多巴胺受体对胰岛素受体、内皮素B受体的调节及在高血压发生中的作用研究
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摘要
一、研究背景
高血压(essential hypertension, EH)及其并发症已成为影响人们健康的主要原因,明确EH的发生机理对于EH的防治具有重要的意义。调节血压的部位包括中枢、血管和肾脏。血管平滑肌细胞(vascular smooth muscle cells, VSMCs)异常增殖可引起血管张力增高,并且是高血压、动脉粥样硬化和血管成形术后再狭窄、斑块形成过程中共同的病理、生理性改变。大量的血管活性物质和生长因子参与了上述疾病中VSMCs的异常增殖,其中胰岛素为促进VSMCs增殖的重要因子,此外胰岛素也可通过刺激细胞内钙离子浓度升高引起血管收缩、周围血管阻力增加参与血压的调控。肾脏通过影响尿钠重吸收在血压的调节过程中发挥重要作用,在肾脏各部分中,肾近曲小管(RPT)的作用尤为明显,该段对尿钠的重吸收能力超过了肾脏总吸收能力的66%。
交感神经在EH发生中的作用得到人们的公认。近年来,作为去甲肾上腺素、肾上腺素的前体物质-多巴胺及其受体在高血压发生中的作用逐渐受到重视,多巴胺与相应的多巴胺受体结合,通过调节肾脏利钠排尿、舒张血管、调控氧化应激等影响机体血压水平。多巴胺受体(dopamine receptors, DR)分为D1类(D1、D5)、D2类(D2、D3、D4)两大受体类型。多巴胺受体的5个亚型,无论敲除何种受体亚型均引起基因敲除小鼠血压明显增高,EH状态下,多巴胺受体介导的利尿排钠作用明显障碍。除多巴胺外,肾脏的利尿、排钠作用还受到其它众多体液因子的调节,交感神经(包括多巴胺)、内皮素、肾素-血管紧张素系统等均在其中发挥重要的作用。按对尿钠的影响可将以上体液因子分为两大类,一类促进尿钠排泄,另一类则抑制尿钠排泄。两大类体液因子及其受体的相互作用、控制尿钠排泄在适当的范围内,从而保持血压在正常水平。
我们在前期研究中发现:D1样受体、D3受体可抑制NE介导的促VSMCs增殖作用;刺激D1样或D3受体亦可抑制VSMCs胰岛素受体蛋白表达;在D1样受体、D3受体激活的情况下,胰岛素介导的促VSMCs增殖作用被部分抑制;作为D2类受体的主要亚型D4受体是否亦发挥对VSMCs增殖作用的调节?刺激D2受体可降低女性肥胖患者血浆胰岛素水平;高胰岛素血症动物和非胰岛素依赖型糖尿病患者多巴胺受体功能出现异常;D4受体在动脉外膜和外中膜结合处表达,在肠系膜动脉、肺动脉血管平滑肌细胞表达丰富;因而我们设想多巴胺受体与促血管平滑肌细胞(VSMCs)的增殖因素(胰岛素受体)之间存在相互作用,该调节作用可能在EH发生中发挥一定功能。
我们前期的实验中发现肾动脉灌注D3受体激动剂可促进WKY(Wistar-Kyoto, WKY)大鼠尿钠排泄,给予内皮素B(Endothelin B receptor, ETB)受体阻断剂,可部分抑制D3受体介导的尿钠排泄;刺激WKY肾脏近曲小管上皮细胞D4受体可影响胰岛素受体、AT1(Angiotensin II typeⅠreceptor, AT1)受体、ETB受体的蛋白表达和功能;基于上述工作基础,我们推测:刺激D3受体亦对肾脏ETB受体的表达和功能具有影响,该影响异常可能参与了高血压的发生、发展的病理生理过程;以上问题的解决有助丰富多巴胺受体/血管胰岛素受体/肾脏ETB受体相互作用的网络学说,为下一步深入探讨控制该网络的共同影响因素奠定基础。
二、研究目的研究多巴胺受体对胰岛素受体/ETB的调节作用,探讨该作用与高血压发生的关系。
三、研究内容
1.D4受体对胰岛素介导的促血管平滑肌细胞增殖的影响。
2.D4受体对血管胰岛素受体表达的影响和及机制。
3.研究D3受体对肾脏ETB受体表达的影响和机制及Na+-K+ ATP酶活性的变化。
4.明确以上研究内容在EH状态下是否异常,探讨其与EH发生之间的关系。
四、研究结果
1、尽管D4受体本身对VSMCs增殖无影响,但可抑制胰岛素介导的促VSMCs增殖作用,该作用呈现浓度依赖性;在WKY、SHR(spontaneously hypertensive rats, SHRs)大鼠VSMCs,D4受体对胰岛素介导的VSMCs增殖抑制作用依然存在,但无论基础状态或刺激D4受体后,SHR VSMCs增殖作用水平均高于WKY大鼠VSMCs。
2、刺激D4受体可能通过增加胰岛素受体衰减而抑制VSMCs胰岛素受体蛋白的表达,该作用呈现浓度和时间依赖性;D4受体经由PKA(protein kinase A, PKA)信号发挥抑制胰岛素受体表达作用。
3、D4受体与胰岛素受体存在共表达和生理连接,基础状态下SHR大鼠VSMCs的D4受体/胰岛素受体共沉淀程度明显高于WKY大鼠VSMCs,刺激D4受体可降低D4受体/胰岛素受体之间的共沉淀程度。
4、刺激D3受体可增加WKY细胞ETB受体的蛋白表达,该调节作用依赖于钙通道,在SHR细胞,刺激D3受体反而使ETB受体蛋白表达量进一步下降。
5、D3 /ETB受体间亦存在共存和生理连接,刺激D3受体可提高WKY和SHR细胞D3/ETB受体共沉淀程度,WKY细胞D3/ETB受体升高的绝对值明显高于SHR细胞,并且WKY细胞基础D3/ETB受体共沉淀程度明显高于SHR细胞。
6、在WKY RPT细胞,预先刺激D3受体可增强ETB受体对Na+-K+ ATP酶活性的抑制作用,但在SHR细胞,该抑制作用丧失,并且基础状态下SHR细胞Na+-K+ ATP酶的活性明显高于WKY细胞。
五、结论
多巴胺受体对血管平滑肌细胞胰岛素受体和肾脏内皮素B受体的表达和功能具有调节作用,D4受体调节胰岛素受体功能在高血压状态依然存在,提示D4受体可能是降低高胰岛素状态下胰岛素对血管危害的靶点之一;高血压状态下D3受体对肾脏ETB受体调节作用受损导致尿钠排泄异常可能参与了高血压发生的病理生理过程。
Background
Essential hypertension is heterogeneous disease involved in which both genetics and environment factors influences blood pressure, which is a major risk factor for the cardiovascular disease. Therefore , to uncover the pathogenesis of essential hypertension is becoming more and more important. Epidemiological evidence supports a link between insulin resistance and hypertension. Insulin resistance leads to hyperinsulinemia. The high levels of insulin may play an important role in the pathogenesis of hypertension by stimulating the proliferation of vascular smooth muscle cells (VSMCs). Besides of the artery, the kidney plays a role in the regulation of blood pressure by affecting sodium reabsorption, Among all segments, the effect of renal proximal tubule (RPT) is evident, which takes charge of 66% renal sodium reabsorption.
It’s known that sympathetic system takes an important position in the pathogenesis of essential hypertension. Dopamine is an endogenous catecholamine that regulates/modu- lates many cellular functions, including effects on renal hemodynamics, ion and water transport and by regulation of hormones and humoral agent release, such as aldosterone, catecholamine, endothelin, prolactin, proopiomelanocortin, renin, and vasopressin. In addition, dopamine can control blood pressure by acting on cardiovascular centers, heart, and arterial and venous vessels. Dopamine exerts its actions by occupation of the D1-like (D1 and D5) and D2-like (D2, D3, and D4) receptors. The natriuretic and diuretic effects of dopamine receptors are confirmed in receptor null mice. In the hypertensive states, the dopamine receptors mediated-natriuresis and diuresis are impaired. Apart from dopamine, the renal natriuresis and diuresis are also regulated by other humoral factors, such as sympathetic nervous system (including dopamine), endothelin, renin-angiotensin system. According to the effects on sodium excretion, those humoral factors are divided into two subfamilies, one family enhances; while the other inhibits sodium excretion. The interaction of those two subfamilies of humoral factors keeps sodium excretion and blood pressure in normal extent.
Our previous study showed that, although D1 or D3 receptor had no effect on VSMC proliferation, it reduced norepinephrine or insulin-mediated VSMCs proliferation, stimulation of D1 or D3 receptor decreases insulin receptor expression, As a major subtype of D2-like receptors, D4 receptor exists in the heart, renal afferent artery, efferent artery, pulmonary artery, mesenteric artery, Whether D4 receptor has some effect on insulin receptor is not known, we hypothesized that D4 receptor may have an inhibitory effect on insulin receptor expression and function.
We also found that D3 receptor agonist (PD128907) induced natriuresis in WKY rats on normal or high NaCl diet, An ETB receptor antagonist (BQ788), which, by itself, did not have a significant effect on sodium excretion, partially blocked the natriuretic effect of the D3 receptor agonist; Activation of D4 receptor affected ETB, AT1, insulin receptror expression in RPT cells. Based on those evidences, we hypothesize that D3 receptors, may have effects on ETB receptor expression and function in RPT cells, the impaired interaction might be involved into the pathogenesis of essential hypertension. Uncover of these issues will be helpful to elucidate the renal and arterial receptors interactions, to provide therapeutic target for normalization of the impaired receptor interaction in hypertensive states.
Objective
To uncover the interactions between dopamine receptor and other receptors (ETB receptor, insulin receptor), to explore the role of impaired interaction in essential hypertension.
Methods
1. Effects of D4 receptor on vascular insulin receptor expression and function and its mechanisms by cell number counting, uptake of MTT, reverse transcription-PCR, immunoblotting, and immunohistochemistry .
2. Effects of D3 receptor on renal ETB receptor expression and function, its mechanisms by immunoblotting and measuring Na+-K+-ATPase activity.
3. The interaction between D4 receptor and insulin receptor, D3 receptor and ETB receptor were determined by laser confocal microscopy and co-immunoprecipitation.
Results
1. D4 receptor, by itself, had no effect on VSMC proliferation. However, it could reduce insulin-mediated VSMC proliferation in A10 cells. The inhibitory effect of D4 receptor still existed in spontaneously hypertensive rats (SHRs).
2. D4 receptor agonist, PD168077, decreased insulin receptor expression in a concentration-dependent and time-dependent manner, the inhibitory effect was via PKA. When actinomycin D was used to block the synthesis of new RNA, we found that D4 receptor increased insulin receptor degradation, which might be involved into the mechanism of D4 inhibitory of insulin receptor expression.
3. There was co-localization and co-immunoprecipitation between D4 and insulin receptors, stimulation of D4 receptor decreased the linkage between D4 and insulin receptors.
4. In WKY RPT cells, the D3 receptor agonist, PD128907, increased ETB receptor protein expression in a concentration-dependent and time-dependent manner, the effect was blocked in the presence of nicardipine. In contrast, in SHR RPT cells, PD128907, decreased ETB receptor protein expression.
5. Basal D3/ETB receptor co-immunoprecipitation was greater in WKY than SHRs, The absolute amount of D3/ETB receptor co-immunoprecipitation induced by D3 receptor agonist was also greater in WKY than SHRs.
6. Stimulation of ETB receptor decreased Na+-K+-ATPase activity in WKY but not in SHR cells. Pretreatment with PD128907 augmented the inhibitory effect of BQ3020 on Na+-K+-ATPase activity in WKY but not in SHR cells.
Conclusions
Dopamine receptor can affected vascular insulin receptor, renal ETB receptor expression and founction. Activation of D4 receptor inhibits insulin receptor expression and insulin-mediated VSMC proliferation both in Wistar-Kyoto and SHRs, which might be a new target to reduce the effects of insulin resistance on artery in hypertensive states. D3 receptor regulate ETB receptor by physical receptor interaction and govern receptor expression and founction, D3 receptor regulation of ETB receptor is aberrant in RPT cells from SHRs.
高血压(essential hypertension, EH)及其并发症已成为影响人们健康的主要原因,明确EH的发生机理对于EH的防治具有重要的意义。调节血压的部位包括中枢、血管和肾脏。血管平滑肌细胞(vascular smooth muscle cells, VSMCs)异常增殖可引起血管张力增高,并且是高血压、动脉粥样硬化和血管成形术后再狭窄、斑块形成过程中共同的病理、生理性改变。大量的血管活性物质和生长因子参与了上述疾病中VSMCs的异常增殖,其中胰岛素为促进VSMCs增殖的重要因子,此外胰岛素也可通过刺激细胞内钙离子浓度升高引起血管收缩、周围血管阻力增加参与血压的调控。肾脏通过影响尿钠重吸收在血压的调节过程中发挥重要作用,在肾脏各部分中,肾近曲小管(RPT)的作用尤为明显,该段对尿钠的重吸收能力超过了肾脏总吸收能力的66%。
交感神经在EH发生中的作用得到人们的公认。近年来,作为去甲肾上腺素、肾上腺素的前体物质-多巴胺及其受体在高血压发生中的作用逐渐受到重视,多巴胺与相应的多巴胺受体结合,通过调节肾脏利钠排尿、舒张血管、调控氧化应激等影响机体血压水平。多巴胺受体(dopamine receptors, DR)分为D1类(D1、D5)、D2类(D2、D3、D4)两大受体类型。多巴胺受体的5个亚型,无论敲除何种受体亚型均引起基因敲除小鼠血压明显增高,EH状态下,多巴胺受体介导的利尿排钠作用明显障碍。除多巴胺外,肾脏的利尿、排钠作用还受到其它众多体液因子的调节,交感神经(包括多巴胺)、内皮素、肾素-血管紧张素系统等均在其中发挥重要的作用。按对尿钠的影响可将以上体液因子分为两大类,一类促进尿钠排泄,另一类则抑制尿钠排泄。两大类体液因子及其受体的相互作用、控制尿钠排泄在适当的范围内,从而保持血压在正常水平。
我们在前期研究中发现:D1样受体、D3受体可抑制NE介导的促VSMCs增殖作用;刺激D1样或D3受体亦可抑制VSMCs胰岛素受体蛋白表达;在D1样受体、D3受体激活的情况下,胰岛素介导的促VSMCs增殖作用被部分抑制;作为D2类受体的主要亚型D4受体是否亦发挥对VSMCs增殖作用的调节?刺激D2受体可降低女性肥胖患者血浆胰岛素水平;高胰岛素血症动物和非胰岛素依赖型糖尿病患者多巴胺受体功能出现异常;D4受体在动脉外膜和外中膜结合处表达,在肠系膜动脉、肺动脉血管平滑肌细胞表达丰富;因而我们设想多巴胺受体与促血管平滑肌细胞(VSMCs)的增殖因素(胰岛素受体)之间存在相互作用,该调节作用可能在EH发生中发挥一定功能。
我们前期的实验中发现肾动脉灌注D3受体激动剂可促进WKY(Wistar-Kyoto, WKY)大鼠尿钠排泄,给予内皮素B(Endothelin B receptor, ETB)受体阻断剂,可部分抑制D3受体介导的尿钠排泄;刺激WKY肾脏近曲小管上皮细胞D4受体可影响胰岛素受体、AT1(Angiotensin II typeⅠreceptor, AT1)受体、ETB受体的蛋白表达和功能;基于上述工作基础,我们推测:刺激D3受体亦对肾脏ETB受体的表达和功能具有影响,该影响异常可能参与了高血压的发生、发展的病理生理过程;以上问题的解决有助丰富多巴胺受体/血管胰岛素受体/肾脏ETB受体相互作用的网络学说,为下一步深入探讨控制该网络的共同影响因素奠定基础。
二、研究目的研究多巴胺受体对胰岛素受体/ETB的调节作用,探讨该作用与高血压发生的关系。
三、研究内容
1.D4受体对胰岛素介导的促血管平滑肌细胞增殖的影响。
2.D4受体对血管胰岛素受体表达的影响和及机制。
3.研究D3受体对肾脏ETB受体表达的影响和机制及Na+-K+ ATP酶活性的变化。
4.明确以上研究内容在EH状态下是否异常,探讨其与EH发生之间的关系。
四、研究结果
1、尽管D4受体本身对VSMCs增殖无影响,但可抑制胰岛素介导的促VSMCs增殖作用,该作用呈现浓度依赖性;在WKY、SHR(spontaneously hypertensive rats, SHRs)大鼠VSMCs,D4受体对胰岛素介导的VSMCs增殖抑制作用依然存在,但无论基础状态或刺激D4受体后,SHR VSMCs增殖作用水平均高于WKY大鼠VSMCs。
2、刺激D4受体可能通过增加胰岛素受体衰减而抑制VSMCs胰岛素受体蛋白的表达,该作用呈现浓度和时间依赖性;D4受体经由PKA(protein kinase A, PKA)信号发挥抑制胰岛素受体表达作用。
3、D4受体与胰岛素受体存在共表达和生理连接,基础状态下SHR大鼠VSMCs的D4受体/胰岛素受体共沉淀程度明显高于WKY大鼠VSMCs,刺激D4受体可降低D4受体/胰岛素受体之间的共沉淀程度。
4、刺激D3受体可增加WKY细胞ETB受体的蛋白表达,该调节作用依赖于钙通道,在SHR细胞,刺激D3受体反而使ETB受体蛋白表达量进一步下降。
5、D3 /ETB受体间亦存在共存和生理连接,刺激D3受体可提高WKY和SHR细胞D3/ETB受体共沉淀程度,WKY细胞D3/ETB受体升高的绝对值明显高于SHR细胞,并且WKY细胞基础D3/ETB受体共沉淀程度明显高于SHR细胞。
6、在WKY RPT细胞,预先刺激D3受体可增强ETB受体对Na+-K+ ATP酶活性的抑制作用,但在SHR细胞,该抑制作用丧失,并且基础状态下SHR细胞Na+-K+ ATP酶的活性明显高于WKY细胞。
五、结论
多巴胺受体对血管平滑肌细胞胰岛素受体和肾脏内皮素B受体的表达和功能具有调节作用,D4受体调节胰岛素受体功能在高血压状态依然存在,提示D4受体可能是降低高胰岛素状态下胰岛素对血管危害的靶点之一;高血压状态下D3受体对肾脏ETB受体调节作用受损导致尿钠排泄异常可能参与了高血压发生的病理生理过程。
Background
Essential hypertension is heterogeneous disease involved in which both genetics and environment factors influences blood pressure, which is a major risk factor for the cardiovascular disease. Therefore , to uncover the pathogenesis of essential hypertension is becoming more and more important. Epidemiological evidence supports a link between insulin resistance and hypertension. Insulin resistance leads to hyperinsulinemia. The high levels of insulin may play an important role in the pathogenesis of hypertension by stimulating the proliferation of vascular smooth muscle cells (VSMCs). Besides of the artery, the kidney plays a role in the regulation of blood pressure by affecting sodium reabsorption, Among all segments, the effect of renal proximal tubule (RPT) is evident, which takes charge of 66% renal sodium reabsorption.
It’s known that sympathetic system takes an important position in the pathogenesis of essential hypertension. Dopamine is an endogenous catecholamine that regulates/modu- lates many cellular functions, including effects on renal hemodynamics, ion and water transport and by regulation of hormones and humoral agent release, such as aldosterone, catecholamine, endothelin, prolactin, proopiomelanocortin, renin, and vasopressin. In addition, dopamine can control blood pressure by acting on cardiovascular centers, heart, and arterial and venous vessels. Dopamine exerts its actions by occupation of the D1-like (D1 and D5) and D2-like (D2, D3, and D4) receptors. The natriuretic and diuretic effects of dopamine receptors are confirmed in receptor null mice. In the hypertensive states, the dopamine receptors mediated-natriuresis and diuresis are impaired. Apart from dopamine, the renal natriuresis and diuresis are also regulated by other humoral factors, such as sympathetic nervous system (including dopamine), endothelin, renin-angiotensin system. According to the effects on sodium excretion, those humoral factors are divided into two subfamilies, one family enhances; while the other inhibits sodium excretion. The interaction of those two subfamilies of humoral factors keeps sodium excretion and blood pressure in normal extent.
Our previous study showed that, although D1 or D3 receptor had no effect on VSMC proliferation, it reduced norepinephrine or insulin-mediated VSMCs proliferation, stimulation of D1 or D3 receptor decreases insulin receptor expression, As a major subtype of D2-like receptors, D4 receptor exists in the heart, renal afferent artery, efferent artery, pulmonary artery, mesenteric artery, Whether D4 receptor has some effect on insulin receptor is not known, we hypothesized that D4 receptor may have an inhibitory effect on insulin receptor expression and function.
We also found that D3 receptor agonist (PD128907) induced natriuresis in WKY rats on normal or high NaCl diet, An ETB receptor antagonist (BQ788), which, by itself, did not have a significant effect on sodium excretion, partially blocked the natriuretic effect of the D3 receptor agonist; Activation of D4 receptor affected ETB, AT1, insulin receptror expression in RPT cells. Based on those evidences, we hypothesize that D3 receptors, may have effects on ETB receptor expression and function in RPT cells, the impaired interaction might be involved into the pathogenesis of essential hypertension. Uncover of these issues will be helpful to elucidate the renal and arterial receptors interactions, to provide therapeutic target for normalization of the impaired receptor interaction in hypertensive states.
Objective
To uncover the interactions between dopamine receptor and other receptors (ETB receptor, insulin receptor), to explore the role of impaired interaction in essential hypertension.
Methods
1. Effects of D4 receptor on vascular insulin receptor expression and function and its mechanisms by cell number counting, uptake of MTT, reverse transcription-PCR, immunoblotting, and immunohistochemistry .
2. Effects of D3 receptor on renal ETB receptor expression and function, its mechanisms by immunoblotting and measuring Na+-K+-ATPase activity.
3. The interaction between D4 receptor and insulin receptor, D3 receptor and ETB receptor were determined by laser confocal microscopy and co-immunoprecipitation.
Results
1. D4 receptor, by itself, had no effect on VSMC proliferation. However, it could reduce insulin-mediated VSMC proliferation in A10 cells. The inhibitory effect of D4 receptor still existed in spontaneously hypertensive rats (SHRs).
2. D4 receptor agonist, PD168077, decreased insulin receptor expression in a concentration-dependent and time-dependent manner, the inhibitory effect was via PKA. When actinomycin D was used to block the synthesis of new RNA, we found that D4 receptor increased insulin receptor degradation, which might be involved into the mechanism of D4 inhibitory of insulin receptor expression.
3. There was co-localization and co-immunoprecipitation between D4 and insulin receptors, stimulation of D4 receptor decreased the linkage between D4 and insulin receptors.
4. In WKY RPT cells, the D3 receptor agonist, PD128907, increased ETB receptor protein expression in a concentration-dependent and time-dependent manner, the effect was blocked in the presence of nicardipine. In contrast, in SHR RPT cells, PD128907, decreased ETB receptor protein expression.
5. Basal D3/ETB receptor co-immunoprecipitation was greater in WKY than SHRs, The absolute amount of D3/ETB receptor co-immunoprecipitation induced by D3 receptor agonist was also greater in WKY than SHRs.
6. Stimulation of ETB receptor decreased Na+-K+-ATPase activity in WKY but not in SHR cells. Pretreatment with PD128907 augmented the inhibitory effect of BQ3020 on Na+-K+-ATPase activity in WKY but not in SHR cells.
Conclusions
Dopamine receptor can affected vascular insulin receptor, renal ETB receptor expression and founction. Activation of D4 receptor inhibits insulin receptor expression and insulin-mediated VSMC proliferation both in Wistar-Kyoto and SHRs, which might be a new target to reduce the effects of insulin resistance on artery in hypertensive states. D3 receptor regulate ETB receptor by physical receptor interaction and govern receptor expression and founction, D3 receptor regulation of ETB receptor is aberrant in RPT cells from SHRs.
引文
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