人类脑动脉瘤血管平滑肌细胞表型蛋白与雌激素受体表达

目的 探讨正常人体动脉血管和脑动脉瘤的血管平滑肌(VSMC)雌激素受体(ERs)和α-平滑肌肌动蛋白(α-SMA)和结蛋白(Desmin)表达变化的关系.方法 临床取材8例非动脉瘤患者手术区头部动脉血管和脑动脉瘤切除标本18例.采用免疫组织化学和形态学分析方法 检测正常动脉血管和脑动脉瘤ERs、α-SMA和Desmin的表达.结果 脑动脉瘤VSMC的ERα和ERβ表达水平均高于对照动脉血管表达水平,差异有统计学意义(P<0.01).脑动脉瘤VSMC的α-SMA和Desmin的表达水平均低于对照动脉血管表达水平,差异有统计学意义(P<0.01).脑动脉瘤VSMCERs的表达与α-SMA和Desmin的表达水平明显相关.结论 人类脑动脉瘤VSMC的ERs表达水平普遍升高,其VSMC表型蛋白表达水平普遍降低,两者之间呈负相关,这可能与脑动脉瘤的形成有关.     

Effects of ATP-sensitive potassium channel openers on the contractile and phosphatidylinositol responses of the rat trachea

Purpose. Although ATP-sensitive potassium channel openers suppress airway smooth muscle contraction, their potencies are different and the mechanisms involved are not fully understood. We examined the effects of cromakalim and Y-26763, a novel ATP-sensitive potassium channel opener, on the contractile and phosphatidylinositol responses of the rat trachea. Methods. Thirty-six male Wistar rats, weighing 250–350 g, were used. In the experiment on contractile response, active contraction was induced with 0.55 μM carbachol in the presence or absence of cromakalim or Y-26763. In the experiment on phosphatidylinositol response, the tracheal slices were incubated with [³H]myo-inositol, 0.55 μM carbachol, and cromakalim or Y-26763, and the formation of [³H]inositol monophosphate (IP₁), a degradation product of phosphatidylinositol response, was measured with a liquid scintillation counter. Statistical significance (P < 0.05) was determined by analysis of variance (ANOVA). Results. Carbachol-induced tension was attenuated by both cromakalim and Y-26763, the latter displaying significantly greater potency. Carbachol-induced IP₁ accumulation was influenced neither by cromakalim nor by Y-26763. Conclusion. Both cromakalim and Y-26763 have effects on airway smooth muscle relaxation. Carbachol-induced IP₁ accumulation was influenced neither by cromakalim nor by Y-26763, suggesting that phosphatidylinositol response may not be a common pathway for the effect of ATP-sensitive potassium channel openers. Received: January 15, 2002 / Accepted: June 14, 2002 Acknowledgment. This study was supported in part by Grant-in-Aid for Scientific Research C, no. 10671421, from the Ministry of Education, Science, and Culture, Japan. Address correspondence to: O. Shibata     

α1 adrenergic receptor agonist,phenylephrine, actively contracts early rat rib fracture callus ex vivo

Early, soft fracture callus that links fracture ends together is smooth muscle‐like in nature. We aimed to determine if early fracture callus could be induced to contract and relax ex vivo by similar pathways to smooth muscle, that is, contraction via α₁ adrenergic receptor (α₁AR) activation with phenylephrine (PE) and relaxation via β₂ adrenergic receptor (β₂AR) stimulation with terbutaline. A sensitive force transducer quantified 7 day rat rib fracture callus responses in modified Krebs–Henseliet (KH) solutions. Unfractured ribs along with 7, 14, and 21 day fracture calluses were analyzed for both α₁AR and β₂AR gene expression using qPCR, whilst 7 day fracture callus was examined via immunohistochemistry for both α₁AR and β₂AR‐ immunoreactivity. In 7 day callus, PE (10^?6 M) significantly induced an increase in force that was greater than passive force generated in calcium‐free KH (n = 8, mean 51% increase, 95% CI: 26–76%). PE‐induced contractions in calluses were attenuated by the α₁AR antagonist, prazosin (10^?6 M; n = 7, mean 5% increase, 95% CI: 2–11%). Terbutaline did not relax callus. Gene expression of α₁ARs was constant throughout fracture healing; however, β₂AR expression was down‐regulated at 7 days compared to unfractured rib (p < 0.01). Furthermore, osteoprogenitor cells of early fibrous callus displayed considerable α₁AR‐like immunoreactivity but not β₂AR‐like immunoreactivity. Here, we demonstrate for the first time that early fracture callus can be pharmacologically induced to contract. We propose that increased concentrations of α₁AR agonists such as noradrenaline may tonically contract callus in vivo to promote osteogenesis. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:740–745, 2011     

A new enzymic method for the isolation and culture of human bladder body smooth muscle cells

Cultured cells of the human urinary bladder smooth muscle are useful for investigating bladder function, but methods for culturing them are not well developed. We have now established a novel enzymic technique. The smooth muscle layer was separated out and incubated with 0.2% trypsin for 30 min at 37 degrees C. The samples were then minced and incubated with 0.1% collagenase for 30 min and centrifuged at 900 g. The pellets were resuspended in RPMI-1640 medium containing 10% fetal calf serum (FCS) and centrifuged at 250 g. The smooth muscle cells from the supernatant were cultured in RPMI-1640 containing 10% FCS. The cells grew to confluence after 7-10 days, forming the "hills and valleys" growth pattern characteristic of smooth muscle cells. Immunostaining with anti-alpha-actin, anti-myosin, and anti-caldesmon antibodies demonstrated that 99% of the cells were smooth muscle cells. To investigate the pharmacological properties of the cultured cells, we determined the inhibitory effect of muscarinic receptor antagonists on the binding of [3H]N-methylscopolamine to membranes from cultured cells. The pKi values obtained for six antagonists agreed with the corresponding values for transfected cells expressing the human muscarinic M2 subtype. Furthermore, carbachol produced an increase in the concentration of cytoplasmic free Ca2+ an action that was blocked by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 selective antagonist. This result suggests that these cells express functional M3 muscarinic receptors, in addition to M2 receptors. The subcultured cells therefore appear to be unaffected by our new isolation method.     

Effects of three different L-type Ca2+ entry blockers on airway constriction induced by muscarinic receptor stimulation

Background. The crucial role of L-type Ca²⁺ channels in airwaysmooth muscle contraction suggests that these channels couldbe an important therapeutic target. There are three separatedrug binding sites on this channel: those for dihydropyridines,benzothiazepines and phenyl alkylamines. In this study, we examinedthe effects of the dihydropyridines nifedipine and nicardipine,the benzothiazepine diltiazem, and the phenylalkylamine verapamilon airway constriction. Methods. Tension of guinea-pig tracheal strips was measuredisometrically in vitro with a force displacement transducer.Strips were precontracted with carbachol 10^–7 M with orwithout 4-aminopyridine 10^–3 M, a voltage-sensitive K⁺channel blocker. Then, nifedipine 10^–8–10^–4M, diltiazem 10^–8–3x10^–4 M or verapamil 10^–8–3x10^–4M was added cumulatively to the organ bath (n=6 each). The bronchialcross-sectional area of pentobarbital-anaesthetized dogs wasassessed using a bronchoscopy method. Bronchoconstriction waselicited with methacholine 0.5 µg kg^–1 plus 5 µgkg^–1 min^–1, and then nicardipine 0–1000 µgkg^–1, diltiazem 0–3000 µg kg^–1 or verapamil0–3000 µg kg^–1 were given i.v. (n=7 each). Results. In the in vitro experiments, nifedipine and diltiazemfully reversed carbachol-mediated tracheal contraction withlogIC₅₀ values of 4.76 (SEM 0.22) (mean 17.5 µM) and 4.60(0.33) (mean 24.8 µM), respectively. Although verapamil10^–6–10^–4 M reversed the contraction by 87.2%,strip tension re-increased by 18.1% following maximal relaxationwith verapamil 3x10^–4 M. This re-increase was almost fullyabolished by pretreatment with 4-aminopyridine. In the in vivoexperiments, nicardipine and diltiazem dose-dependently reversedmethacholine-induced bronchoconstriction, with logID₅₀ valuesof 3.22 (0.05) (mean 0.60 mg kg^–1) and 1.85 (0.32) (mean14.0 mg kg^–1), respectively. Verapamil worsened methacholine-inducedbronchoconstriction. Conclusions. Although supraclinical doses of dihydropyridinesand benzothiazepines can produce airway relaxant effects, theseagents are unlikely to be used in the treatment of bronchoconstriction.In addition, verapamil may aggravate airway constriction. Br J Anaesth 2003; 90: 671–5     

Chronic high pressure potentiates the antiproliferative effect and abolishes contractile phenotypic changes caused by endothelial cells in cocultured smooth muscle cells

High in vitro pressures have been reported to alter smooth muscle cell (SMC) and endothelial cell (EC) phenotype, while endothelial cells (ECs) can influence the proliferation, phenotype, and contractile features of smooth muscle cells (SMC) in coculture systems. However, little is known about the in vitro effects of pressure on EC/SMC cocultures. We therefore sought to compare SMC proliferation in independent and EC coculture under ambient and high pressure, and identify changes in the contractile phenotype of SMCs by measuring levels of the L-type Ca(2+) channel a(1) subunit (dihydropyridine-DHP receptor) which is critical for Ca(2+) transients, differentiation and contractility in SMC. METHODS: Rat aortic SMCs in independent culture (SMC/0) and coculture with ECs (SMC/EC) were maintained in 5% CO(2) under either atmospheric or high pressure (130 mmHg). SMC were counted at 0, 1, 3, and 5 days and compared to initial cell counts of day 0 before the exposure to experimental conditions. DHP receptor levels were quantitated by Western blotting (three similar studies). RESULTS: ECs suppressed SMC proliferation on day 1 of coculture in both atmospheric and high pressure (20% inhibition vs independent culture, P < or = 0.05). By day 3, cocultured SMC under atmospheric pressure displayed no EC-mediated inhibition, and at day 5, atmospheric cocultured SMCs revealed statistically significant enhanced proliferation as compared with SMCs in independent cultures. However, cocultured SMCs exposed to 130 mmHg pressure displayed sustained sensitivity to EC growth inhibition at both days 3 and 5 of the experiment. Coculture decreased SMC DHP-receptor levels under atmospheric pressure. However, this effect was abolished in cocultures exposed to high pressure. CONCLUSIONS: High pressure substantially alters the regulatory influence of EC on SMC proliferation and contractile potential. This pressure/coculture model should increase our understanding of cellular interaction in hypertensive vasculopathy.     

Correlation of in vitro pressure generation with urinary bladder function

Urinary bladder function consists of a filling and storage phase followed by an active coordinated expulsion phase. A large part of our knowledge of bladder function, as well as bladder physiology and pharmacology, comes from a variety of in-vivo and in-vitro experimental animal models. Of the in-vitro methodologies available for the study of bladder smooth muscle, the isolated bladder smooth muscle strip techniques and isolated whole bladder techniques are two of the most popular. In general, the ability to generate pressure in the non-emptying whole bladder model is directly related to phasic contraction of the bladder smooth muscle, and can be equated with the response of isolated strips. Although both techniques generate useful and important information, one must be very careful in making conclusions concerning bladder function based solely on contractile data. We have studied the volume-pressure relationship of the bladders of control rabbits and of those subjected to bladder outlet obstruction (2 week) and unilateral ischemia (2 week). For each bladder, in-vitro cystometry, pressure generation, and the ability of the bladder to empty (in response to both bethanechol and field stimulation) was determined. Although it was clear that outlet obstruction and unilateral ischemia induced marked alterations in bladder compliance, capacity, and the ability to empty, bladder pressure generation was not significantly affected. These studies support the view that contractile studies in and of themselves cannot be used to describe the functional state of the bladder. Only through the application of several techniques including studies on bladder compliance, capacity, and the ability of the bladder to empty can the functional state of the bladder be evaluated.     

Vascular effects of poly-N-acetylglucosamine in isolated rat aortic rings.

BAACKGROUND: Poly-N-acetylglucosamine (p-GlcNAc) is a secretion of marine diatoms that is known to be useful in controlling bleeding. As a component of promoting hemostasis, p-GlcNAc is thought to exert vasoconstrictor effects in arteries. The present study was undertaken to determine whether p-GlcNAc induced a significant vasoconstrictor effect and, if so, what the mechanism of this effect might be. MATERIALS AND METHODS: We examined vascular effects of p-GlcNAc on isolated aortic rings obtained from Sprague-Dawley rats. The rings were suspended in organ baths and precontracted with U46619, a thromboxane A2 mimetic. RESULTS: p-GlcNAc produced a concentration-dependent vasoconstriction over the range of 14 to 100 microg/ml. At a concentration of 100 microg/ml, p-GlcNAc significantly contracted aortic rings by 133 +/- 20 mg of developed force (P < 0.01). Neither a deacetylated derivative of p-GlcNAc nor a structurally related macromolecule, chitin, contracted rat aortic rings, indicating a specificity for p-GlcNAc. The vasoconstriction to p-GlcNAc was totally abolished in deendothelialized rat aortic rings, suggesting that an endothelial component is essential to the vasoconstriction. Pretreatment with the endothelin ET(A) receptor antagonist, JKC-301 (0.5 and 1 microM), significantly diminished p-GlcNAc-induced vasoconstriction by 57 to 61% (P < 0.01). However, p-GlcNAc did not significantly diminish nitric oxide release from rat aortic endothelium. CONCLUSION: These results provide evidence that p-GlcNAc significantly contracts isolated rat aortic rings via an endothelium-dependent mechanism, partly via enhancement of endothelin-1 release from endothelial cells.     

Mast cell migration to Th2 stimulated airway smooth muscle from asthmatics

BACKGROUND: Mast cell microlocalisation within the airway smooth muscle (ASM) bundle is an important determinant of the asthmatic phenotype. We hypothesised that mast cells migrate towards ASM in response to ASM derived chemokines. METHODS: Primary ASM cultures from subjects with and without asthma were stimulated with interleukin (IL)-1beta, IL-4, and IL-13 alone and in combination. Mast cell chemotaxis towards these ASM supernatants was investigated, and the chemotaxins mediating migration by using specific blocking antibodies for stem cell factor (SCF) and the chemokine receptors CCR3, CXCR1, 3 and 4 as well as the Gi inhibitor pertussis toxin and the tyrosine kinase inhibitor genistein were defined. The concentrations of CCL11, CXCL8, CXCL10, TGF-beta, and SCF in the supernatants were measured and the effect of non-asthmatic ASM supernatants on the mast cell chemotactic activity of asthmatic ASM was examined. RESULTS: Human lung mast cells and HMC-1 cells migrated towards Th2 stimulated ASM from asthmatics but not non-asthmatics. Mast cell migration was mediated through the combined activation of CCR3 and CXCR1. CCL11 and CXCL8 expression by ASM increased markedly after stimulation, but was similar in those with and without asthma. ASM supernatants from non-asthmatics inhibited mast cell migration towards the asthmatic ASM supernatant. CONCLUSION: Th2 stimulated ASM from asthmatics is chemotactic for mast cells. Non-asthmatic ASM releases a mediator or mediators that inhibit mast cell migration towards stimulated asthmatic ASM. Specifically targeting mast cell migration into the ASM bundle may provide a novel treatment for asthma.     

利多卡因对气管平滑肌舒张作用的研究

利多卡因是临床上常用的局部麻醉药和抗室性心律失常药物;除此之外,利多卡因还具有较强的扩张气道、抑制气道炎症、降低气道高反应性的作用。此方面的研究最早可追溯到上世纪60年代,利多卡因在围术期预防和处理支气管痉挛中所占的地位已得到充分肯定,现将近10余年来利多卡因对气道平滑肌影响的主要研究成果综述如下。     

Characterization of the control of intracellular [Ca2+] and the contractile phenotype of cultured human detrusor smooth muscle cells

PURPOSE: We measured the functional properties of cultured human detrusor myocytes with respect to their ability to regulate their intracellular [Ca2+] and generate force in collagen matrices. MATERIALS AND METHODS: Human detrusor biopsies were dissociated into single cells by collagenase treatment and used immediately or cultured in D-valine medium and subsequently used after culture trypsinization. Intracellular [Ca2+] was measured in Fura-2 loaded myocytes. Cell force development was measured by incorporating cells into a collagen gel and attaching it to an isometric strain gauge. RESULTS: Carbachol was equally effective in generating Ca transients in freshly isolated and cultured cells. Carbachol potency (pEC50) and the magnitude of Ca2+ transients were similar. Adenosine triphosphate potency was decreased in cultured cells and Ca2+ transients showed properties consistent with a purinoceptor shift from a purinergic subtype. Temporal restitution of Ca2+ transients was similar in the 2 groups, indicative of retained intracellular Ca2+ stores in cultured cells. Cultured cells (approximately 10(6)) embedded in collagen gel generated a force about 10 times greater than that generated by gel alone. The cell dependent force could be further increased by adding carbachol. CONCLUSIONS: Cultured cells retain the ability to generate agonist induced intracellular Ca2+ transients. There was no evidence that the cell culture altered the properties of muscarinic receptors, although purinoceptor mediated properties were altered. Restitution experiments indicated that functional intracellular Ca2+ stores were retained in cultured cells. Cultured cells also retained a contractile phenotype, especially in response to carbachol. The magnitude of force was attenuated, which may be a function of the biomechanical properties of the gel used to embed the cells.     

不同浓度的咪唑安定对兔离体气管平滑肌收缩的影响

目的研究不同浓度的咪唑安定对离体气管平滑肌的舒张作用,以明确咪唑安定在10-5mol/L浓度时是否能够产生较为明显的舒张作用。方法用高浓度氯化钾、乙酰胆碱、电脉冲三种刺激因素诱发兔离体气管平滑肌收缩,并观察三种不同浓度的咪唑安定对其的影响。结果1.5×10-5mol/L的咪唑安定对三种刺激因素诱发的气管平滑肌收缩不产生明显抑制作用;1.5×10-4mol/L和3.0×10-4mol/L的咪唑安定可显著抑制上述三种刺激因素诱发的气管平滑肌收缩(P<0.05或P<0.01),普萘洛尔和中枢性苯二氮卓艹类受体阻断药氟马西尼不能拮抗咪唑安定对气管平滑肌的舒张作用。结论咪唑安定在10-5mol/L左右的浓度时对兔离体气管平滑肌收缩不能产生明显的抑制作用。     

Estrogen induced functional hypertrophy and increased force generation of the female rabbit bladder

AIMS: Estrogen is essential for physiological maintenance of the female urogenital tract. It is believed that alterations in female sex hormones play a major role in the etiology and response to urinary tract dysfunctions. In animal studies, ovariectomy (Ovx) results in smooth muscle (SM) weakness and atrophy whereas estrogen supplementation reverses these effects. Our study seeks to establish the mechanisms by which estrogen augmentation results in increased contractility. METHODS: Twenty New Zealand White female rabbits were separated into five groups of four each. Group 1 served as control, rabbits of groups 2-5 were ovariectomized, group 2 ovariectomized received no estradiol, groups 3-5 were given 17-beta estradiol (1 mg/kg/day) by subcutaneous slow release tablet implant for 1, 3, and 7 days, respectively, beginning 2 weeks after Ovx. At the end of the experimental period, each rabbit was anesthetized and the urinary bladder was removed for contractile, histological, and biochemical studies. RESULTS: Ovx resulted in significantly decreased bladder contractile function, whereas bladders tested after estradiol administration showed increased contractility. Ovx resulted in a decrease in SM/collagen ratio, whereas estrogen resulted in an increase. The estrogen receptor (ER) density significantly increased following Ovx. After 1 day of estrogen treatment, the ER density decreased significantly below control levels, but rose progressively during the estrogen treatment. CONCLUSION: The present study demonstrates that estrogen supplementation mediates a "functional hypertrophy," that is a hypertrophy characterized by increased contractile responses to all forms of stimulation, and an increased ratio of SM/collagen.     

Localized contractions in the normal human bladder and in urinary urgency

OBJECTIVE: To describe an observational study to establish whether localized activity arises in the normal human bladder, and whether there is any correspondence between changes in such activity and reported sensation. PATIENTS, SUBJECTS AND METHODS: The generation of sensory information by the bladder depends on afferent stimulation by increased tension within the bladder wall. Autonomous bladder activity is apparent in several species, which is often localized and multifocal, giving rise to localized areas of stretch. Thus afferent activity may partly result from localized distortions of the bladder wall. Fourteen women patients presenting with increased bladder sensation during filling-phase cystometry were compared with six asymptomatic women volunteers. Localized bladder activity was assessed by the micromotion detection (MMD) method, using eight electrodes mounted on a Silastic balloon; local displacements of the electrodes were recorded as changes in electrical resistance, which were used to compute changes in the distance between each pair of electrodes. RESULTS: In two of the six volunteers, micromotions were seen in the extraperitoneal (ventral) portion of the bladder. Women with increased sensation on filling cystometry had a significantly higher prevalence of localized activity than the control group during MMD recording. The localized activity was more sustained and at a higher frequency than in asymptomatic women. All nine women reporting urinary urgency during MMD recording had localized contractile activity, while only four had phasic increases in detrusor pressure during the episodes of urgency. CONCLUSIONS: By measuring localized contractions within the bladder wall, we established a significant difference in the prevalence of localized activity between the groups studied, but there was no objective difference with conventional urodynamic studies. There was also a difference in the character of the localized contractions, with the exaggerated activity in the symptomatic group corresponding with the reported sensations. These findings suggest that localized distortion of the bladder wall stimulates afferent activity, and that the human detrusor may be functionally modular.     

Effect of partial urethral obstruction on force development of the guinea pig bladder

We created gradual partial urethral obstruction in 20 guinea pigs using silver jeweler's jump rings. After 4 or 8 weeks obstruction all animals underwent cystometry and were assigned to one of five urodynamic categories: normal, high pressure voiding, unstable, low compliance, or decompensated. After sacrifice, the contractile responses of bladder strips to electrical field stimulation of intramural nerves, direct electrical muscle stimulation, 0.1 mM carbachol, and high K + solution were sampled by computer for phase plot analysis. Following 8 weeks obstruction, the value of the phase plot parameter Fiso, indicative of the number of contractile muscle units, was reduced to 60% of the control response to nerve stimulation (P < 0.05) and to 77% of the control response to carbachol stimulation (P < 0.05). Parameter C, the slope of the phase plot (indicative of unit recruitment during force development), was unchanged for all forms of stimulation. Although in the latter case not statistically significant, obstruction affected responses to nerve and muscle stimulation similarly suggesting that muscle change may possibly be a common denominator of dysfunction. In view of the reduction in Fiso and the increase in bladder weight, instability may represent a more advanced form of dysfunction due to obstruction than high pressure voiding.