Subtype identification and functional characterization of ryanodine receptors in rat cerebral artery myocytes.
Subtype identification and functional characterization of ryanodine receptors in rat cerebral artery myocytes.
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大鼠脑动脉肌细胞兰尼碱受体的亚型鉴定和功能特征。
DOI：
10.1152/ajpcell.00318.2009
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Dopico,AlejandroM
中科院分区：
文献类型：
--
作者：
Vaithianathan,Thirumalini;Narayanan,Damodaran;Asuncion-Chin,MariaT;Jeyakumar,LoiceH;Liu,Jianxi;Fleischer,Sidney;Jaggar,JonathanH;Dopico,AlejandroM
Ryanodine受体（RyRs）调节阻力大小的脑动脉平滑肌的收缩性，但其分子身份，亚细胞位置和表型在这个组织中仍然未知。在大鼠电阻大小的脑动脉肌细胞肌浆网（SR）纯化并掺入POPE-POPS-POPC（5：3：2; wt/wt）双层后，在对称的300 mM Cs+中通常检测到110 ± 8、334 ± 15和441 ± 27 pS的单位电导。当Cs+被Ca 2+取代时，最常见的（34/40）双层电导（334 pS）降至≤100 pS。主导电导显示66次爆发/分钟，至少有三个打开状态和三个关闭状态。稳态活性（NPo）-电压曲线呈钟形，当电压从−30 mV切换到−40 mV时，NPo急剧下降。在0.1-100 μM范围内，胞内钙（Ca ~（2+）i）升高时，NPO增加，但随着Ca ~（2+）i的升高，NPO突然减少。因此，在生理条件下，在大鼠脑动脉肌细胞中发现的Ca 2+范围内发生最大活性。钌红（80 μM）可降低NPo，咖啡因（0.1-5 mM）或兰尼碱（0.05-5 μM）可单调增加NPo，肝素（2 mg/ml）对NPo无影响。这种表型类似于心脏RyR和重组RyR 2。RT-PCR检测脑动脉肌细胞RyR 1、RyR 2和RyR 3转录本。然而，实时荧光定量PCR表明，RyR 2的丰度分别是RyR 1和RyR 3的4倍和1.5倍。Western blotting结果显示，RyR 2表达量较高。免疫荧光显示，每个RyR亚型分布差异的亚细胞室。特别是，RyR 2是强烈的subplasmalemma比在其他车厢，强调RyR 2的优势，SR是丰富的车厢。同样，来自SR富集膜的RyR显示出RyR 2的典型药理学特异性，被地高辛（1 μM）激活，对丹曲林（100 μM）具有抗性，并被新霉素（100 nM）转移到亚电导。因此，RyR 2是主要的分子和功能的RyR，在大鼠阻力大小的脑动脉肌细胞的SR膜中表达。
Ryanodine receptors (RyRs) regulate contractility in resistance-size cerebral artery smooth muscle, yet their molecular identity, subcellular location, and phenotype in this tissue remain unknown. Following rat resistance-size cerebral artery myocyte sarcoplasmic reticulum (SR) purification and incorporation into POPE-POPS-POPC (5:3:2; wt/wt) bilayers, unitary conductances of 110 ± 8, 334 ± 15, and 441 ± 27 pS in symmetric 300 mM Cs+were usually detected. The most frequent (34/40 bilayers) conductance (334 pS) decreased to ≤100 pS when Cs+was replaced with Ca2+. The predominant conductance displayed 66 bursts/min with at least three open and three closed states. The steady-state activity (NPo)-voltage curve was bell shaped, with NPodrastically decreasing when voltage was switched from −30 to −40 mV. NPoincreased when intracellular calcium (Ca2+i) was raised within 0.1–100 μM to abruptly diminish with higher Ca2+i. Thus maximal activity occurred within the Ca2+irange found in rat cerebral artery myocytes under physiological conditions. NPowas reduced by ruthenium red (80 μM), increased monotonically by caffeine (0.1–5 mM) or ryanodine (0.05–5 μM), and unaffected by heparin (2 mg/ml). This phenotype resembles that of cardiac RyR and recombinant RyR2. RT-PCR detected RyR1, RyR2, and RyR3 transcripts in cerebral artery myocytes. However, real-time PCR indicated that RyR2 was 4 and 1.5 times more abundant than RyR1 and RyR3, respectively. Consistently, Western blotting showed that the RyR2 product was very abundant. Immunofluorescence showed that each RyR isoform distributed differentially among subcellular compartments. In particular, RyR2 was drastically stronger in the subplasmalemma than in other compartments, underscoring the predominance of RyR2 in a compartment where SR is abundant. Consistently, RyR from SR-enriched membranes displayed pharmacological specificity typical of RyR2, being activated by digoxin (1 μM), resistant to dantrolene (100 μM), and shifted to a subconductance by neomycin (100 nM). Therefore, RyR2 is the predominant molecular and functional RyR that is expressed in the SR membrane of rat resistance-size cerebral artery myocytes.
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二氢吡啶激动剂激活纯化的心脏兰尼碱受体。
DOI：
10.1152/ajpheart.2001.280.3.h1201
发表时间：
2001
期刊：
American journal of physiology. Heart and circulatory physiology
影响因子：
--
作者：
Sagawa,T;Nishio,M;Sagawa,K;Kelly,JE;Lokuta,AJ;Tsai,J;Kan,E;Wasserstrom,JA
通讯作者：
Wasserstrom,JA
DOI：
10.1152/ajpheart.2001.280.5.h2399
发表时间：
2001-05-01
影响因子：
4.8
作者：
Mauban, JRH;Lamont, C;Wier, WG
通讯作者：
Wier, WG
影响因子：
56.9
作者：
Marx, SO;Ondrias, K;Marks, AR
通讯作者：
Marks, AR
DOI：
10.2741/meissner
发表时间：
2002-11-01
期刊：
FRONTIERS IN BIOSCIENCE
影响因子：
--
作者：
Meissner, G
通讯作者：
Meissner, G
DOI：
--
发表时间：
2003
期刊：
American journal of physiology. Cell physiology
影响因子：
--
作者：
A. Dopico
通讯作者：
A. Dopico