Signalling In Space And Time: Intracellular Cyclic AMP Dynamics In Human Vascular Smooth Muscle

血管不断改变其直径，以使血流量与组织对氧气的需求相匹配。这些调节是通过血管壁内肌肉细胞的收缩和松弛来实现的。这使得了解控制肌肉收缩的机制对于了解身体周围的正常血液流动，以及在运动过程中，随着年龄或糖尿病或高血压等疾病的这种变化非常重要。当一个组织缺氧并需要更多血液时，它会向动脉肌细胞发出“放松”信号。这些信号通过一种名为环状AMP的可扩散的小信使分子从细胞表面传递到细胞内部，该分子的功能是将信息分发到细胞内的多个位置，从而诱导松弛。一个根本的问题是，可以在细胞内自由移动的高度扩散的信使如何将信息传递到正确的细胞内“地址”？解决这个问题的一个方法是，如果循环AMP在血管肌肉细胞内以复杂的“波”形式移动，协调松弛信号正确到达不同的细胞目标。这些模式可以由称为磷酸二酯酶(PDE)的酶产生，这种酶降解环状AMP并限制其在细胞内的自由运动。PDE的屏障，就像防洪一样，可以将循环AMP引导到预定的目的地，确保消息以正确的顺序到达正确的细胞内目标。动脉细胞具有许多不同类型的PDE酶，这应该允许它们产生这些复杂的环状AMP扩散模式，但对血管平滑细胞中的这一点知之甚少。这是我们对血管生理学的理解上的一个重大差距，制药业尤其感兴趣，因为PDE活性(以及产生环状AMP的酶)的遗传差异与高血压和中风的易感性有关。靶向PDE的药物可能对许多疾病有用，但由于我们对这些酶在正常细胞中如何工作的了解有限，目前它们的使用受到严重副作用的限制。在这个项目中，我们将使用最先进的分子传感器锚定在人类动脉细胞内的特定点，以跟踪细胞内环磷酸腺苷的实时流动。这些感应器激活时间的不同将使我们能够在任何时候确定细胞内循环AMP‘波’的位置。我们还可以使用选择性抑制不同类型的PDE的药物来告诉我们，这些酶中的哪一种在引导环状AMP信号方面是重要的。我们认为，来自不同激素和神经递质的不同细胞表面信号会产生不同的环状AMP扩散模式，这些模式的维持对正常的血管松弛至关重要。我们将在两条不同动脉--冠状动脉和肺动脉--的人体细胞中进行实验。这些动脉扮演着截然不同的生理角色：冠状动脉为心肌提供含氧血液，而肺动脉将含氧血液从心脏输送到肺部，以获取更多氧气。重要的是，我们必须确定PDE在不同动脉之间的作用方式的任何潜在差异，因为这将指导未来的研究，旨在确定能够扩张一条动脉而不影响另一条动脉的药物，从而减少旨在调节体内血流的治疗的副作用。该项目的总体成果将是：1)确定确保我们的动脉扩张以优化全身血液和氧气流动的分子机制；2)解释循环AMP信号蛋白活性的遗传变异如何导致血流和血压的差异；以及3)最终帮助开发针对循环AMP信号轴的未来疗法。

Blood vessels constantly change their diameter to match blood flow to tissue needs for oxygen. These adjustments are made by the contraction and relaxation of muscle cells within blood vessel walls. This makes understanding the mechanisms that control muscle contractility important for understanding normal blood flow around the body and how this changes during exercise, with age or in diseases like diabetes or high blood pressure. When a tissue becomes starved of oxygen and needs more blood it sends 'relaxation' signals to the arterial muscle cells. These signals are relayed from the cell surface to the cell interior by a small diffusible messenger molecule called cyclic AMP which functions to distribute the message to multiple sites within the cell to induce relaxation. A fundamental question is how a highly diffusive messenger that can move freely in the cell manages to deliver information to the correct intracellular 'address'?One way to solve the problem would be if cyclic AMP moved about within vascular muscle cells in complex 'waves' that co-ordinated the correct arrival of the relaxation signal at different cellular targets. These patterns can be generated by enzymes called phosphodiesterases (PDEs) that degrade cyclic AMP and restrict its free movement in the cell. Barriers of PDEs, like flood defences, could channel cyclic AMP towards its intended destination ensuring the message reaches the correct intracellular targets in the correct order. Arterial cells possess many different types of PDE enzyme that should allow them to generate these complex cyclic AMP dispersal patterns, but little is known about this in vascular smooth cells. This is a major gap in our understanding of blood vessel physiology and of particular interest to the pharmaceutical industry since genetic differences in the activity of PDEs (and also the enzymes that produce cyclic AMP) are linked to susceptibility to high blood pressure and stroke. Drugs that target PDEs could be useful in a number of diseases, but their usage is currently restricted due to serious side-effects because of our limited knowledge about how these enzymes work in normal cells.In this project we will use state-of-the-art molecular sensors anchored at specific points within human arterial cells to track the real-time flow of cyclic AMP around the cell. Differences in the timing of activation of these sensors will allow us to determine where the cyclic AMP 'wave' is at any one time within the cell. We can also use drugs that selectively inhibit different types of PDE to tell us which of these enzymes is important in channelling the cyclic AMP signal. We believe that different cell-surface signals from different hormones and neurotransmitters generate distinct patterns of cyclic AMP dispersal and that the maintenance of these patterns is crucial to normal blood vessel relaxation. We will carry out experiments in human cells from two different arteries, the coronary artery and the pulmonary artery. These arteries carry out very different physiological roles: the coronary artery feeds the heart muscle with oxygenated blood, while the pulmonary artery carries deoxygenated blood from the heart to the lungs to pick up more oxygen. It is important that we identify any potential differences in how PDEs work between different arteries as this will direct future research aimed at identifying drugs that can dilate one artery while leaving other unaffected, thus reducing the side effects of therapies aimed at modulating blood flow in the body. The overall outcome of this project will be to: 1) identify the molecular mechanisms that ensure that our arteries dilate to optimise the flow of blood and oxygen around the body; 2) explain how genetic variation in cyclic AMP signalling protein activity can result in differences in blood flow and blood pressure, and 3) ultimately help in the development of future therapies that target the cyclic AMP signalling axis.