P20-Mediated Suppression of Vascular Force

描述（由申请人提供）：血管平滑肌松弛通常被假设为激活的逆转，即肌浆[Ca2+]肌球蛋白调节轻链（MRLC）去磷酸化的减少。我们将这种形式的放松称为“失活”，因为它是激活机制的逆转。一些刺激诱导的放松不涉及“失活”机制。尽管MRLC磷酸化水平持续升高，但[cGMP]或[cAMP]的升高可降低平滑肌张力。我们将这一过程称为“力抑制”，以将其与通过减少MRCL磷酸化来降低力的失活机制分开。在本研究中，我们提出的证据表明，“力抑制”的机制涉及磷酸化p20（也称为热休克蛋白20或HSP20）与平滑肌细丝的结合。我们有初步的数据，重组磷酸化p20抑制力在皮肤颈动脉。我们也有初步的数据表明，力抑制与缩短速度的减少无关。这表明力抑制是在交叉桥水平介导的。为了进一步研究这一现象，我们建议在剥皮颈动脉中研究重组突变体p20，并在完整颈动脉中进行详细的肌肉力学研究。这三个目标是：验证磷酸化的p20通过肽p20中包含的肌动蛋白结合域特异性抑制皮肤平滑肌力的假设（110-121）。这个p20结构域与肌钙蛋白1中称为抑制肽的结构域具有高度的序列同源性。目标2。验证力抑制受p20丝氨酸16磷酸化调控的假设。目标3。检验力抑制是在跨桥水平介导的假设。在发达国家，大多数发病率和死亡率与平滑肌反应不当有关。许多目前的治疗针对平滑肌，以减少张力。针对力抑制途径（即p20）可能会提供类似的好处。

DESCRIPTION (provided by the applicant): Vascular smooth muscle relaxation is typically hypothesized to be the reversal of activation, i.e. reduction of myoplasmic [Ca2+] myosin regulatory light chain (MRLC) dephosphorylation. We term this form of relaxation as "deactivation" because it is the reversal of activation mechanisms. Some stimuli induce a relaxation that dose not involve "deactivation" mechanisms. Elevations in [cGMP] or [cAMP] can reduce smooth muscle tone despite persistent elevations in MRLC phosphorylation levels. We term this process 'force suppression' to separate it from deactivation mechanisms which reduce force by reducing MRCL phosphorylation.In this proposal, we present evidence suggesting that the mechanism responsible for 'force suppression' involves binding of phosphorylate p20 (also known as heat shock protein 20 or HSP20) to smooth muscle thin filaments. We have preliminary data that recombinant phosphorylated p20 suppresses force in skinned carotid artery. We also have preliminary data that force suppression is not associated with reductions in shortening velocity. This suggests that force suppression is mediated at the crossbridge level. To further study this phenomenon, we propose to study recombinant mutant p20 in skinned carotid artery and to perform detailed muscle mechanics in intact carotid artery. Thee aims are:Aim 1. Test the hypothesis that phosphorylated p20 specifically suppresses skinned smooth muscle force via the actin binding domain contained in the peptide p20 (110-121). This p20 domain has a high sequence homology with a domain in troponin 1 called the inhibitory peptide.Aim 2. Test the hypothesis that force suppression is regulated by p20 phosphorylation at serine 16.Aim 3. Test the hypothesis that force suppression is mediated at the crossbridge level.Inappropriate responses of smooth muscle are involved in most morbidity and mortality in the developed world. Many current treatments target smooth muscle to reduce tone. It is possible that targeting the force suppression pathway (i.e. p20) might provide comparable benefits.