 10 mg/kg, no significant damage was observed in gastric, intestinal mucosa, and heart muscle, but pronounced damages were found in the control group after dosing free doxorubicin. The results demonstrate that the pegylated liposomes improve the efficacy of toxics and reduce the toxicity, therefore providing favorable evidence for building a pegylated liposomal platform.

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The effect of captopril on neurally evoked bradycardia and tachycardia was investigated in pithed rats. Captopril enhanced the vagal nerve stimulation-evoked bradycardia. Angiotensin I reduced the vagal bradycardia, which was reversed by subsequent administration of captopril. Bradykinin did not affect the neurally evoked bradycardia. Captopril and angiotensin I affected neither the exogenous acetylcholine-evoked bradycardia nor the sympathetic nerve stimulation-evoked tachycardia. These results suggest that the interruption of angiotensin II formation by captopril causes less presynaptic inhibition of acetylcholine release via angiotensin II receptors without affecting cardiac sympathetic neurotransmission.

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We examined the combined effects of the calcium channel blockers 1,4-dihydropyridine (benidipine) and benzothiazepine (diltiazem) on vasopressin-induced myocardial ischemia in anesthetized rats, an experimental model of angina. Benidipine (3, 10 μg/kg, i.v.) and diltiazem (300, 1000 μg/kg, i.v.) caused dose-related inhibition of vasopressin-induced S-wave depression, an index of myocardial ischemia. Co-administration of low doses of benidipine (3 μg/kg) and diltiazem (300 μg/kg) almost completely inhibited the S-wave depression, where the efficacy was similar to that obtained with the use of high doses of benidipine (10 μg/kg) or diltiazem (1000 μg/kg). These results suggest that the administration strategy employed may be useful in the treatment of angina pectoris.

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Cilnidipine, a dihydropyridine Ca²⁺ channel antagonist, is known to have inhibitory effects on both L- and N-type Ca²⁺ currents. In the present study, we examined the effect of cilnidipine on myocardial L- and T-type Ca²⁺ currents and sinoatrial node action potential configuration. In voltage clamped guinea pig ventricular myocytes, cilnidipine concentration-dependently decreased L- and T-type Ca²⁺ currents. In rabbit sinoatrial node tissue, cilnidipine increased cycle length through reduction of phase 4 depolarization slope. In conclusion, cilnidipine has inhibitory effects on T-type Ca²⁺ current, which may contribute to its negative chronotropic potency.

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The last name of the third author in the above-referenced article was misspelled in the text. It should have appeared as "Tobo" and not "Tobe". Wrong:Masayuki Tobe
Right:Masayuki Tobo

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