in是什么意思，词典释义与在线翻译：
prep.(介词)
穿着，戴着
在...之内，在...里
在...中，处在...中
在（某段时间）内， 在...期间， 在...当儿
从事于，参加着
adv.(副词)
在家， 在屋里
入，进，进入
在内， 在里头，在里面
（已）到达，已来临
【板】轮到击球
【网】在界内
adj.(形容词)
时髦的 ，流行的，赶时髦的
在里面的， 内部的
朝里面的，朝内的
限于小圈子的
抵港的，进港的
在朝的，执政的，当权的
被完成的，被集中的，被计算的
当朝派， 执政者，在朝派，执政党
（特殊）关系
详情，全部细节
【体】攻球的一方
位置特殊的人
从事遴选工作的人
abbr.(缩略词)
=Indiana 美国印第安那州邮递区号
=Information Network 情报网路
=Intelligent Network 【计】智能网
【化】元素铟的符号（=indium）
收集，集拢
把…封入，围住
prep.(介词)
(表示位置)在…里面〔内,中〕; 在,于; 在…部位上 at a point within the area or volume of (sth)
(表示时间)在…时期,在…之后,在过程中 during (a period of time); after (a maximum length of time)
(表示方向)往…内,朝…方向 toward
(表示状态)处于…之中,在…情况下,在…身上,穿着 (indicating physical surroundings, circumstances)
(表示方式)用,以,按,乘,以…形式 (indicating form, shape, arrangement or quantities)
(表示原因)由于,为了 for
(表示领域,范围)在…以内 within the shape of
(表示结果)当作,作为 as
(表示目的)为了 as to
(表示方面)就…而言,关于,在…方面 with reference to (sth); regarding
(表示比率)每,以…为单位 (indicating ratio)
(表示职业)在…中服务,从事于,忙于 (indicating sb's occupation, activity)
(表示材料)用,以 (indicating the medium, means, material, etc.)
adv.(副词)
进入,入内 away from the open air, the outside, etc.to be contained or surrounded
在家,在里面 at home, indoors
到达,来临 arriving
当政; 当选 elected
正当时令; 正在流行 in fashion
提示：各行业词典APP中含有本词条的独家正版内容，在手机上可看到更多释义内容。
in&:&在…时, 在… ...
在&&中查看更多...
a unit of length equal to one twelfth of a foot
a rare soft silv occurs in small quantities in sphalerite
a state in midwestern United States
Adjective:
"the in party"
"took the in bus"
"the in basket"
"the in thing to do"
"large shoulder pads are in"
"smash in the door"
in的用法和样例：
用作介词 (prep.)
The telephone was in the little study on the ground floor.
电话在底楼的小书房里。
The books are printed in Hong Kong.
这些书在香港印刷。
They are in research of this new technique.
他们正在研究这种新工艺。
He is a layman in economics.
他对经济学一窍不通。
I invited him in for a drink.
我邀请他进屋喝一杯。
I am sorry that my room is in such a mess.
不好意思,我的房间太乱了。
She looked so beautiful in her wedding dress.
她穿上婚纱看起来真美。
You have to pay in cash.
你得付现金。
We are trying to teach mathematics in a more interesting way.
我们正在努力尝试着用一种有趣的方式来教数学。
We all sat around in a circle.
我们围成一圈坐在一起。
用作副词 (adv.)
He is not in at the moment.
这个时候他不在家。
He opened the door and stepped in.
他打开门走了进去。
She looked in the window and found a baby sleeping on the bed.
她朝窗户里看,发现有个婴儿在床上睡觉。
The manager is in before anyone else.
经理在别人来之前就到了
They are having a heated argument. You'd better not wade in.
他们讨论地很激烈，你最好不要介入。
He got in bad with his roommate.
他与室友相处得不好。
用作形容词 (adj.)
The in part of the machine is very complex.
该机器内部结构非常复杂。
He arrived in time to take the in bus.
他来得及时,正好赶上了新开来的汽车。
He applied for a membership in the in party.
他申请成为当前执政党中的一员。
Thar kind of skirt has been in for a long time.
那种式样的短裙流行了很长时间。
用作名词 (n.)
The election made him an in.
这次选举使他成为执政者。
The businessman had an in with the authorities.
这个生意人与当局有特殊的关系。
The job itself is really boring, but it's an in to a career in publishing.
那份工作本身真的很无聊,不过却是以后从事出版业的一块跳板。
用作介词 (prep.)
I could feel the tension in the room.
我可以感觉到房间里的紧张气氛。
They live in France.
他们住在法国。
The children are playing in the garden.
孩子们正在花园里玩耍。
The man was wounded in the leg.
那人腿部受伤。
One of the pistons in the engine had jammed.
引擎上的一个活塞被堵塞住了。
She dies in the last act.
她在最后一幕中死去。
In her twenties and thirties she had no difficulty getting jobs.
她在二三十岁时找工作一点也不困难。
A fast train does the journey to London in three hours.
快车在3小时以内走完去伦敦的旅程。
I cannot see you now, come back in half an hour.
我现在不能见你,半小时后回来。
He hasn't had a good meal in weeks.
他已几个星期没有饱餐一顿了。
I saw him go in the shop.
我看到他走进了商店。
He found himself walking in the direction of the zoo.
他发现自己不知不觉地往动物园方向走去。
Martin was in his pyjamas.
马丁穿着睡衣。
She hated the bully in him.
她憎恶他恃强欺弱的个性。
He left in a temper.
他怒气冲冲地走开了。
They were living in terrible poverty.
他们生活在极度贫困之中。
They were speaking in Italian.
他们在讲意大利语。
He wore his hair in the prevailing fashion.
他的头发梳的是当时盛行的发型。
They went up in the lift.
他们乘电梯上楼了。
They stood in a queue.
他们站成一队。
He went in fear of his life.
他为自己的性命担忧,所以走了。
It is not in my power to do that.
做那事非我力所能及。
In these circumstances prices and profits would remain stable.
在这种情况下价格和利润会保持稳定。
What did you give him in return?
你给他什么作为报答呢?
They set off in search of the lost child.
他们出发去寻找走失的孩子。
They accepted the plan in principle.
他们原则上同意了这项计划。
She's an expert in children's literature.
她是儿童文学专家。
One child in twenty suffers from this disease.
每20名儿童中,有一名患有这种疾病。
Pack them in tens.
把它们按每件十份打包。
Would you like to serve in a shop?
你愿意当一名营业员吗?
She's in television.
她在电视台工作。
Yesterday, he was occupied in translating last week's report.
昨天，他忙于翻译上周的报告。
Don't write your name in ordinary handwriting.
不要用普通字体写你的姓名。
用作副词 (adv.)
The door being opened, they came in at once.
门一打开,他们就马上进来了。
She opened the purse and put her wages in.
她打开钱包,把工资放了进去。
My wife won't be in until five o'clock.
我妻子要到五点钟才在家。
Is the ship in yet?
船到港了吗?
Summer is in.
夏天来了。
This year the Conservative Party are in.
今年保守党执政。
Honey peaches are in now and we can eat them every day.
现在水蜜桃正上市,我们每天都可以吃到。
Short skirts are in again this year.
短裙子今年又流行了。
用作介词 (prep.)
抵达,到达,乘车到达
相信,信奉,信仰,信任
突然出现,突然流露出
绊倒…,给…绊了一下,遇上雨,被雨淋
在…中作弊
在…上失势
使(某人)加强…,认可(某人)…
淹死在,用…掩盖,用(调料)浸泡食品,充满
使忙于,使从事于
在…失败,衰退,缺少,未充分具有
使参与,陷入,牵扯到
使继续处于…,提供,抚养,维持
平放于,使处于,以…为场景
领着做,在…上领先
在…上的成就
在…上的帮助
在…上的改进
在某种意义〔程度〕上,以…方式
乱七八糟,陷入窘境,在食堂
不整洁,破损,焦躁,烦恼
有几分,在某种程度上
简而言之,一言以蔽之
在…之前,超过
无论如何,不管怎样
事实上,实际上
关于,在…方面
用作副词 (adv.)
完好无损地收进
The bar of Jim's Garage..was currently an 'in' place in downtown Detroit.
出自：A. Hailey
I went in and sat down.
出自：J. Conrad
He must have a boat to take him..to the rock face where he plunged in.
出自：G. Household
Jane was sitting up in bed drinking..tea.
出自：M. Keane
Sarah was in Australia with her husband.
出自：I. Murdoch
He talked exactly like the balloons in comic strips.
出自：L. Bruce
in的详细讲解：
prep.(介词)
参见about条。
in, according to, by, from, on, under
这组词的共同意思是“根据”“按照”。其区别是：
according to是这组词中最常用的一个,可指依据某人的话语、说法、指示,也可指按照某计划、法规等,可以是正式的,也可以是非正式的; by的本意是“凭借某种手段、方式”,由此引申出“依据,按照”,其宾语常为某种规则、法规、标准、习俗等; from的本意是“来源”,由此引申出“根据事物的来源判断”; in指“依据”时,仅见于与eye, opinion, view等表示观点的名词或order, sequence等表示顺序的名词连用,还见于in law, in practice, in theory等短语中; on是根据事物发生和存在所依靠的基础这一用法,由此引申为“根据,按照”,与之连用的词一般是表示忠告、规则、命令、指示、协议、建议、原理的名词; under通常指根据正式的协议、法令、条款、合同等,含有在其控制或约束之下的意味。
in, after, since
这三个介词都可指示时间关系,表示“在…之后”。其区别是：
after可表示从过去时间算起的一段时间之后,往往与过去时态连用（若接表示时间点的词，也可与将来时态连用）; in则表示从现在时间算起推移到将来的一段时间之后,一般与将来时态连用; since所表示的时间往往是从过去某一点时间一直延续到说话的时间,因而一般与完成时态连用。
这两个介词都可表示“在”“在…里〔上〕”。其区别在于:among往往强调处于同类人或事物当中,因而常与复数名词或着重于个体的集体名词连用; in往往强调人或事物所处的位置或环境,因而常与单数名词或着重于整体环境的集体名词连用。
in, at, on
1.at,in和on都可表示时间。at通常指确切的某个时刻,如at six o' in通常表示一天的某一部分,如in the morning〔evening,afternoon〕; on则具体说明哪一天的上午或下午,如on Monday morning,on a summer's day。在谈论节日时,at通常指整个的节〔假〕日,不只指一天,如at Easter 在复活节,at Christmas 在圣诞节; on可具体指节日的某一天,如on Easter Monday 在复活节后的星期一,on Christmas Day 在圣诞节(那天)。在谈论月份、季节、年份或世纪时,通常用介词in,如in the eighteenth century 在18世纪,in summer 在夏天,in March 在三月,in 1985 在1985年。
2.at,in和on都可表示地方、位置。at多用于指空间某一点; in用来指有大小、体积和幅度的一个地方或位置。
3.at the weekend 在周末(英国用法); on the weekend 在周末(美国用法)。
in between
参见between条。
这两个词都有“不迟于”的意思,但in表明的是一段时间; 而by只是用在日历上确定的时间,或者日历上的一点。
for和in都可表示时间。
1.for表示一整段时间,其动作或状态由这段时间的开始持续到这段时间的终了,因此for本身就有“持续”的含意; 而in只是划定一个时间界限,而其动作或状态只局限在这个范围之内,但不一定贯穿始终。
2.表示将来的一个时期,在肯定句中用 但在否定句中只能用for。
in, inside
这两个词都可表示“在…里面”。
1.inside更强调被包围的意义,语气也比in强。
2.表示位置的对比时,一般用inside。表示“在…内侧”时,一般也只用inside,不用in。
in表示“在里面”的静止状态或一定范围内的动作; 而into表示由外向里的动作。二者有时可通用。
in, on, to
这三个词均可表示方位。to表示在某范围之外,两者之间可以互相连接,也可以不连接; on表示在某范围之外,两者之间一般互相连接; in表示在某范围之内,是其中的一部分。
1.with表示使用某工具; in表示使用某材料。
2.如同时表示工具和材料时,应用 某人用某种语言或某种语调说或唱时,用in。
in, within
这两个介词均可表示“在…(时间)以内”。within着重一段时间终结之前,用于正式场合; in着重一段时间的过程,常用于重复动作或延续动作。
这些前置词在表示地点或时间时均含“在……”之意。
:at表地点时，指空间位置上的某一点；表时间时，指在时间上的某一时刻。
:in表地点时，指在某一立体空间范围内；表时间时，指一段时间或与年、月、季节时间连用。
:on表地点时，指某物与另一物表面相接触，或与某地方接壤等；表时间时，指在某一天或某一天的某个时间，尤指在星期几。
☆ 直接源自古英语的in；最初源自原始日耳曼语的in，意为在……里面。
in的海词问答与网友补充：
in的相关资料：
in&:&〈D〉 a.
在&&中查看更多...
in&:&铟 ...
在&&中查看更多...
in&:&铟(indium) ...
在&&中查看更多...
in&:&(表示地点 ...
在&&中查看更多...
【近义词】
在(某段时间、距离或范围...
流行的(漂亮的)...
在 ... 之中
在 ... 期间
当 ... 的时候...
被 ... 围住的...
[计算机]前导字符...
因为，由于，既然...
动词boss的第三人称单...
(刀片的)刃口...
(物)被据有
与 ... 合伙(与 ...
被 ... 环绕着的...
印第安纳州（美国中北部的...
印第安纳州(In...
在 ... 之上
【反义词】
in的相关缩略词，共有62条
印地安那州(美国邮政的缩写)
铟（化学元素）
in:In 词缀:进入的意思 英文名： Indium中文名： 铟 相对原子质量： 114.82常见化合价： 1, 2, 3电负性： 1.5外围电子排布： 5s2 5p1核外电子排布： 2,8,18,18,3…
相关词典网站：in tension是什么意思，词典释义与在线翻译：
处于紧张状态
名词 intension:
what you must know in order to determine the reference of an expression
in tension的用法和样例：
An instrument for recording variations in pressure, as of the blood, or in tension, as of a muscle, by means of a pen or stylus that marks a rotating drum.
压力记录器，记波器一种记录压力(如血压)或张力(如肌肉张力)变化的仪器，它通过一支笔或记录针的方式描出旋转的轨迹
in tension的海词问答与网友补充：
in tension的相关资料：
相关词典网站：Heterozygous Deficiency of Manganese Superoxide Dismutase in Mice (Mn-SOD+/-): A Novel Approach to Assess the Role of Oxidative Stress for the Development of 2006年第68卷第3期 | 39康复网 | 医源世界
当前位置：&&&&&&&&&&&&&&&Heterozygous Deficiency of Manganese Superoxide Dismutase in Mice (Mn-SOD+/-): A Novel Approach to Assess the Role of Oxidative Stress for the Development of
Heterozygous Deficiency of Manganese Superoxide Dismutase in Mice (Mn-SOD+/-): A Novel Approach to Assess the Role of Oxidative Stress for the Development of
来源：分子药理学杂志 作者：Andreas Daiber, Matthias Oelze, Silke Sulyok, Meik
摘要: Effects of Mn-SOD Deficiency on Mitochondrial ALDH-2 Dehydrogenase, Esterase Activity, and Vascular ALDH Dehydrogenase Activity。 Heterozygous Mn-SOD Deficiency Does Not Affect NO-Signaling/Endothelial Function but Makes Vessels More Susceptible to Nitrate Tolerance and Cross-Tolerance。 Heterozy......
专题推荐：
&&& Klinikum der Johannes Gutenberg-Universitt Mainz, Medizinische Klinik II, Kardiologie, Mainz, Germany (A.D., M.O., E.S., U.H., A.M., T.M.)&&& University of Ulm, Department of Dermatology and Allergology, Ulm, Germany (S.S., N.T., K.S.-K.)&&& Universittsklinikum Hamburg-Eppendorf, Medizinische Klinik III, Angiologie und Kardiologie, Hamburg, Germany (M.C.)
&&& Abstract
&&& Nitroglycerin (GTN)-induced tolerance was reported to be associated with increased levels of reactive oxygen species (ROS) in mitochondria. In the present study, we further investigated the role of ROS for the development of nitrate tolerance by using heterozygous manganese superoxide dismutase knock-out mice (Mn-SOD+/-). Mn-SOD is acknowledged as a major sink for mitochondrial superoxide. Vasodilator potency of mouse aorta in response to acetylcholine and GTN was assessed by isometric tension studies. Mitochondrial ROS formation was detected by 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012)-enhanced chemiluminescence and mitochondrial aldehyde dehydrogenase (ALDH-2) activity was determined by a high-performance liquid chromatography-based assay. Aortic rings from Mn-SOD+/- mice showed normal endothelial function and vasodilator responses to GTN. In contrast, preincubation of aorta with GTN or long-term GTN infusion caused a marked higher degree of tolerance as well as endothelial dysfunction in Mn-SOD+/- compared with wild type. Basal as well as GTN-stimulated ROS formation was significantly increased in isolated heart mitochondria from Mn-SOD+/- mice, correlating well with a marked decrease in ALDH-2 activity in response to in vitro and in vivo GTN treatment. The data presented indicate that deficiency in Mn-SOD leads to a higher degree of tolerance and endothelial dysfunction associated with increased mitochondrial ROS production in response to in vitro and in vivo GTN challenges. These data further point to a crucial role of ALDH-2 in mediating GTN bioactivation as well as development of GTN tolerance and underline the important contribution of ROS to these processes.
&&& Although organic nitrates such as nitroglycerin (glyceryl trinitrate, GTN) have been used for over a century in the therapy of cardiovascular diseases such as stable and unstable angina (Abrams, 1995) the underlying mechanisms of nitrate bioactivation and development of nitrate tolerance remain elusive. The anti-ischemic effects of organic nitrates are due largely to venous and coronary artery dilation as well as improvement of collateral blood flow, which all decrease myocardial oxygen consumption and are mediated by nitric oxide or a related species. However, the use of organic nitrates is limited because of the rapid development of tolerance and cross-tolerance to endothelium-dependent and -independent vasodilators. Impairment of the NO-signaling pathway by increased formation of reactive oxygen species (ROS) (Munzel et al., 1995b) as well as an impaired biotransformation of organic nitrates may contribute to the development of tolerance and cross-tolerance. The mitochondrial aldehyde dehydrogenase (ALDH-2), which is subjected to an oxidative mechanism-based inactivation, has recently been identified as a GTN-metabolizing enzyme and a possible important component in the processes leading to tolerance (Chen et al., 2002). Our laboratory further substantiated this concept in an animal model of in vivo tolerance and extended previous observations by demonstrating that mitochondria are a major source of ROS formation in response to short- and long-term GTN challenges (Daiber et al., 2004b; Sydow et al., 2004). These results provided indeed the missing link between tolerance and cross-tolerance, the oxidative stress concept, and the concept that tolerance is secondary to decreased GTN biotransformation. Because mitochondrial ROS formation seems to play a major role for development of tolerance and cross-tolerance, one could hypothesize that a deficiency in mitochondrial superoxide dismutase (Mn-SOD) would render vascular tissue more susceptible for the development of tolerance.
&&& There are two different types of Mn-SOD-deficient mice. Removal of exon 1 and 2 shows lethality at 21 days as a result of neuronal abnormalities (Lebovitz et al., 1996), whereas removal of exon 3 shows lethality at 10 days with dilated ventricular cardiomyopathy (Li et al., 1995). The lack of Mn-SOD causes an increase in mitochondrial superoxide levels, which in turn leads to destruction of iron-sulfur-cluster [4Fe-4S] proteins (Flint et al., 1993). In Mn-SOD-/- mice, aconitase activity in the heart is decreased by 42.6%, which, combined with a decrease in succinic acid dehydrogenase activity, impairs the citric acid cycle and lead to lipid accumulation in the liver and muscle (Li et al., 1995). The estimation that 1 to 2% of all electrons transported by the respiratory chain will ultimately result in the formation of superoxide justifies the importance of Mn-SOD for survival in all mammals (Robinson, 1998). The importance of Mn-SOD was also underlined by the finding that Cu,Zn-SOD overexpression could not compensate for the lack of Mn-SOD: the lethality of these animals was unchanged (Copin et al., 2000). The expression of Mn-SOD, in contrast to Cu,Zn-SOD isoforms (cytosolic and extracellular), can be induced by cytokines (Hennet et al., 1993) and oxidative stress (Shull et al., 1991), but Mn-SOD is also subject to oxidative inactivation, namely nitration and dimerization of essential tyrosine residues. Overexpression of Mn-SOD in mice protected from myocardial ischemia/reperfusion injury (Jones et al., 2003), in cells, however, this condition was associated with a hydrogen peroxide-induced up-regulation of matrix-degrading metalloproteinase-1 (Wenk et al., 1999).
&&& This is the first study to use heterozygous Mn-SOD deficiency (Mn-SOD+/-) in mice as a tool to assess the role of oxidative stress for the development of in vitro nitrate tolerance and cross-tolerance upon short-term GTN treatment of isolated murine aortic rings. The expression of Mn-SOD in Mn-SOD+/- mice is decreased by approximately 50% compared with wild-type (wt) animals, leading to distinct ultrastructural damage of the myocard, with swelling and disruption of mitochondria and accumulation of lipid droplets, increased nitrotyrosine formation and lipid peroxidation as well as activation of apoptosis signaling pathways in the heart in vivo (Strassburger et al., 2005). Cu,Zn-SOD deficiency is well characterized with respect to the vascular system and endothelial dysfunction (Lynch et al., 1997; Didion et al., 2002), but little is known about vascular consequences of Mn-SOD deficiency. One report presented data seeming to indicate that endothelial function (response to acetylcholine) in Mn-SOD+/- mice was not altered compared with wt animals (Andresen et al., 2004). With the present studies, we sought to focus on 1) the short-term GTN responsiveness as well as on GTN tolerance development in wt mice and Mn-SOD+/- mice in response to in vitro and in vivo GTN challenges, and 2) whether ALDH-2 activity and the GTN bioactivation are affected by Mn-SOD deficiency.
&&& Materials and Methods
&&& Materials. For induction of in vitro tolerance and isometric tension studies, GTN was used from a Nitrolingual infusion solution (1 mg/ml) from G. Pohl-Boskamp (Hohenlockstedt, Germany). For induction of in vivo tolerance, GTN was used from a solution in ethanol (102 g/liter), which was obtained from UNIKEM (Copenhagen, Denmark). L-012 was purchased from Wako Pure Chemical Industries (Osaka, Japan). Dihydroethidium (hydroethidine, DHE) was obtained from Molecular Probes (Eugene, OR). All other chemicals were of analytical grade and were obtained from Sigma Chemie (Deisenhofen, Germany), Fluka (Buchs, Switzerland), or Merck (Darmstadt, Germany).
&&& Animal Model, In Vitro and In Vivo Nitrate Tolerance. In the present study, we used female mice aged 4 to 8 months on a mixed genetic background (C57BL/6 x 129/Ola). Experiments were performed with 16 wt and 16 Mn-SOD+/- mice. Mn-SOD+/- mice were generated according to a published procedure (Strassburger et al., 2005) in the laboratory of author K.S.-K. In brief, male mice carrying two SOD2flox alleles were crossed to K14Cre females that, because of Keratin 14 expression in oocytes, also express Cre recombinase in maturing oocytes (Hafner et al., 2004). Because Cre recombinase remains active in oocytes until the paternally inherited SOD2flox allele becomes accessible after fertilization, all animals derived from such breedings carry a stably deleted SOD2 allele (SOD2-). SOD2+/- mice were further bred with wt animals of the outbred strain more than 10 times. The absence of the K14Cre allele in the heterozygous offspring was proven by Southern blot analysis. The deletion of exon 3 of the Mn-SOD gene was determined by Southern blot analysis. Because exon 3 of the Mn-SOD gene codes for the domain important for tetramer formation of the Mn-SOD, deletion of this domain results in a complete loss of the activity of the enzyme. The deficiency of the Mn-SOD activity was determined using a specific activity assay, as described recently (Strassburger et al., 2005).
&&& In vitro tolerance development as a result of GTN treatment was assessed by ex vivo incubation of murine vessels with 200 e GTN for 30 min at 37°C in Krebs-HEPES buffer (5.78 g/liter NaCl, 0.35 g/liter KCl, 0.37 g/liter CaCl2, 0.30 g/liter MgSO4, 2.1 g/liter NaHCO3, 0.14 g/liter K2HPO4, 5.21 g/liter HEPES, and 2.0 g/liter D-glucose) followed by a 1-h wash-out phase and subsequent recording of concentration-response curves with GTN. In vivo tolerance was induced by long-term infusion of mice with GTN by implanted micro-osmotic pumps (0.5 e/h for 7 model 1007D; ALZET Osmotic Pumps, Cupertino, CA). Infusion of the solvent ethanol served as a control. To determine the infusion rate of GTN that causes tolerance, female wt mice (C57BL, 5-6 months old) were infused with either ethanol or GTN at high (100 e/h, 220 nmol/min/kg) and low (16 e/h, 35 nmol/min/kg) dosages for 3 d. Based on these results, four female wt (C57BL/6 x 129/Ola) and four female Mn-SOD+/- mice (all 6-8 months old) were infused with ethanol, and the same number of animals was infused with GTN (16 e/h, 35 nmol/min/kg) for 4 d. After this period, the animals were sacrificed, and aortas as well as hearts were subjected to further analysis. All animals were treated in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health and was granted by the Ethics Committee of the University Hospital Eppendorf and of the University Hospital Mainz.
&&& Isometric Tension Studies. Vasodilator responses to GTN and acetylcholine (ACh) were assessed with endothelium-intact isolated murine aortic rings mounted for isometric tension recordings in organ chambers, as described previously (Munzel et al., 1995a). In brief, mice were anesthetized using CO2. Thoracic aortas were rapidly removed and cut into ring segments of approximately 3-mm length and mounted in organ chambers for isometric tension recording. Preliminary studies revealed that the optimum resting tension for tone development in response to 80 mM KCl was 1.00 g, which was achieved by gradual stretching over 1 h. After preconstriction with prostaglandin F2 to reach 50 to 80% of maximal tone induced by KCl, a concentration-response to increasing concentrations of the endothelium-dependent vasodilator ACh (1 nM to 3 e) and the endothelium independent vasodilator GTN (1 nM to 30 e) was established as described.
&&& Western Blot Analysis. Aortic segments (1 cm) from wt and Mn-SOD+/- mice were incubated for 5 min with GTN (0.1 e), frozen, and homogenized in liquid nitrogen. The expression of the phosphorylated vasodilator stimulated phosphoprotein (P-VASP) was determined as described previously (Oelze et al., 2000). Immunoblotting was performed with a mouse monoclonal P-VASP phosphoserine 239 antibody (clone 16C2, 1.5 e/ Calbiochem, Schwalbach, Germany). Detection was performed by enhanced chemiluminescence with peroxidase conjugated anti-rabbit/mouse secondary antibodies (1:10,000; Vector Laboratories, Burlingame, CA).
&&& ALDH-2 Dehydrogenase and Esterase Activity in Isolated Mouse Heart Mitochondria and Dehydrogenase Activity in Isolated Aortic Segments. The activity of ALDH in isolated mitochondria was determined by measuring the conversion of benzaldehyde to benzoic acid. Mouse heart mitochondria were prepared according to a previously published method (Raha et al., 2000) that was slightly modified (Daiber et al., 2004b). The mitochondrial fraction (total protein, approximately 5-10 mg/ml) was kept on ice and diluted to approximately 1 mg/ml protein in 0.25 ml of PBS and preincubated for 30 min at room temperature. In some experiments, mitochondria were incubated with GTN (5 or 50 e) for 30 min before ALDH substrate addition. For measurement of ALDH-2 dehydrogenase activity, benzaldehyde (400 e) was added to the mitochondrial suspension, and the samples were incubated for another 30 min at 37°C. For determination of vascular dehydrogenase activity, aortic rings of 3 to 4 mm in length were incubated with benzaldehyde (400 e) for 30 min at 37°C. For measurement of ALDH-2 esterase activity, methylbenzoate (1 mM) was added, and the samples were incubated for another 30 min at 37°C. Mitochondrial samples were sonicated, centrifuged at 20,000g (4°C) for 20 min, and the supernatant was purified by size-exclusion centrifugation through a Microcon YM-10 filter device from Millipore (Bedford, MA). Two hundred microliters of each sample was subjected to high-performance liquid chromatography analysis. The details were published recently (Daiber et al., 2004b).
&&& Measurement of Reactive Oxygen Species Production from Isolated Heart Mitochondria and from Isolated Aortic Segments. Mitochondrial stock solutions were diluted to final total protein concentrations of approximately 0.1 mg/ml in 0.5 ml of PBS. The dye L-012 (100 e) was used as described previously (Daiber et al., 2004a) to quantify ROS after addition of the complex II substrate succinate (final concentration, 4 mM). Chemiluminescence was monitored over 5 min using a Lumat LB9507 from Berthold Technologies (Bad Wildbad, Germany), and the signal at 5 min was expressed in counts per minute. ROS production was quantified in mitochondria from wt and Mn-SOD+/- mice in the presence or absence of GTN (50 e) or antimycin A (20 e/ml). Vascular ROS production was qualitatively detected by DHE (0.1 e)-derived fluorescence in aortic tissue sections as described previously (Hink et al., 2001) and by L-012 (100 e)-derived chemiluminescence from isolated aortic rings (length, 3-4 mm) in Krebs-HEPES buffer (composition as described above). Chemiluminescence was monitored over 20 min using a Lumat LB9507 and the signal at 20 min expressed in counts per minute.
&&& Statistical Analysis. Results are expressed as mean ± S.E.M. One-way analysis of variance (with Bonferroni's or Dunn's correction for comparison of multiple means) was used for comparisons of vasodilator potency and efficacy, L-012-derived chemiluminescence, ALDH-2 dehydrogenase and esterase activity, and protein expression. The EC50 value for each experiment was obtained by log-transformation.
&&& Results
&&& Vasodilator Responses. The basal response to ACh was almost identical in both wt and Mn-SOD+/- mice (Fig. 1A; Table 1). Upon pretreatment of vessels with 200 e GTN, those from Mn-SOD+/- mice showed a highly significant degree of endothelial dysfunction (cross-tolerance to ACh) that was absent in those from wt mice. The efficacy of ACh in Mn-SOD+/- aorta was dramatically changed upon GTN pretreatment (maximal relaxation, 52 ± 4% versus 69 ± 4% in untreated wt tissue). Similar results were obtained for the response to GTN (Fig. 1B; Table 1). The Mn-SOD+/- aorta showed a decrease in maximal relaxation compared with wt aorta. Both groups showed a significant degree of tolerance upon pretreatment with 200 e GTN that was significantly more pronounced in aortas from Mn-SOD+/- mice (Fig. 1B; Table 1).
&&& In vitro GTN indicates that vessels in these groups were incubated for 30 min in the presence of 200 e GTN, followed by a 1-h washout period before isometric tension measurement. EC50 values were normalized to maximal relaxation.
&&& To assess the role of Mn-SOD deficiency on development of in vivo tolerance, mice were subjected to long-term infusion with GTN. In a preceding experiment, wt mice were treated with high and low doses of GTN or with solvent alone to determine the infusion rate of GTN required to induce tolerance in mice. As determined by isometric tension studies with aortic rings in organ baths, the low dose of GTN (16 e/h) induced neither nitrate tolerance nor cross-tolerance and had a tendency to shift the ACh and GTN dose-response curve slightly to the right (Fig. 1, C and D; Table 2). In contrast, the high dose of GTN (100 e/h) induced a marked degree of nitrate tolerance and cross-tolerance, as predicted by the right-shifted dose-response curves to ACh and GTN, and significantly decreased efficacy of both vasodilators as well as a significantly reduced potency of GTN (Fig. 1, C and D; Table 2).
&&& Presented are a separate set of experiments in which relaxation of aorta from wt mice treated in vivo with ethanol or GTN (low and high dose) was assessed. EC50 values were normalized on maximal relaxation.
&&& To test the GTN-induced NO-signaling, we performed Western blots to determine the phosphorylation state of VASP, a ubiquitous substrate of the cGMP-dependent protein kinase. No significant difference in P-VASP levels between ethanol and GTN (16 e/h) in vivo infusion of wt mice was observed, whereas P-VASP expression was significantly decreased in GTN-infused Mn-SOD+/- mice compared with the ethanol-infused control mice, respectively (Fig. 1E). P-VASP levels in both Mn-SOD-deficient groups were significantly decreased compared with the wt groups, indicating an impaired GTN bioactivation and/or NO-signaling in the deficient animals.
&&& Mitochondrial and Vascular Reactive Oxygen Species Formation. The formation of mitochondrial ROS was detected by a chemiluminescence (CL)-based assay using the luminol analog L-012. Isolated murine heart mitochondria were assessed for basal ROS production and for ROS production in the presence of GTN and antimycin A. ROS formation was significantly increased by 80% in Mn-SOD+/- mitochondria compared with wt mitochondria (56,802 ± 3919 versus 31,368 ± 2871 see Fig. 2A). In the presence of 50 e GTN, the CL signal in Mn-SOD+/- mitochondria increased by 100% and that in wt mitochondria by 150% (113,611 ± 9736 versus 78,616 ± 5071 cpm). The presence of antimycin A, which preferentially induces generation of mitochondrial superoxide, significantly increased the CL signal in Mn-SOD+/- mitochondria by 58%, whereas the CL signal in wt mitochondria increased by 88% (89,863 ± 9550 versus 58,972 ± 5192 cpm) (Fig. 2A). Mitochondrial ROS were also detected in isolated heart mitochondria from in vivo ethanol or GTN (16 e/h)-treated wt and Mn-SOD+/- mice. GTN infusion had no effect on mitochondrial ROS production of wt mice, whereas it significantly increased that in Mn-SOD+/- mice (Fig. 2B). In vitro challenges of isolated mitochondria from in vivo-treated animals with GTN (25 e) elevated ROS formation in both groups, but the absolute increase was higher in Mn-SOD-deficient mice (Fig. 2B). The ROS-induced signals in ethanol, GTN (16 e/h) in vivo, and GTN in vivo plus in vitro treated Mn-SOD+/- mice were significantly higher than those in similarly treated wt mice, indicating the increased basal oxidative stress in deficient animals (Fig. 2B). Differences in the signal intensities between Fig. 2, A and B may be due to long-term ethanol infusion in the second set of experiments.
&&& Vascular ROS formation was detected by DHE-derived fluorescence and L-012-derived chemiluminescence. Vascular ROS production from isolated aortic segments of wt mice was not significantly altered upon infusion with GTN (16 e/h) in vivo compared with the ethanol-treated control mice (Fig. 2C). Likewise, vascular ROS formation in ethanol infused deficient mice was not significantly changed compared with the wt animals, but GTN in vivo infusion elevated the signal significantly compared with the wt treatment groups (Fig. 2C). Figure 2D shows representative DHE stainings of tissue sections from wt and Mn-SOD+/- aorta. The staining of Mn-SOD+/- material is more intense compared with wt material, indicating an increased basal production of vascular superoxide in Mn-SOD+/- mice.
&&& Effects of Mn-SOD Deficiency on Mitochondrial ALDH-2 Dehydrogenase, Esterase Activity, and Vascular ALDH Dehydrogenase Activity. Basal enzyme activities were not significantly altered in Mn-SOD+/- compared with wt mitochondria (Fig. 3, A and B). The addition of 5 e GTN attenuated the dehydrogenase activity in both groups. The decrease amounted to 31% in Mn-SOD+/- mitochondria and to only 21% in those from wt mice (decreases from 13.3 ± 0.8 to 9.2 ± 0.8 e and 14.8 ± 0.8 to 11.7 ± 0.9 e, respectively) (Fig. 3A). In contrast, the esterase activity in wt mitochondria was almost unaffected by pretreatment with 50 e GTN (43.6 ± 4.9 versus 38.5 ± 6.9 e), whereas esterase activity in Mn-SOD+/- mitochondria was significantly decreased by 32% in the presence of GTN (45.3 ± 4.9 versus 30.8 ± 3.4 e) (Fig. 3B).
&&& Vascular dehydrogenase activity in isolated aortic segments from ethanol-infused deficient mice was not significant different compared with similarly treated wt mice (Fig. 3C). GTN (16 e/h) in vivo infusion caused no significant decrease of ALDH activity compared with ethanol controls of the same animal group, but GTN-infused Mn-SOD+/- mice showed a significantly lower vascular ALDH activity compared with ethanol-treated wt mice (Fig. 3C). Long-term infusion with either ethanol or an ethanolic solution of GTN increased mitochondrial ALDH-2 activity significantly (p & 0.001 for wt versus wt/EtOH) in comparison with noninfused animals, and ALDH-2 activity increased in deficient mice (Fig. 3, compare D and A). In vivo GTN treatment decreased ALDH-2 dehydrogenase activity in both animal groups, and the decrease was significantly stronger in deficient mice (Fig. 3D).
&&& Discussion
&&& We (Sydow et al., 2004) and others (Chen et al., 2002; de la Lande et al., 2004b; Kollau et al., 2004; Zhang et al., 2004) have shown previously that ALDH-2 biotransforms GTN in vitro and in vivo and that inhibition of this enzyme markedly decreases the vasodilator potency of GTN. So far, only one study has questioned the contribution of ALDH-2 to GTN bio-activation (DiFabio et al., 2003). The loss of ALDH-2 activity was associated with or was secondary to mitochondrial ROS formation upon long- or short-term challenges to GTN in vitro and in vivo (Daiber et al., 2004b; Sydow et al., 2004). With the present studies, we can demonstrate for the first time that increased oxidative stress within mitochondria from mice with heterozygous Mn-SOD deficiency (Mn-SOD+/-) predisposes vascular tissue to develop tolerance as well as cross-tolerance (endothelial dysfunction) in response to in vitro and in vivo GTN challenges. These results point to a crucial role of ROS within mitochondria in determining vascular GTN biotransformation and vascular responsiveness to endothelium dependent and independent nitrovasodilators, respectively.
&&& It is noteworthy that the degree of tolerance and cross-tolerance was markedly higher in vessels from Mn-SOD+/- versus wt mice (Fig. 1, A and B; Tables 1 and 2). Moreover, basal mitochondrial and vascular ROS formation and ROS production, in response to in vitro and in vivo challenges of GTN and the complex III inhibitor antimycin A, was substantially increased in Mn-SOD+/- animals (Fig. 2). Therefore, the redox-sensitive ALDH-2 was found to be inhibited upon in vitro and in vivo challenges with GTN, and this effect was more obvious in Mn-SOD+/- mice compared with wt animals (Fig. 3). In particular, the ALDH-2 esterase activity, which has been proposed to be crucial for GTN bio-activation (Chen et al., 2002) was strikingly more susceptible to GTN-mediated inactivation in Mn-SOD-deficient mice (Fig. 3).
&&& Heterozygous Mn-SOD Deficiency Does Not Affect NO-Signaling/Endothelial Function but Makes Vessels More Susceptible to Nitrate Tolerance and Cross-Tolerance. The vasodilator potency and efficacy in response to the endothelium-dependent vasodilator ACh was not significantly different in Mn-SOD+/- compared with wild-type mice. This observation was in agreement with recent reports from Andresen et al. (2004) that neither the basal response to ACh was changed in Mn-SOD-deficient mice nor the ACh response upon treatment with the complex III inhibitor antimycin A, which probably would yield mitochondrial ROS. This is even more surprising because DHE staining clearly demonstrated increased ROS production throughout the vascular wall. It is interesting, however, that upon preincubation of the vessels with GTN (200 e), there was a quite marked loss of ACh efficacy in the Mn-SOD+/- mice that was absent in the wt animals, indicating that GTN treatment―presumably by inducing oxidative stress in mitochondria (Daiber et al., 2004b; Sydow et al., 2004)―influenced the ACh response and therefore caused cross-tolerance (endothelial dysfunction) in Mn-SOD-deficient mice (Fig. 1A; Table 1).
&&& It is interesting that we could not detect significantly decreased responsiveness to the endothelium-independent nitrovasodilator GTN in vessels from Mn-SOD-deficient mice compared with wt control mice. Upon preincubation of the isolated vessels from wt and Mn-SOD+/- with GTN (200 e), however, there was a significant loss of GTN potency in both animal groups, but it was more pronounced in the Mn-SOD+/- mice (Fig. 1B; Table 1). There was no significant difference in the efficacy of GTN pretreated Mn-SOD+/- and wt vessels (Table 1). These observations were further supported by the effects of in vivo GTN infusion (16 e/h, 35 nmol/min/kg) in deficient mice on GTN-dependent NO-signaling (as measured by P-VASP Fig. 1E). This indicates that Mn-SOD deficiency makes the vessels more susceptible to in vitro and in vivo nitrate tolerance. To address whether this phenomenon might be related to increased oxidative stress in Mn-SOD+/- mice, we measured superoxide production in mitochondria from wt and Mn-SOD+/- in the presence and absence of GTN.
&&& Heterozygous Mn-SOD Deficiency Increases Basal, GTN-, and Antimycin A-Triggered Mitochondrial and Vascular ROS Formation, Which May Be a Key Event for the Development of Nitrate Tolerance and Cross-Tolerance. Oxidative stress plays an important role in the development of nitrate tolerance and cross-tolerance (Munzel et al., 1995b; Hink et al., 2003; Schwemmer and Bassenge, 2003). We have previously identified superoxide and/or peroxynitrite as the reactive species formed in tolerant vessels. In addition, scavengers of peroxynitrite and derived free radicals, such as ebselen and uric acid, normalized luminol-enhanced chemiluminescence in vessels from GTN-treated animals, restored the activity of the cGMP-dependent kinase I, and subsequently improved GTN tolerance in isolated rings (Hink et al., 2003). Increased vascular peroxynitrite formation also led to increased protein tyrosine nitration of the prostacyclin synthase and was associated with an almost complete inhibition of vascular prostaglandin I2 formation in the setting of tolerance (Warnholtz et al., 2002; Hink et al., 2003). Nitration of prostacyclin synthase is a specific foot-print of peroxynitrite in vivo formation (Zou et al., 1999).
&&& Herein, we provide further evidence that a considerable part of the organic nitrate-induced oxidative stress may originate from mitochondrial nitrate metabolism. Using L-012-dependent chemiluminescence (Daiber et al., 2004a), we detected peroxynitrite and superoxide in isolated rat heart mitochondria under basal conditions as well as upon in vitro and in vivo administration of GTN or antimycin A. As expected, basal mitochondrial ROS formation was significantly increased in mitochondria from Mn-SOD+/- mice compared with the wt group (Fig. 2, A and B). As expected, the absolute increase upon stimulation with bolus or long-term GTN as well as antimycin A was significantly more pronounced in mitochondria from Mn-SOD+/--deficient animals (Fig. 2, A and B). This indeed indicates that the antioxidant defense system in Mn-SOD-deficient mitochondria is impaired and that ROS formation is increased under basal conditions. Vascular ROS formation was detected by two methods (DHE-dependent oxidative fluorescent microtopography and L-012-derived chemiluminescence) and also showed that basal as well as GTN-triggered vascular ROS formation was increased in tissue from Mn-SOD+/- mice (Fig. 2, C and D).
&&& Heterozygous Mn-SOD Deficiency Decreases ALDH-2 Dehydrogenase and Esterase Activity in Response to GTN and Thereby Impairs the Mitochondrial GTN Bioactivation Leading to Tolerance and Endothelial Dysfunction. The mitochondrial isoform of ALDH (ALDH-2) was previously identified as a GTN bioactivating enzyme (Chen et al., 2002). We recently demonstrated that ALDH-2 is sensitive to oxidative inactivation by different organic nitrates as well as by oxidants such as hydrogen peroxide or peroxynitrite (Daiber et al., 2004b). ALDH-2 contains three cysteine residues in the catalytic center, rendering the dehydrogenase activity highly sensitive toward oxidative inactivation (Senior and Tsai, ). In addition to its dehydrogenase activity, ALDH-2 also exhibits esterase activity, which has been proposed to be essential for the bioactivation of GTN (Chen et al., 2002). This activity also involves oxidation-sensitive cysteine residues (Tsai and Senior, 1991). Because of increased oxidative stress in the Mn-SOD+/- mitochondria, we found that ALDH-2 dehydrogenase activity and esterase activity was significantly decreased in mitochondria but also in isolated aortic rings upon in vitro or in vivo treatment with GTN (Fig. 3).
&&& Mechanistic Implications. Mn-SOD together with glutathione peroxidase make up the most important antioxidant defense enzymes in mitochondria. Complete deficiency is disastrous for the organism and causes death within 2 to 3 weeks (Li et al., 1995; Lebovitz et al., 1996). We report herein that heterozygous Mn-SOD deficiency increases the basal but also the GTN- and antimycin A-induced formation of mitochondrial ROS. Because of GTN-induced ROS production, the GTN bioactivating enzyme ALDH-2 (Daiber et al., 2004b) or its repair system, which may involve mitochondrial lipoic acid stores and/or the glutathione/glutathione reductase system (A. Daiber, unpublished observation), will be impaired. Inactivation of ALDH-2 will subsequently slow down the mitochondrial bioactivation of GTN, which will be manifested by the phenomenon of nitrate tolerance and further point to a crucial role of this enzyme in the bioactivation process of GTN. A hypothetical unifying scheme is shown in Fig. 4. With respect to the importance of oxidative stress for the development of nitrate tolerance, it is worth mentioning that not all organic nitrates induce oxidative stress. Less potent nitrates, such as isosorbide dinitrate and isosorbide-5-mononitrate, will probably generate less mitochondrial ROS than GTN (Daiber et al., 2004b). In addition, for the highly potent pentaerythritol tetranitrate (PETN), studies have shown that this nitrate induces neither oxidative stress nor nitrate tolerance (Jurt et al., 2001). This is because of intrinsic antioxidative responses triggered by PETN, such as increased expression of the protective proteins heme oxygenase-1 and ferritin (Oberle et al., 2003).
&&& Although GTN-triggered ROS formation within mitochondria explains the tolerance phenomenon caused by impaired GTN biotransformation, it is difficult to understand why this process should also cause the phenomenon of endothelial dysfunction, because ROS formed within mitochondria will not easily cross the mitochondrial membrane. It is possible, however, that GTN-derived ROS (superoxide and peroxynitrite) might react with iron-sulfur cluster proteins, disrupting the respiratory chain (Flint et al., 1993). Therefore, it seems conceivable to conclude that GTN initiates a vicious cycle of mitochondrial ROS formation that could further be exaggerated by oxidative inactivation of Mn-SOD (MacMillan-Crow et al., 1996). Some of these ROS might escape the mitochondrial space and impair NO-signaling by direct reaction with NO or by an oxidative inactivation of soluble guanylyl cyclase (Brune et al., 1990; Mulsch et al., 1997). On the other hand, GTN-triggered mitochondrial ROS might lead to opening of mitochondrial ATP-dependent potassium channels (Zhang et al., 2001) and thereby trigger further ROS production (Lebuffe et al., 2003). The exact components of this molecular cascade are still not well determined, and this hypothesis remains rather speculative. However, it is interesting to note that mitochondrial ROS production and subsequent K-ATP channel opening might determine both GTN-induced protective (preconditioning-mimetic) effect (Dawn and Bolli, 2002) and, upon long-term GTN treatment, increased oxidative damage (Munzel et al., 1995b), leading to tolerance and endothelial dysfunction. Finally, we would like to emphasize that nitrate tolerance and cross-tolerance are probably multifactorial phenomena, and other processes contribute to the degree of tolerance as demonstrated by the marked effects of endothelium denudation of tolerant vessels on GTN responsiveness (de la Lande et al., 2004a; Munzel et al., 1995b). With respect to the "oxidative stress concept," there are also other sources of ROS that may trigger the development of tolerance, such as GTN-activated NADPH oxidases (Munzel et al., 1995a; Schwemmer and Bassenge, 2003), and probably an uncoupled NO-synthase.
&&& Acknowledgements
&&& We thank Dr. Tommaso Gori for helpful discussions. The expert technical assistance of Claudia Kuper and Yasamin Nazirizadeh is gratefully acknowledged.
&&& A.D. and M.O. contributed equally to this work
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