NEAT1-SOD2 Axis Confers Sorafenib and Lenvatinib Resistance by Activating AKT in Liver Cancer Cell Lines.

This study investigated the effects of a long noncoding RNA, nuclear paraspeckle assembly transcript 1 (NEAT1) variant 1 (NEAT1v1) on drug resistance in liver cancer cell lines. NEAT1 knockdown activated mitogen-activated protein kinase (MAPK) signaling pathways, including MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK), but suppressed AKT. Moreover, NEAT1 knockdown sensitized liver cancer cells to sorafenib and lenvatinib, both clinically used for treating hepatocellular carcinoma, whereas it conferred resistance to an AKT-targeted drug, capivasertib. NEAT1v1 overexpression suppressed MEK/ERK and activated AKT, resulting in resistance to sorafenib and lenvatinib and sensitization to capivasertib. Superoxide dismutase 2 (SOD2) knockdown reverted the effects of NEAT1v1 overexpression on the sensitivity to the molecular-targeted drugs. Although NEAT1 or SOD2 knockdown enhanced endoplasmic reticulum (ER) stress, concomitant with the suppression of AKT, taurodeoxycholate, an ER stress suppressor, did not restore AKT activity. Although further in vivo and clinical studies are needed, these results suggested that NEAT1v1 switches the growth modality of liver cancer cell lines from MEK/ERK-dependent to AKT-dependent mode via SOD2 and regulates sensitivity to the molecular-targeted drugs independent of ER stress.

NEAT1 Confers Radioresistance to Hepatocellular Carcinoma Cells by Inducing PINK1/Parkin-Mediated Mitophagy.

A long noncoding RNA, nuclear paraspeckle assembly transcript 1 (NEAT1) variant 1 (NEAT1v1), confers radioresistance to hepatocellular carcinoma (HCC) cells by inducing autophagy via γ-aminobutyric acid A receptor-associated protein (GABARAP). Radiation induces oxidative stress to damage cellular components and organelles, but it remains unclear how NEAT1v1 protects HCC cells from radiation-induced oxidative stress via autophagy. To address this, we precisely investigated NEAT1v1-induced autophagy in irradiated HCC cell lines. X-ray irradiation significantly increased cellular and mitochondrial oxidative stress and mitochondrial DNA content in HCC cells while NEAT1v1 suppressed them. NEAT1v1 concomitantly induced the phosphatase and tensin homolog-induced kinase 1 (PINK1)/parkin-mediated mitophagy. Interestingly, parkin expression was constitutively upregulated in NEAT1v1-overexpressing HCC cells, leading to increased mitochondrial parkin levels. Superoxide dismutase 2 (SOD2) was also upregulated by NEAT1v1, and GABARAP or SOD2 knockdown in NEAT1v1-overexpressing cells increased mitochondrial oxidative stress and mitochondrial DNA content after irradiation. Moreover, it was suggested that SOD2 was involved in NEAT1v1-induced parkin expression, and that GABARAP promoted parkin degradation via mitophagy. This study highlights the unprecedented roles of NEAT1v1 in connecting radioresistance and mitophagy in HCC.

NEAT1 Confers Radioresistance to Hepatocellular Carcinoma Cells by Inducing Autophagy through GABARAP.

A long noncoding RNA (lncRNA), nuclear enriched abundant transcript 1 (NEAT1) variant 1 (NEAT1v1), is involved in the maintenance of cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). CSCs are suggested to play important roles in therapeutic resistance. Therefore, we investigated whether NEAT1v1 is involved in the sensitivity to radiation therapy in HCC. Gene knockdown was performed using short hairpin RNAs, and NEAT1v1-overexpressing HCC cell lines were generated by stable transfection with a NEAT1v1-expressing plasmid DNA. Cells were irradiated using an X-ray generator. We found that NEAT1 knockdown enhanced the radiosensitivity of HCC cell lines and concomitantly inhibited autophagy. NEAT1v1 overexpression enhanced autophagy in the irradiated cells and conferred radioresistance. Gamma-aminobutyric acid receptor-associated protein (GABARAP) expression was downregulated by NEAT1 knockdown, whereas it was upregulated in NEAT1v1-overexpressing cells. Moreover, GABARAP was required for NEAT1v1-induced autophagy and radioresistance as its knockdown significantly inhibited autophagy and sensitized the cells to radiation. Since GABARAP is a crucial protein for the autophagosome-lysosome fusion, our results suggest that NEAT1v1 confers radioresistance to HCC by promoting autophagy through GABARAP.

Dried Saliva Spot (DSS) as a Convenient and Reliable Sampling for Bioanalysis: An Application for the Diagnosis of Diabetes Mellitus.

This paper proposes the dried saliva spot (DSS) as a convenient sampling technique for bioanalysis. The analytical method with the DSS was used for the determination of D,L-lactic acid (D,L-LA) and the D/L ratio of diabetic patients and prediabetic persons for the simple screening of the disease. The D,L-LA in the DSS was labeled with a chiral reagent (DMT-3(S)-Apy) for carboxylic acids and determined by UPLC-ESI-MS/MS. The limits of detection (signal-to-noise ratio (S/N) = 3) for the DSS analysis were on the amol level (∼30 amol). Because good stability, recovery, accuracy, and precision of the D,L-LA for the DSS method was also obtained from the proposed procedure, the DSS method was applied to the determination of the D- and L-isomers of LA of diabetic patients, and prediabetic and healthy persons. The D/L-LA ratio by the present DSS method and the HbA1c value in blood were well-correlated to the serious diabetic patients, whereas the relation in the prediabetic persons was not very good. The reason seems to be due to the rough saliva sampling, and not to the DSS method, because strict regulation was not requested for the prediabetic and healthy persons. In order to have a successful DSS analysis, the stability of the target molecule, the detection sensitivity to the target molecule, and the validated determination method are important.

Profiling of chiral and achiral carboxylic acid metabolomics: synthesis and evaluation of triazine-type chiral derivatization reagents for carboxylic acids by LC-ESI-MS/MS and the application to saliva of healthy volunteers and diabetic patients.

Novel triazine-type chiral derivatization reagents, i.e., (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine (DMT-3(S)-Apy) and (S)-4,6-dimethoxy-N-(pyrrolidin-3-yl)-1,3,5-triazin-2-amine (DMT-1(S)-Apy), were developed for the highly sensitive and selective detection of chiral carboxylic acids by UPLC-MS/MS analysis. Among the synthesized reagents, DMT-3(S)-Apy was a more efficient chiral reagent for the enantiomeric separation of chiral carboxylic acids in terms of separation efficiency by reversed-phase chromatography and detection sensitivity by ESI-MS/MS. The DMT-3(S)-Apy was used for the determination of 13 carboxylic acids in human saliva of healthy volunteers and diabetic patients. Various biological carboxylic acids including chiral carboxylic acids, and mono- and di-carboxylic acids were clearly identified in the saliva of healthy persons and diabetic patients. The concentrations of carboxylic acids detected in the saliva of diabetic patients were relatively higher than those in the healthy persons. Furthermore, the concentration of D-lactic acid (LA) and the ratio of D/L-LA in the diabetic patients were significantly higher than those in the healthy persons. The low ratio of D/L-LA in healthy persons was also identified to be independent of age and sex. These results suggest that the determination of the D/L-LA ratio in saliva might be applicable for the diagnosis of diabetes. Based on these observations, DMT-3(S)-Apy seems to be a useful chiral derivatization reagent for the determination not only of chiral carboxylic acids but also achiral ones. In conclusion, the proposed method using DMT-3(S)-Apy is useful for the carboxylic acid metabolomics study of various specimens.

Conjunctivitis caused by Neisseria gonorrhoeae isolates with reduced cephalosporin susceptibility and multidrug resistance.

We report two cases of conjunctivitis caused by Neisseria gonorrhoeae with reduced cephalosporin susceptibility. Patients showed no response to cefmenoxime eye drops and intravenous ceftriaxone administration. The patients' condition improved after the addition of oral minocycline. The isolates contained the mosaic penA for reduction of ß-lactam susceptibility.

Simultaneous determination of DL-lactic acid and DL-3-hydroxybutyric acid enantiomers in saliva of diabetes mellitus patients by high-throughput LC-ESI-MS/MS.

A simultaneous determination method for the enantiomers of chiral carboxylic acids by the combination of ultraperformance liquid chromatography and mass spectrometry (UPLC-MS/MS) has been developed. (S)(+)-1-(2-Pyrrolidinylmethyl)-pyrrolidine (S-PMP) was used as the derivatization reagent for the high-throughput determination of biological chiral carboxylic acids, i.e., lactic acid (LA) and 3-hydroxybutyric acid (HA). The S-PMP efficiently reacted with the carboxylic acids under mild conditions at room temperature in the presence of 2,2'-dipyridyl disulfide and triphenylphosphine. The resulting S-PMP derivatives were highly responsive in the electrospray ionization (ESI)-MS operating in the positive-ion mode and gave characteristic product ions during the MS/MS, which enabled the sensitive detection using selected reaction monitoring. The derivatization was effective for the enantiomeric separation of the chiral carboxylic acids, and the resolution values of DL-LA and DL-HA were 4.91 and 9.37, respectively. Furthermore, a rapid separation of the derivatives of DL-LA and DL-HA within 7 min was performed using the UPLC system. The limits of detection on the column were in the low femtogram range (5-12 fg). The proposed procedure was successfully applied for the determination of the D- and L-isomers of LA and HA in the saliva of diabetes mellitus (DM) patients and healthy volunteers. The D-LA in DM patients was clearly higher than that in normal subjects. The derivatization followed by UPLC-ESI-MS/MS enabled the enantiomeric separation and detection of trace amounts of LA and HA in human saliva with a simple pretreatment and small sample volume.

Augmentation of serotonin release by sustained exposure to MDMA and methamphetamine in rat organotypic mesencephalic slice cultures containing raphe serotonergic neurons.

Several lines of evidence suggest the involvement of the raphe-serotonergic neurons in addiction to psychostimulants and some recreational drugs. In this study, we established rat organotypic mesencephalic slice cultures containing the raphe nuclei and examined the effects of sustained exposure to 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (METH). Immunostaining for tryptophan hydroxylase (TPH) studies revealed that serotonergic neurons were abundant in the slice cultures. Sustained exposure to MDMA and METH (1-1000 microM) for 4 days had little effect on the serotonin tissue content, [(3)H]citalopram binding, or expression/phosphorylation of TPH. Treatment with MDMA or METH for 30 min increased serotonin release in a concentration-dependent manner. Slice cultures were exposed to MDMA for 4 days following a 1-day withdrawal period and then challenged with MDMA (10 microM). Sustained MDMA exposure augmented MDMA-induced serotonin release in a concentration-dependent manner, indicating serotonergic sensitization. Similar serotonergic sensitization was observed for METH. The development of MDMA-induced serotonergic sensitization was attenuated by the NMDA receptor antagonist, MK-801 (10 microM). These results suggest that in mesencephalic slice cultures sustained MDMA or METH exposure induces serotonergic sensitization through activation of NMDA receptors without serotonergic neurotoxicity. The in vitro model system could help to elucidate the mechanisms underlying drug addiction.

Case of lipoatrophic diabetes induced by juvenile dermatomyositis.

Lipodystrophy is a rare condition that is often accompanied by one or more metabolic diseases. Here, we report a case of lipoatrophic diabetes induced by juvenile dermatomyositis. Although pioglitazone was not effective for lowering blood glucose levels, our observation suggested that it improved liver function slightly. The effectiveness of metreleptin for lowering blood glucose levels could not be determined, as we administered it in a short period. Liver biopsy showed burned-out non-alcoholic steatohepatitis. The present results show that the successful treatment of lipoatrophic diabetes induced by juvenile dermatomyositis requires an early diagnosis and therapeutic intervention.

Aziridination of Activated Imines with Monocarbonyl Iodonium Ylides Generated from (Z)-(2-Acetoxyvinyl)iodonium Salts via Ester Exchange: Stereoselective Synthesis of 2-Acylaziridines.

Monocarbonyl iodonium ylides, generated in situ from (Z)-(2-acetoxyvinyl)iodonium salts via an ester exchange reaction with EtOLi, undergo alkylidene transfer reactions to activated imines yielding 2-acylaziridines in good yields. The stereochemical outcome of this aziridination was shown to be dependent on both the activating groups of the imines and the reaction solvents: that is, the aziridination of N-(2,4,6-trimethylbenzenesulfonyl)imines in THF affords cis-aziridines as a major product while that of N-benzoylimines in THF-DMSO or THF gives the trans isomer stereoselectively.

alpha-Vinylation of 1,3-Dicarbonyl Compounds with Alkenyl(aryl)iodonium Tetrafluoroborates: Effects of Substituents on the Aromatic Ring and of Radical Inhibitors.

Direct alpha-vinylations of enolate anions derived from 1,3-dicarbonyl compounds with 4-tert-butyl-1-cyclohexenyl(aryl)iodonium 2 and 1-cyclopentenyl(aryl)iodonium tetrafluoroborates 3 are reported. Frequently, alpha-phenylations compete with the vinylations in the reaction of 1,3-dicarbonyl compounds with alkenyl(phenyl)iodonium salts 2a and 3a. Use of alkenyl(p-methoxyphenyl)iodonium salts 2b and 3b, however, leads to selective alpha-vinylation at the expense of the competing arylation of 1,3-dicarbonyl compounds. Use of an efficient aryl radical trap, 1,1-diphenylethylene, inhibits radical-induced decomposition of the alkenyl(aryl)iodonium salts, thereby improving the yields of alpha-vinylations of enolate anions derived from 1,3-dicarbonyl compounds.

Nucleophilic Vinylic Substitutions of (Z)-(beta-Haloalkenyl)phenyliodonium Salts with Sodium Benzenesulfinate: First Evidence of a Michael Addition of Nucleophiles to Alkenyliodonium Salts at the C(beta) Atom.

Evidence for a Michael addition of a nucleophile to alkenyl(phenyl)iodonium salts at the C(beta) atom is reported here for the first time. Nucleophilic vinylic substitutions of (Z)-(beta-chloroalkenyl)- 2b and (Z)-(beta-bromoalkenyl)iodonium tetrafluoroborates 3b with sodium benzenesulfinate in THF afforded stereoselectively (Z)-1,2-bis(benzenesulfonyl)alkene 5b with retention of configuration. Intermediate formation of (Z)-(beta-(benzenesulfonyl)alkenyl)iodonium salt 9b in these reactions was established by (1)H NMR experiments in CDCl(3). The formation of (Z)-9b involves a hitherto unobserved Michael addition of benzenesulfinate anion to the alkenyliodonium salts at the C(beta) atom, followed by halogen extrusion. The formation of a stereoisomeric mixture of (Z)- and (E)-bis-sulfones 5b, and 1-(benzenesulfonyl)cyclopentene 11 that was observed in the reaction of (Z)-(beta-fluoroalkenyl)iodonium salt 4b in CDCl(3), strongly suggests the intermediacy of 9b in this nucleophilic vinylic substitution.

Postextrasystolic contractility normally decays in alternans in canine in situ heart.

We have reported that the postextrasystolic (PES) potentiation of left ventricular (LV) contractility usually decays in alternans at heart rates above 80-100 beats/min in the canine excised, cross-circulated heart. We examined whether the PES contractility would also decay in alternans even in the canine in situ heart presumably more physiological than the excised heart. In anesthetized, ventilated, and open-chest mongrel dogs, we measured LV pressure and volume with a micromanometer and a conductance catheter cannulated into the LV and obtained LV end-systolic maximum elastance (E(max)) as the reasonably load-independent contractility index. We inserted an extrasystole followed by a compensatory pause into steady-state regular beats at heart rates above 90 beats/min and analyzed the PES decay pattern of E(max). We found that E(max) potentiated in the first PES beat decayed in alternans within 5-6 PES beats. This indicates that PES contractility also decays in alternans in the normal canine in situ heart.

Determination of acetone in saliva by reversed-phase liquid chromatography with fluorescence detection and the monitoring of diabetes mellitus patients with ketoacidosis.

BACKGROUND: In diabetes mellitus (DM) patients with ketoacidosis, ketone bodies, i.e., acetone, acetoacetic acid (AA) and ß-hydroxybutyric acid (HA), are increased in the blood and urine. Acetone is also excreted by breathing due to the spontaneous decomposition of AA. Thus, the increase in acetone has been considered as one of the biomarkers for the diagnosis of DM. However, the determination of acetone in one's breath is not recommended because of the sample handling difficulty. We measured acetone in saliva by reversed-phase liquid chromatography (LC) with fluorescence (FL) detection. The proposed method was applied to the determination of acetone in the saliva of healthy volunteers and DM patients with and without ketoacidosis. METHODS: 3-Pentanone (I.S.) and DBD-H in acetonitrile were added to freshly collected saliva and reacted at room temperature for 20 min in the presence of trifluoroacetic acid. After the reaction, the solution was centrifuged at 10,000 × g and 4 °C for 5 min. The supernatant was separated by reversed-phase LC and the FL detected at 550 nm (excitation at 460 nm). RESULTS: The concentrations of acetone in the DM patients with ketoacidosis were significantly higher than those of the normal subjects and DM patients without ketoacidosis. Furthermore, the total contents of the ketone bodies in the blood correlated with acetone in the saliva of the DM patients. The concentrations of acetone in the saliva of an emergency patient also correlated with the ketone bodies in the blood at each sampling time. CONCLUSION: The proposed method using LC-FL seems to be useful for the determination of acetone in the saliva of DM patients with ketoacidosis. The method offers a new option for the diagnosis and monitoring of DM patients with ketoacidosis.

Roles of MED1 in quiescence of hair follicle stem cells and maintenance of normal hair cycling.

MED1 (mediator complex subunit 1) is expressed by human epidermal keratinocytes and functions as a coactivator of several transcription factors. To elucidate the role of MED1 in keratinocytes, we established keratinocyte-specific Med1-null (Med1(epi-/-)) mice using the K5Cre/LoxP system. Development of the epidermis and appendages of Med1(epi-/-) mice were macroscopically and microscopically normal until the second catagen of the hair cycle. However, the hair cycle of Med1(epi-/-) mice was spontaneously repeated after the second telogen, which does not occur in wild-type (WT) mice. Hair follicles of Med1(epi-/-) mice could not enter anagen after 6 months of age, resulting in sparse pelage hair in older Med1(epi-/-) mice. Interfollicular epidermis (IFE) of Med1(epi-/-) mice was acanthotic and more proliferative than that of WT mice, whereas these findings were less evident in older Med1(epi-/-) mice. Flow cytometric analysis revealed that the numbers of hair follicle bulge stem cells were reduced in Med1(epi-/-) mice from a few months after birth. These results suggest that MED1 has roles in maintaining quiescence of keratinocytes and preventing depletion of the follicular stem cells.

Utility of organotypic raphe slice cultures to investigate the effects of sustained exposure to selective 5-HT reuptake inhibitors on 5-HT release.

BACKGROUND AND PURPOSE: Selective 5-hydroxytryptamine (5-HT, serotonin) reuptake inhibitors (SSRIs) are widely used antidepressants and their therapeutic effect requires several weeks of drug administration. The delayed onset of SSRI efficacy is due to the slow neuroadaptive changes of the 5-hydroxytryptaminergic (5-HTergic) system. In this study, we examined the acute and chronic effects of SSRIs on the 5-HTergic system using rat raphe slice cultures. EXPERIMENTAL APPROACH: For organotypic raphe slice cultures, mesencephalic coronal sections containing dorsal and median raphe nuclei were prepared from neonatal Wistar rats and cultured for 14-16 days. KEY RESULTS: Acute treatment with citalopram, paroxetine or fluoxetine (0.1-10 µM) in the slice cultures slightly increased extracellular 5-HT levels, while sustained exposure for 4 days augmented the elevation of 5-HT level in a time-dependent manner. Sustained exposure to citalopram had no effect on tissue contents of 5-HT and its metabolite, expression of tryptophan hydroxylase or the membrane expression of 5-HT transporters. The augmented 5-HT release was attenuated by Ca(2+) -free incubation medium or treatment with tetrodotoxin. Experiments with 5-HT(1A/B) receptor agonists and antagonists revealed that desensitization of 5-HT(1) autoreceptors was not involved in the augmentation of 5-HT release. Finally, co-treatment with an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate, but not an N-methyl-d-aspartate, receptor antagonist, suppressed this augmentation. CONCLUSION AND IMPLICATIONS: These results suggest that sustained exposure to SSRIs induces the augmentation of exocytotic 5-HT release, which is caused, at least in part, by the activation of AMPA/kainate receptors in the raphe slice cultures.

Isoproterenol-induced hypertrophied rat hearts: does short-term treatment correspond to long-term treatment?

In consideration of clinical implications, it is often complained that short-term experimental diseased heart models do not mimic long-term diseased hearts that are often clinically encountered. The aim of the present study was (i) to compare the left ventricular function between rat cardiac hypertrophy models treated with isoproterenol for 3 days (Iso 3d) and 7 days (Iso 7d) by pressure-volume measurements with a catheter method, and (ii) to follow up the left ventricular function in the same model treated with Iso up to 16 weeks with a less-invasive echocardiography. An infusion of either Iso (1.2 mg x kg(-1) x day(-1) for 3 days-16 weeks) or vehicle (saline 24 microl x day(-1) for 3 days-16 weeks; Sa group) was performed by subcutaneously implanting an osmotic minipump. There were no significant differences in the systolic pressure-volume area at midrange left ventricular volume (PVA(mLVV): a mechanical work capability index) between Iso 3d and 7d groups, though PVA(mLVV) in both groups was significantly reduced from that in the Sa group. From echocardiography, the left ventricular function of the hypertrophy models at 3 days, 1 week, and 2 weeks was unchanged, but the model at a term longer than 4 weeks resulted in prolonged systolic failure. The results indicated that (i) no marked differences in the left ventricular mechanical work capability were found between the Iso 3d and 7d groups, and that (ii) only a 3-day Iso infusion induced the hypertrophy model similar in shape and function to that induced by a 2-week Iso infusion. We concluded that the 3-day model was sufficient.

Effects of a novel histone deacetylase inhibitor, N-(2-aminophenyl) benzamide, on a reversible hypertrophy induced by isoproterenol in in situ rat hearts.

The aim of the present study was performed to determine whether a novel histone deacetylase (HDAC) inhibitor, N-(2-aminophenyl)-4-{[benzyl(2-hydroxyethyl)amino]methyl} benzamide (K-183), prevents a reversible cardiac hypertrophy induced by isoproterenol and improves left ventricular (LV) dysfunction in rats. Either isoproterenol or vehicle was infused for 3 days by osmotic minipump. One hour prior to the implantation of isoproterenol, K-183 or trichostatin A (TSA) was injected twice a day for 3 days. We recorded continuous LV pressure-volume (P-V) loops of in situ hearts one hour after removal of the osmotic minipump. LV work capability (systolic P-V area at midrange LV volume: PVA(mLVV)) and hemodynamics were evaluated. K-183 per se induced neither cardiac hypertrophy nor collagen production. Although K-183 did not prevent the hypertrophy, where PVA(mLVV) remained decreased, K-183, differently from TSA, significantly attenuated the decrease of cardiac output and the increase of effective arterial elastance in the hypertrophied heart. These results indicate that the novel HDAC inhibitor K-183 has some beneficial effects on hemodynamics, although K-183 has no effects of anti-hypertrophic modalities.

Positive inotropic effect of nicorandil in adult rats in situ.

PURPOSE: Although the cardiac protective and negative inotropic effects of nicorandil via opening the sarcolemmal or mitochondrial K(ATP) channel and NO-like action are well known, positive inotropic effects of nicorandil in normal hearts have not been reported. The aim of the current study was to investigate how nicorandil affects left ventricular (LV) function of in situ adult rat hearts through the entire cardiovascular system. METHODS: We performed simultaneous and continuous measurements of LV pressure (P) by a catheter-tip micromanometer and LV volume (V) using a conductance catheter. We obtained steady-state LV P-V loops and intermittent curvilinear LV end-systolic pressure-volume relations (ESPVRs), LV end-systolic pressure (ESP(mLVV)), and systolic pressure-volume area (PVA(mLVV)) at midrange LVV (mLVV). We evaluated the effects of nicorandil on LV function by these mechanical indexes sensitively reflecting changes in LV contractility and work capability, preload (end-diastolic volume, EDV), and afterload (effective arterial elastance, Ea). RESULTS: Nicorandil (10 and 20 microg.kg(-1).min(-1)) (blood concentration: 0.53 +/- 0.14 and 1.48 +/- 0.21 microg/ml) largely shifted ESPVR upward and thus significantly increased ESP(0.06), PVA(0.06), stroke volume due to increase in EDV, and ejection fraction without significant changes in Ea and heart rate. In contrast, an NO donor, nitroglycerin (1 microg.kg(-1) . min(-1)), significantly decreased Ea but did not change ESP(0.06), PVA(0.06), and EDV. Furthermore, either a nonselective K(ATP) channel blocker, glybenclamide, or a mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate, abolished these nicorandil-induced positive inotropic actions. CONCLUSION: These results suggest that nicorandil exhibited positive inotropic actions on LV function of in situ hearts in adult rats with resultant increased preload (EDV).