Nicorandil restores the downregulated eNOS expression and attenuates the upregulated caspase-3 expression in MCT-injured lungs.

<p>(<b>A</b>) The expressions of eNOS and cleaved caspase-3 in lungs were determined by western blotting. MCT significantly downregulated the expression of eNOS and inversely upregulated the expression of cleaved caspase-3 (<i>see</i> vehicle). Nicorandil and ZVAD-fmk restored eNOS expression and attenuated caspase-3 expression in MCT-injured lungs, which were blocked by glibenclamide and l-NAME. Bands were quantified using a lumino-analyzer, and the data of eNOS (<b>B</b>) and caspase-3 (<b>C</b>) expressions were expressed as fold increases normalized to β-actin expression. ^#<i>P</i><0.01 vs. normal control; <b>^*</b><i>P</i><0.05 vs. vehicle; <b>^†</b><i>P</i><0.05 and <b>^‡</b><i>P</i><0.01 vs. nicorandil (5.0 mg·kg⁻¹·day⁻¹).</p

Nicorandil prevents the induction of vascular endothelial cell apoptosis in vitro.

<p>(<b>A</b>) The HUVECs were incubated in the serum-free medium in the absence or presence of nicorandil (10–1000 µmol/L) for 48 h. The serum-starved HUVECs exhibited apoptotic morphology, which is characterized by cell shrinkage (<i>see</i> “serum starved”). (<b>B</b>) The viability of the HUVECs was measured with the MTS assay, and the percent cell death was calculated. Stimulation with nicorandil and diazoxide partially restored cell viability in a concentration-dependent manner. VEGF, vascular endothelial growth factor (positive control). <b>^*</b><i>P</i><0.05 and <b>^**</b><i>P</i><0.01 vs. control (no drug). (<b>C</b>) TUNEL staining revealed that a large number of the serum-starved HUVECs exhibited apoptotic morphology (left), and the apoptotic effects induced by serum starvation were attenuated by nicorandil (middle). TUNEL (green); nuclei (blue). (<b>D</b>) The TUNEL-positive nuclei in the serum-starved HUVECs were counted in 10 randomly selected fields and expressed as a percentage of the total number of nuclei. <b>^**</b><i>P</i><0.01 vs. control (no drug).</p

The effects of nicorandil in the reversal protocol.

<p>The RVSP (<b>A</b>) and the RV/LV ratio (<b>B</b>) in the vehicle group increased at 21 days after the MCT injection (MCT-21) as compared to the baseline, and additionally increased in the next 2 weeks (MCT-35). Late treatment with nicorandil on days 21–35 prevented the additional increase in these parameters, while these effects were blocked by glibenclamide and l-NAME. ^#<i>P</i><0.05 and ^##<i>P</i><0.01 vs. normal control; <b>^*</b><i>P</i><0.05 and <b>^**</b><i>P</i><0.01 vs. vehicle (MCT-35); <b>^†</b><i>P</i><0.05 vs. nicorandil (5.0 mg·kg⁻¹·day⁻¹). (<b>C</b>) The histological findings of the PAs (arrows) in the reversal protocol. Top, HE staining; bottom, EVG staining. Scale bar, 50 µm. (<b>D</b>) Late treatment with nicorandil on days 21–35 prevented further increase in the percent medial wall thickness of the PAs. The indications of the symbols are the same as those in panel (A). (<b>E</b>) Survival analysis in the reversal protocol. Each group comprised 14–15 rats. <b>^*</b><i>P</i><0.05 vs. vehicle.</p

The effects of nicorandil in the prevention protocol.

<p>The RVSP (<b>A</b>) and the RV/LV ratio (<b>B</b>) 28 days after MCT injection are shown. Treatment with nicorandil and ZVAD-fmk attenuated the MCT-induced increase in both these parameters, while these effects of nicorandil were blocked by glibenclamide and l-NAME. Each group comprised 8–10 rats. ^#<i>P</i><0.01 vs. normal control; <b>^*</b><i>P</i><0.05 and <b>^**</b><i>P</i><0.01 vs. vehicle; <b>^†</b><i>P</i><0.05 and <b>^‡</b><i>P</i><0.01 vs. nicorandil (5.0 mg·kg⁻¹·day⁻¹). (<b>C</b>) Histological findings of the PAs (arrows). Top, hematoxylin and eosin (HE) staining; bottom, elastic Van Gieson (EVG) staining. Scale bar, 50 µm. (<b>D</b>) MCT markedly increased the percent medial wall thickness of the PAs (#), and nicorandil and ZVAD-fmk attenuated MCT-induced medial wall thickening (<b>**</b>). In contrast, the effects of nicorandil were inhibited by glibenclamide and l-NAME (<b>‡</b>). (<b>E</b>) Survival analysis in the prevention protocol. Each group comprised 12–13 rats. <b>^*</b><i>P</i><0.05 vs. vehicle.</p

Nicorandil improves the histopathological findings of MCT-injured lungs.

<p>(<b>A</b>) Immunohistochemical staining for αSMA (<b>a</b>), CD68 (<b>b</b>), and eNOS (<b>c</b>) in lung sections are shown. Histopathologically, MCT injury induced the thickened medial wall of PAs (arrows) that was composed of αSMA-positive cells, the recruitment of CD68-positive macrophages into the perivascular areas, and the deficiency of eNOS expression in the endothelium of the pulmonary vasculature (<i>see</i> vehicle). Nicorandil and ZVAD-fmk improved these deleterious histopathological changes in the MCT-injured lungs, whereas glibenclamide and l-NAME blocked the effects of nicorandil. Scale bar, 50 µm. (<b>B</b>) MCT readily induced thromboemboli formation that occluded the small PAs (arrows; <i>see</i> vehicle), which was attenuated by nicorandil. HE staining. Scale bars, 100 µm (top) and 50 µm (bottom). (<b>C</b>) The number of small PAs that were occluded by thromboemboli was counted in the lung sections. (<b>D</b>) TUNEL staining with immunofluorescence staining for αSMA in lung sections. MCT readily induced endothelial cell apoptosis (arrowheads) in a number of PAs (arrows; <i>see</i> vehicle), which was attenuated by nicorandil. TUNEL (green); αSMA (red); and nuclei (blue). Scale bars, 100 µm (top) and 20 µm (bottom). (<b>E</b>) The number of small PAs having TUNEL-positive endothelial cell(s) [EC(s)] was counted in lung sections, and the proportion of PAs with TUNEL-positive EC(s) in a total of equally sized PAs was calculated. ^#<i>P</i><0.01 vs. normal control; <b>^*</b><i>P</i><0.05 and <b>^**</b><i>P</i><0.01 vs. vehicle; <b>^†</b><i>P</i><0.05 and <b>^‡</b><i>P</i><0.01 vs. nicorandil (5.0 mg·kg⁻¹·day⁻¹).</p

Nidorandil attenuates proliferation of PA-SMCs in MCT-injured lungs.

<p>Immunofluorescent double staining of lung frozen sections for Ki67 and αSMA was performed. The number of proliferating PA-SMCs with Ki67 positive nuclei expressed as the percentage of Ki67-positive cells over the total number of αSMA-positive SMCs in the media of 30–40 PAs (external diameter, 20–100 µm) per rat was significantly reduced by treatment with nicorandil and ZVAD-fmk. In contrast, glibenclamide and l-NAME diminished the effect of nicorandil, respectively. ^#<i>P</i><0.01 vs. normal control; <b>^*</b><i>P</i><0.05 and <b>^**</b><i>P</i><0.01 vs. vehicle; <b>^‡</b><i>P</i><0.01 vs. nicorandil (5.0 mg·kg⁻¹·day⁻¹).</p

Nicorandil activates the PI3K/Akt and ERK pathways in HUVECs.

<p>(<b>A</b>) Nicorandil and diazoxide increased Akt serine-473 and GSK-3 phosphorylation in the serum-starved HUVECs, while these effects of nicorandil were blocked by glibenclamide and the PI3K inhibitor LY294002. (<b>B</b>) The Akt phosphorylation (p-Akt) data are expressed as fold increases normalized to the total Akt (t-Akt) expression. IGF, insulin-like growth factor (positive control). <b>^**</b><i>P</i><0.01 vs. control (no drug); <b>^‡</b><i>P</i><0.01 vs. 60-min nicorandil treatment. (<b>C</b>) Nicorandil and diazoxide also increased ERK1/2 threonine-202/204 and Bad serine-112 phosphorylation in the HUVECs, while these effects of nicorandil were blocked by glibenclamide and the MEK inhibitor PD98059. (<b>D</b>) The ERK phosphorylation (p-ERK) data are expressed as fold increases normalized to the total ERK (t-ERK) expression. <b>^*</b><i>P</i><0.05 and <b>^**</b><i>P</i><0.01 vs. control (no drug); <b>^†</b><i>P</i><0.05 and <b>^‡</b><i>P</i><0.01 vs. 15-min nicorandil treatment. (<b>E</b>) In serum-starved HUVECs treated with nicorandil for 24 h, the expression of eNOS and Bcl-2 was upregulated in a concentration-dependent manner, while these effects were blocked by glibenclamide, LY294002 (data not shown), and PD98059. (<b>F</b>) eNOS and Bcl-2 expression data are expressed as fold increases normalized to the β-actin expression. <b>^*</b><i>P</i><0.05 and <b>^**</b><i>P</i><0.01 vs. control (no drug); <b>^‡</b><i>P</i><0.01 vs. 100-µmol/L nicorandil treatment.</p

Nicorandil restores eNOS expression in the pulmonary vascular endothelium in established PAH.

<p>(<b>A</b>) Immunohistochemical staining for eNOS in lung sections in the reversal protocol. Arrows indicate the PAs. Scale bar, 50 µm. (<b>B</b>) Detection of eNOS expression in lungs by western blotting in this protocol. MCT downregulated eNOS expression in a time-dependent manner, whereas late treatment with nicorandil resulted in partial restoration of eNOS expression. (<b>C</b>) Bands in panel (B) were quantified, and eNOS expression data were expressed as fold increases normalized to β-actin expression. ^#<i>P</i><0.01 vs. normal control; ^##<i>P</i><0.01 vs. normal control and <i>P</i><0.05 vs. MCT-14; <b>^*</b><i>P</i><0.01 vs. MCT-35; <b>^†</b><i>P</i><0.01 vs. nicorandil (5.0 mg·kg⁻¹·day⁻¹).</p

Modulation of Luminescence Intensity of Lanthanide Complexes by Photoinduced Electron Transfer and Its Application to a Long-Lived Protease Probe

Luminescent lanthanide complexes (Tb3+, Eu3+, etc.) have excellent properties for biological applications, including extraordinarily long lifetimes and large Stokes shifts. However, there have been few reports of lanthanide-based functional probes, because of the difficulty in designing suitable complexes with a luminescent on/off switch. Here, we have synthesized a series of complexes which consist of three moieties:  a lanthanide chelate, an antenna, and a luminescence off/on switch. The antenna is an aromatic ring which absorbs light and transmits its energy to the metal, and the switch is a benzene derivative with a different HOMO level. If the HOMO level is higher than a certain threshold, the complex emits no luminescence at all, which indicates that the lanthanide luminescence can be modulated by photoinduced electron transfer (PeT) from the switch to the sensitizer. This approach to control lanthanide luminescence makes possible the rational design of functional lanthanide complexes, in which the luminescence property is altered by a biological reaction. To exemplify the utility of our approach to the design of lanthanide complexes with a switch, we have developed a novel protease probe, which undergoes a significant change in luminescence intensity upon enzymatic cleavage of the substrate peptide. This probe, combined with time-resolved measurements, was confirmed in model experiments to be useful for the screening of inhibitors, as well as for clinical diagnosis

Highly Sensitive Near-Infrared Fluorescent Probes for Nitric Oxide and Their Application to Isolated Organs

Novel near-infrared (NIR) fluorescent probes for nitric oxide (NO) have been designed, synthesized, and evaluated. Their NIR fluorescence was increased in an NO concentration-dependent manner under physiological conditions, and their reaction efficiency with NO was at least 53 times higher than that of a widely used NO probe, DAF-2. They were confirmed to function in isolated intact rat kidneys. Because NIR light can penetrate deeply into tissues, these probes may have potential for in vivo NO imaging