Matomo Nishio

Increased basal levels of plasma nitric oxide in Type 2 diabetic subjects Relationship to microvascular complications

To assess the underlying mechanisms of decreased endothelial function and advanced vascular complications in patients with Type 2 diabetes, we determined basal levels of plasma nitric oxide (NO(x): NO(2)(-) and NO(3)(-)) using a newly developed high-performance liquid chromatography (HPLC)-Griess method in hospitalized 129 diabetic and 76 nondiabetic subjects, and examined their clinical characteristics. Serum lipid peroxide and advanced glycation end products (AGEs) as markers of oxidative stress were also measured, and intima-media thickness (IMT) of the carotid artery was evaluated as a marker of atherosclerosis. In diabetic subjects, microvascular complications were newly evaluated during their admission. There were no differences in age or sex between the diabetic and nondiabetic subjects. Although there was no difference in basal plasma NO(2)(-) levels between the two groups, the basal levels of plasma NO(3)(-) in diabetic subjects were significantly higher than those in nondiabetic subjects. Plasma NO(x) levels in neither diabetic nor nondiabetic subjects correlated with serum lipids, HbA1c, or IMT. In diabetic subjects, plasma NO(3)(-) levels were related not only to the presence of hypertension but also to advanced microvascular complications. Moreover, plasma NO(3)(-) levels were positively correlated with both serum lipid peroxide and AGEs. Multiple regression analysis revealed that serum AGEs level was strongly associated with plasma NO(3)(-) level. Thus, the findings are consistent with the hypothesis that decreased endothelium-dependent vasodilation in diabetic subjects is associated with the impaired action of NO secondary to its inactivation resulting from increased oxidative stress, rather than decreased NO production from vascular endothelium, and that abnormal NO metabolism is related to advanced diabetic microvascular complications.

Single ionic channels induced by palytoxin in guinea-pig ventricular myocytes.

1 Mechanisms of palytoxin‐induced ion permeability were examined in isolated single ventricular cells of guinea‐pig under whole‐cell‐attached patch clamp conditions. 2 Palytoxin (1–2 × 10−11 m, dissolved in Tyrode solution and put in the patch electrode) induced an elementary current flowing through single channels. Direction of the current was inward and the amplitude was 0.65 ± 0.03 pA (mean ± s.e. mean) at the resting membrane potential. The amplitude increased linearly with membrane hyperpolarization and decreased with depolarization; the single channel conductance was 9.5 ± 0.5 pS. 3 Palytoxin‐induced single channel current was resistant to tetrodotoxin (5 × 10−5 m) or cobalt ions (2 × 10−3 m) and was observed under Ca‐free conditions. However, no channel current was induced by palytoxin (10−11‐10−9 m) dissolved in Na+‐free, choline‐Tyrode solution. 4 Palytoxin also induced single channel currents in Na+‐free, NH+4‐, Li+‐ or Cs+‐Tyrode solution, and the slope conductances were 16.5 ± 1.6 pS, 9.2 ± 0.7 pS and 11.0 ± 0.7 pS, respectively. 5 These results indicate that palytoxin forms a new type of ionic channel with unique ion selectivity and gating behaviour.

Negative NO3− difference in human coronary circulation with severe atherosclerotic stenosis

To examine whether or not the levels of NOx (nitrite; NO2- and nitrate; NO3-) in coronary circulating blood reflect endothelial dysfunction due to coronary atherosclerosis, NOx levels in plasma obtained from ostium of left coronary artery and coronary sinus of patients who complained of chest pain were evaluated in relation to their coronary angiographic findings. Prior to the study, a HPLC-Griess system for NOx measurement was critically evaluated. This system has a detection limit of 0.1 microM of NO2- and NO3- by 10 microl of loading and was able to distinguish a difference of 0.1-0.2 microM of these substances. Heparin (1 U/10 microl) did not affect the detective and discriminative abilities. NO3- difference, calculated from sino-arterial difference of NO3-, was almost zero (-0.2 +/- 0.2 microM) in patients with either normal coronary arteries or mild organic coronary stenosis (< or = 20% narrowing), while a significant negative value (-5.9 +/- 1.7 microM) was obtained from patients with significant stenosis (> or = 70% narrowing) in the left coronary arteries. These results demonstrate reliable ability on the HPLC-Griess system in evaluating NO2- and NO3- in biological samples, and that the negative NO3- difference through coronary circulation may reflect endothelial dysfunction in the patients with coronary atherosclerosis with severe organic stenosis.

Antiproliferative effect of Ca2+ channel blockers on human epidermoid carcinoma A431 cells

The effects of Ca(2+) channel blockers on the proliferation of human epidermoid carcinoma A431 cells were investigated by microtiter tetrazolium (MTT) proliferation assay and bromodeoxyuridine (BrdU) incorporation assay. Dihydropyridine derivatives, such as amlodipine, nicardipine, and nimodipine inhibited A431 cell growth and the incorporation of BrdU into cells with IC(50) values of 20-30 microM, while verapamil, diltiazem and dihydropyridine nifedipine inhibited neither the cell growth nor BrdU incorporation at the same concentration. Though extracellular Ca(2+) is indispensable to the cell growth, an L-type Ca(2+) channel agonist, 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl) phenyl]pyridine-3-carboxylic acid methyl ester (200 nM), did not affect the antiproliferative action of amlodipine. Thapsigargin, an inhibitor of Ca(2+)-ATPase of the endoplasmic reticulum, inhibited itself the growth of A431 cells and also showed a synergistic effect with the antiproliferative action of amlodipine. In the fluorimetric measurement of intracellular free Ca(2+) concentration in fura-2 or fluo-3 loaded A431 cells, amlodipine blunted the thapsigargin- or cyclopiazonic acid-induced Ca(2+) release from endoplasmic reticulum and the ensuing Ca(2+) influx through Ca(2+)-permeable channels. The effect on the thapsigargin-induced Ca(2+) responses could be reproduced by nicardipine and nimodipine but not by nifedipine or verapamil, lacking antiproliferative potency. These findings suggest that the intracellular Ca(2+) control system responsible for thapsigargin- and cyclopiazonic acid-sensitive endoplasmic reticulum, but not L-type Ca(2+) channels, may be modulated by amlodipine, which results in the inhibition of A431 cell growth.

Lung deflation impairs alveolar epithelial fluid transport in ischemic rabbit and rat lungs

BACKGROUND Because the fluid transport capacity of the alveolar epithelium after lung ischemia with and without lung deflation has not been well studied, we carried out experimental studies to determine the effect of lung deflation on alveolar fluid clearance. METHODS After 1 or 2 hr of ischemia, we measured alveolar fluid clearance using 125I-albumin and Evans blue-labeled albumin concentrations in in vivo rabbit lungs in the presence of pulmonary blood flow and in ex vivo rat lungs in the absence of any pulmonary perfusion, respectively. RESULTS The principal results were: (1) lung deflation decreased alveolar fluid clearance while inflation of the lungs during ischemia preserved alveolar fluid clearance in both in vivo and ex vivo studies; (2) alveolar fluid clearance was normal in the rat lungs inflated with nitrogen (thus, alveolar gas composition did not affect alveolar fluid clearance); (3) amiloride-dependent alveolar fluid clearance was preserved when the lungs were inflated during ischemia; (4) terbutaline-simulated alveolar fluid clearance was preserved in the hypoxic rat lungs inflated with nitrogen; (5) lecithinized superoxide dismutase, a scavenger of superoxide anion, and N(omega)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide, preserved normal alveolar fluid clearance in the deflated rat lungs. CONCLUSION Lung deflation decreases alveolar fluid clearance by superoxide anion- and nitric oxide-dependent mechanisms.

Evaluation of NOx in the cardiovascular system: relationship to NO-related compounds in vivo.

Diverse attention should be paid to evaluating NOx (NO2- and NO3-) in plasma as an index of endothelial nitric oxide (NO) formation in vivo. Nitric oxide, which subsequently appears as NOx, originates from different types of NO synthase and from nonenzymatic reactions. NOx also comes from exogenous sources such as food and gastrointestinal microorganisms. The fate of the NO incorporated into activation of guanylate cyclase, formation of nitrosyl hemoglobin (or nitrosohemoglobin), nitrosothiols, peroxynitrite and its derivatives and other possible compounds is not clear at present. However, some of these compounds would produce NOx as by-products or as final products through metabolism. Therefore, plasma NOx contains information about these pathways, although how extensively these factors contribute to plasma NOx has not been quantitatively defined. A theoretical simulation of NOx in the systemic circulation indicates that only small changes are expected by inhibition or stimulation of endothelial NO production. Measuring NOx production during coronary circulation has the advantage that some degree of NOx accumulation is expected from intact endothelial cells because an excretion system is absent in the heart.

Electrophysiological and mechanical effects of calcitonin gene‐related peptide on guinea‐pig atria

1 The effects of calcitonin gene‐related peptide (CGRP) on mechanical and electrophysiological responses were studied in the guinea‐pig atrial muscle preparations and in single cells. 2 CGRP (>10−9m) enhanced the twitch contraction in a concentration‐dependent manner in electrically driven left atria and increased heart rate in spontaneously beating right atria. The positive inotropic and chronotropic effects of CGRP were not inhibited by propranolol but were attenuated by reduction of the calcium concentration in the bathing medium. 3 In single left atrial cells, CGRP slightly hyperpolarized the resting potential but did not affect the other action potential parameters significantly. 4 Under whole‐cell voltage‐clamp conditions, CGRP increased the calcium inward current. The peptide also increased the steady inward current elicited by hyperpolarization and the late outward current by depolarization. 5 These results suggest that CGRP may produce the positive inotropic and presumably chronotropic effects by increasing calcium inward current. CGRP also increases the potassium permeability. Such effects on ionic currents may not produce any apparent change in the action potential conformation, due to their opposite directional actions and relatively weak potencies.

Effects of chronic cigarette smoking on endothelial function in young men

The aim of this study was to elucidate endothelial dysfunction due to chronic cigarette smoking in young smokers and to determine practical markers of the functional derangement. The subjects were young, healthy, male non-smokers (n=11) and smokers (n=9). Endothelium-dependent and -independent vasodilation was assessed by flow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation (NID), respectively, and possible markers of endothelial function were measured. FMD in smokers was significantly lower than in control subjects (5.0 ± 2.6% and 9.5 ± 5.2%, p<0.05). Plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator levels were significantly (p<0.05) higher in smokers (6.7 ± 4.5 ng/ml and 4.3 ± 2.0 ng/ml) compared with control subjects (2.9 ± 1.9 ng/ml and 3.0 ± 0.6 ng/ml). Furthermore, PAI-1 levels correlated inversely with FMD (r=-0.451, p<0.05). No significant differences were observed for NID, or plasma NO(2)(-), NO(X), thrombomodulin, von Willebrand factor, and tissue factor pathway inhibitor levels. Chronic cigarette smoking-induced endothelial dysfunction and the PAI-1 level could be a good marker of endothelial dysfunction in young smokers.

Knockdown of stromal interaction molecule 1 (STIM1) suppresses store-operated calcium entry, cell proliferation and tumorigenicity in human epidermoid carcinoma A431 cells

Store-operated calcium (Ca(2+)) entry (SOCE) is important for cellular activities such as gene transcription, cell cycle progression and proliferation in most non-excitable cells. Stromal interaction molecule 1 (STIM1), a newly identified Ca(2+)-sensing protein, monitors the depletion of endoplasmic reticulum (ER) Ca(2+) stores and activates store-operated Ca(2+) channels at the plasma membrane to induce SOCE. To investigate the possible roles of STIM1 in tumor growth in relation to SOCE, we established STIM1 knockdown (KD) clones of human epidermoid carcinoma A431 cells by RNA interference. Thapsigargin, an inhibitor of ER Ca(2+)-ATPase, -induced and phospholipase C-coupled receptor agonist-induced SOCEs were reduced in two STIM1 KD clones compared to a negative control clone. Re-expression of a KD-resistant full-length STIM1, but not a Ca(2+) release-activated Ca(2+) channel activation domain (CAD)-deleted STIM1 mutant, in the KD clone restored the amplitude of SOCE, suggesting the specificity of the STIM1 knockdown. The cell growth of the STIM1 KD clones was slower than that of the negative control clone. DNA synthesis assessed by BrdU incorporation, as well as EGF-stimulated EGF receptor activation, decreased in the STIM1 KD clones. Xenograft growth of the STIM1 KD clones was significantly retarded compared with that of the negative control. Cell migration was attenuated in the STIM1 KD clone and the STIM1 silencing effect was reversed by transient re-expression of the full-length STIM1 but not CAD-deletion mutant. These results indicate that STIM1 plays an important role in SOCE, cell-growth and tumorigenicity in human epidermoid carcinoma A431cells, suggesting the potential use of STIM1-targeting agents for treating epidermoid carcinoma.

Novel contribution of cell surface and intracellular M1-muscarinic acetylcholine receptors to synaptic plasticity in hippocampus.

Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1‐mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligand‐binding experiments with cell‐permeable and ‐impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1‐mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic‐binding sites were abolished in M1‐mAChR‐gene‐knockout mice. Activation of cell surface M1‐mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long‐term potentiation. On the other hand, activation of intracellular M1‐mAChRs phosphorylated extracellular‐regulated kinase 1/2 and gradually enhanced long‐term potentiation. Our data thus demonstrate that M1‐mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system.