Hiromi Nobe

Novel diacylglycerol kinase inhibitor selectively suppressed an U46619-induced enhancement of mouse portal vein contraction under high glucose conditions

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. The conventional DG kinase inhibitor, 6‐[2‐(4‐[(4‐fluorophenyl)phenyl‐methylene]‐1‐piperidinyl)ethyl]‐7‐methyl‐5H‐thiazolo [3,2‐α] pyrimidine‐5‐one (R59022) (0.1–30 μM), inhibited thromboxane A2 analogue 9,11‐dideoxy‐11α,9α‐epoxymethanoprostaglandin F2α (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 μM) 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. 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. 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. 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.

Dysfunction of aorta involves different patterns of intracellular signaling pathways in diabetic rats.

Rat models of insulin-dependent (streptozotocin-induced) and independent (Otsuka Long-Evans Tokushima Fatty (OLETF)) diabetes had sustained and transient increases in blood glucose levels. Over-contraction due to norepinephrine was seen exclusively in streptozotocin rat aorta. Contraction was enhanced under high-glucose conditions in OLETF rats. In order to understand the association between these patterns of changes, total diacylglycerol was measured as a key element of phosphatidylinositol-turnover due to the conversion of some incorporated glucose into diacylglycerol. Streptozotocin rats had enhanced basal diacylglycerol. Both diacylglycerol kinase (metabolic enzyme of diacylglycerol) and total phosphatidylinositol turnover activities also increased on norepinephrine stimulation, independent of extracellular glucose level. On the other hand, diacylglycerol, diacylglycerol kinase and phosphatidylinositol turnover in OLETF rats increased under high glucose conditions in the absence of norepinephrine treatment. These results indicated that diacylglycerol and diacylglycerol kinase-mediated phosphatidylinositol turnover acceleration was influenced by an increase in glucose levels in OLETF rats or by receptor-mediated signals in streptozotocin rats including glucose desensitization based on submaximal incorporation. We suggest that the alteration of vascular dysfunction is induced by different factors in each type of diabetes.

Preferential role of intracellular Ca2+ stores in regulation of isometric force in NIH 3T3 fibroblast fibres

1 Fibroblast contraction plays a major role in wound repair, but the regulatory mechanisms are not well known. We investigated the relations between isometric force and intracellular calcium concentration ([Ca2+]i) in fibroblast fibres. These fibres were made with mouse NIH 3T3 fibroblasts cultured with native collagen in a three‐dimensional matrix. 2 Calf serum (CS; 30 %) elicited a monotonic increase in force that attained a maximum within 15 min and could be sustained indefinitely. In contrast, [Ca2+]i increased to a peak at 3 min after CS stimulation, then returned to baseline levels by 10 min. Pretreatment with Ca2+‐free medium or the Ca2+‐channel antagonist nicardipine (10 μM) blocked the CS‐induced [Ca2+]i increase, but force was not affected. 3 KCl (50 mM) stimulation on the other hand, elicited a prolonged increase in [Ca2+]i but did not increase force. 4 Inhibition of the endoplasmic reticulum Ca2+ release with Ca2+‐ATPase inhibitors cyclopiazonic acid (5 μM) or thapsigargin (5 μM) nearly abolished (< 20 % control) the increase in [Ca2+]i and force response to CS. Treatment with ryanodine (10 μM) and caffeine (20 mM) had a similar effect. The phospholipase C inhibitor U73122 (3 μM) reduced the CS‐induced increases in [Ca2+]i and force by 70 and 40 %, respectively. 5 We conclude that fibroblast isometric force is not coupled to Ca2+ arising from transmembrane influx but is correlated with the transient [Ca2+]i increase due to release from intracellular stores. Store‐released Ca2+ may initiate activation pathways for fibroblast force development, but is not required for force maintenance.

Rho A and the Rho kinase pathway regulate fibroblast contraction: Enhanced contraction in constitutively active Rho A fibroblast cells

Fibroblast cells play a central role in the proliferation phase of wound healing processes, contributing to force development. The intracellular signaling pathways regulating this non-muscle contraction are only partially understood. To study the relations between Rho A and contractile responses, constitutively active Rho A (CA-Rho A) fibroblast cells were reconstituted into fibers and the effects of calf serum (CS) on isometric force were studied. CS-induced force in CA-Rho A fibroblast fibers was twice as large as that in wild type (NIH 3T3) fibroblast fibers. During this response, the translocation of Rho A from the cytosol to the membrane was detected by Rho A activity assays and Western blot analysis. Pre-treatment with a Rho specific inhibitor (C3-exoenzyme) suppressed translocation as well as contraction. These results indicate that Rho A activation is essential for fibroblast contraction. The Rho kinase inhibitor (Y27632) inhibited both NIH 3T3 and CA-Rho A fibroblast fiber contractions. Activation of Rho A is thus directly coupled with Rho kinase activity. We conclude that the translocation of Rho A from the cytosol to the membrane and the Rho kinase pathway can regulate wound healing processes mediated by fibroblast contraction.