NO-cGMP信号转导系统的上调参与阿片类药物耐受和戒断的生化机制

目的　观察阿片激动剂长时程作用对NO-cGMP信号转导系统的影响。方法　选用iNOS cDNA稳定表达的NG-LNCXiNOS细胞,采用NOS活性和cGMP放免测定,Western杂交和NADPH黄递酶组化染色技术。结果阿片类药物长时程作用剂量依赖性增高胞浆相iNOS活性和胞内cGMP含量,药物作用强弱顺序是DPDPE> DADLE>吗啡,EC₅₀都在nmol.L^-1数量级。用纳洛酮急性戒断阿片耐受细胞,造成酶活性和cGMP水平增加更显著。DPDPE长时程作用还引起iNOS基因表达增强和NADPH黄递酶染色阳性细胞增多。结论　提示NO-cGMP信号转导系统上调可能是阿片耐受和成瘾的重要生化改变。     

前胡丙素对Ang II致离体血管平滑肌细胞肥厚及胞内钙、NO含量和信号转导的影响

目的前胡丙素(Pra-C)对血管紧张素II(Ang II)致离体培养大鼠血管平滑肌细胞(SMCs)肥厚模型胞内游离钙浓度、NO含量和信号转导的影响。方法以Ang II刺激SMCs形成肥厚模型,用倒置显微镜测定SMCs面积;用Fura-2/AM测定单细胞内［Ca²⁺］i,Griess法测定NO含量;在PMA和ST(PKC激动剂及抑制剂)、PTX(Gi蛋白敏感毒素)作用下观察Pra-C对KCl和NE所致胞内［Ca²⁺］i浓度变化的影响。结果Pra-C组SMCs细胞面积较肥厚组减小39.01%,并接近正常细胞水平;NO含量明显增加;胞内［Ca²⁺］i对KCl和NE激动的反应明显低于肥厚组。PMA使肥厚SMCs［Ca²⁺］i升高,而ST及PTX则使之降低,Pra-C均使之恢复正常。结论Pra-C抑制Ang II致体外培养细胞SMCs肥厚,改善肥厚细胞因PKC和Gi蛋白的信号转导改变所致的［Ca²⁺］i改变。     

羟苯氨酮强心作用的生化机理研究

目的：研究羟苯氨酮(oxyphenamone, Oxy)强心作用的生化机理。方法：采用Na⁺,K⁺-ATP酶活性和cAMP-PDE活性、肌浆网Ca²⁺-ATP酶活性和cAMP含量以及心肌肌原纤维Ca²⁺,Mg²⁺-ATP酶活性等测定法,研究Oxy对它们的影响,并与milrinone和MCI-154作比较。 结果：Oxy对Na⁺,K⁺-ATP酶和PDE无抑制作用,也不影响心肌cAMP含量,但能显著增强心肌肌原纤维对Ca²⁺的敏感性,高浓度时轻度抑制心肌肌浆网Ca²⁺-ATP酶活性。结论：Oxy的强心作用方式不同于强心苷、β受体激动剂和PDE抑制剂等强心药,可能为一种新的钙增敏性强心药物。     

体外液压冲击伤对大鼠神经细胞内游离钙和pH值的影响

目的研究体外液压冲击伤后大鼠神经细胞内游离钙和pH值的变化及其药物的保护作用。方法培养新生乳鼠的大脑皮层神经细胞,给予2.5kPa,20ms的液压冲击伤,通过激光扫描共聚焦显微镜检测伤后单个神经细胞内游离［Ca²⁺］i和pH值的变化,并分别给予尼莫地平和D-AP-5,观察药物对上述变化的影响。结果伤后细胞内［Ca²⁺］i迅速升高,持续12h达高峰,随后逐渐下降,48h接近正常;pH值下降较慢,于12h达低谷,48h未恢复正常。尼莫地平和D-AP-5均可明显抑制细胞内［Ca²⁺］i的升高和pH值的下降。结论可根据液压冲击伤后神经细胞内游离［Ca²⁺］i及pH值的变化规律指导用药。     

β受体激动剂异丙肾上腺素对小鼠子宫cAMP水平的影响

异丙肾上腺素(ISP)可明显升高子宫cAMP水平,此作用可被心得安能完全拮抗。在去极化小鼠子宫中,ISP升高cAMP的作用与外Ca²⁺浓度呈反相关系。利血平化后,ISP作用明显增强。提示ISP引起小鼠子宫松驰作用,可能与其激动β受体从而升高cAMP有关;ca²+)与cAMP间有互相拮抗作用,而利血平化后,ISP作用增强可能与此时子宫平滑肌β受体对外源性激动剂效应增敏有关。     

环维黄杨星D对分离大鼠心室肌细胞内Ca2+和L型钙电流的影响

目的研究环维黄杨星D（CD）对大鼠心室肌细胞内Ca²⁺动员和L型钙电流(I_{Ca-L/sub>)的影响。方法采用全细胞膜片钳和激光扫描共聚焦显微术研究CD对心肌细胞ICa-L/sub>以及氯化钾、咖啡因诱发心肌细胞内Ca²⁺动员的影响。结果CD浓度依赖性抑制ICa-L/sub>。指令电压为10 mV时，1和10 μmol·L^-1 CD分别使ICa-L/sub>电流密度从（-9.9±1.8）pA/pF降至（-6.4±1.4）pA/pF和（-4.2±0.6）pA/pF。共聚焦实验显示1和10 μmol·L^-1 CD不影响静息心肌细胞［Ca²⁺］i?/sub>，对氯化钾诱发［Ca²⁺］i?/sub>升高水平无明显抑制作用；咖啡因引起的细胞内Ca²⁺动员可被CD进一步增强。结论CD浓度依赖性抑制大鼠心室肌细胞ICa-L/sub>，并有促进咖啡因诱发心肌细胞内Ca²⁺释放的作用。}     

小檗碱对培养大鼠心肌细胞胞内游离Ca2+的作用

利用Fura-2技术和AR-CM-MIC阳离子测定系统,直接观察了小檗碱对培养大鼠心肌细胞胞内[Ca²⁺]i的影响。结果显示:小檗碱可明显升高心肌细胞静息[Ca²⁺]i且具饱合性,维拉帕米和CoCl₂对其有一定的抑制作用;小檗碱与高K⁺,高Ca²⁺,去甲肾上腺素,哇巴因合用比单用上述激动剂更能明显增高[Ca²⁺]i;维拉帕米对其有抑制作用;在胞外无外Ca²⁺和无外Ca²⁺,外K⁺,外Na⁺时,小檗碱30～200μmol·L^-1仍能升高静息[Ca²⁺]i,维拉帕米只对前者有一定抑制作用。结果提示:小檗碱可能通过促胞外Ca²⁺内流和胞内Ca²⁺释放等途径有限度地增高心肌细胞内游离Ca²⁺浓度,显示强心作用。     

三七总皂苷对大鼠左心室构型重建的影响

目的探讨三七总皂苷(total saponins of Panax notoginseng,PNS)对大鼠左室重构中一氧化氮(NO)和环磷酸鸟苷(cGMP)含量的影响及其相关机制。方法异丙肾上腺素(isoproterenol,ISO)5 mg·kg^-1·d^-1,sc,连续7 d,建立大鼠心肌肥厚模型。造模第二天开始大鼠腹腔注射PNS 25和50 mg·kg^-1·d^-1,连续14 d,测定心脏重量参数;分光光度法检测左心室心肌组织中NO含量和一氧化氮合酶(NOS)活力;放免分析法检测左心室心肌组织中环磷酸鸟苷(cGMP)和环磷酸腺苷(cAMP)含量。结果ISO模型组大鼠心脏重量参数明显增大;左心室诱生型NOS(iNOS)活力和cAMP含量显著升高;NO、cGMP含量和结构型NOS(cNOS)活力显著降低。PNS能明显提高心肌组织NO水平、cNOS活力和cGMP的含量;降低iNOS活力和cAMP含量;减轻心脏重量;抑制左室重构。结论PNS改善大鼠左心室构型重建的作用与其提高NO和cGMP含量有关。     

γ-羟基丁酸受体在大鼠局灶性脑缺血再灌注损伤中的作用

研究γ-羟基丁酸(gamma-hydroxybutyric acid，GHB)受体在大鼠局灶性脑缺血再灌注损伤中的作用及其机制。选用GHB受体选择性激动剂NCS-356和特异性拮抗剂NCS-382作为工具药，采用改良的Longa法制备大鼠大脑中动脉栓塞(MCAO)模型。缺血2 h再灌注2 h后，动物进行Longa法行为功能评分；再灌注24 h后，部分动物用TTC染色法测定大鼠脑梗死体积；部分动物应用流式细胞仪测定神经细胞内游离钙离子浓度；分光光度法测定缺血侧大脑皮质中总一氧化氮合酶(tNOS)、诱导型一氧化氮合酶(iNOS)活性和一氧化氮(NO)含量；放射免疫法测定大鼠缺血侧大脑皮层环磷酸鸟苷(cGMP)含量。Isc/R组大鼠行为功能评分、脑梗死体积、神经细胞内游离Ca²⁺浓度、cGMP和NO含量、tNOS及iNOS活性均显著高于假手术组；NCS-356 160 μg·kg^-1(N₁)、NCS-356 320 μg·kg^-1(N₂)、 NCS-356 640 μg·kg^-1(N₃)和尼莫地平600 μg·kg^-1(Nim)组的上述各指标均不同程度地低于Isc/R组，而NCS-382 640 μg·kg^-1+NCS-356 640 μg·kg^-1 (NCS-382+N₃)组则能显著对抗N₃组的作用。激动GHB受体对大鼠局灶性脑缺血再灌注损伤具有一定的保护作用，其作用机制可能与降低神经细胞内游离Ca²⁺浓度，减少NO及cGMP含量有关。     

银杏叶提取物对低氧复氧、H2O2和谷氨酸损伤时谷氨酸引起的大鼠星形胶质细胞［Ca2+］i变化的影响

目的研究银杏叶提取物对低氧复氧、H₂O₂和L-谷氨酸损伤时谷氨酸升高大鼠星形胶质细胞［Ca²⁺］_i的影响。方法钙荧光探针Fluo-3/AM标记胞浆内游离钙离子，激光扫描共聚焦显微镜测定［Ca²⁺］_i的变化。结果 在低氧复氧、H₂O₂以及高浓度的L-谷氨酸损伤后，外源性谷氨酸（27 μmol·L^-1）均不能引起培养乳大鼠星形胶质细胞正常的［Ca²⁺］_i升高，反而使［Ca²⁺］_i分别降低(3.3±1.6)%，(81±11)%和(81±7)%；损伤前预先给予GbE（10 mg·L^-1）不能明显改善星形胶质细胞的谷氨酸反应，但预先给予GbE（100 mg·L^-1）后，27 μmol·L^-1谷氨酸可使损伤的星形胶质细胞［Ca²⁺］_i分别升高(135±98)%，(117±93)%和(89±36)%。结论低氧复氧、H₂O₂以及高浓度的L-谷氨酸均能损伤星形胶质细胞的谷氨酸反应，影响神经细胞与胶质细胞的双向交流。GbE能明显逆转不同损伤后谷氨酸诱导星形胶质细胞［Ca²⁺］_i的异常变化，使星形胶质细胞在不同损伤时能维持正常功能，该作用可能与GbE的脑保护作用有关。     

NO-cGMP信息通路在阿片类药物耐受和依赖中的调节作用

ＮＯ合酶抑制剂能阻断或逆转μ、δ激动剂耐受和戒断反应,而对κ激动剂无明显作用,其药理效应类似于阿片成瘾治疗的常用药物可乐定。甲基兰抑制鸟苷酸环化酶活性,使ｃＧＭＰ生成减少,对阿片类药物耐受和戒断症状均有阻断作用,提示ＮＯ ｃＧＭＰ通路在阿片耐受和依赖发生中起重要作用,为寻找和设计治疗阿片类耐受成瘾的药物提供新的策略。     

阿片类药物对诱导型NO合酶稳定表达神经细胞受体介导AC-cAMP系统的影响

目的:利用稳定表达诱导型NO合酶的NG LNCXiNOS细胞,观察阿片类药物对受体介导AC cAMP系统的影响。方法:竞争性蛋白结合法和完整细胞受体结合实验测定cAMP含量和受体结合力。结果:阿片类药物短时程作用抑制AC活性,受阿片受体介导的、百日咳毒素敏感的G蛋白信号通路调节。阿片类药物长时程作用和纳洛酮急性戒断引起受体脱敏,呈时间和剂量依赖性,伴有受体下调。结论:成功地建立阿片耐受和依赖细胞模型,可用于研究AC cAMP系统和NO cGMP系统在阿片耐受和成瘾中的调节。     

Inhibition of guinea pig aortic sarcolemmal Ca2+-Mg2+ ATPase and cAMP phosphodiesterase activity by milrinone

Milrinone is a positive inotrope/vasodilator that inhibits cardiovascular low K_m cAMP phosphodiesterase (PDE) and not Na⁺?K⁺ ATPase activity. To explore other possible mechanisms of action, we quantitated the effects of milrinone on Ca²⁺-stimulated Mg²⁺ ATPase activity in guinea pig aortic smooth muscle plasma membranes. Milrinone inhibited Ca²⁺- stimulated activity, but not basal activity, in aortic microsomes. Maximum inhibition (70%) occurred at 1 μM, which coincided with the inflection of a parabolic dose-response curve. In a sarcolemmal-enriched (F1) aortic preparation, 1 μM cAMP, 1 μM Cl-930 (another low K_m cAMP PDE inhibitor), and 100 μM W-7 (a calmodulin antagonist) all inhibited Ca² -stimulated Mg²⁺ ATPase activity. This F1 preparation contained cAMP PDE activity which was inhibited by 1 μM milrinone (26%) and 1 μM Cl-930 (40%) but not by 100 μM W-7. The inhibition of F1 Ca²⁺ -Mg²⁺ ATPase activity by 1 μM milrinone could be diminished by increasing the concentration of CaCl₂ in reaction mixtures. In sum, these studies show that milrinone can inhibit vascular sarcolemmal Ca²⁺ -stimulated Mg²⁺ ATPase activity. However, inhibition may be direct or direct or may be secondary to cAMP PDE inhibition in vascular sarcolemma, since inhibition also occurs with cAMP and another low-K_m cAMP PDE inhibitor, Cl-930.     

Relaxation of rat thoracic aorta induced by 2,2′,2″-tripyridine

The pharmacological and toxicological activity of 2,2′,2″-tripyridine was determined in rat thoracic aorta. 2,2′,2″-Tripyridine inhibited norepinephrine (3 μM)-induced phasic and tonic contractions in the thoracic aorta as well as the endothelium-denuded aorta of the rat. The tonic pre-contraction elicited by norepinephrine was also relaxed by the addition of 2,2′,2″-tripyridine and this relaxing effect was not affected by indomethacin (20 μM) but partially antagonized by methylene blue (50 μM). In high-K⁺ medium (80 mM), 2,2′,2″-tripyridine inhibited the Ca²⁺ concentration dependent vasocontraction. Moreover, in Ca²⁺-free medium, the phasic contraction induced by either norepinephrine (3 μM) or caffeine (10 mM) was also supressed by 2,2′,2″-tripyridine. Although the cAMP level of rat aorta was not changed by 2,2′,2″-tripyridine, cGMP level was significantly increased by 2,2′,2″-tripyridine. The increase in cGMP level caused by 2,2′,2″-tripyridine was completely blocked by methylene blue (50 μM). The ⁴⁵Ca²⁺ influx elicited by either norepinephrine or high-K⁺ was inhibited by 2,2′,2″-tripyridine. All of these findings indicate that 2,2′,2″-tripyridine relaxes rat thoracic aorta by virtue of its Ca²⁺-channel blocking properties and by elevating cGMP levels in vascular smooth muscle.     

Inhibition of nitric oxide-activated guanylyl cyclase by calmodulin antagonists

Background and purpose:

Nitric oxide (NO) controls numerous physiological processes by activation of its receptor, guanylyl cyclase (sGC), leading to the accumulation of 3′-5′ cyclic guanosine monophosphate (cGMP). Ca²⁺-calmodulin (CaM) regulates both NO synthesis by NO synthase and cGMP hydrolysis by phosphodiesterase-1. We report that, unexpectedly, the CaM antagonists, calmidazolium, phenoxybenzamine and trifluoperazine, also inhibited cGMP accumulation in cerebellar cells evoked by an exogenous NO donor, with IC₅₀ values of 11, 80 and 180 µM respectively. Here we sought to elucidate the underlying mechanism(s).

Experimental approach:

We used cerebellar cell suspensions to determine the influence of CaM antagonists on all steps of the NO-cGMP pathway. Homogenized tissue and purified enzyme were used to test effects of calmidazolium on sGC activity.

Key results:

Inhibition of cGMP accumulation in the cells did not depend on changes in intracellular Ca²⁺ concentration. Degradation of cGMP and inactivation of NO were both inhibited by the CaM antagonists, ruling out increased loss of cGMP or NO as explanations. Instead, calmidazolium directly inhibited purified sGC (IC₅₀= 10 µM). The inhibition was not in competition with NO, nor did it arise from displacement of the haem moiety from sGC. Calmidazolium decreased enzyme V_max and K_m, indicating that it acts in an uncompetitive manner.

Conclusions and implications:

The disruption of every stage of NO signal transduction by common CaM antagonists, unrelated to CaM antagonism, cautions against their utility as pharmacological tools. More positively, the compounds exemplify a novel class of sGC inhibitors that, with improved selectivity, may be therapeutically valuable.     

Xanthine‐analog,KMUP‐2, enhances cyclic GMP and K+ channel activities in rabbit aorta and corpus cavernosum with associated penile erection

The pharmacological properties of KMUP‐2 were examined in isolated rabbit aorta and corpus cavernosum smooth muscle (CCSM). KMUP‐2 caused relaxations that were attenuated by removed endothelium, high K⁺, and pretreatment with the soluble guanylate cyclase (sGC) inhibitors methylene blue (10 μM) and ODQ (1 μM), a NOS inhibitor, L‐NAME (100 μM), a K⁺ channel blocker TEA (10 mM), a K_ATP channel blocker glibenclamide (1 μM), a voltage‐dependent K⁺ channel blocker 4‐AP (100 μM), and the Ca²⁺‐dependent K⁺ channel blockers apamin (1 μM) and charybdotoxin (ChTX, 0.1 μM). The relaxant responses of KMUP‐2 (0.01, 0.05, 0.1 μM) together with a PDE inhibitor, IBMX (0.5 μM), had additive effects on rabbit aorta and CCSM. Additionally, KMUP‐2 (100 μM) also affected cGMP metabolism, due to its inhibiting activity on PDE in human platelets. KMUP‐2 (0.1–100 μM) further induced an increase of intracellular cGMP levels in the primary cultured rabbit aortic and CCSM cells. These increases in cGMP content were abolished in the presence of methylene blue (100 μM) and ODQ (10 μM). Obviously, the relaxant effects of KMUP‐2 on rabbit isolated tissues are more sensitive in CCSM than in aorta. Moreover, KMUP‐2 also stimulated NO/sGC/cGMP pathway and subsequent elevation of cGMP by blockade of PDE and enhanced opening of K⁺ channels in rabbit aorta and CCSM. KMUP‐2 (0.2, 0.4, 0.6 mg/kg), similar to KMUP‐1 and sildenafil, caused increases of intracavernous pressure (ICP) and duration of tumescene (DT) in a dose‐dependent manner. It is concluded that both the increases of cGMP and the opening activity of K⁺ channels play prominent roles in KMUP‐2‐induced aortic smooth muscle and CCSM relaxation and increases of ICP in rabbits. Drug Dev. Res. 55:162–172, 2002. © 2002 Wiley‐Liss, Inc.     

The xanthine derivative KMUP-1 inhibits models of pulmonary artery hypertension via increased NO and cGMP-dependent inhibition of RhoA/Rho kinase

Background and purpose:

KMUP-1 is known to increase cGMP, enhance endothelial nitric oxide synthase (eNOS) and suppress Rho kinase (ROCK) expression in smooth muscle. Here, we investigated the mechanism of action of KMUP-1 on acute and chronic pulmonary artery hypertension (PAH) in rats.

Experimental approach:

We measured pulmonary vascular contractility, wall thickening, eNOS immunostaining, expressions of ROCK II, RhoA activation, myosin phosphatase target subunit 1 (MYPT1) phosphorylation, eNOS, soluble guanylyl cyclase (sGC), protein kinase G (PKG) and phosphodiesterase 5A (PDE-5A), blood oxygenation and cGMP/cAMP, and right ventricular hypertrophy (RVH) in rats.

Key results:

In rings of intact pulmonary artery (PA), KMUP-1 relaxed the vasoconstriction induced by phenylephrine (10 µM) or the thromboxane A₂-mimetic U46619 (0.5 µM). In endothelium-denuded PA rings, this relaxation was reduced. In acute PAH induced by U46619 (2.5 µg·kg⁻¹·min⁻¹, 30 min), KMUP-1 relaxed vasoconstriction by enhancing levels of eNOS, sGC and PKG, suppressing those of PDE-5A, RhoA/ROCK II activation and MYPT1 phosphorylation, and restoring oxygenation in blood and cGMP/cAMP in plasma. Incubating smooth muscle cells from PA (PASMCs) with KMUP-1 inhibited thapsigargin-induced Ca²⁺ efflux and angiotensin II-induced Ca²⁺ influx. In chronic PAH model induced by monocrotaline, KMUP-1 increased eNOS and reduced RhoA/ROCK II activation/expression, PA wall thickening, eNOS immunostaining and RVH. KMUP-1 and sildenafil did not inhibit monocrotaline-induced PDE-5A expression.

Conclusion and implications:

KMUP-1 decreased PAH by enhancing NO synthesis by eNOS, with consequent cGMP-dependent inhibition of RhoA/ROCK II and Ca²⁺ desensitization in PASMCs. KMUP-1 has the potential to reduce vascular resistance, remodelling and RVH in PAH.     

ROLE OF CYCLIC NUCLEOTIDES IN THE CONTROL OF CYTOSOLIC Ca2+ LEVELS IN VASCULAR ENDOTHELIAL CELLS

• 1 Endothelial cells have a key role in the cardiovascular system. Most endothelial cell functions depend on changes in cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) to some extent and Ca²⁺ signalling acts to link external stimuli with the synthesis and release of regulatory factors in endothelial cells. The [Ca²⁺]_i is maintained by a well‐balanced Ca²⁺ flux across the endoplasmic reticulum and plasma membrane.
• 2 Cyclic nucleotides, such as cAMP and cGMP, are very important second messengers. The cyclic nucleotides can affect [Ca²⁺]_i directly or indirectly (via the actions of protein kinase (PK) A or PKG‐mediated phosphorylation) by regulating Ca²⁺ mobilization and Ca²⁺ influx. Fine‐tuning of [Ca²⁺]_i is also fundamental to protect endothelial cells against damaged caused by the excessive accumulation of Ca²⁺.
• 3 Therapeutic agents that control cAMP and cGMP levels have been used to treat various cardiovascular diseases.
• 4 The aim of the present review is to discuss: (i) the functions of endothelial cells; (ii) the importance of [Ca²⁺]_i in endothelial cells; (iii) the impact of excessive [Ca²⁺]_i in endothelial cells; and (iv) the balanced control of [Ca²⁺]_i in endothelial cells via involvement of cyclic nucleotides (cAMP and cGMP) and their general effectors.