ATP通过P2Y1受体引发脊髓背角星形胶质细胞[Ca2+]i升高和GFAP表达上调

目的 观察体外培养的背角星形胶质细胞P2Y1受体激活对其[Ca2 ]i的变化和GFAP表达的影响.方法 培养并纯化脊髓背角星形胶质细胞,采用免疫组织化学染色观察背角星形胶质细胞P2Y1受体及GFAP的表达,激光共聚焦技术观察星形胶质细胞[Ca2 ]i的变化.结果 体外培养的大鼠脊髓背角星形胶质细胞大多表达P2Y1受体;P2Y受体激动剂ATP、ADP、ADP-βs剂量依赖性促进星形胶质细胞[Ca2 ]i升高;10 μg/mL的ATP、ADP和ADP-βs显著增加胞内[Ca2 ]i,此作用可被特异性P2Y1受体拮抗剂MRS2179所阻断,并具量效关系.免疫组织化学染色结果显示,100 μg/mL的ATP、ADP和ADP-βs作用下,星形胶质细胞GFAP表达上升,此效应可被100 μg/mL的MRS2179所抑制.结论 体外培养的大鼠脊髓背角星形胶质细胞表达P2Y1受体;P2Y1受体介导了ATP、ADP及ADP-βs促进星形胶质细胞[Ca2]i升高和GFAP表达增强的过程.     

膀胱二聚体Cajal间质细胞的超微结构及其对ATP的电反应

目的:为证明膀胱二聚体Cajal间质细胞(interstitial cells of Cajal,ICC)发挥起搏作用提供超微结构和功能学证据。方法:电子显微镜下鉴定组织和培养的豚鼠膀胱二聚体ICC并观察ICC之间的超微联系;体外分离、培养豚鼠膀胱ICC,运用细胞膜片钳技术观察ATP(100μmol/L)刺激下二聚体ICC和单体ICC的兴奋性差别。结果:二聚体ICC中两个ICC细胞胞体紧密相连,细胞之间的间隙小于20 nm,存在大量缝隙连接,局部细胞膜融合,形成联胞体。在ATP(100μmol/L)的刺激下体外培养的二聚体ICC的内向电流幅度为286.9±26.4 pA,显著大于单体ICC的内向电流幅度163.8±18.6 pA(P<0.01)。结论:二聚体ICC具有形成高兴奋起搏电流的超微结构基础,高兴奋性的二聚体ICC可能才是逼尿肌的起搏细胞。     

人红细胞抗高血压因子对人脐静脉VSMC胞浆及核内Ca2+水平的影响

采实验用Fluo-3／AM染色在激光扫描共聚焦显微镜下观察了红细胞抗高血压因子（antihypertensivefactor,AHF）对人脐静脉VSMC胞浆（［Ca2＋］i）及核内（［Ca2＋］n）游离钙离子水平的影响,结果表明：AHF（10-4g／mL）明显抑制Bayk8644（10-6mol／L）,KCl（60mmol／L）,AngⅡ（10-6mol／L）,CPA（10-5mol／L）及IP3（10-5mol／L）引起的人脐静脉VSMC［Ca2＋］i与［Ca2＋］n升高。结果提示：1．AHF通过抑制Ca2＋内流、胞内钙库、放等机制阻断［Ca2＋］i的升高：2．AHF有益于核内钙稳态的维持。     

豚鼠膀胱中二聚体Cajal间质细胞的分布特点及意义

目的　观察二聚体Cajal间质细胞(interstitial cells of Cajal,ICC)在豚鼠膀胱不同组织及部位的分布特点,并探讨其意义。 方法　电子显微镜下观察豚鼠膀胱壁组织切片黏膜层,黏膜下层,肌层内二聚体ICC分布情况;对膀胱组织切片进行免疫荧光染色,用c-Kit抗体标记ICC,激光共聚焦显微镜下观察二聚体ICC在膀胱顶部、体部、颈部的分布特点。 结果　在电子显微镜下见二聚体ICC主要分布于黏膜下层,而肌层主要以单体ICC为主。免疫荧光染色发现每高倍镜视野下膀胱顶部二聚体ICC平均数量为（3.47±0.53）个,体部和颈部为（1.57±0.45）个和(0.49±0.19)个。膀胱顶部二聚体ICC数量明显高于体部和颈部（P<0.01）。 结论　二聚体ICC主要分布于豚鼠膀胱顶部的黏膜下层,可能是感受黏膜张力刺激,引发顶部膀胱自发兴奋的起搏细胞。     

外源性锌对缺氧神经元的保护作用

目的:观察不同浓度外源性锌对神经元细胞内游离钙([Ca2 ]i)的影响,探讨低浓度外源性锌对于缺氧神经元保护作用的可能机制.方法:原代培养大鼠皮层神经元,加入不同浓度的外源性锌,激光扫描共聚焦显微镜检测神经元[Ca2 ]i的变化;建立细胞缺氧模型,检测10 μmol/L外源性锌对于缺氧神经元[Ca2 ]i和[Zn2 ]i的影响.结果:神经元的[Ca2 ]i,μmol/L锌组与对照相比无明显变化;100μmol/L锌组一过性升高,后降至对照水平;500 μmol/L锌组,明显高于对照水平.除正常加锌组外,其余各组神经元[Zn2 ]i和[Ca2 ]i均较正常对照组增高;但正常加锌组、缺氧加锌组神经元[Zn2 ]i和[Ca2 ]i均较缺氧组降低.结论:外源性锌对于神经元的作用随浓度而不同;10 μmol/L外源性锌维持了神经元的钙稳态,它对于缺氧神经元的保护作用,可能通过抑制钙超载实现.     

阿司匹林对化学性缺氧状态下新生大鼠脑神经细胞[Ca~(2+)]i和NGB的影响

目的 探讨阿司匹林(ASA)对化学性缺氧大鼠脑神经细胞的保护作用及细胞外钙离子对ASA神经保护作用的影响。方法 分别在含钙和去钙培养液中加入连二亚硫酸钠（Na2S2O4）诱导化学性缺氧,用ASA预处理体外培养的大鼠脑细胞,激光共聚焦显微镜观察[Ca2+]i和脑红蛋白（NGB）的变化。结果 化学性缺氧使大鼠脑细胞[Ca2+]i和NGB表达增高（P＜0.05, n=5）。ASA预处理可明显缓解缺氧组和无钙缺氧组大鼠脑细胞[Ca2+]i和NGB的增高（P＜0.05, n=5）。结论 ASA可能通过减少大鼠脑细胞的钙超载,抑制缺氧诱导的NGB表达增高,发挥神经细胞保护作用。     

P2Y_(13)受体激活促进脊髓背角小胶质细胞钙浓度升高

目的:观察ADP对培养的脊髓背角小胶质细胞内Ca2+浓度([Ca2+]i)的影响及作用机制。方法:培养纯化新生SD大鼠脊髓背角小胶质细胞,免疫组化观察P2Y13受体表达,激光共聚焦显微镜检测ADP作用下,小胶质细胞[Ca2+]i的变化。结果:大鼠脊髓背角小胶质细胞表达P2Y13受体,ADP可导致培养的脊髓背角小胶质细胞[Ca2+]i快速增高,且呈现剂量依赖性;P2Y13受体拮抗剂MRS2211(100μmol/L)能基本阻断ADP的作用,而P2Y1受体拮抗剂MRS2179(100μmol/L)、P2Y12受体拮抗剂MRS2395(100μmol/L)均不影响ADP致小胶质细胞[Ca2+]i升高的效应。结论:ADP可能通过P2Y13受体途径,导致培养的脊髓背角小胶质细胞[Ca2+]快速升高。     

钙敏感受体在缺氧诱导的大鼠肺动脉平滑肌细胞增殖中的作用

目的:观察钙敏感受体(CaSR)在缺氧诱导的大鼠肺动脉平滑肌细胞增殖中的作用。方法:Ⅱ型胶原酶消化法提取、培养大鼠肺动脉平滑肌细胞。应用Western blotting技术及免疫荧光染色分析CaSR蛋白在肺动脉平滑肌的表达;采用激光共聚焦扫描技术检测不同处理因素对细胞内钙离子浓度([Ca2+]i)的影响,应用MTT法分析不同处理因素对细胞存活率的影响。结果:大鼠肺动脉平滑肌细胞有CaSR蛋白表达。缺氧能够增加CaSR和增殖细胞核抗原(PCNA)的表达、细胞存活率及[Ca2+]i(与对照组比较,P0.05)。GdCl3(CaSR激动剂)能够增强缺氧的上述作用,NPS2390(CaSR抑制剂)则能够减弱缺氧的作用。结论:缺氧诱导的CaSR表达增加参与了缺氧性肺动脉平滑肌细胞增殖。     

腺相关病毒载体介导的PLB基因反义RNA对大鼠心肌细胞肌浆网Ca2+-ATPase活性和［Ca2+］i的作用

目的：探讨腺相关病毒载体介导的磷酸受纳蛋白（PLB）反义RNA对肌浆网Ca2+-ATPase活性以及细胞内钙浓度（［Ca2+］i）的作用。 方法： 构建PLB反义RNA的重组腺相关病毒载体（rAAV-asPLB）和携带报告基因LacZ的重组腺相关病毒载体（rAAV-LacZ）。分别转染培养的大鼠心肌细胞，测定PLB mRNA和蛋白质表达，检测肌浆网Ca2+-ATPase活性和［Ca2+］i。 结果： RT-PCR和Western blotting显示，感染rAAV-asPLB的心肌细胞的PLB mRNA和蛋白质表达低于正常对照和感染rAAV-LacZ组；而Ca2+-ATPase活性大于正常对照和感染rAAV-LacZ组。激光共聚焦显微镜检测显示，静息状态时，rAAV-asPLB感染组［Ca2+］i降低；异丙肾上腺素刺激后，各组［Ca2+］i均升高，但是rAAV-asPLB感染组［Ca2+］i变化幅度大。 结论： 腺相关病毒介导的PLB反义RNA对大鼠心肌细胞PLB表达具有抑制作用，增强Ca2+-ATPase活性，降低静息状态的［Ca2+］i ，增加异丙肾上腺素刺激后［Ca2+］i 的变化幅度。     

尾加压素Ⅱ对大鼠肾小球系膜细胞内游离钙浓度影响

目的 探讨尾加压素Ⅱ（UmtensinⅡ，UⅡ）对肾小球系膜细胞（GMC）内游离钙浓度的影响及其作用机制。方法 以体外培养的SD乳鼠GMC为研究对象，用UⅡ刺激GMC，Fluo3／AM荧光标记细胞内游离钙离子，激光共聚焦显微技术动态测定GMC内游离钙浓度变化。结果 UⅡ激发GMC内[Ca^2＋]i升高作用分2个时相，即瞬时峰值升高时相和缓慢持久升高时相，对峰值升高呈剂量依赖方式，在10^-10mol／L～10^-7mol／L范围内引起峰值升高，且随着剂量增加，幅度增大。硝苯地平和无钙缓冲液对UⅡ激发GMC内[Ca^2＋]i瞬时峰值升高无作用，但可完全阻止缓慢持久升高时相出现。结论 UⅡ激发GMC内[Ca^2＋]i浓度瞬时峰值升高时相是由细胞内储存钙释放介导的，而缓慢持久升高时相是由细胞外钙流人增加引起的。     

Glevic抑制ICCs功能对ATP诱导大鼠盆神经放电的影响

目的探讨Glevic抑制ICCS功能对ATP诱导大鼠盆神经传入电活动的影响。方法20只SD大鼠随机分为ATP组和Glevic组。ATP组通过膀胱内灌注ATP诱发盆神经放电评估ATP介导的膀胱感觉功能,Glevic组则通过Glevic预处理抑制膀胱ICCs功能后,重复ATP组实验。结果两组大鼠给药前膀胱灌注生理盐水均可诱发盆神经进行性增强的簇状放电。膀胱内灌注20mmol／LATP可诱发大鼠盆神经放电,频率为（27．36±2．53）次／分,并伴有较微弱的逼尿肌收缩。100μmol／LGlevic抑制ICCs功能后,ATP诱发的盆神经放电频率为（2．46±0．38）次／分,与ATP组相比差异非常显著（P〈0．01）。结论Glevic对ATP诱发的大鼠盆神经放电的抑制作用表明ICCs可能在ATP介导的膀胱感觉形成中发挥了重要作用。     

Expression and function of bradykinin B1 and B2 receptors in normal and inflamed rat urinary bladder urothelium

The bladder urothelium exhibits dynamic sensory properties that adapt to changes in the local environment. These studies investigated the localization and function of bradykinin receptor subtypes B1 and B2 in the normal and inflamed (cyclophosphamide (CYP)-induced cystitis) bladder urothelium and their contribution to lower urinary tract function in the rat. Our findings indicate that the bradykinin 2 receptor (B2R) but not the bradykinin 1 receptor (B1R) is expressed in control bladder urothelium. B2R immunoreactivity was localized throughout the bladder, including the urothelium and detrusor smooth muscle. Bradykinin-evoked activation of this receptor elevated intracellular calcium  (EC₅₀= 8.4 n m )  in a concentration-related manner and evoked ATP release from control cultured rat urothelial cells. In contrast, B1R mRNA was not detected in control rat urinary bladder; however, following acute (24 h) and chronic (8 day) CYP-induced cystitis in the rat, B1R mRNA was detected throughout the bladder. Functional B1Rs were demonstrated by evoking ATP release and increases in [Ca²⁺]_i in CYP (24 h)-treated cultured rat urothelial cells with a selective B1 receptor agonist (des-Arg⁹-bradykinin). Cystometry performed on control anaesthetized rats revealed that intravesical instillation of bradykinin activated the micturition pathway. Attenuation of this response by the P2 receptor antagonist PPADS suggests that bradykinin-induced micturition facilitation may be due in part to increased purinergic responsiveness. CYP (24 h)-treated rats demonstrated bladder hyperactivity that was significantly reduced by intravesical administration of either B1 (des-Arg¹⁰-Hoe-140) or B2 (Hoe-140) receptor antagonists. These studies demonstrate that urothelial expression of bradykinin receptors is plastic and is altered by pathology.     

Quantitative and ultrastructural characteristics of immunocompetent cells in the mammary gland during pregnancy and lactation

The quantity and ultrastructure of immunocompetent cells (ICC) were evaluated in the of rat mammary gland in dynamics of pregnancy and lactation. ICC found between the alveolar epithelial cells were represented by monocytes, small and medium lymphocytes, including some granule-containing lymphocytes. The quantity of ICC was found to increase in dynamics of pregnancy and to reach a its maximum on day 3 of lactation. This was accompanied by the appearance of macrophages and plasma cells. Similar changes were found in stromal ICC. It is suggested that quantitative and structural dynamics of mammary ICC is associated with provision of immune reactivity of the newborn and its adaptive immunity.     

A possible role of the cholinergic and purinergic receptor interaction in the regulation of the rat urinary bladder function

The contractile activation of the upper (dome) and lower (base) parts of the urinary bladder show some differences. Cellular mechanisms that might be responsible for cholinergic effects blocking non-adrenergic non-cholinergic contractions in the base of the rat urinary bladder were investigated. Smooth muscle cells were thus freshly isolated or cultured both from the dome and the base of the rat urinary bladder and the contribution from cholinergic and purinergic pathways to their Ca²⁺ homeostasis was examined. The expression of nicotinic acetylcholine (nAChR) and P2X2 purinergic receptors on the cultured cells and on tissue sections was investigated. The ATP-evoked Ca²⁺ transients in rat smooth muscle cells did not show any desensitization. However, when ATP was administered together with carbamylcholine (CCh), the latter essentially prevented ATP from evoking Ca²⁺ transients in smooth muscle cells from the base (suppression to 12 ± 2.5% of control, n = 57; p < 0.01), but not from the dome (99 ± 5% of control, n = 52; p > 0.05) of the rat urinary bladder. While atropine was unable to modify (6 ± 3% of control, n = 14; p < 0.05), α-bungarotoxin (118 ± 12% of control, n = 20; p > 0.05) blocked the inhibitory effects of CCh. Additionally, α7 subunits of nAChR and P2X2 purinergic receptors were identified using immunocytochemistry, immunohistochemistry, and Western blot in cultured urinary bladder smooth muscle cells, in urinary bladder sections, and in urinary bladder muscle strips, respectively, suggesting that the activation of nAChR modifies the action of ATP.     

Rhythmic spontaneous contractions in the rat proximal colon

C-kit immunoreactive cells are known to be interstitial cells of Cajal (ICCs), and they generate pacemaker activity of the gastrointestinal tract. Recently a large number of special smooth muscle cells corresponding to c-kit immunoreactive cells were found in the proximal colon of the guinea pig. We learned that the rat proximal colon showed tetrodotoxin-insensitive regular rhythmic spontaneous contractions (RSCs) and hypothesized that RSCs are generated and/or regulated by ICCs. To prove our hypothesis, we investigated whether RSCs are absent in homozygous Ws/Ws mutant rats, since c-kit positive ICCs along the submucosal surface of the circular muscle (ICC(SM)) and myenteric plexus (ICC(MY)) are lacking. In contrast to our hypothesis, we found that RSCs were still present in the proximal colon of the Ws/Ws mutant rats. A recent study has reported that c-kit negative ICC(SM) remains in Ws/Ws mutant rats. Taken together, RSCs may be generated by c-kit negative ICC(SM) in the rat proximal colon. The blockade of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA) (10(-6)M) or by thapsigargin (10(-6)M) increased the frequency of RSCs. The increasing effects of CPA on the frequency of RSCs were more prominent in Ws/Ws mutant rats than in +/+ rats. We concluded that the functional coordination between c-kit negative ICC(SM) and other mutationally impaired c-kit positive ICC(MY) and ICC(SM) may be required for moderate regulation in the frequency of spontaneous activity.     

ATP, beta-gamma-methylene-ATP, and adenosine inhibit non-cholinergic non-adrenergic transmission in rat urinary bladder

ATP and adenosine caused a dose-dependent and reversible inhibition of the atropine-resistant contraction response to transmural nerve stimulation in the rat urinary bladder. Both purines also inhibited contraction responses to acetylcholine and direct muscle stimulation, indicating a postjunctional effect on the transmission. It seems as ATP per se inhibits the excitatory transmission, because the stable ATP-analogue beta-gamma-methylene-ATP was inhibitory as well, and because exogenous adenosine deaminase annulled the inhibition by adenosine but not that by ATP or beta-gamma-methylene-ATP. Blockade of purine inactivation enhanced the inhibitory action of ATP and adenosine, and by itself inhibited the transmission. These results are consistent with the possibility that endogenous purines may modulate non-cholinergic non-adrenergic excitatory transmission in the rat urinary bladder.     

Altered urinary bladder function in mice lacking the vanilloid receptor TRPV1

In the urinary bladder, the capsaicin-gated ion channel TRPV1 is expressed both within afferent nerve terminals and within the epithelial cells that line the bladder lumen. To determine the significance of this expression pattern, we analyzed bladder function in mice lacking TRPV1. Compared with wild-type littermates, trpv1(-/-) mice had a higher frequency of low-amplitude, non-voiding bladder contractions. This alteration was accompanied by reductions in both spinal cord signaling and reflex voiding during bladder filling (under anesthesia). In vitro, stretch-evoked ATP release and membrane capacitance changes were diminished in bladders excised from trpv1(-/-) mice, as was hypoosmolality-evoked ATP release from cultured trpv1(-/-) urothelial cells. These findings indicate that TRPV1 participates in normal bladder function and is essential for normal mechanically evoked purinergic signaling by the urothelium.     

ATP, β-γ-methylene-ATP,and adenosine inhibit non-cholinergic non-adrenergic transmission in rat urinary bladder

ATP and adenosine caused a dose-dependent and reversible inhibition of the atropine-resistant contraction response to transmural nerve stimulation in the rat urinary bladder. Both purines also inhibited contraction responses to acetylcholine and direct muscle stimulation, indicating a postjunctional effect on the transmission. It seems as ATP per se inhibits the excitatory transmission, because the stable ATP-analogue β-γ-methylene-ATP was inhibitory as well, and because exogenous adenosine deaminase annulled the inhibition by adenosine but not that by ATP or β-γ-methylene-ATP. Blockade of purine in-activation enhanced the inhibitory action of ATP and adenosine, and by itself inhibited the transmission. These results are consistent with the possibility that endogenous purines may modulate non-cholinergic non-adrenergic excitatory transmission in the rat urinary bladder.