C型钠尿肽对心房机械活动的影响及其机制探讨
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摘要
心脏作为内分泌腺,生成和分泌钠尿肽激素。钠尿肽激素以钠尿肽家族(family of natriuretic peptides, NPs)形式存在于人和哺乳动物体内,其成员有ANP (atrial natriuretic peptide, ANP)、BNP (brain natriuretic peptide, BNP)、CNP (C-type natriuretic peptide, CNP)和DNP (dendroaspis natriuretic peptide, DNP).这类激素共同的生物学作用是排钠利尿,参与水盐平衡和血压调节。
C-型钠尿肽(C-type natriuretic peptide, CNP)是1990年Sudoh等发现的钠尿肽家族的第三个成员。其结构和生理学功能类似于ANP和BNP,但它有独到之处。CNP最早在中枢神经系统中发现,并认为是参与调节血压和水盐平衡的神经肽,但后来逐渐发现亦广泛存在于人和动物的肾、肠、肺和心血管等器官系统中,表明CNP可能由多个器官合成和释放。CNP以自分泌／旁分泌方式通过多种途径发挥广泛的生物学效应,如可扩张血管,抑制平滑肌细胞增殖、迁移及细胞外基质形成,抑制血管内膜及心肌细胞增殖,减少动脉硬化及斑块的形成,抑制重塑,抑制炎症反应等,但对各自作用及效果尚存在争议。
现有的研究表明,CNP对心脏具有负性肌力或正性肌力作用或双重作用,并受多种因素的影响,但其作用及机制尚不清楚。因此,本研究采用离体搏动的心房灌流模型,利用实时评价环核苷酸代谢方法及反转录聚合酶链式反应(RT-PCR)法观察和探讨CNP对心房机械活动的影响及其作用机制,为探讨钠尿肽激素对心脏功能的调节作用以及临床诊治相应疾病提供理论基础。
本研究共分四个部分,其结果如下：
1.C型钠尿肽对家兔心房机械活动的影响
①不同浓度的CNP(10.0 nmol·L-1、30.0 nmol·L-1和300.0 nmol·L-1)明显降低心房输出量和心房搏动压(与对照循环比较,P<0.05和P<0.01),并呈现浓度依赖性特征。
②CNP(30.0 nmol·L-1)显著增加cGMP含量(与对照循环比较P<0.01)的同时明显降低心房搏出量和搏动压(与对照循环比较,均P<0.01),但cAMP含量则无明显变化(与对照循环比较P>0.05)。
③B型钠尿肽受体(NPR-B)阻断剂HS-142-1(400.0μg·mL-1)显著抑制CNP诱导的心房肌细胞cGMP的生成(与对照循环比较P<0.001)。
2.磷酸二酯酶亚类型对CNP诱导家兔心房机械活动的影响
①磷酸二酯酶(phosphodiesterase, PDE)非选择性抑制剂IBMX(1.0 mmol·L-1)显著增加心房搏出量、心房搏动压、cAMP及cGMP的含量(与对照循环比较,均P<0.001)。在IBMX存在下再处理CNP(30.0 nmol·L-1)时,心房搏出量、搏动压和cAMP及cGMP含量仍高于对照循环水平(与对照循环比较,均P<0.001),但与IBMX的第2循环比较则无显著变化(均P>0.05)。
②PDE2选择性抑制剂EHNA(0.1mmol·L-1)明显降低心房搏出量和心房搏动压(与对照循环比较,均P<0.05),但cAMP含量显著增加(与对照循环比较P<0.05)。在EHNA的存在下再处理CNP(30.0 nmol·L-1)时,心房搏出量和搏动压进一步降低(与EHNA的第3循环比较,均P<0.01),但cAMP含量进一步增多(与EHNA的第3循环比较,均P<0.05)。另外,与单独处理CNP时比较,心房搏出量及搏动压的抑制性效果有减缓趋势。
③PDE3选择性抑制剂milrinone(10.0μmol·L-1)明显增加心房肌组织cAMP含量(与对照循环比较P<0.05),而心房搏出量、搏动压及cGMP含量则无显著变化(与对照循环比较,均P>0.05)；在milrinone存在下同时处理CNP(30.0nmol·L-1)时,心房肌组织cGMP含量明显增加(P<0.001),但未能改变milrinone诱导cAMP含量增多的效应。心房搏出量和搏动压在milrinone作用下有所增加,但无显著差异(与对照循环比较,均P>0.05)：而在CNP的作用下,心房搏出量和搏动压明显受到抑制(均P<0.001)。
3. Forskolin对CNP诱导家兔心房机械活动的影响
①腺苷酸环化酶直接激活剂forskolin(0.1μmol·L-1)逆转CNP(30.0 nmol·L-1)对心房搏出量及搏动压的抑制效应,并使CNP通过进一步增加心房cAMP含量发挥正性肌力作用。
②Milrinone(10.0μmol·L-1)可阻断forskolin逆转CNP对心房搏出量及搏动压的抑制而发挥正性肌力作用的效应,并促使CNP恢复其对心房的抑制性调节作用；但EHNA(0.1mmol·L-1)则无此效应。
③EHNA(0.1mmol·L-1)和milrinone(10.0μmol·L-1)同时起作用时,不仅可阻断forskolin逆转CNP对心房搏出量及搏动压的抑制而发挥正性肌力作用的效应,也可阻止forskolin作用下milrinone恢复CNP对心房机械活动的抑制性调节作用。4. Forskolin对CNP诱导大鼠心房搏动压及其细胞PDE3A基因表达的影响
①CNP(0.5μmol·L-1)明显降低心房搏动压(与对照循环比较P<0.01)。
②Forskolin(7.0μmol·L-1)可逆转CNP对心房搏动压的抑制效应,并使其发挥正性肌力作用。
③CNP(0.5μmol·L-1)明显降低心房肌细胞PDE3A的基因表达(与对照组比较P<0.001)；forskolin(7.0μmol·L-1)显著增加心房肌细胞PDE3A的基因表达(与对照组比较P<0.01)；与forskolin组比较,forskolin+CNP明显降低心房肌细胞PDE3A基因表达(P<0.01)。
以上结果提示：
1.CNP对家兔心房具有负性肌力效应,其作用是通过CNP-GC-cGMP信号转导途径实现的。
2.PDE不同亚类型参与调节CNP对家兔心房机械活动的作用。
3.腺苷酸环化酶直接激活剂forskolin可逆转CNP对家兔心房的负性肌力作用,使其发挥正性肌力效应,其作用与CNP-GC-cGMP-PDE3A信号转导途径有关。
As an endocrine gland, the heart synthesis and secretion of natriureitc peptides (NPs) include atrial natriureitc peptide (ANP), brain ntriuretic peptide (BNP), C-type natriuretic peptide (CNP) and dendroaspis natriuretic peptide (DNP). These hormones elicit natriuretic, diuretic, hypotensive effects, regulate body fluid and blood pressure.
CNP, a third member of the NPs, was first isolated from the porcine brain and recognized that it was a neuropeptide, and late, it was demonstrated that the distribution of CNP was wide and immunoreactivity has been found in kidney, intestines, lung, and cardiovascular system. Therefore, it was indicated that several tissues could synthesis and release of CNP. Because the plasma levels of CNP are very low, the presence of the peptide in tissues is considered to perform a paraceine/autocrine function as a local regulator. CNP has been shown to produce a variety of effects including vascular dilation, antiproliferative and antimigative effects on vascular smooth muscle cells, reduce to form of arteriosclerosis and plaque, and inhibition of remodeling and inflammatory reaction.
Recently, a regulatory function of CNP has been recognized in the hart, i. e., CNP-induced negative and/or positive inotropic effects in the heart. However, the effects and the mechanism of the CNP on the heart are not well known. Therefore, the purpose of the present study was to define the effects of CNP on atrial dynamics and regulatory roles of intracellular cGMP and cAMP levels in perfused beating atria.
The results of the present study were shown that:
1. Effect of CNP on atrial dynamics
1) Different doses of CNP (10.0 nmol/L,30.0 nmol/L and 300.0 nmol/L) significantly inhibited atrial stroke volume and pulse pressure (P＜0.05 and P＜0.01 vs control) in dose dependent manner.
2) CNP (30.0 nmol/L) increased atrial cGMP production (P＜0.001 vs control) and decreased atrial stroke volume and pulse pressure (both P＜0.01 vs control) without changes in atrial cAMP levels (P>0.05 vs control).
3) CNP-induced increase of atrial cGMP was blocked by HS-142-1, an inhibitor of NPR-B receptor.
2. Effects of subtype phosphodiesterase on CNP-induced atrial dynamics
1) 3-Isobutyl-l-methylxanthine (IBMX,1.0 mmol/L), a non-specific inhibitor of phosphodiesterses (PDEs) significantly increased atrial stroke volume, pulse pressure, cAMP and cGMP levels (all P＜0.001 vs Control). In the presence of IBMX, CNP (30.0 nmol/L) failed to modulation of IBMX-induced increase of atrial dynamics and cyclic nucleotides (P＜0.001 vs control and P＞0.05 vs the period of second cycle treated with IBMX alone).
2) [erythro-9-(2-Hydroxy-3-nonyl) adenine-HCl] (EHNA,0.1 mmol/L), a specific inhibitor of PDE2, decreased atrial dynamics (P＜0.05 vs control) with increased atrial cAMP levels (P<0.05 vs control). In the presence of EHNA, CNP (30.0 nmol/L) augmented the effects of EHNA-induced decrease of atrial dynamics (P＜0.01 vs the period of third cycle treated with EHNA alone) and the EHNA-induced increase of atrial cAMP levels (P＜0.05 vs the period of third cycle treated with EHNA alone). However, EHNA+CNP attenuated inhibitory effect of CNP the atrial dynamics.
3) Milrinone (10.0μmol/L), a specific inhibtor of PDE3, significantly increased atrial cAMP levels (P<0.05 vs control) without changes in atrial dynamics and cGMP levels. In the presence of milrinone, CNP (30.0 nmol/L) significantly decreased atrial dynamics (P＜0.001 vs control) with increased cGMP levels (P＜0.001 vs control).
3. Effects of forskolin on CNP-induced atrial dynamics
1) Forkolin (0.1μmol/L), an activator of adenylyl cyclase (AC), reversed the inhibitory effects of CNP (30.0 nmol/L) on atrial dynamics and sacrificed the CNP play a positive inotropism via an augmented increase in atrial cAMP levels.
2) In the presence of forskolin, the augmented increase of atrial dynamics and c AMP levels by CNP were blockade by combined treatment with mirinone, an inhibitor of PDE3, and recovered the inhibitory effect of CNP on atrial dynamics again.
3) In the presence of forskolin and milrinone, the augmented increase of atrial dynamics and cAMP levels by CNP were blockade by combined treatment with EHNA (0.1 mmol/L), and the inhibitory effect of CNP on atrial dynamics also attenuated by EHNA.
1) CNP (0.5μmol/L) also inhibited rat atrial dynamics.
2) Forskolin (7.0μmol/L) also reversed inhibitory effect of CNP on rat atrial dynamics and then CNP augmented rat altrial dynamics.
3) PDE3A gene expression was decreased by CNP and increased by forskolin respectively. In the presence of forskolin, the PDE3A gene expression was also decreased by combined treatment of CNP.
The results of the present study indicate that:
1. CNP inhibits atrial dynamics via GC-cGMP signaling in beating rabbit atria.
2. Subtype of PDEs show the distinct roles in regulation of atrial dynamics with CNP.
3. In the presence and absence of forskolin, CNP can play positive and/or negative inotropic effects on atrial dynamics via GC-cGMP-PDE signaling pathway.
C-型钠尿肽(C-type natriuretic peptide, CNP)是1990年Sudoh等发现的钠尿肽家族的第三个成员。其结构和生理学功能类似于ANP和BNP,但它有独到之处。CNP最早在中枢神经系统中发现,并认为是参与调节血压和水盐平衡的神经肽,但后来逐渐发现亦广泛存在于人和动物的肾、肠、肺和心血管等器官系统中,表明CNP可能由多个器官合成和释放。CNP以自分泌／旁分泌方式通过多种途径发挥广泛的生物学效应,如可扩张血管,抑制平滑肌细胞增殖、迁移及细胞外基质形成,抑制血管内膜及心肌细胞增殖,减少动脉硬化及斑块的形成,抑制重塑,抑制炎症反应等,但对各自作用及效果尚存在争议。
现有的研究表明,CNP对心脏具有负性肌力或正性肌力作用或双重作用,并受多种因素的影响,但其作用及机制尚不清楚。因此,本研究采用离体搏动的心房灌流模型,利用实时评价环核苷酸代谢方法及反转录聚合酶链式反应(RT-PCR)法观察和探讨CNP对心房机械活动的影响及其作用机制,为探讨钠尿肽激素对心脏功能的调节作用以及临床诊治相应疾病提供理论基础。
本研究共分四个部分,其结果如下：
1.C型钠尿肽对家兔心房机械活动的影响
①不同浓度的CNP(10.0 nmol·L-1、30.0 nmol·L-1和300.0 nmol·L-1)明显降低心房输出量和心房搏动压(与对照循环比较,P<0.05和P<0.01),并呈现浓度依赖性特征。
②CNP(30.0 nmol·L-1)显著增加cGMP含量(与对照循环比较P<0.01)的同时明显降低心房搏出量和搏动压(与对照循环比较,均P<0.01),但cAMP含量则无明显变化(与对照循环比较P>0.05)。
③B型钠尿肽受体(NPR-B)阻断剂HS-142-1(400.0μg·mL-1)显著抑制CNP诱导的心房肌细胞cGMP的生成(与对照循环比较P<0.001)。
2.磷酸二酯酶亚类型对CNP诱导家兔心房机械活动的影响
①磷酸二酯酶(phosphodiesterase, PDE)非选择性抑制剂IBMX(1.0 mmol·L-1)显著增加心房搏出量、心房搏动压、cAMP及cGMP的含量(与对照循环比较,均P<0.001)。在IBMX存在下再处理CNP(30.0 nmol·L-1)时,心房搏出量、搏动压和cAMP及cGMP含量仍高于对照循环水平(与对照循环比较,均P<0.001),但与IBMX的第2循环比较则无显著变化(均P>0.05)。
②PDE2选择性抑制剂EHNA(0.1mmol·L-1)明显降低心房搏出量和心房搏动压(与对照循环比较,均P<0.05),但cAMP含量显著增加(与对照循环比较P<0.05)。在EHNA的存在下再处理CNP(30.0 nmol·L-1)时,心房搏出量和搏动压进一步降低(与EHNA的第3循环比较,均P<0.01),但cAMP含量进一步增多(与EHNA的第3循环比较,均P<0.05)。另外,与单独处理CNP时比较,心房搏出量及搏动压的抑制性效果有减缓趋势。
③PDE3选择性抑制剂milrinone(10.0μmol·L-1)明显增加心房肌组织cAMP含量(与对照循环比较P<0.05),而心房搏出量、搏动压及cGMP含量则无显著变化(与对照循环比较,均P>0.05)；在milrinone存在下同时处理CNP(30.0nmol·L-1)时,心房肌组织cGMP含量明显增加(P<0.001),但未能改变milrinone诱导cAMP含量增多的效应。心房搏出量和搏动压在milrinone作用下有所增加,但无显著差异(与对照循环比较,均P>0.05)：而在CNP的作用下,心房搏出量和搏动压明显受到抑制(均P<0.001)。
3. Forskolin对CNP诱导家兔心房机械活动的影响
①腺苷酸环化酶直接激活剂forskolin(0.1μmol·L-1)逆转CNP(30.0 nmol·L-1)对心房搏出量及搏动压的抑制效应,并使CNP通过进一步增加心房cAMP含量发挥正性肌力作用。
②Milrinone(10.0μmol·L-1)可阻断forskolin逆转CNP对心房搏出量及搏动压的抑制而发挥正性肌力作用的效应,并促使CNP恢复其对心房的抑制性调节作用；但EHNA(0.1mmol·L-1)则无此效应。
③EHNA(0.1mmol·L-1)和milrinone(10.0μmol·L-1)同时起作用时,不仅可阻断forskolin逆转CNP对心房搏出量及搏动压的抑制而发挥正性肌力作用的效应,也可阻止forskolin作用下milrinone恢复CNP对心房机械活动的抑制性调节作用。4. Forskolin对CNP诱导大鼠心房搏动压及其细胞PDE3A基因表达的影响
①CNP(0.5μmol·L-1)明显降低心房搏动压(与对照循环比较P<0.01)。
②Forskolin(7.0μmol·L-1)可逆转CNP对心房搏动压的抑制效应,并使其发挥正性肌力作用。
③CNP(0.5μmol·L-1)明显降低心房肌细胞PDE3A的基因表达(与对照组比较P<0.001)；forskolin(7.0μmol·L-1)显著增加心房肌细胞PDE3A的基因表达(与对照组比较P<0.01)；与forskolin组比较,forskolin+CNP明显降低心房肌细胞PDE3A基因表达(P<0.01)。
以上结果提示：
1.CNP对家兔心房具有负性肌力效应,其作用是通过CNP-GC-cGMP信号转导途径实现的。
2.PDE不同亚类型参与调节CNP对家兔心房机械活动的作用。
3.腺苷酸环化酶直接激活剂forskolin可逆转CNP对家兔心房的负性肌力作用,使其发挥正性肌力效应,其作用与CNP-GC-cGMP-PDE3A信号转导途径有关。
As an endocrine gland, the heart synthesis and secretion of natriureitc peptides (NPs) include atrial natriureitc peptide (ANP), brain ntriuretic peptide (BNP), C-type natriuretic peptide (CNP) and dendroaspis natriuretic peptide (DNP). These hormones elicit natriuretic, diuretic, hypotensive effects, regulate body fluid and blood pressure.
CNP, a third member of the NPs, was first isolated from the porcine brain and recognized that it was a neuropeptide, and late, it was demonstrated that the distribution of CNP was wide and immunoreactivity has been found in kidney, intestines, lung, and cardiovascular system. Therefore, it was indicated that several tissues could synthesis and release of CNP. Because the plasma levels of CNP are very low, the presence of the peptide in tissues is considered to perform a paraceine/autocrine function as a local regulator. CNP has been shown to produce a variety of effects including vascular dilation, antiproliferative and antimigative effects on vascular smooth muscle cells, reduce to form of arteriosclerosis and plaque, and inhibition of remodeling and inflammatory reaction.
Recently, a regulatory function of CNP has been recognized in the hart, i. e., CNP-induced negative and/or positive inotropic effects in the heart. However, the effects and the mechanism of the CNP on the heart are not well known. Therefore, the purpose of the present study was to define the effects of CNP on atrial dynamics and regulatory roles of intracellular cGMP and cAMP levels in perfused beating atria.
The results of the present study were shown that:
1. Effect of CNP on atrial dynamics
1) Different doses of CNP (10.0 nmol/L,30.0 nmol/L and 300.0 nmol/L) significantly inhibited atrial stroke volume and pulse pressure (P＜0.05 and P＜0.01 vs control) in dose dependent manner.
2) CNP (30.0 nmol/L) increased atrial cGMP production (P＜0.001 vs control) and decreased atrial stroke volume and pulse pressure (both P＜0.01 vs control) without changes in atrial cAMP levels (P>0.05 vs control).
3) CNP-induced increase of atrial cGMP was blocked by HS-142-1, an inhibitor of NPR-B receptor.
2. Effects of subtype phosphodiesterase on CNP-induced atrial dynamics
1) 3-Isobutyl-l-methylxanthine (IBMX,1.0 mmol/L), a non-specific inhibitor of phosphodiesterses (PDEs) significantly increased atrial stroke volume, pulse pressure, cAMP and cGMP levels (all P＜0.001 vs Control). In the presence of IBMX, CNP (30.0 nmol/L) failed to modulation of IBMX-induced increase of atrial dynamics and cyclic nucleotides (P＜0.001 vs control and P＞0.05 vs the period of second cycle treated with IBMX alone).
2) [erythro-9-(2-Hydroxy-3-nonyl) adenine-HCl] (EHNA,0.1 mmol/L), a specific inhibitor of PDE2, decreased atrial dynamics (P＜0.05 vs control) with increased atrial cAMP levels (P<0.05 vs control). In the presence of EHNA, CNP (30.0 nmol/L) augmented the effects of EHNA-induced decrease of atrial dynamics (P＜0.01 vs the period of third cycle treated with EHNA alone) and the EHNA-induced increase of atrial cAMP levels (P＜0.05 vs the period of third cycle treated with EHNA alone). However, EHNA+CNP attenuated inhibitory effect of CNP the atrial dynamics.
3) Milrinone (10.0μmol/L), a specific inhibtor of PDE3, significantly increased atrial cAMP levels (P<0.05 vs control) without changes in atrial dynamics and cGMP levels. In the presence of milrinone, CNP (30.0 nmol/L) significantly decreased atrial dynamics (P＜0.001 vs control) with increased cGMP levels (P＜0.001 vs control).
3. Effects of forskolin on CNP-induced atrial dynamics
1) Forkolin (0.1μmol/L), an activator of adenylyl cyclase (AC), reversed the inhibitory effects of CNP (30.0 nmol/L) on atrial dynamics and sacrificed the CNP play a positive inotropism via an augmented increase in atrial cAMP levels.
2) In the presence of forskolin, the augmented increase of atrial dynamics and c AMP levels by CNP were blockade by combined treatment with mirinone, an inhibitor of PDE3, and recovered the inhibitory effect of CNP on atrial dynamics again.
3) In the presence of forskolin and milrinone, the augmented increase of atrial dynamics and cAMP levels by CNP were blockade by combined treatment with EHNA (0.1 mmol/L), and the inhibitory effect of CNP on atrial dynamics also attenuated by EHNA.
1) CNP (0.5μmol/L) also inhibited rat atrial dynamics.
2) Forskolin (7.0μmol/L) also reversed inhibitory effect of CNP on rat atrial dynamics and then CNP augmented rat altrial dynamics.
3) PDE3A gene expression was decreased by CNP and increased by forskolin respectively. In the presence of forskolin, the PDE3A gene expression was also decreased by combined treatment of CNP.
The results of the present study indicate that:
1. CNP inhibits atrial dynamics via GC-cGMP signaling in beating rabbit atria.
2. Subtype of PDEs show the distinct roles in regulation of atrial dynamics with CNP.
3. In the presence and absence of forskolin, CNP can play positive and/or negative inotropic effects on atrial dynamics via GC-cGMP-PDE signaling pathway.
引文
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