Chronic Volume Overload Caused by Abdominal Aorto-Venocaval Shunt Provides Arrhythmogenic Substrates in the Rat Atrium.

Atrial enlargement is thought to provide arrhythmogenic substrates, leading to the induction of atrial fibrillation (AF). In this study, we investigated the anatomical, molecular biological, and electrophysiological characteristics of remodeled atria in an animal model with chronic volume overload. We used rats that underwent abdominal aorto-venocaval shunt (AVS) surgery. In the in vivo studies, marked changes in electrocardiogram parameters, such as the P-wave duration, PR interval, and QRS width, as well as prolongation of the atrial effective refractory period were observed 12 weeks after the creation of AVS (AVS-12W), which were undetected at 8 weeks postoperative (AVS-8W) despite obvious atrial and ventricular enlargement. Moreover, the duration of AF induced by burst pacing in the AVS-12W rats was significantly longer than that in the Sham and AVS-8W rats. In the isolated atria, a longer action potential duration at 90% repolarization was detected in the AVS-12W rats compared with that in the Sham group. The mRNA levels of the Kv and Kir channels in the right atrium were mostly upregulated in the AVS-8W rats but were downregulated in the AVS-12W rats. These results show that chronic volume overload caused by abdominal AVS provides arrhythmogenic substrates in the rat atrium. The difference in gene expression in the right atrium between the AVS-8W and AVS-12W rats may partly explain the acquisition of arrhythmogenicity.

Fluorescein Permeability of the Blood-Brain Barrier Is Enhanced in Juvenile- but Not Young Adult-Onset Type 1 Diabetes in Rats.

Clinically, neurological disorders, such as cognitive impairments and dementia, have been reported as diabetic complications, which are remarkable, especially in children with diabetes. The blood-brain barrier (BBB) is a physiologically dynamic regulatory barrier that maintains the consistency of the fluid microenvironment composition of the brain. However, the differences in BBB conditions between children and adults and the contribution of the BBB to the severity of cognitive impairments remain unclear. We generated adult-onset diabetes mellitus (DM) and juvenile-onset diabetes mellitus (JDM) diabetic rat models and investigated BBB functions in these models during the early stages of type 1 diabetes. We performed a BBB permeability assay using sodium fluorescein, a small-molecule fluorescent dye, to evaluate endothelial transport from the blood to the central nervous system. One week after diabetes onset, BBB permeability increased in the hippocampus and striatum of JDM rats, but no changes were observed in the frontal cortex and hypothalamus of JDM rats or for any region of DM rats. The double staining of tight junction proteins and astrocytes revealed no changes in the hippocampus and striatum of JDM rats. These results suggested that the observed increase in BBB permeability during early-stage diabetes onset in JDM rats, which did not depend on the expression of the interendothelial tight junction protein, claudin-5, may affect stylized neural development and cognitive function.

Hypoalgesia and recovery in methylmercury-exposed rats.

Methylmercury (MeHg), the causal substrate in Minamata disease, can lead to severe and chronic neurological disorders. The main symptom of Minamata disease is sensory impairment in the four extremities; however, the sensitivity of individual sensory modalities to MeHg has not been investigated extensively. In the present study, we performed stimulus-response behavioral experiments in MeHg-exposed rats to compare the sensitivities to pain, heat, cold, and mechanical sensations. MeHg (6.7 mg/kg/day) was orally administered to 9-week-old Wistar rats for 5 days and discontinued for 2 days, then administered daily for another 5 days. The four behavioral experiments were performed daily on each rat from the beginning of MeHg treatment for 68 days. The pain sensation decreased significantly from day 11 onwards, but recovered to control levels on day 48. Other sensory modalities were not affected by MeHg exposure. These findings suggest that the pain sensation is the sensory modality most susceptive to MeHg toxicity and that this sensitivity is reversible following discontinuation of the exposure.

Systematic Review and Meta-Analysis of In Vitro Anti-Human Cancer Experiments Investigating the Use of 5-Aminolevulinic Acid (5-ALA) for Photodynamic Therapy.

5-Aminolevulinic acid (5-ALA) is an amino acid derivative and a precursor of protoporphyrin IX (PpIX). The photophysical feature of PpIX is clinically used in photodynamic diagnosis (PDD) and photodynamic therapy (PDT). These clinical applications are potentially based on in vitro cell culture experiments. Thus, conducting a systematic review and meta-analysis of in vitro 5-ALA PDT experiments is meaningful and may provide opportunities to consider future perspectives in this field. We conducted a systematic literature search in PubMed to summarize the in vitro 5-ALA PDT experiments and calculated the effectiveness of 5-ALA PDT for several cancer cell types. In total, 412 articles were identified, and 77 were extracted based on our inclusion criteria. The calculated effectiveness of 5-ALA PDT was statistically analyzed, which revealed a tendency of cancer-classification-dependent sensitivity to 5-ALA PDT, and stomach cancer was significantly more sensitive to 5-ALA PDT compared with cancers of different origins. Based on our analysis, we suggest a standardized in vitro experimental protocol for 5-ALA PDT.

Novel Photosensitizer ß-Mannose-Conjugated Chlorin e6 as a Potent Anticancer Agent for Human Glioblastoma U251 Cells.

A photosensitizer is a molecular drug for photodynamic diagnosis and photodynamic therapy (PDT) against cancer. Many studies have developed photosensitizers, but improvements in their cost, efficacy, and side effects are needed for better PDT of patients. In the present study, we developed a novel photosensitizer ß-mannose-conjugated chlorin e6 (ß-M-Ce6) and investigated its PDT effects in human glioblastoma U251 cells. U251 cells were incubated with ß-M-Ce6, followed by laser irradiation. Cell viability was determined using the Cell Counting Kit-8 assay. The PDT effects of ß-M-Ce6 were compared with those of talaporfin sodium (TS) and our previously reported photosensitizer ß-glucose-conjugated chlorin e6 (ß-G-Ce6). Cellular uptake of each photosensitizer and subcellular distribution were analyzed by fluorescence microscopy. ß-M-Ce6 showed 1000× more potent PDT effects than those of TS, and these were similar to those of ß-G-Ce6. ß-M-Ce6 accumulation in U251 cells was much faster than TS accumulation and distributed to several organelles such as the Golgi apparatus, mitochondria, and lysosomes. This rapid cellular uptake was inhibited by low temperature, which suggested that ß-M-Ce6 uptake uses biological machinery. ß-M-Ce6 showed potent PDT anti-cancer effects compared with clinically approved TS, which is a possible candidate as a next generation photosensitizer in cancer therapy.

Possible mechanism of heme oxygenase-1 expression in rat malignant meningioma KMY-J cells subjected to talaporfin sodium-mediated photodynamic therapy.

BACKGROUND: We previously demonstrated that heme oxygenase-1 (HO-1) induction may contribute to a protective response against photodynamic therapy (PDT) using talaporfin sodium (TS) in rat malignant meningioma KMY-J cells. In the present study, we examined the mechanism of HO-1 induction by PDT with TS (TS-PDT) in KMY-J cells. METHODS: KMY-J cells were incubated with 25 µM TS for 2 h and then exposed to 664 nm diode laser irradiation at 1 J/cm2. The gene and protein expression levels of HO-1 and hypoxia-inducible factor-1α (HIF-1α) were determined by real-time RT-PCR and western blot analysis, respectively. Cell viability was measured using the cell counting kit-8 assay. RESULTS: mRNA and protein levels of HO-1 in KMY-J cells were increased significantly at 3, 6, and 9 h after laser irradiation and the increased mRNA level of HO-1 was decreased by antioxidant N-acetyl cysteine treatment. The protein level of HIF-1α, which mediates transcriptional activation of the HO-1 gene, was increased significantly at 1 h after laser irradiation. Additionally, induction of mRNA expression of HO-1 by TS-PDT was diminished by HIF-1α inhibitor echinomycin. We also demonstrated that echinomycin significantly augmented the cytotoxic effect of TS-PDT. CONCLUSIONS: Our findings indicate that TS-PDT may induce HO-1 expression via reactive oxygen species production and then HIF-1 pathway activation in KMY-J cells, and the HO-1 induction may cause attenuation of the therapeutic effect of TS-PDT.

Synergistic effect of dichloroacetate on talaporfin sodium-based photodynamic therapy on U251 human astrocytoma cells.

BACKGROUND: Talaporfin sodium (TS) is an authorized photosensitizer for photodynamic therapy (PDT) against some tumors in Japan; however, the drawbacks of the drug include its high cost and side effects. Thus, reducing the dose of TS in each round of TS-PDT against tumors is important for reducing treatment costs and improving patients' quality of life. Dichloroacetate (DCA) is approved for treating lactic acidosis and hereditary mitochondrial diseases, and it is known to enhance reactive oxygen species production and induce apoptosis in cancer cells. Therefore, DCA has the potential to enhance the effects of TS-PDT and permit the use of lower TS doses without reducing the anti-cancer effect. METHODS: U251 human astrocytoma cells were simultaneously incubated with TS and DCA using different concentrations, administration schedules, and treatment durations, followed by laser irradiation. Cell viability was determined using the CCK-8 assay. RESULTS: The combinational use of DCA and TS resulted in synergistically enhanced TS-PDT effects in U251 cells. The duration of DCA treatment before TS-PDT slightly enhanced the efficacy of TS-PDT. The intensity of laser irradiation was not associated with the synergistic effect of DCA on TS-PDT. In addition, the relationship between the elapsed time after TS/DCA combination treatment and PDT ineffectiveness was identical to that of TS monotherapy. CONCLUSIONS: DCA synergistically enhanced the anti-cancer effect of TS-PDT, illustrating its potential for drug repositioning in cancer therapy in combination with PDT.

Benzothiazepines, diltiazem and JTV-519, exert an anxiolytic-like effect via neurosteroid biosynthesis in mice.

We investigated whether benzothiazepines could produce anxiolytic effects via allopregnanolone (ALLO) biosynthesis in mice. We compared the behavioral effects caused by benzothiazepines to those caused by carbamazepine and sodium valproate. We found that a pretreatment with finasteride (a 5 alpha-reductase inhibitor) suppressed carbamazepine-induced anxiolytic effects but not the effects of sodium valproate. Similar to carbamazepine, diltiazem and JTV-519 displayed anxiolytic effects that were suppressed by a pretreatment with finasteride. We clearly demonstrate that the benzothiazepines, diltiazem and JTV-519, exert an anxiolytic-like effect via ALLO biosynthesis in mice.

Permissive role of reduced inwardly-rectifying potassium current density in the automaticity of the guinea pig pulmonary vein myocardium.

The electrophysiological properties underlying the automaticity of the guinea pig pulmonary vein myocardium were studied. About 30% of the isolated pulmonary vein tissue preparations showed spontaneous electrical activity, as shown by glass microelectrode recordings from their myocardial layer. The remaining quiescent preparations had a resting membrane potential less negative than that in the left atria. Blockade of the acetylcholine activated potassium current (IK-ACh) by tertiapin induced a depolarizing shift of the resting membrane potential and automatic electrical activity in the pulmonary vein, but not in the atria. The tertiapin-induced electrical activity, as well as the spontaneous activity, was inhibited by the application of carbachol or by chelation of intracellular Ca2+ by BAPTA. The isolated pulmonary vein cardiomyocytes had an IK-ACh density similar to that of the atrial cardiomyocytes, but a lower density of the inwardly-rectifying potassium current (IK1). Spontaneous Ca2+ transients were observed in about 30% of the isolated pulmonary vein cardiomyocytes, but not in atrial cardiomyocytes. The Ca2+ transients in the pulmonary vein cardiomyocytes were induced by tertiapin and inhibited by carbachol. These results indicate that the pulmonary vein cardiomyocytes have a reduced density of the inwardly-rectifying potassium current, which plays a permissive role in their intracellular Ca2+-dependent automaticity.

Involvement of alpha- and beta-adrenoceptors in the automaticity of the isolated guinea pig pulmonary vein myocardium.

We examined the involvement of adrenoceptors in the automaticity of the pulmonary vein myocardium, which probably plays a crucial role in the generation of atrial fibrillation. The automatic activity of the myocardium in guinea pig pulmonary vein tissue preparations were monitored by contractile force or membrane potential measurement. In quiescent preparations, application of noradrenaline induced an automatic activity. The firing frequency was reduced by prazosin or atenolol. Methoxamine induced an automatic activity of low frequency, which was accelerated by further application of isoproterenol. In preparations driven at a constant frequency, noradrenaline, in the presence of atenolol, caused a depolarizing shift of the resting membrane potential and an increase in the slope of the diastolic depolarization. In contrast, in the presence of prazosin, noradrenaline had no effect on the slope, but caused acceleration of the late repolarization and a hyperpolarizing shift of the maximum diastolic potential. At clinically relevant concentrations, carvedilol significantly inhibited the noradrenaline-induced activity but bisoprolol did not. It was concluded that α1- and ß1-adrenoceptor stimulation enhance automaticity through different mechanisms in the guinea pig pulmonary vein myocardium. Dual blockade of these adrenoceptors appears to be effective for suppressing noradrenaline-induced pulmonary vein automaticity and probably atrial fibrillation.

Enhancement of Automaticity by Mechanical Stretch of the Isolated Guinea Pig Pulmonary Vein Myocardium.

The effects of mechanical stretch on the automaticity of the guinea pig isolated pulmonary vein preparations were studied in microelectrode experiments. The application of cumulative mechanical stretch to the preparations resulted in increases in the firing rate of spontaneous electrical activity in the myocardial layer. This effect was significantly inhibited by stretch-activated channel blockers such as gadolinium or streptomycin. These results suggest that acute mechanical stretch enhances the automaticity of the guinea pig pulmonary vein myocardium through the opening of the stretch-activated channels.

Manifestation of automaticity in the pulmonary-vein myocardium of rats with abdominal aorto-venocaval shunt.

Effect of abdominal aorto-venocaval shunt (AVS) on the automaticity of the pulmonary-vein myocardium was studied in the rat. Spontaneous electrical activity was observed in one third of the isolated pulmonary-vein preparations from the AVS rats, but scarcely in those from sham-operated rats; the activity was induced by tertiapin and suppressed by carbachol or chelation of intracellular Ca(2+). The evoked action potentials in AVS rats had less negative resting membrane potential and longer action potential duration than those in sham-operated rats. These results suggest that the automaticity of the rat pulmonary-vein myocardium is manifested under chronic volume overload.

Epac1-dependent phospholamban phosphorylation mediates the cardiac response to stresses.

PKA phosphorylates multiple molecules involved in calcium (Ca2+) handling in cardiac myocytes and is considered to be the predominant regulator of ß-adrenergic receptor-mediated enhancement of cardiac contractility; however, recent identification of exchange protein activated by cAMP (EPAC), which is independently activated by cAMP, has challenged this paradigm. Mice lacking Epac1 (Epac1 KO) exhibited decreased cardiac contractility with reduced phospholamban (PLN) phosphorylation at serine-16, the major PKA-mediated phosphorylation site. In Epac1 KO mice, intracellular Ca2+ storage and the magnitude of Ca2+ movement were decreased; however, PKA expression remained unchanged, and activation of PKA with isoproterenol improved cardiac contractility. In contrast, direct activation of EPAC in cardiomyocytes led to increased PLN phosphorylation at serine-16, which was dependent on PLC and PKCε. Importantly, Epac1 deletion protected the heart from various stresses, while Epac2 deletion was not protective. Compared with WT mice, aortic banding induced a similar degree of cardiac hypertrophy in Epac1 KO; however, lack of Epac1 prevented subsequent cardiac dysfunction as a result of decreased cardiac myocyte apoptosis and fibrosis. Similarly, Epac1 KO animals showed resistance to isoproterenol- and aging-induced cardiomyopathy and attenuation of arrhythmogenic activity. These data support Epac1 as an important regulator of PKA-independent PLN phosphorylation and indicate that Epac1 regulates cardiac responsiveness to various stresses.

Cardiohemodynamic and electrophysiological effects of anti-influenza drug oseltamivir in vivo and in vitro.

Electropharmacological effects of oseltamivir were studied in comparison with pilsicainide using halothane-anesthetized dogs (n = 4) and isolated left atrium of guinea pigs (n = 5). Oseltamivir (0.3, 3 and 30 mg/kg, i.v.) or pilsicainide (1 and 3 mg/kg, i.v.) was additionally administered to the dogs. The low dose of oseltamivir provided clinically relevant plasma concentrations with C max of 4 µM. The low and middle doses of oseltamivir increased cardiac output, whereas the middle dose increased blood pressure and delayed intra-atrial conduction and ventricular repolarization. The high dose of oseltamivir exerted negative chronotropic, inotropic and hypotensive effects, while it delayed intra-atrial, atrioventricular nodal and intra-ventricular conduction and ventricular repolarization. Use-dependent delay of ventricular repolarization was observed after oseltamivir, whereas reverse use-dependent prolongation was induced by pilsicainide. Moreover, oseltamivir more selectively suppressed intra-atrial conduction than intra-ventricular conduction, which was less selective for pilsicainide. Action potential assay using isolated atrium indicated that oseltamivir (10 µM) decreased V max more than pilsicainide (10 µM) and that oseltamivir (10-100 µM) prolonged action potential duration, which was not induced by pilsicainide (1-10 µM). Thus, oseltamivir in clinically relevant to its 10 times higher doses is relatively safe, whereas 10-100 times higher doses possess unique electrophysiological profile.

Electrophysiological and pharmacological properties of the pulmonary vein myocardium.

The pulmonary vein contains a myocardial layer extending from the left atrium, which is receiving attention as the source of ectopic electrical activity underlying atrial fibrillation. Electrophysiological and pharmacological analysis of the pulmonary vein myocardium performed in various experimental animal species have revealed characteristics such as presence of intracellular Ca(2+) oscillations and low repolarizing potency. The automaticity of the pulmonary vein myocardium is affected by various neurotransmitters, hormones and pharmacological agents. Clarification of the mechanisms and regulation of pulmonary vein automaticity would lead to the development of novel therapeutic strategies and pharmacological agents for atrial fibrillation.

Electrical activity of the mouse pulmonary vein myocardium.

We recorded the electrical activity from the myocardial layer of isolated mouse pulmonary veins with the glass microelectrode technique. Spontaneous electrical activity was observed in about half of the preparations, which appeared either as constant firing or as repetitive bursts. Noradrenaline enhanced, while acetylcholine reduced, automatic activity. The action potentials evoked in quiescent preparations showed a resting membrane potential less negative than the atria and an extremely rapid early repolarization followed by a late plateau. The present study revealed that the mouse pulmonary vein myocardium shows diverse electrical activity, which is influenced by autonomic neurotransmitters.

Electrophysiological effects of the class Ic antiarrhythmic drug pilsicainide on the guinea-pig pulmonary vein myocardium.

The pulmonary vein is known as an important source of ectopic beats, initiating frequent paroxysms of atrial fibrillation. We compared effects of the class Ic antiarrhythmic drug pilsicainide on the electrophysiological parameters in the isolated pulmonary vein preparation from guinea pigs with those in the left atrium. Three pairs of bipolar electrodes were attached to the left atrium, pulmonary vein, and junctional region of the left atrium and pulmonary vein to measure intra-atrial and intra-pulmonary vein conduction velocity and effective refractory period. Pilsicainide (10 µM) decreased the conduction velocity in the pulmonary vein as well as the left atrium, whose effect on the pulmonary vein was relatively greater than that on the left atrium. The drug prolonged the effective refractory period in the pulmonary vein as well as the left atrium, and the effect of the drug on the pulmonary vein was less than that on the left atrium. The currently observed electrophysiological property of pilsicainide suggests that its effects on reentry within the pulmonary vein are estimated to be weaker than within the left atrium, which may be one of the key considerations for understanding its antiarrhythmic mechanisms in the atrium and pulmonary vein.

Developmental changes in action potential prolongation by K+-channel blockers in chick myocardium.

The effects of K(+)-channel blockers on the action potential duration of the myocardium were examined in isolated right ventricles from the 7 - 10-day-old, 11 - 13-day-old, and 14 - 20-day-old embryo and 1 - 7-day-old hatched chicks. E-4031 significantly prolonged action potential duration at all developmental stages examined; the prolongation was largest in the 11 - 13-day-old embryo and was accompanied by early after-depolarizations. Chromanol 293B showed smaller prolongation at all stages examined. Terfenadine prolonged action potential duration in the 11 - 13-day-old embryo, but not in other stages. Thus, the chick ventricular myocardium changes its repolarization properties during development.

Blocking effect of NIP-142 on the KCNQ1/KCNE1 channel current expressed in HEK293 cells.

We examined the effect of NIP-142, a benzopyran compound with terminating effect on experimental atrial arrhythmia, on the KCNQ1/KCNE1 channel, which underlies the slow component of the cardiac delayed rectifier potassium channel (I(Ks)). NIP-142, as well as chromanol 293B, showed concentration-dependent blockade of the current expressed in HEK293 cells; the EC(50) value of NIP-142 and chromanol 293B for the inhibition of tail current was 13.2 µM and 4.9 µM, respectively. These results indicate that NIP-142 has blocking effect on the KCNQ1/KCNE1 channel current.

Electrophysiological and pharmacological characteristics of triggered activity elicited in guinea-pig pulmonary vein myocardium.

The pulmonary vein is known as an important source of ectopic beats, initiating frequent paroxysms of atrial fibrillation. We analyzed electrophysiological and pharmacological characteristics of triggered activity elicited in the isolated pulmonary vein from the guinea pig. Immediately after the termination of train stimulation (pacing cycle length of 100 ms), spontaneous activities accompanied with phase-4 depolarization were detected in 43 out of 45 pulmonary vein preparations. Such triggered activities were not observed in the isolated left atrium. The incidence of triggered activity was higher at a shorter pacing cycle length (100 - 200 ms), and the coupling interval was shorter at a shorter pacing cycle length. Verapamil (1 µM), ryanodine (0.1 µM), and pilsicainide (10 µM) suppressed the occurrence of triggered activities. The resting membrane potential of the pulmonary vein myocardium was more positive than that of the left atrium. Carbachol (0.3 µM) hyperpolarized the resting membrane potential and completely inhibited the occurrence of triggered activities. These results suggest that the pulmonary veins have more arrhythmogenic features than the left atrium, possibly through lower resting membrane potential. The electrophysiological and pharmacological characteristics of triggered activity elicited in the pulmonary vein myocardium were similar to those previously reported using ventricular tissues.