[H]Spermine binding to synaptosomal membranes from the chick retina

The binding of [³H]spermine to synaptosomal membranes from chick retina was examined. Saturable specific binding of [³H]spermine to synaptosomal membranes from plexiform layers of retina (P₁ and P₂) has been characterized, and found to concentrate in the inner plexiform layer compared to the outer plexiform layer (B_max=9.3 and 37 pmol/mg protein for P₁ and P₂, respectively). Kinetics of specific [³H]spermine binding yield a sigmoidal saturation curve, indicating positive cooperativity (nH: 2.4 and 3.2 for P₁ and P₂, respectively) with high affinity: K_app=61 and 67 nM for P₁ and P₂. The time required to attain equilibrium at room temperature was less than 5 min in both fractions. Dose–response curves for spermine, spermidine, and diethylene–triamine (DET) show different potencies for inhibiting [³H]spermine binding: spermine>spermidine>DET. Our results support a role for polyamines (PA) as neurotransmitters or neuromodulators in the vertebrate retina.     

Calcium-independent release of [H]spermine from chick retina

Spermine has been shown to influence NMDA receptor function through an interaction at the coagonist site for glycine in the central nervous system (CNS) and the retina. In order to support a role for spermine as neurotransmitter or neuromodulator in the chick retina, specific stimulated-release of spermine should be demonstrated. Isolated chick retinas, preloaded with [³H]spermine, were stimulated with 1 mM NMDA and other glutamate agonists at ionotropic receptors, in a continuous superfusion system. [³H]spermine was released from the retina by depolarization with 50 mM KCl, in a Ca²⁺-independent manner. Inhibition of Na⁺/K⁺-ATPase by ouabain or digitoxigenin also induced spermine release following 36 min in the presence of the drugs; such effect seems unrelated to changes in Na⁺ electrochemical gradients, since nigericin and veratrine did not induce release in Na⁺ containing medium. The lack of effect of glutamate, NMDA and kainate at 1 mM concentration, suggests that release of spermine in the retina is mediated by the reversal of uptake and not necessarily linked to EAA-receptor activation.     

Gene delivery by pullulan derivatives in brain capillary endothelial cells for protein secretion

The blood-brain barrier (BBB) formed by brain capillary endothelial cells protects the brain against potentially harmful substances present in the circulation, but also restricts exogenous substances such as pharmacologically acting drugs or proteins from entering the brain. A novel and rather unchallenged approach to allow proteins to enter the brain is gene therapy based on delivery of genetic material into brain capillary endothelial cells. In theory in vivo transfection will allow protein expression and secretion from brain capillary endothelial cells and further into the brain. This would denote a new paradigm for therapy to transport proteins across the BBB. The aim of this study was to investigate the possibility to use brain capillary endothelial cells as factories for recombinant protein production. Non-viral gene carriers were prepared from pullulan, a polysaccharide, and spermine, a naturally occurring polyamine that were additionally conjugated with plasmid DNA. We were able to transfect rat brain endothelial cells (RBE4s) and human brain microvascular endothelial cells (HBMECs). Transfection of HBMECs with pullulan-spermine conjugated with plasmid DNA bearing cDNA encoding human growth hormone 1 (hGH1), led to secretion of hGH1 protein into the growth medium. Hence, the pullulan-spermine delivery system is a very promising method for delivering DNA to brain endothelial cells with potential for using these cells as factories for secretion of proteins.     

线粒体钙单向转运体在二氮嗪预处理脑保护中的作用

目的探讨线粒体钙单向转运体是否参与了二氮嗪预处理所发挥的脑保护作用方法将52只健康雄性Wistar大鼠按照完全随机方法分为4组（每组13只）：A组,假手术组;B组,脑缺血厢獾注组;C组,脑缺血,再灌注＋二氮嗪组;D组,脑缺血,再灌注＋二氮嗪＋精胺组。采用线栓法建立大鼠大脑中动脉闭塞模型,缺血2h再灌注24h后观测各组神经行为变化,缺血侧脑组织线粒体钙含量及超氧化物歧化酶（superoxide dismutase,SOD）活性、丙二醛（malondialdehyde,MDA）含量、一氧化氮（nitric oxide,NO）含量和谷胱甘肽过氧化物酶（glutathione peroxidase,GSH-Px）活性。结果与B组比较,C组二氮嗪预处理可以改善大鼠脑缺血／再灌注引起的神经行为障碍（13．1±0．8,P〈0．01）,降低缺血侧脑线粒体钙含量（293±7）nmol／mgprot,（P〈0．05）,提高SOD（143±18）U／mgprot及GSH-Px（84±8）U／mgprot活性、降低MDA（0．799±0．092）nmol／mgprot,及NO（0．216±0．138）nmol／mgprot,含量（脚．05）。与c组比较,D组精胺可减弱二氮嗪预处理改善神经行为障碍（8．0±0．7,P〈0．01）、降低脑线粒体钙含量（322±10）nmol／mgprot,提高SOD（128±7）U／mgprot和GSH-Px（731-6）U／mgprot活性以及降低MDA（0．955±0．141）nmo]]mgprot,和NO（0．575±0．292）nmol／mgprot,含量的效应（P〈0．05）。结论二氮嗪预处理对抗大鼠脑缺血/再灌注损伤所发挥的脑保护效应,可能与其再灌注时减少线粒体钙单向转运体的活动以减少线粒体内钙含量及过氧化损伤有关。     

棉酚对雄性大鼠生殖系统多胺的影响

将分离后的雄性大鼠生殖系统的组织和细胞制备成多胺的丹酰化衍生物,采用多阶梯度反相高效液相色谱荧光检测法,研究不同剂量的棉酚对大鼠前列腺、附睾精子、生殖细胞和间质细胞中多胺的影响及其时效关系。结果表明:(1)棉酚ig 30,60mg/kg,连续给药6d,与对照组相比,生殖细胞、间质细胞中精脒和精胺均显著下降,下降率分别为29.0%和54.5%,32.3%和56.3%;精囊中精脒也明显降低,下降率分别为30.2%和61.7%,精囊中精胺,低剂量无明显变化,高剂量明显降低,下降率为42.7%;附睾精子中精胺急剧升高,升高率分别为99.7%和140.2%。(2)ig 30mg/kg,分别连续给药3,6,9,12或16d,同法测定生殖系统各组织中精脒和精胺,与对照组相比,精囊在第3天、附睾精子在第6天显著增加,前列腺中在第3天也升高,但不如前两者明显。继续给药,精脒和精胺都显著降低。棉酚干扰了雄性大鼠生殖系统多胺的正常水平,这可能是棉酚抗生育作用的较为重要的生化机理之一。     

生物组织和细胞中多胺的高效液相色谱测定

本文对分离所得的生物样品经丹酰化反应在μ—Bondapak—C_(18)柱上,以甲醇-四氢呋喃-醋酸-水系统为流动相,采用多阶梯度反相高效液相色谱荧光检测法,测定了未成熟的大鼠前列腺、附睾、精囊等组织及生殖细胞和间质细胞中多胺的含量。其中流速为1ml/min,激发波长为370nm,发射波长为530nm,纸速为5mm/min。内标为1,10-二氨基癸烷。本法测定腐胺,精胺和精脒的精密度分别为3.63%,1.65%,2.11%;生物样品的回收率为99.6%～100.3%;三种多胺的最低检出量依次为0.2ng,0.4ng和0.8ng。     

Spermine depresses NMDA, K/AMPA and GABAA-mediated synaptic transmission in the rat hippocampal slice preparation

The effects of spermine, an endogenous polyamine, were examined in area CA1 of the rat hippocampal slice preparation. Spermine, at low millimolar concentrations, rapidly and potently depressed NMDA and K/AMPA-mediated population EPSPs, and GABA-mediated monosynaptic population IPSPs. These effects contrast with its well-known potentiation of NMDA currents at lower concentrations. Our results raise the possibility that the large intracellular stores of spermine that are released after various neural insults could act as an endogenous neuroprotective mechanism by limiting excessive calcium entry.     

Effects of polyamines on voltage-activated calcium channels in guinea-pig intestinal smooth muscle

Effects of polyamines on the spontaneous mechanical and electrical activity of guinea-pig intestinal smooth muscle were studied. Spermine and spermidine inhibited action potential generation and contractions, while putrescine had no effect. Single smooth muscle cells were isolated from the longitudinal muscle layer of the guinea-pig ileum. Whole-cell voltage-clamp experiments were carried out to investigate the effects of polyamines on current through voltage-activated Ca²⁺ channels. Spermine and spermidine (0.1–1 mM) reduced the inward current in a concentration-dependent manner. Spermine blocked current activated by the dihydropyridine agonist BAY K 8644 (1 M), whereas no additional inhibition by spermine was seen after blockage of dihydropyridine-sensitive channels by nifedipine (0.1 M). Inhibition by spermine or spermidine did not shift the peak of the current voltage relation of the inward current. Steady-state activation and inactivation relationships were not affected and thus the amplitude, but not the voltage dependence, of the window current responsible for Ca²⁺ inflow during sustained depolarization was affected. Putrescine (1 mM) had no significant effect on the inward current. These results suggest that spermine and spermidine inhibit contraction in spontaneously active intestinal smooth muscle by inhibiting Ca²⁺ current responsible for generation of action potentials.     

UV-Induced changes in urinary polyamine excretion in the mouse

Summary Exposure of hairless mice to the light of a germicidal lamp (254 nm) under conditions which are known to induce epidermal DNA synthesis, cell proliferation, and polyamine metabolism produced a marked increase of polyamine excretion in the urine which lasted for many days. The increase was about the same for free and acetylated polyamines. Although the ratio of N¹-acetylspermidine/N⁸-acetylspermidine increased somewhat in the urine of animals exposed to UV, the increase was not significant enough to be useful as a marker of enhanced cell proliferation. A single topical dose of -difluoromethylornithine, a selective inhibitor of ornithine decarboxylase, prevented the UV-induced increase of polyamine excretion in agreement with its effect on UV-induced epidermal polyamine turnover.     

Comparison of responses to novel nitric oxide donors in the feline pulmonary vascular bed

Pulmonary vascular responses to the novel diazeniumdiolate nitric oxide (NO) donors diethylamine/NO, diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt, were investigated and compared in the intact-chest cat. Under conditions of controlled blood flow, when tone in the pulmonary vascular bed had been raised to a high steady level, intralobar injections of diethylamine/NO (0.3–10 μg), diethylenetriamine/NO (10–30 μg), spermine/NO (10–30 μg), sulfite/NO (10–30 μg), and angeli's salt (10–30 μg) caused dose-related decreases in lobar arterial pressure without changing left atrial pressure. In terms of relative vasodilator activity in the pulmonary vascular bed, the dose of the compounds that decreased lobar arterial pressure 4 mm Hg (ED₄ mm Hg) was significantly lower for diethylamine/NO compared to S-nitroso-N-acetylpenicillamine which was significantly less than diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt. The half-life of the vasodilator responses, as measured by 50% response recovery time, to diethylamine/NO, diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt was similar for doses with similar magnitudes of vasodilation, while the half-life to S-nitroso-N-acetylpenicillamine was significantly less than the diazeniumdiolate NO donors. The present data demonstrate that the diazeniumdiolate NO donors diethylamine/NO, diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt have potent but relatively short-lasting vasodilator activity in the pulmonary vascular bed of the cat.