人骨肉瘤细胞株MG63功能性N-甲基-D-天冬氨酸受体的表达

目的:研究人骨肉瘤细胞株MG63上功能性N-甲基-D-天冬氨酸(NMDA)受体的表达。方法:利用钙离子成像系统检测NMDA或谷氨酸对MG63细胞内游离钙离子浓度([Ca2+]i)的影响。结果:仅6.4%MG63细胞对NMDA产生[Ca2+]i升高反应,对谷氨酸则没有反应。多数MG63细胞呈现自发性的钙离子震荡现象,且该现象不能被MK801所阻断。结论:MG63细胞表达的功能性NMDA受体很少。     

Specific receptors for leukotriene C4 on a smooth muscle cell line.

Specific receptors for leukotriene C4 (LTC4) have been identified on an intact smooth muscle cell line, DDT1 MF-2 cells derived from the Syrian hamster vas deferens. Specific [3H]LTC4 binding at a fixed input at 4 degrees C was rapid, reached a plateau at 86% of total binding at 60 min, and was reversible upon addition of excess homoligand. With incremental inputs of radioligand and a constant cell number, specific [3H]LTC4 binding reached a plateau indicative of saturable binding sites. LIGAND analysis of the Scatchard plot demonstrated a single high affinity binding site with a dissociation constant (Kd) of 5 nM. With incremental inputs of unlabeled LTC4, LIGAND analysis of the Scatchard plot demonstrated a single high affinity site with a Kd of 4.4 nM and in some experiments an additional low affinity site with a Kd of 634 nM. The myotonically active structural analogues of LTC4, 5(R),6(S)-LTC4, 11-trans-LTC4, and C1-monoamide-LTC4, competed effectively with radiolabeled LTC4 such that the relative Kd values of these heteroligands were within one log of that of the homoligand. In contrast, the other native sulfidopeptide leukotrienes, leukotriene D4 and leukotriene E4, exhibited relative Kd values that were 2-3 logs less than that of LTC4. Thus, the high affinity receptor on the DDT1 smooth muscle cell line is specific for a single constituent, LTC4, of slow reacting substance of anaphylaxis.     

Pharmacological and biochemical evidence for metabolism of peptide leukotrienes by guinea-pig airway smooth muscle in vitro

It has been demonstrated that leukotriene (LT)C4 is metabolized to LTD4 via the action of the enzyme gamma-glutamyl transpeptidase. LTD4 is, in turn, converted by the enzyme aminopeptidase to LTE4. In the present study, the pharmacological effects of the aminopeptidase inhibitor, L-cysteine and the gamma-glutamyl transpeptidase inhibitor, L-serine borate, on peptide LT concentration-response curves were evaluated in isolated guinea-pig trachea. L-Cysteine (3 mM) enhanced the contractile activity of both LTC4 and LTD4. L-Serine borate (45 mM) enhanced the contractile activity of LTC4 without altering the response to LTD4. In contrast, neither L-cysteine nor L-serine borate consistently altered the concentration-response curves to LTE4, histamine or carbachol, which rules out a nonspecific effect of these inhibitors on airway smooth muscle. In the absence of enzyme inhibitors the peptide LTs were equipotent; whereas in their presence the relative order of potency was LTC4 = LTD4 greater than LTE4. Incubation of isolated guinea-pig trachea with [3H]LTC4 resulted in the formation of [3H]LTD4 and [3H]LTE4 with a proportional decrease in [3H]LTC4. The bioconversion of [3H]LTC4 was blocked by L-serine borate in a concentration-related manner (IC50 = 3.4 +/- 0.5 mM, mean +/- S.E.M., n = 3) and the formation of LTE4 was blocked by L-cysteine (10 mM). The results suggest that LTC4 is converted to LTD4 and subsequently to LTE4 by isolated guinea-pig trachea. The potency of LTC4 and LTD4 is increased when their transformation to LTE4 is prevented.     

Differentiation of a human B cell hybridoma with interleukin-2 receptor

A 6-thioguanine-neo-resistant clone, Raji 6-TGR-neoR was fused with pokeweed mitogen-stimulated B cells (PWM-B cells) obtained from a normal healthy donor, and selected by a new selection medium (HAT-neo). Two hybrid clones, HYNI-1 and HYNI-2, were established. These clones expressed surface IgG and HLA class II antigens derived from PWM-B cells, and they had about 20 more chromosomes than Raji 6-TGR-neoR. More significantly, one clone, HYNI-2, expressed interleukin-2 receptor on the cell surface, and generated IgG when stimulated with recombinant interleukin-2. These findings indicate that the HYNI-2 is a good model for studying human B cell differentiation.     

低强度超声诱导人产后离体子宫平滑肌收缩

目的 探讨低强度超声对人产后离体子宫平滑肌收缩的影响. 方法 以人产后离体子宫平滑肌条为研究对象,利用频率为0.8 MHz,声强为2 W/cm2的低强度超声辐照标本10 min,采用BL-410生物机能实验系统记录子宫平滑肌的收缩情况,并用光镜观察辐照前后子宫平滑肌形态变化. 结果 低强度超声辐照人产后离体子宫平滑肌收缩频率、幅度、张力及子宫活动力均明显高于辐照前(P<0.05). 结论 低强度超声可诱导人产后离体子宫平滑肌收缩.     

Bcl-2和p53蛋白在子宫平滑肌肿瘤的表达和意义

目的　了解Bcl- 2和p5 3蛋白在子宫平滑肌肿瘤 (USMTs)的表达和意义。方法　在 2 0例良性子宫平滑肌瘤 (UL)、18例子宫平滑肌肉瘤 (LMS)及 70例交界性子宫平滑肌瘤 (BLM) :包括 4 0例富于细胞型子宫平滑肌瘤 (CL)、13例奇异型子宫平滑肌瘤 (BL)、4例核分裂活跃型子宫平滑肌瘤 (ML)、10例不典型子宫平滑肌瘤 (AL)及 3例恶性潜能未定型子宫平滑肌瘤 (STUMP)应用免疫组织化学法检测Bcl- 2和p5 3蛋白的表达。结果　UL组bcl- 2蛋白表达阳性率明显高于LMS组 (P <0 .0 5 ) ;BLM组bcl- 2蛋白表达阳性率显著高于LMS组 (P <0 .0 1)。LMS组p5 3蛋白表达阳性率显著高于UL组 (P <0 .0 1)和BLM组 (P <0 .0 1)。结论　本研究结果提示 :细胞凋亡旁路的抑制即通过Bcl- 2和p5 3蛋白的过度表达可能在USMT的发生、发展起重要作用     

Pharmacological evidence for a distinct leukotriene C4 receptor in guinea-pig trachea

The effect of the leukotriene (LT) antagonist, FPL55712, on the contractile activity of peptide leukotrienes was evaluated in the presence and absence of enzyme inhibitors of leukotriene metabolism. L-Cysteine, an inhibitor of aminopeptidase, prevents the formation of LTE4 from LTD4 and LTC4. L-Serine borate, an inhibitor of gamma-glutamyl transpeptidase prevents the conversion of LTC4 to LTD4. L-Cysteine (3mM) enhanced the contractile activity of LTC4 and LTD4. L-Serine borate (45 mM) increased selectively the contractile activity of LTC4. FPL55712 (10 microM) antagonized the contractile activity of LTC4, LTD4 and LTE4 in the absence of enzyme inhibitors. In the presence of L-serine borate, FPL55712 (10-30 microM) failed to antagonize the contractile activity of LTC4 but did antagonize LTD4 and LTE4. However, the dissociation constant (KB) for FPL55712 against LTD4 was increased by L-serine borate whereas the KB against LTE4 was not altered. In the presence of L-cysteine, FPL55712 antagonized the contractile activity of the peptide LTs. However, FPL55712 was more effective (P less than .05) in antagonizing LTD4 and LTE4 than LTC4 in the presence of L-cysteine. The data suggest that when the conversion of LTC4 to LTD4 and subsequently to LTE4 is blocked by L-serine borate FPL55712 is ineffective in antagonizing the actions of LTC4. This would indicate that LTC4 occupies a distinct LT receptor in guinea-pig trachea which is insensitive to the actions of FPL55712.     

CD117在子宫平滑肌肿瘤中表达的探讨

目的研究胃肠道外间质肿瘤(子宫平滑肌肿瘤),c-kit(CD117)的阳性表达率,以证实子宫平滑肌肉瘤是否能表达CD117,是否具备胃肠道间质肿瘤细胞中所含的c-kit(酪氨酸激酶)。旨在寻找新的治疗途径。方法55例子宫平滑肌肿瘤均测定SMA及CD117,并选用经病理确诊的胃肠道间质肿瘤5例为阳性对照。结果55例子宫平滑肌肿瘤测定SMA证实均来源子平滑肌,CD117阳性率从低到高依次为平滑肌瘤、多细胞、奇异性、恶性潜能未定及平滑肌肉瘤。结论CD117阳性且不可切除者或转移性子宫平滑肌肉瘤患者可考虑分子靶向治疗。     

子宫交界性平肌肿瘤增殖细胞核抗原表达的研究

目的 探讨子宫交界性平滑肌肿瘤（ＢＬＭ）细胞的增殖活性。方法 应用抗ＰＣＮＡ单克隆抗体,采用Ｓ－Ｐ免疫组化方法对６３例子宫交界性平滑肌瘤和２０例普通子宫平滑肌瘤（ＵＬ）和１０例子宫平滑肌肉瘤（ＬＭＳ）进行研究。结果 ９６.８％的子宫平滑肌肿瘤（usMT)PCNA阳性,但ＰＣＮＡ随恶性度增高强阳性率增加。结论 在usMT中,PCNA的强阳性随病理程度加重有增加趋势。提示ＰＣＮＡ强阳性有助于ＢＬＭ及ＬＭＳ的诊断。     

Prevention of smooth muscle cell outgrowth from human atherosclerotic plaque by a recombinant cytotoxin specific for the epidermal growth factor receptor.

Smooth muscle cell proliferation in the intima of arteries is a principal event associated with vascular narrowing after balloon angioplasty and bypass surgery. Techniques for limiting smooth muscle cell proliferation, however, have not as yet yielded any therapeutic benefit for these conditions. This may reflect the present lack of sufficiently potent and specific inhibitors of smooth muscle cell proliferation. DAB389 EGF is a genetically engineered fusion protein in which the receptor-binding domain of diphtheria toxin has been replaced by human epidermal growth factor. We evaluated the effect of this fusion toxin on human vascular smooth muscle cells in culture. Incubation of proliferating cells with DAB389 EGF yielded a dose-dependent inhibition of protein synthesis, as assessed by uptake of [3H]leucine, with an IC50 of 40 pM. The cytotoxic effect was inhibited in the presence of excess EGF or with monoclonal antibody to the EGF receptor. We further studied the effect of the fusion toxin on smooth muscle cell outgrowth from human atherosclerotic plaque. Outgrowth was markedly inhibited after as little as 1 h of exposure to the fusion protein. Furthermore, complete inhibition of proliferation of cells within the plaque could be attained. These results demonstrate that DAB389 EGF is highly cytotoxic to human smooth muscle cells proliferating in culture and can prevent smooth muscle cell outgrowth from "growth-stimulated" human atherosclerotic plaque. DAB389 EGF may therefore be of therapeutic value in vascular diseases characterized by smooth muscle cell accumulation.     

Pharmacological characterization of [Leu-13-psi-CH2NH-Leu14]-bombesin as a specific bombesin receptor antagonist on isolated smooth muscle cells

Isolated smooth muscle cells from guinea pig stomach were used to study the pharmacological characteristics of a newly synthetized bombesin analog, [Leu13-psi-CH2NH-Leu14]-bombesin (psi 13,14-BN) to function as antagonist of bombesin-induced contractile response. The antagonism caused by this new analog was compared to that obtained with the substance P analog [D-Arg1,D-Pro2,D-Trp7,9,Leu11]Substance P [( APTTL]SP), which has been used until now to characterize bombesin receptors on smooth muscle cells. psi 13,14-BN resulted to be more potent than [APTTL]SP as antagonist of bombesin action on smooth muscle. Comparing the IC50, psi 13,14-BN (IC50 70 nM) was 8 times more potent than [APTTL]SP (IC50 600 nM). In contrast to [APTTL] SP, the action of psi 13,14-BN was shown to be specific toward bombesin receptors in that it does not interfere with receptors for other agents (i.e., cholecystokinin, acetylcholine or substance P). The antagonism induced by both compounds was competitive inasmuch as the slope of the regression lines obtained by Schild plot analysis were not significantly different from the unity. The apparent affinity for the bombesin receptor was 0.8 nM for psi 13,14-BN and 7.8 nM for [APTTL]SP. These results indicate that psi 13,14-BN acts on isolated gastric smooth muscle cells as a competitive bombesin receptor antagonist, with a higher affinity and specificity than the substance P analog used previously.     

Effects of N-ethylmaleimide on arginine vasopressin-induced responses in an established smooth muscle cell line

We have previously reported that 8-arginine vasopressin (AVP) stimulates phosphatidylinositol turnover and Ca++ mobilization in rat aortic smooth muscle cells (A10). In the present study, N-ethylmaleimide (NEM) was used to further characterize the putative guanine nucleotide binding protein that transduces the V1 receptor effects on phosphatidylinositol turnover and Ca++ efflux in these cells. Pretreatment of the cells with low concentrations of NEM did not affect the basal levels of the inositol phosphates and Ca++ efflux but significantly inhibited the AVP-induced increases. NEM pretreatment did not significantly affect [3H]AVP binding to intact cells. Guanyl-5'-yl imidodiphosphate reduced the apparent binding affinity of AVP to cell membranes. NEM pretreatment abolished this guanyl-5'-yl imidodiphosphate effect. AVP stimulated a specific GTPase activity in cell membranes; this effect was also abolished by NEM pretreatment. The results suggest that in A10 cells a guanine nucleotide binding protein sensitive to NEM couples vasopressin receptors to phospholipase C.     

Characterization of serotonin-1B receptors negatively coupled to adenylate cyclase in OK cells, a renal epithelial cell line from the opossum

We have previously reported the presence of a 5-HT1 (serotonin)-like receptor coupled in an inhibitory manner to adenylate cyclase in the opossum kidney cell line, which is derived from the kidney of a North American opossum. Pharmacological data from binding and cyclic AMP production studies indicate that this receptor does not have characteristics of a 5-HT1A, 5-HT1C or 5-HT1D receptor, but is similar to 5-HT1B receptors found in rodent tissues. Many serotonergic drugs, including 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyrindinyl)1H-indol, 5-HT and methysergide, but not (+/-)-8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide or buspirone, were full agonists at this receptor as defined by the inhibition of bovine parathyroid hormone peptide fragment 1-34-stimulated cyclic AMP production in an intact cell assay. Several classical beta adrenergic antagonists including propranolol and cyanopindolol were also full agonists at this receptor. Radioligand binding studies using [125I](-)-iodocyanopindolol gave a Bmax of 88 fmol/mg of protein and a KD of 47 pM for saturation experiments carried out in the presence of GTP. In the absence of GTP, the binding data were significantly better fit by a two-site model with KD values of 10 and 345 pM. Inhibition binding experiments were consistent with the results of the cyclic AMP experiments. The identification of 5-HT1B receptors in a tissue derived from the opossum kidney suggests that these receptors may be distributed more widely than previously thought, inasmuch as other studies have found them only in neuronal tissues of rodents.(ABSTRACT TRUNCATED AT 250 WORDS)     

血管平滑肌β-肾上腺能受体对细胞凋亡的影响

目的探讨刺激生理状态及过表达的血管平滑肌细胞 β2 肾上腺能受体 (VSMCβ2 AR)对细胞凋亡的影响。方法应用腺病毒介导的转基因技术过表达VSMCβ2 AR ,采用Hoechst 3 3 3 42染色和流式细胞仪分析生理状态下和 β2 AR过表达活化后细胞凋亡情况。结果应用异丙基肾上腺素刺激VSMCβ2 AR 2 4h后 ,流式细胞仪检测存活VSMC数量与对照组相比较并无明显减少 ,48h后较对照组明显减少 (P <0 0 1) ;过表达 β2 AR的VSMC应用异丙基肾上腺素刺激 2 4h后 ,流式细胞仪检测细胞存活率明显下降(P <0 0 1) ,Hoechst染色显示凋亡细胞率明显升高 (P <0 0 1)。结论刺激生理状态及过表达的VSMCβ2 AR具有促进凋亡的效应。     

Pharmacological properties of potassium currents in swine tracheal smooth muscle.

The pharmacological properties of large conductance Ca(++)-activated K+ channels and spontaneous transient outward currents (STOCs) in swine tracheal smooth muscle (TSM) were studied using enzymatically dissociated single cells and patch clamp techniques. Recording from inside-out patches showed that extracellular tetraethylammonium caused a concentration-dependent decrease in single channel current amplitude. Acetylcholine (ACh; 10(-8) M) induced repetitive outward current transients in cell-attached patches due to simultaneous opening of many large conductance Ca(++)-activated K+ channels. Whole-cell recordings revealed the presence of STOCs in TSM membrane that were inhibited by tetraethylammonium at concentrations similar to those which inhibited the large conductance Ca(++)-activated K+ channels. Caffeine (5 mM) stimulated and then inhibited STOCs, suggesting that they are induced by the release of calcium from internal stores. ACh (1 microM) initially increased STOC frequency followed by an inhibition of STOCs. The effects of ACh occurred in control solution (2 mM Ca++) or when calcium influx was eliminated (0 Ca++, 2 mM Mn++). ACh also induced an inward current at negative membrane potentials associated with an increase in conductance. We conclude that inhibition of STOCs (i.e., potassium channel activity) and induction of an inward cation current by ACh may give rise to the depolarization observed in the presence of ACh and be important in the development of ACh-induced contractions in TSM.