Tomoyuki Ono

Thromboxane A2 activates phospholipase C in astrocytoma cells via pertussis toxin-insensitive G-protein

The properties of thromboxane A2 (TXA2) receptors were examined in 1321N1 human astrocytoma cells. 9,11-Epithio-11,12-methanothromboxane A2 (STA2), a stable analogue of TXA2, stimulated the accumulation of inositol phosphates (IPs) with an EC50 of about 50 nM. The STA2-induced accumulation of IPs was inhibited concentration dependently by ONO3708, a TXA2 receptor antagonist, with an inhibition constant (Ki) of about 10 nM. Inositol trisphosphate (IP3) was accumulated more rapidly than inositol bisphosphate (IP2) in response to STA2. HPLC analysis indicated that inositol 1,4,5-trisphosphate accumulated in the presence of STA2. STA2 alone had no effect on the accumulation of IPs in membrane preparations but it potentiated the accumulation induced by GTP gamma S. [3H]SQ29548, a TXA2 receptor antagonist, bound specifically to TXA2 receptors, expressing a single binding site with a dissociation constant (Kd) of 10.9 nM. The competition curve for STA2 inhibition of [3H]SQ29548 binding was shifted to the right and was steeper in the presence of GTP gamma S. Pertussis toxin (IAP) elicited ADP-ribosylation of 41KD protein but had no effect on the sensitivity to GTP of the STA2 inhibition of SQ29548 binding or of STA2-induced accumulation of IPs. It is concluded from these results that the stimulation of TXA2 receptors results in activation of phospholipase C via a GTP binding protein and that the protein is not a substrate for IAP.

Inhibitory effect of 2,3‐butanedione monoxime (BDM) on Na+/Ca2+ exchange current in guinea‐pig cardiac ventricular myocytes

The effect of 2,3‐butanedione monoxime (BDM), a ‘chemical phosphatase’, on Na+/Ca2+ exchange current (INCX) was investigated using the whole‐cell voltage‐clamp technique in single guinea‐pig cardiac ventricular myocytes and in CCL39 fibroblast cells expressing canine NCX1. INCX was identified as a current sensitive to KB‐R7943, a relatively selective NCX inhibitor, at 140 mM Na+ and 2 mM Ca2+ in the external solution and 20 mM Na+ and 433 nM free Ca2+ in the pipette solution. In guinea‐pig ventricular cells, BDM inhibited INCX in a concentration‐dependent manner. The IC50 value was 2.4 mM with a Hill coefficients of 1. The average time for 50% inhibition by 10 mM BDM was 124±31 s (n=5). The effect of BDM was not affected by 1 μM okadaic acid in the pipette solution, indicating that the inhibition was not via activation of okadaic acid‐sensitive protein phosphatases. Intracellular trypsin treatment via the pipette solution significantly suppressed the inhibitory effect of BDM, implicating an intracellular site of action of BDM. PAM (pralidoxime), another oxime compound, also inhibited INCX in a manner similar to BDM. Isoprenaline at 50 μM and phorbol 12‐myristate 13‐acetate (PMA) at 8 μM did not reverse the inhibition of INCX by BDM. BDM inhibited INCX in CCL39 cells expressing NCX1 and in its mutant in which its three major phosphorylatable serine residues were replaced with alanines. We conclude that BDM inhibits INCX but the mechanism of inhibition is not by dephosphorylation of the Na+/Ca2+ exchanger as a ‘chemical phosphatase’.

Mastoparan elicits prostaglandin E2 generation and inhibits inositol phosphate accumulation via different mechanisms in rabbit astrocytes.

The effects of mastoparan on phosphoinositide hydrolysis and prostaglandin E2 (PGE2) generation were investigated in astrocytes cultured from rabbit brain. Mastoparan inhibited the accumulations of [3H]inositol phosphates induced by bradykinin (1 microM) in a time- and concentration-dependent manner. Mastoparan (3-30 microM) also released PGE2 in a time- and concentration-dependent manner. Mastoparan-induced release of PGE2 was inhibited by indomethacin, a cyclooxygenase inhibitor, by dexamethasone, a steroidal anti-inflammatory drug, and by pertussis toxin, an inactivator of some G proteins, such as Gi and Go. Mastoparan also caused [3H]arachidonic acid liberation, which was inhibited by dexamethasone or pertussis toxin. In contrast, indomethacin, dexamethasone and pertussis toxin failed to attenuate mastoparan-induced inhibition of [3H]inositol phosphate accumulation induced by bradykinin. Thus, mastoparan-induced inhibition of phosphoinositide hydrolysis does not involve pertussis toxin-sensitive G protein nor arachidonic acid metabolites. In addition to the inhibition of phospholipase C, mastoparan activates phospholipase A2 through pertussis toxin-sensitive G protein.

Adenosine Inhibits Histamine‐Induced Phosphoinositide Hydrolysis Mediated via Pertussis Toxin‐Sensitive G Protein in Human Astrocytoma Cells

The effect of adenosine on phosphoinositide hydrolysis was examined in 1321N1 human astrocytoma cells. Adenosine, L‐N6‐phenylisopropyladenosine (l‐PIA), and 5‐ (N‐ethylcarboxamido)adenosine (NECA) inhibited histaminc‐stimulated accumulation of inositol phosphates in a concentration‐dependent manner. The potency order of adenosine analogues for inhibition of inositol phosphate accumulation was l‐PIA > adenosine > NECA, a finding indicating that A1‐class adenosine receptors are involved in the inhibition. The reduction in inositol phosphate accumulation by l‐PIA was blocked by an adenosine receptor antagonist, 8‐phenyltheophylline. Stimulation of A1‐class adenosine receptors inhibited isoproterenol‐stimulated cyclic AMP accumulation as well as histamine‐induced inositol phosphate accumulation. Both inhibitory effects were blocked by pretreatment of the cells with pertussis toxin [islet‐activating protein (IAP)]. l‐PIA also inhibited guanosine 5′‐(γ‐thio)triphosphate (GTPγ)‐stimulated accumulation of inositol phosphates in membrane preparations, and 8‐phenyltheophylline antagonized the inhibition. l‐PIA could not inhibit GTPγS‐induced accumulation of inositol phosphates in IAP‐treated membranes. Gi/G0, purified from rabbit brain, inhibited GTPγS‐stimulated accumulation of inositol phosphates in a concentration‐dependent manner in membrane preparations. These results suggest that stimulation of Ar class adenosine receptors interacts with the IAP‐sensitive G protein(s), resulting in the inhibitions of phospholipase C as well as adenylate cyclase in human astrocytoma cells.

Inhibition of aggregation of rabbit and human platelets induced by adrenaline and 5-hydroxytryptamine by KB-R7943, a Na+/Ca2+ exchange inhibitor

We investigated the effect of KB‐R7943, a Na+/Ca2+ exchange inhibitor, on the aggregation response induced by adrenaline and 5‐hydroxytryptamine (5‐HT), alone or in combination in human and rabbit platelets in the presence or absence of ouabain. KB‐R7943 inhibited aggregation induced by the combination of adrenaline and 5‐HT in a concentration‐dependent manner. The IC50 values of KB‐R7943 were 4.2±2.0 or 3.0±0.7 μM with washed rabbit platelets with or without ouabain pretreatment, respectively. In platelet‐rich human plasma, the aggregation was biphasic. The IC50 value of KB‐R7943 was 17.2±4.4 μM for the first phase aggregation. KB‐R7943 did not inhibit the first phase of aggregation induced by adrenaline alone, or the monophasic aggregation induced by 5‐HT alone. The aggregation of rabbit platelets depended on the presence of K+ in the medium, and K+‐dependent and K+‐independent Ca2+ influx were observed in resting platelets. Ouabain treatment increased only the K+‐dependent but not the K+‐independent Ca2+ influx. KB‐R7943 inhibited K+‐dependent Ca2+ influx with or without ouabain pretreatment, but not K+‐independent Ca2+ influx. From these results, we conclude that KB‐R7943 inhibits the adrenaline plus 5‐HT induced aggregation of rabbit and human platelets by inhibiting K+‐dependent Na+/Ca2+ exchange (NCKX). Our results suggest that NCKX plays an important role in platelet aggregation.

Pharmacokinetic and Pharmacodynamic Interaction of Nadolol With Itraconazole, Rifampicin and Grapefruit Juice in Healthy Volunteers

To evaluate effects of itraconazole, rifampicin and grapefruit juice on pharmacokinetics and pharmacodynamics of a hydrophilic non‐selective β‐adrenoceptor blocker nadolol, we conducted an open‐label, four‐way crossover study in 10 healthy male volunteers. A single oral dose of 30 mg nadolol was administered with water (control), itraconazole (100 mg), or grapefruit juice (300 mL), or after a 6‐day pretreatment with rifampicin (450 mg/day). Plasma concentrations and urinary excretions of nadolol were measured over 48 hours after its dosing. Systolic and diastolic blood pressures and pulse rate were periodically recorded after nadolol administration as pharmacodynamic parameters. Itraconazole increased the peak plasma concentration and the area under the plasma concentration–time curve (AUC0−∞) of nadolol by 468% and 224% of control, respectively (P < .001). A slight, but not statistically significant, decrease in AUC0−∞ of nadolol was observed in rifampicin and grapefruit juice phases as compared to control. Elimination half‐life for nadolol did not differ among the four phases. During itraconazole phase, nadolol reduced pharmacodynamic parameters to a greater extent than the other phases. These results suggest that itraconazole substantially increases the oral availability of nadolol possibly by the inhibition of intestinal P‐glycoprotein, whereas grapefruit juice has little effect on nadolol pharmacokinetics.

Dual Roles of 5-Hydroxytryptamine in Ischemia-Reperfusion Injury in Isolated Rat Hearts

Background: 5-Hydroxytryptamine (5-HT) has been shown to be involved in exacerbating cardiac ischemia-reperfusion injury; however, the role of 5-HT in the injury has yet to be established. This study demonstrates that 5-HT has dual roles in ischemia-reperfusion injury. Methods and Results: The role of 5-HT in cardiac ischemia-reperfusion injury was examined in isolated rat hearts perfused with oxygenated Krebs-Henseleit solution. A 30-minute global ischemia and 30-minute reperfusion exacerbated functional cardiac parameters such as left ventricular end-diastolic pressure, coronary flow, heart rate, and total lactate dehydrogenase release. The 5-HT2A receptor antagonist sarpogrelate (0.3-1.0 μM) improved cardiac function during ischemia-reperfusion. High-performance liquid chromatography analysis revealed an elevation in the level of 5-HT in the coronary effluent immediately after ischemia, suggesting that 5-HT is released from the ischemic heart and that sarpogrelate protects the heart from ischemia-reperfusion injury by blocking 5-HT2A receptors. However, 5-HT (0.3-1.0,μM) applied exogenously unexpectedly improved the cardiac mechanical parameters during ischemia-reperfusion, increased coronary flow, and increased the level of NO in the effluent, which was inhibited by L-NG-nitro-arginine methyl ester, a NO synthase blocker. Conclusions: Present results suggest dual roles of 5-HT in ischemia-reperfusion injury. During ischemia, 5HT is released endogenously, constricts coronary smooth muscles via 5-HT2A receptors, and aggravates cardiac function. In contrast, 5-HT applied exogenously affects predominantly non-5HT2A receptors on the endothelium and induces coronary vasodilatation via endothelial NO production, which is protective against ischemia-reperfusion injury.

Mechanisms of Impaired Neutrophil Migration by MicroRNAs in Myelodysplastic Syndromes

In myelodysplastic syndromes (MDS), functional defects of neutrophils result in high mortality because of infections; however, the molecular basis remains unclear. We recently found that miR-34a and miR-155 were significantly increased in MDS neutrophils. To clarify the effects of the aberrant microRNA expression on neutrophil functions, we introduced miR-34a, miR-155, or control microRNA into neutrophil-like differentiated HL60 cells. Ectopically introduced miR-34a and miR-155 significantly attenuated migration toward chemoattractants fMLF and IL-8, but enhanced degranulation. To clarify the mechanisms for inhibition of migration, we studied the effects of miR-34a and miR-155 on the migration-regulating Rho family members, Cdc42 and Rac1. The introduced miR-34a and miR-155 decreased the fMLF-induced active form of Cdc42 to 29.0 ± 15.9 and 39.7 ± 4.8% of that in the control cells, respectively, although Cdc42 protein levels were not altered. miR-34a decreased a Cdc42-specific guanine nucleotide exchange factor (GEF), dedicator of cytokinesis (DOCK) 8, whereas miR-155 reduced another Cdc42-specific GEF, FYVE, RhoGEF, and PH domain-containing (FGD) 4. The knockdown of DOCK8 and FGD4 by small interfering RNA suppressed Cdc42 activation and fMLF/IL-8–induced migration. miR-155, but not miR-34a, decreased Rac1 protein, and introduction of Rac1 small interfering RNA attenuated Rac1 activation and migration. Neutrophils from patients showed significant attenuation in migration compared with healthy cells, and protein levels of DOCK8, FGD4, and Rac1 were well correlated with migration toward fMLF (r = 0.642, 0.686, and 0.436, respectively) and IL-8 (r = 0.778, 0.659, and 0.606, respectively). Our results indicated that reduction of DOCK8, FGD4, and Rac1 contributes to impaired neutrophil migration in MDS.

Maturity-dependent fractionation of neutrophil progenitors: A new method to examine in vivo expression profiles of differentiation-regulating genes

To investigate differentiation-dependent gene expression during granulopoiesis, we established a new method to isolate six sequential differentiation stages of neutrophil progenitors from bone marrow. Neutrophil progenitors were divided into three populations by density centrifugation, followed by depletion of other lineages, and further separated by fluorescence-activated cell sorting based on the expressions of CD34, CD11b, and CD16: CD34(+) fraction from a low-density population (F1), CD11b(-)/CD16(-) (F2), CD11b(+)/CD16(-) (F3), and CD11b(+)/CD16(low) (F4) fractions with intermediate density, and CD11b(+)/CD16(int) (F5) and CD11b(+)/CD16(high) (F6) fractions from a high-density population. To examine whether this fractionation was applicable to the study of in vivo gene expression profiles during granulopoiesis, we analyzed messenger RNA levels of AML-1 and CCAAT/enhancer binding protein (EBP)-ε and two target genes of C/EBP-ε, granulocyte-macrophage colony-stimulating factor receptor common β subunit and lactoferrin, in the six fractions and peripheral blood-derived neutrophils (F7). Expression of AML-1 and C/EBP-ε peaked at F1 and F4, respectively, followed by a gradual decrease. Although granulocyte-macrophage colony-stimulating factor receptor common β subunit messenger RNA levels remained low from F1 through F6 and elevated at F7, lactoferrin messenger RNA showed a drastic increase at F3 and dropped at F5. The difference in the expression profiles of the two C/EBP-ε target genes suggests the involvement of regulators other than C/EBP-ε in the induction of the two genes. The new fractionation method is able to provide new information on maturation-dependent gene expression during granulopoiesis.

EFFECT OF EXTRACELLULAR pH ON CONTRACTILE RESPONSES OF THE GUINEA‐PIG VAS DEFERENS

1. Effects of changing the pH of the bathing solution (7.0, 7.4 and 7.8) on the contractile response of the guinea‐pig isolated vas deferens to ATP, noradrenaline (NA) and ATP in the presence of NA or electrical field stimulation (EFS) were investigated.