【作者】 陶金；

【导师】 李胜男；

【作者基本信息】 南京医科大学 ， 药理学， 2007， 博士

【摘要】 背景促肾上腺皮质激素释放因子（CRF）属于一类结构相关的肽类家族，其中还包括尿紧张素，蛙皮降压肽，以及最近在哺乳动物中发现的尿紧张素类似肽urocortin 1-3（UCN1-3）。据报道，CRF肽类家族主要通过与相应的七个跨膜片段的G-蛋白偶联CRF受体（CRFR1及CRFR2）相结合，调节机体内分泌、自律性以及对应急的行为反应等生理功能。实验证明尿紧张素，蛙皮降压肽，以及UCN对CRFR2的结合比CRF高1000倍左右。UCN2和UCN3被认为仅仅是哺乳动物CRFR2的内在配体，而UCN1是CRFR1以及CRFR2的内在配体。UCN是40个氨基酸的小分子CRF类肽，与CRF有43％的结构相似性，参与哺乳动物应激反应。其最早在中枢神经系统内发现并作用，近年越来越多的研究表明UCN可以通过自分泌和／或旁分泌的方式与CRF受体相结合在各个系统发挥重要作用，并且它的这种作用在某些系统，如心血管，神经，生殖系统，比CRF更为显著。在体及离体实验研究发现，在缺血-再灌注损伤以及高血压等心血管病症中，UCN能保护心肌细胞免受损伤，起到心血管保护作用。此外，UCN剂量依赖性的增加主动脉和睾丸动脉的血流量，抑制食欲，维持胎盘功能，并能激活子宫肌肉收缩。报道表明，在男性生殖系统内也有UCN的表达，提示UCN可能通过影响精子的功能而跟着床和妊娠有关。此外，UCN2通过结合CRFR2在神经及心血管保护方面，比如，缺血再灌注，缺氧损伤过程中保护心肌，病理性抑制凋亡，保护神经细胞等。然而，也有报道显示，UCN2能激活不表达CRFR2的神经细胞，导致不依赖于CRFR2活性的神经元放电。这种CRF受体依赖的和非依赖的UCN的作用，其机制迄今为止尚不是很明确。文献显示，UCN能抑制缺血再灌注可能跟PKC／K_ATP有关，保护缺氧损伤跟PKA／MAPK有关，神经保护跟PKC及PLC信号通路相关，CRF受体非依赖性自由基及PKA／K_ATP信号通路在心肌缺氧保护中起一定作用。电压门控性钙通道对细胞内钙的调节起很重要的作用。细胞内钙离子浓度迅速升高能诱发神经递质释放，精子获能，肌肉收缩等。病理性的钙超载导致心肌重构，血管结构紊乱，细胞凋亡等。此外，在病理性心脏重构中，钙离子通道也表达异常。大量的文献报道显示，电压依赖性钙通道对于体内各个系统病理及生理调节起非常重要的作用。根据电生理和药理特性的不同，钙离子通道分为长时程型L钙通道，短时程型T通道，神经源性N型，浦肯野（氏）细胞P型，粒细胞型Q型，以及毒素耐受型R型。体内L型，T型，以及N型电压门控钙通道据报道能被与跨膜的G蛋白藕联的受体，以及下游的PKA，PKC，PLC以及钙调蛋白2型酶广泛调控，然而，对于CRF类肽，UCN及UCN2是否CRF受体依赖或非依赖的作用于钙离子通道，而发挥体内调节功能，目前领域研究还是个空白。目的1．研究促肾上腺皮质激素释放因子（CRF）家族类肽UCN及其受体在小鼠生殖系统中的表达和定位，阐述UCN在雄性小鼠中的生理功能，并探讨其与细胞内钙相关的发生机制；2．通过进一步研究UCN对小鼠生精细胞T型钙通道电流的影响，进一步阐述其在小鼠生殖功能中的作用；3．从亚细胞水平探讨CRFR1调节T型钙通道亚单位的信号通路，阐述其调节正常神经生殖内分泌功能的信号机制；4．探讨CRF家族类肽UCN2在神经系统内的表达并阐述其神经保护的离子机制；5．探讨CRF家族类肽UCN2的血管保护作用，阐述其抗高血压血管重构过程中与L-型钙通道、细胞内钙离子浓度相关的离子作用机制。方法1．UCN在雄性小鼠生殖系统中的表达及影响：逆转录PCR（RT-PCR）法及WESTERN BLOT检测小鼠生精细胞UCN，CRFR1及CRFR2的表达，免疫组织化学定位UCN及CRF受体的定位，检测UCN对小鼠精子功能及顶体反应的作用，流式细胞术检测细胞内钙在UCN生殖功能中的变化。2．UCN对雄性小鼠生殖功能影响的机制研究：分离小鼠生精细胞，采用全细胞膜片钳技术观察UCN对T型钙通道的影响。3．CRFR1激活对T通道亚单位的影响及机制：RT-PCR，Westernblotting，及激光共聚焦定位CRFR1在HEK293转染系统的表达。cAMP检测CRFR1的功能。全细胞膜片钳技术观察UCN及CRF对转染的3个T型钙通道的亚单位的影响并用药理学方法（使用PKA，PKC，PLC，CaMK，QEHA，及SKEE等阻断剂）阐明其信号通路作用机制。克隆CRFR1，全细胞膜片钳技术观察UCN及CRF对CRFR1共转染后的Cav3.2的影响。4．UCN2在神经细胞中的表达及其神经保护机制：RT-PCR检测PC12 UCN及CRF受体的表达，激光共聚焦检测UCN2对PC12细胞内钙浓度的影响，全细胞膜片钳技术检测UCN2对PC12细胞电流的影响，药理学方法（CRFR阻断剂，硝苯地平，P／Q通道毒素阻断剂等）观察UCN2的通道作用类型，全细胞膜片钳技术检测UCN2对急性分离大鼠神经细胞大脑皮层神经元的影响。5．UCN2高血压血管系统中的作用及其作用机制：分离高血压大鼠平滑肌细胞，TUNNEL检测UCN2对平滑肌凋亡的影响，激光共聚焦检测UCN2对SHR中VSMC细胞内钙浓度的影响，全细胞膜片钳技术检测UCN2对平滑肌细胞钙通道电流的影响，并观察UCN2对重组L型钙通道Cav1.2的影响。结果1．UCN及CRF受体在RNA水平和蛋白水平在小鼠睾丸组织有不同定位与表达：UCN在精子细胞上表达，CRFR1在精母细胞表达，而CRFR2在精原和精母细胞中都有表达，UCN的蛋白表达随顶体反应的进行而减少。2．UCN对雄性小鼠的生殖功能，如精子活动，顶体反应等，有抑制作用。3．UCN对男性生殖细胞细胞内钙的浓度有降低作用，并阻断生精细胞T型钙离子通道电流。4．UCN及CRF选择性阻断Cav3.2，对Cav3.1和Cav3.3没有影响。5．HEK293细胞内源性表达CRFR1，蛋白定位在细胞膜，并发挥激活腺苷酸环化酶的作用。6．UCN左移Cav3.2的失活曲线，对激活曲线却没有影响。7．阻断G蛋白作用能消除UCN对Cav3.2的作用，UCN对Cav3.2的抑制不受PKC，PKA及PLC的影响，却被QEHA所阻断。8．UCN2内源性表达于PC12细胞中，降低PC12细胞内钙，并选择性抑制L型钙电流，这种阻断不被CRFR阻断剂astressin消除，同时UCN2能抑制急性分离的大脑皮质神经细胞电压依赖性钙通道电流。9．UCN2抑制高血压平滑肌细胞凋亡，降低Bay K8644和KCL诱导的平滑肌细胞内钙浓度的增加，UCN2抑制平滑肌细胞L型钙电流，CRFR阻断剂astressin对UCN2的L型钙通道的阻断作用没有影响，UCN2对人工重组Cav1.2有相似于平滑肌凋亡抑制的作用。结论1．UCN及其受体内源性表达于小鼠睾丸组织，并抑制男性生殖功能。2．UCN激活G蛋白藕联的CRFR1能抑制T型钙通道电流产生。3．CRFR1的激活选择性抑制Cav3.2是通过诱导beta-gamma复合体的产生。4．HEK293表达系统能功能性的内源产生CRFR1，可以提供一种基于CRFR1表达的转染模式。5．UCN2能通过非依赖于CRF受体而发挥神经保护作用，这种保护作用是非依赖于CRFR的作用于L型钙离子通道。6．UCN2的非依赖于CRF受体的L型阻断功能，可以阐述其抗高血压血管重构的基本机制。更多还原

【Abstract】 BACKGROUNDCorticotropin-releasing factor （CRF） belongs to a family of structurally relatedpeptides that includes fish urotensin 1, amphibian sauvagine, and the recentlyidentified urotensin homologue, urocortins, which includes urocortin 1-3. CRF familyhas two receptors, CRFR1 and CRFR2. Urotensin 1, sauvagine, and urocortin 1-3bind with up to 1000-fold higher affinities to the CRFR2 than species homologues ofCRF. UCN2 and UCN3 are generally considered to represent endogenous ligands formammalian CRFR2 variants, whereas UCN is thought to be an endogenous ligand forboth CRFR1 and CRFR2. UCN shares 43％homology with CRF at the amino acidlevel. Previous reports showed that UCN was involved in the modulation of a varietyof biological activities, such as causing a dose-dependent increase in coronary,testicular artery blood flow suppression of appetite, and maintenance of the placentalfunction & labor. CRF was observed to have modulating effects on male reproductionearly in late 80’s. It was reported that UCN could activate the myometrial contractility.In addition, presence of UCN in the seminal fluid could be of relevance duringfertilization and pregnancy by influencing sperm transport through the female genitaltract. Therefore, these findings suggest that the CRF family, including UCN, CRF, andits receptors, may play important roles in various biological functions during the course of reproduction, which is highly associated with calcium channels. Previousreports also have showed that urocortin 2 had a number of physiological properties byinteracting with its CRFR2. However, urocortin 2 could also cause neural activationin cell groups that are involved in autonomic activation, but do not express either typeof CRF receptor. However, to date, the exact mechanisms for UCNs’ effects viaCRFR-dependent or independent manner are not very clear. It has been demonstratedthat UCNs, espcically UCN and UCN2 protected neonatal cardiac myocytes in vitrowhen administered before hypoxia/ischemia, which suggests that calcium channelsmay play some roles since ischemia and hypoxia damage is highly associated withCa²⁺-overload. Furthermore, it was reported that UCN2 played the cardiovascularprotective role via protein kinase A （PKA）, PKC, PLC, and mitogen-activated proteinkinase （MAPK） pathway, which might lead to changes in the intracellular Ca²⁺, anddownstream regulation of the calcium channels.Voltage-gated calcium channels （VGCCs） first described in excitable cells aregenerally classified according to their electrophysiological, pharmacological andinactivation properties as T-type, long-lasting L-type, neuronal N-type, purkinje cellP-type, granular cell Q-type and toxin/drug-resistant R-type. So far, however, fewreports addressed UCNs’ effects on VGCC. In addition, whether the effecs are CRFreceptor dependent or independent is still unknown.PURPOSE1. The aim of this study was to investigate the expression of UCN & its receptors and the effects of UCN on male reproductive functions, and identify the mechanismsof these effects relevant to intracellular calcium.2. The second goal is to investigate the effects of UCN on T-type calcium channels,and explore the mechanisms of UCN’s role in male reproductive functions,especially in acrosome reaction （AR） and sperm motility.3. Thirdly, we aim to clarify the subcellular signaling pathway between CRFR1 andthe three isoforms of T-type calcium channels, and explore the mechanisms forneuroendrine systems.4. Another goal of this study is to observe the expressions of UCN2 in neural system,and the ion mechnisms for UCN2’s neuroprotective effects.5. The last aim of this study is to explore the vascular protection of UCN2, charify theinhibition of UCN2 on vascular remodeling, and investigate the ion mechanismsrelevant to L-type calcium channels.METHODS1. Separate locations of UCN and its receptors in mouse testis and functions of UCNin male reproduction and the relevant mechanisms: Preparation of spermatozoaand spermatogenic cells, Reverse transcription-polymerase chain reaction（RT-PCR）, Immunohistochemical staining, Double immunostaining for UCN,Measurement of sperm motility, Assay for acrosome reaction （AR）, Determinationof [Ca²⁺]_i by flow cytometry2. Mechanisms of UCN’s effects on male reproductive in mice: Spermatogenic cell preparation and electrophysiology3. Activation of CRFR1 selectively inhibits Ca_v3.2 calcium channels: RT-PCR,Western, and Confocal Microscopy detect the expression of CRFR1 in HEK293,cAMP detection, Whole-cell patch clamp, Pharmacological methods （PKA, PKC,PLC, CaMK, QEHA, and SKEE etc.）, Construct rat CRFR14. Expression of UCN2 and mechanisms of its neuroprotection: Cell culture forrat undifferentiated pheochromocytoma （PC12） cells, Acute isolation of cerebralcortex neurons, Reverse transcription-polymerase chain reaction （RT-PCR）,Measurements of [Ca²⁺]_i by confocal laser scanning microscopy, Whole-CellPatch Clamp Recording5. Effects of UCN2 on vascular remodeling and the relevant mechanisms: Isolationof mesenteric arteries smooth muscle cells （MASMC） from SHR, TUNEL assayto detect the apoptosis of VSMC in SHR, Measurement of nitrite oxide （NO）,Intracellular Ca²⁺ concentration ([Ca²⁺]_i) measurements, Transient expression ofhuman Cav1.2 in HEK293 Cells, Whole-cell patch clamp recordingRESULTS1. UCN expressed only in mature sperm while CRFR1 and CRFR2 existed inspermatocytes and spermatogonia, respectively. With AR proceeding, theexpression of UCN declined, and after ascrosome reaction, UCN could not bedetected any more.2. UCN significantly inhibited the motility of the sperm and AR in a concentration-dependent manner.3. UCN reduced the [Ca²⁺]_i increase induced by high potassium and T-type calciumcurrents.4. Activation of CRFR1 selectively inhibited Cav3.2, instead of Cav3.1 and Cav3.3.5. HEK293 cells endogenously expressed functional CRFR1. The CRFR1 waslocalized in cell membrane, and activated the increase of cAMP.6. CRFR1 activation affected Cav3.2 channels by negatively shifting steady-stateinactivation properties.7. UCN inhibited Ca_v3.2 T-type calcium channels independent of PKA, PKC andPLC. UCN inhibited Ca_v3.2 T-type calcium channels via G_βγ.8. UCN2 reduced the [Ca²⁺]_i levels via inhibiting L-type VGCC in undifferentiatedPC12 cells, where both UCN2 and CRFR2β, but not CRFR1 or CRFR2α, wereexpressed.9. UCN2 reduced [Ca²⁺]_i, inhibited MASMC apoptosis induced by hypoxia in SHR,and blocked Cav1.2.CONCLUSIONS1. UCN and its recepotrs endogenously express in mouse testis. UCN inhibits themale reproductive functions.2. Activation of CRFR1 inhibits T-type calcium channels, which may be responsiblefor its male reproductive inhibition.3. Activation of CRFR1 inhibits Ca_v3.2 T-type calcium channels via G_βγ subunits. 4. HEK 293 cells endogenously express functional CRFR1, which may be used as anew transfection model for CRFR1.5. We provides evidence that UCN2, co-expressed with CRFR2βin undifferentiatedPC12 cells, decreases [Ca²⁺]_i levels via inhibiting the L-type VGCC. It may be apotential endogenous protective agent under some pathophysiological conditions,such as brain ischemia and some neuronal degenerative diseases like Alzheimer’sand Parkinson’s diseases, in which Ca²⁺-overload plays very important roles.6. Our results provide convincing evidence of a possible link between theapoptosis-inhibitory effects of UCN2 and L-type calcium channels in SHR.Based on the theory that VSMC apoptosis plays a key role in hypertensivevasculopathy, these findings highly imply that UCN2 might be a beneficial agentin vascular pathology where Ca²⁺-overload plays an important role.更多还原