柴胡皂甙(I)对胰腺腺泡的拟膜受体激动剂作用

应用检测淀粉酶分泌和单细胞[Ca^2 ]的技术,研究了Bt2-cGMP和GDP对柴胡皂甙（Ⅰ）[SA(I)]和CCK－8促大鼠胰腺腺泡分泌和增加[Ca^2 ]i的抑制作用。Bt2-cGMP对SA（I）和CCK－8促酶分泌的抑制有相似的剂量依赖性。Bt2-cGMP对SA（I）刺激的酶分泌动力学的抑制较对CCK－8滞后并持续。SA（I）诱发的胰腺腺泡单细胞[Ca^2 ]i的变化与CCK－8的作用有所不同;[Ca^2 ]i峰值上升较慢且持续较长,并在峰后[Ca^2 ]i再次升高。GDP亦抑制SA（I）刺激的酶分泌和[Ca^2 ]i增加的峰值。结果表明,SA（I）可激活胰腺腺泡细胞膜受体从而升高[Ca^2 ]i和促酶分泌。     

GTPγs对柴胡皂甙(I)刺激胰腺腺泡酶分泌的影响

为了解柴胡皂甙(I)[SA(I)]刺激大鼠胰腺腺泡酶分泌的信号传导通路,研究了GTPγs对SA(I)剌激通透腺泡细胞酶分泌的影响.用SLO通透细胞的同时,加入GTPγs在15min期间能诱发酶分泌,10'mol·L-1GTPγs有最大促泌效应.GTPγs浓度依赖性的增强SA(I)促酶分泌作用,l0-7 mol·L-1 GTPγs导致10-5mol·L-1 SA(I)刺激酶分泌量增加到1.6倍.用SLO预通透腺泡10min后,加入GTPγs使SA(I)刺激酶分泌的量-效曲线左移,SA(I)的EC50从2 0×10-5mol·L-1减小到1 0×10-5mol·L-1.以上结果提示,SA(I)活化受体偶联的G蛋白包括在其刺激酶分泌的信号传导通路中.     

大黄素升高豚鼠结肠带细胞[Ca~(2 )]_i的特征和GDP的抑制作用

利用Fluo -3荧光探针检测细胞内自由Ca2 浓度([Ca2 ]i),研究了大黄素升高豚鼠结肠带细胞[Ca2 ]i 的量—效关系和动态变化特征,及GDP和胞外Ca2 浓度对其的影响。较低浓度大黄素随药物浓度增加使[Ca2 ]i 显著升高 ,更高浓度大黄素有超最大抑制效应。GDP对大黄素升高细胞[Ca2 ]i 的抑制作用随其浓度增加而增强。GDP和胞外Ca2 浓度影响大黄素诱发的[Ca2 ]i 动态变化的结果表明 :GDP使[Ca2 ]i 峰消失 ,胞外无Ca2 导致[Ca2 ]i 随时间显著下降 ,大黄素升高[Ca2 ]i 作用趋向消失。     

对牛胰多肽抑制胰酶分泌作用的离体研究

为探讨胰多肽抑制胰酶分泌的机制,我们利用大鼠离体胰腺泡制备观察了牛胰多肽(BPP)在细胞受体水平对氨甲酰胆碱等促分泌物作用的影响。实验结果显示,BPP 对氨甲酰胆碱诱导的胰腺泡淀粉酶分泌具有抑制作用,并存在剂量反应关系。BPP0.1μmol/L 和0.2μmol/L,可分别使氨甲酰胆碱诱导淀粉酶分泌的效价降低3倍和10倍;BPP 还可抑制氨甲酰胆碱刺激胰腺泡释放~(45)Ca。以上结果提示,BPP 对胰腺泡的胆碱能 M 受体具有拮抗作用。此外,BPP 对促胰液素及其同类激动剂和氨甲酰胆碱协同作用诱导的胰腺泡淀粉酶分泌具有抑制作用,提示胰多肽在整体对促胰液素诱导的胰酶分泌的抑制,可能是通过拮抗胰腺泡细胞上的 M 受体而抑制了促胰液素和胆碱能刺激协同作用引起的胰酶分泌。     

电离辐射后大鼠离体胰腺腺泡淀粉酶释放及M受体数量变化

我们曾观察到大鼠经γ-射线照射后胰淀粉酶活性降低和分泌减少[1],为进一步探讨照射后胰酶分泌减少的机制,本研究制备出分散的大鼠胰腺腺泡悬液并以不同浓度的~3H-二苯羟乙酸-3-喹咛环酯(~3Hquinuclidinyll benzilatc,简称~3H-QNB)进行M受体结合测定,同时观察胆碱能介质氨甲酰胆碱刺激腺泡所引起的淀粉酶释放反应。结果表明,γ-射线10Gy照射后3天,大鼠分散的胰腺腺泡在氨甲酰胆碱刺激时淀粉酶释放量减少到对照的50％,腺泡M受体与~3H-QNB最大结合量(Bmax)减少到对照的38％,伋M受体与~3H-QNB结合的解离常数(K_D)无改变,说明胰腺腺泡细胞M受体数量的减少可能是照射后胰腺腺泡分泌淀粉酶减少的原因之一。     

大黄素影响巨噬细胞升高[Ca~(2+)]_i和释放TNF-a的作用特征

为了研究大黄素(emodin)对正常的和细菌脂多糖(LPS)刺激的大鼠腹腔巨噬细胞(PM准)释放肿瘤坏死因子琢(TNF-琢)和升高[Ca2+]i的影响,应用L929细胞系和MTT法检测TNF-琢量,同时用激光共焦扫描显微术检测单细胞[Ca2+]i变化动力学。结果显示大黄素能轻度促进正常PM准释放TNF-琢,并发现大黄素诱发PM准[Ca2+]i变化呈振荡波模式。大黄素显著抑制LPS刺激PM准过度释放TNF-琢和升高[Ca2+]i,10-5mol/L大黄素抑制了10mg/LLPS刺激的TNF-琢峰值的50%和[Ca2+]i峰值的68%。LPS诱发PM准[Ca2+]i变化呈现高幅值的“平台期”,大黄素使之转变为低幅值的波动变化。以上结果说明,大黄素对PM准释放TNF-琢和升高[Ca2+]i表现出的双向调节作用之间有一定的相关性,大黄素对LPS诱发的[Ca2+]i升高的调制,可能是抑制LPS刺激PM准释放TNF-琢的信号传导通路中的重要环节。     

发现一种蛙皮素受体阻断剂

蛙皮素是最早由蛙的皮肤中提取出来的一种14肽,它的类似物已在哺乳动物的胃肠道、脑和肺中发现。这些肽有广泛而强烈的生物学效应,并且在胰腺腺泡、脑膜和垂体细胞的膜上存在受体,至今尚未发现其阻断剂。美国R.T.Jensen等最近发现了一种蛙皮素受体的竞争性阻断剂,它是人工合成的P物质(SP)类似物和拮抗剂,即[D-精,D-脯,D-色,亮]SP(简记作ASP)。他们用豚鼠的游离胰腺腺泡进行实验。已知腺泡上存在蛙皮素、P物质、胆碱(M型)、CCK、VIP和促胰液素的受体。其中蛙皮素受体仅对蛙皮素及其类似物有亲和力,它的激动可动员胞内钙并刺激胰酶分泌。蛙皮素(0.03～1nmol)可使胰淀粉酶释放增加(最大达10倍),剂量效应曲线呈S型。ASP本身(即使     

垂体腺苷酸环化酶激活多肽对鲤鱼脑垂体细胞内cAMP和Ca^2＋的影响

采用环腺苷酸 (cAMP)放射免疫测定法和活细胞内Ca2 荧光探针Indo 1,研究绵羊垂体腺苷酸环化酶激活多肽 (oPACAP)对原代培养的鲤鱼脑垂体细胞内cAMP和游离Ca2 ([Ca2 ]i)的影响 ,以期探讨PACAP调节脑垂体生长激素 (GH)分泌的机制受体后。oPACAP 38和oPACAP 2 7以剂量依存方式促进脑垂体细胞内cAMP释放和合成。oPACAP 38和oPACAP 2 7也能升高脑垂体细胞内 [Ca2 ]i 水平 ,该作用会因用EGTA消竭细胞外Ca2 ([Ca2 ]e)而迅速消失 ;L型电位敏感性Ca2 通道 (VSCC)阻断剂硝苯吡啶可抑制oPACAP 38诱导的 [Ca2 ]i 水平的升高 ,而当用硝苯吡啶预处理脑垂体细胞 ,oPACAP 38诱导 [Ca2 ]i 水平升高作用完全被抑制。可见 ,PACAP刺激鲤鱼脑垂体GH分泌机制包括依赖于cAMP和依赖于通过L型VSCC内流的 [Ca2 ]e 的机制。     

小檗碱对豚鼠结肠平滑肌细胞内游离钙浓度的影响

采用Ca2 荧光示踪剂Fura 2 AM和双波长荧光分光光度法 ,观察小檗碱 (berberine ,Ber)对酶法分离的豚鼠结肠平滑肌细胞内游离钙 ([Ca2 ]i)的影响并探讨其机制。在含 1 5mmol/LCaCl2 的HEPES Ringer缓冲液中 ,豚鼠结肠平滑肌细胞 [Ca2 ]i为 10 8± 9 4nmol/L (n =7) ,Ber对静息 [Ca2 ]i 无明显影响 ,Ber呈浓度依赖性抑制 ,6 0mmol/LKCl引起的 [Ca2 ]i 增高 ,IC50 值为 34 0 9μmol/L。在含 1 5mmol/LCa2 和无Ca2 的缓冲液中 ,30、10 0μmol/LBer均显著抑制 10 μmol/LACh所诱发的 [Ca2 ]i 的增高 ,且有浓度依赖性 ;同样Ber对环匹阿尼酸 (CPA)所致的 [Ca2 ]i 增高也有浓度依赖性抑制作用 ,有钙和无钙条件下IC50 分别为 37 97μmol/L和 49 70 μmol/L。结果提示 ,Ber对结肠平滑肌细胞外Ca2 内流和细胞内钙释放均有抑制作用。     

八肽胆囊收缩素激活钙离子通道和酪氨酸激酶诱导豚鼠心肌细胞内的游离钙增高

探讨八肽胆囊收缩素(CCK-8)对豚鼠单个心肌细胞内游离钙浓度([Ca2+]i的影响及其信号转导机制.Fluo 3-AM标记酶消化法分离的单个心室肌细胞,用激光共聚焦显微镜测定细胞内[Ca2+]i的浓度.[Ca2+]i的变化用荧光强度(Fi)和相对荧光强度(Fi/F0%)表示.实验结果如下(1)在含Ca2+1.0 mmol/L的Tyrode's液中,CCK-8(1～104pmoVL)均可引起[Ca2+]i快速显著上升(P＜0.01).(2)用钙离子鳌合剂EGTA(3 mmol/L)和钙离子通道阻断剂nisoldipine(0.5μmol/L)预孵育心肌细胞5 min,CCK-8(102pmol/L)仅可引起[Ca2+]i缓慢轻度上升(P＜0.01).(3)用非选择性CCK受体拮抗剂丙谷胺(proglumide 6μmo1/L)或酪氨酸激酶抑制剂genistein(1 μmol/L)预孵育心肌细胞5 min,则完全抑制CCK-8诱导的[Ca2+]i升高(P＜0.01).CCK-8可通过激活其受体控制的Ca2+通道,引起Ca2+内流,诱导细胞内Ca2+释放,引起豚鼠单个心肌细胞内[Ca2+]i上升,此作用可能由酪氨酸激酶介导.     

Cyclic GMP does not inhibit protein kinase C-mediated enzyme secretion in rat pancreatic acini

In pancreatic acini, cGMP can be increased by secretagogues such as cholecystokinin (CCK), cholinergic agents, and bombesin, whose actions on enzyme secretion are believed to be mediated by protein kinase C. However, the role of cGMP in acinar cell function has been unclear. A recent paper by Rogers et al. (Rogers, J., Hughes, R.G., and Matthews, E. K. (1988) J. Biol. Chem. 263, 3713-3719) reported that two analogues of cGMP, N2,O2-dibutyl guanosine 3':5'-monophosphate (Bt2cGMP) and 8-bromoguanosine 3':5'-monophosphate (8Br-cGMP), at concentrations in the nanomolar range, inhibited the stimulation of amylase secretion caused by CCK-8, bethanechol, bombesin, and 12-O-tetradecanoylphorbol-13-acetate (TPA). Rogers et al. also reported that sodium nitroprusside inhibited the stimulation of enzyme secretion caused by CCK-8 or TPA. These authors concluded that cGMP inhibits protein kinase C-mediated secretion in pancreatic acini. In the present study we attempted to confirm the findings of Rogers et al., We found, however, that Bt2cGMP inhibited CCK-8-stimulated amylase release only at concentrations of the nucleotide above 10 microM. Moreover, there was a close correlation between the ability of Bt2cGMP to inhibit CCK-8-stimulated amylase release and its ability to inhibit binding of 125I-CCK-8. Bt2cGMP, at concentrations as high as 3 mM, did not alter the stimulation of amylase release caused by carbachol, bombesin, TPA, or A23187. 8Br-cGMP, at concentrations up to 1 mM, did not inhibit the stimulation of amylase release caused by CCK-8 or TPA. At concentrations above 0.1 mM, 8Br-cGMP augmented the stimulation of amylase release caused by CCK-8, carbachol, bombesin, or TPA. Sodium nitroprusside, at a concentration that causes a 60-fold increase in cGMP, did not inhibit the stimulation of amylase release caused by CCK-8, carbachol, bombesin, or TPA. Our results do not confirm the findings of Rogers et al. and indicate that cGMP does not inhibit protein kinase C-mediated secretion in pancreatic acini.     

Involvement of cellular calcium in exocrine pancreatic insufficiency during streptozotocin-induced diabetes mellitus

This study investigates the effects of the islet hormones insulin (Ins), glucagon (Glu), and somatostatin (Som) with nerve stimulation (EFS) acetylcholine (ACh) and cholecytokinin-octapeptide (CCK-8) on amylase secretion and intracellular free calcium concentration [Ca(2+)](i) in the pancreas of age-matched control and diabetic rats. Either Ins, Glu or Som elicited small increases in amylase secretion from the pancreas of age-matched control animals compared to a much larger increase in amylase secretion with either EFS, ACh or CCK-8. Combining the islet hormones with either EFS, ACh or CCK-8 resulted in marked potentiation of amylase output. In the diabetic pancreas, the islet hormones had no effect on amylase secretion compared to diabetic control. Moreover, either EFS, ACh or CCK-8 evoked a much smaller increase in amylase output compared to age-matched control. In addition, the islet hormones failed to potentiate the secretory effects of either EFS, ACh or CCK-8. In fura-2 loaded acinar cells from age-matched control pancreas either Ins or Glu elicited a small increase in [Ca(2+)](i) whereas Som had no effect. Both ACh and CCK-8 evoked large increases in [Ca(2+)](i) compared to control. Combining either Ins, Glu or Som with either ACh or CCK-8 resulted in a marked elevation in [Ca(2+)](i) compared to the responses obtained with either the islet hormones, ACh or CCK-8 alone. In diabetic fura-2 loaded pancreatic acinar cells, the islet hormones had no effect on [Ca(2+)](i) compared to control and moreover, the responses were much smaller than those obtained in acinar cells from age-matched control. Both ACh and CCK-8 induced large increases in [Ca(2+)]( i) in diabetic acinar cells. However, combining the islet hormones with either ACh or CCK-8 failed to enhance [Ca(2+)](i) compared to the reponses obtained in acinar cells from age-matched control. The results suggests that [Ca(2+)](i) homeostasis is deranged during diabetes mellitus and this in turn is probably associated with reduced pancreatic amylase secretion.     

Effects of reactive oxygen species on actin filament polymerisation and amylase secretion in mouse pancreatic acinar cells

The present study investigates the effect of reactive oxygen species (ROS) on actin filament reorganisation and its relevance to exocytosis in pancreatic acinar cells. Treatment of pancreatic acini with cholecystokinin (CCK-8) induced spatial and temporal changes in actin filament reorganisation with an initial depolymerisation of the apical actin barrier followed by an increase in the actin filament content in the subapical area leading to amylase release. Hydrogen peroxide (H(2)O(2)) increased actin filament content and potentiated the polymerizing effects of CCK-8 in these cells but abolished the disruption of the apical actin layer and amylase release induced by CCK-8. Similar to CCK-8, ROS generated by the oxidation of hypoxanthine (HX) with xanthine oxidase (XOD) induced an initial decrease in actin filaments located under the apical membrane followed by a smaller increase in the content of actin filaments in the subapical area. XOD-generated ROS are able to increase amylase release in pancreatic acini although combination with CCK-8 leads to abnormal exocytosis. We provide evidence that indicates that CCK-8- and ROS-induced actin reorganisation is entirely dependent on Ca(2+) mobilisation and independent of PKC activation. The regulation of the actin cytoskeleton by ROS might be involved in radical-induced cell injury in pancreatic acinar cells.     

Phenylarsine oxide evokes intracellular calcium increases and amylase secretion in isolated rat pancreatic acinar cells

The effects of the thiol reagent, phenylarsine oxide (PAO, 10(-5)-10(-3) M ), a membrane-permeable trivalent arsenical compound that specifically complexes vicinal sulfhydryl groups of proteins to form stable ring structures, were studied by monitoring intracellular free calcium concentration ([Ca2+]i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. PAO increased [Ca2+]i by mobilizing calcium from intracellular stores, since this increase was observed in the absence of extracellular calcium. PAO also prevented the CCK-8-induced signal of [Ca2+]i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF-4). In addition to the effects of PAO on calcium mobilization, it caused a significant increase in amylase secretion and reduced the secretory response to either CCK-8 or AlF-4. The effects of PAO on both [Ca2+]i and amylase release were reversed by the sulfhydryl reducing agent, dithiothreitol (2 mM). Pretreatment of acinar cells with high concentration of ryanodine (50 microM) reduced the PAO-evoked calcium release. However, PAO was still able to release a small fraction of Ca2+ from acinar cells in which agonist-releasable Ca2+ pools had been previously depleted by thapsigargin (0.5 microM) and ryanodine receptors were blocked by 50 microM ryanodine. We conclude that, in pancreatic acinar cells, PAO mainly releases Ca2+ from the ryanodine-sensitive calcium pool and consequently induces amylase secretion. These effects are likely to be due to the oxidizing effects of this compound.     

Phosphatidic acid production,required for cholecystokinin octapeptide-stimulated amylase secretion from pancreatic acinar AR42J cells,is regulated by a wortmannin-sensitive process

To investigate the role of phospholipids in exocytotic secretory events, we utilized rat pancreatic acinar AR42J cells that secreted amylase in response to cholecystokinin octapeptide (CCK-8). Wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), was found to inhibit the secretion in a dose-dependent manner. When changes in cell membrane phospholipids were investigated before and after CCK-8 stimulation using [32P]orthophosphoric acid-labeled AR42J cells, we observed a rapid increase in phosphatidic acid (PtdOH) levels right after stimulation, which was not observed in non-stimulated cells. The increase, however, was suppressed by wortmannin pre-treatment, which also inhibited amylase secretion. Changes in other major phospholipids were not significant. These results indicate that CCK-8 induces amylase secretion through PI3K-regulated production of PtdOH in cell membranes.     

Effects of an inhibitor of myosin light chain kinase on amylase secretion from rat pancreatic acini

Ca(2+)/calmodulin-dependent protein (CaM) kinases play an important role in Ca(2+)-mediated secretory mechanisms. Previously, we demonstrated that a CaM kinase II inhibitor KN-62 had a small inhibitory effect on amylase secretion stimulated by CCK. In the present study, we investigated the effects of a myosin light chain kinase (MLCK) inhibitor on amylase secretion and Ca(2+) signaling in rat pancreatic acini. A specific inhibitor of MLCK, wortmannin, inhibited amylase secretion stimulated by CCK-8 (30 pM) in a concentration-dependent manner. Wortmannin (10 microM) had no effects on basal secretion but reduced amylase secretion stimulated by CCK-8 (30 pM) by 67 +/- 3%. Wortmannin inhibited amylase secretion stimulated by calcium ionophore (A23187) and phorbol ester (TPA). Wortmannin also inhibited amylase response to thapsigargin by 76 +/- 8% and to both thapsigargin and TPA by 52 +/- 10%. Ca(2+) oscillations evoked by CCK-8 (10 pM) were inhibited by wortmannin (10 microM). Wortmannin had a little inhibitory effect on an initial rise in [Ca(2+)](i), and abolished a subsequent sustained elevation of [Ca(2+)](i) evoked by 1 nM CCK-8. In conclusion, MLCK plays a crucial role in amylase secretion from pancreatic acini and regulates Ca(2+) entry from the extracellular space.     

Melatonin modulates Ca2+ mobilization and amylase release in response to cholecystokinin octapeptide in mouse pancreatic acinar cells

In the present work, we have evaluated the effect of an acute addition of melatonin on cholecystokinin octapeptide (CCK-8)-evoked Ca²⁺ signals and amylase secretion in mouse pancreatic acinar cells. For this purpose, freshly isolated mouse pancreatic acinar cells were loaded with fura-2 to study intracellular free Ca²⁺ concentration ([Ca²⁺]_c). Amylase release and cell viability were studied employing colorimetric methods. Our results show that CCK-8 evoked a biphasic effect on amylase secretion, finding a maximum at a concentration of 0.1 nM and a reduction of secretion at higher concentrations. Pre-incubation of cells with melatonin (1 μM–1 mM) significantly attenuated enzyme secretion in response to high concentrations of CCK-8. Stimulation of cells with 1 nM CCK-8 led to a transient increase in [Ca²⁺]_c, followed by a decrease towards a constant level. In the presence of 1 mM melatonin, stimulation of cells with CCK-8 resulted in a smaller [Ca²⁺]_c peak response, a faster rate of decay of [Ca²⁺]_c and lower values for the steady state of [Ca²⁺]_c, compared with the effect of CCK-8 alone. Melatonin also reduced the oscillatory pattern of Ca²⁺ mobilization evoked by a physiological concentration of CCK-8 (20 pM), and completely inhibited Ca²⁺ mobilization induced by 10 pM CCK-8. On the other hand, Ca²⁺ entry from the extracellular space was not affected in the presence of melatonin. Finally, melatonin alone did not change cell viability. We conclude that melatonin, at concentrations higher than those found in blood, might regulate exocrine pancreatic function via modulation of Ca²⁺ signals.     

Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca2+ entry in mouse pancreatic acinar cells

We recently reported that store-operated Ca²⁺ entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca²⁺ channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as "secretion-like coupling." As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca²⁺ entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca²⁺ influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca²⁺ store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type. synaptosome-associated protein; vesicle-associated membrane protein; pancreatic acinar cells; cytoskeleton; calcium entry