高血压大鼠红细胞膜钙泵功能障碍机理的初步分析

本工作比较自发性高血压大鼠(SHR)和肾性高血压大鼠(RHR)及对照大鼠红细胞膜Ca~(2+),Mg~(2+)-ATP酶(钙泵)活性及其对CaM、TFP、川芎嗪和硝苯吡啶(Nifedipine、Nif)等的反应,目的是分析高血压时钙泵功能障碍的机理,并寻找能有效提高钙泵活性的药物,为高血压防治提供有效措施。 结果表明SHR及RHR基础钙泵活性明显低于相应对照大鼠钙泵活性;原因之一可能与高血压动物质膜钙泵对钙泵抑制剂TFP的敏感性增加有关,对RHR,川芎嗪作用很类似TFP。提示此药有CaM拮抗作用。Nif除了公认为钙通道阻断剂外,本实验表明对RHR它还有激活钙泵的作用。     

慢性给锂盐对大鼠脑内钙调素活性的影响

Wistar大鼠24只,分为两组:锂盐组:腹腔注射锂盐七天(血浆锂浓度 1.368mM/1)。对照组:腹腔注射生理盐水七天。采用磷酸二酯酶法(PDE)测定慢性给锂盐后大鼠脑内CaM活性的改变。实验结果表明,与对照组比较,锂盐组大鼠的端脑,中脑和脑干的CaM含量明显升高(P<0.01),纹状体内CaM明显降低(P<0.05);下丘脑内CaM含量升高,但P>0.05。从实验结果可见,慢性给锂盐能明显改     

抗钙调素抗体的测定与临床应用

<正> 钙调素(Calmodulin,CaM)参与细胞内钙离子介导的多种生理过程,与免疫功能有密切关系。Ikeda等发现某些自身免疫病患者(肝炎、SLE等)体内存在抗CaM抗体,推测这种自身抗体与胞内CaM结合后可干扰后者对免疫活性细胞的调节作用。我们建立了检测血清抗CaM抗体的ELISA技术,并进行了初步临床观察。     

高血压患者血清钙调素测定及其与钙拮抗剂的相关探讨

高血压病是心血管疾病中最常见的病种。钙拮抗剂是能有效减低细胞内钙含量,松弛血管平滑肌,扩张周围血管,从而治疗高血压的药物。钙调素(Camodulin,CaM)作为钙离子主要的细胞内受体,调节着高血压发生过程中许多酶的活性及细胞反应过程,是一种重要的钙结合蛋白,因此近年来在高血压病的诊治中受到越来越多的重视。本文根据部分患者服用钙拮抗剂(心痛定)的不同情况分组测定了82例高血压患者的血浆CaM水平,用以探讨钙拮抗剂与CaM的关系,现报告如下。     

心肌缺血及再灌注后心肌钙调蛋白及环磷酸腺苷含量的变化

本文应用放免技术对家兔心肌急性缺血及再灌注后心肌组织钙调蛋白(CaM)和环磷酸腺苷(cAMP)的变化进行了初步观察。缺血20分钟时,心肌组织CaM和cAMP含量均升高,缺血40分钟时,二者与缺血20分钟时比无明显变化。缺血20分钟时再灌注,心肌组织CaM含量下降,cAMP含量上升,缺血40分钟时再灌注与缺血20分钟再灌注相比CaM含量升高,cAMP含量下降。文章讨论了这些变化的可能机制。     

胞苷环3’,5’一磷酸对正常人淋巴细胞分泌IL-2和IL-2受体表达的抑制作用

0.2mM 3’,5’-cCMP不仅能抑制正常人外周血淋巴细胞IL-2的诱生(P<0.01),而且能抑制IL-2受体的表达(P<0.01)。对后者的抑制率为28.9±1.9%。但是在充分IL-2存在的条件下,0.00625mM至0.2mM的3’,5’-cCMP对CTLL-2细胞的增殖有促进作用,这种促进作用在其浓度较低时更为明显。     

川芎嗪对离体血管舒张的机理

用离体器官浴的方法,在家兔主动脉及肺动脉血管条制备上,观察了川芎嗪(四甲基吡嗪Tetramethyl-pyrazine)松驰血管平滑肌作用与Ca~(++)的关系。 氯化钾(50mM)使主动脉条(n=6)及肺动脉条(n=5)收缩后,川芎嗪10~(-4)g能引起明显地松驰作用,并能被提高浴液中Ca~(++)浓度所拮抗,显示了川芎嗪与Ca~(++)之间存在     

吗啡依赖对小鼠睾丸组织的影响及其相关机制分析

为了解吗啡依赖对小鼠睾丸发育及其功能的影响 ,研究了吗啡依赖对睾丸生精细胞凋亡的影响及其可能机制。以剂量递增法皮下注射吗啡建立吗啡成瘾小鼠模型 ,用纳洛酮催促戒断 ,观察戒断症状 ,采用原子吸收、放射免疫分析和原位缺口平移 (ISNT)技术观察睾丸细胞锌和钙调素 (CaM)含量、睾丸超氧歧化酶 (SOD)、还原型谷胱甘肽过氧化物酶 (GSH Px)活性和睾丸组织中凋亡细胞数目。结果表明 ,与对照组相比 ,吗啡依赖组和纳洛酮催促戒断组躯体运动神经功能和植物神经功能亢进 ,睾丸锌和CaM含量减少 ,睾丸SOD、GSH Px活性降低 ,睾丸组织中生精细胞凋亡数目明显增多 (P <0 0 5或P <0 0 1)。吗啡依赖小鼠可诱导睾丸组织中细胞凋亡 ,这种变化可能与睾丸组织中锌和CaM含量减少、睾丸SOD和GSH Px活性降低有关。     

哌唑嗪对原发性高血压病人红细胞Ca~(2+),Mg~(2+)-ATP酶和钙调素的影响

本实验观察了原发性高血压(EH)病人红细胞Ca~(2+)Mg~(2+)-ATP酶活性,红细胞和血浆钙调素(CaM)的变化及哌唑嗪对其影响。结果表明:EH病人红细胞基础和最大Ca~(2+),Mg~(2+)-ATP酶活性显著下降,并与血压呈显著负相关;红细胞CaM活性也显著降低并与最大Ca~(2+),Mg~(2+)-ATP酶活性呈显著正相关;经哌唑嗪治疗后,EH病人红细胞Ca~(2+),Mg~(2+)-ATP酶活性和CaM变化不显著,但血浆CaM有所降低。提示EH病人红细胞钙代谢异常与外周阻力增高有密切关系,哌唑嗪对细胞和体液CaM有不同影响。     

强磁重力环境对MG63成骨样细胞钙离子/钙调蛋白信号的影响

目的:研究强磁重力环境对MG63成骨样细胞钙离子浓度和钙离子下游信号分子表达的影响。方法:利用大梯度强磁场提供μg(12T),1g(16T)和2g(12T)三组不同强磁重力复合环境处理MG63成骨样细胞后,经Fluo-3/AM标记的细胞用激光共聚焦显微镜检测处理0.5 h对细胞胞内游离钙离子浓度([Ca2+]i)的影响;用Western blot检测处理3 h对钙调蛋白(CaM)和肌球蛋白轻链激酶(MLCK)表达以及钙离子/钙调蛋白依赖蛋白激酶Ⅱ(CaMKⅡ)活性的变化。结果:钙离子浓度检测结果表明,与对照组细胞(1 g,地磁)相比,1 g(16 T)组细胞Fluo-3荧光强度增加,结果显示强磁场导致[Ca2+]i增加;与2 g(12 T)组相比,μg(12 T)组细胞Fluo-3荧光强度下降,结果显示模拟失重导致[Ca2+]i降低,抑制钙离子信号。蛋白质表达的检测结果表明,与对照组相比,1 g组细胞CaM和MLCK表达以及CaMKⅡ活性没有明显变化;与2 g(12 T)组相比,μg(12 T)组细胞CaM表达以及CaMK活性下降,结果显示模拟失重抑制CaM/CaMKⅡ信号。结论:强磁场导致MG63成骨样细胞胞内游离钙离子浓度增加,模拟失重抑制成骨样细胞钙离子/钙调蛋白信号。     

Evidence against a role of cyclic nucleotides in the regulation of anaphylactic histamine release in isolated rat mast cells

The effect of different phosphodiesterase (PDE) inhibitors on the antigen or 48/80 induced histamine release from isolated Hooded Lister rat mast cells was tested. The unselective PDE inhibitors theophylline (2.5 mM) and IBMX (0.2 mM) and the selective cyclic GMP PDE inhibitor M & B 22948 (0.1 mM) inhibited the antigen induced histamine release by 50% while 48/80 induced release was inhibited by about 25%. The cyclic AMP selective PDE inhibitors ICI 63197 (0.5 mM) or Ro 20-1724 (0.2 mM) had no effect on 48/80 induced histamine release but tended to enhance antigen induced release. There was no correlation between the measured levels of cyclic AMP and the effect on histamine release by the investigated PDE inhibitors. Cyclic AMP or cyclic GMP up to 10(-3) M did not affect the anaphylactic histamine release. Dibutyryl-cAMP and dibutyryl-cGMP (10(-4) M) both inhibited the release about 20% but this effect could be explained by the effect of butyric acid as sodium butyrate (2 X 10(-4) M) also inhibited the release by 20%. The presence results suggest that cyclic nucleotides are not important regulators of histamine release from isolated mast cells.     

Effects of thiazinamium chloride on histamine release from rat peritoneal mast cells and on phosphodiesterase activity in guinea pig lung

The effect of thiazinamium Cl (TCl) on histamine release from rat peritoneal mast cells (RPMC) was investigated. Although TCl inhibited compound 48/80-induced histamine release moderately (IC50 value 40 microM), the drug was a weaker inhibitor of ovalbumin-induced histamine release (100 microM, -21%). In contrast, promethazine HCl (PHCl) was more effective against antigen-induced histamine release (IC50 value 13 microM) than against compound 48/80-induced histamine release (100 microM, -53%). Disodium cromoglycate (DSCG) was effective against both antigen and compound 48/80-induced release of histamine with IC50 values of 7 and 1 microM, respectively. Neither TCl nor DSCG at 1 mM increased spontaneous release of histamine from RPMC, whereas PHCl induced spontaneous release by over 50% at 1 mM. TCl did not inhibit phosphodiesterase (PDE) activity in guinea pig lung at 1 mM, whereas theophylline and DSCG inhibited PDE with IC50 values of 1.1 and 0.32 mM, respectively. These data suggest that high local concentrations of TCl may reduce histamine release during an asthmatic attack and improve its effectiveness as a bronchoprotectant.     

Sexual Dimorphism and Electrophoretic Variation in the Form I Cyclic Nucleotide Phosphodiesterase From Drosophila melanogaster

We have investigated the form I cyclic nucleotide phosphodiesterase (PDE) from Drosophila melanogaster and shown that whereas heads and male thoraces and abdomens contain high levels of Ca^{2 +} -stimulated enzyme, female thoraces and abdomens contain little Ca^{2 +}-stimulated activity. The electrophoretic patterns of form I PDE from these 3 sources have also been studied and reveal that heads, and male thoraces and abdomens, produce two bands of form I PDE both of which are stimulated by Ca²⁺. Extracts of female thoraces and abdomens, on the other hand, show only a single, faster running band of PDE activity which is only marginally stimulated by Ca²⁺, if at all. Surveying wild-type strains of Drosophila has revealed that one strain, Swedish, shows altered electrophoretic mobility of the PDE band from female thoraces and abdomens. The alteration is such that the Swedish PDE band runs more anodally than the Oregon-R and Canton-S PDE activities. Mixing experiments, using co-homogenization of heads with female thoraces and abdomens, yeild a single faster running band on electrophoresis. This band contains only Ca²⁺ -insensitive PDE. Attempts to reconstruct this loss of Ca²⁺ -sensitive PDE without electrophoresis have failed. The Swedish electrophoretic variation of the PDE from female thoraces and abdomens has been found to be recessive with respect to the Canton-S phenotype, but the variation is observed to re-emerge and segregate with the third chromosome in the F₂ generation. The results indicate that electrophoretic variation in the form I PDE is, by itself, insufficient to allow the location of the structural gene for this enzyme.     

Effect of catecholamines on the activity of rat brain dipeptidyl aminopeptidase

The inhibitory effect of catecholamines, their metabolic intermediates and derivatives on the activity of rat brain dipeptidyl aminopeptidase was studied. The enzyme was extracted from a crude mitochondrial fraction containing lysosomes (and also synaptosomes) and purified by three-step column chromatography. The activity of the purified enzyme was inhibited by a low concentration (IC50 2 approximately 5 mM) of dopamine, norepinephrine and epinephrine. Dihydroxyphenylalanine or dihydroxyphenylacetic acid, each having a carboxyl group, showed only a weak inhibitory effect (IC50 greater than 10 mM). These results suggest the possibility that brain dipeptidyl aminopeptidase activity may be regulated by the changing of the concentration of catecholamines in the central nervous system.     

Inhibition of cGMP accumulation in mesangial cells by bradykinin and tyrosine kinase inhibitors.

We examined the effect of bradykinin (BK) on the accumulation of cGMP of the mesangial cell (MC), a smooth muscle-like cell of the renal glomerulus. BK caused a time- and concentration dependent reduction of the cGMP concentration. In addition, BK inhibited total protein tyrosine kinase (PTK) activity. Two tyrosine kinase inhibitors (TKI) genistein and tyrphostin also reduced the cGMP concentration. The inhibition of BK and TKI were not additive. The inhibition of PTK by BK, mediated through activation of the B2-receptor, was unaffected by inhibitors of Gi/o proteins, phospholipase C, protein kinase C, cyclooxygenase and Ca2+ release from intracellular stores. Only IBMX a broad spectrum inhibitor of phosphodiesterases (PDE) and 8-methoxymethyl IBMX a specific type-1 PDE inhibitor prevented the inhibitory effects of BK and TKI indicating the involvement of type-1 PDE. In addition, BK had no effect on soluble guanylate cyclase (sGC) and nitric oxide synthase activity. In freshly isolated glomeruli, which represent the physiological environment of MC, BK also reduced the cGMP concentration. Like in MC, the inhibitory effect was suppressed by IBMX. These data demonstrate that BK suppresses a PTK-dependent pathway of cGMP production in rat MC at a level downstream of NO synthase and sGC. It is suggested that BK and TKI inhibitors decrease cGMP levels by preventing tyrosine phosphorylation of type-1 PDE activity, thereby leading to enzyme activation.     

Regulation of cyclic adenosine monophosphate synthesis in human ejaculated spermatozoa. II. The role of calcium and bicarbonate ions on the activation of adenylyl cyclase.

In this study, we have applied our previous data describing the experimental conditions necessary for expression of cyclic adenosine monophosphate (cAMP)-synthesizing adenylyl cyclase in human ejaculated spermatozoa, to investigate the direct effects of calcium (Ca2+) and bicarbonate (HCO3-) upon activation of the enzyme in vitro. We report that the effects of Ca2+ and HCO3- were significantly dependent on the status of the enzyme activity. Thus, at a near saturating (10 mM) concentration of MnCl2 giving high enzyme activity, addition of less than 10 mM HCO3- did not affect adenylyl cyclase activity and higher concentrations inhibited the enzyme, with 50 mM HCO3- reducing the activity by 33%. Also, addition of less than 20 mM CaCl2 alone or in combination with 10 mM HCO3- did not significantly change the enzyme activity. In great contrast, enzyme activation was highly responsive to Ca2+ and HCO3- when MnCl2 was present at a concentration giving submaximal enzyme activity. Thus, at 2 mM MnCl2, adenylyl cyclase was markedly increased by CaCl2 concentrations between 10 and 100 mM. The activation was further enhanced by increasing concentrations of HCO3-, with 50 mM HCO3- giving the highest activity at 50-100 mM CaCl2. Activation by CaCl2 was also observed in the absence of added MnCl2, being significantly greater than basal activity at 10 mM CaCl2 and maximal at 100 mM CaCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemotactic activity generated in human serum from the fifth component of complement by hydrogen peroxide.

Exposure of normal human serum to various concentrations of hydrogen peroxide as low as 68.8 microM resulted in the generation of chemotactic activity for human neutrophils, which was inhibited by adding catalase prior to the exposure. Maximum chemotactic activity was obtained by hydrogen peroxide at a concentration of 1.1 mM, and the concentration greater than 1.1 mM expressed decreased activity. On the contrary, hydrogen peroxide less than 1.1 mM produced dose-dependent chemotactic activity. The generation of chemotactic activity is initiated at an incubation time of 5 minutes, and subsequently increases with up to 90 minutes. The suppression of chemotactic activity was minimum even after 180 minutes. The maximum activity was obtained by 1024-fold dilution of serum treated with 13.8 mM hydrogen peroxide. These results suggest that the disappearance of chemotactic activity at the high-dose range of hydrogen peroxide is due to neutrophil deactivation rather than inactivation of the chemotactic activity after it was generated. Similarly, purified human C5 exposed to hydrogen peroxide generated chemotactic activity. The chemotactic activity was inhibitable by antiserum to human C5. The molecular weight of chemotactically active substance was approximately 15,000. The generation of chemotactic activity was not inhibited by addition of EGTA or EDTA. These results imply that hydrogen peroxide generates C5a-like chemotactic factor through hydrolysis of C5.     

Effect of calcium on neurohumoral stimulation of feline colonic smooth muscle

The purpose of this study was to compare the effect of altering the extracellular calcium ion concentration on bethanechol or octapeptide of cholecystokinin (OP-CCK) stimulation of the isolated transverse colon of the cat. Myoelectric activity was recorded with monopolar glass-pore electrodes. Bethanechol (10(-6) M) stimulated an increase in the number of slow waves with superimposed spike potentials to 85.5 +/- 5.3% (P less than 0.001) compared with the basal spike activity (8.9 +/- 1.4%). OP-CCK (4 x 10(-9)) also increased spike activity (80.7 +/- 3.8%, P less than 0.001), which was not inhibited by atropine, phentolamine, or propranolol. Addition of 0.0 mM calcium solution to the colonic smooth muscle abolished both slow-wave and spike activity, which returned after replacing 0.25 mM calcium in the solution. Bethanechol stimulated a greater increase in spike activity as the concentration of calcium was increased. OP-CCK stimulation of colonic spike activity was more sensitive to the extracellular calcium concentration than bethanechol stimulation. Verapamil had a minimal effect on bethanechol stimulation of colonic spike activity, but it inhibited the OP-CCK stimulation. These studies suggest that 1) OP-CCK appears to stimulate colonic smooth muscle directly and 2) OP-CCK requires the presence of a greater amount of extracellular ionic calcium in order to stimulate colonic spike activity compared with bethanechol.     

Anion-stimulated ATPase activity of brush border from rat small intestine.

Differential centrifugation of rat small intestinal homogenates produced a crude brush border (BB) fraction that was enriched 15-fold for the marker enzymes, alkaline phosphatase and sucrase; contamination with mitochondrial enzymes, monoamine oxidase and succinate dehydrogenase, was minimal. ATP hydrolysis by this BB fraction was stimulated by addition of several anions to the incubation medium: HCO3 and Cl were equally effective in this regard, with NO3, NO2, SO4, and acetate being less stimulatory. SCN and CNO inhibited ATPase activity, whereas the divalent anion SO3 was stimulatory at low concentrations (less than 25 mM) but inhibitory at 100 mM. Maximum anion stimulation was observed at a Mg concentration of 0.5 mM, and pH optimum was 8.5. Kinetic analysis showed that HCO3 increased the Vmax without altering the Km for ATP; the Ka for this effect of HCO3 was 35 mM. This enzyme activity was completely inhibited by 20 mM L-phenylalanine, 10 mM L-cysteine, and 3 mM EDTA, compounds that also inhibited intestinal alkaline phosphatase. These results demonstrate the presence of anion-stimulated ATPase activity in rat small intestinal brush border and suggest that this activity may be related to intestinal alkaline phosphatase. The role of this enzyme in intestinal transport is not known, but could relate to the regulation of intestinal absorption and secretion.     

Xanthine derivatives: comparison between suppression of tumour necrosis factor-alpha production and inhibition of cAMP phosphodiesterase activity.

Several in vitro and in vivo studies have demonstrated suppression of tumour necrosis factor-alpha (TNF-alpha) synthesis by pentoxifylline. In the present study we compared the effect of pentoxifylline with that of five other xanthine derivatives. We addressed two questions. First, what is the relative potency of those chemically related compounds in suppressing the lipopolysaccharide (LPS)-induced production of TNF-alpha in human mononuclear cells? Second, does suppression of TNF-alpha production by these xanthine derivatives correlate with their capacity to inhibit 3',5'-cAMP phosphodiesterase (PDE) activity? The experimental drug A 80 2715 [1-(5-hydroxy-5-methylhexyl)-3-methyl-7-propylxanthine] was identified as the most potent agent with an IC50 (concentration exerting 50% suppression of LPS-induced TNF-alpha production) of 41 microM (mean of 13 individuals). The IC50 values of the other substances ranged between 106 microM for HWA 138 and 419 microM for theophylline. The LPS-induced interleukin-1 beta (IL-1 beta) production was not influenced by all substances tested at comparable concentrations. Inhibition of PDE activity was determined in a cell-free system using PDE isolated from bovine heart. All xanthine derivatives dose-dependently inhibited PDE activity. Furthermore, with the exception of theophylline, there was a high degree of correlation between the potency to suppress TNF-alpha production in the cell culture system and the potency to inhibit PDE activity in the cell-free enzymatic assay. This argues for a crucial role of PDE inhibition in the suppression of TNF-alpha synthesis by xanthine derivatives.