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Notes: Summary Effects of amezinium on postganglionic sympathetic neurones were studied in the heart and pulmonary artery of the rabbit. 1. In isolated perfused hearts, amezinium increased the rate of beat. The effect was antagonized by propranolol or cocaine and by pretreatment with reserpine or 6-hydroxydopamine. 2. In hearts pre-perfused with 3H-noradrenaline, amezinium 0.003 μM reduced the outflow of 3H-DOPEG by 50%. 0.1 μM and higher concentrations in addition reduced the outflow of 3H-MOPEG and increased the outflow of 3H-noradrenaline and 3H-NMN. The outflow of 3H-DOMA and 3H-VMA was not changed. Cocaine prevented the effects of amezinium. When hearts were first perfused with amezinium and then with 3H-noradrenaline, the subsequent outflow of 3H-DOMA was abolished. 3. Hearts were perfused with 3H-noradrenaline 50 nM, and the arterio-venous difference of the 3H-amine was determined. Amezinium 0.29 μM reduced the arterio-venous difference by 50%. 4. Rabbits received intravenous injections of amezinium and were killed 30 min later. The hearts were perfused with 3H-noradrenaline. Pretreatment with amezinium 10 μg/kg diminished the outflow of 3H-DOPEG during the perfusion with 3H-noradrenaline, whereas pretreatment with 1 mg/kg was necessary to decrease the arterio-venous difference of 3H-noradrenaline. 5. Amezinium 4 μM caused 50% inhibition of MAO in crude homogenates and mitochondrial preparations from rabbit hearts, with 3H-noradrenaline as substrate. 6. In pulmonary artery strips preincubated with 3H-noradrenaline, the effects of amezinium on the outflow of 3H-compounds were similar to its effects in the heart. In addition, amezinium reduced the overflow of 3H-DOPEG and enhanced contractions elicited by electrical stimulation. 7. Amezinium is a relatively weak inhibitor of MAO in cell-free preparations. However, in the intact tissue it is a very potent inhibitor of the MAO inside noradrenergic neurones because it is concentrated in these neurones by the noradrenaline uptake mechanism. Being a substrate of uptake, amezinium also inhibits the uptake of noradrenaline. In contrast to bretylium which also inhibits intraneuronal MAO, amezinium is not an adrenergic neurone blocking agent. Concentrations higher than those needed to block intraneuronal MAO in addition release noradrenaline and thereby increase the rate of cardiac contraction.

Notes: Summary The effect of bradykinin on postganglionic sympathetic neuroeffector transmission was studied in superfused strips of rabbit pulmonary artery and in perfused rabbit hearts. 1. In pulmonary artery strips preincubated with 3H-noradrenaline, bradykinin (1–100 nM) diminished the overflow of total tritiated compounds evoked by transmural stimulation at 2 Hz and simultaneously reduced stimulation-evoked contractions. The inhibition was stronger, the less time was allowed to elapse between addition of bradykinin and stimulation. The effect of bradykinin was not changed by atropine, but was abolished by indometacin and 5,8,11,14-eicosatetraynoic acid. 2. Separation of individual 3H-compounds showed that bradykinin and prostaglandin E2 (PGE2) caused proportionate reduction of the stimulation-evoked overflow of total radioactive material, 3H-noradrenaline, 3H-3,4-dihydroxyphenylglycol, and 3H-normetanephrine. 3. Bradykinin (1–100 nM) greatly increased the outflow of PGE from the tissue. The outflow peaked in the 3-min period after addition of bradykinin and then declined rapidly. PGF2α and the metabolites 15-keto-PGF2α, 13,14-dihydro-15-keto-PGF2α and 13,14-dihydro-15-keto-PGE2 were not detected. 4. In the heart, bradykinin (100 nM) diminished the overflow of endogenous noradrenaline evoked by sympathetic nerve stimulation at 3 Hz. Similar results were obtained in hearts perfused with atropine-containing medium. Indometacin, on the other hand, abolished the effect of bradykinin. 5. Bradykinin (100 mM) greatly increased the venous outflow of PGE. PGE2α and the metabolites 15-keto-PGF2α, 13,14-dihydro-15-keto-PGF2α and 13,14-dihydro-15-keto-PGE2 were not detected. 6. It is concluded that bradykinin inhibits the nerve impulse-evoked release of noradrenaline, and consequently postganglionic sympathetic neuroeffector transmission, by enhancing the biosynthesis of prostaglandins of the E series; however, a contribution to the inhibition by other products of the fatty acid cyclooxygenase pathway cannot be ruled out. No prostaglandin-independent presynaptic effect of bradykinin was found.

Notes: Summary Effects of dopamine receptor agonists and antagonists on the release of dopamine were studied in the caudate nucleus of the rabbit. The nucleus contained 6.7 μg/g of dopamine, but negligible levels of noradrenaline and dopamine-β-hydroxylase. No formation of 3H-noradrenaline was detected in caudate slices preincubated with 3H-dopamine, and more than 95% of the tritium content of the tissue consisted of 3H-dopamine. When caudate slices were preincubated with 3H-dopamine and then superfused with amine-free medium, there was a basal outflow of tritium that was not or only slightly changed by tetrodotoxin (10−7 and 10−6 M), apomorphine (up to 10−5 M), bromocriptine (up to 10−6 M), chlorpromazine (up to 10−6 M), haloperidol (up to 10−7 M), or omission of calcium. Electrical stimulation (3 Hz, 24 mA, 2 ms pulse duration, 2-min periods) greatly increased the outflow of tritium. The stimulation-evoked overflow was abolished by tetrodotoxin (10−7 and 10−6 M) and in calcium-free medium. Apomorphine (10−8–10−5 M) and bromocriptine (10−8–10−6 M) reduced, whereas chlorpromazine (10−7 and 10−6 M) and haloperidol (10−8 and 10−7 M) enhanced the evoked overflow. The inhibitory effect of apomorphine and bromocriptine was antagonized by chlorpromazine and haloperidol, but not by phentolamine. Silicone tubings that had been in contact with 3H-haloperidol retained tritiated material that was slowly eluted during perfusion with water or physiological salt solution. The material was identified as 3H-haloperidol. When silicone tubings pretreated with unlabelled haloperidol were used in subsequent dopamine release experiments, the inhibitory effect of apomorphine was not reproduced. It is concluded that, in the caudate nucleus of the rabbit, apomorphine and bromocriptine depress, whereas chlorpromazine and haloperidol facilitate action potential-evoked release of dopamine. The effects are mediated by specific receptors which may be located on the dopaminergic nerve terminals. The receptors appear to be normally activated by released dopamine itself, which thus inhibits its own further release. Part of the discrepancies in the literature concerning dopaminergic modulation of dopamine release may be due to retention of neuroleptic drugs in superfusion assemblies, followed by slow elution and interference with subsequent experiments.

Notes: Summary Slices of the rabbit caudate nucleus were preincubated with 3H-dopamine or 3H-choline and then superfused and stimulated electrically. DiPr-5,6-ADTN reduced the stimulation-evoked overflow of tritium over the same concentration range, independently of whether slices had been preincubated with 3H-dopamine or 3H-choline, and the same was true for apomorphine, NPA and pergolide. Three other putative dopamine receptor agonists, namely 3-PPP, DPI and SKF 38393, failed to decrease the evoked overflow of tritium. Each of six antagonists — (−)-sulpiride, (+)-sulpiride, CGP 11109 A, cis-flupentixol, domperidone and corynanthine —increased the evoked overflow over the same concentration range in experiments with 3H-dopamine and in those with 3H-choline. For each of these antagonists except cis-flupentixol, and also for chlorpromazine, haloperidol and rauwolscine, the pA2 values against apomorphine obtained in 3H-dopamine and in 3H-choline experiments were closely similar. The antagonist effect of cis-flupentixol against apomorphine was not purely competitive. (−)-Sulpiride was a more potent antagonist than (+)-sulpiride, and cis-flupentixol was more potent than trans-flupentixol. This study supplements a previous one in which (±)-sulpiride, metoclopramide and molindone were used as antagonists. It is a functional in vitro approach to receptor characterization, as opposed to radioligand binding studies or in vivo investigations. The results show that a large number of dopamine receptor agonists and antagonists are unable to distinguish between the presynaptic, release-inhibiting dopamine autoreceptors and those postsynaptic dopamine receptors which, when activated, depress the release of acetylcholine. Both receptors can be classified as D2. There was an excellent correlation between pA2 values and the — log K i values of antagonists, taken from the literature, for inhibition of the binding of 3H-spiperone to rat striatal membrane fragments. The correlation supports the view that the sites labelled by 3H-spiperone are true receptors, although the affinities in the binding experiments were consistently lower than the functionally determined affinities in the intact tissue.

Notes: Summary The rabbit pulmonary artery contains postsynaptic α-adrenoceptors which mediate smooth muscle contraction; its noradrenergic nerves contain presynaptic α-adrenoceptors which mediate inhibition of the release of the transmitter evoked by nerve impulses. Dose-response curves for the pre- and postsynaptic effects of eight α-receptor agonists were determined on superfused strips of the artery in the presence of cocaine, corticosterone and propranolol. 1. According to the concentrations which caused 20% of the maximal contraction (EC20 post), the postsynaptic rank order of potency was: adrenaline 〉 noradrenaline 〉 oxymetazoline 〉 naphazoline 〉 phenylephrine 〉 tramazoline 〉α-methylnoradrenaline 〉 methoxamine. The pA2 values of phentolamine against oxymetazoline, phenylephrine, α-methylnoradrenaline and methoxamine were 7.43, 7.48, 7.59 and 7.69, respectively. 2. For the investigation of presynaptic effects, the arteries were preincubated with 3H-noradrenaline. All agonists inhibited the overflow of tritium evoked by transmural sympathetic nerve stimulation. According to the concentrations which reduced the stimulation-induced overflow by 20% (EC20 pre), the rank order of potency was: adrenaline 〉 oxymetazoline 〉 tramazoline 〉 α-methylnoradrenaline 〉 noradrenaline 〉 naphazoline 〉 phenylephrine 〉 methoxamine. 10−5 M phentolamine shifted the presynaptic dose-response curves for noradrenaline and oxymetazoline to the right. 3. The ratio EC20 pre/EC20 post was calculated for each agonist as an index of its relative post- and presynaptic potency. According to the ratios, the agonists were arbitrarily classified into three groups. Group 1 (ratio about 30; preferentially postsynaptic agonists) comprised methoxamine and phenylephrine; group 2 (ratio near 1; similar pre- and postsynaptic potencies) comprised noradrenaline, adrenaline and naphazoline; group 3 (ratio below 0.2; preferentially presynaptic agonists) comprised oxymetazoline, α-methylnoradrenaline and tramazoline (as well as clonidine). 4. Preferentially presynaptic and preferentially postsynaptic agonists had opposite effects on the vasoconstrictor response to nerve stimulation. Methoxamine and phenylephrine either did not change or enhanced, but never reduced, the response. In contrast, oxymetazoline, α-methylnoradrenaline and tramazoline at low concentrations selectively inhibited the response to stimulation at low frequency (0.25–2 Hz). 5. It is concluded that α-adrenoceptor agonists vary widely in their relative pre- and postsynaptic potencies, possibly because of structural differences between pre- and postsynaptic α-receptors. Pre- and postsynaptic components contribute to their overall postsynaptic effect in actively transmitting synapses. The preferential activation of presynaptic α-receptors results in α-adrenergic inhibition of synaptic transmission.

Notes: Summary 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.

Notes: Summary Slices of the head of the rabbit caudate nucleus were preincubated with 10−7 M 3H-dopamine and then superfused, and the effect of unlabeled dopamine on the outflow of tritium was investigated. In most experiments, nomifensine was added throughout superfusion in order to block uptake of the unlabeled amine. Nomifensine was a potent inhibitor of the uptake of 3H-dopamine into rabbit caudate synaptosomes, with an IC50 of 5·10−8 M at a 3H-dopamine concentration of 4·10−8 M. In the absence of nomifensine, unlabeled dopamine (10−7 M and higher concentrations) accelerated the basal outflow of tritium from preincubated slices. 10−5 M nomifensine strongly counteracted the acceleration. In the presence of nomifensine, unlabeled dopamine (10−7 to 10−6 M) caused a concentrationdependent decrease of the overflow of tritium evoked by electrical stimulation at 0.1 Hz. Chlorpromazine and haloperidol (in the presence of nomifensine) increased the stimulation evoked overflow and antagonized the inhibitory effect of dopamine. It is concluded that extracellular dopamine shares with other dopaminergic agonists the ability to inhibit action potential-evoked release of intraneuronal dopamine. The inhibition is mediated by specific receptors. The results support the hypothesis that previously released dopamine, by an action on these receptors, can inhibit further release of dopamine.

Notes: Summary Slices of the caudate nucleus of rabbits were preincubated with 3H-choline and then superfused. Stimulation by electrical pulses at 3 Hz or by 25 mmol/l potassium elicited an increase in tritium outflow which was calcium-dependent and, in the case of electrical stimulation, tetrodotoxin-sensitive. The dopamine receptor agonist apomorphine (0.01–1 μmol/l) decreased, whereas the antagonist haloperidol increased the electrically evoked overflow of tritium. Nomifensine and cocaine, used at concentrations known to inhibit the re-uptake of dopamine, also reduced the evoked overflow of tritium, and this reduction was antagonized by haloperidol. Combined pretreatment with reserpine and α-methyltyrosine methylester (α-MT), which lowered dopamine levels by 99.5%, increased the electrically evoked overflow, as did bretylium which is shown here to block action potential-induced release of dopamine. The facilitation by haloperidol and bretylium as well as the inhibition by nomifensine and cocaine were diminished or abolished after pretreatment with reserpine plus α-MT. Apomorphine decreased, and haloperidol increased, the potassium-evoked overflow of tritium; the effects were not changed by tetrodotoxin. The results indicate that the striatal dopamine receptors which, when activated, depress the release of acetylcholine, are akin to the D-2 type. Endogenous dopamine also acts on the receptors as shown by several manipulations with known effects on dopaminergic transmission. A large fraction of these dopamine receptors may be located on the cholinergic axon terminals.