Role of nitric oxide in modulating neurotransmitter release from rat striatum

The effect of the nitric oxide synthase inhibitor N-nitro- -arginine methyl ester (L-NAME) on the basal and stimulation-evoked release of dopamine (DA) and acetylcholine (ACh) was investigated in rat striatum. The experiments were carried out in isolated superfused striatal slices, loaded with either [³H]-dopamine or [³H]-choline.We have found that L-NAME reduced the elecrical field stimulation-evoked release of DA, while its enantiomer N-nitro-D-arginine methyl ester (D-NAME) was ineffective. In the presence of the nitric oxide (NO) precursor -arginine L-NAME failed to influence DA release. Furthermore, treatment with the N-methyl- -aspartate (NMDA) receptor antagonist MK-801 completely reversed the effect of L-NAME on striatal DA release. In contrast, L-NAME had no effect on either the basal or the stimulation-evoked ACh release in any experimental conditions studied.Our data indicate that endogenously produced NO is involved in the modulation of striatal DA, but not in ACh release. Furthermore, it seems likely that the modulatory effect of NO is linked to activation of presynaptic NMDA receptors located on the striatal dopaminergic nerve terminals.     

Inhibition of N-, P/Q- and other types of Ca channels in rat hippocampal nerve terminals by the adenosine A1 receptor

The effects of the adenosine A₁ receptor agonist, N⁶-cyclopentyladenosine (CPA), on both the increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and on the release of endogenous glutamate in rat hippocampal synaptosomes were studied. The inhibitory effect of CPA on the increase in [Ca²⁺]_i stimulated with 4-aminopyridine was neutralized by the adenosine A₁ receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The inhibitory effect of CPA was greater in synaptosomes from the CA1 subregion than in whole hippocampal synaptosomes. The inhibitory effects of both CPA and of the Ca²⁺ channel blockers, ω-conotoxin GVIA, ω-conotoxin MVIIC or ω-conotoxin GVIA plus ω-conotoxin MVIIC, were greater than those caused by the Ca²⁺ channel blockers. The release of endogenous glutamate was inhibited by 41% by CPA. The inhibition observed when CPA and ω-conotoxin GVIA or CPA and ω-conotoxin MVIIC were present was also greater than the inhibition by the Ca²⁺ channel blockers alone. The presence of both ω-conotoxin GVIA and ω-conotoxin MVIIC did not completely inhibit the release of glutamate, and CPA significantly enhanced this inhibition. The membrane potential and the accumulation of [

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]tetraphenylphosphonium of polarized or depolarized synaptosomes was not affected by CPA, suggesting that adenosine did not increase potassium conductances. The present results suggest that, in hippocampal glutamatergic nerve terminals, adenosine A₁ receptor activation partly inhibits P/Q- and other non-identified types of Ca²⁺ channels.     

Feedback Stimulation of Somatodendritic Serotonin Release: A 5-HT3 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-HT₃ receptor agonist 2-methyl-5-HT (1 to 10 μmol/l) increased the resting tritium outflow in superfused raphe nuclei slices, EC₅₀ 5.3 μmol/l. The 2-methyl-5-HT-induced increase of tritium outflow was an external Ca²⁺-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-HT₃ 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 (EC₅₀ 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-HT₃ 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-HT₃ 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.     

Niflumic acid-induced increase in potassium currents in frog motor nerve terminals: effects on transmitter release

The actions of the nonsteroidal antiinflammatory drug niflumic acid were studied on frog neuromuscular preparations by conventional electrophysiological techniques. Niflumic acid reduced the amplitude and increased the latency of endplate potentials in a concentration-dependent manner. Neuromuscular junctions pretreated with niflumic acid (0.05–0.5 mM) showed much less depression than control when they were stimulated with trains of impulses. Inhibition of acetylcholine release was reverted by raising the extracellular Ca²⁺ concentration but not by simply washing out the preparations with niflumic acid-free solutions. Pretreatment with indomethacin (0.1 mM), another nonsteroidal antiinflamatory drug, did not affect the niflumic acid-induced inhibition of evoked responses. Niflumic acid (0.1 mM) did not change the amplitude of miniature endplate potentials and had a dual action on the frequency of miniatures: it decreased their frequency at 0.1 mM whereas it produced an enormous increase in the rate of spontaneous discharge at 0.5 mM. Niflumic acid (0.1–1 mM) reversibly increased the amplitude and affected the kinetics of presynaptic voltage-activated K⁺ current and Ca²⁺-activated K⁺ current in a concentration-dependent manner. Niflumic acid (0.1–1 mM) irreversibly decreased the amplitude and reversibly affected the kinetics of the nodal Na⁺ current. Indomethacin (0.1 mM) had no effect on presynaptic currents. In conclusion, niflumic acid reduces acetylcholine release by increasing presynaptic K⁺ currents. This may shorten the depolarizing phase of the presynaptic action potential and may reduce the entry of Ca²⁺ with each impulse.     

Insulin release by glucose anomers in a rat model of non-insulin-dependent diabetes

Summary The effects of the and anomers of D-glucose on insulin release were studied in a rat model of non-insulin-dependent diabetes, which was induced by streptozotocin injection at 2 days of age. Glucose tolerance of the streptozotocin-treated rats at 8–10 weeks of age was mildly diabetic. Insulin release from the isolated perfused pancreas of the diabetic rats in response to 10 mmol/l -D-glucose was markedly impaired, while insulin response to 10 mmol/l -D-glucose in the diabetic pancreas was only slightly reduced as compared to that in the control pancreas.     

Only activated but not non-activated presynaptic α2-autoreceptors interfere with neighbouring presynaptic receptor mechanisms

Summary Experiments were carried out in rabbit cerebrocortical slices in order to find out whether the attenuation by presynaptic ₂-autoreceptors of effects mediated by presynaptic opioid - and adenosine A₁-receptors requires activation of the ₂-receptors. The slices were preincubated with ³H-noradrenaline and then superfused with medium containing desipramine 1 mol/l. They were stimulated electrically either with single pulses or with trains of 32 pulses at 1 Hz.The overflow of tritium elicited by a single pulse amounted to 0.21% of the tritium content of the tissue. It was Ca²⁺-dependent and tetrodotoxin-sensitive and not changed by rauwolscine 1 mol/l or yohimbine 0.3 mol/l. Ethylketocyclazocine (EK; 0.1–10 nmol/l) and R-(–)-N⁶-phenylisopropyladenosine (PIA; 1–1,000 nmol/1) potently inhibited the overflow evoked by a single pulse, and their effects were not changed by yohimbine. — The overflow of tritium elicited by trains of 32 pulses at 1 Hz amounted to 0.92% of the tritium content of the tissue and was increased approximately fourfold by yohimbine 0.3 mol/l. EK and PIA were less potent inhibitors than in the one pulse experiments. Yohimbine greatly enhanced the effects of EK and PIA. The enhancement was even more pronounced when the Ca²⁺ concentration in the medium was reduced in order to obtain a control tritium overflow similar to that evoked by 32 pulses in the absence of yohimbine.The results demonstrate that there is no ₂-adrenergic autoinhibition when noradrenaline release is elicited by a single pulse. Under these conditions, the non-activated presynaptic ₂-adrenoceptor does not interfere with presynaptic opioid - and adenosine A₁-receptor mechanisms. It is only when the autoreceptor is activated by released noradrenaline that it attenuates neighbouring presynaptic receptor mechanisms, and this attenuation is removed by ₂-adrenoceptor antagonists.Send offprint requests to N. Limberger at the above address     

Stimulation of spontaneous transmitter release at the frog neuromuscular junction by 12-O-tetradecanoylphorbol-13-acetate occurs in the absence of extracellular Ca2+ and is enhanced by depolarization

Summary The effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the release of transmitter at the frog neuromuscular junction has been investigated electrophysiologically. TPA (100 nmol/l) caused a gradual rise in miniature end-plate potential (MEPP) frequency. After 20–30 min MEPP frequency had risen by approximately 40%. This action of the drug was not inhibited by bathing preparations in either Ca²⁺-free medium (0 Ca²⁺-1 mmol/l EGTA) or high Mg²⁺ medium, or by pretreatment with verapamil (5 mol/l). The inactive TPA analogue 4--TPA had no effect on release rate. There was no indication of any positive correlation between resting MEPP frequency and the size of the subsequent response to TPA treatment. Any synergism between [Ca²⁺]_i and TPA treatment is therefore likely to occur at a site other than that which determines spontaneous release rate.The stimulatory effect of TPA was enhanced 2-fold by carrying out the experiments in a partially depolarising saline (10 mmol/l K⁺). When TPA was applied to preparations bathed in Ca²⁺-free depolarising saline, the response to the drug was still significantly greater than that in non-depolarised preparations. It is concluded that responsiveness to TPA is enhanced by depolarisation, but that little, if any, of this enhancement can be attributed to the consequent influx of Ca²⁺.Send offprint requests to S. J. Publicover at the above addressPEL was in receipt of an S.E.R.C. postgraduate awardZYS was in receipt of financial support from Umm Al Qura University, Saudi Arabia     

Potential involvement of a baclofen-sensitive autoreceptor in the modulation of the release of endogenous GABA from rat brain slices in vitro

Summary The effects of the GABA_A agonist, muscimol, and of the enantiomers of the GABA_B agonist, baclofen, on the release of endogenous GABA from slices of the rat cerebral cortex, striatum and hippocampus were measured by means of a HPLC method with electrochemical detection. Moreover, the effect of the GABA_A antagonist, bicuculline, and of the frequency of stimulation were studied in cortical slices. The amount of endogenous GABA released per impulse from cortical slices decreased by about 50% when the frequency was increased from 0.25 Hz to 1 Hz. This might indicate that GABA inhibited its own release. (–)-Baclofen at 1 and 10 M, but not its (+)-enantiomer, markedly inhibited the release of endogenous GABA, to a similar extent in all 3 areas investigated. The effect of (–)-baclofen was dependent on the frequency of stimulation: at lower frequencies (0.25 and 0.5 Hz) it was more marked than at a higher one (4 Hz). This would be expected from the results showing that the release of endogenous GABA decreases with increasing frequency, which suggests that this amino acid inhibits its own release. Muscimol at 10 M, on the other hand, was ineffective in all 3 areas at a stimulation frequency of 0.5 Hz. Bicuculline (10 M) at 4 Hz, at which autosuppression of GABA release is maximal did not enhance the release of endogenous GABA from cortical slices. With cerebellar or nigral slices, no adequate stimulation-induced release of endogenous GABA could be obtained under comparable conditions. These data are compatible with, but do not prove the existence of GABAB-type presynaptic autoreceptors modulating the release of this amino acid. More definite conclusions may possibly be drawn when a GABAB antagonist becomes available, which is expected to enhance GABA release under appropriate conditions.Presented in part at the 3rd Brit. Meeting on Electrochemical detection in Pharmacology and Neurochemistry, Cambridge, March 30–April 1, 1987Send offprint requests to P. C. Waldmeier     

Presynaptic regulation of the electrically evoked release of endogenous dopamine from the isolated neurointermediate lobe or isolated neural lobe of the rat pituitary gland in vitro

Summary Isolated neurointermediate lobes (NILs) or isolated neural lobes (NLs) of the rat pituitary gland were incubated in Krebs-HEPES solution which contained pargyline and the dopamine uptake inhibitor GBR 12921. The release of endogenous dopamine was determined by HPLC with electrochemical detection. Electrical stimulation of the pituitary stalk induced a frequency-dependent release of dopamine.The release of dopamine from the combined NIL evoked by stimulation at 15 Hz was increased by 130% in the presence of the dopamine D2 receptor antagonist, (–)-sulpiride; the (+)-enantiomer of sulpiride had virtually no effect. When the stimulation frequency was 3 Hz (–)-sulpiride caused an increase in dopamine release by 230%. A similar increase was observed in the presence of domperidone, another dopamine D2 receptor antagonist.The dopamine receptor agonists, apomorphine and quinpirole, had no significant effects on the evoked release of dopamine indicating that under the present incubation conditions endogenous dopamine may have been maximally activating the autoinhibition. However, in the presence of 1 mol/l (–)-sulpiride, apomorphine as well as quinpirole reduced the evoked release of dopamine in a concentration-dependent manner.The dopamine D1 receptor selective antagonist, SCH 23390, had no effect on the evoked release of dopamine at a concentration of 1 mol/1. Only at a concentration of 10 mol/l did SCH 23390 cause a small increase in dopamine release; this effect was, however, abolished in the presence of 1 mol/1(–)-sulpiride.In the presence of 1 mol/l (–)-sulpiride neither clonidine, yohimbine, 5-methoxytryptamine nor metitepine significantly affected the release of dopamine from the NIL evoked by stimulation at 3 Hz.In the NL, the release of dopamine is inhibited by endogenous opioids. For this reason, naloxone 1 or 10 mol/1 was present in the experiments on isolated NLs. Domperidone and (–)-sulpiride, but not (+)-sulpiride, increased the release of dopamine from the NL evoked by electrical stimulation at 15 Hz by about 90%. SCH 23390 caused a significant increase in dopamine release at 10 mol/l, but not at 1 mol/lIn conclusion, the release of endogenous dopamine from the neurons terminating in the intermediate and neural lobe of the pituitary gland is inhibited via dopamine receptors of the D2 type.Abbreviations DOPAC dihydroxyphenylacetic acid - 5-HT 5-hydroxytryptamine - HPLC high performance liquid chromatography - IL intermediate lobe - NIL neurointermediate lobe - NL neurallobe Send offprint requests to K. Racké at the above address     

ACTH increases noradrenaline release in the rabbit heart

Summary The effects of ACTH on the release of noradrenaline and the increase of heart rate produced by sympathetic nerve stimulation (1 Hz) were studied in isolated perfused rabbit hearts. ACTH-(1–24) 0.1–100 nmol/l increased the stimulation-evoked overflow of noradrenaline concentration-dependently, reversibly and up to two-fold. The basal outflow of noradrenaline, the basal heart rate and the stimulation-evoked increase in heart rate were not changed. Human ACTH-(1–39) also increased the evoked overflow of noradrenaline. The effect of ACTH-(1–24) 0.3 nmol/l persisted after blockade of -adrenoceptors with propranolol and blockade of neuronal catecholamine uptake by cocaine. ACTH-(1–24) 3 nmol/l did not change the removal of noradrenaline from the perfusion fluid, when hearts were perfused with medium containing 59 nmol/l noradrenaline. The results show that ACTH increases the action potential-evoked release of noradrenaline from cardiac postganglionic sympathetic neurones, probably by activating specific presynaptic ACTH receptors. The high potency of ACTH suggests that these presynaptic receptors may be activated in vivo by circulating ACTH under certain pathophysiological conditions.Send offprint requests to B. Szabo at the above address