ATP敏感钾通道亚单位在大鼠组织中的表达

目的　研究ATP敏感钾通道 (KATP)亚单位在大鼠组织中的表达。方法　逆转录多聚酶链式反应 (RT PCR)检测通道mRNA的表达。结果　左心室有Kir 6 1,Kir 6 2和SUR 2A的表达 ,大脑皮层中Kir 6 1,Kir 6 2 ,SUR 1,SUR2B均有表达 ,主动脉平滑肌有Kir 6 1,Kir 6 2 ,SUR 2B的表达 ,膀胱平滑肌中有Kir 6 1,Kir 6 2 ,SUR 2B的表达。结论　各组织的KATP组成有所不同 ,Kir 6 1、Kir 6 2和SUR 2B亚单位在多组织中广泛表达。     

细胞内pH对胆碱能受体介导的Kir3.1/3.4电流的调节

目的　研究细胞内pH对胆碱能受体介导的Kir3 1/3 4电流的调节作用。方法　应用AzideNa、KHCO3 和通过灌流直接降低细胞内 pH ,用双电极电压钳和膜片钳方法观察在蛙卵细胞中表达的Kir3 1/ 3 4钾离子通道电流的变化和M受体激活对Kir3 1/ 3 4电流调节的变化。结果　细胞内 pH降低能抑制Kir3 1/ 3 4的电流 ;Kir3 1/ 3 4对细胞内pH的敏感性介于另外两种Kir通道Kir2 1和Kir2 3之间 ,即这三种通道对细胞内 pH的敏感性依次为Kir2 3>Kir3 1/ 3 4 >Kir2 1;细胞内pH降低能够减弱M1受体激活对Kir3 1/ 3 4的抑制作用 ,加强M2 受体激活Kir3 1/ 3 4电流后的去敏作用。结论　在维持细胞静息电位方面起重要作用的Kir3 1/ 3 4通道在细胞内pH降低的情况下基础电流和M受体激活调节电流均发生了变化 ,这些变化在缺血缺氧引起的细胞 (如心肌细胞 )兴奋性改变中可能有重要的生理学意义     

Kir2.1/Kir2.3嵌合体的构建与表达

目的构建Kir2.1/Kir2.3通道嵌合体,为进一步研究Kir2.1和Kir2.3通道的调控机制打下基础。方法利用重叠延伸PCR方法构建不同的Kir2.1/Kir2.3嵌合体质粒:N1P3C3,N3P1C1,N3P3C1,N1P1C3,NIP3C1,N3P1C3。将不同的嵌合体质粒分别用NheI线性化后,转录为cRNA表达于非洲爪蟾卵母细胞,用双电极电压钳记录电流。结果不同的Kir2.1/Kir2.3嵌合体质粒构建成功,在非洲爪蟾卵母细胞上可以记录到电流表达。结论成功构建Kir2.1/Kir2.3嵌合体并完成了嵌合体通道的异源性表达和电压钳记录。     

Iptkalim inhibits cocaine challenge—induced enhancement of dopamine levels in nucleus accumbens and striatum of rats by up—regulating Kir6.1 and Kir6.2 mRNA expression

目的:研究钾通道开放剂埃他卡林对急慢性可卡因应用引起的伏隔核、纹状体和额叶皮层的多巴胺和谷氨酸水平变化的影响及其机制。方法:采用高效液相色谱与电化学检测、荧光检测联用的方法测定各脑区谷氨酸和多巴胺的含量;采用半定量逆转录聚合酶链反应(RT-PCR)研究ATP敏感性钾通道亚单位Kir6.1、Kir6.2、SUR1和SUR2 mRNA表达的变化。结果:埃他卡林不影响急性可卡因应用引起纹状体和伏隔核中多巴胺和谷氨酸水平的增高(P>0.05),能够逆转激发剂量可卡因诱导的慢性成瘾大鼠纹状体和伏隔核的多巴胺含量增高(P<0.05),对激发后皮层和伏隔核谷氨酸水平增高有降低趋势但差异无显著性(P>0.05)。激发剂量可卡因能提高可卡因预处理组和埃他卡林预处理组纹状体和伏隔核的Kir6.1和Kir6.2 mRNA表达以及皮层的Kir6.2 mRNA表达,而且IPT预处理组的升高幅度显著高显著高于可卡因慢性处 理组。结论:埃他卡林通过上调Kir6.1和Kir6.2 mRNA表达抑制可卡因激发引起的纹状体和伏隔核的多巴胺水平的增高。     

TGF—β1和Smad3在子宫肌瘤组织中的表达及意义

目的检测转化生长因子β1（transforming growth factor β1,TGF—β1）和Smad3在子宫肌瘤组织中的表达,探讨两者的相关性。方法采用RT—PCR法分别检测子宫肌瘤组织以及邻近正常子宫平滑肌组织中TGF—β1和Smad3的mRNA表达情况。结果①TGF—β1在子宫肌瘤组织和正常子宫平滑肌组织中都有表达,且肌瘤组中TGF—β1的mRNA的表达（1．51±0．34）明显高于正常组（1．29±0．35）,差异有统计学意义（t=5．73,P=0．000）;②Smad3在肌瘤组和正常组都有表达,且肌瘤组中Smad3表达（1．36±0．20）明显高于正常组（1．13±0．27）,差异有统计学意义（t=7．34,P=0．000）;③肌瘤组中TGF—β1和Smad3的mRNA的表达呈正相关（rs=0．769,P=0．000）。结论TGF—β1能诱导子宫肌瘤纤维化,信号蛋白Smad3在其中发挥了重要作用。     

高胆固醇血症改变大鼠血管功能及动脉平滑肌细胞钾通道基因表达(英文)

目的:研究高胆固醇血症对大鼠主动脉功能及主动脉平滑肌上内向整流和ATP敏感钾通道基因表达的影响。方法:大鼠灌胃高胆固醇乳剂两周,制备带内皮和去内皮的主动脉血管环检测收缩和舒张反应,应用RT-PCR观察基因表达的变化。结果:高胆固醇血症明显损伤血管内皮功能,但不影响血管内皮非依赖性舒张反应;在高胆固醇血症血管平滑肌中Kir6.2mRNA表达明显升高(P<0.05),而Kir3.1mRNA表达却显著降低(P<0.05)。结论:高胆固醇血症可改变血管功能,并影响某些内向整流和ATP敏感钾通道亚型的基因表达。     

膀胱癌组织TGF-β1mRNA表达及其意义

目的:探讨TGF-β1 mRNA表达与膀胱癌临床生物学行为的关系。方法;采用Northern杂交及斑点杂交方法检测了42例膀胱移行细胞癌及9例正常膀胱黏膜组织标本中的TGF-β1　mRNA表达情况,GAPDH作为内对照。结果:肿瘤组织中TGF-β1 mRNA含量高于正常膀胱黏膜组织(P<0.01)。从病理学分级看,G3级组中TGF-β1 mRNA含量高于正常黏膜组(P<0.01),但G1、G2、G3级3组间TGF-β1mRNA表达量无显著差别。从临床分期看,T2、T3期组中TGF-β1 mRNA含量均高于正常黏膜组(均P<0.05),T2期与T3期组也均高于T1期组(P<0.05和P<0.01),但T2期和T3期组间没有差别(P>0.05)。浸润性肿瘤组(T2期和T3期组)中TGF-β1mRNA含量高于表浅性肿瘤组(Ta和T1期组)。结论:TGF-β1 mRNA的表达量可作为判断膀胱癌浸润程度和预后的指标之一。     

心房颤动患者心房组织ATP敏感性钾通道基因转录表达的研究

目的 探讨心房颤动患者心房组织ATP敏感性钾通道(Kir6.2)基因转录的变化。方法 35例风湿性心瓣膜病患者,心外科手术时被取右心耳组织。通过逆转录-聚合酶链反应,以三磷酸甘油醛脱氢酶(GAPDH)为内参照,测量心耳组织Kir6.2通道的mRNA表达量。结果 窦性心律组和阵发性房颤组心房组织Kit6.2的mRNA水平均显著高于慢性房颤组(P<0.05);而阵发性房颤组心房组织Kir6.2的mRNA又显著高于窦性心律组(P<0.05)。结论 Kir6.2通道转录水平的改变可能是相应ATP敏感性钾通道产生的内向整流钾电流(IKATP)重构的分子基础,Kir6.2基因转录改变可能是促进房颤发生和持续的因素之一。     

新型K_(ATP)开放剂埃他卡林对慢性低氧诱导大鼠体内肺动脉平滑肌K_(ATP)通道mRNA表达变化的影响

目的研究慢性低氧对大鼠肺动脉平滑肌KATP通道mRNA表达的影响及新型KATP开放剂埃他卡林(Iptakalim,IPT)的作用。方法将清洁级SD大鼠30只随机分成对照组、低氧组、IPT组,每组10只。将低氧组与IPT组放入常压低氧舱制备动物模型;采用右心导管测量大鼠肺动脉压;用实时荧光定量PCR检测(Real time PCR)技术分析各组肺动脉平滑肌KATP通道Kir6.1与SUR2B mRNA表达。结果低氧组大鼠肺动脉平均压高于对照组和IPT组(P<0.05),低氧组SUR2B亚基mRNA水平低于对照组(P<0.05),IPT可逆转慢性低氧对SUR2B的作用,各组Kir6.1亚基mRNA表达水平无差异(P>0.05)。结论慢性缺氧导致大鼠肺动脉平滑肌KATP通道SUR2B mRNA表达减少,IPT能拮抗慢性缺氧对KATP通道SUR2B mRNA基因表达的抑制作用。     

不同年龄大鼠心血管组织中钙调神经磷酸酶活性的变化

目的通过检测大鼠心肌及主动脉中钙调神经磷酸酶(CaN)活性探讨:①大鼠心脏和主动脉组织中CaN活性有无差异。②不同月龄大鼠心脏和主动脉CaN活性的变化。方法随机抽取1月龄组大鼠12只作为幼年组、6月龄组大鼠12只为青年组和24月龄组大鼠12只为老年组。用CAN测试盒采用发色底物法测定其心脏及主动脉组织中CaN活性。结果①三组大鼠心肌组织CaN活性显著高于同月龄组主动脉平滑肌CaN活性(P<0.05);②大鼠心肌组织CaN活性随月龄增加而依次降低,三组间相比差异有显著性(P<0.05);③主动脉平滑肌CaN活性也是随月龄增加而依次降低,三组间相比差异有显著性(P<0.05)。结论①大鼠心肌CaN活性比主动脉组织中高,显示CaN分布有组织学差异,这与其生理分工不同有关。②大鼠心肌和主动脉组织中的CaN活性随年龄的增长而降低,CaN可以作为心血管老化的一个指标。     

Selective expression of Kir6.1 protein in different vascular and non-vascular tissues

K(ATP) channels are composed of pore-forming subunits Kir6.x and auxiliary subunits SURx. These channels play important roles in modulating the contractility of vascular smooth muscle cells (SMCs) by altering membrane potentials. The molecular basis of K(ATP) channels in vascular SMCs is unclear and the expression of different K(ATP) channel subunits at protein level in various tissues still undetermined. In this study, using an anti-Kir6.1 antibody, we detected the expression of Kir6.1 proteins in rat vascular tissues including mesenteric artery, pulmonary artery, aorta, and tail artery. Kir6.1 proteins were also identified in heart and other non-vascular tissues including spleen and brain, but they were undetectable in liver and kidney. Immunocytochemical study revealed the expression of Kir6.1 proteins in cultured rat thoracic aortic SMCs. Using the whole-cell patch-clamp technique, it was found that the intracellularly applied anti-Kir6.1 antibody significantly inhibited K(ATP) channel currents in HEK-293 cells that were stably transfected with Kir6.1 cDNA. A better understanding of differential expression of Kir6.1 proteins in various vascular and non-vascular tissues may help discern different molecular basis and functions of K(ATP) channel complexes in these tissues.     

Similarities and differences in the autonomic control of airway and urinary bladder smooth muscle

The airways and the urinary bladder are both hollow organs serving very different functions, i.e. air flow and urine storage, respectively. While the autonomic nervous system seems to play only a minor if any role in the physiological regulation of airway tone during normal breathing, it is important in the physiological regulation of bladder smooth muscle contraction and relaxation. While both tissues share a greater expression of M2 than of M3 muscarinic receptors, smooth muscle contraction in both is largely mediated by the smaller M3 population apparently involving phospholipase C activation to only a minor if any extent. While smooth muscle in both tissues can be relaxed by beta-adrenoceptor stimulation, this primarily involves beta2-adrenoceptors in human airways and beta3-adrenoceptors in human bladder. Despite activation of adenylyl cyclase by either subtype, cyclic adenosine monophosphate plays only a minor role in bladder relaxation by beta-agonists; an important but not exclusive function is known in airway relaxation. While airway beta2-adrenoceptors are sensitive to agonist-induced desensitization, beta3-adrenoceptors are generally considered to exhibit much less if any sensitivity to desensitization. Gene polymorphisms exist in the genes of both beta2- and beta3-adrenoceptors. Despite being not fully conclusive, the available data suggest some role of beta2-adrenoceptor polymorphisms in airway function and its treatment by receptor agonists, whereas the available data on beta3-adrenoceptor polymorphisms and bladder function are too limited to allow robust interpretation. We conclude that the distinct functions of airways and urinary bladder are reflected in a differential regulation by the autonomic nervous system. Studying these differences may be informative for a better understanding of each tissue.     

Orphan-receptor ligand human urotensin II: receptor localization in human tissues and comparison of vasoconstrictor responses with endothelin-1

We have determined the distribution of receptors for human urotensin-II (U-II) in human and rat CNS and peripheral tissues. In rat, [(125)I]-U-II binding density was highest in the abducens nucleus of brainstem (139.6+/-14 amol mm(-2)). Moderate levels were detected in dorsal horn of spinal cord and lower levels in aorta (22. 5+/-6 amol mm(-2)). In human tissues density was highest in skeletal muscle and cerebral cortex ( approximately 30 amol mm(-2)), with lower levels (<15 amol mm(-2)) in kidney cortex and left ventricle. Little binding was identified in atria, conducting system of the heart and lung parenchyma. Receptor density was less in human coronary artery smooth muscle (14.6+/-3 amol mm(-2), n=10) than rat aorta with no significant difference between normal and atherosclerotic vessels. In human skeletal muscle [(125)I]-U-II bound to a single receptor population with K(D)=0.24+/-0.17 nM and B(max)=1.97+/-1.1 fmol mg(-1) protein (n=4). U-II contracted human coronary, mammary and radial arteries, saphenous and umbilical veins with sub-nanomolar EC(50) values. U-II was 50 times more potent in arteries and <10 times more potent in veins than endothelin-1 (ET-1). The maximum response to U-II ( approximately 20% of control KCl) was significantly less than to ET-1 ( approximately 80% KCl). In contrast, in rat aorta, U-II and ET-1 were equipotent with similar maximum responses. This is the first report of high affinity receptors for [(125)I]-U-II in human CNS and peripheral tissues. This peptide produces potent, low efficacy, vasoconstriction in human arteries and veins. These data suggest a potential role for U-II in human physiology.     

Tissue distribution and renal developmental changes in rat organic cation transporter mRNA levels.

Organic cation transporters (OCTs) are responsible for excretion of cationic substances into urine. Tissue OCT expression may be important for the disposition and excretion of xenobiotics. Therefore, OCT1, OCT2, OCT3, OCTN1, and OCTN2 mRNA levels were measured in adult rat tissues and rat kidney tissue at various stages of development from day 0 to 45. OCT1 mRNA expression was highest in kidney and spleen, moderate in skin, and low in the gastrointestinal tract, brain, lung, thymus, muscle, and prostate. OCT2 mRNA levels were highest in kidney, with low expression in other tissues, and with renal OCT2 levels being approximately 4 times higher in males than that in females. In gonadectomized males, OCT2 mRNA levels were attenuated to female levels, suggesting a role for testosterone in OCT2 expression. OCT3 was moderately expressed in kidney and was highest in blood vessel, skin, and thymus. OCTN1 was expressed in most of the tissues examined, with relatively higher expression in kidney and ileum and lower levels in thymus. Lastly, OCTN2 was expressed abundantly in kidney and ileum, moderately in large intestine, dorsal prostate, bladder, duodenum, and cerebellum, and minimally in thymus, spleen, and cerebral cortex. Renal OCT1, OCTN1, and OCTN2 mRNA levels increased gradually from postnatal day 0 through day 45 in both genders. Renal OCT2 levels remained the same in males and females through day 25 and then dramatically increased only in male kidney after day 30. In summary, OCT mRNA was detected primarily in kidney, and the high level of renal OCT expression may explain why the kidney is a target organ for xenobiotics with cationic properties.     

PTEN在哮喘大鼠气道平滑肌细胞增生中的作用

目的:探讨哮喘大鼠肺组织中PTEN表达的变化及其在气道平滑肌细胞增生中的作用.方法:清洁级雄性Wistar大鼠16只随机分为对照组和哮喘组,每组8只.用卵蛋白(OVA)致敏与激发建立大鼠哮喘模型,末次激发后HE染色法观察哮喘组大鼠支气管-肺组织中炎性细胞浸润情况及气道形态学改变,免疫组化法检测增生细胞核抗原(PCNA)及PTEN蛋白在大鼠肺组织中的表达,RT-PCR法检测肺组织PTEN mRNA表达.结果:哮喘组大鼠气道壁和肺组织中可见大量炎症细胞浸润,气道平滑肌增生肥厚;与对照组比较,哮喘组PCNA蛋白表达显著增强(P < 0.05),PTEN蛋白及其mRNA表达则显著减弱(P < 0.05).结论:PTEN基因失活可能在哮喘气道平滑肌增生中起重要作用,该作用可能与PI3K信号传导通路有关.     

Quantification of the Na,K-Pumps in Mammalian Skeletal Muscle

Abstract: The muscarinic cholinergic receptors in the urinary bladders of man, guinea pig, rat and rabbit were studied by means of a receptor binding technique, with L-quinuclidinyl [phenyl 4-³H]benzilate, (-)³H-QNB, as radioligand. The potential role of the receptors in the supersensitivity of the rat bladder to muscarinic agonists, following parasympathetic denervation, hypertrophy and urinary diversion, was also investigated. In addition, the binding of various unlabelled antimuscarinic drugs in the guinea pig bladder was compared to that in other tissues in order to study the putative muscarinic receptor subtypes, commonly referred to as M₁ and M₂. According to this classification the putative M₁ receptors prevail in discrete areas of the brain, whereas the M₂-receptors predominate in peripheral tissues, such as the exocrine glands and smooth muscles. The receptor density (but not the qualitative properties of the receptors) in the bladder differed between the species. The affinities of various antimuscarinic drugs were virtually identical in the guinea pig and human bladders. In both species, the binding data were found to correlate with functional in vitro data. In the rat bladder, the receptor density was increased after denervation but decreased, below control values, when the denervation was combined with urinary diversion. A decrease was also found after urinary diversion of innervated bladders, whereas the receptor density was unaffected by hypertrophy. These results suggest that the receptors are not involved in the development of supersensitivity and that the receptor levels may be influenced by the functional state of the bladder. Binding studies with classical muscarinic antagonists indicated that the receptors in the guinea pig bladder are indistinguishable from those in the ileum, heart, parotid gland and cerebral cortex. However, four drugs - namely, oxybutynin, dicyclomine, benzhexol and pirenzepine had a much higher affinity for the receptors in the parotid gland and cortex than for those in the other tissues. Moreover, dicyclomine and benzhexol, like pirenzepine, seemed in the cortex to distinguish between two classes of sites exhibiting high and low affinity. The high affinity sites could be selectively labelled with ³H-benzhexol. The ability of oxybutynin, dicyclomine, benzhexol and pirenzepine to discriminate between the receptors in the parotid gland and those in smooth muscle provides further evidence that the M₁/M₂ concept is inaccurate. The present data indicate that there may be three classes of muscarinic antagonist binding sites.