15-hydroxy-11-alpha-9-alpha-(epoxymethano)prosta-5-13-dienoic-acid and 1-2-dioctanoylglycerol

1. Diacylglycerol kinase (DG kinase) is a key enzyme in vascular contraction; however, alterations of the regulatory mechanisms in vascular dysfunction are poorly understood. In this study, the effect of a novel DG kinase inhibitor, stemphone, on vascular contraction was investigated. 2. The conventional DG kinase inhibitor, 6-[2-(4-[(4-fluorophenyl)phenyl-methylene]-1-piperidinyl)ethyl]-7-methyl-5H-thiazolo [3,2-alpha] pyrimidine-5-one (R59022) (0.1-30 microm), inhibited thromboxane A(2) analogue 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F(2alpha) (U46619)-induced sustained contractions in mouse aorta and porcine coronary artery in a dose-dependent manner. Treatment with stemphone did not affect contractions in these tissues. However, stemphone significantly inhibited (>0.3 microm) U46619-induced spontaneous phasic contraction in mouse portal vein. This inhibitory effect was not detected following R59022 treatment in portal vein. Therefore, stemphone demonstrated selectivity in terms of portal vein contraction. 3. Under high glucose (22.2 mm) conditions, U46619-induced contraction was enhanced in these three types of vascular tissue. Inhibitory effects of R59022 were attenuated under these conditions; however, effects of stemphone were observed. These results indicated that stemphone could inhibit portal vein contraction under high glucose conditions, for example, diabetes. These data suggested the possibility that DG kinase may be a target of hyperportal pressure. 4. Total mass of DG was enhanced under high glucose conditions. DG was derived from incorporated glucose via de novo synthesis in the absence of phospholipase C pathway mediation. This enhanced DG under high glucose conditions activated a calcium-independent protein kinase C (PKC). This PKC was associated with calcium-independent DG kinase activation. Treatment with stemphone also inhibited calcium-independent DG kinase. These signal transduction pathways were distinguishable from a DG-PKC pathway under normal glucose conditions. 5. The present investigation suggested that stemphone selectively inhibited overcontraction of portal vein induced by high glucose levels. This phenomenon was attributable to inhibition of calcium-independent DG kinase activation that occurred under high glucose conditions mediated by both DG synthesized from glucose and calcium-independent PKC activation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aorta, Thoracic; Benzoquinones; Diacylglycerol Kinase; Diglycerides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glucose; In Vitro Techniques; Isometric Contraction; Male; Mice; Muscle Contraction; Muscle, Smooth, Vascular; Portal Vein; Subcellular Fractions; Type C Phospholipases; Vasoconstrictor Agents

Defects in glycoprotein (GP)IIb-IIIa or in its activation may cause abnormal platelet aggregation and a bleeding diathesis. We report studies in a 67-year-old man with a myeloproliferative disease and markedly abnormal platelet responses. By flow cytometry, platelet binding of two complex-specific anti-GPIIb-IIIa monoclonal antibodies (mAbs), A2A9 and 10E5, was approximately 50% of normal. An enzyme-linked immunosorbent assay (ELISA) using immobilized kistrin showed 18% of normal membrane GPIIb-IIIa complex. By immunoblot analysis, GPIIb and GPIIIa levels in platelet lysates and membranes were near normal. Activation of GPIIb-IIIa, monitored with mAb PAC-1, was markedly decreased (< 20% of normal) in response to ADP, thrombin and platelet-activating factor (PAF); expression of ligand-induced binding sites (LIBS) was < or = 30% of normal. Signal transduction-independent LIBS expression, induced by echistatin, was approximately 60% of normal, suggesting that the integrin present had intact ligand-binding capability. Sequence analysis of GPIIb and GPIIIa cDNA, and platelet mRNA levels for both subunits, were normal. These findings document an acquired combined defect in membrane expression (secondary to a defect in post-translational processing of the complex) and inside-out signalling-dependent activation of the GPIIb-IIIa complex.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Aged; Antibodies, Monoclonal; Binding Sites; Blood Platelets; Calcimycin; Diglycerides; DNA, Complementary; Enzyme Activators; Flow Cytometry; Humans; Immunoblotting; Intercellular Signaling Peptides and Proteins; Ionophores; Macrophage-1 Antigen; Male; Myeloproliferative Disorders; Neutrophils; Peptides; Platelet Activating Factor; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Polymerase Chain Reaction; Protein Binding; Protein Kinase C; Receptors, Thrombin; RNA, Messenger; Sequence Analysis, DNA; Serotonin; Signal Transduction