Hydrogen peroxide-induced impairment of reactivity in rat isolated aorta: potentiation by 3-amino-1,2,4-triazole

1. In this study the impairment induced by hydrogen peroxide of vascular reactivity and the role of endogenous catalase in protection against this impairment was assessed in isolated rings of rat aorta.
2. Incubation with hydrogen peroxide at 1 mM, but not at 0.1 mM, for 15, 30 or 60 min followed by washout depressed, in a time-dependent manner, the subsequent ability of endothelium-containing and endothelium-denuded rings to contract to phenylephrine.
3. Incubation with 3-amino-1,2,4-triazole (50 mM, 90 min, followed by washout) to inhibit endogenous catalase had no effect by itself on subsequent phenylephrine-induced contraction. However, pretreatment with 3-amino-1,2,4-triazole did lead to a profound enhancement of the ability of hydrogen peroxide (1 mM, present for the final 30 min of the 90 min incubation, followed by washout) to depress phenylephrine-induced contraction in both endothelium-containing and endothelium-denuded rings.
4. Incubation with hydrogen peroxide at 1 mM, but not at 0.1 mM, for 15, 30 or 60 min followed by washout inhibited, in a time-dependent manner, the subsequent ability of acetylcholine (10 nM–3 μM) to induce endothelium-dependent relaxation. Furthermore, incubation with hydrogen peroxide 1 mM (30 min, followed by washout) also inhibited relaxation induced by glyceryl trinitrate (1–100 nM) or isoprenaline (10 nM–3 μM) in endothelium-denuded rings.
5. Incubation with 3-amino-1,2,4-triazole (50 mM, 90 min, followed by washout) had no effect by itself on relaxation induced by acetylcholine, glyceryl trinitrate or isoprenaline. In contrast, pretreatment with 3-amino-1,2,4-triazole led to profound enhancement of the ability of hydrogen peroxide (1 mM, present for final 30 min of the 90 min incubation) to block relaxation to acetylcholine, glyceryl trinitrate or isoprenaline.
6. On the basis of the actions of 3-amino-1,2,4-triazole, it is likely that endogenous catalase plays an important role in the protection of vascular reactivity of rat aorta against oxidant damage by high (1 mM) but not lower (0.1 mM) concentrations of hydrogen peroxide. The data are consistent with the promotion of non-selective damage to the vascular smooth muscle cells by hydrogen peroxide, but endothelial damage may also be sustained.

     

Role of Ca2+-activated K+ channel in epithelium-dependent relaxation of human bronchial smooth muscle

1. To elucidate whether K⁺ channels play a role in the action of epithelium-dependent bronchodilatation, we studied responses in human bronchial strips in the presence of indomethacin and N^G-nitro-L-arginine methylester under isometric conditions, in vitro.
2. Mechanical removal of the epithelium increased the contractile responses to acetylcholine; the pD₂ values increased from 5.0±0.2 to 5.9±0.3 (P<0.001). This potentiation was abolished by iberiotoxin but not by apamin or glibenclamide.
3. In cascade bioassay, application of the bathing medium from dispersed, bronchial epithelial cells to epithelium-denuded bronchial strips decreased acetylcholine-induced contraction by 44±6%. This effect was reduced to 10±3% (P<0.01) when the epithelial cells were pretreated with iberiotoxin, and to 4±1% (P<0.001) when the epithelial cells were incubated with Ca²⁺-free medium containing [1,2-bis (2) aminophenoxy] ethane N,N,N′,N′-tetraacetic acid-acetomethoxy ester.
4. In contrast, the bronchodilator effect of the medium bathing epithelial cells was not altered by the direct addition of iberiotoxin to epithelium-denuded tissues.
5. These results suggest that the Ca²⁺-activated K⁺ channel may play a role in the synthesis and/or release of smooth muscle relaxing factor, which is neither nitric oxide nor a cyclo-oxygenase product, from airway epithelial cells.

     

用125I-M胆碱受体亚型特异抗体测定大鼠脏器M受体亚型的分布

目的人类多种疾病在Ｍ胆碱受体（Ｍ－ＡｃｈＲ）亚型的分布上有差别。本研究旨在根据Ｍ受体的氨基酸序列,合成人工多肽抗原,研究大鼠主要脏器Ｍ受体亚型的分布。方法制备特异性Ｍ１和Ｍ２受体抗体,用１２５标记两种抗体,注射到大鼠体内。结果研究显示Ｍ１和Ｍ２受体在大鼠心脏、肝脏、气管平滑肌、肾脏及肺组织中分布不同。结论人工抗体抗原诱发的Ｍ受体亚型抗体特异性好,为进行Ｍ受体亚型检测提供比较简便、可靠的方法。     

大鼠纹状体边缘区神经元中乙酰胆碱激活的离子通道特性

用细胞贴附式膜片钳技术研究了急性分离的新生SD大鼠纹状体边缘区神经元中乙酰胆碱激活的离子通道的特性。发现通道有25PS和60PS两种电导状态,25PS通道为主要类型,因此,本文主要报道25PS通道的特性。ACh激活的单通道电流为快速内向电流,随着超极化程度增加而增加,反转电位约为0mV。通道开放具单个开放和簇状开放,其开放时间均可权指数拟和,单个开放时间常数为0．29ms和1.84ms,簇状开放时间常数为1．96ms和18.24ms。平均关闭时间也可双指数拟和,两个时间常数为1．70ms和54．00ms。通道开放概率为0．012,未表现出电压依赖性。提示纹状体边缘区神经元上存在乙酸胆碱受体离子通道。根据特性证实为烟碱型离子通道。     

A 'long-term-potentiation-like' facilitation of hippocampal synaptic transmission induced by the nootropic nefiracetam

Nefiracetam, a nootropic agent, enhanced the slope of field excitatory postsynaptic potentials in the CA1 region of rat hippocampal slices to about 170% of basal levels, being evident still at 4-h washing-out of the drug. A similar sustained enhancement (>/=16 h after i.m. injection with nefiracetam) was observed in the population spikes recorded from the granular cell layer of the intact mouse hippocampus. Saturation of the enhancement in the synaptic strength occluded potentiation obtained with long-term potentiation (LTP) induced by high-frequency (tetanic) stimulation, and vice versa. Interestingly, the facilitatory action of nefiracetam was blocked by either the nicotinic acetylcholine (ACh) receptor antagonists, alpha-bungarotoxin and mecamylamine, or the selective protein kinase C (PKC) inhibitor, GF109203X, but in contrast, it was not affected by D-2-amino-5-phosphonovaleric acid (APV), a selective N-methyl-D-aspartate (NMDA) receptor antagonist. The results of the present study suggest that nefiracetam, whereas the action is independent of NMDA receptors, induces an 'LTP-like' facilitation of hippocampal synaptic transmission as a consequence of modulation of nicotinic ACh receptors and PKC. This may represent a likely mechanism underlying the cognition-enhancing actions of nefiracetam.     

Cells defective in sphingolipids biosynthesis express low amounts of muscle nicotinic acetylcholine receptor

The properties of the nicotinic acetylcholine receptor (AChR) are modulated by its lipid microenvironment. Studies of such modulation are hampered by the cell's homeostatic mechanisms that impede sustained modification of membrane lipid composition. We have devised a novel strategy to circumvent this problem and study the effect of changes in plasma membrane lipid composition on the functional properties of AChR. This approach is based on the stable transfection of AChR subunit cDNAs into cells defective in a specific lipid metabolic pathway. In the present work we illustrate this new strategy with the successful transfection of a temperature-sensitive Chinese hamster ovary (CHO) cell line, SPB-1, with the genes corresponding to the four adult mouse AChR subunits. The new clone, SPB-1/SPH, carries a mutation of the gene coding for serine palmitoyl transferase, the enzyme that catalyses the first step in sphingomyelin (Sph) biosynthesis. This defect causes a decrease of Sph de novo synthesis at non-permissive temperatures. The IC50 for inhibition of alpha-BTX binding with the agonist carbamoylcholine exhibited values of 3.6 and 2.7 microm in the wild-type and Sph-deficient cell lines, respectively. The corresponding IC50 values for the competitive antagonist D-tubocurarine (D-TC) were 2.8 and 3.4 microm, respectively. No differences in single-channel properties were observed between wild-type and mutant cell lines grown at the non-permissive, lipid defect-expressing temperature using the patch-clamp technique. Both cells exhibited two open times with mean values of 0.35 +/- 0.05 and 1.78 +/- 0.2 ms at 12 degrees C. Taken together, these results suggest that the AChR is expressed as the complete heteroligomer. However, only 10-20% of the total AChR synthesized reached the surface membrane in the mutant cell line and exhibited a higher metabolic turnover, with a half-life about 50% shorter than the wild-type cells. When control CHO-K1/A5 cells were treated with fumonisin B1, an inhibitor of sphingosine (sphinganine) N-acetyltransferase (ceramide synthase), a 45.5% decrease in cell surface AChR expression was observed. The results suggest that sphingomyelin deficiency conditions AChR targeting to the plasma membrane.     

Regional differences of the effect of σ receptor ligands on the acetylcholine release in the rat brain

Summary We found that a receptor ligands differentially regulated the acetylcholine (ACh) neurotransmission in the rat brain. Acute administration of (+)-N-allylnormetazocine [(+)-SKF-10,047], a prototype ₁ receptor ligand, and 1,3-di(2-tolyl)guanidine (DTG), a non-specific receptor ligand, increased the extracellular ACh level in the rat hippocampus. This increase of hippocampal extracellular ACh level elicited by (+)-SKF-10,047 was more potent than that elicited by DTG. On the other hand, the striatal extracellular ACh level was slightly affected by (+)-SKF-10,047. In addition, DTG did not affect the striatal extracellular ACh level. Our previous studies have shown that both (+)-SKF-10,047 and DTG increased the extracellular ACh level in the rat frontal cortex. Taking all these data into consideration, the regulation of ACh neurotransmission by receptor ligands are different depending upon the brain region.     

Role of Ca2+-activated K+ channels in acetylcholine-induced dilatation of the basilar artery in vivo

1. We tested the hypothesis that activation of large conductance calcium-activated potassium channels is involved in dilator responses of the basilar artery to acetylcholine in vivo. Using a cranial window in anaesthetized rats, we examined responses of the basilar artery to acetylcholine.
2. Topical application of acetylcholine (10⁻⁶ and 10⁻⁵ M) increased diameter of the basilar artery from 238±7 μm to 268±7 and 288±7 μm, respectively (P<0.05 vs. baseline diameter). Iberiotoxin (10⁻⁸ M), an inhibitor of large conductance calcium-activated potassium channels, did not affect baseline diameter of the basilar artery. In the presence of 10⁻⁸ M iberiotoxin, 10⁻⁶ and 10⁻⁵ M acetylcholine increased diameter of the basilar artery from 239±7 μm to 246±7 and 261±7 μm, respectively. Thus, iberiotoxin attenuated acetylcholine-induced dilatation of the basilar artery (P<0.05).
3. Sodium nitroprusside (10⁻⁷ and 10⁻⁶ M) increased diameter of the basilar artery from 242±9 μm to 310±12 and 374±13 μm, respectively (P<0.05 vs. baseline diameter). In the presence of iberiotoxin (10⁻⁸ M), sodium nitroprusside (10⁻⁷ and 10⁻⁶ M) increased diameter of the basilar artery from 243±6 μm to 259±9 and 311±12 μm, respectively. Thus, iberiotoxin attenuated dilator responses of the basilar artery to sodium nitroprusside (P<0.05).
4. Iberiotoxin partly inhibited dilator responses of the basilar artery to forskolin, a direct activator of adenylate cyclase, but did not affect vasodilatation produced by levcromakalim, a potassium channel opener.
5. These results suggest that dilator responses of the basilar artery to acetylcholine and sodium nitroprusside are mediated, in part, by activation of large conductance calcium-activated potassium channels. Because both acetylcholine and sodium nitroprusside have been shown to activate guanylate cyclase via nitric oxide, activation of large conductance calcium-activated potassium channels may be one of the major mechanisms by which cyclic GMP causes dilatation of the basilar artery in vivo.

     

The role of Ca2+ in the elimination of polyneuronal innervation of rat soleus muscle fibres

The mechanism by which nerve - muscle contacts are reduced during postnatal development of the rat soleus muscle was investigated using electrophysiological methods. Between days 7 and 9 after birth, soleus muscle fibres lose 0.19–0.24 terminals per muscle fibre within 24 h. A much more rapid loss of contacts is seen when muscles are exposed in vitro to acetylcholine (10⁻³ g/ml). In this case 0.67–0.87 terminals per muscle fibre lose contact within 2 h. The loss of neuromuscular contacts induced by acetylcholine can be reduced by preincubating the muscles in solutions containing acetoxymethyl ester of 1,2-bis(2-amino-phenoxylethane-N,N₁;N₁-tetraacetic acid (BAPTA-AM), a Ca²⁺ chelating agent that enters cells and reduces the Ca²⁺ transients inside the cell. Treatment of muscles with nifedipine, which blocks dihydropyridine-sensitive (L-type) Ca²⁺ channels, also reduced the acetylcholinesterase-induced loss of neuromuscular contacts. The results indicate that transient increases in Ca²⁺ inside nerve terminals contribute to loss of neuromuscular contacts, and that these increases occur by Ca²⁺ entry through L-type channels.