CORONARY MUSCLE CELL FREE CALCIUM BUFFERING
冠状肌细胞无钙缓冲
基本信息
- 批准号:6110388
- 负责人:
- 金额:$ 28.25万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1999
- 资助国家:美国
- 起止时间:1999-05-01 至 2000-04-30
- 项目状态:已结题
- 来源:
- 关键词:buffers calcium channel calcium flux confocal scanning microscopy coronary artery coronary disorder digital imaging disease /disorder model electrophysiology exercise fluorimetry immunofluorescence technique miniature swine muscle contraction potassium chloride ryanodine sarcolemma sarcoplasmic reticulum vascular smooth muscle voltage /patch clamp
项目摘要
The sarcoplasmic reticulum (SR) may buffer (attenuate) increases in
averaged mycoplasmic free Ca (Ca/m) in quiescent (resting) cells by
slowly releasing Ca from the superficial SR to be extruded (unloaded)
into the extracellular space. This process is termed "SR Ca unloading"
because it results in attenuated agonist-induced ca release. SR Ca
unloading suggests close functional association (localization) of SR Ca
release sites and sarcolemmal Ca efflux mechanisms, such as the Ca pump
and/or Na-Ca exchanger, which would result in subsarcolemmal Ca (Ca/s)
localization. We have shown that coronary smooth muscle cells of
exercise trained (EX), not sedentary (SED), pigs show SR Ca unloading
that is dependent on ryanodine-sensitive Ca release channels. The
general hypothesis of this proposal is that the SR in EX cells releases
Ca from superficial SR, thus resulting in net Ca efflux (Ca unloading)
from the cell with almost no increase in Ca/m. Specific aims are to test
the following hypotheses: 1) EX-induced SR Ca unloading requires
ryanodine-sensitive Ca release channels and occurs with minimal change
in Ca/m, but an increase in free Ca in a subsarcolemmal compartment
(Ca/s). Cells will be dispersed from epicardial coronary arteries
(conduit size) of EX and SED Yucatan miniature pigs. Whole-cell voltage
clamp will allow the use of Ca-activated K current or membrane-bound
fura-2 (FFP18) for monitoring Ca/s; mycoplasmic fura-2 salt will monitor
Ca/m. 2) EX-induced SR Ca unloading results in net Ca efflux from the
cell, not translocation (redistribution) to a caffeine-insensitive store.
Net Ca efflux from suspensions of cells will be monitored by
extracellular, membrane-impermeant fura-2 3) EX-induced SR Ca unloading
requires localized efflux of Ca from the cell at superficial SR sites.
The relative localization of Ca efflux from an intact cell will be
monitored with digital imaging using extracellular fluo-3 and with an
excised (inside-out) patch of Ca-activated K channels used as a "Ca
electrode patch". SR locations will be visualized with the green
fluorescent SR marker DiOC6. 4) EX-induced SR Ca unloading requires
localized Na-Ca exchange. It will be determined whether in EX cells Na-
Ca exchange immunofluorescence co-localizes with Ca efflux sites
determined with the Ca electrode patch and extracellular fluo-3. These
data will provide evidence for a tight functional coupling between slow
Ca release from localized, superficial SR sites and Ca extrusion via Na-
Ca exchange. 5) SR Ca release is proportional to the contractile
response of arterial rings. Simultaneous fura-2 measures of Ca/m and
contraction recordings in arterial rings will determine the relevance of
SR Ca release to vascular contractile function in intact arteries. The
main significance of this work is the integration of functional data
obtained from studies at the tissue and subcellular levels of coronary
artery smooth muscle.
肌浆网(SR)可以缓冲(减弱)
静止(静息)细胞中平均支原体游离Ca(Ca/m),
从表面SR中缓慢释放钙以挤出(卸载)
进入细胞外空间 这一过程被称为“SR Ca卸载”
因为它导致减弱的激动剂诱导的Ca释放。 网Ca
卸载表明SR Ca的密切功能关联(定位)
释放位点和肌膜钙外流机制,如钙泵
和/或Na-Ca交换器,这将导致肌膜下Ca(Ca/s)
本地化 我们已经证明,
运动训练(EX),非久坐(SED),猪显示SR Ca卸载
依赖于ryanodine敏感的Ca释放通道。 的
该建议的一般假设是EX细胞中的SR释放
Ca从浅表SR流出,从而导致净Ca流出(Ca卸载)
从细胞中几乎没有增加Ca/m。 具体目标是测试
以下假设:1)EX诱导的SR Ca卸载需要
Ryanodine敏感的钙释放通道,发生变化极小
但肌膜下室游离钙增加
(Ca/秒)。 细胞将从心外膜冠状动脉分散
(导管尺寸)的EX和SED尤卡坦小型猪。 全细胞电压
钳将允许使用钙激活的钾电流或膜结合
fura-2(FFP 18)用于监测Ca/s;支原体fura-2盐将监测
Ca/m。 2)EX诱导的SR Ca卸载导致从细胞膜的净Ca流出。
细胞,而不是转移(重新分配)到咖啡因不敏感的储存。
将通过以下方法监测细胞悬液的净Ca流出量:
细胞外,膜不渗透的fura-2 3)EX诱导的SR Ca卸载
需要在表面SR位点从细胞局部流出Ca。
来自完整细胞的Ca流出的相对定位将是
使用细胞外fluo-3进行数字成像监测,
切除的(由内而外)钙激活钾通道片,用作“钙激活钾通道”。
电极贴片”。 SR位置将显示为绿色
荧光SR标记DiOC 6。 4)EX诱导的SR Ca卸载需要
局部钠钙交换 将确定在EX细胞中Na-
Ca交换免疫荧光与Ca流出位点共定位
用Ca电极贴片和细胞外Fluo-3测定。 这些
数据将提供证据,证明缓慢的
局部浅表SR部位的Ca释放和Na-
换卡。 5)SR Ca释放与收缩成正比
动脉环的反应。 钙/米和钙/米的同时fura-2测量
动脉环中的收缩记录将确定
完整动脉中SR Ca释放对血管收缩功能的影响。 的
这项工作的主要意义在于功能数据的整合
从冠状动脉组织和亚细胞水平的研究中获得,
动脉平滑肌
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Michael Sturek其他文献
Michael Sturek的其他文献
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{{ truncateString('Michael Sturek', 18)}}的其他基金
Swine Core - Regional/National Shared Resources Core
养猪核心 - 区域/国家共享资源核心
- 批准号:
10155472 - 财政年份:2015
- 资助金额:
$ 28.25万 - 项目类别:
PORCINE MODELS OF CORONARY ARTERY DISEASE IN DIABETES
糖尿病冠状动脉疾病的猪模型
- 批准号:
7621683 - 财政年份:2007
- 资助金额:
$ 28.25万 - 项目类别:
EXERCISE, DIABETES, AND CORONARY SMOOTH MUSCLE CALCIUM
运动、糖尿病和冠状动脉平滑肌钙
- 批准号:
6184686 - 财政年份:1999
- 资助金额:
$ 28.25万 - 项目类别:
EXERCISE, DIABETES, AND CORONARY SMOOTH MUSCLE CALCIUM
运动、糖尿病和冠状动脉平滑肌钙
- 批准号:
6390349 - 财政年份:1999
- 资助金额:
$ 28.25万 - 项目类别:
EXERCISE, DIABETES, AND CORONARY SMOOTH MUSCLE CALCIUM
运动、糖尿病和冠状动脉平滑肌钙
- 批准号:
6537584 - 财政年份:1999
- 资助金额:
$ 28.25万 - 项目类别:
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