Kenro Imaeda

Endothelium-dependent hyperpolarization and intercellular electrical coupling in guinea-pig mesenteric arterioles.

1 Using the conventional whole‐cell clamp method, the electrical responses of individual smooth muscle and endothelial cells to acetylcholine (ACh) were observed in multicellular preparations where the two types of cells remained in close apposition. 2 In both types of cells, ACh induced similar hyperpolarizing responses which, when recorded in current clamp mode, had two phases (an initial fast and a second slower phase). 3 After blocking gap junctions, including myoendothelial junctions, with 18β‐glycyrrhetinic acid, ACh induced an outward current with two phases in voltage‐clamped endothelial cells. The outward current appeared around −90 mV and increased linearly with the membrane depolarization. 4 In smooth muscle cells, ACh failed to induce a membrane current after gap junctions had been blocked with 18β‐glycyrrhetinic acid. The inhibition of ACh‐induced response by 18β‐glycyrrhetinic acid was observed using either sharp or patch electrodes. 5 Nominally Ca2+‐free solution reduced the initial phase and abolished the second phase of ACh‐induced responses of endothelial cells. Both phases were also reduced by charybdotoxin (CTX). 6 Our results indicate that in guinea‐pig mesenteric arterioles, ACh hyperpolarizes endothelial cells by activating Ca2+‐activated K+ channels which are sensitive to CTX. On the other hand, hyperpolarizing responses detected in smooth muscle cells seem to originate in endothelial cells and conduct to the muscle layer via myoendothelial gap junctions.

Glucagon-like peptide-1 (GLP-1) protects against methylglyoxal-induced PC12 cell apoptosis through the PI3K/Akt/mTOR/GCLc/redox signaling pathway.

Patients with long-standing diabetes commonly develop diabetic encephalopathy, which is characterized by cognitive impairment and dementia. Oxidative stress-induced neuronal cell apoptosis is a contributing factor. Glucagon-like peptide (GLP)-1 has recently become an attractive treatment modality for patients with diabetes. It also readily enters the brain, prevents neuronal cell apoptosis, and improves the cognitive impairment characteristic of Alzheimers disease. Therefore, we investigated whether GLP-1 could protect against oxidative stress-induced neuronal cell apoptosis in pheochromocytoma (PC12) cells. PC12 cells were exposed to 1 mM methylglyoxal (MG) or MG plus 3.30 microg/ml GLP-1. Cell apoptosis, expression and phosphorylation of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin/gamma-glutamylcysteine ligase catalytic subunit (GCLc), and redox balance were then determined. The data showed that MG induced PC12 apoptosis in accordance with the redox (glutathione (GSH) and GSH/glutathione disulfide [GSSG]) imbalance. GLP-1 protected against this MG-induced apoptosis, which corresponded to the phosphorylation of PI3K, Akt, and mTOR, as well as the upregulation of GCLc and the restoration of the redox imbalance. Inhibitors of PI3K (LY294002), Akt (Akt-I), and mTOR (rapamycin) reduced the GLP-1-induced GCLc upregulation and its protection against MG-induced PC12 apoptosis. The GLP-1-induced redox restoration was also attenuated by rapamycin. In conclusion, the neuroprotective effect of GLP-1 is due to an enhancement of PI3K/Akt/mTOR/GCLc/redox signaling.

Statins inhibit high glucose-mediated neutrophil-endothelial cell adhesion through decreasing surface expression of endothelial adhesion molecules by stimulating production of endothelial nitric oxide.

Neutrophil-endothelial adhesion is a crucial step in vascular inflammation, which is recognized as the direct cause of atherosclerosis-mediated serious diseases. We demonstrated previously that high glucose increased adhesion in a protein kinase C (PKC)-dependent manner within 48 h through increasing surface expression of endothelial adhesion molecules. On the other hand, statins, used for patients with hypercholesterolemia, have been shown to decrease the incidence of atherosclerosis-mediated diseases, but direct effects of statins on endothelial cells remain unclear. In this study, we examined the effects of these compounds on high glucose-mediated neutrophil-endothelial adhesion with respect to the participation of PKC and nitric oxide (NO). After human endothelial cells were cultured for 48 h in high glucose medium, neutrophils from healthy volunteers were added and allowed to adhere for 30 min. Adhered neutrophils were quantified by measuring their myeloperoxidase activities, and surface expression of endothelial adhesion molecules was determined with an enzyme immunoassay. Both pravastatin (0.05 microM) and fluvastatin (0.5 microM) significantly attenuated the adhesion mediated by 27.8 mM glucose for 48 h through decreasing surface expression of endothelial adhesion molecules (intercellular adhesion molecule-1, P-selectin, and E-selectin). NO synthase inhibitors reduced the inhibitory effects of statins, whereas statins did not affect the adhesion mediated by a PKC activator. These data suggest that statins act directly on endothelial cells to inhibit expression of adhesion molecules and neutrophil adhesion mediated by high glucose through increasing endothelial NO production, but not by inhibiting PKC.

Cerivastatin ameliorates high insulin-enhanced neutrophil-endothelial cell adhesion and endothelial intercellular adhesion molecule-1 expression by inhibiting mitogen-activated protein kinase activation.

BACKGROUND AND AIMS There is growing evidence that hyperinsulinemia is linked to the development of atherosclerosis in patients with diabetes. We demonstrated previously that high insulin exacerbates neutrophil-endothelial cell adhesion and endothelial intercellular adhesion molecule (ICAM)-1 expression through activation of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase. Though 3-hydroxymethyl-3-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been employed as therapeutic agents in the treatment of dyslipidemia, which is frequently accompanied by diabetes mellitus; it is not known whether statins protect against leukocyte-endothelial interactions, especially in hyperinsulinemia. In this study, we determined which statin(s) could protect against endothelial reactions to high insulin. METHODS Studies of adhesion between neutrophils from healthy volunteers and human umbilical vein endothelial cells incubated in regular insulin-rich medium with or without statins were performed. Adhered neutrophils were quantified by measuring their myeloperoxidase (MPO) activities, and endothelial expression of ICAM-1 was examined using an enzyme immunoassay. RESULTS Both the increased neutrophil-endothelial cell adhesion and ICAM-1 expression caused by high insulin (100 microU/ml) for 48 h were significantly attenuated by pretreatment with cerivastatin (0.01 microM), but not by fluvastatin (0.5 microM) or pravastatin (0.05 microM). These protective actions of cerivastatin were attenuated by a key intermediate in the cholesterol biosynthesis pathway, mevalonate (400 microM). In addition, cerivastatin attenuated both neutrophil-endothelial cell adhesion and endothelial ICAM-1 expression enhanced by a MAP kinase activator, anisomycin (1 microM) but not by a PKC activator, PMA (10 nM). CONCLUSIONS These results suggest that through inhibiting MAP kinase but not PKC activation therapy with cerivastatin would be promising strategy for inhibiting neutrophil-endothelial cell adhesion and endothelial ICAM-1 expression enhanced by high insulin, which is closely correlated with atherosclerosis.

The mechanisms of inhibitory actions of gliclazide on neutrophils–endothelial cells adhesion and surface expression of endothelial adhesion molecules mediated by a high glucose concentration

BACKGROUND We previously reported that culture of endothelial cells in the presence of high glucose concentrations (27.8 and 55.5 mM) increase neutrophils adhesion because of the increase in endothelial adhesion molecules expression via activation of a protein kinase C (PKC) pathway. The antidiabetic sulfonylurea gliclazide, but not glibenclamide, inhibited these events, but the mechanisms involved were not clarified then. We present hereafter the results of further investigations of that effect with special reference to PKC activation. METHODS Human umbilical vein endothelial cells (HUVEC) were cultured for 48 h in a glucose-rich medium and neutrophils from healthy volunteers were then added and allowed to adhere for 30 min. Adhered neutrophils were quantified by measuring myeloperoxidase (MPO) activities and the surface expression of endothelial adhesion molecules was determined by enzyme immunoassay. RESULTS Culture in the presence of a high glucose concentration (27.8 mM for 48 h) increased neutrophils-endothelial cells adhesion and the surface expression of intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin on the endothelial cells. These phenomena were significantly inhibited by gliclazide (20 microM). On the other hand, phorbol 12-myristate 13-acetate (PMA), a PKC activator, had an effect similar to a high glucose concentration and that effect was also inhibited by gliclazide. CONCLUSIONS These data suggest that gliclazide inhibits high glucose-mediated neutrophils-endothelial cells adhesion and expression of endothelial adhesion molecules through inhibition of a PKC pathway.

Mechanisms of inhibitory activity of the aldose reductase inhibitor, epalrestat, on high glucose-mediated endothelial injury: Neutrophil–endothelial cell adhesion and surface expression of endothelial adhesion molecules

BACKGROUND We have previously reported that endothelial cells cultured in the presence of high concentrations of glucose (27.8 and 55.5 mM) exhibited enhanced neutrophil adhesion through increased expression of endothelial adhesion molecules via the activation of a protein kinase C (PKC)-dependent pathway. We also found that the aldose reductase inhibitor, epalrestat, inhibited these events, but the mechanisms for this inhibition remained unclear. In this study, we further investigated the inhibitory mechanisms of epalrestat with reference to PKC activation and nitric oxide (NO) production. METHODS Human umbilical vein endothelial cells (HUVECs) were cultured for 48 h in glucose-rich medium and neutrophils from healthy volunteers were then added and allowed to adhere for 30 min. Adhered neutrophils were quantified by measuring myeloperoxidase (MPO) activity and surface expression of endothelial adhesion molecules was determined by enzyme immunoassay. RESULTS Culture in the presence of a high concentration of glucose (27.8 mM for 48 h) increased neutrophil-endothelial cell adhesion and surface expression of intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin on endothelial cells. These phenomena were significantly inhibited by epalrestat (10 microM), while NO synthase (NOS) inhibitors reduced the inhibitory effects of this compound. In contrast, 10 nM phorbol 12-myristate 13-acetate (PMA), a PKC activator, showed similar effects as high glucose, and these effects were also inhibited by epalrestat. CONCLUSIONS Our data suggested that epalrestat inhibited high glucose-mediated neutrophil-endothelial cell adhesion and expression of endothelial adhesion molecules not only through inhibition of a PKC-dependent pathway, but also through increased endothelial NO production.

Hyperpolarization‐induced dilatation of submucosal arterioles in the guinea‐pig ileum

The effects of inhibition of acetylcholine (ACh)‐induced hyperpolarization on dilatation of submucosal arterioles were investigated in the guinea‐pig ileum. In smooth muscles of the arterioles depolarized by Ba2+ (0.5 mM) to about −40 mV, ACh (3 μM) repolarized the membrane to about −65 mV (hyperpolarization), irrespective of the absence or presence of L‐Nω‐nitroarginine (L‐NOARG, 0.1 mM) and diclofenac (1 μM), and increased the diameter (dilatation). Combined application of charybdotoxin (CTX, 50 nM) and apamin (0.1 μM), inhibitors of some types of K+‐channels, abolished the ACh‐induced hyperpolarization and dilatation. 18β‐Glycerrhetinic acid (18β‐GA, 30 μM), a known inhibitor of gap junctions, depolarized the membrane to about −36 mV, either in the absence or in the presence of Ba2+, with no associated contraction of the arterioles. In the presence of 18β‐GA, ACh‐induced hyperpolarization was abolished, however the dilatation was inhibited only partially, with associated inhibition of constriction produced by Ba2+ and NA. 18β‐GA inhibited the dilatation produced by sodium nitroprusside, an NO donor. The ACh‐induced hyperpolarization and dilatation were abolished in the presence of 2‐aminoethoxydiphenyl borate (30 μM), an inhibitory modulator of inositol trisphosphate receptor‐mediated Ca2+ release from intracellular stores. It is concluded that in submucosal arterioles, hyperpolarizations produced by ACh have causal relationship to the arteriolar dilatation. 18β‐GA did not induce parallel relationship between hyperpolarization and dilatation produced by ACh. 18β‐GA may have unidentified inhibitory effects on agonist‐mediated actions, in addition to the inhibition of gap junctions.

Alteration of the properties of gastric smooth muscle in the genetically hyperglycemic OLETF rat

Membrane responses were recorded from isolated gastric smooth muscle of Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Otsuka (LETO) rats, using microelectrode techniques. At the age of 68-76 weeks, the blood sugar level was 181 mg/dl in LETO rats and 350 mg/dL in OLETF rats. In both rats, the membrane potential was stable in fundus muscle and spontaneously active with generation of slow waves in antrum muscle. The resting membrane potential was about - 46 mV in fundus and - 55 mV in antrum muscles of LETO rats, and the values were 3-7 mV lower in OLETF rats. The slow waves were generated regularly in LETO rats, while they were irregular and of small amplitude in OLETF rats. Transmural nerve stimulation evoked a cholinergic excitatory junction potential and following inhibitory junction potential in LETO rats, and only an inhibitory junction potential of smaller size was generated in most of OLETF rats. The acetylcholine-induced depolarization was greater in OLETF than in LETO rats. The level of hyperpolarization produced by noradrenaline was similar between OLETF and LETO rats. Thus, the reduction of the resting membrane potential, weakening of spontaneous activity, impairment of cholinergic transmission and cholinergic supersensitivity were associated with hyperglycemia. These alterations were considered due to the development of diabetes mellitus.

Epidermal Growth Factor Receptor Transactivation Is Necessary for Glucagon-Like Peptide-1 to Protect PC12 Cells from Apoptosis

Aim: Patients with long-standing diabetes commonly develop diabetic encephalopathy, which is characterized by cognitive impairment and dementia. To identify potential treatments for diabetic encephalopathy, we focused on the protective action of glucagon-like peptide-1 (GLP-1) against neural cell apoptosis. In this study, we evaluated whether exposure of cells to GLP-1 leads to epidermal growth factor receptor (EGFR) transactivation and signaling through the PI3K/Akt/mTOR/GCLc/redox pathway, which we previously reported. Methods: We monitored the phosphorylation of EGFR and Akt in PC12 cells exposed to MG and GLP-1 that had been first incubated in the presence or absence of various inhibitors of EGFR transactivation. Results: DAPI staining revealed that pretreatment of cells with BiPS, HB-EGF and anti-TGF-α neutralization antibodies or AG1478 abrogated the ability of GLP-1 to rescue cells from MG-induced apoptosis. We show that exposure of PC12 cells to GLP-1 induces EGFR phosphorylation and that this effect was inhibited by prior exposure of the cells to BiPS, HB-EGF and anti-TGF-α neutralization antibodies or AG1478. Interestingly, these agents also diminished the capacity of GLP-1 to protect cells from MG-induced apoptosis. Moreover, these agents reduced GLP-1-induced phosphorylation of Akt. EGF itself also protected the cells from MG-induced apoptosis and induced phosphorylation of Akt, which was inhibited by LY294002. Conclusion: The neuroprotective effects of GLP-1 against MG-induced apoptosis are mediated by EGFR transactivation, which signals through the PI3K/Akt/mTOR/GCLc/redox pathway in PC12 cells.

The antidiabetic agent, gliclazide, reduces high insulin–enhanced neutrophil‐transendothelial migration through direct effects on the endothelium

Many lines of evidence indicate that hyperinsulinemia might be associated with coronary athrosclerosis, and, currently, there are no effective strategies for preventing this. We previously reported that high insulin enhances neutrophil‐transendothelial migration, a process that involves increased surface presentation of platelet endothelial cell adhesion molecule‐1 (PECAM‐1) through a mitogen‐activated protein (MAP) kinase–dependent event. In this current study, we examined if antidiabetic agents, especially KATP channel blockers, might similarly protect against the leukocyte‐endothelial cell interactions enhanced by high insulin.