Tritium Efflux Research Articles

Feedback Stimulation of Somatodendritic Serotonin Release: A 5-HT 3 Receptor-Mediated Effect in the Raphe Nuclei of the Rat

Slices from rat midbrain containing the raphe nuclei and from hippocampus were prepared, loaded with [ 3H]5-HT and superfused and the resting and the electrically stimulated [ 3H]5-HT release was measured. The 5-HT 3 receptor agonist 2-methyl-5-HT (1 to 10 μmol/l) increased the resting tritium outflow in superfused raphe nuclei slices, EC 50 5.3 μmol/l. The 2-methyl-5-HT-induced increase of tritium outflow was an external Ca 2+-independent process and was not altered by reserpine pretreatment but it was reversed by addition of the 5-HT uptake inhibitor fluoxetine (1 μmol/l). The 5-HT 3 receptor antagonists ondansetron and GYKI-46 903 (1 μmol/l) did not antagonize the stimulatory effect of 2-methyl-5-HT on resting tritium outflow. 2-Methyl-5-HT in lower concentration increased the electrically induced tritium overflow from raphe nuclei slices (EC 50 0.56 μmol/l) and also from hippocampal slices preloaded with [ 3H]5-HT. These effects were reversed by 1 μmol/l of ondansetron and GYKI-46903. The 5-HT 3 receptor antagonists (1 μmol/l) were without effects on depolarization-evoked [ 3H]5-HT release at 2 Hz stimulation, when 10 Hz stimulation was used, ondansetron and GYKI-46 903 reduced the tritium overflow from raphe nuclei slices. These data indicate that 5-HT 3 receptors positively alter depolarization-induced somatodendritic 5-HT release in the raphe nuclei. They also show that 2-methyl-5-HT is able to evoke 5-HT release not only from vesicles but also from cytoplasmic stores via a transporter-dependent exchange process.

Mechanism of Peroxide-Induced Potentiation of Sympathetic Neurotransrnission in Bovine Irides: Role of Extracellular Calcium

Hydrogen peroxide (H2O2) and enzymes that regulate its metabolism are present in tissues of the anterior segment of the eye. We have previously shown that in vitro, H2O2 can enhance sympathetic neurotransmis-sion in irides from several mammalian species. In the present study, we investigated the role of extracellular calcium in H2O2--induced potentiation of sympathetic neurotransmission in the bovine isolated iris. Isolated bovine hemiirides were incubated in a bicarbonate-buffered, carbogen-gassed Krebs buffer solution containing [3H]-norepinephrine ([3H]NE) for 60min. After incubation, tissues were prepared for studies of [3H]NE release using the superfusion method. Release of [3H]NE was elicited by consecutive trains of electrical field stimulation. Removal of calcium from the buffer solution attenuated field-stimulated [3H]NE overflow in isolated, superfused bovine irides without affecting basal tritium efflux. H2O2 (1 mM) enhanced evoked [3H]NE release to the same extent in tissues exposed to buffer solutions containing normal calcium (1.3 mM) as in those containing low calcium (0.13 mM) or zero calcium. However, in the presence of zero-calcium buffer solution containing the chelator, EDTA (lmM), H2O2 (1 mM) caused a gradual and sustained increase in basal tritium efflux. In buffer solutions containing high calcium (1.95 mM), the magnitude of H2O2--induced increase in field-stimulated [3H]NE release was significantly (P < 0.05) attenuated. Although the neuronal calcium channel antagonist w-conotoxin (20 nM) inhibited [3H]NE by 25%, it had no effect on H2O2 (1 mM)-induced potentiation of evoked [3H]NE overflow. We conclude that while trace amounts of extracellular calcium are necessary for H2O2--induced enhancement of sympathetic neurotransmission, increasing extracellular (buffer) calcium concentration impaired peroxide-induced enhancement of [3H]NE release. Furthermore, voltage-activated calcium channels may not be directly involved in peroxide-induced alteration of adrenergic neurosecre-tion in bovine isolated irides.

Prejunctional alpha2-adrenoceptors and peroxide-induced potentiation of norepinephrine release from the bovine iris.

Peroxides can enhance field-stimulated [3H]norepinephrine ([3H]NE) release in isolated irides from several mammalian species. In the present study, we investigated the role of prejunctional alpha2-adrenoceptors in peroxide-induced potentiation of sympathetic neurotransmission in bovine isolated irides. Isolated hemi-irides were incubated in a Krebs buffered-solution containing [3H]NE and prepared for studies of neurotransmitter release using the superfusion method. Alpha2-adrenoceptor agonists, oxymetazoline, UK-14304 and clonidine inhibited field-stimulated [3H]NE overflow without affecting basal tritium efflux. Pretreatment of tissues with H2O2 (300 microM) had no effect on inhibition of evoked [3H]NE release caused by the alpha2-adrenergic agonists. However, H2O2 (300 microM) caused significant (P < 0.01) leftward shifts of excitatory concentration-response curves to yohimbine (10 nM-1 microM). In contrast, yohimbine (1 microM) did not prevent the enhancement of evoked [3H]NE overflow induced by H2O2 (300 microM). In conclusion, excitatory effects of peroxides on sympathetic neurotransmission in bovine irides are not mediated by prejunctional alpha2-adrenoceptors.

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.

The muscarinic modulation of [ 3H] d-aspartate efflux and [Ca 2+] i levels in rat cerebellar granule cells

The effects of ACh on [ 3H] d-aspartate efflux and on calcium levels ([Ca 2+] i) were studied at the same time in sister cultures of rat cerebellar granule cells stimulated with electrical pulses (5–20 Hz) or depolarized with KCl (15–40 mM). ACh, 0.3–1000 nM, greatly facilitated the 10-Hz-evoked tritium efflux while its effect on 20 mM KCl-evoked efflux was significantly smaller. ACh, 10–1000 nM, enhanced [Ca 2+] i levels to a limited extent under both experimental conditions. Therefore, ACh facilitation was evident above all on the electrically evoked [ 3H] d-aspartate efflux. The ACh-mediated responses depended on the activation of M 3-muscarinic receptors since these responses were blocked by 4-DAMP. ACh, 50 μM, reduced the [Ca 2+] i plateau, determined by prolonged electrical or KCl stimulation. This effect was due to its action of M 2-receptors being blocked by AF-DX 116. In conclusion, at very low concentrations, ACh greatly facilitated the electrically evoked [ 3H] d-aspartate efflux through M 3-receptors, while at a higher concentrations, it inhibited, through M 2-receptors, the rise in [Ca 2+] i caused by prolonged cell depolarization.

Identification of histamine H3 receptors in the tail artery from normotensive and spontaneously hypertensive rats.

We examined the possible existence of prejunctional histamine H3 receptors on sympathetic nerve fibers innervating rat tail artery. The stimulation-evoked tritium outflow from isolated vessels preincubated with [3H]-noradrenaline and perfused/superfused in the presence of the alpha2-adrenoceptor antagonist rauwolscine, 3 microM, was inhibited by histamine 10 microM (by 8%) and the H3 agonists R-(-)-alpha-methylhistamine, 10 microM (by 18%), and imetit, 0.1-10 microM (by < or =20%). The inhibitory effect of imetit, which did not occur in the absence of rauwolscine, was counteracted by thioperamide, 1 microM. In the presence of rauwolscine, 3 microM, the inhibitory effect of imetit also occurred when the current strength or the Ca2+ concentration in the medium was reduced to compensate for the increase in tritium overflow elicited by rauwolscine, indicating that the inhibitory action of imetit is not associated with the increase in noradrenaline release produced by rauwolscine. In spontaneously hypertensive rats (SHRs), imetit also inhibited the overflow of tritium. This inhibitory effect was comparable to that observed in Wistar-Kyoto (WKY) rats and indicates that the sympathetic nerves of the rat tail artery in SHRs, like those in normotensive rats, are endowed with prejunctional histamine H3 receptors.

Effects of mercuric chloride and methyl mercury on cholinergic neuromuscular transmission in the guinea-pig ileum.

The effects of mercuric chloride (HgCl2) and methyl mercury (MeHg) were examined on basal mechanical activity and electrically-induced neurogenic cholinergic contractions (twitch contractions) in longitudinal muscle-myenteric plexus strips from guinea-pig distal ileum. Both compounds at 0.33 microM slightly enhanced the amplitude of twitch contractions in approximately 50% preparations. This effect was probably due to facilitation of acetylcholine (ACh) release since 0.1 and 1 microM mercurials increased electrically-evoked tritium outflow from [3H]choline preloaded muscle layer with attached myenteric plexus. Conversely, higher mercury concentrations inhibited twitch contractions (HgCl2 IC50 = 21.3 +/- 6.4 microM; MeHg IC50 = 45.1 +/- 5.5 microM), as well as contractions to exogenous ACh (0.1 microM) in resting preparations, and concomitantly increased the basal tone. The former effects possibly reflected an antimuscarinic activity of mercury, while the latter was related to alterations of calcium homeostasis in the effector cells. Indeed, the effect of HgCl2 on basal tone was antagonized by the Ca2+ entry blocker nifedipine (3, 10, 30 nM), indicating Hg-induced facilitation of Ca2+ influx through voltage-dependent channels. On the whole, our results suggest that cholinergic neuromuscular transmission and Ca(2+)-dependent mechanisms underlying smooth muscle contractility are targets for mercury toxicity in the intestine.

Influence of dopamine on GABA release in striatum: evidence for D 1–D 2 interactions and non-synaptic influences

Striatal slices from the rat were preincubated with [ 3H]GABA and superfused in the presence of nipecotic acid and aminooxyacetic acid, inhibitors of high-affinity GABA transport and GABA aminotransferase, respectively. GABA efflux was estimated by monitoring tritium efflux, 98% of which was in the form of [ 3H]GABA. The following three major observations were made: (1) The overflow of GABA evoked by electrical field stimulation (8 Hz) was increased two-fold by SKF-38393 (10 μM), an agonist at the D 1 family of dopamine receptors. This increase was completely blocked by the D 1 receptor antagonist SCH-23390 (10 μM). However, SCH-23390 had no effect on GABA overflow when given alone. Thus, dopamine agonists appear to exert an excitatory influence on GABA release; however, this effect was not elicited by endogenous dopamine under the conditions of this experiment. (2) Electrically evoked GABA overflow was reduced 50% by quinpirole (10 μM), an agonist at the D 2 family of dopamine receptors, and this effect was blocked by the D 2 antagonist sulpiride (10 μM). Moreover, exposure to sulpiride alone caused a 60% increase in GABA overflow, and this effect was abolished by 3-iodotyrosine (2 mM), a dopamine synthesis inhibitor. Thus, D 2 agonists appear to exert an inhibitory influence on dopamine release, an effect that can be exerted by endogenous stores of dopamine. (3) The stimulatory effect of SKF-38393 was attenuated by quinpirole, whereas the sulpiride-induced increase in GABA efflux was attenuated by SCH-23390. Sulpiride also increased [ 3H]GABA efflux during KCl-induced depolarization, an effect that was antagonized by SCH-23390 as in the case of electrical stimulation. However, although tetrodotoxin did not alter the stimulatory effect of sulpiride, it did block the ability of SCH-23390 to antagonize the sulpiride-induced increase in GABA overflow. These latter results suggest that there is an interaction between D 1 and D 2 receptors whereby the effects of dopamine mediated via D 1 sites are inhibited by an action on D 2 sites. In conclusion, our results suggest that (i) dopamine agonists can exert an excitatory influence on depolarization-induced GABA release within neostriatum via D 1 receptors and an inhibitory influence via D 2 receptors; (ii) under the conditions of these experiments, endogenous dopamine fails to act on D 1 sites but does exert an inhibitory influence via D 2 sites; and (iii) there is an interaction between D 1 and D 2 receptors such that the actions of dopamine mediated via D 1 sites are inhibited as a result of the concomitant actions exerted via D 2 sites.

Effect of 1-aminocyclopropanecarboxylic acid on N-methyl-D-aspartate-stimulated [3H]-noradrenaline release in rat hippocampal synaptosomes.

1. The effect of 1-aminocyclopropanecarboxylic acid (ACPC), a partial agonist at the glycine site of the N-methyl-D-aspartate (NMDA) receptor complex that exhibits neuroprotective, anxiolytic and antidepressant-like actions, was investigated in a functional assay for presynaptic NMDA receptors. 2. NMDA (100 microM) produced a 36% increase of tritium efflux above basal efflux in rat hippocampal synaptosomes preincubated with [3H]-noradrenaline ([3H]-NA), reflecting a release of tritiated noradrenaline. This effect was prevented by 10 microM 7-chlorokynurenic acid, an antagonist of the glycine site of the NMDA receptor. 3. Glycine enhanced the effect of NMDA with Emax and EC50 values of 84 +/- 11% and 1.82 +/- 0.04 microM, respectively. ACPC potentiated the effect of NMDA on tritium overflow with a lower EC50 (43 +/- 6 nM) and a lower maximal effect (Emax = 40 +/- 9%) than glycine. Furthermore, ACPC (0.1 microM) shifted the EC50 of glycine from 1.82 microM to > or = 3 mM. 4. These results show that ACPC can reduce the potentiation by glycine of NMDA-evoked [3H]-NA release and hence, may act as an antagonist at the glycine site of presynaptic hippocampal NMDA receptors when the concentration of glycine is high.

Progesterone in vitro increases NMDA-evoked [ 3H]dopamine release from striatal slices in proestrus rats

The dopaminergic nerve terminals in rat striatum appear to be an important target for progesterone (Pg) and the excitatory amino acid glutamate. In the present study the possible interaction between glutamate and Pg upon [ 3H]DA release in Striatal slices from rats in proestrus was examined. [ 3H]DA release was augmented by NMDA in a concentration-dependent manner. The presence of Pg (400 nM) in the perfusion medium produced an amplification of the responses to NMDA (50 μM) as shown by significant increase in the tritium outflow. The NMDA selective antagonists AP-7 (100 μM) and MK-801 (0.1 μM) prevented the effects of both NMDA and NMDA plus Pg on [ 3H]DA release. In contrast, the AMPA/kainate receptor antagonist CNQX (10 and 20 μM) was ineffective. Furthermore, AP-7 (100 μM) attenuated the enhancing effect of 400 nM Pg on [ 3H]DA release evoked by 28 mM K +. The antagonist was unable to alter the effect produced by K + alone. These results indicate a specific action of Pg on dopaminergic terminals mediated by NMDA receptors and suggest a close interaction between glutamate and dopamine systems in the striatum, apparently modulated by progesterone.

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release.

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [3H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium efflux from raphe nuclei slices preloaded with [3H]serotonin, and this inhibition was reversed by the 5-HT1A receptor antagonist (+)WAY-100135. The 5-HT1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [3H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubated with [3H]serotonin was completely external Ca(2+)-dependent, and omega-conotoxin GVIA and Cd2+, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced tritium release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT1A and 5-HT1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca2+ channels; (3) the 5-HT1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K+ channels.

Alpha-Bungarotoxin, kappa-bungarotoxin, alpha-cobratoxin and erabutoxin-b do not affect [3H]acetylcholine release from the rat isolated left hemidiaphragm.

Endplate preparations of the rat left hemidiaphragm were incubated with [3H]choline to label neuronal transmitter stores. Nerve evoked release of newly-synthesized [3H]acetylcholine was measured in the absence of cholinesterase inhibitors to investigate whether snake venom neurotoxins by blocking presynaptic nicotinic autoreceptors affect evoked transmitter release. Contractions of the indirectly stimulated hemidiaphragm were recorded to characterize the blocking effect of alpha-neurotoxins at the post-synaptic nicotinic receptors. Neither the long chain neurotoxins alpha-cobratoxin (1 microgram ml-1) and alpha-bungarotoxin (5 microgram ml-1) nor the short chain neurotoxin erabutoxin-b (0.1, 1 and 10 micrograms ml-1) affected the nerve-evoked release of [3H]acetylcholine. kappa-Bungarotoxin (1 and 5 micrograms ml-1), a toxin preferentially blocking neuronal nicotinic receptors, did also not affect evoked [3H]acetylcholine release, whereas (+)-tubocurarine (1 microM) under identical conditions reduced the release by about 50%. alpha-Bungarotoxin, alpha-cobratoxin and erabutoxin-b concentration-dependently (0.01-0.6 micrograms ml-1) inhibited nerve-evoked contractions of the hemidiaphragm. All neurotoxins except erabutoxin-b enhanced the basal tritium efflux immediately when applied to the endplate preparation or to a non-innervated muscle strip labelled with [3H]choline. This effect was attributed to an enhanced efflux of [3H]phosphorylcholine, whereas the efflux of [3H]choline and [3H]acetylcholine was not affected. It is concluded that the alpha-neurotoxins and kappa-bungarotoxin do not block presynaptic nicotinic receptors of motor nerves. These nicotinic autoreceptors differ from nicotinic receptors localized at the muscle membrane and at autonomic ganglia.

Mucosa-dependent muscarinic liberation of prostaglandins from rat isolated trachea.

1. The present study examined whether cholinoceptor stimulation modulates the release of arachidonic acid-derived mediators from rat isolate tracheae. 2. Tracheae were preincubated with [3H]-arachidonic acid and the outflow of 3H-compounds was determined. Acetylcholine and the muscarinic agonist, carbachol but not nicotine, increased the rate of tritium outflow maximally by about 30%. The M3 receptor-preferring antagonist rho-fluoro-hexahydrosiladiphenidol was more effective than pirenzepine and methoctramine in antagonizing the effect of acetylcholine. 3. High performance liquid chromatography analysis (methanol gradient) of the released 3H-compounds showed that one peak, co-eluting with [14C]-prostaglandin E2([14C]-PGE2) and [3H]-PGD2, was enhanced almost 10 fold following muscarinic receptor activation, whereas the outflow of [3H]-arachidonic acid remained unaffected. 4. Using an acetonitril gradient separation it was shown that [3H]-PGE2, [3H]-PGD2 and [3H]-PGF2alpha are released spontaneously, but [3H]-PGE2 represented the major fraction of 3H-prostaglandins. Acetylcholine enhanced the release of all three 3H-prostaglandins, but the effect on PGE2 was most pronounced and most consistent. 5. After removal of the mucosa the muscarinic effect of acetylcholine on total tritium and on that of the 3H-prostaglandins ([3H]-PGE2/PGD2 peak) was abolished. 6. Acetylcholine also enhanced the outflow of radioimmunologically determined PGE2 in a mucosa-dependent manner. 7. After inhibition of cyclo-oxygenase by 3 microM indomethacin, the outflow of 3H-prostaglandins was reduced to almost undetectable levels and acetylcholine evoked a marked release [3H]-arachidonic acid. The phospholipase A2 inhibitor, quinacrine (up to 100 microM) also blocked the effect of acetylcholine on the outflow of 3H-prostaglandins, but this was not followed by a compensatory increase in the outflow of [3H]-arachidonic acid. 8. In conclusion, activation of muscarinic receptors which have characteristics of the M3 subtype can evoke release of prostaglandins from the airway mucosa.

Cultured chick sympathetic neurons: ATP-induced noradrenaline release and its blockade by nicotinic receptor antagonists.

The ATP-induced increase in tritium outflow from cultured chick sympathetic neurons prelabelled with [3H]-noradrenaline was investigated. Seven days-old dissociated cell cultures of embryonic paravertebral ganglia, loaded with [3H]-noradrenaline (0.05 microM), were superfused in the presence of (+)-oxaprotiline and exposed to ATP, ATP-analogues, or 1,1-dimethyl-4-piperazinium (DMPP) for 2 min. ATP (3 microM-3 mM), 2-methylthio-ATP (3-100 microM), as well as DMPP (10 and 100 microM) induced a significant overflow of tritium. The EC50-value of ATP was 20 microM. Both the ATP-induced and the DMPP-induced tritium overflow was Ca(2+)-dependent and sensitive to tetrodotoxin (0.3 microM) and omega-conotoxin (0.1 microM); in addition, it was inhibited by the alpha 2-adrenoceptor agonist 5-bromo-6-(2-imidazoline-2-ylamino)-quinoxaline (UK-14,304; 1 microM). The effects of ATP and DMPP were not additive. The ATP-induced as well as the DMPP-induced overflow of tritium was diminished by the P2-purinoceptor antagonists suramin (300 microM) and reactive blue 2 (3 microM); in all 4 cases, the inhibition amouted to approximately 40%. The tritium overflow induced by ATP or DMPP was almost abolished by the nicotinic receptor antagonist mecamylamine (10 microM) and markedly inhibited by hexamethonium (100 microM). Neither ATP nor electrical stimulation caused an overflow of tritium from cultures loaded with [3H]-choline. The results suggest that ATP at mumolar concentrations induces noradrenaline release from cultured chick sympathetic neurons via an action on a subclass of the nicotinic cholinoceptor.

Neuronal localization and evoked release of norepinephrine in the cnidarian Renilla koellikeri

AbstractNeuronal release of norepinephrine (NE) in the sea pansy Renilla koellikeri was investigated by searching for evidence 1) of neuronal localization of endogenous NE and 2) of synaptic‐like release of exogenously supplied NE in sea pansy tissues. Measurements of endogenous NE by high‐performance liquid chromatography with electrochemical detection corresponded with the visualization of specific NE immunoreactivity in six individual colonies thus sampled. Peroxidase‐antiperoxidase immunohistochemistry with antisera against NE‐aldehyde‐protein conjugates revealed that cellular NE immunostaining was predominantly associated with neurons and amoebocytes constituting the mesogleal nerve net. Transient increases of tritium outflow above background levels were evoked by field electrical stimulation of colonial (rachis) tissues pre‐loaded with 1 μM [3H]NE, under conditions that elicited nerve net‐mediated bioluminescence. This evoked release was largely reduced or abolished in a reversible manner by substituting high‐magnesium or calcium‐free seawater to normal seawater perfusate. Evoked release was significantly enhanced by exogenous norepinephrine (50 μM) and reduced in half or less by the α‐adrenergic blockers phentolamine and yohimbine (10 μM), thus providing support for the existence of an autoregulatory mechanism associated with release. These results suggest that neuronal, synaptic‐like release of NE occurs in the nerve‐net of the sea pansy, a representative of the phylum (Cnidaria) in which the first nervous systems of multicellular organisms are believed to have originated. © 1995 Wiley‐Liss, Inc.

Release of [3H]acetylcholine in human isolated bronchi. Effect of indomethacin on muscarinic autoinhibition.

Receptor-mediated regulation of acetylcholine release in the airways, particularly in humans, remains unclear. In the present study, the tissue content of acetylcholine and release of [3H]acetylcholine were measured in freshly dissected human bronchi obtained at thoracotomy. Large (main and lobar bronchi) and small (segmental and subsegmental bronchi) airways contained considerable amounts of endogenous acetylcholine (300 +/- 50 pmol/100 mg wet weight), whereas significantly less was found in lung parenchyma (60 +/- 30 pmol/100 mg). Isolated small bronchi incubated in an organ bath with the precursor [3H]choline synthesized significant amounts of [3H]acetylcholine (26,000 +/- 4,000 dpm/100 mg). Subsequent transmural stimulation (four 20 s trains at 15 Hz) of radiolabeled bronchi caused an enhanced tritium outflow that was abolished by removal of extracellular calcium or by tetrodotoxin. HPLC analysis of the medium collected before, during, and after transmural stimulation showed that the electrically stimulated tritium outflow represented exclusively [3H]acetylcholine, whereas the outflow of [3H]choline and [3H]phosphorylcholine was not affected by electrical stimulation. Oxotremorine (0.1 and 1 mumol/L) inhibited evoked [3H]acetylcholine release in a concentration-related manner, whereas atropine (0.03 mumol/L) enhanced evoked [3H]acetylcholine release. Inactivation of cyclooxygenase activity by 3 mumol/L of indomethacin did not impair the inhibitory effect of 0.1 or 1 mumol/L of oxotremorine. In conclusion, the present experiments indicate a considerable cholinergic innervation of human large and small airways.

Cardiovascular effects of agmatine, a "clonidine-displacing substance", in conscious rabbits.

Agmatine has been identified as a "clonidine-displacing substance" in extracts from bovine brain. We studied its effect on cardiovascular regulation and the role played in this effect by alpha 2-adrenoceptors. In conscious rabbits, agmatine 10 micrograms kg-1 injected intracisternally (i.c.) caused no change, whereas agmatine 30, 100 and 300 micrograms kg-1 i.c. increased renal sympathetic nerve firing, the plasma concentration of noradrenaline and adrenaline and arterial blood pressure. Heart rate tended to be decreased. Yohimbine 1.5 micrograms kg-1 i.c. caused no change, whereas yohimbine 5, 15 and 50 micrograms kg-1 increased renal sympathetic nerve activity, the plasma concentration of noradrenaline and adrenaline, blood pressure and heart rate. In rabbit brain cortex slices preincubated with [3H]-noradrenaline, agmatine 1 to 100 microM did not modify the electrically evoked overflow of tritium (either 4 pulses at 100 Hz or 36 pulses at 3 Hz). The evoked overflow was reduced by 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304) 0.03 to 30 nM (4 pulses at 100 Hz), and this inhibition was not affected by agmatine 10 and 100 microM. Agmatine did not change the basal efflux of tritium. The results show that agmatine, like yohimbine, causes central sympathoexcitation when given i.c., but agmatine differs from yohimbine in that it does not increase heart rate. Agmatine acts neither as an agonist nor as an antagonist at the alpha 2-autoreceptors in rabbit brain cortex. alpha 2-Adrenoceptors, therefore, are probably not involved in its cardiovascular effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Benzimidazolones and renzapride facilitate acetylcholine release from guinea-pig myenteric plexus via 5-HT4 receptors.

The effects of the 5-HT4 receptor agonists BIMU 8, BIMU 1, renzapride and of the 5-HT1p receptor agonist 5-hydroxyindalpine on basal and electrically evoked outflow of tritium were studied in guinea-pig longitudinal muscle myenteric plexus preparations preincubated with [3H]choline. Muscle contractions were recorded simultaneously. BIMU 8 caused a calcium dependent and tetrodotoxin sensitive increase in basal [3H]outflow that was assumed to represent release of [3H]acetylcholine. In addition, BIMU 8 enhanced the release of [3H]acetylcholine and twitch contractions evoked by submaximal electrical stimulation. Ondansetron (1 mumol/l) did not change the effects of BIMU 8, but DAU 6285 and tropisetron (each 1 mumol/l) competitively antagonized the various facilitatory effects of BIMU 8 with pA2 values of 7.0-7.2 (DAU 6285) and 7.0-7.3 (tropisetron). The phosphodiesterase inhibitors IBMX and rolipram did not increase the effects of BIMU 8. BIMU 1 and renzapride also concentration-dependently increased basal release of acetylcholine, and release and contractions caused by submaximal stimulation. The effects of BIMU 1 and renzapride were competitively antagonized by 1 mumol/l tropisetron (pA2 6.6-7.1). The EC50 values for the increase in the evoked [3H]acetylcholine release and contractions were closely similar. 5-Hydroxyindalpine did not change basal release and slightly inhibited the evoked release of [3H]acetylcholine. Release of acetylcholine and contractions elicited by submaximal stimulation were strongly inhibited by (+)-tubocurarine which indicates that nicotine ganglionic transmission is involved in this kind of release. The results suggest that BIMU 8, BIMU 1 and renzapride stimulate 5-HT4 receptors at cholinergic interneurones and thereby facilitate nicotinic ganglionic transmission in the myenteric plexus.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid, agonist-induced desensitization of alpha 2-autoreceptors modulating transmitter release.

1. The release of previously incorporated [3H]-noradrenaline was investigated in cultures of dissociated chick or rat sympathetic neurones and in cerebrocortical slices from neonatal or adult rats. Noradrenaline, in the presence of 10 mumol l-1 of the uptake inhibitor, cocaine, or the selective alpha 2-adrenoceptor agonist, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK 4,304), was applied for different periods of time in order to detect a possible time-dependence of the alpha 2-adrenoceptor-mediated inhibition of electrically evoked tritium outflow. 2. In chick sympathetic neurones, stimulation-evoked overflow was reduced to 30%, 42%, or 56% of control when noradrenaline (1 mumol l-1) was present for 2, 8, or 16 min, respectively. Likewise, UK 14,304 (1 mumol l-1) present for these periods of time reduced 3H overflow to 35%, 51%, and 53% of control, respectively. Addition of 1 nmol l-1 to 10 mumol l-1 UK 14,304 for either 2 or 16 min did not produce significantly different IC50 values, but the inhibitory effects were smaller with 16 min as compared to 2 min exposure at concentrations > or = 10 nmol l-1. 3. In rat sympathetic neurones, noradrenaline (100 nmol l-1) reduced stimulation-evoked overflow to 33%, 56%, or 57% of control, when present for 2, 8, or 16 min, respectively. Addition of UK 14,304 (1 mumol l-1) for these periods of time caused inhibition to 11%, 41%, and 46% of control. Applying UK14,304 for either 2 or 16 min did not result in significantly different IC5o values, but the inhibition induced by 16 min as compared to 2 min exposure was smaller at concentrations > 10 nmol 1-1.4. In cerebrocortical slices from either neonatal or adult rats, exposure to 0.1 to 1.0 micromol 1-1 UK14,304 for 16 min never caused a smaller inhibition than a corresponding 3 min exposure, although various experimental conditions were investigated.5 The results demonstrate that alpha 2-adrenoceptors which regulate noradrenaline release from sympathetic neurones undergo agonist-induced desensitization within minutes. Such rapid desensitization of alpha 2-autoreceptors was not detected in brain slice preparations.

The effect of ibogaine on κ-opioid- and 5-HT 3-induced changes in stimulation-evoked dopamine release in vitro from striatum of C57BL/6BY mice

Ibogaine is an kndole alkaloid that has bean suggested to have potential efficacy for internrpdng dependermy on stimulant drugs. The κ-opiold and serotonkr 5-HT 3 systems may be involved in the action of Ibogaine, related to their modulation of dopaminergic transmission. The κ-opioid agonist U 62066 attenuated the in vitrostimulation-evoked efflux of tritium label from striatal tissue prelabeled with ( 3H)dopamine. In mice protreated with Ibogaine·HCl (40 mg/kg IP given 2 h prior or 2 × 40 mg/kg and animals killed 18 h later), the Inhibitory effect of U 62066 on stimulation-evoked release of tritium was eliminated. The 5-HT 3 agonist phernylbiguanide had a biphasic effect on stimulation-evoked release of tritium; at 10 −6 M phenylbiguanide, stimulation-evoked release was attenuated. At 10 −5 M the basal outflow of tritium was increased. Ibogaine pretreatment had no effect on basal or stimulation-evokedrelease in the presence of 10 −6 M phenylbiguanide, but increased the stimulation-evoked outflow of bftkan in the presence of 10 −5 M phenylbiguanide. Cocaine (10 −6 M), a dopanlne uptake blocker, increased the electrically-evoked release of dopamine; ibogaine pretreatment did not affect the enhanced electrically-induced release of ( 3H]dopamine by in vitro cocalne. The effects of ibogaine on the κ-opioid and 5-HT 3 receptors, located presynaptically on stristal dopamine terminals, modulating dopamine release may partly underlie its putative antiaddictive properties.