Junko Sugatani

High-density lipoprotein inhibits the synthesis of platelet-activating factor in human vascular endothelial cells

The regulation of platelet-activating factor (PAF) synthesis by serum lipoproteins was investigated in human umbilical vein endothelial cells. High-density lipoprotein (HDL) inhibited PAF synthesis in agonist (thrombin, histamine, and A23187)-stimulated endothelial cells, that was determined by incorporation of [3H]acetate into PAF and by bioassay. The inhibition by HDL was increased in a concentration-dependent manner, but was reversed as the concentration of thrombin increased. HDL did not affect the time course of PAF production. HDL lipids suppressed the PAF production to a lesser extent than HDL. The reduction of PAF accumulation in HDL, did not result from degradation of PAF but inhibition of PAF synthesis, which was mainly mediated via the blockade of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase activation. HDL did not prevent the release of [3H]arachidonic acid in thrombin-stimulated endothelial cells. The binding of 125I-HDL to endothelial cells and its uptake were not enhanced by thrombin stimulation. These results demonstrate that HDL may inhibit the activation of acetyltransferase by thrombin at the cell surface. This observation may explain a part of mechanism of HDL action.

Development of a novel method for determination of acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine acetyltransferase activity and its application to screening for acetyltransferase inhibitors: Inhibition by magnolol and honokiol from Magnoliae cortex

A method was developed for determining the activity of acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine acetyltransferase (EC 2.3.1.67), a key enzyme in the biosynthesis of platelet-activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine). The assay involves measurement of the radioactivity in the trichloroacetic acid (TCA)-precipitated complex of radioactive product and albumin after incubation of 1-alkyl-sn-glycero-3-phosphocholine and [3H]acetyl-CoA with rat spleen microsomes or membrane fractions of human polymorphonuclear leukocytes (PMNs). The radioactive product associated with the precipitate was identified as PAF using an ultrahigh-sensitivity TV camera system after extraction and separation by TLC. This TCA method was then used to screen the components of crude preparations that inhibited acetyltransferase activity. Major components from the cortex of Magnoliae (magnolol and honokiol), which have anti-inflammatory and anti-bacterial actions, inhibited the acetyltransferase activity in rat spleen microsomes (IC50, 150 and 150 microM, respectively) and membrane fractions of human PMNs (IC50, 70 and 60 microM, respectively). The inhibitory action of magnolol and honokiol was reversible, and similar to or higher than that of nordihydroguaiaretic acid. PAF production in human PMNs stimulated by the ionophore A23187 was also suppressed dose dependently by magnolol and honokiol. These activities may be relevant to the claimed therapeutic effects of the extract from Magnoliae cortex.

Absence of prostaglandin E2-induced hyperalgesia in NMDA receptor ε subunit knockout mice

We have previously found that intrathecal administration of prostaglandins E2 (PGE2) and D2 (PGD2) into conscious mice induced hyperalgesia by the hot plate test. The present study investigated the involvement of N‐methyl‐d‐aspartate (NMDA) receptor in the prostaglandin‐induced hyperalgesia by use of mice lacking NMDA receptor ε1, ε4, or ε1/ε4 subunits. PGE2 induced hyperalgesia over a wide range of doses from 50 pg to 500 ng kg−1 in wild‐type mice. But PGE2 could not induce hyperalgesia in ε1, ε4, or ε1/ε4 subunit knockout mice. The NMDA receptor antagonist d‐(−)‐2‐amino‐5‐phosphonovaleric acid (d‐AP5), the non‐NMDA receptor antagonist γ‐d‐glutamylaminomethyl sulphonic acid (GAMS), and the nitric oxide synthase inhibitor Nω‐nitro‐l‐arginine methyl ester (l‐NAME) inhibited the PGE2‐induced hyperalgesia in wild‐type mice. PGD2 induced hyperalgesia at doses of 25 ng to 250 ng kg−1 in both wild‐type and ε1/ε4 subunit knockout mice. The substance P receptor antagonist CP 96,345 blocked the PGD2‐induced hyperalgesia in wild‐type and ε1/ε4 subunit knockout mice. These results demonstrate that the pathways leading to hyperalgesia are different between PGD2 and PGE2 and that both ε1 and ε4 subunits of the NMDA receptor are involved in the PGE2‐induced hyperalgesia.

Properties and localization of phospholipase A2 activity in rat stomach.

In the glandular stomach of rat, phospholipase A2 activity was detected and characterized by the use of sonicated 1-acyl-2-[1-14C]oleoyl-sn-glycero-3-phosphocholine (1 mM in 200 mM glycylglycine buffer/2 mM CaCl2) as substrate. The specific activity was 4.9 X 10(-2) mumol/min per mg protein in the whole glandular stomach homogenate (n = 8). It showed individual variation among rats. The optimal pH was 8.0. The activity was relatively heat-resistant and calcium-dependent. More than 90% of phospholipase A2 activity was located in the corpus and less than 10% was in the antrum. In the corpus wall, which consists of mucosa, submucosa, muscularis externa and serosa, the mucosal part (mucosa and submucosa) contained 80-90% of total activity. The effect of some clinical agents (ulcerogenic and anti-ulcer agents) on the phospholipase A2 activity was examined. The activity in the corpus was inhibited by cimetidine (50% inhibition at 5 X 10(-3) M) and stimulated by indomethacin (50% stimulation at 5 X 10(-5) M). Cetraxate hydrochloride (10(-6)-10(-3) M), 16,16-dimethylprostaglandin E2 (10(-7)-10(-4) M) and dexamethasone (10(-7)-10(-3) M) had no effect.

Involvement of prostaglandin E2 in rabbit corneal injury by anterior segment ischaemia

The involvement of prostaglandins (PGs) in the development of anterior segment ischaemia after occlusion of the bilateral long posterior ciliary arteries was investigated in rabbit eyes. In this experimental ischaemia, the tissue weight and protein content in the peripheral cornea and the protein content in the aqueous humour increased on the first postoperative day. Topically applied cyclooxygenase inhibitor diclofenac (0.1%) reduced corneal inflammation and further suppressed the elevation in the tissue weight and protein content in the peripheral cornea on day 1 after ischaemia, but did not affect the changes in the aqueous humour. Subconjunctivally administered PGE1 and PGE2 induced corneal oedema and increased corneal protein content in diclofenac-treated and ischaemia-induced eyes, but PGD2, PGF2alpha, and the stable PGI2 analogue cicaprost did not evoke any change. In fact, PGE2 content was markedly increased in the aqueous humour on day 1 after ischaemia, and diclofenac suppressed the increase. In addition, CPT-cAMP increased the corneal tissue weight and protein content in organ culture. These observations suggest that PGE2 may play an important role in developing corneal oedema at the initial stage of ischaemic damage, possibly through the cAMP-mediated pathway.

Auditory pathway and auditory brainstem response in mice lacking NMDA receptor ϵ1 and ϵ4 subunits

Abstract There is considerable evidence that the N -methyl- d -aspartate receptor ((NMDAR) is a component of excitatory amino acid synapses in the ascending auditory pathway. The availability of mice that are defective in NMDAR ϵ 1 or NMDAR ϵ 4 subunit paves the way for investigations on the role of this receptor in auditory function. Non-radioactive in situ hybridization was used in the parent C57/6J wild strain to determine if these subunits are normally expressed in cochlear nucleus (CN) and superior olivary complex (SOC) and to confirm their absence in the respective mutant mice. Evoked auditory brainstem response (ABR) to normal acoustic stimulation was investigated to assess function. In situ hybridization revealed the expression of NMDAR ϵ 1 and ϵ 4 subunits mRNAs in major neuronal types in the CN and SOC of the wild type mice while ϵ 1 and ϵ 4 expression were absent in their respective mutant mice. The ABR threshold for the ϵ 1 mutant mice was similar to that of wild type mice however the threshold for the ϵ 4 mutant mice was significantly elevated. These results suggest a role for the NMDAR ϵ 4 in normal auditory functions while the NMDAR ϵ 1 may have a less critical function under normal conditions.