JP2004537251A - Anchor for safety rope - Google Patents

Abstract

The present invention relates to the regulation of the activity of the enzyme nitric oxide synthase, and in particular to the regulation of the activity of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS and nNOSμ). According to a first aspect, the present invention provides a method for identifying a modulator of AMPK-mediated activation of eNOS, comprising a putative modulator of the ability to increase or decrease eNOS phosphorylation by calmodulin and calcium ion concentrations. A method comprising testing In an alternative aspect, the invention provides a method of identifying a modulator of AMPK-mediated suppression of eNOS, comprising a putative modulator of the ability to reduce or increase AMPK-mediated phosphorylation of eNOS in the presence of limiting calcium ions. A method comprising the steps of: Preferably, specific phosphorylation of threonine 495 is assessed. According to a second aspect, the present invention provides a method for identifying a modulator that promotes or suppresses phosphorylation of nNOS and nNOSμ at Ser-1417. Activated protein kinase, a compound that activates AMP, is known to stimulate glucose and fatty acid metabolism and to increase NOS activity to improve nutrient and oxygen supply to muscle cells and reduce mechanical activity. It is expected to be useful for treating ischemic heart disease. These compounds also have utility in treating pulmonary hypertension and obstructive airway disease.

Description

【００２５】
^３２Ｐ−リン酸放出シーケンシング（Ｍｉｔｃｈｅｌｈｉｌｌ ｅｔ ａｌ， １９９７ａ）により、ペプチドＡ、ＴＱＸＦＳＬＱＥＲ中のリン酸化部位の位置を同定した。ＡＭＰＫ−α１によりリン酸化されたｅＮＯＳは、ｅＮＯＳ ＣＯＯＨ−末端に対する抗体によってもはや認識されなかった、あるいは１００ｍＭのＮＡＤＰＨによりＡＤＰ−セファロースアフィニティーカラムから溶離されなかった。これらの特性は、ｉｎ ｓｉｔｕにおけるＳｅｒ−１１７７のリン酸化の直接的確証を妨げた。これは、Ｖｅｎｅｍａ等（１９９６）に説明されている。
【００２６】
Ｃａ^２＋−ＣａＭの非存在下で、またはＥＧＴＡが存在する場合に、第二の部位であるＴｈｒ−４９５をリン酸化した。これを、図２の下部パネルＢで説明する。この残基は、ｅＮＯＳのオキシダーゼおよびレダクターゼドメイン間のＣａＭ結合配列：
ＴＲＫＫＴ^４９５ＦＫＥＶＡＮＡＶＫＩＳＡＳＬＭ
中にある（Ｖｅｎｅｍａ ｅｔ ａｌ， １９９６）。ｅＮＯＳのＮ末端領域中のＳｅｒ−１０１を、リン酸化の副次的部位として同定した（図２の下部パネルのＣ）。
【００２７】
ＡＭＰＫにより、Ｔｈｒ−４９５またはＳｅｒ−１１７７を含有する合成ペプチドを、容易にリン酸化した。カイネティック値はＳＡＭＳペプチド基質と同様であった。Ｔｈｒ−４９５を含有するペプチドＧＴＧＩＴＲＫＫＴＦＫＥＶＡＮＡＶＫは、３９±１０μＭのＫｍおよび６．７±０．６μｍｏｌ／分／ｍｇのＶｍａｘでリン酸化され、一方、Ｓｅｒ−１１７７を含有するペプチドＲＩＲＴＱＳＦＳＬＱＥＲＱＬＲＧは、５４±６μＭのＫｍおよび５．８±０．３μｍｏｌ／分／ｍｇのＶｍａｘでリン酸化された。これらは、３３±３μＭのＫｍおよび８．１±１．５μｍｏｌ／分／ｍｇのＶｍａｘを有する十分特性化されたＳＡＭＳペプチド基質を用いて得られた結果（Ｍｉｃｈｅｌｌ ｅｔ ａｌ， １９９６）に匹敵する。ペプチドのｉｎ ｖｉｔｒｏリン酸化は、リン酸化の同定部位を確証する。
【００２８】
実施例３：ＡＭＰＫによるｅＮＯＳのリン酸化に及ぼすＣａ^２＋−ＣａＭの効果
Ｂａｌｌｉｇａｎｄ等，１９９５の方法を用いてＬ−［^３Ｈ］−シトルリン産生を測定することにより、ｅＮＯＳ活性を確定した。Ｒｏｄｒｉｇｕｅｚ−Ｃｒｅｓｐｏ ａｎｄ Ｏｒｉｔｉｚ ｄｅ Ｍｏｎｔｅｌｌａｎｏ， １９９６に記載されたように、組換え体ｅＮＯＳをＣａＭと同時に発現させた。部分精製ラット心臓ｅＮＯＳは、多少のＣａ^２＋−ＣａＭを含有した。付加的ＥＧＴＡの非存在下で、ＣａＭ依存性を０〜１００ｎＭの付加的ＣａＭ存在下で観察した。Ｃａ^２＋−ＣａＭの非存在下および存在下でのリン酸化に伴うＮＯＳ活性の変化を調べるために、ＥＧＴＡ緩衝を用いて、０〜１μＭの範囲でＣａＭ用量反応曲線を作製した。ルーチンに、７〜１５μＭのＥＧＴＡを付加して、付加的ＣａＭに依存させてｅＮＯＳ活性を生じさせた。ｅＮＯＳ検定前にリン酸化反応にＣａ^２＋−ＣａＭを用いる場合、余分のＣａ^２＋−ＣａＭがごくわずかであるように、または指示濃度が付加的Ｃａ^２＋−ＣａＭの総最終濃度を表すように、試料を稀釈する。
【００２９】
心臓ｅＮＯＳを、以下のように部分精製した。２０匹のラットの心臓を、８０ｍｌの氷冷緩衝液Ａ［５０ｍＭ トリス塩酸、ｐＨ７．５、１ｍＭ ＥＤＴＡ、１ｍＭ ＥＧＴＡ、１ｍＭ ＤＴＴ、５０ｍＭ ＮａＦ、５ｍＭ ピロリン酸ナトリウム、１０μｇ／ｍｌ トリプシン阻害剤、２μｇ／ｍｌ アプロチニン、１ｍＭ ベンズアミジン、１ｍＭ ＰＭＳＦ、１０％ グリセロール、１％ トリトン−Ｘ−１００］中でホモジネートした。ホモジネートを３０分間氷上に載せて、１６，０００×ｇで３０分間遠心分離した。上清を２ｍｌの２’，５’−ＡＤＰ−セファロース（Ｂｒｅｄｔ ａｎｄ Ｓｎｙｄｅｒ， １９９０）とともにインキュベートした。懸濁液を２０ｍｌの緩衝液Ａおよび０．５Ｍ ＮａＣｌを含有する２０ｍｌの緩衝液Ａを用いて、次に２０ｍｌの緩衝液Ｂ［５０ｍＭ トリス塩酸、ｐＨ７．５、１ｍＭ ＤＴＴ、１０％ グリセロール、０．１％ トリトン−Ｘ−１００］を用いて、フリットカラム中で洗浄する前に、１時間インキュベートした。２ｍＭ ＮＡＤＰＨを含有する緩衝液ＢでｅＮＯＳを溶離し、次に遠心分離濾過（ＵＬＴＲＡＦＲＥＥ−ＭＣ ＭＩＬＬＩＰＯＲＥ）を施して、ＮＡＤＰＨを除去した。ｎＮＯＳよりｅＮＯＳの選択的検出のために、免疫ブロッティングを行った。
【００３０】
図３の上部パネルに示したように、Ｃａ^２＋−ＣａＭの存在下でのＡＭＰＫによるｅＮＯＳのリン酸化は、活性化を起こすが、しかしＣａＭ依存性は保持された。活性化により、用量反応曲線がＣａＭに関して左にシフトした。ホスホペプチドマッピングは、図３の下部パネルに示したように、ｅＮＯＳの活性化がＳｅｒ−１１７７のリン酸化と相関するが、Ｔｈｒ−４９５のリン酸化とは相関しない、ということを明示した。付加的なＣａ^２＋−ＣａＭを伴わないリン酸化はＴｈｒ−４９５リン酸化を増強し、Ｓｅｒ−１１７７リン酸化を抑制し、ｅＮＯＳ活性を阻害した（図３上部パネル）。Ｔｈｒ−４９５リン酸化によるｅＮＯＳ活性の阻害は、プロテインキナーゼＣによるこの領域に対応する合成ペプチドのリン酸化がＣａＭ結合を阻害するという初期報告と一致する（Ｍａｔｓｕｂａｒａ ｅｔ ａｌ， １９９６）。同様の結果は、ｎＮＯＳに関して報告されている（Ｌｏｃｈｅ ｅｔ ａｌ， １９９７）。
【００３１】
実施例４：ＡＭＰＫ−α１、ＡＭＰＫ−α２およびｅＮＯＳの活性に及ぼす虚血の効果
単離された還流ラット心臓のＬａｎｇｅｎｄｏｒｆ調製物を、Ｋｕｄｏ等（１９９５）の方法により虚血させた。ＡＭＰＫ−α１およびＡＭＰＫ−α２アイソフォームを、α２（４９０−５１６）またはα１（２３１−２５１）抗体を用いて免疫沈降させ、ＳＡＭＳペプチド基質を用いて検定した（Ｍｉｃｈｅｌｌ ｅｔ ａｌ， １９９６； Ｈａｒｄｉｅ ａｎｄ Ｃａｒｌｉｎｇ， １９９７）。ｅＮＯＳ活性を、実施例３に記載されているように測定した。結果を図４に示す。α１およびα２アイソフォームはともに、図４Ａに示したように活性化されるが、これは、ＡＭＰＫが、主にα２アイソフォームを有する毛管内皮細胞、および主にα１アイソフォームを有する心筋細胞の両方で活性化されることを示す。虚血中のＡＭＰＫ活性化はｅＮＯＳ活性化も伴い、図４Ｂおよび４Ｃに示したようにＣａＭ依存性を変化させ、図３に示したようなｉｎ ｖｉｔｒｏでのＡＭＰＫによるｅＮＯＳリン酸化の作用に似ている。
【００３２】
ｅＮＯＳ配列：ＲＩＲＴＱＳｐＦＳＬＱＥＲおよびＧＩＴＲＫＫＴｐＦＫＥＶＡＮＣＶを基礎にした合成ホスホペプチドに対して、ポリクローナル抗体を生じさせた。慣用的方法を用いて、鍵穴吸着ヘモシアニンと結合したホスホペプチドを用いてウサギを免疫感作し、次にフロイントの完全アジュバント中で乳化させた。デホスホペプチドアフィニティーカラムを用いて予備精製後に、対応するホスホペプチドアフィニティーカラムを用いて、抗体を精製した。ＥＩＡおよび免疫ブロッティングの両方を用いて精製抗体の特異性を確証し、それらが組換え脱リン酸−ｅＮＯＳを認識しなかったことを確証した。
【００３３】
Ｓｅｒ−１１７７およびＴｈｒ−４９５リン酸化部位に対する抗ホスホペプチド抗体を用いて、Ｓｅｒ−１１７７のリン酸化が虚血により約３倍増大されたが、これらの条件下ではＴｈｒ−４９５リン酸化の検出可能な変化は認められなかったことが観察された。心筋は、毛管内皮細胞と心筋細胞の両方にｅＮＯＳを含有し（Ｂａｌｌｉｇａｎｄ ｅｔ ａｌ， １９９５）、低レベルのｎＮＯＳμアイソフォームを有する（Ｓｉｌｖａｇｎｏ ｅｔ ａｌ， １９９６）。
【００３４】
３つの型のＮＯＳの配列を図５で比較しているが、これは、ＣａＭ結合領域およびＣ末端を示す。ｎＮＯＳでは、Ｓｅｒ−１４１７はｅＮＯＳのＳｅｒ−１１７７に対応し、一方ｉＮＯＳは切断され、この領域にＧｌｕを有する。ｉＮＯＳおよびｎＮＯＳはともに、ＣａＭ結合領域においてＴｈｒ−４９５に相当するリン酸化可能残基を欠く。
【００３５】
実施例５：ｅＮＯＳリン酸化に及ぼすプロテインキナーゼＣの刺激の効果
０．１％ウシ胎仔血清中（血清飢餓）で２０時間培養したウシ大動脈内皮細胞に、プロテインキナーゼＣ活性化物質である０．１μＭ フォルボール−１２−ミリスチン酸−１３−酢酸（ＰＭＡ）による処置を５分間施した。ＰＭＡ処置は、抗ホスホペプチド特異的抗体を用いて測定した場合、Ｔｈｒ−４９５でのｅＮＯＳのリン酸化を増大し、Ｓｅｒ−１１７７でのリン酸化を低減した。使用した抗体は、実施例４に記載したものと同一であった。結果を図６に示す。カルシウムを含有しない培地中で培養した細胞において、Ｓｅｒ−１１７７リン酸化が４分の１に低減したことが観察された。さらに、カルシウムを含有する標準培地中で細胞をインキュベートした場合、カルシウムイオノフォアＡ２３１８７の付加（９０秒間１０μＭ）は、Ｓｅｒ−１１７７リン酸化をさらに７倍増大させた。０．５μＭのオカダ酸を用いた細胞の予備インキュベーションは、ＰＭＡ処理によるＳｅｒ−１１７７の脱リン酸化を妨げ、Ｔｈｒ−４９５のリン酸化を大いに増大させた（結果は平均±ＳＥＭ、ｎ＝６）。オカダ酸はプロテインホスファターゼＰＰ２Ａを抑制するため、結果は、ＰＰ２ＡがＳｅｒ−１１７７の脱リン酸化に関与することを示す。ＰＭＡおよびオカダ酸による処理に応答してＴｈｒ−４９５およびＳｅｒ−１１７７リン酸化で観察された変化は、ｅＮＯＳの活性に反映された。ＰＭＡまたはＰＭＡ＋オカダ酸によるＴｈｒ−４９５のリン酸化増大は、ｅＮＯＳ活性の低減に関連した。オカダ酸単独では、Ｓｅｒ−１１７７リン酸化を増大し、Ｔｈｒ−４９５リン酸化への変化を伴わず、ｅＮＯＳ活性増大に関連した（図６の上部パネル）。
【００３６】
実施例６：ｅＮＯＳのリン酸化に及ぼすホスホジエステラーゼおよびホスファターゼの抑制の効果
実験の詳細は、実施例５と同様であった。ウシ大動脈内皮細胞を、１０ｎＭ ホスファターゼ阻害物質カリキュリンＡを用いてまたは用いずに１０分間、予備インキュベートし、次に０．５ｍＭ ホスホジエステラーゼ阻害物質３−イソブチル−１−メチルキサンチン（ＩＢＭＸ）を用いてまたは用いずに５分間、インキュベートした。図７に示したように、ＩＢＭＸ処理はＳｅｒ−１１７７のリン酸化およびＴｈｒ−４９５の脱リン酸化の増強を引き起こした。カリキュリンＡを用いた予備インキュベーションは、Ｔｈｒ−４９５の脱リン酸化を妨げた（結果は平均±ＳＥＭ、ｎ＝６）。カリキュリンＡはプロテインホスファターゼＰＰ１を抑制するため、結果は、ＰＰ１がＴｈｒ−４９５の脱リン酸化に関与することを示す。
【００３７】
考察
本発明人によるＳｅｒ−１１７７リン酸化部位の同定以来、他のプロテインキナーゼがこの部位でリン酸化をすることが認識されてきた。特に、プロテインキナーゼＡｋｔ（ＰＫＢとも呼ばれる）は、血管内皮細胞増殖因子（ＶＥＧＦ）による内皮細胞の刺激（Ｆｕｌｔｏｎ ｅｔ ａｌ， １９９９； Ｍｉｃｈｅｌｌ ｅｔ ａｌ．， １９９９）に、あるいは流体剪断応力（Ｄｉｍｍｅｌｅｒ ｅｔ ａｌ．， １９９９； Ｇａｌｌｉｓ ｅｔ ａｌ．， １９９９）に応答してＳｅｒ−１１７７をリン酸化する。Ｇａｌｌｉｓ等（１９９９）による研究では、流体剪断応力は、配列ＫＬＱＴＲＰＳＰＧＰＰＰＡ中のＳｅｒ−１１６のリン酸化を刺激する、ということが報告された。ｅＮＯＳ上のこの部位に関与するキナーゼも、リン酸化の機能的効果も未だ同定されていない。このリン酸化部位は、オキシダーゼドメイン中に存在する。
【００３８】
プロテインキナーゼＣによるＴｈｒ−４９５でのｅＮＯＳのリン酸化は、血清飢餓で、フォルボールエステルＰＭＡの存在下で無カルシウム培地中でインキュベートさせた内皮細胞中で起こる、ということを我々は見出した。内皮細胞中のＳｅｒ−１１７７とＴｈｒ−４９５でのリン酸化の間には、相互補足的な関係が存在する。プロテインキナーゼＣは、ｉｎ ｖｉｔｒｏで両部位をリン酸化するが、しかしフォルボールエステルによる内皮細胞中のプロテインキナーゼＣの刺激は、Ｔｈｒ−４９５リン酸化の増強、しかしＳｅｒ−１１７７の顕著なリン酸化を引き起こす。Ｓｅｒ−１１７７の脱リン酸化は、オカダ酸により妨げられるが、しかしカリキュリンＡによっては妨げられず、これはホスファターゼＰＰ２Ａが関与することを示す。オカダ酸は、フォルボールエステルに応答してＴｈｒ−４９５のリン酸化も大いに増強する。プロテインキナーゼＣを介して作用するトロンビンも、Ｔｈｒ−４９５のリン酸化およびＳｅｒ−１１７７の脱リン酸化を刺激する。
【００３９】
これに対比して、ホスホジエステラーゼ阻害物質である３−イソブチル−１−メチルキサンチン（ＩＢＭＸ）による内皮細胞の処理は、Ｔｈｒ−４９５でのｅＮＯＳの顕著な脱リン酸化およびＳｅｒ−１１７７リン酸化増強を引き起こす。ＩＢＭＸに応答するＴｈｒ−４９５の脱リン酸化はカリキュリンＡを用いた処理により遮断されるが、これは、ホスファターゼＰＰ１がＴｈｒ−４９５の脱リン酸化に関与することを示唆する。
【００４０】
これらの関係を図８に要約する。骨格筋の運動は、ｅＮＯＳのＳｅｒ−１１７７に対応する部位であるｎＮＯＳμのＳｅｒ−１４１７のリン酸化を生じることを我々は確認した（図５参照）。ラット長指伸筋（ＥＤＬ）の電気的刺激は、ＡＭＰＫを活性化し、アセチルＣｏＡカルボキシラーゼをＳｅｒ−７９（阻害部位）でリン酸化し、およびｎＮＯＳμをＳｅｒ１４１７でリン酸化することが判明した。同様に、３０秒間自転車スプリントのような激しい運動後のヒト骨格筋の生検では、アセチルＣｏＡカルボキシラーゼ中のＳｅｒ−７９でのリン酸化の１０倍増大、ならびにｎＮＯＳμのＳｅｒ−１４１７でのリン酸化の７．５倍増大が認められる（図９参照）。
【００４１】
内皮由来ＮＯは、血栓形成をもたらす未熟血小板の付着および凝集防止に重要な役割を有する（Ｒａｄｏｍｓｋｉ ａｎｄ Ｍｏｎｃａｄａ， １９９３）。心臓血管系に及ぼす高密度リポタンパク質（ＨＤＬ）増大の防御作用は、血小板ＮＯ産生増大により仲介され得る、という証拠がある。ＨＤＬの一構成成分であるアポリポタンパク質Ｅは、血小板中に存在する受容体（アポＥＲ２）に作用して、ＮＯシグナル伝達経路を刺激する（Ｒｉｄｄｅｌｌ ｅｔ ａｌ．， １９９７； Ｒｉｄｄｅｌｌ ａｎｄ Ｏｗｅｎ， １９９９）。
【００４２】
そのＣＯＯＨ末端のリン酸化によるｅＮＯＳの活性化は、ｅＮＯＳ自己阻害に新規の見解を与える。Ｓｅｒ−１１７７でのリン酸化に伴う活性化増大およびＣａＭ用量依存性のシフトは、ｅＮＯＳにおいて、そしておそらくはｎＮＯＳにおいても、ＣＯＯＨ末端が、ＣａＭ依存性プロテインキナーゼにおけるのと同様に部分自己調節配列として作用することを示唆する（Ｋｅｍｐ ａｎｄ Ｐｅａｒｓｏｎ， １９９１； Ｋｏｂｅ ｅｔ ａｌ， １９９６）。
【００４３】
ｅＮＯＳのＣＯＯＨ末端は、Ｃａ^２＋−ＣａＭが結合した場合にのみ完全にＡＭＰＫが接触可能であり、これはこの領域がＣａＭの非存在下で埋没していることと一致する。図５から分かるように、それらのＣＯＯＨ末端においてｅＮＯＳとｎＮＯＳとの間の高レベルの類似性が存在するが、一方でｉＮＯＳは異なっている。低Ｃａ^２＋依存性による特性化されるｉＮＯＳ ＣａＭ結合は標準的ＣａＭ結合配列、ならびにｎＮＯＳキメラでは満たされ得ないＣＯＯＨ末端中の遠位残基の両方を必要とする（Ｒｕａｎ ｅｔ ａｌ， １９９６）。いかなる提案されたメカニズムとも結びつけずに考えると、ｅＮＯＳおよびｎＮＯＳはそれらのＣＯＯＨ末端により自己阻害され、末端部におけるＣａＭ結合およびリン酸化の両方による完全活性のための２段階活性化工程を要するが、一方、ｉＮＯＳはＣａＭ結合のみを必要とする。近年、Ｓａｌｅｒｎｏ等（１９９７）は、ＦＭＮ結合ドメインにおける挿入配列も自己調節に重要であり得ることを提唱した。
【００４４】
従来の研究は、ｅＮＯＳがｉｎ ｖｉｔｒｏおよびｉｎ ｖｉｖｏの両方でリン酸化され得ることを示しているが、しかし、リン酸化の正確な部位およびリン酸化事象の機能は、これまで完全には特性化されていない（Ｍｉｃｈｅｌ ａｎｄ Ｆｅｒｏｎ， １９９７で検討されている）。ｅＮＯＳは、ＡＭＰＫにより活性化される酵素として同定される最初の例であり、そしてリン酸化が、Ｃａ^２＋−ＣａＭの獲得可能性によって、活性化または抑制をもたらすため、一般的ではない。その他の酵素、特にサイクリン依存性プロテインキナーゼは、リン酸化により活性化または抑制されるが、しかしこれは、異なるプロテインキナーゼにより触媒される。プロテインキナーゼＣはｅＮＯＳ中のＴｈｒ−４９５をリン酸化するが、このことは、Ｔｈｒ−４９５またはＳｅｒ−１１７７のリン酸化によるｅＮＯＳへの作用の調節経路が交差していることを実証する。例えば糖尿病により誘導される高血糖症に応答するプロテインキナーゼＣの持続的活性化がＳｅｒ−１１７７でのｅＮＯＳのリン酸化を長期的に抑制し、それによりその活性を低減し得るということも考えられる。
【００４５】
ＡＭＰＫによるｅＮＯＳの調節は、酵母ｓｎｆｌｐキナーゼとＡＭＰＫとの間の概念上の関係を拡張する。ｓｎｆｌｐキナーゼは、インベルターゼを分泌することにより環境からのグルコースの供給を調節し、一方哺乳類ＡＭＰＫは循環系の制御に伴って代謝ストレスシグナリングを統括する。したがって、内皮細胞および筋細胞内の細胞内代謝ストレスシグナルは、栄養供給改善を引き起こし、筋肉の機械的活動を抑制し得る。
【００４６】
本発明を分かり易くし、理解するために多少詳細に本発明を説明してきたが、本明細書中に開示された本発明の概念の範囲を逸脱しない限り、本明細書中に記載した実施態様および方法に対する種々の修正および変更が成され得る、ということは当業者には明らかである。
【００４７】
本明細書中に引用した参考文献を以下のページに列挙するが、これらの記載内容は、参照により本明細書中に含まれる。
【表２】

【表３】

【表４】

【表５】

【図面の簡単な説明】
【図１】
心臓および前脛骨筋におけるＡＭＰＫ−α２の蛍光免疫局在を示す。
パネルＡは、対照ウサギＩｇＧおよび対照マウスＩｇＧで染色し、抗ウサギＦＩＴＣおよび抗マウステキサスレッドで染色したラット心臓の陰性対照切片を示す。
パネルＢは、ＡＭＰＫ−α２（４９１−５１４）に対するアフィニティー精製ウサギポリクローナル抗体および抗ウサギＦＩＴＣで染色したラット心臓の切片を示す。
パネルＣは、ラット内皮ｒｅｃＡ−１に対するモノクローナル抗体および抗マウステキサスレッドで染色した、パネルＢと同一の切片を示す。
パネルＤは、パネルＢおよびＣの上層を示す。同時染色により、共局在化が観察され得る。矢印は、両抗体により染色される特異的内皮細胞を示している。
パネルＥは、対照ウサギＩｇＧおよび対照マウスＩｇＧで染色し、抗ウサギＦＩＴＣおよび抗マウステキサスレッドで染色したラット前脛骨筋の陰性対照切片を示す。
パネルＦは、ＡＭＰＫ−α２（４９１−５１４）に対するアフィニティー精製ウサギポリクローナル抗体および抗ウサギＦＩＴＣで染色した切片を示す。
パネルＧは、ラット内皮ｒｅｃＡ−１に対するモノクローナル抗体および抗マウステキサスレッドで染色した、パネルＢと同一切片を示す。
パネルＨは、パネルＥおよびＦの上層を示す。同時染色により、共局在化が観察され得る。
【図２】
ＡＭＰＫによる組換えｅＮＯＳのリン酸化を示す。
上部パネル：ｅＮＯＳをラット肝臓ＡＭＰＫ−α１および［γ−^３２Ｐ］ＡＴＰとともにインキュベートした。
レーン１：クーマシー染色ＳＤＳ−ＰＡＧＥ、
レーン２：オートラジオグラフ。
下部パネル：ｅＮＯＳの^３２Ｐ−トリプシンホスホペプチドマップ。
【図３】
Ｃａ^２＋−ＣａＭを付加した場合と付加しない場合の、ＡＭＰＫによるｅＮＯＳのリン酸化の効果を示す。２’，５’−ＡＤＰ−セファロースアフィニティークロマトグラフィーにより精製されたラット心臓ｅＮＯＳを、０．８μＭのＣａＭ／３．２μＭのＣａ^２＋の存在下（中黒丸）で、Ｃａ^２＋−ＣａＭの非存在下（中黒三角）でＡＭＰＫを用いて、そしてＡＭＰＫを用いず（中白四角）に、リン酸化した。リン酸化後、試料を稀釈し、ｅＮＯＳ活性を測定した。下部パネルは、Ｃａ^２＋−ＣａＭの存在下および非存在下でリン酸化されたラット心臓ｅＮＯＳに関するホスホペプチドマップを示す。
【図４】
ＡＭＰＫ−α１、ＡＭＰＫ−α２およびｅＮＯＳの活性に及ぼす虚血の作用を示す。
パネルＡは、ＳＡＭＳペプチド基質を用いて検定される、ＡＭＰＫ−α１およびＡＭＰＫ−α２に特異的な抗体を用いた免疫沈降の結果を示す。示された結果は、平均±ＳＥＭ（ｎ＝５）である。
パネルＢは、５００ｎＭのＣａＭで測定されたｅＮＯＳ活性を示す。
パネルＣは、代表的実験に関する全ＣａＭ−用量応答を有するｅＮＯＳ活性を示す。虚血時：０分（中白丸）、１分（中黒菱形）、１０分（中黒丸）および２０分（中白三角）。４回の反復実験の結果は、２０分虚血ｅＮＯＳ ＣａＭ−依存性が１０分と同様の値を残存した１回の場合以外は、同一であった。
【図５】
ＮＯＳ配列の比較を示す。ｅＮＯＳおよびｎＮＯＳに関するリン酸化部位配列が、ＮＯＳの模式図モデルで示されている。ＣａＭ結合領域（ｅＮＯＳのＴｈｒ−４９５リン酸化部位周辺）から、ＣＯＯＨ末端（ｅＮＯＳのＳｅｒ−１１７７リン酸化部位周辺）に関する配列が示されている。
【図６】
ｅＮＯＳ活性（上部パネル）ならびにＳｅｒ−１１７７およびＴｈｒ−４９５でのリン酸化（下部パネル）に及ぼすフォルボールエステル（ＰＭＡ）およびオカダ酸によるウシ大動脈内皮細胞の処理の効果を示す。
【図７】
Ｓｅｒ−１１７７およびＴｈｒ−４９５でのリン酸化に及ぼす３−イソブチル−１−メチルキサンチン（ＩＢＭＸ）およびカリキュリンＡによるウシ大動脈内皮細胞の処理の効果を示す。
【図８】
プロテインキナーゼＰＫＣ、ＡＭＰＫおよびＡｋｔにより仲介されるＴｈｒ−４９５およびＳｅｒ−１１７７でのリン酸化によるｅＮＯＳの調節の要約を示す。これらの部位でのリン酸化の逆転は、それぞれＩＢＭＸおよびＰＭＡによる細胞の処理に応答してタンパク質ホスファターゼＰＰ１およびＰＰ２Ａにより仲介される。
【図９】
ヒト筋肉中でのｎＮＯＳリン酸化に及ぼす３０秒自転車スプリント運動の効果を示す。ｎＮＯＳは生検物質から抽出され、抗ホスホペプチド抗体を用いてＳｅｒ−１４１７でのリン酸化に関してプローブされた。左パネルは免疫ブロットを示し、右パネルは５個体の定量分析を示す。[0001]
The present invention relates to the regulation of the activity of the enzyme nitric oxide synthase, and in particular to the regulation of the activity of endothelial and neuronal nitric oxide synthase. We have found that phosphorylation of endothelial and neuronal nitric oxide synthase by several protein kinases, including protein kinase C and AMP-activated protein kinase, regulates their activity.
[0002]
(Background of the Invention)
Nitric oxide (NO) has recently been recognized as an important mediator of a very wide variety of cellular functions and is present in most, if not all, mammalian cells (Moncada, S. and Higgs, A., 1993). It is associated with a range of diseases, hypertension, hypocholesterolemia, diabetes, heart disease, aging, inflammation and smoking effects, and is of particular importance in vascular biology. It regulates systemic blood pressure, as well as angiogenesis in response to vascular remodeling (Rudic et al., 1998) and tissue ischemia (Murohara et al., 1998). NO is synthesized from the amino acid L-arginine by the enzyme nitric oxide synthase (NOS).
[0003]
Three isoforms of NOS have been identified: neuronal NOS (nNOS) found in neuronal tissue, and those found in skeletal muscle (nNOSμ isoform); very broad Inducible NOS (iNOS) found in a variety of mammalian tissues, such as activated macrophages, cardiomyocytes, glial cells and vascular smooth muscle cells; and endothelial NOS found in vascular endothelium, cardiomyocytes and platelets ( endothial NOS (eNOS). Endothelial cells produce NO in response to shear stress generated by blood flow over the endothelial layer.
[0004]
The three isoforms of NO synthase have about 55% amino acid sequence homology, with strong sequence conservation in the regions involved in catalysis. For all three isoforms, the mechanism of NO synthesis involves binding of the ubiquitous calcium regulatory protein calmodulin (CaM) to enzymes. However, the conditions under which CaM is bound appear to be different for iNOS, at least as far as calcium concentration is concerned. These three NOS enzymes have been intensively studied and the field has been recently reviewed (see, for example, Michel and Feron (1997); Harrison (1997); and Mayer and Hellens (1997)). Initial studies have shown that eNOS can be variably phosphorylated, but the mechanisms of these phosphorylation events, including the enzymes involved in phosphorylation, and the role of phosphorylation in modulating eNOS function are unknown. Was.
[0005]
AMP-activated protein kinase (AMPK) has a key role in the regulation of acetyl-CoA carboxylase and is a metabolic stress sensitive protein kinase known to result in accelerated fatty acid oxidation during intense exercise or ischemia It is. AMPK is well known as a regulator of lipid metabolism, and in particular is known to have a role in cholesterol synthesis, as reviewed in Hardie and Carling (1997). AMPK is also thought to have an important role in exercise-enhanced glucose transport (Hayashi et al., (1998)), which is distinct from the insulin-mediated glucose uptake mechanism. AMPK has been primarily studied in liver, heart and skeletal muscle. AMPK has been purified and the gene encoding the enzyme subunit has been cloned (see International Patent Applications PCT / GB94 / 01093 and PCT / US97 / 00270 and published WO 97/25341).
[0006]
Mammalian AMPK (Mitchellhill et al. 1994) is correlated with Saccharomyces cerevisiae SNF1 protein kinase. It is required for the expression of the glucose repressor gene in response to the nutrient stress required for growth on alternative carbon sources (Celenza and Carlson, 1986), and both mammalian and yeast kinases are activated by upstream kinases (Hardie and Carling, 1997). AMPK is involved in the metabolic stress response by phosphorylation at Ser-79, and in suppressing the co-localization of acetyl-CoA carboxylase and HMG-CoA reductase (Hardie and Carling, 1997). A variety of AMPK isoforms result. They comprise the α1 or α2 catalytic subunits (Stapleton et al, 1996; Stapleton et al, 1996) along with the non-catalytic subunits β and γ (Mitchellhill et al, 1994; Carling et al, 1994; Stapleton et al, 1994). ) Comprising the αβγ heterotrimers consisting of yeast sip1p and snf4p, respectively.
[0007]
The AMPKα2 subunit gene is on chromosome 1 (Beri et al., 1994), the α1 subunit gene is on chromosome 5, ₁ And γ ₁ The subunit gene is on chromosome 12 ₂ The subunit gene is on chromosome 1 and ₂ The subunit gene is on chromosome 7 (Stapleton et al, 1997). γ ₃ The gene was detected using an expressed sequence tag (EST) generated by genomic sequencing (accession number AA178898).
[0008]
One of the genes encoding eNOS is on chromosome 7 and AMPK γ ₂ In the immediate vicinity of the gene corresponding to the subunit. Another gene encoding nNOS is found on chromosome 12 (human gene map; see http://www.ncbi.nlm.nih.gov/cgi-bin/SCIENCE96/tsrch?QTEXT=nitric+oxide+synthase).
[0009]
Recent studies have demonstrated that AMPK in cardiac and skeletal muscle is activated by intense exercise or by ischemic stress (Winder and Hardie, 1996; Vavvas et al, 1997; Kudo et al, 1995). ). This led us to study the localization of AMPK isoforms in these tissues. The AMPK-α2 isoform is present in capillary endothelial cells in cardiac and skeletal muscle, and the AMPK-α1 isoform occurs in cardiomyocytes and vessels. The presence of AMPK in endothelial cells led us to test bacterially expressed eNOS as a substrate and found that it was readily phosphorylated by AMPK-α1 or AMPK-α2.
[0010]
We have now surprisingly found that AMP-activated protein kinase phosphorylates and regulates endothelial NO synthase. AMPK was found to phosphorylate eNOS at two sites. In the presence of calcium and calmodulin, Ser-1177 in the human sequence and Ser-1179 in the bovine sequence are phosphorylated at the COOH terminus of the enzyme, causing eNOS activation by shifting calmodulin dose dependence. In the absence of additional calcium and calmodulin, phosphorylation also occurs at Thr-495 in the eNOS calmodulin binding sequence, repressing the enzyme. Cardiac ischemia mimics the effect of phosphorylation at Ser-1177, causing activation of AMPK and eNOS. A phosphopeptide-specific antibody against phosphorylated Ser-1177 was used to confirm that this site was phosphorylated during ischemia. Our results show that the linkage between metabolic stress that reduces ATP and increases AMP and signals eNOS to control nutrient availability (by arterial vasodilation) as well as to suppress myocardial contraction, It is particularly interesting because it can be identified. This links the metabolic state of endothelial and muscle cells to vascular supply and mechanical demands. Our results provide new insights into the post-translational regulation of eNOS, which has particular significance in the cardiovascular and skeletal muscle fields. In addition, structural and functional similarities between eNOS and nNOS were identified, allowing us to identify modulators of the activity of both these enzymes.
[0011]
(Summary of the Invention)
According to a first aspect, the present invention relates to a method for identifying a modulator of AMPK-mediated activation of a nitric oxide synthase enzyme selected from the group consisting of eNOS, nNOS and nNOSμ, comprising phosphorylation of said enzyme. Testing the ability of the putative modulator to increase or decrease the concentration of the protein, wherein the increase or decrease is dependent on calmodulin and calcium ion concentration.
[0012]
Preferably, the specific phosphorylation of Ser-1177 is assessed in the presence of calcium and calmodulin.
[0013]
In an alternative aspect, the present invention is a method of identifying a modulator of AMPK-mediated suppression of eNOS, wherein the putative method is for its ability to reduce or increase AMPK-mediated phosphorylation of eNOS in the presence of limiting calcium ions. A method comprising testing a modulator is provided. Preferably, the specific phosphorylation of Thr-495 is assessed.
[0014]
Compounds that can increase the phosphorylation of Ser-1177 and decrease the phosphorylation of Thr-495 are referred to herein as activators and reduce the phosphorylation of Ser-1177 and reduce the phosphorylation of Thr-495. Compounds that can increase oxidation are called inhibitors.
[0015]
In both aspects of the invention, one or more of the following activities may optionally be additionally assessed for each putative activator or inhibitor identified by the method of the invention:
(A) effects on smooth muscle contraction,
(B) effects on the inotropic activity of the heart,
(C) an effect on the cycle altering activity of the heart, or
(D) Effect on platelet function.
[0016]
The phosphorylation site corresponding to Thr-495 in the eNOS calmodulin binding site is missing from the neuronal form of NOS, and it is predicted that the inhibitors and activators identified by the method of the invention will have at least some tissue specificity Is done.
[0017]
Compounds that activate AMP-activated protein kinases may increase glucose and fatty acid metabolism and increase NOS activity to improve nutrient and oxygen supply to muscle cells and reduce mechanical activity. Expected to be useful for bloody heart disease. These compounds have utility in pulmonary hypertension and also in obstructive airway disease.
[0018]
As used herein, the term "comprising" is intended to mean "including" but not limited to, and the term "comprising" is understood to have the corresponding meaning. Obviously.
[0019]
(Detailed description of the invention)
The present invention will now be described in detail with reference only to the following non-limiting examples and drawings.
[0020]
Ca ²⁺ -In the presence of calmodulin (CaM), eNOS is phosphorylated by AMPK at Ser-1177 resulting in activation, while CaNO ²⁺ It has been surprisingly found that phosphorylation of eNOS in the absence of is preferentially caused at Thr-495, a site in the CaM binding sequence, resulting in suppression. It was previously thought that phosphorylation was simply inhibitory. We have also found that cardiac ischemia results in rapid activation of both isoforms of the metabolic stress sensitive enzymes AMPK and eNOS. These data indicate that AMPK manipulates a “flip-over” signaling pathway that leads to reduced arterial vasodilation and myocardial contraction, and therefore can link the metabolic state of endothelial and muscle cells to vascular supply and mechanical activity. Suggests.
[0021]
Example 1: Fluorescent immunolocalization of AMPK-α2 in cardiac and skeletal muscle
Confocal fluorescence immunomicroscopy was performed on AMPK-α2 using affinity purified rabbit polyclonal antibody (antibodies 491-414). Staining with a fluorescently labeled anti-rabbit antibody showed that the α2 isoform was found preferentially in capillary endothelial cells in both myocardial and skeletal muscle, while in cardiomyocytes and blood vessels, It showed a strong but diffuse staining. In skeletal muscle, the α2 isoform was found in capillary endothelial cells and fast twitch muscle fibers, while the α1 isoform was found in type I aerobic fibers. The localization of AMPK-α2 in capillary endothelial cells in both cardiac and skeletal muscle is illustrated in FIG.
[0022]
Example 2: AMPK phosphorylates recombinant eNOS
Bacterial eNOS co-expressed with CaM by the method of Rodriguez-Crespo et al. (1996) was phosphorylated by AMPK-α1 or AMPK-α2 as shown in the upper panel of FIG. Recombinant eNOS phosphorylation by immunoprecipitated AMPK-α2 was detected. Since the highly specific activity of AMPK-α2 could not be purified, no further characterization of sites of eNOS regulation or phosphorylation by α2 isoforms was attempted. Analysis of the phosphorylation sites in eNOS after trypsin digestion revealed four phosphopeptides arising from three distinct sites (A, A ', B, C in the lower panel of FIG. 2). Identification of phosphorylation sites by mass spectrometry and Edman sequencing using a modification of the method described by Mitchellhill and Kemp, 1999, was performed using Ser-1177 as shown in the lower panel A, A 'of FIG. Is the most prominent phosphorylation site, and its phosphorylation is Ca ²⁺ -Demonstrated that it depends on the presence of CaM.
[0023]
Isolation of phosphopeptides from in-gel tryptic digests was performed as described by Mitchellhill et al. (1997a). More than 98% of the radioactivity was recovered from the gel. The peptides isolated and characterized by mass spectrometry and Edman sequencing are described in Table 1.
[0024]
[Table 1]

[0025]
³² The position of the phosphorylation site in peptide A, TQXFSLQER, was identified by P-phosphate release sequencing (Mitchellhill et al, 1997a). ENOS phosphorylated by AMPK-α1 was no longer recognized by antibodies to the eNOS COOH-terminus or was eluted from the ADP-Sepharose affinity column by 100 mM NADPH. These properties prevented direct confirmation of phosphorylation of Ser-1177 in situ. This is described in Venema et al. (1996).
[0026]
Ca ²⁺ -The second site, Thr-495, was phosphorylated in the absence of CaM or in the presence of EGTA. This will be described with reference to the lower panel B of FIG. This residue is a CaM binding sequence between the oxidase and reductase domains of eNOS:
TRKKT ⁴⁹⁵ FKEVANAVKISASLM
(Venema et al, 1996). Ser-101 in the N-terminal region of eNOS was identified as a secondary site of phosphorylation (C in the lower panel of FIG. 2).
[0027]
Synthetic peptides containing Thr-495 or Ser-1177 were readily phosphorylated by AMPK. Kinetic values were similar to the SAMS peptide substrate. The peptide GTGITRKKTFKEVANAVK containing Thr-495 is phosphorylated with a Km of 39 ± 10 μM and a Vmax of 6.7 ± 0.6 μmol / min / mg, while the peptide RIRTQSSFSLQERQLRG containing Ser-1177 has 54 ± 6 μM. It was phosphorylated at Km and Vmax of 5.8 ± 0.3 μmol / min / mg. These are comparable to the results obtained with a well-characterized SAMS peptide substrate with a Km of 33 ± 3 μM and a Vmax of 8.1 ± 1.5 μmol / min / mg (Michell et al, 1996). In vitro phosphorylation of the peptide confirms the site of phosphorylation identification.
[0028]
Example 3: Effect of Ca on phosphorylation of eNOS by AMPK ²⁺ -Effect of CaM
Using the method of Balligand et al., 1995, L- [ ³ ENOS activity was determined by measuring [H] -citrulline production. Recombinant eNOS was co-expressed with CaM as described in Rodriguez-Crespo and Oriziz de Montellano, 1996. The partially purified rat heart eNOS contains some Ca ²⁺ -Contains CaM. In the absence of additional EGTA, CaM dependence was observed in the presence of 0-100 nM additional CaM. Ca ²⁺ -To investigate the change in NOS activity with phosphorylation in the absence and presence of CaM, CaM dose response curves were generated using EGTA buffer in the range of 0-1 μM. The routine was supplemented with 7-15 μM EGTA to generate eNOS activity dependent on additional CaM. Ca before phosphorylation reaction before eNOS assay ²⁺ -When using CaM, extra Ca ²⁺ -CaM is negligible or the indicated concentration is ²⁺ -Dilute sample to represent total final concentration of CaM.
[0029]
Heart eNOS was partially purified as follows. The hearts of 20 rats were treated with 80 ml of ice-cold buffer A [50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 50 mM NaF, 5 mM sodium pyrophosphate, 10 μg / ml trypsin inhibitor, 2 μg / ml ml aprotinin, 1 mM benzamidine, 1 mM PMSF, 10% glycerol, 1% Triton-X-100]. The homogenate was placed on ice for 30 minutes and centrifuged at 16,000 xg for 30 minutes. The supernatant was incubated with 2 ml of 2 ', 5'-ADP-Sepharose (Bredt and Snyder, 1990). The suspension was prepared using 20 ml of buffer A and 20 ml of buffer A containing 0.5 M NaCl, followed by 20 ml of buffer B [50 mM Tris-HCl, pH 7.5, 1 mM DTT, 10% glycerol, 0% .1% Triton-X-100] for 1 hour before washing in a frit column. The eNOS was eluted with buffer B containing 2 mM NADPH, followed by centrifugal filtration (ULTRAFREE-MC MILLIPORE) to remove NADPH. Immunoblotting was performed for selective detection of eNOS over nNOS.
[0030]
As shown in the upper panel of FIG. ²⁺ Phosphorylation of eNOS by AMPK in the presence of -CaM resulted in activation, but CaM dependence was retained. Activation shifted the dose response curve to the left with respect to CaM. Phosphopeptide mapping revealed that eNOS activation correlated with Ser-1177 phosphorylation but not Thr-495 phosphorylation, as shown in the lower panel of FIG. Additional Ca ²⁺ -Phosphorylation without CaM enhanced Thr-495 phosphorylation, suppressed Ser-1177 phosphorylation and inhibited eNOS activity (Fig. 3, upper panel). Inhibition of eNOS activity by Thr-495 phosphorylation is consistent with earlier reports that phosphorylation of a synthetic peptide corresponding to this region by protein kinase C inhibits CaM binding (Matsubara et al, 1996). Similar results have been reported for nNOS (Loche et al, 1997).
[0031]
Example 4: Effect of ischemia on the activity of AMPK-α1, AMPK-α2 and eNOS
The isolated Langendorff preparation of perfused rat heart was ischemic by the method of Kudo et al. (1995). AMPK-α1 and AMPK-α2 isoforms were immunoprecipitated using α2 (490-516) or α1 (231-251) antibodies and assayed using a SAMS peptide substrate (Michell et al, 1996; Hardie and Carling). , 1997). eNOS activity was measured as described in Example 3. FIG. 4 shows the results. Both the α1 and α2 isoforms are activated as shown in FIG. 4A, which indicates that AMPK is active in both capillary endothelial cells with predominantly α2 isoforms and cardiomyocytes with predominantly α1 isoforms. Indicates that it is activated. AMPK activation during ischemia also accompanies eNOS activation and alters CaM dependence as shown in FIGS. 4B and 4C, mimicking the effects of eNOS phosphorylation by AMPK in vitro as shown in FIG. ing.
[0032]
Polyclonal antibodies were raised against synthetic phosphopeptides based on eNOS sequences: RIRTQSpFSLQER and GITRKKTpFKEVANCV. Rabbits were immunized using a phosphopeptide coupled to keyhole-adsorbed hemocyanin using conventional methods, and then emulsified in Freund's complete adjuvant. After preliminary purification using a dephosphopeptide affinity column, the antibody was purified using the corresponding phosphopeptide affinity column. The specificity of the purified antibodies was confirmed using both EIA and immunoblotting, confirming that they did not recognize the recombinant dephosphorylated-eNOS.
[0033]
Using anti-phosphopeptide antibodies against the Ser-1177 and Thr-495 phosphorylation sites, Ser-1177 phosphorylation was increased about 3-fold by ischemia, but under these conditions Thr-495 phosphorylation was detectable. No significant change was observed. Myocardium contains eNOS in both capillary endothelial cells and cardiomyocytes (Balligand et al, 1995) and has low levels of the nNOSμ isoform (Silvagno et al, 1996).
[0034]
The sequences of the three types of NOS are compared in FIG. 5, which shows the CaM binding region and the C-terminus. In nNOS, Ser-1417 corresponds to Ser-1177 of eNOS, while iNOS is truncated and has Glu in this region. Both iNOS and nNOS lack phosphorylatable residues corresponding to Thr-495 in the CaM binding region.
[0035]
Example 5: Effect of stimulation of protein kinase C on eNOS phosphorylation
Treatment of bovine aortic endothelial cells cultured in 0.1% fetal calf serum (serum starved) for 20 hours with 0.1 μM phorbol-12-myristate-13-acetic acid (PMA), a protein kinase C activator For 5 minutes. PMA treatment increased phosphorylation of eNOS at Thr-495 and reduced phosphorylation at Ser-1177 as measured using an anti-phosphopeptide specific antibody. The antibodies used were the same as those described in Example 4. FIG. 6 shows the results. It was observed that Ser-1177 phosphorylation was reduced by a factor of 4 in cells cultured in medium without calcium. In addition, addition of the calcium ionophore A23187 (10 μM for 90 seconds) further increased Ser-1177 phosphorylation by 7-fold when cells were incubated in standard medium containing calcium. Preincubation of cells with 0.5 μM okadaic acid prevented the dephosphorylation of Ser-1177 by PMA treatment and greatly increased the phosphorylation of Thr-495 (results mean ± SEM, n = 6) . Since okadaic acid suppresses the protein phosphatase PP2A, the results indicate that PP2A is involved in Ser-1177 dephosphorylation. The changes observed in Thr-495 and Ser-1177 phosphorylation in response to treatment with PMA and okadaic acid were reflected in eNOS activity. Increased phosphorylation of Thr-495 by PMA or PMA + okadaic acid was associated with reduced eNOS activity. Okadaic acid alone increased Ser-1177 phosphorylation and was associated with increased eNOS activity without change to Thr-495 phosphorylation (FIG. 6, upper panel).
[0036]
Example 6: Effect of inhibition of phosphodiesterase and phosphatase on eNOS phosphorylation
The details of the experiment were the same as in Example 5. Bovine aortic endothelial cells are preincubated for 10 minutes with or without 10 nM phosphatase inhibitor calyculin A, and then with or with 0.5 mM phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). For 5 minutes. As shown in FIG. 7, IBMX treatment caused an increase in Ser-1177 phosphorylation and Thr-495 dephosphorylation. Preincubation with calyculin A prevented Thr-495 dephosphorylation (results mean ± SEM, n = 6). Since caliculin A suppresses protein phosphatase PP1, the results indicate that PP1 is involved in the dephosphorylation of Thr-495.
[0037]
Consideration
Since the identification of the Ser-1177 phosphorylation site by the present inventors, it has been recognized that other protein kinases phosphorylate at this site. In particular, the protein kinase Akt (also referred to as PKB) may stimulate endothelial cells by vascular endothelial growth factor (VEGF) (Fulton et al, 1999; Michell et al., 1999) or fluid shear stress (Dimmerler et al.). , 1999; phosphorylates Ser-1177 in response to Gallis et al., 1999). A study by Gallis et al. (1999) reported that fluid shear stress stimulated phosphorylation of Ser-116 in the sequence KLQTRPSPGPPPA. Neither the kinase involved in this site on eNOS nor the functional effect of phosphorylation has yet been identified. This phosphorylation site is in the oxidase domain.
[0038]
We have found that phosphorylation of eNOS at Thr-495 by protein kinase C occurs in serum-starved endothelial cells incubated in calcium-free medium in the presence of phorbol ester PMA. There is a complementary relationship between phosphorylation at Ser-1177 and Thr-495 in endothelial cells. Protein kinase C phosphorylates both sites in vitro, but stimulation of protein kinase C in endothelial cells by phorbol ester enhances Thr-495 phosphorylation, but marked Ser-1177 phosphorylation. cause. Dephosphorylation of Ser-1177 was prevented by okadaic acid, but not by caliculin A, indicating that phosphatase PP2A is involved. Okadaic acid also greatly enhances phosphorylation of Thr-495 in response to phorbol esters. Thrombin, which acts via protein kinase C, also stimulates phosphorylation of Thr-495 and dephosphorylation of Ser-1177.
[0039]
In contrast, treatment of endothelial cells with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) causes significant dephosphorylation of eNOS and enhanced Ser-1177 phosphorylation with Thr-495. . Dephosphorylation of Thr-495 in response to IBMX is blocked by treatment with calyculin A, suggesting that phosphatase PP1 is involved in dephosphorylation of Thr-495.
[0040]
These relationships are summarized in FIG. We confirmed that skeletal muscle movement resulted in phosphorylation of Ser-1417 in nNOSμ, a site corresponding to Ser-1177 in eNOS (see FIG. 5). Electrical stimulation of rat extensor digitorum longisus (EDL) was found to activate AMPK, phosphorylate acetyl-CoA carboxylase at Ser-79 (inhibition site), and phosphorylate nNOSμ at Ser1417. Similarly, biopsies of human skeletal muscle after strenuous exercise such as a 30 second bicycle splint show a 10-fold increase in phosphorylation at Ser-79 in acetyl-CoA carboxylase, as well as phosphorylation of nNOSμ at Ser-1417. A 7.5-fold increase is observed (see FIG. 9).
[0041]
Endothelial-derived NO has an important role in preventing the adhesion and aggregation of immature platelets that lead to thrombus formation (Radomski and Moncada, 1993). There is evidence that the protective effects of increased high density lipoprotein (HDL) on the cardiovascular system can be mediated by increased platelet NO production. Apolipoprotein E, a component of HDL, acts on a receptor (apoER2) present in platelets to stimulate the NO signaling pathway (Riddell et al., 1997; Riddell and Owen, 1999).
[0042]
Activation of eNOS by phosphorylation of its COOH terminus gives a new perspective on eNOS autoinhibition. The increased activation and CaM dose-dependent shift associated with phosphorylation at Ser-1177 indicates that the COOH terminus acts as a partial autoregulatory sequence in eNOS, and probably also in nNOS, as in CaM-dependent protein kinase. (Kemp and Pearson, 1991; Kobe et al, 1996).
[0043]
The COOH end of eNOS is Ca ²⁺ Only AMPK is accessible when CaM is bound, which is consistent with this region being buried in the absence of CaM. As can be seen from FIG. 5, there is a high level of similarity between eNOS and nNOS at their COOH termini, while iNOS is different. Low Ca ²⁺ INOS CaM binding, characterized by dependence, requires both a standard CaM binding sequence, as well as distal residues in the COOH terminus that cannot be satisfied by nNOS chimeras (Ruan et al, 1996). Without considering any proposed mechanism, eNOS and nNOS are autoinhibited by their COOH termini, requiring a two-step activation step for full activity by both CaM binding and phosphorylation at the termini, On the other hand, iNOS requires only CaM binding. Recently, Salerno et al. (1997) proposed that the insertion sequence in the FMN binding domain may also be important for autoregulation.
[0044]
Previous studies have shown that eNOS can be phosphorylated both in vitro and in vivo, but the exact site of phosphorylation and the function of phosphorylation events have, to date, been completely characterized. (Discussed in Michel and Feron, 1997). eNOS is the first example identified as an enzyme activated by AMPK, and phosphorylation is ²⁺ -Is not common as it leads to activation or suppression depending on the availability of CaM. Other enzymes, especially cyclin-dependent protein kinases, are activated or repressed by phosphorylation, but they are catalyzed by different protein kinases. Protein kinase C phosphorylates Thr-495 in eNOS, demonstrating that the regulatory pathways of action on eNOS by phosphorylation of Thr-495 or Ser-1177 are crossed. For example, it is also possible that sustained activation of protein kinase C in response to diabetes-induced hyperglycemia may long-term suppress eNOS phosphorylation at Ser-1177, thereby reducing its activity. .
[0045]
Regulation of eNOS by AMPK extends the conceptual relationship between yeast snflp kinase and AMPK. Snflp kinase regulates the supply of glucose from the environment by secreting invertase, whereas mammalian AMPK regulates metabolic stress signaling with control of the circulatory system. Thus, intracellular metabolic stress signals in endothelial cells and muscle cells can cause improved nutritional supply and suppress muscle mechanical activity.
[0046]
Although the present invention has been described in some detail for purposes of clarity and understanding, the embodiments described herein have been described without departing from the scope of the inventive concepts disclosed herein. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and methods.
[0047]
References cited in the present specification are listed on the following pages, and the contents of these descriptions are incorporated herein by reference.
[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Brief description of the drawings]
FIG.
FIG. 4 shows the fluorescent immunolocalization of AMPK-α2 in heart and tibialis anterior muscle.
Panel A shows a negative control section of a rat heart stained with control rabbit IgG and control mouse IgG and stained with anti-rabbit FITC and anti-mouse Texas Red.
Panel B shows a section of a rat heart stained with affinity purified rabbit polyclonal antibody to AMPK-α2 (491-514) and anti-rabbit FITC.
Panel C shows the same section as panel B, stained with a monoclonal antibody against rat endothelial recA-1 and anti-mouse Texas Red.
Panel D shows the upper layers of panels B and C. By co-staining, co-localization can be observed. Arrows indicate specific endothelial cells stained by both antibodies.
Panel E shows a negative control section of rat tibialis anterior muscle stained with control rabbit IgG and control mouse IgG and stained with anti-rabbit FITC and anti-mouse Texas Red.
Panel F shows sections stained with affinity purified rabbit polyclonal antibody to AMPK-α2 (491-514) and anti-rabbit FITC.
Panel G shows the same section as panel B, stained with a monoclonal antibody against rat endothelial recA-1 and anti-mouse Texas Red.
Panel H shows the upper layer of panels E and F. By co-staining, co-localization can be observed.
FIG. 2
FIG. 4 shows phosphorylation of recombinant eNOS by AMPK.
Upper panel: eNOS was administered to rat liver AMPK-α1 and [γ- ³² [P] ATP.
Lane 1: Coomassie stained SDS-PAGE,
Lane 2: autoradiograph.
Lower panel: eNOS ³² P-trypsin phosphopeptide map.
FIG. 3
Ca ²⁺ The effect of AMPK phosphorylation of eNOS with and without CaM is shown. Rat heart eNOS purified by 2 ', 5'-ADP-Sepharose affinity chromatography was converted to 0.8 μM CaM / 3.2 μM Ca ²⁺ In the presence of ²⁺ -Phosphorylated with AMPK in the absence of CaM (filled triangles) and without AMPK (filled squares); After phosphorylation, samples were diluted and eNOS activity was measured. The lower panel is Ca ²⁺ -Shows phosphopeptide maps for rat heart eNOS phosphorylated in the presence and absence of CaM.
FIG. 4
Figure 3 shows the effect of ischemia on the activity of AMPK-α1, AMPK-α2 and eNOS.
Panel A shows the results of immunoprecipitation using antibodies specific for AMPK-α1 and AMPK-α2, assayed using a SAMS peptide substrate. The results shown are means ± SEM (n = 5).
Panel B shows eNOS activity measured at 500 nM CaM.
Panel C shows eNOS activity with total CaM-dose response for a representative experiment. At ischemia: 0 minutes (solid white circle), 1 minute (solid black diamond), 10 minutes (solid black circle) and 20 minutes (filled white triangle). The results of the four replicates were identical except for one case where the 20 minute ischemic eNOS CaM-dependence remained similar to 10 minutes.
FIG. 5
3 shows a comparison of NOS sequences. Phosphorylation site sequences for eNOS and nNOS are shown in a schematic model of NOS. The sequence from the CaM binding region (around the phosphorylation site of Thr-495 of eNOS) to the COOH terminal (around the phosphorylation site of Ser-1177 of eNOS) is shown.
FIG. 6
Figure 4 shows the effect of treatment of bovine aortic endothelial cells with phorbol ester (PMA) and okadaic acid on eNOS activity (top panel) and phosphorylation at Ser-1177 and Thr-495 (bottom panel).
FIG. 7
FIG. 4 shows the effect of treatment of bovine aortic endothelial cells with 3-isobutyl-1-methylxanthine (IBMX) and calyculin A on phosphorylation at Ser-1177 and Thr-495.
FIG. 8
1 shows a summary of the regulation of eNOS by phosphorylation at Thr-495 and Ser-1177 mediated by protein kinases PKC, AMPK and Akt. Reversal of phosphorylation at these sites is mediated by protein phosphatases PP1 and PP2A in response to treatment of cells with IBMX and PMA, respectively.
FIG. 9
Figure 3 shows the effect of a 30 second bicycle splint exercise on nNOS phosphorylation in human muscle. nNOS was extracted from biopsies and probed for phosphorylation at Ser-1417 using anti-phosphopeptide antibodies. The left panel shows the immunoblot and the right panel shows the quantitative analysis of 5 individuals.

Claims (13)

A method for identifying a modulator of AMPK-mediated activation of a nitric oxide synthase enzyme selected from the group consisting of eNOS, nNOS and nNOSμ, wherein the putative modulator has the ability to increase or decrease phosphorylation of the enzyme. Testing, wherein said increase or decrease is dependent on calmodulin and calcium ion concentration. 2. The method of claim 1, wherein the specific phosphorylation of Ser-1177 is assessed in the presence of calcium and calmodulin. A method of identifying a modulator of AMPK-mediated suppression of eNOS, comprising testing a putative modulator for the ability to reduce or increase AMPK-mediated phosphorylation of eNOS in the presence of limiting calcium ions. 4. The method of claim 3, wherein specific phosphorylation of Thr-495 is assessed. One or more of the following activities:
(A) effects on smooth muscle contraction,
(B) effects on the inotropic activity of the heart,
5. The method according to any one of claims 1 to 4, wherein (c) the effect on the cycle altering activity of the heart or (d) the effect on platelet function is further assessed. The method according to any one of claims 1 to 5, wherein the modulator is an activator described herein. 7. The method of claim 6, wherein said activator promotes both glucose and fatty acid metabolism. The method of any one of claims 1 to 5, wherein the modulator is an inhibitor described herein. 9. The method according to any one of claims 3 to 8, wherein the modulator acts preferentially on non-neuronal cells. The method according to claim 1 or 2, wherein the modulator promotes dephosphorylation of Ser-1177 and suppresses eNOS activity. 4. The method of claim 3, wherein said modulator promotes Thr-495 dephosphorylation and stimulates eNOS activity. The method according to claim 1 or 2, wherein the modulator promotes phosphorylation of nNOS or nNOSμ at Ser-1417. 3. The method of claim 1 or claim 2, wherein the modulator promotes dephosphorylation of nNOS or nNOS [mu] at Ser-1417.

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