突触功能必需基因参与线虫衰老的调控(英文)

目的鉴定参与线虫衰老的神经内分泌调控的新基因。方法鉴于神经系统在衰老调控中的重要作用,通过寿命分析和脂褐质自发荧光的检测,从编码突触蛋白的遗传位点中筛选参与衰老调控的基因。我们还进一步检查了这些遗传位点相应的突变体的永久性幼虫形成情况,探讨它们是否可能受胰岛素样信号通路的调控。结果遗传位点 unc-10,syd-2,hlb-1,dlk-1,mkk-4,scd-2,snb-1,ric-4,nrx-1,unc-13,sbt-1,unc-64 可能参与线虫衰老的调控。而且在衰老的调控中,unc-10,syd-2,hlb-1,dlk-1,mkk-4,scd-2,snb-1,ric-4,nrx-1 的功能可能与unc-13,sbt-1,unc-64相反。肠道脂褐质自发荧光的检测进一步证明了筛选出的各基因对应突变体的长寿或短寿表型,是由减慢或缩短的组织衰老所致。在筛选出的基因中,syd-2,hlb-1,mkk-4,scd-2,snb-1,ric-4,unc-64 也参与了永久性幼虫形成的调控。另外,daf-2突变增强了syd-2和hlb-1的表达,降低了mkk-4,nrx-1,ric-4,sbt-1,rpm-1,unc-10,dlk-1,unc-13 的表达。daf-16突变提高了syd-2和 hlb-1 的表达,降低了mkk-4,nrx-1,sbt-1,rpm-1,unc-10,dlk-1,unc-13 的表达. 结论突触功能可能在个体寿命和永久性幼虫形成的调控机制中具有重要的作用。     

Electrical responses of the muscularis externa to distension of the isolated guinea-pig distal colon

Abstract Enteric reflex pathways were studied in isolated segments of guinea-pig distal colon by recording the electrical responses to distension from the muscularis externa with suction electrodes. The end of the electrode wire were in the circular muscle and thus the recordings discussed below are deduced to be primarily from this layer. Moreover, intracellular microelectrodes in circular muscle cells and suction electrodes recorded similar events. Spontaneous activity consisted of myogenic slow waves at about 25 min ^-1 and transient biphasic potentials at about 6 min^-1 and 3-sec duration which were dependent on a stimulus from the enteric nervous system as they were blocked by tetrodotoxin (0.5 μM), d-tubocurarine (30 μM) and hexa-methonium (100 μM). Atropine (0.8 μM) blocked the depolarizing part of the biphasic potentials and unmasked transient spontaneous inhibitory junction potentials (IJPs) (～2-sec duration) which appeared to be responsible for the hyperpolarizing part of the biphasic potential. Three different responses were observed at sites oral to distension of the colon: a transient depolarizing response that was cholinergic (blocked by atropine (0.8 μM); ascending cholinergic excitation) and, after atropine, a transient IJP (ascending inhibition) which was followed by a transient non-cholinergic depolarizaton (ascending non-cholinergic excitation) that was sometimes followed by several cycles of slow wave activity. The oral responses to anal distension were also blocked by the nicotinic antagonists and were similar to the neurogenic spontaneous events, which also appeared to originate from activity in ascending nervous pathways. Four different responses were observed following distension of the oral end of the segment: an IJP followed by a prolonged phase of hyperpolarization that lasted for the duration of the distension (descending inhibition); a burst of depolarizing potentials (for up to 30 sec) that followed the termination of distensions up to 25 sec and was blocked by atropine (0.8 μM) (delayed cholinergic excitation), and a transient non-cholinergic response that immediately followed the termination of distension (non-cholinergic ‘off’ response). Apamin (0.5 μM) reduced the amplitude of the spontaneous IJPs and evoked IJPs. After apamin, distension evoked a small transient hyperpolarization at oral sites, which was similar to spontaneous events, and the prolonged hyperpolarization at anal sites. A second distension given within 20 sec of the first evoked an IJP of reduced amplitude at oral sites in every preparation. In contrast, the amplitudes of the oral Cholinergic excitation and descending inhibition were relatively unaffected by reducing the interval between distensions. Thus distension stimulates excitatory and inhibitory motor neurons supplying the circular muscle both oral and anal to the stimulus. The polarity of the reflex relies in part on the differences in timing and duration of responses as well as the transmission characteristics of the nervous pathways.     

Synaptic and extrasynaptic NMDA receptors: Problems and prospects

This review is devoted to the problem of induction of differently directed NMDA-dependent longterm plasticity in the central nervous system and their transformation into pathological changes. Their hypothetical mechanisms and the corresponding experimental data are discussed. Special attention is paid to the functional characteristics of synaptic and extrasynaptic NR1/NR2A and NR1/NR2B receptors, their different involvement in the spatiotemporal organization of Ca²⁺ signaling, and peculiarities of biochemical reactions of the cell. The possible structural reorganization of NMDA receptors as one of the kinds of plasticity is also analyzed.     

Differential Distribution of Syntaxin Isoforms 1A and 1B in the Rat Central Nervous System

Syntaxin 1 binds to several proteins of the synaptic terminal and is a central component in the pathway of protein–protein interactions that underlies docking and fusion of synaptic vesicles. Molecular studies revealed the occurrence of two isoforms, syntaxin 1A and syntaxin 1B, which coexpress in neural tissues. However, they display differential expression patterns in endocrine cell types. We generated isoform-specific antibodies that were used in Western blotting and immunocytochemical studies. First, we confirmed the sole presence of syntaxin 1A in endocrine pituitary cells. Second, we found distinctive immunolabelling patterns of each isoform in the rat olfactory system, hippocampus, striatum, thalamus and spinal cord. In addition, the principal white matter commissures displayed distinct immunoreactivity for each isoform. This report shows, for the first time, major differences between the distributions of syntaxin 1A and syntaxin 1B isoforms in the rat central nervous system.     

NANC transmitters in the female pig urethra–localization and modulation of release via α2-adrenoceptors and potassium channels

1. To investigate further the release, localization and identity of a non-nitrergic mediator of smooth muscle relaxation in the female pig urethra, we studied the effects of drugs acting at α₂-adrenoceptors or K⁺ channels, the effects of capsaicin and chemical sympathectomy, and the actions of several transmitter candidates.
2. Electrical field stimulation (EFS; frequencies above 12 Hz) of spontaneously contracted smooth muscle strips from the female pig urethra evoked long-lasting, frequency-dependent relaxations in the presence of prazosin, scopolamine, and N^G-nitro-L-arginine. Treatment with the selective α₂-adrenoceptor agonist UK-14 304 markedly reduced the relaxations evoked by EFS at all frequencies tested (16–30 Hz). The inhibitory effect of UK-14 304 was completely antagonized by the α₂-adrenoceptor antagonist rauwolscine. The muscarinic M₁ receptor antagonist, pirenzepine, or exogenously administered carbachol, did not have any effects on the electrically evoked relaxations.
3. Inhibition of high conductance Ca²⁺ activated K⁺ channels by iberiotoxin or charybdotoxin significantly enhanced the relaxations evoked by EFS at all frequencies. However, inhibition of voltage-sensitive K⁺ channels with 4-aminopyridine or dendrotoxin-1, treatment with the ATP-sensitive K⁺ channel blocker, glibenclamide, or treatment with the high and low conductance Ca²⁺ activated K⁺ channel blockers, tetraethylammonium chloride and apamin, had no effect on the relaxations evoked by EFS.
4. Electrically evoked relaxations were not affected by adrenergic denervation with 6-hydroxydopamine (6-OHDA) at any frequency. However, treatment with 6-OHDA abolished prazosin-sensitive electrically induced contractions, and a long-lasting relaxation was revealed. Treatment with capsaicin, believed to damage selectively a subpopulation of primary afferent fibres, did not affect basal tone or relaxations evoked by EFS.
5. Exogenously applied vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP)-27, PACAP-38, adenosine, ATP and 5-hydroxy-tryptamine caused relaxations of the urethral preparations, whereas prostaglandin E₂ and calcitonin gene-related peptide had no effects. VIP 10-28, α, β-methylene-ATP, reactive blue-2, suramin or indomethacin did not reduce the electrically-evoked relaxations at any frequency. However, the relaxations were slightly reduced by trypsin or α-chymotrypsin.
6. The present results suggest that the release of the unknown mediator in the female pig urethra can be modulated via α₂-adrenoceptors and high conductance Ca²⁺ activated K⁺ channels. Furthermore, the mediator does not appear to be localized to or released from adrenergic or capsaicin-sensitive sensory nerve-endings. The identity of the transmitter remains to be established.

     

Opposing effects on blood pressure following the activation of metabotropic and ionotropic glutamate receptors in raphe obscurus in the anaesthetized rat

The microinjection of l-glutamate (1–6 nmol/rat) and N-methyl-d-aspartate (NMDA 1–10 nmol/rat), ionotropic glutamate receptor (iGluR) agonists, into the nucleus raphe obscurus caused a concentration-dependent increase of arterial blood pressure. In contrast, (±)-1-aminocyclopentane-trans-1, 3-dicarboxylic acid (t-ACPD, 14–42 nmol/rat), a metabotropic glutamate receptor (mGluRs) agonist, caused a concentration-dependent decrease in blood pressure. Pretreatment with D, L-2-amino-phosphono valeric acid (2-APV, 5 nmol/rat) a selective NMDA iGluR antagonist, and (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a, b] cyclohepten-5,10-imine hydrogen maleate (MK801, 0.9 nmol/rat), a noncompetitive NMDA iGluR antagonist, blocked both the glutamate and NMDA pressor responses, while pretreatment with (+)--methyl-4-carboxyphenylglycine (MCPG, 0.05 nmol/rat), a mGluR₁ antagonist, increased the glutamate-induced pressor effects and blocked the fall in blood pressure induced by t-ACPD. 6-Cyano-7-nitroquinoxaline-2,3dione-(CNQX, 0.4 nmol/rat) a non-NMDA iGluR antagonist, did not affected the glutamate-induced hypertension. These observations indicate opposing roles for ionotropic and metabotropic receptors in the glutamate-induced blood pressure changes elicited from the nucleus raphe obscurus. Moreover, we suggest that the glutamate-induced hypertension may be due to the activation of NMDA ionotropic receptor subtypes and the metabotropic receptors may influence this activaction through a reduction of excitability at level of synapses.     

Pertussis toxin does not affect the adenosine-induced inhibition of the efferent function of cardiac capsaicin-sensitive nerves

Summary The negative inotropic effect of adenosine (1–100 M) was abolished in isolated guinea-pig atria obtained from pertussis toxin-pretreated guinea pigs electrically driven at 4 Hz. However, the inhibitory effect of the same concentrations of adenosine on the cardiac response to stimulation of non-adrenergic non-cholinergic (NANC), capsaicin-sensitive sensory nerves, was not modified by the toxin.These results suggest that, while pertussis toxin-sensitive G proteins are involved in the negative inotropic effect of adenosine, they do not mediate the inhibitory effect of adenosine on cardiac NANC neurotransmission.     

In vitro studies on 6-fluoronoradrenaline at several peripheral sympathetic neuroeffector junctions

Summary A comparison of the effects of noradrenaline and 6-fluoronoradrenaline has been made at several peripheral sympathetic neuroeffector junctions. In the rat vas deferens preparation in the presence of 1 M cocaine, 6-fluoronor-adrenaline was found to be about 9 times more potent than noradrenaline as an agonist at presynaptic inhibitory ₂-adrenoceptors. In the rabbit aorta, 6-fluoronoradrenaline had approximately one tenth of the potency of noradrenaline in stimulating the postsynaptic ₁-adrenoceptors. Furthermore 6-fluoronoradrenaline, in contrast to previous reports, appears to be a substrate for the neuronal uptake process since exposure to cocaine potentiated the inhibition of the twitch response of the vas deferens by 6-fluoronoradrenaline. In addition, 6-fluoronoradrenaline increased the spontaneous outflow of radioactivity from rabbit pulmonary artery strips prelabelled with ³H-noradrenaline and this increase was blocked by cocaine (30M).These results demonstrate that 6-fluoronoradrenaline is a preferential ₂-adrenoceptor agonist which is a substrate for the neuronal uptake process in peripheral sympathetically innervated smooth muscle preparations.     

Effect of age on the prejunctional α-adrenoceptor-mediated feedback in the heart of spontaneously hypertensive rats and normotensive Wistar Kyoto rats

Summary The prejunctional ₂-adrenoceptor-mediated feed-back in the heart of pithed young and adult spontaneously hypertensive rats (SHR) and corresponding normotensive Wistar Kyoto rats (WKY) was studied. After electrical stimulation of the sympathetic outflow from the spinal cord to the heart, B-HT 920 induced an inhibition of the cardiac response, which was significant at stimulation frequencies up to 1 Hz in young SHR and WKY and up to 2 Hz in the adult animals. Rauwolscine produced a potentiation of the cardiac response to electrical stimulation in SHR, which was significant from 0.2–10 Hz in young SHR and from 0.1–10 Hz in adult SHR. In young WKY, rauwolscine did not potentiate the increase in heart rate to sympathetic nerve stimulation, whereas in adult WKY ₂-adrenoceptor blockade by rauwolscine significantly potentiated the cardiac response to electrical stimulation at frequencies in the range of 0.2–10 Hz. In SHR the potentiation of the cardiac response to sympathetic nerve stimulation by rauwolscine was much stronger than in WKY.These results suggest that in adult animals the prejunctional ₂-adrenoceptor mediated feedback is more developed than in young rats. In contrast with young WKY, a significant endogenous feedback can be demonstrated in adult WKY. In SHR, however, the physiological role of prejunctional ₂-adrenoceptors is much more important.     

Analysis of the potentiating action of NG-nitro-l-arginine on the contraction of the dog temporal artery elicited by transmural stimulation of noradrenergic nerves

Summary Dog temporal artery strips without endothelium responded to transmural electrical stimulation with a contraction which was potentiated by N^G-nitro-l-arginine (l-NNA). The noradrenaline-induced contraction and the release of 3H-noradrenaline were not affected. The stimulation-induced contraction was reversed to a relaxation by phentolamine. The relaxation was not influenced by timolol and atropine but inhibited by l-NNA; l-arginine abolished the inhibition. Transmural stimulation released NO_x from the arteries, the release being abolished by l-NNA. Potentiation by l-NNA of the neurally-induced contraction appears to be due to elimination of NO produced by non-adrenergic, non-cholinergic vasodilator nerve activation. Send offprint requests to N. Toda at the above address