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Novel mechanism of increasing cerebral vascular constriction in acute hyperglycemia and diabetes through adenylyl cyclase 5-generated cyclic adenosine monophosphate.
Journal of Diabetes Investigation ( IF 3.1 ) Pub Date : 2019-11-23 , DOI: 10.1111/jdi.13186
Kenichi Sakamoto 1, 2 , Koutaro Yokote 1, 3
Affiliation  

The recent increase in the global incidence of type 2 diabetes mellitus poses major threats to societies worldwide. Patients with diabetes are at heightened risk of vascular complications, such as cardiovascular diseases (CVD), including stroke. Many studies showed that glycemic control improves cardiovascular outcome in diabetes patients; however, the relationship linking diabetes to CVD is more complex and multifaceted in nature. Although the improvement of strategy to prevent CVD induced by diabetes is required, the molecular mechanism altering vascular reactivity in diabetes has not yet been well defined. Hypertension is extremely common in patients with diabetes. In observational studies, patients with both diabetes and hypertension have approximately twice the risk of CVD as non‐diabetic people with hypertension. Epidemiological and functional studies show that impaired arterial myocyte contractility contributes to vascular complications in diabetes patients and animal models of diabetes1. Therefore, to clarify the mechanism of how hyperglycemia increases vascular tone is an important subject of diabetes research.

Several types of potassium (K+) channels and L‐type Ca2+ channels have important roles in regulating arterial myocyte excitability and myogenic tone. The expression and function of these channels alter diabetic hyperglycemia, which induces arterial myocyte contractility and myogenic tone.

Navedo et al .2 reported that L‐type Ca2+ channel activity in arterial myocytes and myogenic tone is elevated in diabetes. This was found to be mediated by phosphorylation of the L‐type Ca2+ channel pore‐forming CaV1.2 subunit at serine 1928 by protein kinase A (PKA). In addition, they reported that the binding of PKA to A‐kinase anchoring protein 150 is a key determinant to activate the CaV1.2 subunit at serine 1928.

The fact that PKA induces vasoconstriction in hyperglycemia is important, because this changes the way we think of the role of PKA in the pathophysiology of artery. Historically, we thought of PKA primarily as a function of vasodilation, but now we have to consider the opposite function of PKA. This raises the possibility that regulating PKA activity could be a potential therapeutic application in vascular disease induced by diabetes. Although the prior work of Navedo et al .2 showing that glucose could mediate vasoconstriction through a PKA pathway is very impressive, the mechanism by which elevated extracellular glucose leads to increased PKA activity is not well understood.

Syed et al. 3 recently reported convincing evidence in the Journal of Clinical Investigation that elevating extracellular glucose stimulates cyclic adenosine monophosphate (cAMP) production in arterial myocytes, and that this was specifically dependent on adenylyl cyclase 5 (AC5) activity.

First, the authors found that acute elevation in extracellular glucose increases myogenic tone and cAMP synthesis, which requires adenylyl cyclase (AC) activity in arterial myocytes. To evaluate cAMP synthesis in myocytes, the authors used a membrane‐targeted Epac1–cAMP‐based fluorescence resonance energy transfer (FRET) sensor (ICUE3‐PM). FRET sensor is a well‐established method to measure cAMP levels in real‐time and in living cells. In ICUE3‐PM‐expressing arterial myocytes, extracellular high glucose induces FRET signal change, which means that high glucose increases cAMP synthesis. The authors carried out voltage ramps using patch clamp electrophysiology and showed L‐type Ca2+ channel activity in freshly dissociated cerebral arterial myocytes is increased after exposure to high glucose. These responses are reversed by treating with the broad AC inhibitor, 2′,5′‐dideoxyadenosine.

The general belief is that AC activity increases cAMP in cells, which is the key driver of PKA activation. AC has nine isoforms, and AC3, AC5 and AC6 are most abundantly expressed in arterial myocytes. Although AC6 and AC3 have been reported as key pathways of vasodilation, AC5 has not been well defined. Therefore, they focused on the function of AC5 in mediating the glucose effects on cAMP synthesis. The authors use AC5 knockout (AC5−/−) mice, and analyze arterial myocytes and arteries. In ICUE3‐PM‐expressing arterial myocytes from AC5−/− mice, extracellular high glucose cannot induce FRET signal change. In addition, high glucose failed to stimulate L‐type Ca2+ channel activity in arterial myocytes from AC5−/− mice by patch clamp electrophysiology. To evaluated the effects of high glucose on vascular reactivity in vivo , the authors used an open cranial window, which is the way to expose middle cerebral arteries in living animals. Permeation of the cranial window with high glucose solution induces a robust sustained constriction of cerebral arteries in wild‐type mice, but not in AC5−/− mice. On the contrary, high glucose induces vasoconstriction to the same extent as in wild‐type mice arteries in a subset of experiments using arteries from AC6−/− mice. These results suggested that AC5 is a key driver for PKA‐dependent L‐type Ca2+ channel activity and constriction in cerebral arteries during hyperglycemia.

To further investigate the role of glucose‐induced AC5 activation to regulate L‐type Ca2+ channel activity and constriction in the artery, the authors focused on spatial organization between AC5 and L‐type Ca2+ channel. They used super‐resolution nanoscopy and proximity ligation assay analysis, which showed that a subpopulation of AC5 is located in close proximity to the L‐type Ca2+ channel pore‐forming subunit CaV1.2 in arterial myocytes. Because AC6 is not close to the CaV1.2 in the previous report, this intimate spatial organization might be an important role for local PKA‐dependent regulation of L‐type Ca2+ channels in arterial myocytes. In addition, their group also reported a similar arrangement between CaV1.2 and PKA, which are closely associated with each other2. These data suggest that a close CaV1.2–PKA–AC5 association is required for glucose‐mediated potentiation of L‐type Ca2+ channels, and vasoconstriction in arterial myocytes.

Finally, the authors analyzed the function of AC5 in diabetes model mice. A diet‐induced diabetic mouse model and the streptozotocin‐induced (STZ‐induced) diabetic mouse model are well‐established chronic hyperglycemia models. It was reported that L‐type Ca2+ channels activity increased in diabetes. They used these models to evaluate the function of AC5 to regulate L‐type Ca2+ channels and vasoconstriction in diabetes. L‐type Ca2+ channel activity and increases in arterial myocytes were seen in high‐fat diet (HFD)‐fed mice and STZ mice. Interestingly, there was an increased association between CaV1.2 and AC5 in HFD‐fed mice and STZ mice in proximity ligation assay. The myogenic tone of dissected arteries in HFD‐fed mice is significantly increased, and similarly, in vivo imaging of cerebral arteries using cranial windows showed that myogenic tone increased in STZ mice. These changes of HFD mice and STZ mice are inhibited in AC5−/− mice, indicating that AC5 has a key function of regulating L‐type Ca2+ channel activity and myogenic tone induced by hyperglycemia in HFD‐fed mice and STZ mice.

The research summarized here broadens our understanding of vasoconstriction in hyperglycemia (Figure 1). This gives us an insight into the pathophysiology of vascular complication in diabetes. However, further research is still required to fully clarify the underpinnings of this process.

image
Figure 1
Open in figure viewerPowerPoint
Schematic model of increasing vascular constriction in acute hyperglycemia through adenylyl cyclase 5 (AC5)‐generated cyclic adenosine monophosphate (cAMP). Elevations in extracellular glucose might activate the purinergic receptor (P2Y receptor) through nucleotide signaling. This induces AC5 activation, which catalyzes the conversion of adenosine triphosphate (ATP) to cAMP. The localized increase of cAMP might stimulate A‐kinase anchoring protein 150‐anchored protein kinase A (AKAP150), which activates Ca2+ channel activity. This will result in increasing arterial myocyte contraction and myogenic tone. PKA, protein kinase A.

First, it is still unclear how AC5 is regulated by hyperglycemia. In general, AC is regulated by Gαs protein‐coupled receptors. Their group reported that G protein‐coupled purinergic receptor (P2Y receptor) regulated hyperglycemia‐induced Ca2+ influx in the arterial myocyte4. There are eight isoforms of P2Y receptor, and P2Y11 is the only isoform that is coupled to G protein. Several studies showed that extracellular high glucose increases autocrine secretion of nucleotides. Adenosine 5´‐triphosphate (ATP) can be transported to the outside of cells through ATP‐binding cassettes, vesicular exocytosis, plasma membrane F1F0‐ATPase, connexin hemichannels and pannexin channels. P2Y11 can be activated by ATP/ATP‐derived nucleotides. It is speculated that one of the secreted ATP/ATP‐derived nucleotides is induced by high‐glucose activated AC5, but further investigation is required to clarify the question as to how nucleotides are released and activate vasoconstriction through activation of AC5 in response to hyperglycemia.

Second, the discrepancy of canonical function of PKA is still unknown. In contrast to this report, PKA activation traditionally induces arterial myocyte dilation in response to endogenous and exogenous vasodilatory agents5. Indeed, the authors found that application of forskolin, which is an adenylyl cyclase activator, induces vasodilation, even in the condition of elevated glucose3. The authors predict that localization of cAMP signaling makes it possible to specifically stimulate a pool of PKA and determine the effect of vascular reactivity. They showed there is a close association between the L‐type Ca2+ channel and AC5, in contrast to less association between the L‐type Ca2+ channel and AC63. Together with a previous report, they consider A‐kinase anchoring protein 150 as essential to anchor AC5, PKA and L‐type Ca2+ channel, but it is still unclear why the association between AC5 and L‐type Ca2+ channel increases in diabetic model mice3. Further investigation is required into this macromolecular complex in arterial myocytes.

In summary, the authors found a novel role of AC5 in PKA‐dependent activation of L‐type Ca2+ channel activation and vasoconstriction during acute hyperglycemia and diabetes. This report might contribute to the clinical implications and novel targets for therapeutic intervention, as they add to several mechanisms for hyperglycemia‐induced vasoconstriction through this pathway in a number of pathophysiological conditions.



中文翻译:


通过腺苷酸环化酶 5 生成环单磷酸腺苷增加急性高血糖和糖尿病脑血管收缩的新机制。



最近全球 2 型糖尿病发病率的增加对全世界社会构成了重大威胁。糖尿病患者发生血管并发症的风险较高,例如心血管疾病(CVD),包括中风。许多研究表明,血糖控制可以改善糖尿病患者的心血管结局;然而,糖尿病与心血管疾病之间的关系本质上更加复杂和多方面。尽管需要改进预防糖尿病诱发CVD的策略,但改变糖尿病血管反应性的分子机制尚未明确。高血压在糖尿病患者中极为常见。在观察性研究中,同时患有糖尿病和高血压的患者患心血管疾病的风险大约是非糖尿病高血压患者的两倍。流行病学和功能研究表明,动脉肌细胞收缩力受损会导致糖尿病患者和糖尿病动物模型的血管并发症1 。因此,阐明高血糖如何增加血管张力的机制是糖尿病研究的重要课题。


几种类型的钾 (K + ) 通道和 L 型 Ca 2+通道在调节动脉肌细胞兴奋性和肌源性张力方面具有重要作用。这些通道的表达和功能改变糖尿病高血糖,从而诱导动脉肌细胞收缩性和肌源性张力。


纳维多等人2报告称,糖尿病患者动脉肌细胞中的 L 型 Ca 2+通道活性和肌源性张力升高。研究发现,这是由蛋白激酶 A (PKA) 对 L 型 Ca 2+通道成孔 Ca V 1.2 亚基丝氨酸 1928 磷酸化介导的。此外,他们报告说,PKA 与 A 激酶锚定蛋白 150 的结合是激活 Ca V 1.2 丝氨酸 1928 亚基的关键决定因素。


PKA 在高血糖中诱导血管收缩这一事实很重要,因为这改变了我们对 PKA 在动脉病理生理学中的作用的看法。历史上,我们认为 PKA 主要是一种血管舒张功能,但现在我们必须考虑 PKA 的相反功能。这提出了调节 PKA 活性可能成为糖尿病引起的血管疾病的潜在治疗应用的可能性。尽管纳维多等人之前的工作。图2显示葡萄糖可以通过PKA途径介导血管收缩,这是非常令人印象深刻的,但细胞外葡萄糖升高导致PKA活性增加的机制尚不清楚。


赛义德等人。 3最近在《临床研究杂志》上报道了令人信服的证据,表明细胞外葡萄糖的升高会刺激动脉肌细胞中环磷酸腺苷 (cAMP) 的产生,并且这特别依赖于腺苷酸环化酶 5 (AC5) 的活性。


首先,作者发现细胞外葡萄糖的急剧升高会增加肌原性张力和 cAMP 合成,这需要动脉肌细胞中的腺苷酸环化酶 (AC) 活性。为了评估肌细胞中的 cAMP 合成,作者使用了基于膜靶向 Epac1-cAMP 的荧光共振能量转移 (FRET) 传感器 (ICUE3-PM)。 FRET 传感器是一种行之有效的实时测量活细胞中 cAMP 水平的方法。在表达 ICUE3-PM 的动脉肌细胞中,细胞外高葡萄糖诱导 FRET 信号变化,这意味着高葡萄糖会增加 cAMP 合成。作者使用膜片钳电生理学进行了电压斜坡测试,结果表明,新鲜分离的脑动脉肌细胞中的 L 型 Ca 2+通道活性在暴露于高葡萄糖后增加。通过使用广泛的 AC 抑制剂 2',5'-双脱氧腺苷治疗可以逆转这些反应。


人们普遍认为 AC 活性会增加细胞中的 cAMP,这是 PKA 激活的关键驱动因素。 AC 有九种亚型,AC3、AC5 和 AC6 在动脉肌细胞中表达最丰富。尽管 AC6 和 AC3 已被报道为血管舒张的关键途径,但 AC5 尚未明确定义。因此,他们重点关注 AC5 在介导葡萄糖对 cAMP 合成的影响中的功能。作者使用 AC5 敲除 (AC5 −/− ) 小鼠,分析动脉肌细胞和动脉。在来自 AC5 −/−小鼠的表达 ICUE3-PM 的动脉肌细胞中,细胞外高葡萄糖不能诱导 FRET 信号变化。此外,通过膜片钳电生理学检测,高葡萄糖未能刺激AC5 −/−小鼠动脉肌细胞中的L型Ca 2+通道活性。为了评估高葡萄糖对体内血管反应性的影响,作者使用了开放的颅窗,这是暴露活体动物大脑中动脉的方法。高葡萄糖溶液渗透颅窗会导致野生型小鼠脑动脉强烈持续收缩,但 AC5 -/−小鼠则不会。相反,在使用 AC6 −/−小鼠动脉的实验子集中,高葡萄糖诱导血管收缩的程度与野生型小鼠动脉相同。这些结果表明,AC5 是高血糖期间 PKA 依赖性 L 型 Ca 2+通道活性和脑动脉收缩的关键驱动因素。


为了进一步研究葡萄糖诱导的 AC5 激活在调节动脉中 L 型 Ca 2+通道活性和收缩方面的作用,作者重点关注了 AC5 和 L 型 Ca 2+通道之间的空间组织。他们使用超分辨率纳米显微镜和邻近连接分析,结果表明 AC5 的一个亚群位于动脉肌细胞中靠近 L 型 Ca 2+通道成孔亚基 Ca V 1.2 的位置。由于 AC6 与之前报告中的 Ca V 1.2 并不接近,因此这种紧密的空间组织可能对动脉肌细胞中 L 型 Ca 2+通道的局部 PKA 依赖性调节发挥重要作用。此外,他们的研究小组还报道了Ca V 1.2 和PKA之间的类似排列,它们彼此密切相关2 。这些数据表明,葡萄糖介导的 L 型 Ca 2+通道增强和动脉肌细胞血管收缩需要密切的 Ca V 1.2–PKA–AC5 关联。


最后,作者分析了 AC5 在糖尿病模型小鼠中的功能。饮食诱导的糖尿病小鼠模型和链脲佐菌素诱导的(STZ诱导的)糖尿病小鼠模型是成熟的慢性高血糖模型。据报道,糖尿病患者 L 型 Ca 2+通道活性增加。他们使用这些模型来评估 AC5 在糖尿病中调节 L 型 Ca 2+通道和血管收缩的功能。在高脂饮食 (HFD) 喂养的小鼠和 STZ 小鼠中观察到 L 型 Ca 2+通道活性和动脉肌细胞增加。有趣的是,在邻近连接试验中,HFD 喂养的小鼠和 STZ 小鼠中 Ca V 1.2 和 AC5 之间的关联性增强。 HFD 喂养的小鼠解剖动脉的肌源性张力显着增加,同样,使用颅窗的脑动脉体内成像显示 STZ 小鼠的肌源性张力增加。 HFD 小鼠和 STZ 小鼠的这些变化在 AC5 −/−小鼠中受到抑制,表明 AC5 具有调节 L 型 Ca 2+通道活性和 HFD 喂养小鼠和 STZ 小鼠高血糖诱导的肌原性张力的关键功能。


这里总结的研究拓宽了我们对高血糖时血管收缩的理解(图 1)。这让我们深入了解糖尿病血管并发症的病理生理学。然而,仍需要进一步的研究来充分阐明这一过程的基础。

image
 图1

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通过腺苷酸环化酶 5 (AC5) 生成的环磷酸腺苷 (cAMP) 增加急性高血糖中的血管收缩的示意图。细胞外葡萄糖升高可能通过核苷酸信号激活嘌呤能受体(P2Y 受体)。这会诱导 AC5 激活,从而催化三磷酸腺苷 (ATP) 转化为 cAMP。 cAMP 的局部增加可能会刺激 A 激酶锚定蛋白 150 锚定蛋白激酶 A (AKAP150),从而激活 Ca 2+通道活性。这将导致动脉肌细胞收缩和肌源张力增加。 PKA,蛋白激酶A。


首先,目前尚不清楚AC5是如何受高血糖调节的。一般来说,AC 受 Gαs 蛋白偶联受体调节。他们的小组报告称,G 蛋白偶联嘌呤能受体(P2Y 受体)调节高血糖诱导的动脉肌细胞中的 Ca 2+流入4 。 P2Y 受体有八种亚型,P2Y 11是唯一与 G 蛋白偶联的亚型。多项研究表明,细胞外高葡萄糖会增加核苷酸的自分泌分泌。 5´-三磷酸腺苷 (ATP) 可通过 ATP 结合盒、囊泡胞吐作用、质膜 F1F0-ATP 酶、连接蛋白半通道和潘联蛋白通道转运到细胞外。 P2Y 11可以被 ATP/ATP 衍生的核苷酸激活。据推测,一种分泌的 ATP/ATP 衍生核苷酸是由高血糖激活的 AC5 诱导的,但需要进一步研究来澄清核苷酸如何释放并通过激活 AC5 来响应高血糖的问题。


其次,PKA 的规范功能的差异仍然未知。与该报告相反,PKA 激活传统上会诱导动脉肌细胞扩张,以响应内源性和外源性血管舒张剂5 。事实上,作者发现,即使在葡萄糖升高的情况下,应用毛喉素(一种腺苷酸环化酶激活剂)也会诱导血管舒张3 。作者预测,cAMP 信号传导的定位使得特异性刺激 PKA 库并确定血管反应性的影响成为可能。他们表明,L 型 Ca 2+通道与 AC5 之间存在密切关联,而 L 型 Ca 2+通道与 AC6 3之间的关联较少。结合之前的报告,他们认为 A 激酶锚定蛋白 150 对于锚定 AC5、PKA 和 L 型 Ca 2+通道至关重要,但仍不清楚为什么 AC5 和 L 型 Ca 2+通道之间的关联在糖尿病模型小鼠3 .需要进一步研究动脉肌细胞中的这种大分子复合物。


总之,作者发现 AC5 在急性高血糖和糖尿病期间 L 型 Ca 2+通道激活和血管收缩的 PKA 依赖性激活中具有新作用。该报告可能有助于治疗干预的临床意义和新目标,因为它们在许多病理生理条件下通过该途径增加了高血糖诱导的血管收缩的几种机制。

更新日期:2019-11-23
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