Local Ca2+ Signaling in Smooth Muscle

平滑肌中的局部 Ca2 信号传导

• 批准号: 7333225
• 负责人: JOHN V WALSH
• 金额: $ 48.21万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7333225
• 关键词: Address; Affect; Amphibia; Blood Pressure; Blood Vessels; Blood flow; Calcium ion; Cardiac; Cells; Cerebrum; Characteristics; Classification; Cyclic GMP; Cytosol; Dependence; Dialysis procedure; Diffusion; Egtazic Acid; Endothelin; Endothelin-1; Equilibrium; Event; Face; Feedback; Functional disorder; Generations; Genes; Hypertension; Image; Ion Channel; Ions; Kinetics; Localized; Maps; Measurement; Measures; Membrane Potentials; Methodology; Methods; Monitor; Mus; Muscle function; Myocardium; Nature; Nitric Oxide; Numbers; Phase; Physical Dialysis; Physiology; Preparation; Process; Rattus; Reaction; Regulation; Relaxation; Role; RyR3; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Signal Transduction; Site; Skeletal system; Smooth Muscle; Smooth Muscle Myocytes; Speed; Standards of Weights and Measures; Stroke; Study Subject; Subarachnoid Hemorrhage; System; Thinking; Transgenic Animals; Vascular Smooth Muscle; Vasospasm; Work; arteriole; cerebral artery; falls; interest; large-conductance calcium-activated potassium channels; mitochondrial membrane; novel; patch clamp; phospholamban; receptor; simulation; voltage

DESCRIPTION (provided by applicant): Calcium ions are universal messengers, regulating many processes within cells, including cerebral vascular smooth muscle (VSM) cells, a single layer of which lines the walls of arteriolar blood vessels. These cerebral arteriolar VSM cells, the subject of this study, are key determinants of blood pressure and local blood flow and thus are crucial in the pathophysiology of hypertension, subarachnoid hemorrhage, vasospasm and stroke. In VSM highly localized, brief cytosolic Ca2+ transients (Ca2+ sparks), emanating from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs), govern nearby Ca2+-activated ion channels, both large-conductance K+(BK) channels and CI- (CI(Ca)) channels. Activation of a cluster of BK channels in the spark "microdomain" causes a spontaneous transient outward current (STOC); activation of nearby CI(Ca) channels results in a spontaneous transient inward current (STIC). Virtually all smooth muscle cells display STOCs, and very many display STICs, including the mouse pial arteriolar VSM cells to be studied here. This proposal has two fundamental objectives: first, to advance basic, biophysical understanding of sparks, STOCs, STICS and the nature of the spark microdomain from which they arise; and second, to more clearly understand the physiology of these important cerebral arteriolar VSM cells. The spark micoromain will be studied with a unique, high-speed widefield imaging system in conjunction with simultaneous patch clamp recordings. The nature of the spark and the underlying Ca2+ current will be analyzed using a novel "signal mass" methodology. The influence of SR Ca2+ stores on events within the microdomain will be examined using direct measurements of free SR [Ca2+] and the use of a phospholamban KO mouse. Of the three types of RyRs, encoded by different genes, type 3 (RyR3) is found only in certain smooth muscle cells, among them arteriolar VSM cells employed here. The effect of RYR3 on events within the microdomain (i.e., sparks, STOCs and STICs) will be examined by use of a RyR3-KO mouse. STOCs are thought to hyperpolarize VSM cells leading to relaxation, whereas STICs should have the opposite effect. Since the same sparks can elicit both, an apparent paradox, we have postulated that sparks have a stabilizing effect on membrane potential and hence on the contractile state of VSM cells. We hypothesize further that those relaxing or contractile agents which affect sparks act like a switch by affecting STICs and STOCs in opposite ways. This hypothesis will be evaluated by examining the mechanisms of action of endothelin and nitric oxide, key contractile and relaxing agents, respectively. The existence of functional Ca2+ microdomains strongly suggests local control of Ca2+ sparks by events within the microdomain, which is critical in regulation of cardiac cells. We postulate an analogous local regulation in VSM cells. We will examine how Ca2 +,sparks are regulated by nearby voltage-activated Ca2vchannels and the how feedback from Ca2+ sparks in turn affects the Ca2+ channels.

描述（由申请人提供）：钙离子是通用信使，调节细胞内的许多过程，包括脑血管平滑肌（VSM）细胞，其单层排列在小动脉血管壁上。这些脑小动脉 VSM 细胞是本研究的主题，是血压和局部血流的关键决定因素，因此在高血压、蛛网膜下腔出血、血管痉挛和中风的病理生理学中至关重要。在 VSM 中，通过兰尼碱受体 (RyR) 从肌浆网 (SR) 发出的高度局部化的短暂胞质 Ca2+ 瞬变（Ca2+ 火花）控制附近的 Ca2+ 激活离子通道，包括大电导 K+(BK) 通道和 CI- (CI(Ca)) 通道。火花“微域”中一组 BK 通道的激活会导致自发瞬态外向电流 (STOC)；附近 CI(Ca) 通道的激活会导致自发瞬态内向电流 (STIC)。事实上，所有平滑肌细胞都显示 STOC，而且很多平滑肌细胞显示 STIC，包括本文要研究的小鼠软脑膜小动脉 VSM 细胞。该提案有两个基本目标：首先，推进对火花、STOC、STICS 以及它们产生的火花微域性质的基本生物物理学理解；其次，更清楚地了解这些重要的脑小动脉VSM细胞的生理机能。火花微结构将通过独特的高速宽场成像系统结合同步膜片钳记录进行研究。将使用一种新颖的“信号质量”方法来分析火花的性质和潜在的 Ca2+ 电流。将通过直接测量游离 SR [Ca2+] 并使用受磷蛋白 KO 小鼠来检查 SR Ca2+ 储存对微域内事件的影响。在由不同基因编码的三种类型的 RyR 中，3 型 (RyR3) 仅在某些平滑肌细胞中发现，其中包括此处使用的小动脉 VSM 细胞。将使用 RyR3-KO 小鼠检查 RYR3 对微域内事件（即火花、STOC 和 STIC）的影响。 STOC 被认为会使 VSM 细胞超极化，从而导致松弛，而 STIC 应该具有相反的效果。由于相同的火花可以引发这两种现象，这是一个明显的悖论，因此我们假设火花对膜电位具有稳定作用，从而对 VSM 细胞的收缩状态具有稳定作用。我们进一步假设那些影响火花的松弛剂或收缩剂通过以相反的方式影响 STIC 和 STOC，起到开关的作用。该假设将通过检查内皮素和一氧化氮（分别是关键的收缩剂和松弛剂）的作用机制来评估。功能性 Ca2+ 微域的存在强烈表明微域内事件对 Ca2+ 火花的局部控制，这对于心肌细胞的调节至关重要。我们假设 VSM 细胞中存在类似的局部调节。我们将研究附近电压激活的 Ca2v 通道如何调节 Ca2+ 火花，以及 Ca2+ 火花的反馈如何反过来影响 Ca2+ 通道。

项目成果

Spontaneous transient outward currents arise from microdomains where BK channels are exposed to a mean Ca(2+) concentration on the order of 10 microM during a Ca(2+) spark.

• DOI: 10.1085/jgp.20028571
• 发表时间: 2002-07
• 期刊: JOURNAL OF GENERAL PHYSIOLOGY
• 影响因子: 3.8
• 作者: Zhuge, Ronghua;Fogarty, Kevin E;Tuft, Richard A;Walsh, John V Jr
• 通讯作者: Walsh, John V Jr

Recombinant expression of the voltage-dependent anion channel enhances the transfer of Ca2+ microdomains to mitochondria.

• DOI: 10.1083/jcb.200205091
• 发表时间: 2002-11-25
• 期刊: JOURNAL OF CELL BIOLOGY
• 影响因子: 7.8
• 作者: Rapizzi, Elena;Pinton, Paolo;Szabadkai, Gyorgy;Wieckowski, Mariusz R;Vandecasteele, Gregoire;Baird, Geoff;Tuft, Richard A;Fogarty, Kevin E;Rizzuto, Rosario
• 通讯作者: Rizzuto, Rosario

Dynamics of signaling between Ca(2+) sparks and Ca(2+)- activated K(+) channels studied with a novel image-based method for direct intracellular measurement of ryanodine receptor Ca(2+) current.

• DOI: 10.1085/jgp.116.6.845
• 发表时间: 2000-12
• 期刊: The Journal of general physiology
• 作者: ZhuGe R;Fogarty KE;Tuft RA;Lifshitz LM;Sayar K;Walsh JV Jr
• 通讯作者: Walsh JV Jr