Modulation by endogenous ATP of the light-evoked release of ACh from retinal cholinergic neurones

The retina is an area of the central nervous system that possesses intrinsic cholinergic neurones which release acetylcholine (ACh) in response to stimulation with flickering light. Using an eye-cup preparation in anaesthetized rabbits we found that when the retina was exposed to the P2-purinoceptor antagonist, PPADS, the light-evoked release of ACh was strikingly increased (by over 40%). In contrast, ATP reduced the light-evoked release of ACh by 20%. The inhibitory effect of ATP was not due to its catabolism to adenosine because it was not affected by the A1-adenosine receptor antagonist, DPCPX, in combination with adenosine deaminase. The actions of both ATP and PPADS were completely blocked by strychnine. We conclude that during physiological stimulation of the retina with light, ATP is co-released with ACh and partially inhibits ACh release by activating (with ACh) an inhibitory glycinergic feedback loop.     

Nocistatin reverses nociceptin inhibition of glutamate release from rat brain slices

We have examined the effects of the recently described heptadecapeptide nocistatin on K+-evoked glutamate release from rat cerebrocortical slices in vitro. In vivo, nocistatin reverses the action of nociceptin. Nocistatin (100 nM, n = 7) did not inhibit K+-evoked glutamate release alone. Nociceptin (100 nM) inhibited glutamate release by 51.7 +/- 8.3% (P < 0.05, n = 6) and this was fully reversed by nocistatin (100 nM). Nocistatin also appears to be an antagonist of nociceptin action in vitro.     

Inhibition of glycine currents by dextromethorphan in neurones dissociated from the guinea-pig nucleus tractus solitarii

1. The effect of dextromethorphan (DM) on the current induced by glycine in acutely dissociated nucleus tractus solitarii (NTS) neurones of guinea-pigs was studied by use of the whole-cell patch clamp technique. The effect of DM on gamma-aminobutyric acid (GABA)-induced currents (IGABA) was also examined. 2. DM inhibited 30 microM glycine-induced current (IGly), without affecting the current caused by 30 microM GABA. The action of DM was concentration-dependent, with a maximum effect at 100 microM, and reversible. The half-maximum inhibitory concentration (IC50) of DM was 3.3 microM, about 85 times higher than that of strychnine. 3. DM 3 microM shifted the concentration-response curve for glycine to the right without affecting the maximum value. DM 10 microM shifted the curve even more to the right, although it was not a parallel shift. Strychnine at a concentration of 0.1 microM shifted the curve for glycine in a nearly parallel fashion. 4. The effect of 10 microM DM was slightly weak voltage-dependency, but the lower concentration of DM, 3 microM, inhibited IGly equally at -50 mV and +50 mV. The effect of 3 microM DM on IGly showed no use-dependence. Blockade by strychnine 0.1 microM showed no voltage- or use-dependence. 5. The results indicate that DM inhibits IGly in single neurones of NTS, and further suggest that DM at a low concentration may act on the glycine receptor-ionophore complex, but not on the Cl channel of the complex. However, a relatively high concentration of DM may at least partly affect the Cl- channel of the complex.     

Interactions of inhibition and excitation in the light-evoked currents of X type retinal ganglion cells

The excitatory and inhibitory conductances driving the light-evoked currents (LECs) of cat and ferret ON- and OFF-center X ganglion cells were examined in sliced and isolated retina preparations using center spot stimulation in tetrodotoxin (TTX)-containing Ringer. ON-center X ganglion cells showed an increase in an excitatory conductance reversed positive to +20 mV during the spot stimulus. At spot offset, a transient inhibitory conductance was activated on many cells that reversed near ECl. OFF-center X ganglion cells showed increases in a sustained inhibitory conductance that reversed near ECl during spot stimulation. At spot offset, an excitatory conductance was activated that reversed positive to +20 mV. The light-evoked current kinetics of ON- and OFF-center X cells to spot stimulation did not significantly differ in form from their Y cell counterparts in TTX Ringer. When inhibition was blocked, current-voltage relations of the light-evoked excitatory postsynaptic currents (EPSCs) of both ON- and OFF-X cells were L-shaped and reversed near 0 mV. The EPSCs averaged between 300 and 500 pA at -80 mV. The metabotropic glutamate receptor agonist 2-amino-4-phosphonobutyric acid (APB), was used to block ON-center bipolar cell function. The LECs of ON-X ganglion cells were totally blocked in APB at all holding potentials. APB caused prominent reductions in the dark holding current and synaptic noise of ON-X cells. In contrast, the LECs of OFF-X ganglion cells remained in APB. An increase in the dark holding current was observed. The excitatory amino acid receptor antagonist combination of D-amino-5-phosphono-pentanoic acid (D-AP5) and 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)-quinoxalinedione (NBQX) was used to block ionotropic glutamate receptor retinal neurotransmission. The LECs of all ON-X ganglion cells were totally blocked, and their holding currents were reduced similar to the actions of APB. For OFF-X ganglion cells, the antagonist combination always blocked the excitatory current at light-OFF; however, in many cells, the inhibitory current at light-ON remained. ON-center X ganglion cells receive active excitation during center illumination, and a transient inhibition at light-OFF. In contrast OFF-center X ganglion cells experience a sustained active inhibition during center illumination, and a shorter increase in excitation at light-offset. Cone bipolar cells provide a resting level of glutamate release on X ganglion cells on which their light-evoked currents are superimposed [corrected].     

ORL1 receptor-mediated inhibition by nociceptin of noradrenaline release from perivascular sympathetic nerve endings of the rat tail artery

In the present study the effect of the opioid heptadecapeptide nociceptin, also termed orphanin FQ, an endogenous ligand for the orphan receptor named ORL1 (opioid receptor-like 1) receptor, was investigated on [3H]noradrenaline release induced by electrical field stimulation (24 pulses at 0.4 Hz, 200 mA, 0.3 ms duration) in the rat tail artery in the absence and presence of an alpha2-adrenoceptor antagonist, rauwolscine 3 microM. Nociceptin inhibited the electrically-evoked tritiated noradrenaline release in a concentration-dependent manner from rat tail arteries. This inhibitory effect of nociceptin was enhanced in the presence of the alpha2-adrenoceptor antagonist rauwolscine (maximum inhibition by 25% and 50% in the absence and presence of rauwolscine, respectively). At a supramaximal concentration (10 microM), the inhibitory action of DAGO, a selective micro-opioid receptor agonist, was less pronounced than that of nociceptin. The inhibitory effect of nociceptin was counteracted by naloxone benzoylhydrazone (3 microM) which by itself did not change the stimulation-evoked noradrenaline overflow. Naloxone (10 microM), a non-selective opioid receptor antagonist, did not affect the inhibitory effect of nociceptin whereas it abolished that of DAGO. In conclusion, these results suggest that nociceptin modulates noradrenergic neurotransmission by acting on prejunctional ORL1 receptors located on nerve terminals innervating the rat tail artery. They also demonstrate that prejunctional ORL1 receptors interact with prejunctional alpha2-adrenoceptors. The physiological significance of this phenomenon remains to be determined.     

Imaging of glutamate release from the goldfish retinal slice

A fluorometric procedure to image release of the neurotransmitter glutamate from living retinal slices is described. Patterns of endogenous glutamate efflux were imaged with a cooled CCD camera in goldfish retinal slices as NADH fluorescence produced by a cycling glutamate dehydrogenase (GDH). Basal and potassium evoked glutamate effluxes were strongly localized to the outer and inner plexiform layers, supporting the model that photoreceptors and bipolar cells release glutamate as their prime fast neurotransmitter.     

Noradrenaline release from rat sympathetic neurones triggered by activation of B2 bradykinin receptors

1. The role of bradykinin receptors in the regulation of sympathetic transmitter release was investigated in primary cultures of neurones dissociated from superior cervical ganglia of neonatal rats. These cultures were loaded with [3H]-noradrenaline and the outflow of radioactivity was determined under continuous superfusion. 2. Bradykinin (100 nmol l[-1] applied for 10 min) caused a transient increase in tritium outflow that reached a peak within four minutes after the beginning of the application and then declined towards the baseline, despite the continuing presence of the peptide. ATP (100 micromol l[-1]) and nicotine (10 micromol l[-1]) caused elevations in 3H outflow with similar kinetics, whereas outflow remained elevated during a 10 min period of electrical field stimulation (0.5 ms, 50 mA, 50 V cm[-1], 1.0 Hz). 3. When bradykinin was applied for periods of 2 min, the evoked 3H overflow was half-maximal at 12 nmol l(-1) and reached a maximum of 2.3% of cellular radioactivity. The preferential B1 receptor agonist des-Arg9-bradykinin failed to alter 3H outflow. The B2 receptor antagonists, [D-Phe7]-bradykinin (1 micromol l[-1]) and Hoe 140 (10 nmol l[-1]), per se did not alter 3H outflow, but shifted the concentration-response curve for bradykinin-evoked 3H overflow to the right by a factor of 7.9 and 4.3, respectively. 4. Bradykinin-induced overflow was abolished in the absence of extracellular Ca2+ and in the presence of either 1 micromol l(-1) tetrodotoxin or 300 micromol l(-1) Cd2+, as was electrically-induced overflow. Activation of alpha2-adrenoceptors by 1 micromol l(-1) UK 14,304 reduced both bradykinin- and electrically-triggered overflow. The Ca2+-ATPase inhibitor thapsigargin (0.3 micromol l[-1]) failed to alter either type of stimulated overflow. Caffeine (10 mmol l[-1]) enhanced bradykinin-induced overflow, but reduced overflow triggered by electrical field stimulation. 5. Inclusion of Ba2+ (0.1 to 1 mmol l[-1]) in the superfusion medium enhanced electrically induced overflow by approximately 100% and potentiated bradykinin-triggered overflow by almost 400%. Application of 1 mmol l(-1) Ba2+ for periods of 2 min triggered 3H overflow, and this overflow was abolished by 1 micromol l(-1) tetrodotoxin and enhanced by 10 mmol l(-1) caffeine. In contrast, inclusion of tetraethylammonium (0.1 to 1 mmol l[-1]) in the superfusion buffer caused similar increases of bradykinin- and electrically evoked 3H overflow (by about 100%), and tetraethylammonium, when applied for 2 min, failed to alter 3H outflow. 6. Treatment of cultures with 100 ng ml(-1) pertussis toxin caused a significant increase in bradykinin-, but not in electrically-, evoked tritium overflow. Treatment with 100 ng ml(-1) cholera toxin reduced both types of stimulated 3H overflow. 7. These data reveal bradykinin as a potent stimulant of action potential-mediated and Ca2+-dependent transmitter release from rat sympathetic neurones in primary cell culture. This neurosecretory effect of bradykinin involves activation of B2-receptors, presumably linked to pertussis- and cholera toxin-insensitive G proteins, most likely members of the Gq family. Results obtained with inhibitors of muscarinic K+ (KM) channels, like caffeine and Ba2+, indicate that the secretagogue action of bradykinin probably involves inhibition of these K+ channels.     

Polarity of 11-cis retinal release from cultured retinal pigment epithelium

Moyamoya disease is characterized by bilateral stenosis and/or occlusion of the terminal portion of the internal carotid artery. Moyamoya disease is prevalent among patients <10 years of age. Although most cases appear to be sporadic, approximately 10% occur as familial cases. The incidence of familial cases has been increasing because noninvasive diagnostic equipment, such as magnetic-resonance imaging and magnetic-resonance angiography, can detect the disease in almost all affected patients, including asymptomatic patients, during screening studies. In this study, we performed a total genome search to identify the location of a familial moyamoya disease gene in 16 families, assuming an unknown mode of inheritance. A linkage was found between the disease and markers located at 3p24.2-26. A maximum NPL score of 3.46 was obtained with marker D3S3050. This is the first genetic locus found to be involved in the molecular pathogenesis of familial moyamoya disease.     

Membrane delimited and intracellular soluble pathways in the somatostatin modulation of ACh release

The signal transduction cascade between the activation of the somatostatin (SOM) receptor and modulation of transmitter release was study using Acetylcholine (Ach) release measurements and patch clamp recordings of Ca2+ current from acutely dissociated St 40 ciliary ganglion neurons. As in intact synapses, somal ACh release was blocked by 100 nM SOM or 100 microM dibutyril cGMP, and the SOM-mediated inhibition could be reversed by 10 microM 1-NAME (a selective inhibitor of nitric oxide synthase, NOS) or 100 microM Rp-8p-CPT-cGMPs (a selective inhibitor of a cGMP protein dependent kinase, PKG). In whole cell recordings, SOM inhibition of Ca2+ current rapidly relaxes to control levels but is sustained in perforated patch recordings which decreases cell dialysis. Inhibition of NOS or PKG in perforated patch recordings, however caused SOM effects to become transient again. We hypothesize that PKG alters the characteristics of the membrane-delimited G protein inhibition of Ca2+ current. Therefore SOM receptors trigger a membrane-delimited signal transduction cascade that is modulated by soluble messengers, converging on voltage activated Ca2+ channels. When both pathways are active together, SOM causes a sustained inhibition of neuronal Ca2+ current leading to a decrease in transmitter release.     

Are there cholinergic and non-cholinergic paradoxical sleep-on neurones in the pons?

Using microiontophoresis on unanesthetized head-restrained cats, we have found two distinct groups of neurones exhibiting tonic discharge specific to paradoxical sleep (PS) (PS-on neurones) in the mesopontine tegmentum, which contains both cholinergic and non-cholinergic neurones. One group is characterized by a broad action potential, slow conduction velocity and an inhibitory response to a potent cholinergic agonist, carbachol, applied iontophoretically during PS. The other is characterized by a short action potential, fast conduction velocity and an excitatory response to applied carbachol. All PS-on neurones were excited by glutamate. The present findings demonstrate the existence of two types of PS-on neurones and suggest their cholinergic and noncholinergic neurochemical properties.     

Inhibition of nitric oxide synthase slows heart rate recovery from cholinergic activation

The role of nitric oxide (NO) in the cholinergic regulation of heart rate (HR) recovery from an aspect of simulated exercise was investigated in atria isolated from guinea pig to test the hypothesis that NO may be involved in the cholinergic antagonism of the positive chronotropic response to adrenergic stimulation. Inhibition of NO synthesis with NG-monomethyl-L-arginine (L-NMMA, 100 micro M) significantly slowed the time course of the reduction in HR without affecting the magnitude of the response elicited by bath-applied ACh (100 nM) or vagal nerve stimulation (2 Hz). The half-times (t1/2) of responses were 3.99 +/- 0.41 s in control vs. 7. 49 +/- 0.68 s in L-NMMA (P < 0.05). This was dependent on prior adrenergic stimulation (norepinephrine, 1 micro M). The effect of L-NMMA was reversed by L-arginine (1 mM; t1/2 4.62 +/- 0.39 s). The calcium-channel antagonist nifedipine (0.2 micro M) also slowed the kinetics of the reduction in HR caused by vagal nerve stimulation. However, the t1/2 for the reduction in HR with antagonists (2 mM Cs+ and 1 micro M ZD-7288) of the hyperpolarization-activated current were significantly faster compared with control. There was no additional effect of L-NMMA or L-NMMA+L-arginine on vagal stimulation in groups treated with nifedipine, Cs+, or ZD-7288. We conclude that NO contributes to the cholinergic antagonism of the positive cardiac chronotropic effects of adrenergic stimulation by accelerating the HR response to vagal stimulation. This may involve an interplay between two pacemaking currents (L-type calcium channel current and hyperpolarization-activated current). Whether NO modulates the vagal control of HR recovery from actual exercise remains to be determined.     

Inhibition of prostaglandin E release by anti-inflammatory steroids

The aearance of tritium in plasma water was measured after i.p. injection of 2-tritio-L-alanine as an index of alanine transamination in rats. L-Cycloserine (10 mg/kg) and D-cycloserine (150 mg/kg) inhibited tritium release, whereas theophylline (100 mg/kg) stimulated tritium release.     

A critical period in the sensitivity of basal forebrain cholinergic neurones to NGF deprivation

OBJECTIVE: We undertook this study to investigate functional MR imaging as a new clinical method for determining hemispheric language dominance. Seven patients undergoing surgical evaluation for chronic intractable epilepsy were studied. Intracarotid amobarbital injection was also performed and the findings compared with the functional MR imaging results. CONCLUSION: Functional MR imaging studies enabled localization of the frontal and temporal lobe language cortices. The results of functional MR imaging and intracarotid amobarbital testing of hemispheric language dominance agreed in all seven patients, including two right-handed patients with right-hemisphere language dominance. These preliminary results show that functional MR imaging is an accurate noninvasive method of determining language dominance that may replace the amobarbital test for some purposes if confirmed by additional research.     

Flunarizine inhibits both calcium-dependent and -independent release of glutamate from synaptosomes and cultured neurones

This paper discusses the general efficacy of the rat as a model for studying the pathogenicity of respirable fibres in humans. Rats show differences in lung structure from man and these could influence the pulmonary response to some degree, but they do develop interstitial fibrosis, carcinoma and mesothelioma when exposed to respirable fibres and these same types of lesions are found in fibre-exposed humans. Because of the importance of long fibres in causing pathological change, a substantial number of long respirable fibres must be used in inhalation studies if the relative pathogenicity of different fibre types is to be assessed. From data on the fibre burden in human disease and information on build-up of fibres in rat studies in a range of different fibres, a 1 year exposure to a cloud of 200 fibres ml-1 minimum, preferably more, is recommended. The rate of dissolution of the fibres within the lung milieu will be an an important factor in determining the pathogenicity. Many questions remain unanswered as to the mechanism of lung pathogenicity caused by respirable fibres particularly in the areas of durability, fibre shape, fibre surface chemistry and the exact mechanism, at the cellular level whereby fibres can cause pathological change. The use of rat inhalation studies, properly conceived and designed, remain a key approach whereby these questions can be answered.     

Inhibition of insulin release by intraduodenally infused enterostatin-VPDPR in rats

BACKGROUND: Retinopathy of prematurity (ROP) is a retinal vascular disease occurring in infants with short gestational age and low birth weight and can lead to retinal detachment (ROP stages 4 and 5). X-linked familial exudative vitreoretinopathy is phenotypically similar to ROP and has been associated with mutations in the Norrie disease (ND) gene in some cases. OBJECTIVE: To determine if similar mutations in the ND gene may play a role in the development of advanced ROP. METHODS: Clinical examination and molecular genetic analysis were performed on 16 children, including 2 dizygotic and 1 monozygotic twin pairs, and their parents from 13 families. RESULTS: Sequencing of the amplified products revealed missense mutations (R121W and L108P) in the third exon of the ND gene in 4 patients. These mutations were not present in an unaffected premature twin, 2 children with regressed stage 3 ROP, the parents, or in 50 unrelated healthy control subjects. CONCLUSION: These findings suggest that mutations in the ND gene may play a role in the development of severe ROP in premature infants.     

Inhibition by propofol (2,6 di-isopropylphenol) of the N-methyl-D-aspartate subtype of glutamate receptor in cultured hippocampal neurones

1. The effects of propofol (2,6 di-isopropylphenol) on responses to the selective glutamate receptor agonists, N-methyl-D-aspartate (NMDA) and kainate, were investigated in cultured hippocampal neurones of the mouse. Whole cell and single channel currents were recorded by patch-clamp techniques. Drugs were applied with a multi-barrel perfusion system. 2. Propofol produced a reversible, dose-dependent inhibition of whole cell currents activated by NMDA. The concentration of propofol which induced 50% of the maximal inhibition (IC50) was approximately 160 microM. The maximal inhibition was incomplete leaving a residual current of about 33% of the control response. This inhibitory action of propofol was neither voltage- nor use-dependent. 3. Analysis of the dose-response relation for whole cell NMDA-activated currents indicated that propofol caused no significant change in the apparent affinity of the receptor for NMDA. 4. Outside-out patch recordings of single channel currents evoked by NMDA (10 microM) revealed that propofol (100 microM) reversibly decreased the probability of channel opening but did not influence the average duration of channel opening or single channel conductance. 5. Whole-cell currents evoked by kainate (50 microM) were insensitive to propofol (1 microM-1 mM). 6. These results indicate that propofol inhibits the NMDA subtype of glutamate receptor, possibly through an allosteric modulation of channel gating rather than by blocking the open channel. Depression of NMDA-mediated excitatory neurotransmission may contribute to the anaesthetic, amnesic and anti-convulsant properties of propofol.     

Inhibition of anaerobic phosphate release by nitric oxide in activated sludge

Activated sludge not containing significant numbers of denitrifying, polyphosphate [poly(P)]-accumulating bacteria was grown in a fill-and-draw system and exposed to alternating anaerobic and aerobic periods. During the aerobic period, poly(P) accumulated up to 100 mg of P x g of (dry) weight. When portions of the sludge were incubated anaerobically in the presence of acetate, 80 to 90% of the intracellular poly(P) was degraded and released as orthophosphate. Degradation of poly(P) was mainly catalyzed by the concerted action of polyphosphate:AMP phosphotransferase and adenylate kinase, resulting in ATP formation. In the presence of 0.3 mM nitric oxide (NO) in the liquid-phase release of phosphate, uptake of acetate, formation of poly-beta-hydroxybutyrate, utilization of glycogen, and formation of ATP were severely inhibited or completely abolished. In cell extracts of the sludge, adenylate kinase activity was completely inhibited by 0.15 mM NO. The nature of this inhibition was probably noncompetitive, similar to that with hog adenylate kinase. Activated sludge polyphosphate glucokinase was also completely inhibited by 0.15 mM NO. It is concluded that the inhibitory effect of NO on acetate-mediated phosphate release by the sludge used in this study is due to the inhibition of adenylate kinase in the phosphate-releasing organisms. The inhibitory effect of nitrate and nitrite on phosphate release is probably due to their conversion to NO. The lack of any inhibitory effect of NO on adenylate kinase of the poly(P)-accumulating Acinetobacter johnsonii 210A suggests that this type of organism is not involved in the enhanced biological phosphate removal by the sludges used.     

Trypanosoma cruzi antigens down-regulate T lymphocyte proliferation by muscarinic cholinergic receptor-dependent release of PGE2

Here we demonstrate that T. cruzi antigen molecule SAPA (shed acute phase antigen) with neuraminidase-trans sialidase activity triggers down-regulation of T lymphocyte proliferation by interacting with T lymphocyte muscarinic acetylcholine receptors (mAChR). SAPA attachment to mAChR from Lyt 2.2+ T cells resulted in synthesis of cyclic GMP (cGMP) and secretion of PGE2, an immunoregulator effector substance. These T suppressor cell signals were blunted by atropine and by indomethacin. Cell sorter analysis showed that the interaction of SAPA with purified T cells, affected the ratio of L3T4+/Lyt 2.2+ T cells increasing the percentage of Lyt 2.2+ T cells, effect that was inhibited by the mAChR antagonist, atropine. The interaction between SAPA and mAChR from Lyt 2.2+ T cells may result, therefore, in the down-regulation of the host immune response as consequence of T suppressor/cytotoxic cells activation and PGE2 release as they were observed. These results support the theory of an immunosuppressive state that contribute to the chronic course of Chagas' disease.