TMB—8抑制神经递质引起的单个脑细胞内游离钙的升高

AIM: To study the effects of TMB-8 on [Ca2+]i elevation induced by neurotransmitters in dissociated brain cells. METHODS: The brain cell suspension was made using a gentle trituration for 1 min with a polished pipette. The changes of [Ca2+]i were detected by the fluorescent indicator, Fura 2-AM. RESULTS: In the presence of extracellular Ca2+ 1.3 mmol.L-1, sodium glutamate (Glu), histamine (His), and serotonin (5-HT) markedly increased the [Ca2+]i which were reduced by TMB-8 30 mumol.L-1. TMB-8 3 mumol.L-1 produced inhibitory effects on the increase of [Ca2+]i by His and 5-HT in a Ca(2+)-free Hanks' solution. The increase of [Ca2+]i by His and 5-HT was reduced to control level by TMB-8 10 mumol.L-1. CONCLUSION: TMB-8 inhibited the [Ca2+]i elevation induced by Glu, 5-HT, and His in brain cells.     

甲基黄酮醇胺对分离的胎鼠脑细胞内游离钙浓度的影响(英文)

目的:观察甲基黄酮醇胺(MFA)对胎鼠脑细胞内游离钙浓度在静息以及激动剂存在时的作用。方法:用钙离子荧光染料Fura 2-AM负载后,测定分离的胎鼠脑细胞内游离钙浓度([Ca~(2 )]_i)及其变化。结果:在含钙1.3mmoL·L~(-1)的Hanks’液中,[Ca~(2 )]_i为197±20nmol·L~(-1)(n=44)。MFA0.15mmol·L~(-1)对静息脑细胞内钙浓度无明显影响。在细胞外钙1.3mmol·L~(-1)条件下,MFA(0.03—0.3 mmoL·L~(-1))浓度依赖性地抑制高钾去极化导致的[Ca~(2 )]_i升高,IC_(50)为0.14(95％可信限:0.05—0.42)mmoL·L~(-1)。在较高浓度时,MFA(0.15—0.3mmoL·L~(-1))也可抑制谷氨酸兴奋所引起的[Ca~(2 )]_i,IC_(50)为0.20(95％可信限:0.01—3.40)mmoL·L~(-1)。结论:MFA抑制高钾去极化引起的[Ca~(2 )]_i升高,在较高浓度时也拮抗谷氨酸兴奋所致的[Ca~(2 )]_i升高。     

大蒜新素对分离大鼠脑细胞内游离Ca~(2 )的影响

目的:观察大蒜新素对不同刺激剂所致分离大鼠脑细胞内游离钙的影响。方法:以Fura 2-AM为细胞内游离钙的荧光指示剂,用AR-CM-MIC阳离子测定系统,直接测定了分离新生大鼠脑细胞内游离钙([Ca~(2 )]_i)值,观察了大蒜新素的影响。结果:大蒜新素对脑细胞静息[Ca~(2 )]_i无明显影响,大蒜新素1-100μmol·L~(-1)能剂量依赖性地抑制高K~ 和谷氨酸引起的[Ca~(2 )]_i升高,其中IC_(50)分别为59.7和69.9μmol·L~(-1),高剂量大蒜新素100μmol·L~(-1)能抑制去甲肾上腺素引起的[Ca~(2 )]_i升高。结论:大蒜新素对高K~ 、去甲肾上腺素及谷氨酸引起的[Ca~(2 )]_i升高的抑制作用可能是其抗脑缺血作用机制之一。     

TMB-8对去甲肾上腺素和BHQ引起新生大鼠单个脑细胞内游离钙增高的影响

应用AR-CM-MIC阳离子测定系统,研究TMB-8对体外新生SD大鼠单个脑细胞内游离钙的抑制作用及其机制。结果表明,在无细胞外钙情况下,静息[Ca²⁺]i为79±13nmol·L^-1。TMB-810,30μmol·L^-1能明显降低静息[Ca²⁺]i。TMB-8100μmol·L^-1对高钾去极化引起的[Ca²⁺]i显著增高无明显影响。在细胞外钙为1.3mmol·L^-1时,去甲肾上腺素诱导的细胞内[Ca²⁺]i升高可部分被TMB-8抑制;TMB-8(30μmol·L^-1)对BHQ引起的[Ca²⁺]i的升高无明显抑制作用。而当细胞外液[Ca²⁺]i为0时,TMB-8几乎完全抑制了去甲肾上腺素和BHQ的作用。提示TMB-8降低脑细胞内游离钙的作用机制是通过促使细胞内钙进入肌浆网以抑制内钙的释放,并通过饱和肌浆网内Ca²⁺间接地阻滞细胞膜钙通道。     

小檗碱对神经递质引起的新生大鼠脑细胞内游离钙升高的影响

以Ｆｕｒａ－２／ＡＭ为荧光指示剂，利用ＡＲ－ＣＭ－ＭＩＣ阳离子系统测定小檗碱（Ｂｅｒ）对新生大鼠脑细胞静息Ｃａ２＋和神经递质引起的脑细胞内游离钙浓度（［Ｃａ２＋］ｉ）变化的影响．Ｂｅｒ１，１０，３０μｍｏｌ·Ｌ－１对脑静息［Ｃａ２＋］ｉ无明显影响．Ｂｅｒ１～１００μｍｏｌ·Ｌ－１剂量依赖的抑制谷氨酸引起的［Ｃａ２＋］ｉ升高．Ｂｅｒ１０μｍｏｌ·Ｌ－１能降低Ｈａｎｋｓ液有Ｃａ２＋和无Ｃａ２＋时去甲肾上腺素引起的［Ｃａ２＋］ｉ升高，且能降低５－羟色胺引起的［Ｃａ２＋］ｉ升高（在细胞外液有Ｃａ２＋时）．结果提示Ｂｅｒ可降低谷氨酸，去甲肾上腺素和５－羟色胺引起的［Ｃａ２＋］ｉ升高，这可能是其抗脑缺血机理之一．     

汉防己甲素抑制三种神经递质引起的新生大鼠脑细胞内游离钙的升高

应用Ｐｕｒａ－２技术测定游离新生大鼠脑［Ｃａ２＋］ｉ浓度的技术、研究了Ｔｅｔ对静息脑［Ｃａ２＋］ｉ和３种递质引起的脑［Ｃａ２］ｉ变化的影响。Ｔｅｔ（１，１０和２０μｍｏｌ·Ｌ－１）对静息脑［Ｃａ２＋］ｉ无明显影响。Ｔｅｔ（１０μｍｏｌ·Ｌ－１）可降低Ｌ－Ｇｌｎ（０．１、１．０和１０μｍｏｌ·Ｌ－１）引起的脑（Ｃａ２＋）ｉ的升高。在Ｈａｎｋ＇ｓ液Ｃａ２＋为１．３ｍｍｏｌ·Ｌ－１时，Ｔｅｔ１０μｍｏｌ·Ｌ－１可降低Ｈｉｓ（５０和１００μｍｏｌ·Ｌ－１）和５－ＨＴ（０．１、１．０、１０和１００μｍｏｌ·Ｌ－１）引起的脑［Ｃａ２＋］ｉ的升高。但不能降低Ｈａｎｋ＇ｓ液无Ｃａ２＋时Ｈｉｓ和５－ＨＴ引起的脑［Ｃａ２＋］ｉ的升高。研究表明Ｔｅｔ可阻滞Ｌ－Ｇｉｎ、Ｈｉｓ和５－ＨＴ受体调控的钙通道。但对Ｈｉｓ和５－ＨＴ引起的细胞内贮存钙的释放并无明显影响。Ｔｅｔ的这种降低脑［Ｃａ２＋］ｉ的作用可能是其治疗脑缺血性疾病的机理之一。Ｐ＜０．０１在Ｔｅｔ１０μｍｏｌ·Ｌ－１作用下，相同浓度的细胞外液钙和Ｈｉｓ（０、５０和１００μｍｏｌ·Ｌ－１），脑［Ｃａ２＋］ｉ分别是２２１±５、２４５±５和３０２±６ｎｍｏｌ·Ｌ－１。增加了１１．８?     

粉防己碱对培养大鼠心肌细胞胞内游离钙的影响(英文)

目的:研究粉防己碱对心肌的作用。方法:采用Fura-2和AR-CM-MIC阳离子测定系统测定培养大鼠单个心肌细胞胞内游离钙。结果:外钙1.3mmol·L~(-1)时,细胞静息钙为90±12nmol·L~(-1)。粉防己碱不影响静息钙,但可明显抑制CaCl_2,KCl,哇巴因引起的胞内钙增高;对于去甲肾上腺素引起的胞内钙增高,粉防己碱只有在外钙存在时,方对其有抑制作用。结论;粉防己碱抑制钙离子的跨膜运动,但在心肌细胞,它并非是选择性的钙通道阻滞剂。     

Effects of hyperin on free intracellular calcium in dissociated neonatal rat brain cells

目的：研究金丝桃苷（Ｈｙｐ）对新生大鼠的脑细胞内游离钙浓度的作用．方法：分离新生大鼠的脑细胞;用钙离子荧光指示剂Ｆｕｒａ２测定静息和激动剂存在时新生鼠脑细胞内游离钙浓度．结果：在ＣａＣｌ２１３ｍｍｏｌ·Ｌ－１的Ｈａｎｋｓ液中,静息［Ｃａ２＋］ｉ为（２０８±１２）ｎｍｏｌ·Ｌ－１（ｎ＝１７）,Ｈｙｐ对静息［Ｃａ２＋］ｉ无明显影响;Ｈｙｐ１０,４０,１６０μｍｏｌ·Ｌ－１呈浓度依赖性显著抑制ＫＣｌ５０ｍｍｏｌ·Ｌ－１致［Ｃａ２＋］ｉ增高;Ｈｙｐ１６０μｍｏｌ·Ｌ－１显著抑制去甲肾上腺素１,２,４和８μｍｏｌ·Ｌ－１诱发的［Ｃａ２＋］ｉ的增高;Ｈｙｐ１６０μｍｏｌ·Ｌ－１还可显著抑制谷氨酸和５羟色胺致［Ｃａ２＋］ｉ增高．结论：Ｈｙｐ对新生大鼠脑细胞钙内流有阻滞作用．     

小檗碱抑制谷氨酸引起的新生大鼠脑细胞 c-fos 表达及游离钙的升高

目的：研究小檗碱（Ｂｅｒ）对谷氨酸（Ｇｌｕ）引起的新生大鼠脑细胞ｃ－ｆｏｓ表达及游离钙（〔Ｃａ２＋〕ｉ）变化的影响。方法：斑点杂交及Ｆｕｒａ－２技术。结果：Ｇｌｕ能诱导分离的脑细胞ｃ－ｆｏｓ的一过性高表达及〔Ｃａ２＋〕ｉ的显著升高。Ｂｅｒ１０μｍｏｌ·Ｌ－１显著抑制Ｇｌｕ诱导的脑细胞ｃ－ｆｏｓ的高表达，而尼莫地平则没有明显作用。Ｂｅｒ１～１００μｍｏｌ·Ｌ－１能剂量依赖地抑制Ｇｌｕ引起的〔Ｃａ２＋〕ｉ升高。结论：Ｂｅｒ的这种降低脑细胞ｃ－ｆｏｓ基因表达及〔Ｃａ２＋〕ｉ水平的作用可能是其治疗脑缺血性疾病的机制之一。     

TMB-8对大鼠软脑膜动脉收缩的作用(英文)

目的:研究TMB-8对大鼠软脑膜动脉的作用。方法:通过颅窗连续直接地观察软脑膜动脉,用微循环系统记录和分析动脉直径的变化。结果:Nim 1μmol·L~(-1)使PA直径立即增加,在给药后2min时达到最大,而TMB-8 50μmol·L~(-1)对PA无明显影响。预先给予TMB-825,50μmol·L~(-1)则明显抑制KCl引起的PA收缩幅度。在CSF中存在5-HT使PA持续收缩的情况下,TMB-8浓度依赖性地扩张PA,在TMB-8 50μmol·L~(-1)加入前,灌流一氧化氮(NO)合酶抑制剂L-NAME 10μmol·L~(-1)则TMB-8扩张PA的作用减弱。结论:TMB-8能扩张痉挛的脑血管,可能与其钙拮抗和一氧化氮释放作用有关。     

Actions of 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate in vascular smooth muscle cell cultures

Ａｃｔｉｏｎｓｏｆ８（Ｎ，Ｎｄｉｅｔｈｙｌａｍｉｎｏ）ｎｏｃｔｙｌ３，４，５ｔｒｉｍｅｔｈｏｘｙｂｅｎｚｏａｔｅｉｎｖａｓｃｕｌａｒｓｍｏｏｔｈｍｕｓｃｌｅｃｅｌｃｕｌｔｕｒｅｓＺｈｏｕＣＨＥＮ，ＳｈｉｒｌｅｙＸＬＬＩＵ，ＧｅｏｒｇｅＣＹ...     

Inhibitory effect of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) in vascular smooth muscle

The inhibitory effects of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) on vascular smooth muscle contraction and cytosolic Ca2+ level ([Ca2+]i) were examined using isolated rabbit aorta loaded with a fluorescent Ca2+ indicator, fura-2. TMB-8 (100 microM) decreased the high K(+)-induced increase in muscle tension, and [Ca2+]i and 45Ca2+ influx to their respective resting levels. TMB-8 (100 microM) almost completely inhibited the increase in [Ca2+]i and 45Ca2+ influx due to norepinephrine although muscle tension was only partially decreased. A higher concentration of TMB-8 (300 microM) inhibited the remaining portion of the contraction without additional decrease in [Ca2+]i. The inhibitory effect of TMB-8 on high K(+)-induced contraction, but not on the norepinephrine-induced contraction, was antagonized by the increase in external Ca2+ concentrations or by the Ca2+ channel activators, CGP 28,392 and by Bay K8644. In Ca(2+)-free solution, norepinephrine-induced transient increases in [Ca2+]i and muscle tension and 100 microM TMB-8 inhibited these changes. The caffeine-induced transient increases in [Ca2+]i and muscle tension were also inhibited by TMB-8 at concentrations higher than those needed to inhibit the norepinephrine-induced transient changes. In permeabilized smooth muscle, TMB-8 (300 microM) did not inhibit the Ca(2+)-induced contraction. These results suggest that TMB-8 inhibits vascular smooth muscle contractility by inhibiting Ca2+ influx, Ca2+ release and Ca2+ sensitization of contractile elements.     

Stimulation of renin secretion by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8)

The intracellular calcium antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) prevents the release of Ca2+ from cell storage sites. The effect of this compound on renin secretion from rat renal cortical slices in vitro was investigated. TMB-8 was a potent stimulant of renin secretion within the concentration range 10(-5)M to 5 X 10(-4)M with an optimum concentration of 2 X 10(-4)M. TMB-8 overcame the inhibition of renin secretion by angiotensin II, ouabain, 60 mM KCl and A23187. The results add to the existing evidence that Ca2+ is a common inhibitory messenger for a number of compounds which affect renin release and suggest a role for intracellular calcium stores in the regulation of juxtaglomerular cell Ca2+ levels.     

8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) acts as a muscarinic receptor antagonist in the epithelial cell line HT29

8-(N, N-diethyl amino) octyl-3,4,5-trimethoxybenzoate (TMB-8) is a widely used pharmacological tool to investigate the involvement of intracellular Ca²⁺ stores in cellular responses. In this study we investigate the effect of TMB-8 as a putative inhibitor of Ca²⁺ signalling in single fura-2 loaded HT₂₉ coIonic epithelial cells stimulated by ATP, carbachol (CCH) and neurotensin (NT). TMB-8 effectively inhibited the CCH-induced (100 mol/l intracellular Ca²⁺ ([Ca²⁺]_i) transient with an IC₅₀ of 20 mol/l. However, [Ca²⁺]_i transients induced by other phospholipase C coupled agonists ATP (10 mol/l, n = 4) and NT (10 nmol/l, n = 4) remained unaffected by TMB-8 (50 mol/l). The agonist-induced [Ca²⁺]_i transients remained equally unaffected by 100 mol/l TMB-8 when the stimulatory concentration was reduced to 0.5 mol/I for ATP (n = 4) or 1 nmol/l for NT (n = 4). The competitive nature of the TMB-8-induced inhibition of the CCH-induced [Ca²⁺]_i transient was demonstrated by examining the agonist at various concentrations in absence and presence of the antagonist. High TMB-8 concentrations (100 mol/l) alone induced a small [Ca²⁺]_i increase ([Ca²⁺]_i: 40 ± 5 nmol/l, n = 7). We assume that this increase is a consequence of a TMB-8 induced intracellular alkalinization ( pH: 0.1 ± 0.02, n = 7) occurring simultaneously with the increase in [Ca ⁺]_i. From these results we draw the following conclusions: (1) In sharp contrast to a large number of other studies, but in agreement with studies in other types of cells, these results substantially challenge the value of the tool TMB-8 as an intracellular Ca²⁺ antagonist; (2) TMB-8 acts a muscarinic receptor antagonist at the M₃ receptor; (3) TMB-8 does not influence the release of Ca²⁺ from intracellular stores when IP₃ signal transduction is activated by ATP or NT; (4) TMB-8 as a weak organic base alkalinizes the cytosol at high concentrations; and (5) TMB-8 induces small [Ca²⁺]_i transients at higher concentrations.     

8—（N,N—二乙胺）—n—辛基—3,4,5—三甲氧基苯甲酸酯在血管平滑肌细胞 …

AIM: To study the effects of 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8) on vascular smooth muscle (VSM) cells A7r5. METHODS: The effects of TMB-8 were investigated in A7r5 cell cultures with 45CaCl2. RESULTS: TMB-8 reduced the intracellular free Ca2+ concentration, [Ca2+]i in a Ca(2+)-free medium and blocked Ca2+ entry from the extracellular site in a regular Ca2+ medium. The equilibrated total cellular binding of Ca2+ was increased by TMB-8 whereas 45Ca2+ entry activated by both NE and KCl was inhibited. However, the NE-activated Ca2+ entry was not blocked by TMB-8 if TMB-8 was added together with 45Ca2+ at a later time instead of by pretreatment. Similar to actions of NE and KCl, depletion of Ca2+ from sarcoplasmic reticulum (SR) would also activate Ca2+ entry, which was blocked by TMB-8. When TMB-8 was rinsed out alone or together with NE after pretreatment with NE plus TMB-8 in VSM cells, the inhibitory effect of TMB-8 was not affected. CONCLUSION: TMB-8 not only blocks Ca2+ entry from the extracellular site, but also enhances Ca2+ uptake into SR which, indirectly inhibits Ca2+ entry from the extracellular site.