(CA2+), CA2+ SENSITIVITY, AND SMOOTH MUSCLE CONTRACTION

我的研究的广泛目标是确定分子 跨境桥梁和收缩的调控机制(S) 血管平滑肌。收缩刺激最初是假设的 仅通过依赖[Ca~(2+)]_i/钙调蛋白来调节平滑肌收缩 肌球蛋白轻链激酶(MLCK)的激活。已知激活的MLCK 磷酸化肌球蛋白的20kD轻链。肌球蛋白轻链 磷酸化似乎是力量产生的主要决定因素。 然而，我们发现肌球蛋白的磷酸化水平取决于这两种变化。 在[钙]i和所采用的刺激措施中。完整光滑的Kc1去极化 肌肉组织引起相对较大的[Ca~(2+)]i和 肌球蛋白磷酸化的增加比观察到的相对较小 组胺刺激(即去极化与较低的 [CA2]i敏感性高于组胺刺激)。那里 有四种可能的机制可以解释[钙]i的变化 敏感性：1)MLCK的钙敏感性可以改变(例如，通过 2)肌球蛋白轻链磷酸酶可被 受调控，3)[钙]i估计可能不准确，和/或4肌球蛋白可能 被另一种激酶磷酸化。 这项提议的主要目标是确定分子 [Ca~(2+)]i敏感性变化机制(S) 在完整的血管平滑肌中观察到。具体目标是：1) 管理性删除。2)准确检验Aequorin的假设 估计完整组织中的肌浆[钙]。我们将测量肌浆 [CA2]在完整组织中同时含有Aequorin和Fura 2。3)测试 假设G蛋白参与调节[钙]i敏感性。 我们将用百日咳毒素或细胞内负载抑制G蛋白 国内生产总值-β-S可以评估对[钙]i和[钙]i敏感性的影响。

The broad objective of my research is to determine the molecular mechanism(s) responsible for regulation of crossbridges and contraction in vascular smooth muscle. Contractile stimuli were originally hypothesized to regulate smooth muscle contraction only via [Ca2+]i/calmodulin-dependent activation of myosin light chain kinase (MLCK). Activated MLCK is known to phosphorylate the 20 Kd light chain of myosin. Myosin light chain phosphorylation appears to be the primary determinant of force production. However, we found that myosin phosphorylation levels depend on both changes in [Ca2+]i and the stimulus employed. KC1 depolarization of intact smooth muscle tissues induced relatively larger increases in [Ca2+]i and relatively smaller increases in myosin phosphorylation than were observed with histamine stimulation (i.e. depolarization was associated with a lower [Ca2+]i sensitivity than was observed with histamine stimulation). There are four possible mechanisms that could explain changes in [Ca2+]i sensitivity: 1) the Ca2+-sensitivity of MLCK could be altered (e.g. by phosphorylation of MLCK), 2) myosin light chain phosphatase could be regulated, 3) [Ca2+]i estimates may be inaccurate, and/or 4 myosin could be phosphorylated by another kinase. The primary objective of this proposal is to determine the molecular mechanism(s) responsible for the alterations in [Ca2+]i sensitivity observed in intact vascular smooth muscle. The Specific Aims are to: 1) administratively deleted. 2) Test the hypothesis that aequorin accurately estimates myoplasmic [Ca2+] in intact tissues. We will measure myoplasmic [Ca2+] with both aequorin and Fura 2 in intact tissues. 3) Test the hypothesis that a G protein is involved in regulating [Ca2+]i sensitivity. We will inhibit G proteins with pertussis toxin or intracellularly loaded GDP-beta-S an evaluate the effect on [Ca2+]i and [Ca2+]i sensitivity.