Toshiharu Azma

Quantitative measurement of thromboelastography as a function of platelet count.

T hromboelastography (TEG) analyzes the status of blood coagulation, including abnormalities associated with low platelet count (1). Using TEG to guide platelet transfusion has thus been advocated by several investigators (2–6). However, it is not clear whether TEG variables, assessed from the shape of its tracing, are regulated by platelets quantitatively, because these variables are concurrently under the influence of coagulation factors. We examined the modulation of TEG variables by the amount of platelet, which was controlled by the dilution of platelets into the patient’s plasma without affecting other factors. We also attempted to evaluate the critical platelet counts in coagulation by using this technique.

Inhibitory Effect of High Concentration of Glucose on Relaxations to Activation of ATP-Sensitive K+ Channels in Human Omental Artery

Objective—The present study was designed to examine in the human omental artery whether high concentrations of D-glucose inhibit the activity of ATP-sensitive K+ channels in the vascular smooth muscle and whether this inhibitory effect is mediated by the production of superoxide. Methods and Results—Human omental arteries without endothelium were suspended for isometric force recording. Changes in membrane potentials were recorded and production of superoxide was evaluated. Glibenclamide abolished vasorelaxation and hyperpolarization in response to levcromakalim. D-glucose (10 to 20 mmol/L) but not l-glucose (20 mmol/L) reduced these vasorelaxation and hyperpolarization. Tiron and diphenyleneiodonium, but not catalase, restored vasorelaxation and hyperpolarization in response to levcromakalim in arteries treated with D-glucose. Calphostin C and Gö6976 simultaneously recovered these vasorelaxation and hyperpolarization in arteries treated with D-glucose. Phorbol 12-myristate 13 acetate (PMA) inhibited the vasorelaxation and hyperpolarization, which are recovered by calphostin C as well as Gö6976. D-glucose and PMA, but not l-glucose, significantly increased superoxide production from the arteries, whereas such increased production was reversed by Tiron. Conclusions—These results suggest that in the human visceral artery, acute hyperglycemia modulates vasodilation mediated by ATP-sensitive K+ channels via the production of superoxide possibly mediated by the activation of protein kinase C.

Roles of Phosphatidylinositol 3-Kinase-Akt and NADPH Oxidase in Adenosine 5′-Triphosphate–Sensitive K+ Channel Function Impaired by High Glucose in the Human Artery

The present study was designed to examine roles of the phosphatidylinositol 3-kinase-Akt pathway and reduced nicotinamide-adenine dinucleotide phosphate oxidases in the reduced ATP-sensitive K+ channel function via superoxide produced by high glucose in the human artery. We evaluated the activity of the phosphatidylinositol 3-kinase-Akt pathway, as well as reduced nicotinamide-adenine dinucleotide phosphate oxidases, the intracellular levels of superoxide and ATP-sensitive K+ channel function in the human omental artery without endothelium. Levels of the p85-α subunit and reduced nicotinamide-adenine dinucleotide phosphate oxidase subunits, including p47phox, p22phox, and Rac-1, increased in the membrane fraction from arteries treated with d-glucose (20 mmol/L) accompanied by increased intracellular superoxide production. High glucose simultaneously augmented Akt phosphorylation at Ser 473, as well as Thr 308 in the human vascular smooth muscle cells. A phosphatidylinositol 3-kinase inhibitor LY294002, as well as tiron and apocynin, restored vasorelaxation and hyperpolarization in response to an ATP-sensitive K+ channel opener levcromakalim. Therefore, it can be concluded that the activation of the phosphatidylinositol 3-kinase-Akt pathway, in combination with the translocation of p47phox, p22phox, and Rac-1, contributes to the superoxide production induced by high glucose, resulting in the impairment of ATP-sensitive K+ channel function in the human visceral artery.

Propofol Restores Brain Microvascular Function Impaired by High Glucose via the Decrease in Oxidative Stress

Background:Vascular dysfunction induced by hyperglycemia has not been studied in cerebral parenchymal circulation. The current study was designed to examine whether high glucose impairs dilation of cerebral parenchymal arterioles via nitric oxide synthase, and whether propofol recovers this vasodilation by reducing superoxide levels in the brain. Methods:Cerebral parenchymal arterioles in the rat brain slices were monitored using computer-assisted videomicroscopy. Vasodilation induced by acetylcholine (10−6 to 10−4 m) was obtained after the incubation of brain slices for 60 min with any addition of l-glucose (20 mm), d-glucose (20 mm), or propofol (3 × 10−7 or 10−6 m) in combination with d-glucose (20 mm). Superoxide production in the brain slice was determined by dihydroethidium (2 × 10−6 m) fluorescence. Results:Addition of d-glucose, but not l-glucose, reduced arteriolar dilation by acetylcholine, whereas the dilation was abolished by the neuronal nitric oxide synthase inhibitor S-methyl-l-thiocitrulline (10−5 m). Both propofol and the superoxide dismutase mimetic Tempol (10−4 m) restored the arteriolar dilation in response to acetylcholine in the brain slice treated with d-glucose. Addition of d-glucose increased superoxide production in the brain slice, whereas propofol, Tempol, and the nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase inhibitor apocynin (1 mm) similarly inhibited it. Conclusions:Clinically relevant concentrations of propofol ameliorate neuronal nitric oxide synthase–dependent dilation impaired by high glucose in the cerebral parenchymal arterioles via the effect on superoxide levels. Propofol may be protective against cerebral microvascular malfunction resulting from oxidative stress by acute hyperglycemia.

Synthetic peroxisome proliferator-activated receptor-gamma agonists restore impaired vasorelaxation via ATP-sensitive K+ channels by high glucose.

The present study was designed to examine whether in the human artery, synthetic peroxisome proliferator-activated receptor (PPAR)-γ agonists restore vasorelaxation as well as hyperpolarization via ATP-sensitive K+ channels impaired by the high concentration of d-glucose and whether the restoration may be mediated by the antioxidant capacity of these agents. The isometric force and membrane potential of human omental arteries without endothelium were recorded. The production rate of superoxide was evaluated using a superoxide-generating system with xanthine-xanthine oxidase in the absence of smooth muscle cells. Glibenclamide abolished vasorelaxation and hyperpolarization in response to levcromakalim. Addition of d-glucose (20 mM) but not l-glucose (20 mM) reduced this vasorelaxation and hyperpolarization. Synthetic PPAR-γ agonists (troglitazone and rosiglitazone) and/or an inhibitor of superoxide generation (4,5-dihydroxy-1,3-benzene-disulfonic acid, Tiron), but not a PPAR-α agonist (fenofibrate), restored vasorelaxation and hyperpolarization in response to levcromakalim in arteries treated with d-glucose. Troglitazone and rosiglitazone, but not fenofibrate, decreased the production rate of superoxide without affecting uric acid generation. These findings suggest that synthetic PPAR-γ agonists recover the function of ATP-sensitive K+ channels reduced by the high concentration of glucose in human vascular smooth muscle cells and that the effect of these agonists may be mediated in part by their antioxidant capacity.

Inhibitory Effect of Lidocaine on Cultured Porcine Aortic Endothelial Cell-dependent Antiaggregation of Platelets

Background Vascular spasm is a well-known complication during vascular surgery. Topical lidocaine is frequently used to prevent this spasm. However, the effects of lidocaine on the endothelium-dependent antiaggregation are not clear.

Beneficial effect of propofol on arterial adenosine triphosphate-sensitive K+ channel function impaired by thromboxane.

Background:It is not known whether thromboxane A2 impairs adenosine triphosphate (ATP)-sensitive K+ channel function via increased production of superoxide in blood vessels and whether propofol as a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor restores this modification. Methods:Rat aortas without endothelium were used for isometric force recording, measurements of membrane potential, and superoxide production and Western immunoblotting. Vasorelaxation to an ATP-sensitive K+ channel opener levcromakalim was obtained during contraction to phenylephrine (3 × 10−7 m) or a thromboxane A2 analogue U46619 (3 × 10−8 m). In some experiments, aortas were incubated with an ATP-sensitive K+ channel antagonist glibenclamide, a superoxide inhibitor Tiron, a nonselective NADPH oxidase inhibitor apocynin, a hydrogen peroxide scavenger catalase, a xanthine oxidase inhibitor allopurinol, a thromboxane receptor antagonist SQ29548 or propofol (3 × 10−7 to 3 × 10−6 m). Results:Levcromakalim-induced vasorelaxation was abolished by glibenclamide in rings contracted with either vasoconstrictor agent. Tiron, apocynin, and propofol, but not catalase, augmented the vasodilator response as well as the hyperpolarization by levcromakalim in aortas contracted with U46619. Tiron, apocynin, SQ29548, and propofol, but not allopurinol, similarly reduced in situ levels of superoxide within aortic vascular smooth muscle exposed to U46619. Protein expression of a NADPH oxidase subunit p47phox increased in these arteries, and this augmentation was abolished by propofol. Conclusions:Thromboxane receptor activation induces vascular oxidative stress via NADPH oxidase, resulting in the impairment of ATP-sensitive K+ channel function. Propofol reduces this stress via inhibition of a NADPH oxidase subunit p47phox and, therefore, restores ATP-sensitive K+ channel function.

Roles of neuronal nitric oxide synthase, oxidative stress, and propofol in N-methyl-d-aspartate-induced dilatation of cerebral arterioles

BACKGROUND It remains unclear whether N-methyl-D-aspartate (NMDA) receptors contribute to cerebral parenchymal vasodilatation, and any effects of clinically used anaesthetics on the dilatation. The present study was designed to examine whether NMDA induces neuronal nitric oxide synthase (NOS)-mediated dilatation, in the cerebral parenchymal arterioles, and whether propofol and superoxide modulate the dilatation in relation to the NMDA receptor activation. METHODS The cerebral parenchymal arterioles within rat brain slices were monitored by a computer-assisted microscopy, and the vasodilatation in response to NMDA (10(-7) to 10(-5) M) was evaluated. Immunofluorescence analysis to neuronal and endothelial NOS and measurement of levels of superoxide and nitric oxide within the arteriole were simultaneously performed. RESULTS Propofol, an NMDA receptor antagonist MK801, and a neuronal NOS antagonist S-methyl-l-thiocitrulline (SMTC) reduced NMDA-induced dilation, whereas a superoxide inhibitor, Tiron, and NADPH oxidase inhibitor, gp91ds-tat, augmented NMDA-induced dilatation. Immunofluorescence analysis revealed distribution of neuronal NOS in both endothelial and smooth muscle cells in addition to neuronal cells. NMDA-induced superoxide and nitric oxide within the parenchymal arterioles. The increased superoxide within the arteriole was similarly inhibited by MK801, SMTC, gp91ds-tat, propofol, and a neuronal NOS antagonist vinyl-l-NIO, whereas the level of nitric oxide was reduced by MK801, SMTC, propofol, and vinyl-l-NIO, and it was augmented by gp91ds-tat. CONCLUSIONS NMDA dilates cerebral parenchymal arterioles possibly via neuronal NOS activation, whereas it produces superoxide via NADPH oxidase. In these arterioles, propofol reduces both the dilatation and superoxide production in response to NMDA.

The role of 20-hydroxyeicosatetraenoic acid in cerebral arteriolar constriction and the inhibitory effect of propofol

BACKGROUND: We conducted this study to examine, in cerebral parenchymal arterioles, whether 20-hydroxyeicosatetraenoic acid (20-HETE) induces constrictor responses via superoxide and whether propofol reduces this constriction. METHODS: Electrical field stimulation or 20-HETE was applied to rat brain slices monitored by computer-assisted microscopy. In some experiments, a Na+ channel antagonist tetrodotoxin, a 20-HETE synthesis inhibitor HET0016, a superoxide scavenger, Tiron, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors diphenyleneiodonium (DPI) and gp91ds-tat, or propofol was added. The superoxide level in the brain slice and the production rate in the absence of slices were evaluated by dihydroethidium fluorescence or cytochrome c reduction with a superoxide-generating system, respectively. RESULTS: Electrical stimulation induced constriction of the cerebral parenchymal arteriole, whereas this response was abolished by tetrodotoxin, HET0016, Tiron, or DPI. 20-HETE (10−8–10−6 mol/L) produced arteriolar constriction, which was inhibited by Tiron or DPI. Propofol reduced the constriction induced by electrical stimulation or 20-HETE. 20-HETE induced superoxide production in the brain slice, which was reduced by Tiron, gp91ds-tat, or propofol. However, propofol did not alter the superoxide production rate in the absence of brain slices. CONCLUSIONS: Either neuronal transmission-dependent or exogenous 20-HETE seems to induce cerebral parenchymal arteriolar constriction via superoxide production resulting from NADPH oxidase activation. Propofol is likely to prevent this constriction via inhibition of NADPH oxidase, but not by its scavenging effect on superoxide.

The Modulation of Vascular ATP-Sensitive K+ Channel Function via the Phosphatidylinositol 3-Kinase–Akt Pathway Activated by Phenylephrine

The present study examined the modulator role of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway activated by the α-1 adrenoceptor agonist phenylephrine in ATP-sensitive K+ channel function in intact vascular smooth muscle. We evaluated the ATP-sensitive K+ channel function and the activity of the PI3K–Akt pathway in the rat thoracic aorta without endothelium. The PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) (10−5 M) augmented relaxation in response to the ATP-sensitive K+ channel opener levcromakalim (10−8 to 3 × 10−6 M) in aortic rings contracted with phenylephrine (3 × 10−7 M) but not with 9,11-dideoxy-11α,9α-epoxy-methanoprostaglandin F2α (U46619; 3 × 10−8 M), although those agents induced similar contraction. ATP-sensitive K+ channel currents induced by levcromakalim (10−6 M) in the presence of phenylephrine (3 × 10−7 M) were enhanced by the nonselective α-adrenoceptor antagonist phentolamine (10−7 M) and LY294002 (10−5 M). Levels of the regulatory subunits of PI3K p85-α and p55-γ increased in the membrane fraction from aortas without endothelium treated with phenylephrine (3 × 10−7 M) but not with U46619 (3 × 10−8 M). Phenylephrine simultaneously augmented Akt phosphorylation at Ser473 and Thr308. Therefore, activation of the PI3K–Akt pathway seems to play a role in the impairment of ATP-sensitive K+ channel function in vascular smooth muscle exposed to α-1 adrenergic stimuli.