Norimichi Nakahata

Lipid rafts, formed by sphingolipids and cholesterol within the membrane bilayer, are believed to have a critical role in signal transduction. P2Y₂ receptors are known to couple with G_q family G proteins, causing the activation of phospholipase C (PLC) and an increase in intracellular Ca²⁺ ([Ca²⁺]_i) levels. In the present study, we investigated the involvement of lipid rafts in P2Y₂ receptor-mediated signaling and cell migration in NG 108-15 cells. When NG 108-15 cell lysates were fractionated by sucrose density gradient centrifugation, Gα_q/11 and a part of P2Y₂ receptors were distributed in a fraction where the lipid raft markers, cholesterol, flotillin-1, and ganglioside GM1 were abundant. Methyl-β-cyclodextrin (CD) disrupted not only lipid raft markers but also Gα_q/11 and P2Y₂ receptors in this fraction. In the presence of CD, P2Y₂ receptor-mediated phosphoinositide hydrolysis and [Ca²⁺]_i elevation were inhibited. It is noteworthy that UTP-induced cell migration was inhibited by CD or the G_q/11-selective inhibitor YM254890 [(1R)-1-{(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16, 22-hexamethyl-15-methylene-2,5,8,11,14,17,–20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl}-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate]. Moreover CD and YM254890 completely inhibited Rho-A activation. Downstream of Rho-A signaling, stress fiber formation and phosphorylation of cofilin were also inhibited by CD or YM254890. However, UTP-induced phosphorylation of cofilin was not affected by the expression of p115–regulator of G protein signaling, which inhibits the G_12/13 signaling pathway. This implies that UTP-induced Rho-A activation was relatively regulated by the G_q/11 signaling pathway. These results suggest that lipid rafts are critical for P2Y₂ receptor-mediated G_q/11–PLC–Ca²⁺ signaling and this cascade is important for cell migration in NG 108-15 cells.

スズメバチ毒素のマストパランはヒスタミン遊離活性を指標に見出された14個のアミノ酸残基からなるペプチドである．マストパランは三量体Gタンパク質の活性化作用を有し，活性化されたGタンパク質共役型受容体の如く振る舞うことから，薬理学的ツールとして貴重な存在である．しかしながら，マストパランには三量体Gタンパク質の活性化作用以外の作用も知られており，細胞膜リン脂質との相互作用による細胞膜におけるポアの形成は細胞障害性を示すことも知られており，注意を必要とする．その他にも，ガングリオシドのネガティブチャージとの結合や，タンパク質の脂溶性の部分と結合して，タンパク質機能を変化させる．いずれにしても，マストパランは強い生物活性を持ちシグナル伝達に影響を与える，魅力的なペプチドである．

Tohoku J. Exp. Med., 221: 287-298, 2010. In the version of this article published in the August issue, 2010, the affiliation number of the tenth author (Shigeru Tsuchiya) was mistyped (page 287). The correct affiliation is shown above. In addition, the lettering of ‘ZT’ is a misprint for ‘CT’ in panels A, B and C of Figures 6 and 7 (pages 294 and 295).

Background Many bedridden patients develop pressure ulcers, not only in hospital but also at home. Clinical studies have indicated cigarette smoking to be a risk factor for pressure ulcers. However, the contribution of nicotine to pressure ulcer formation has not been identified. Objectives We aimed to clarify the effect of nicotine on pressure ulcer formation, and its mechanism. Methods Ischaemia‐reperfusion (I/R) was performed in rat dorsal skin to induce pressure ulcers. The extent of the resulting necrotic area was determined. To clarify the mechanism of the effect of nicotine, mRNA levels of cyclooxygenase‐2 (COX‐2), interleukin (IL)‐1β, IL‐6 and inducible nitric oxide synthase (iNOS) and protein expression of COX‐2 and iNOS in the necrotic area were investigated by real‐time reverse transcription–polymerase chain reaction and Western blotting, respectively. Furthermore, the effects of the COX‐2 inhibitor NS‐398 and the iNOS inhibitor aminoguanidine on necrosis were examined. Results Skin necrosis in the I/R‐treated area was significantly increased by intraperitoneal administration of nicotine (0·175 mg kg⁻¹ daily). Repeated nicotine administration had little effect on systolic and diastolic blood pressure. I/R treatment increased mRNA levels of COX‐2, IL‐1β, IL‐6 and iNOS, which were further augmented by nicotine in a dose‐dependent manner. Correspondingly, nicotine (0·35 mg kg⁻¹ daily) markedly enhanced the protein expression of COX‐2 and iNOS. Moreover, NS‐398 and aminoguanidine showed a tendency to abrogate the increase of I/R‐induced skin necrosis caused by nicotine. Conclusions These results suggest that the increased risk of pressure ulcers due to cigarette smoking is mediated, in part, by nicotine. They also indicated that the effect of nicotine is not mediated by a change in blood pressure, but is elicited via an increase of inflammatory mediators in the I/R‐treated skin.