Release Of 3H-noradrenaline Research Articles

Morphine and beta-endorphin inhibit release of noradrenaline from cerebral cortex but not of dopamine from rat striatum.

β-ENDORPHIN is a large polypeptide containing 31 amino acid residues, the first five of which are identical with methionine-enkephalin1 which has potent naloxone-sensitive analgesic properties2 and opiate activity in receptor binding assays in guinea pig ileum3,4. It has been shown that morphine depresses the potassium-induced release of 3H-noradrenaline in the rat cerebral cortex5 and of 3H-dopamine in the rat striatum6. These effects are blocked by the opiate antagonist naloxone and are probably due to the activation of presynaptic inhibitory opiate receptors located in noradrenergic and dopaminergic nerve endings of the rat brain. While met-enkephalin reproduced the effects of morphine in rat cortex slices7, it failed to reduce the release of dopamine from the rat striatum6. In view of these results, it was considered of interest to examine under similar experimental conditions, the effects of morphine and of β-endorphin on the potassium-stimulated release of 3H-dopamine from striatal slices and of 3H-noradrenaline from cerebral cortex slices of the rat8. In contrast to Loh et al.6, who more recently have not been able to reproduce their data (E. L. Way, personal communication), we find no evidence for the inhibition of the potassium-stimulated overflow of 3H-dopamine by β-endorphin and morphine in the striatal slices of the rat.

Is histamine involved in the sympathomimetic effect of nicotine?

Experiments were carried out on superfused strips of the main pulmonary artery of the rabbit in order to examine the proposal that histamine mediates the sympathomimetic effect of nicotine in this tissue. Moreover, effects of serotonin on the nerve endings within the artery were compared with those of nicotine, histamine and tyramine. 1. Pretreatment with with 6-hydroxydopamine intravenously lowered the noradrenaline content of heart and pulmonary artery and enhanced contractions of the latter evoked by noradrenaline. The contractile response to nicotine was abolished even though tissue histamine levels (determined in the heart) and the response to histamine were unchanged. 2. (+)-Chlorpheniramine 10−8 M reduced contractions caused by histamine, whereas a concentration of 10−5 M was necessary for a significant antagonism of nicotine effects. This high concentration enhanced the response to exogenous noradrenaline. Although (-)-chlorpheniramine had less than 1/100 the potency of the (+)-enantiomer as an antihistaminic agent, it also reduced the response to nicotine at 10−5 M. 3. Phenoxybenzamine irreversibly antagonized the effects of nicotine as well as of histamine and noradrenaline. Receptor protection by a high concentration of histamine failed to preserve the effect of nicotine. On the other hand, protection of α-adrenoceptors by a high concentration of noradrenaline afforded cross-protection against blockade of the effect of nicotine. 4. At very high concentrations of histamine, the initial contraction was soon followed by a secondary relaxation. The tissue was then refractory to histamine, but normally sensitive to nicotine and noradrenaline. 5. In artery strips preincubated with 3H-noradrenaline, nicotine elicited an immediate increase in the outflow of tritium which peaked in the first 1–2 min and then declined rapidly. In contrast, histamine and serotonin produced slow and progressive increases in tritium outflow. 6. Nicotine elicited a transient release of 3H-noradrenaline which was followed by minor increases of 3H-3,4-dihydroxyphenylglycol (DOPEG) and 3H-normetanephrine. Most of the nicotine-evoked outflow of 3H-noradrenaline and 3H-normetanephrine was calcium-dependent. In contrast, the outflow of 3H-DOPEG caused by nicotine 10−3 M was unchanged even when the release of 3H-noradrenaline was almost completely abolished by superfusing with a calciumfree, hexamethonium-containing medium. 7. Histamine, serotonin and tyramine elicited a gradual acceleration of 3H-DOPEG outflow which was accompanied by only a very small and (with exceptions mentioned below) progressive and calcium-independent increase in 3H-noradrenaline. 8. With serotonin 10−6 and histamine 10−4 peaks of 3H-noradrenaline outflow were obtained in the first 3 min after drug addition. The significance of this finding seems questionable, however, since the peaks were by several orders of magnitude smaller than those caused by nicotine and too small to lead to an increase in the efflux of total tritium. The outflow of 3H-noradrenaline evoked by histamine 10−4 M, but not that evoked by serotonin 10−6 M, was significantly diminished in calcium-free medium. 9. In conclusion, no evidence was obtained for a histaminergic link in the sympathomimetic effect of nicotine. All observations are compatible with the view that only noradrenaline mediates the effect, and that the release of noradrenaline is due to a direct action on the nerve endings. The major effects of histamine and serotonin on the noradrenergic nerve terminals could be classified as tyramine-like. No convincing evidence was found for the presence of presynaptic serotonin receptors similar to those previously detected in the rabbit heart and which, like the nicotine receptors, mediate calcium-dependent release of the transmitter.

Tyrosine hydroxylase activation and transmitter release from central noradrenergic neurons by electrical field stimulation.

Electrical stimulation of the noradrenergic neurons in the locus coeruleus of the rat results in a marked increase in the tyrosine hydroxylase activity of the hippocampus on the stimulated side (Roth et al., 1975). We have developed an in vitro system to further study this interesting phenomenon. Rat hippocampal slices were stimulated in an electrical field using specially built chambers which permitted a continous superfusion with Krebs Ringer Phosphate solution while stimulation was taking place. The slices were then homogenized and tyrosine hydroxylase activity and kinetic parameters were determined in the 104000 g supernate fraction. Electrical field stimulation (60 V, 4 ms, 5–20 Hz, 5 min) induced a stimulus-dependent increase in tyrosine hydroxylase activity. The increase in tyrosine hydroxylase activity was directly dependent on the number of pulses of stimulation applied. Potassium depolarization of hippocampal slices also resulted in a marked increase in the activity of the enzyme. Electrical field stimulation appears to activate tyrosine hydroxylase by causing an increase in its affinity for both substrate and pteridine cofactor and by decreasing its affinity for the endproduct inhibitor, norepinephrine. No change in Vmax was observed. The superfusion system was also used to study spontaneous and electrically evoked release of labelled norepinephrine from hippocampal slices. Electrical field stimulation for 1.0 min produced a marked increase in the release of label. Absence of calcium from the superfusion solution almost completely abolished the electrically evoked release of exogenously taken up 3H-norepinephrine. The release of 3H-norepinephrine was also found to be dependent on the number of pulses of stimulation used. The results reported support the hypothesis that depolarization of central noradrenergic neurons results in an increase in transmitter synthesis mediated in part via a kinetic activation of tyrosine hydroxylase. The concomitant use of superfusion and electrical field stimulation of hippocampal slices provides a simple system to study neurotransmitter synthesis and release in central noradrenergic neurons.

89) - The release of l4c-dopamine and 3H-noradrenaline in rabbit arteries by tyramine

89) - The release of l4c-dopamine and 3H-noradrenaline in rabbit arteries by tyramine

A MODULATING ROLE OF TAURINE ON RELEASE OF ACETYLCHOLINE AND NOREPINEPHRINE FROM NEURONAL TISSUES

Effects of taurine (2-aminoethanesulfonic acid) on the uptake and release of 14C-acetylcholine (14C-ACh) and 3H-norepinephrine (3H-NE) in the superior cervical ganglion and cerebral cortex of the rat were studied. Taurine suppressed high potassium evoked release of 14C-ACh and 3H-NE from the rat superior cervical ganglia and cerebral cortical slices, while the drug did not modify per se the uptake and unstimulated (spontaneous) release of 14C-ACh and 3H-NE in these tissues. Furthermore, taurine inhibited the release of 3H-NE from the crude synaptosomal (P2) fraction of the rat brain without affecting the uptake. These results suggest that taurine may act as a modulator of neuronal activity, possibly by stabilizing excitable membrane and by suppressing the release of neurotansmitter at synapses.

On the mechanism of alpha-receptor mediated modulation of 3H-noradrenaline release from slices of rat brain neocortex

Brain cortical slices were superfused with Krebs-Ringer media and the effects of oxymetazoline (an α-adrenoceptor agonist) and phentolamine (an α-antagonist) on depolarization-induced 3H-NA release were examined. Depolarization was effected by various K+-concentrations or by electrical pulses.

Effects of tetrodotoxin, elevated calcium and calcium antagonists on electrically induced 3H-noradrenaline release from brain slices

Electrically induced release of 3H-noradrenaline from superfused rat brain cortex slices was completely inhibited by tetrodotoxin (1 μg/ml) if 0.5 or 1 V stimuli were used, while inhibition was 97% with 2 V, and nil with 12 V stimuli. 20 mM calcium depressed noradrenaline release at all applied potentials, and shifted the release versus voltage curve to higher potentials. Manganese (5 mM) and D-600 (10–100 μg/ml) also considerably inhibited release. This suggests that stimuli of up to 2 V induce transmitter release by exciting intracortical noradrenergic axons in their non-terminal regions.

ALPHA-ADRENERGIC BLOCKING PROPERTIES OF DROPERIDOL ON ISOLATED BLOOD VESSELS OF THE DOG

Concentrations of droperidol which caused a shift to the right of the dose-response curve to noradrenaline in the pulmonary artery and the saphenous vein of the dog did not affect myogenic activation by K+; they did not inhibit spontaneous activity of portal-mesenteric veins. Droperidol inhibited the contractile response to nerve stimulation, but did not affect the evoked release of 3H-noradrenaline. These experiments indicate that the vasodilator properties of smaller doses of droperidol are a result of its ability to block alpha-adrenergic receptors.

Release of noradrenaline by the ionophore X537A from normal and reserpinized guinea-pig atrium.

The effects of ionophore X537A on the release of 3H-noradrenaline and its metabolites from the superfused guinea-pig left atrium were investigated. Concentrations of ionophore of 10 and 30 mocrometer greatly increased the release of tritium. Of the total increase in radioactivity elicited by X537A 44% was accounted for as noradrenaline and 50% was due to deaminated metabolites. The ionophore-evoked release of tritium was independent of the extracellular calcium ions and was not affected by agents which modify calcium movements such as verapamil, ryanodine, ruthenium red and tetracaine. X537A released 3H-noradrenaline from extragranular sites in MAO-inhibited atria from reserpine-treated animals and this release was also calcium independent. It is concluded that the ability of X537A to release noradrenaline from vesicular or cytoplasmic sites is not related to its ability to couple with and transport calcium ions through membranes. The ionophore might modify the ionic distribution outside and inside the neuronal membrane which would lead to leakage of the transmitter.

Blockade of presynaptic ?-receptors and of amine uptake in the rat brain by the antidepressant mianserine

Mianserine (Org GB 94, Tolvon®) is 1, 2, 3, 4, 10, 14b-hexahydro-2-methyl-dibenzo [c, f] pyrazino [1, 3-a] azepine hydrochloride, a new antidepressant drug. Its effect on noradrenaline release and its capacity to inhibit amine uptake were investigated. Mianserine increased the release of 3H-noradrenaline from field-stimulated cortical slices previously labelled with the tritiated transmitter. The assumption that this effect is due primarily to the blockade of the presynaptic noradrenergic α-receptors is supported by the fact that mianserine failed to augment 3H-noradrenaline release further after blockade of the presynaptic α-receptors by phentolamine. In the reciprocal experiment, phentolamine failed to augment 3H-noradrenaline release after exposure of the slices to mianserine. The hypothesis is further reinforced by the fact that mianserine antagonized the reduction of 3H-noradrenaline release by clonidine in the same manner as the α-blocking drugs phentolamine and phenoxybenzamine. Mianserine inhibited noradrenaline uptake in vitro and in vivo (in the rat heart and midbrain-diencephalon synaptosomes from pretreated rats.) Only a marginal inhibition of serotonin uptake was observed.

Cyclic amp and α-receptor-mediated modulation of noradrenaline release from rat brain slices

Oxymetazoline (an α-receptor agonist) reduced and phentolamine (an α-receptor antagonist) increased depolarization-induced release (potassium or electrical field stimulation) of 3H-noradrenaline (NA) from superfused neocortical slices in a dose-dependent way. Inhibition of phosphodiesterase also reduced NA release; this effect could be reversed by phentolamine. Phosphodiesterase inhibition potentiated the effect of oxymetazoline. It is suggested that stimulation of presynaptic α-receptor may reduce NA release up to about 60% and that increased cyclic AMP formation might be involved in this modulation.

3H-noradrenaline outflow induced from isolated adventitia and intima-media of rabbit aorta by electrical field stimulation

Electrical field stimulation of the isolated adventitial and intima-medial layers of rabbit aorta preloaded with 3H-noradrenaline elicited a 3H outflow. The initial 3H outflow from adventitia was independent of external Ca 2+ concentration. Each of the subsequent 3H outflows was mainly Ca 2+ sensitive. All stimulation-induced 3H outflows from media were Ca 2+ insensitive. It is concluded that the isolated adventitia is a suitable preparation to study electrical field stimulation-evoked release of 3H-noradrenaline. Furthermore, the stimulation-induced 3H outflow from aorta is of neuronal and extraneuronal origin.

Inhibition by botulinum toxin of depolarization-evoked release of (14C)acetylcholine from synaptosomes in vitro.

1. Cerebral-cortex synaptosomes were shown to synthesize (14C)acetylcholine after incubation with (14C)choline, and 25mM-KCl released (14C)acetylcholine (but not (14C)choline) into the medium by a Ca2+-dependent and Mg2+-sensitive process. 2. The K+-stimulated release of (14C)acetylcholine was inhibited by more than 80% after preincubation of the synaptosomes with 10(5) mouse lethal doses of botulinum toxin/ml. (14C)choline uptake, (14C)acetylcholine synthesis, intrasynaptosomal K+ and occluded lactate dehydrogenase were unaffected by the toxin. It also failed to prevent the K+-stimulated release of (3H)noradrenaline and (14C)glycine from synaptosomes. 3. Fractionation of hypo-osmotically shocked synaptosomes revealed that more than 75% of the radioactive acetylcholine was in the cytoplasmic compartment, although the vesicle pellet contained more total acetylcholine than the cytoplasmic pool. Consequently the specific radioactivity of acetylcholine in the cytoplasmic pool was almost 5 times that of the vesicles. This distribution was unaffected by preincubation with botulinum toxin. It is concluded that the toxin acts directly on the release of acetylcholine, rather than influencing its storage. 4. After K+-stimulation, toxin-inhibited synaptosomes contained increased amounts of total acetylcholine, which suggests that its rate of synthesis is controlled by depolarization rather than release.

Effects of ionophores A23187 and X537A on brain calcium, catecholamines and excitability

The ionophores A23187 and X537A inhibit 45Ca uptake by rabbit brain mitochondria and synaptosomes and also stimulate the release of accumulated 45Ca from these preparations, but have no effect on 45Ca binding by synaptic membranes or on total brain Ca in mice. Both agents inhibit uptake and stimulate release of 3H-norepinephrine by rabbit P 2 synaptosomal preparations, while the NE and serotonin levels of mouse brain are depressed by X537A. The changes in Ca activities may be related both to the elevated thresholds for cortical after-discharge produced in cats by these ionophores, and to the ionophore induced reduction of pentylenetetrazol seizures in mice.

Effects of apomorphine in vitro on the uptake and release of catecholamines in crude synaptosomal preparations of rat striatum and hypothalamus

It is currently believed that apomorphine exerts its pharmacological effects primarily by a direct stimulatory action on post-synaptic dopaminergic receptors. In the present study, uptake of 3H-dopamine into crude synaptosomal preparations of rat striatum and uptake of 3H-norepinephrine into crude synaptosomal preparations of rat hypothalamus were inhibited 50 per cent by concentrations of apomorphine of 3.6 ± 0.5 × 10 −5 M and 3.2 ± 0.2 × 10 −5 M respectively. Lineweaver-Burk plots of 3H-catecholamine uptake data revealed that apomorphine exhibited mixed inhibition kinetics for dopamine uptake into striatum ( K i = 1.91 ± 0.19 × 10 −5 M) and mixed inhibition kinetics for norepinephrine uptake into hypothalamus ( K i = 2.0 ± 0.17 × 10 −5 M). In crude synaptosomes of rat striatum, apomorphine elicited a dose-dependent release of 3H-dopamine during a 5-min incubation period at 37°. In contrast, the drug failed to produce a significant release of 3H-norepinephrine from crude synaptosomes obtained from rat hypothalamus. While these results suggest that apomorphine in high concentrations is capable of inhibiting the uptake of catecholamines and stimulating the release of dopamine at nerve endings, it is unlikely that these events contribute significantly to the behavioral effects elicited in animals after administration of the drug, since such effects occur at much lower doses.

Stimulus-coupled release of unmetabolized 3H-norepinephrine from rat brain synaptosomes

Summary The calcium-triggered release of 3H-norepinephrine from prelabelled rat brain synaptosomes was studied by a superfusion technique. It was found that the radioactivity released by the depolarizing stimulus, in the presence of calcium, consisted of unmetabolized 3H-norepinephrine. In contrast, the increased release caused by reserpine was totally accounted for by 3H-deaminated metabolites. The results suggest that calcium triggers a release of the amine directly from synaptic vesicles.

Inhibition by dopamine of 3H-noradrenaline release elicited by nerve stimulation in the isolated cat's nictitating membrane.

After loading the isolated nerve muscle preparation of the cat nictitating membrane with 3H-(+/-) -noradrenaline the effects of exogenous dopamine and (-)- noradrenaline were determined on 3H-transmitter overflow elicited by nerve stimulation in the presence of cocaine, 29 muM. Dopamine, 0.20 muM, and (-)- noradrenaline, 0.18 muM, inhibited 3H-noradrenaline release elicited by nerve stimulation at 4 or 10 Hz. Similar results were obtained with apomorphine 0.03 or 0.1 muM. Chlorpromazine, 1 muM, or pimozide, 1 muM, antagonized selectively the reduction in 3H-noradrenaline release obtained with dopamine or apomorphine, without affecting the inhibition obtained with (-)-noradrenaline. Phentolamine, 1 muM, antagonized more effectively the inhibitory effects of (-)-noradrenaline than those of dopamine. Phenoxybenzamine, 0.29 muM, prevented the inhibition of 3H-transmitter overflow obtained with (-)-noradrenaline, dopamine or apomorphine. In the absence of cocaine neither chlorpromazine nor pimozide were able to increase 3H-transmitter overflow during nerve stimulation. Inhibition by dopamine probably located in the outer surface of adrenergic nerve endings. These dopamine receptors differ from the prejunctional alpha-noradrenaline release by nerve stimulation. The prejunctional inhibitory dopamine receptors are not involved in an endogenously mediated regulatory mechanism for noradrenaline release by nerve stimulation under normal conditions. The possibility that these dopamine receptors are involved in the hypotension commonly observed in patients with chronic L-Dopa treatment is discussed.

Differential effects of electrical stimulation on release of 3H-noradrenaline and 14C-alpha-aminoisobutyrate from brain slices.

Differential effects of electrical stimulation on release of 3H-noradrenaline and 14C-α-aminoisobutyrate from brain slices

D-Amphetamine and the release of 3H-norepinephrine from synaptosomes

The release of 3H-norepinephrine from rat brain synaptosomes was studied by a superfusion technique which prevents re-uptake of the released amine. d-Amphetamine had a minimal stimulatory effect on 3H-norepinephrine release and was a potent uptake inhibitor. It is suggested that d-amphetamine may act primarily by inhibiting norepinephrine re-uptake at adrenergic synapses.

Effect of nicotine and other drugs on the release of 3H-norepinephrine and 3H-dopamine from rat brain slices

Nicotine produced a significant increase in the release of 3H-NE from incubated slices prepared from rat hypothalamus, cortex, and cerebellum. The effect on the hypothalamus was much greater than on the other two regions. Nicotine also produced a release of 3H-NE from superfused slices of rat hypothalamus which was dependent upon extracellular calcium and reduced by prior addition of hexamethonium or acetylcholine to the superfusion medium. Nicotine produced a similar release of 3H-DA from rat striatal slices. This effect was reduced by acetylcholine, methacholine, hexamethonium and lidocaine. Morphine and cocaine were without effect while phenoxybenzamine produced a significant increase in the release of 3H-DA induced by nicotine. It is concluded that nicotine can release monoamines from central tissue; the effect is dependent upon extracellular calcium and is produced by an action on classical nicotinic receptors. In addition, this study provides support for the muscarinic inhibitory hypothesis suggesting that acetylcholine may modulate the release of DA and NE from central neurones in a similar way as NE from peripheral adrenergic neurones.