细胞外液酸碱度改变对自发性高血压大鼠脑动脉平滑肌细胞电生理特性的影响

目的:探讨细胞外液酸碱度(pHo)的改变对自发性高血压大鼠(SHR)脑动脉平滑肌细胞电生理特性的影响。方法:取200~250 g自发性高血压大鼠,应用全细胞膜片钳记录技术观察细胞外液酸碱度改变后对SHR脑动脉平滑肌细胞膜电流的作用,进一步揭示其离子机制。结果:①pHo酸化可电压依赖性的抑制SHR脑动脉平滑肌细胞的外向电流。其主要抑制SHR脑动脉平滑肌细胞0~+60 mV区间的电流幅度;②1 mmol/L TEA可以有效抑制pHo酸化对脑动脉平滑肌细胞外向电流的抑制作用。结论:pHo的改变引起SHR脑动脉平滑肌细胞外向电流变化,其可能与电压依赖性的抑制SHR脑动脉平滑肌细胞BKCa通道电流有关。     

豚鼠不同部位微动脉平滑肌细胞电生理学特性的比较

本研究应用电生理技术在豚鼠离体小脑前下动脉(anterior inferior cerebellar artery,AICA)、肠系膜动脉(mesenteric artery,MA)和耳蜗螺旋动脉(spiral modiolar artery,SMA)分支(直径小于100μm)上比较微动脉平滑肌细胞电生理学特性的异同。结果显示:(1)应用细胞内微电极记录技术测得AICA、MA和SMA细胞静息膜电位分别为(-68±1.8)(n=65)、(-71±2.4)(n=80)和(-66±2.9)mV(n=58),各微动脉间无统计学差异。(2)一段血管微动脉标本全细胞膜片钳记录的平滑肌细胞膜电容和膜电导都远大于单个细胞标本,且微动脉间存在统计学差异,大小顺序为MAAICASMA。应用缝隙连接阻断剂2-APB(100μmol/L)后记录一段微动脉平滑肌细胞膜电容和膜电导与单个细胞十分接近。(3)AICA、MA和SMA单个平滑肌细胞膜电流I/V关系呈明显的外向整流特性,都对1mmol/L4-AP和10mmol/LTEA敏感。当指令电压为+40mV时,AICA、MA和SMA血管平滑肌细胞电流密度分别为(26±2.0)、(24±1.7)和(18±1.3)pA/pF,SMA和AICA、MA间存在统计学差异。上述结果提示,豚鼠不同部位微动脉平滑肌细胞在缝隙连接耦联力和电流密度等电生理特性存在差异。     

咖啡因对大鼠背根神经节急性分离神经元GABA-激活电流的抑制作用

应用全细胞膜片钳记录技术,在大鼠新鲜分离背根神经节(dorsal root ganglion,DRG)神经元上,观察预加咖啡因对GABA-激活电流(IGABA)的调制作用。实验中,大部分受检细胞(97．4％,l13／116)对外加GABA敏感。1-1000μmol／L GABA引起一剂量依赖性、有明显上敏感作用的内向电流。在受检的108个DRG细胞中,约有半数(53．7％,58／108)对胞外加咖啡因(0．1-100μmol／L)敏感．产生一幅值很小的内向电流。倾加咖啡因(0．1～100μmol／L)30s后再加GABA能明显抑制GABA(100μmol／L)激活电流的幅值。预加咖啡因后GABA量效曲线明显下移;GABA-激活电流的最人值较之对照下降约57％;而Kd值(30μmol／L)几乎不变,表示此种抑制为非竞争性的。预加安定(diazepam,1μmol／L)对GABA(100μmol／L)激活电流有增强作用,而预加咖啡因(10μmol／L)有拈抗安定增强IGABA的作用。胞内透析H-8后,几乎可以完全消除咖啡因对,IGABA的抑制作用。已知GABA作用于初级感觉神经元能引起初级传入去极化,因而实验结果提示,咖啡因有可能在初级传入末梢产生对抗突触前抑制的效应。     

GBE对大鼠急性分离海马神经元NMDA-激活电流的调制作用

目的:观察不同制型的银杏叶提取物(GBE)对N-甲基-D-天门冬氨酸(NMDA)受体激活电流的影响,并比较其作用。方法:应用全细胞膜片钳记录技术记录急性分离大鼠海马神经NMDA激活电流,比较加药前后电流幅度的变化。结果:大部分受检细胞(81.8%,90/110)对外加NMDA敏感,引起一去敏感的内向电流(INMDA)。此电流可被NMDA受体特异阻断剂(MK-801)所阻断。预加不同制型的GBE均能明显抑制NMDA激活电流(P<0.01),但制型不同抑制效应不一,GBE纳米制剂(nGBE)对INMDA的抑制作用明显优于微米型(mGBE组),抑制率分别为64%±15%,40%±17%(n=8),两组比较差异具有统计学意义(P<0.05)。结论:预加GBE能抑制NM-DA-激活电流,从而对抗海马神经元兴奋毒性脑损伤,起神经保护作用。nGBE对NMDA受体的调控作用优于mGBE制剂。     

铅对大鼠海马神经细胞H-电流特性的影响

应用全细胞膜片钳技术研究了铅对大鼠海马神经细胞H－电流特性的影响。结果表明：铅抑制了H－电流表达;降低了H－通道电压敏感性和升高了H－通道的激活电压。铅对H－电流的压抑效应有其对H－通道电压敏感性的改变,是铅损伤神经活动的一个因素之一。     

正常大鼠植入高血压大鼠肾脏后动脉血压的变化

本实验对12周龄的自发性高血压大鼠(spontaneouslyhypertensiverat,SHR)及其对照组WistarKyoto(WKY)大鼠进行了肾脏移植的研究,并观察受肾移植大鼠动脉血压的变化以及免疫抑制剂对动脉血压的影响。用尾套法对接受同窝另一同胞WKY大鼠肾脏移植且存活5周的6只WKY大鼠(A组)及接受SHR肾脏移植且存活5周的6只WKY大鼠(B组)的尾动脉收缩压进行检测,移植前A、B两组受肾移植大鼠的尾动脉收缩压分别为180±093和183±068kPa,无统计学显著差异(P>005);移植后3、4、5周时,B组大鼠的尾动脉收缩压显著高于A组大鼠,移植后5周时,A,B两组大鼠的收缩压分别为190±071和230±069kPa(P<0001);所用剂量的免疫抑制剂CsA对双侧肾脏完整以及右侧肾脏切除的SHR、WKY大鼠的动脉血压无显著影响。以上结果表明,SHR的肾脏在高血压的形成中可能起重要作用     

自发性高血压鼠脑底动脉神经肽Y能神经纤维的起源

应用免疫组织化学ABC法和神经节切除术,观察了16只自发性高血压鼠（分手术Ⅰ、Ⅱ、Ⅲ组和对照组）脑底动脉主要分支神经肽Y能神经纤维的起源,对照组自发性高血压鼠大脑前动脉,大脑中动脉,大脑后动脉和基底动脉壁上均可见棕褐色的神经肽Y能阳性纤维,纤维似曲线状,多呈网状走行,密度较高,手术Ⅰ组作双侧颈上神经节切除术,脑底动脉主要分支的阳性纤维明显减少,但不完全消失,手术Ⅱ组作双侧星状神经节切除术,脑底动脉主要分支壁上的阳性纤维明显减少;手术Ⅲ组作双侧颈上神经节和星状神经节切除术,脑底动脉主要分支的阳性纤维完全消失,结果提示;自发性高血压鼠脑底动脉主要分支神经肽Y能神经纤维起源于双侧颈上神经节和星状神经节,讨论了NPY神经在高血压时期在防御脑中风和血脑屏障破裂方面所起的作用。     

蝎毒耐热蛋白对大鼠海马神经元钠通道的抑制作用

应用全细胞膜片钳技术观察蝎毒耐热蛋白（scorpion venom heat resistant protein,SVHRP）对急性分离大鼠海马神经元电压依赖性钠通道的影响。结果表明,急性分离大鼠海马神经元产生的河豚毒素（tetrodotoxin,TTX）敏感的电压依赖性钠电流被SVHRP浓度依赖性地抑制,半数抑制浓度为（0．0034±0．0004）μg／mL,Hill常数为0．4361±0．0318;SVHRP可使钠通道稳态激活曲线向电压的正方向移动,正常TTX敏感的钠通道的半数激活电压（V1/2）为（-34．38±0．62）mV（n=16）,给予0．1μg／mL的SVHRP后V1/2为（-23．96±0．41）mV（n=8,P〈0．05）,斜坡因子（κ）由正常的4．52±0．52变为3．73±0．08（n=8,P〈0．05）。SVHRP亦能改变电压依赖性钠通道的稳态失活曲线,使其向电位的负方向移动,SVHRP处理前钠通道半数失活电压（V1/2）为（-32．60±1．52）mV,κ为6．73±0．51（n=16）;0．1μg／mL的SVHRP处理后V1/2变为（-50．69±2．55）mV（n=8,P〈0．01）,κ为5．49±0．72（n=8,P〈0．05）。结果提示,SVHRP能抑制电压依赖性钠电流,改变钠通道的动力学特性,抑制其激活,促进其失活,从而影响神经元的兴奋性,这可能是其抗癫痫的机制之一。     

自发性高血压大鼠动脉平滑肌细胞内肌醇磷脂代谢的改变

本研究观察了在基础状态下和Ｇ蛋白激活性,自发性高血压大鼠动脉平滑肌细胞内肌醇磷脂代谢的改变。结果显示：在基础状态下自发性高血压大鼠动脉平滑肌细胞内三磷酸肌醇的含量基本正常;ＧＴＰγ激尖Ｇ蛋白后,动脉平滑肌细胞三磷酸肌醇的增加呈剂量和时间依赖性。     

5—HT对大鼠新鲜分离DRG神经元GABA激活电流的调制作用

本实验在新鲜分离的大鼠背根神经节（ＤＲＧ）细胞上应用全细胞膜片钳技术,观察５－ＨＴ对ＧＡＢＡ激活电流的调制作用。结果发现,绝大多数细胞（４９／５４）对ＧＡＢＡ（１０－８～１０－３ｍｏｌ／Ｌ）产生浓度依赖性的内向电流,并有明显的去敏感现象。在多数细胞预加５－ＨＴ（１０－７～１０－４ｍｏｌ／Ｌ）,ＧＡＢＡ激活电流受到明显抑制,抑制率分别为３．０％,１３．５％,１９．７％,２４．７％,５９．１％。５－ＨＴ（１０－７～１０－４ｍｏｌ／Ｌ）本身在部分细胞（２４／４０）可引起浓度依赖性的内向电流,部分细胞（１２／４０）未检测到膜电流,少数细胞（４／４０）可引起微弱的外向电流。此结果提示５－ＨＴ可能作用于初级传入终末,抑制ＧＡＢＡ引起的初级传入去极化从而调节ＧＡＢＡ的突触前抑制效应     

Nifedipine induces apoptosis in cultured vascular smooth muscle cells from spontaneously hypertensive rats

Apoptosis (programmed cell death) of smooth muscle cells (SMC) in blood vessels is an essential process involved in the control of vessel wall structure. Several antihypertensive drugs currently used in therapy may exert their pharmacological effects by promoting SMC apoptosis. The biochemical events which regulate SMC apoptosis in the vessel wall are complex, and not well understood. We therefore investigated whether treatment of cultured SMC from normotensive Wistar-Kyoto rats (WKY) and from spontaneously hypertensive rats (SHR) with selected antihypertensive drugs would induce SMC apoptosis. We treated aortic SMC from WKY and SHR in vitro with the L-type Ca2+ channel antagonist, nifedipine; with the nitric oxide donor, sodium nitroprusside (SNAP); with forskolin (an activator of adenylyl cyclase); or with thapsigargin (a selective inhibitor of the sarcoplasmic reticulum (SR), Ca2+-ATPase); and compared their apoptosis-promoting effects in SMC derived from the two strains of rats. SMC were derived from the thoracic aorta of 3-4-week-old WKY and SHR, and were used in passages 7-10. Apoptotic cells were detected by in-situ end labeling using the terminal deoxynucleotide transferase-mediated dUTP-nick end-labeling (TUNEL) method, and by morphological examination. We found that: 1) Treatment of cultured aortic SMC with the L-type Ca2+ channel antagonist, nifedipine (5 X 10(-5) M) for 24 hours induced a significantly higher level of apoptosis in SHR cells than in SMC from WKY. Cells from WKY, following exposure to nifedipine for 72 hours, exhibited a similar response to the cells from SHR treated for 24 hours. This was detectable by both morphological criteria as well as DNA labeling by the TUNEL technique. 2) Similar treatment of these cells with thapsigargin (1 x 10(-7) M) led to morphological alterations characteristic of apoptotic cells in SMC from both WKY and SHR, and cells from SHR but not WKY were labeled by the TUNEL technique at 24 hours. The TUNEL method did however identify cells from both WKY and SHR as apoptotic after 48 and 72 hours of treatment. 3) The addition of SNAP, or forskolin to the cultured SMC induced significant, but low levels of apoptosis in WKY SMC only. This selective apoptosis-promoting effect of nifedipine in SHR SMC may result from differences in the control of intracellular Ca2+ between the two strains of cells, or it may indicate that the signaling pathways which regulate apoptosis are different in SMC from the normotensive and the hypertensive rats. Our findings imply that SMC apoptosis may be a selective target for pharmacological intervention in hypertension.     

Vasopressin-induced calcium increases in smooth muscle cells from spontaneously hypertensive rats

Cytosolic free Ca2+ concentrations [( Ca2+]i) were measured in smooth muscle cells (SMC) from spontaneously hypertensive rats (SHR) and age and sex matched Wistar-Kyoto rats (WKY). Resting levels of [Ca2+]i were 114 +/- 6 nM and 116 +/- 5 nM in SMC from WKY and SHR, respectively. Angiotensin II (AII) induced a dose-dependent large increases in [Ca2+]i in SMC. There were no significant differences in resting or AII-stimulated levels of [Ca2+]i when SMC from WKY and SHR were compared. Arg-vasopressin (AVP) caused a similar but smaller [Ca2+]i increase than AII in SMC. AVP caused larger [Ca2+]i increases in SMC from SHR than in SMC from WKY. Although concentrations of AVP higher than those ordinarily detected in plasma were necessary to obtain different responses between SHR and WKY, these differences may be related to the pathogenesis of hypertension.     

Morphological changes in cerebral vascular smooth muscle cells in stroke-prone spontaneously hypertensive rats (SHRSP)

The ultrastructure of the vascular smooth muscle cells of the middle cerebral artery in 6-month-old male stroke-prone spontaneously hypertensive rats (SHRSP) was studied by scanning (SEM) and transmission electron microscopy (TEM) and compared with that of age-matched normotensive Wistar Kyoto rats (WKY). Although the smooth muscle cells of WKY rats by SEM had a typical spindle shape and smooth surface texture, those of SHRSP were structurally modified by numerous surface invaginations and projections, bearing some structural resemblance to the myotendinous junction of skeletal muscle. Structural modifications affected more than half the surface of medial smooth muscle cells in SHRSP, but less than 0.6% of the surface of these cells in WKY rats. About 10% of medial smooth muscle cells were necrotic in SHRSP, but no necrotic cells were identified in WKY rats. By TEM, smooth muscle cells in SHRSP were shown to be irregular in profile with deep indentations of the plasma membrane and were surrounded by many layers of basal laminalike material. The present study suggests that most smooth muscle cells in the middle cerebral artery of SHRSP may be modified to adapt to chronic hypertension by increasing the junctional area between muscle cells and connective tissue and that some cells may undergo necrosis.     

Electron-microscopic studies on reaggregate cultures of vascular smooth muscle cells from normotensive and spontaneously hypertensive rats

Summary Vascular smooth muscle cells were taken from the aortae of the WKY (normotensive) and SHR (spontaneously hypertensive) strains of rat by enzymatic dispersion and put into reaggregate culture. Initially the cells became individual spheroids having average diameters of 10 m and surfaces that were either rough or smooth. The cells were far more complex than they appeared on their surfaces; after one day in culture, there was considerable internal variation in these cells. All the cells, whether WKY or SHR, lost the bulk of their cytoplasmic contents (including myofilaments, many mitochondria, and vesicular structures) in the early stages of culture and eventually became flattened. After 14 days in culture, these modified cells collected to form reaggregates that were commonly roughly spherical and several hundred m in diameter. These reaggregates consisted of peripheral regions made up of several layers of flattened cells overlying cores formed by glia-like networks of cells similar in cytological appearance to the cells at the periphery. The meshes formed in this way contained cellular debris derived from dead cells or extrusion of cellular contents. It appears that SHR cells are quicker to form reaggregates than are WKY cells. Yet the SHR cells retained a rounded conformation after five days, whereas the WKY cells were more flattened and formed a more discrete aggregate at this stage of culture. However, by the fourteenth day of culture, differences between the two cell strains were not so pronounced, as far as could be judged by observations made with scanning and transmission electron microscopy. Both WKY and SHR cells at 14 days appeared highly secretory, possessing large Golgi systems as well as numerous ER cisternae and mitochondria. SHR cells produced greater amounts of connective tissue at all stages of culture than did WKY cells, indicating that a similar difference may contribute to the hypertension which develops naturally in situ in SHR animals.     

Resveratrol inhibits AGEs-induced proliferation and collagen synthesis activity in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats

Advanced glycation end-products (AGEs) of plasma proteins and/or matrix proteins are candidate mediators for various vascular complications such as atherosclerosis. We previously reported a significantly larger accumulation of AGEs of the aorta in stroke-prone spontaneously hypertensive rats (SHRSP) than in age-matched Wistar-Kyoto rats (WKY). In this study, we examined the effects of AGEs on vascular smooth muscle cells (VSMC) from SHRSP and WKY rats. We also studied the in vitro effects of resveratrol (3, 4',5-trihydroxystilbene), a natural phytestrogen, on VSMC proliferation, DNA synthesis, and collagen synthesis activity in SHRSP-VSMC. AGEs accelerated the proliferation of SHRSP- or WKY-VSMC in a time- and dose-dependent manner. VSMC from SHRSP were more sensitive to AGEs than VSMC from normotensive WKY. AGEs also significantly increased DNA synthesis and prolyl hydroxylase activity, a marker for collagen synthesis, in SHRSP-VSMC. AGEs-induced increases in TGF-beta1 mRNA in SHRSP-VSMC were significantly greater than in WKY-VSMC. Resveratrol inhibited AGEs-stimulated proliferation, DNA synthesis, and prolyl hydroxylase activity in SHRSP-VSMC in a dose-dependent manner. ICI 182780, a specific estrogen receptor antagonist, partly blocked the inhibitory effects of resveratrol on AGEs-stimulated proliferation, DNA synthesis, and prolyl hydroxylase activity. Resveratrol significantly inhibited AGEs-induced TGF-beta1 mRNA increases in a dose-dependent manner. Thus, resveratrol may confer protective effects on the cardiovascular system by attenuating vascular remodeling and may be clinically useful as a safer substitute for feminizing estrogens in preventing cardiovascular disease.     

Aspirin-induced AMP-activated protein kinase activation regulates the proliferation of vascular smooth muscle cells from spontaneously hypertensive rats

Acetylsalicylic acid (aspirin), used to reduce risk of cardiovascular disease, plays an important role in the regulation of cellular proliferation. However, mechanisms responsible for aspirin-induced growth inhibition are not fully understood. Here, we investigated whether aspirin may exert therapeutic effects via AMP-activated protein kinase (AMPK) activation in vascular smooth muscle cells (VSMC) from wistar kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Aspirin increased AMPK and acetyl-CoA carboxylase phosphorylation in a time- and dose-dependent manner in VSMCs from WKY and SHR, but with greater efficacy in SHR. In SHR, a low basal phosphorylation status of AMPK resulted in increased VSMC proliferation and aspirin-induced AMPK phosphorylation inhibited proliferation of VSMCs. Compound C, an AMPK inhibitor, and AMPK siRNA reduced the aspirin-mediated inhibition of VSMC proliferation, this effect was more pronounced in SHR than in WKY. In VSMCs from SHR, aspirin increased p53 and p21 expression and inhibited the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. These results indicate that in SHR VSMCs aspirin exerts anti-proliferative effects through the induction of AMPK phosphorylation.