l-DOPA induces concentration-dependent facilitation and inhibition of presynaptic acetylcholine release in the guinea-pig submucous plexus

Neurotransmitter- or neuromodulator-like actions ofl-DOPA were investigated with intracellular recordings from submucous plexus neurons of the guinea-pig caecum.l-DOPA at 30 nM augmented the amplitude of fast EPSPs, but did not affect depolarizations elicited by puff application of acetylcholine (ACh). The augmenting effect ofl-DOPA on the fast EPSPs was counteracted byl-DOPA methyl ester. The fast EPSPs were depressed by 10 μMl-DOPA, but transiently augmented after rinsing the drug.l-DOPA methyl ester did not affect the inhibitory action ofl-DOPA on the fast EPSPs, but antagonized the potentiation following the inhibition. The depolarization elicited by exogenously applied. ACh was inhibited by 10 μMl-DOPA. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i) of the neuronal soma were measured with fura-2 microfluorophotometry. The transient increase in the [Ca²⁺]_i evoked by the somatic action potential (Δ[Ca²⁺]_AP) was facilitated by 30 nMl-DOPA, but decreased by the drug at 10 μM. It is concluded thatl-DOPA at low concentrations enhances the Δ[Ca²⁺]_AP, increasing the neurotransmitter release, but at high dose diminishes the Δ[Ca²⁺]_AP, inhibiting the neurotransmission.     

Cerebral type 2 astroglia are heterogeneous with respect to their ability to respond to neuroligands linked to calcium mobilization.

Very little information is available concerning the pharmacology of type 2 astroglia. During the past decade it has become apparent that two distinct lineages of astroglial cells can be defined in vitro. These two lineages are commonly referred to as type 1 and type 2 and are distinguished from each other on the basis of their morphological features and antigenic phenotypes. In contrast to type 1 astroglia, very little is known about the pharmacology of type 2 astroglia. The lack of information concerning the responsiveness of these cells stems primarily from difficulties encountered in isolating large numbers of type 2 astroglia free of other cell types. In the present study video- and photometer-based imaging systems were used to monitor the influence of a series of neuroligands on the intracellular calcium levels of individual cerebral type 2 astroglia in order to assess their expression of calcium-mobilizing receptors. The responses of 85 immunocytochemically identified cerebral type 2 astroglia to bradykinin (BK), norepinephrine (NE), histamine (HIST), carbachol (CARB), 2-methyl-thio ATP (2MT-ATP), glutamate (GLUT), and serotonin (5-HT) were analyzed. Approximately 50% of cerebral type 2 astroglia responded to BK, NE, HIST, CARB, and 2MT-ATP whereas only 16% and 9% of the cells responded to GLUT and 5-HT, respectively. The number of neuroligands that increased calcium in individual cells ranged from 0 to 6. These responses are quite similar to those previously demonstrated in cultured cerebral type 1 astroglia. No pattern of receptor co-expression was observed for the different neuroligands tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

The amphicrine pancreatic cell line AR42J: a model system for combined studies on exocrine and endocrine secretion

Summary Secretory vesicles of both the exocrine and the endocrine pancreas have been isolated and characterized in molecular terms from pancreatic tissue and primary cell cultures. Studies on pancreatic secretory processes could be further facilitated by the use of permanent cell lines that respond to secretory stimuli with a regulated secretory response. We now present biochemical, morphological and secretory studies on the rat pancreatic acinar cell line AR42J. This cell line is characterized by the presence of digestive enzyme-containing dense core vesicles, which are released in response to cholecystokinin. In addition, we present evidence that these cells also contain small neuroendocrine-specific vesicles, as evidenced by the expression of the neuroendocrine-specific vesicle proteins synaptophysin and S.V.2. Corresponding to these mixed exocrine-neuroendocrine features, we also found considerable amounts of the neurotransmitters glycine, glutamine and gammaaminobutyric acid (GABA), as well as the rate-limiting enzyme in GABA synthesis, glutamic acid decarboxylase (GAD) (EC 4.1.1.15) expressed in these cells. We demonstrated a specific uptake mechanism for radioactively-labelled GABA by these cells. In addition, GABA was released from intracellular storage pools by nicotinic receptor stimulation or membrane depolarization. In summary, AR42J cells represent the first amphicrine pancreatic cell line with the combined expression of exocrine and neuroendocrine secretory organelles, both of which follow a regulated secretory pathway in response to various secretory stimuli.Abbreviations DCV dense core vesicles - GABA gammaaminobutyric acid - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Differential Cellular Gene Expression in Ganglioglioma

Summary:  Purpose:  Gangliogliomas (GGs) are neuronal-glial tumors highly associated with epilepsy. We hypothesized that the expression of select gene families including neurotransmitter receptor subunits and growth factors would be distinct in neurons and astrocytes within GG compared with adjacent cortex and that these changes would yield insights into seizure onset and lesion formation.
Methods:  Candidate gene expression was defined in single immunohistochemically labeled neurons and astrocytes microdissected from GG specimens compared with neurons and astrocytes microdissected from morphologically intact cortex adjacent to the GG or normal control cortex.
Results:  Differential expression of 16 genes including glutamate transporter (EAAC1) and receptor (NMDA2C, mGluR5), growth factor (hepatocyte growth factor), and receptor (platelet derived growth factor receptor β, fibroblast growth factor receptor 3) mRNAs was detected in GG neurons compared with control neurons. In astrocytes, altered expression of p75NGF, mGluR3, TGFβ3 and Glt-1 mRNAs was detected. Nestin mRNA, a gene that exhibits enhanced expression in balloon cell cortical dysplasia, was increased in GG neurons. Because of the morphological similarities between GG and cortical dysplasia, we show that there is activation of the mTOR cascade in GG as evidenced by enhanced expression of phospho-p70S6kinase and phosphoribosomal S6 proteins.
Conclusion:  We find differential candidate gene expression in neurons and astrocytes in GG compared with adjacent cortex and show that there is activation of the mTOR pathway. These changes highlight pathways that may be pivotal for epileptogenesis and lesion growth.     

Catecholamine alterations in experimental hydrocephalus

Experimental hydrocephalus was induced in rats by intracisternal injection of kaolin suspension. The amounts of norepinephrine and dopamine were determined in the whole brain and specific brain regions at 1 week (acute phase) and 4 weeks (chronic phase). The turnover of catecholamine, an index of the activity of catecholamine-containing neurons, was determined by measuring the decrease in catechlamine contents 2 h after intraperitoneal injection of -methyl-p-tyrosine (250 mg/kg), an inhibitor of tyrosine hydroxylase. We observed that the catecholamine contents in kaolin-induced hydrocephalus were not significantly different from control values. Following injection of -methyl-p-tyrosine, there was decrease in levels of catecholamines in both control and hydrocephalic rats. This decrease was, however, significantly less in induced hydrocephalus than in control animals. This result suggested that in hydrocephalus, the activities of norepinephrinergic and dopaminergic neurons are reduced.     

Regulation of GABA release by depolarisation-evoked Ca2+ transients at a single hippocampal terminal

We correlated dynamic changes in free cytosolic [Ca²⁺] ([Ca²⁺]_i) within single presynaptic terminals of cultured hippocampal neurones with the postsynaptic GABA-mediated currents. The local changes in [Ca²⁺]_i and evoked inhibitory postsynaptic currents (eIPSCs) were recorded simultaneously using Fura-2 fluorescence and whole-cell patch-clamp respectively. The Ca²⁺ signals and eIPSCs were evoked by direct extracellular electrical stimulation of a single presynaptic terminal by short depolarising pulses. The presynaptic Ca²⁺ transient was graded by varying the amplitude of extracellular stimulating pulses. The probability of the release event, P, estimated for each stimulation strength, reached a maximum (P=1) when the Ca²⁺ signal became maximal and remained at this level at higher stimulation strength, despite the subsequent decrease in the amplitude of the Ca²⁺ transient. A gradual, linear increase in stimulation amplitude (V_stim) resulted in a bell-shaped dependence of the averaged amplitudes of Ca²⁺ signals and corresponding averaged amplitudes of eIPSCs. Analysis of the eIPSC demonstrated that the decrease in both the mean eIPSC amplitude and the mean quantal content of release resulted from a reduction in the probability of multivesicular release, i.e. in the disappearance of failures and in the decrease of individual eIPSC amplitude. The Ca²⁺ signals of similar amplitude resulted in both random and determinate (non-random) neurotransmitter release. We conclude that depolarisation-induced elevation of [Ca²⁺]_i within the terminal is necessary but not sufficient for activation of vesicular release of neurotransmitter.     

Dynamic storage of glutamate in rat brain synaptic vesicles

Storage of [³H]glutamate accumulated by highly purified synaptic vesicles from brain was characterized. [³H]Glutamate was lost with single exponential kinetics with a time constant of minutes after synaptic vesicles were diluted into medium that allowed uptake to continue but that contained unlabeled glutamate in place of [³H]glutamate. This [³H]glutamate efflux occurred at similar rates in media containing 50 and 500 μM glutamate, which suggests that it did not depend on the rate of glutamate transport and was independent of the external and internal glutamate concentrations. All efflux was blocked at 0°C. These results imply that glutamate stored in synaptic vesicles turns over with a half-time of minutes, even during active uptake under physiological conditions.     

The dorsal facial area of the medulla in cats: inhibitory action of serotonin on glutamate release in regulating common carotid blood flow

Whether glutamate and serotonin would release and interact in the dorsal facial area (DFA) of cat medulla to regulate common carotid arterial (CCA) blood flow was explored by placing a microdialysis probe in DFA and employing high performance liquid chromatographic technique. Glutamate concentration was dose-dependently decreased by perfusion with serotonin, or alaproclate, a serotonin reuptake inhibitor. Serotonin and glutamate concentrations were increased by perfusion with KC1, a depolarizing agent. Furthermore, CCA blood flow was decreased when glutamate concentration was reduced by serotonin or alaproclate perfusion, and conversely increased when glutamate concentration was increased by KC1 perfusion. In conclusion, glutamate and serotonin releases in DFA that involve regulation of CCA blood flow are tonically mediated by nerve terminals. The glutamate release is depressed by the serotonin release.     

Effects of verapamil,diltiazem and ryosidine on the release of dopamine and acetylcholine in rabbit caudate nucleus slices

Summary Slices of the rabbit caudate nucleus were preincubated with ³H-dopamine or ³H-choline and then superfused with label-free medium. Release of ³H-dopamine and ³H-acetylcholine was elicited by either electrical stimulation at 8 (in one series 2) Hz, or an increase in the K⁺ concentration by 50 mmol/l, or addition of L-glutamate 1 mmol/l. Verapamil 1 mol/l, diltiazem 1 and 10 mol/l, and ryosidine 1 mol/l failed to the reduce the electrically-, K⁺- and glutamate-evoked overflow of tritium. Verapamil 1 mol/l and diltiazem 10 mol/l also failed to reduce the electricallyevoked overflow (2 Hz) when dopamine receptors, neuronal dopamine uptake, and neuronal choline uptake were blocked by domperidone, nomifensine and hemicholinium, respectively. Inhibition of the evoked overflow of tritium was only obtained when concentrations were increased to verapamil 10 mol/l, diltiazem 100 mol/l and ryosidine 10 mol/l. The inhibition was generally small. It was more evident for slices preincubated with ³H-choline than for those preincubated with ³H-dopamine, because in the latter verapamil, diltiazem and (much less) ryosidine accelerated the basal efflux of tritium. The inhibition of the K⁺-evoked overflow of tritium was probably due to blockade of Ca²⁺ channels because this overflow was Ca²⁺-dependent but tetrodotoxin-resistant. In contrast, the inhibition of the electrically- and glutamateevoked overflow possibly involved blockade of Na⁺ channels as well. The results indicate that three calcium antagonists from different chemical classes are very weak inhibitors of Ca²⁺ entry into, and hence transmitter release from, the terminal axons of central dopaminergic and cholinergic neurones. The function of the high affinity calcium antagonist binding sites that have been identified in brain remains unknown.     

从发病机制概述针刺治疗血管性痴呆的研究进展

血管性痴呆是脑血管病变后所引起的认知功能障碍,是临床中较为常见的痴呆类型。其发病机制源于血管系统损害引起低脑血流灌注、氧化应激等一系列病理过程。针灸作为一种非药物疗法,在脑血管病及相关疾病中有较为广泛的临床应用,许多研究都显示了针灸对认知功能的改善作用,并涉及细胞凋亡、氧化应激、神经炎症、神经递质通路等方面的调节作用。因此,现从血管性痴呆的发病机制角度探讨针刺的治疗作用,以期为进一步揭示针灸在血管性痴呆治疗中的潜在机制提供参考。