Prejunctional effects of a purified toxin from the scorpion Tityus serrulatus

Summary The effects of a purified fraction of the venom of the Brazilian scorpion, Tityus serrulatus, were studied in isolated guinea-pig atria previously labelled with ³H-noradrenaline. Exposure to 0.3 and 1.0 /ml of the scorpion toxin resulted in a long lasting positive chronotropic effect which was concentration-dependent. The increase in atrial rate coincided with an enhancement in spontaneous outflow of radioactivity. The increase in outflow of radioactive products elicited by exposure to 1.0 g/ml of the scorpion toxin was approximately 3-fold. ³H-noradrenaline accounted for 60% of the total increase in outflow of radioactivity elicited by the scorpion toxin and the ³H-deaminated glycol (3,4-dihydroxyphenylglycol) represented the main metabolite formed, accounting for approximately 35% of the total release. 20 min after exposure to 1.0 g/ml of the scorpion toxin the overflow of the labelled transmitter elicited by accelerans nerve stimulation (4 Hz, during 60 sec, supramaximal voltage) was increased 8-fold. This effect of the scorpion toxin appears to be unrelated to inhibition of neuronal uptake, block of -adrenoceptors or stimulation of -adrenoceptors. Consequently, in addition to releasing noradrenaline, the scorpion toxin enhances transmitter overflow elicited by nerve stimulation through a prejunctional effect which appears to reflect a nove mechanism of action.     

Accumulation of 3H-(±)-noradrenaline by isolated rat liver cells

Summary Using hepatocytes isolated by collagenase perfusion, we studied the accumulation of ³-noradrenaline. Cells incubated during 15 min in the presence of 0.4 mol/l ³H-noradrenaline (without inhibition of noradrenaline metabolism) accumulated 8.32 ± 1.77 pmol/10⁶ cells (n = 3). The accumulation of ³H-noradrenaline in isolated parenchymal liver cells was sensitive to 10 mol/l cocaine (inhibition 36.6 ± 7.9%, n = 3) and 1 mol/l desipramine (inhibition 27.2 ± 6.9, n = 3). Accumulation of ³H-noradrenaline was temperature and sodium dependent (inhibition 33.2 ± 9.4%, n = 9, when Na⁺ was replaced by Tris⁺) and was influenced by the inhibition of the membrane Na⁺-K⁺-adenosine triphosphatase (Na⁺-K⁺-ATPase) by 150 mol/l ouabain (34.7 ± 6.9% inhibition, n = 3). Accumulation of 3H-noradrenaline in the hepatocytes was not affected by the presence of uptake₂ inhibitors, normetanephrine (30 mol/l) and corticosterone (30 mol/l), but was reduced by 30 mol/l isoprenaline (76.3 ± 5.0% inhibition, n = 6). Thus, the system that takes up and accumulates noradrenaline in the isolated rat liver cells possesses some characteristics of both, uptake₁ and uptake₂ systems and appears to be different from other extraneuronal cocaine-sensitive systems, such as the one reported for pulmonary endothelial cells. Send offprint request to M. I. Masana at the above address     

Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors and α2D-adrenoceptors modulating release of acetylcholine in guinea-pig ileum

The study was designed to classify in terms of _2A, _2B, _2C and _2D the presynaptic ₂-autoreceptors, as well as the ₂-receptors modulating the release of acetylcholine, in the myenteric plexus-longitudinal muscle (MPLM) preparation of the guinea-pig ileum. A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the MPLM preparation were preincubated with ³H-noradrenaline or ³H-choline and then superfused and stimulated electrically.The stimulation periods used (3H-noradrenaline: 3 trains of 20 pulses, 50 Hz, train interval 60 s; ³H-choline: single trains of 30 pulses, 0.2 Hz) did not lead to ₂-autoinhibition or inhibition of ³H-acetylcholine release by endogenous noradrenaline. The ₂-selective agonist 5-bromo6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced the evoked overflow of tritium in both ³H-noradrenaline and ³H-choline experiments. Most (³H-noradrenaline) or all (³H-choline) of the 10 antagonists shifted the concentration-inhibition curves of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. pK_d values from ³H-noradrenaline experiments correlated with pK_d values from ³H-choline experiments (r = 0.981).It is concluded that ₂-autoreceptors and ₂-heteroreceptors modulating the release of acetylcholine in the MPLM preparation are of the same subtype. Comparison with antagonist affinities for prototypic native ₂ binding sites, binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that both are _2D. The results are consonant with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree, across mammalian or at least across rodent and lagomorph species. The same may hold true for ₂-adrenoceptors on non-noradrenergic neurones.     

Noradrenaline release from rat sympathetic neurons evoked by P2-purinoceptor activation

The effects of ATP and analogues on the release of previously incorporated ³H-noradrenaline were studied in cultured sympathetic neurons derived from superior cervical ganglia of neonatal rats. Electrical field stimulation (40 mA at 3 Hz) of the neurons for 10 s markedly enhanced the outflow of tritium. ATP applied for 5 s to 2 min at concentrations of 0.01 to 1 mmol/l caused a time- and concentration-dependent overflow with half maximal effects at about 10 s and 100 mol/l, respectively. 2-Methylthio-ATP was equipotent to ATP in inducing ³H-overflow. ADP (100 mol/l), when applied for 2 min, also caused a small ³H-overflow, but , -methylene-ATP (100 mol/l), AMP (100 mol/l), R(–)N⁶-(2-phenylsiopropyl)-adenosine (R(–)-PIA; 10 mol/l) and 5-N-ethylcarboxamidoadenosine (NECA; 1 mol/l) did not. The ³H-overflow induced by 10 s applications of 100 mol/l ATP was abolished by suramin (100 mol/l) and reduced by about 70% by reactive blue 2 (3 mol/l). Electrically evoked overflow, in contrast, was slightly enhanced by suramin, but not modified by reactive blue 2. Xanthine amine congener (10 mol/l) and hexamethonium (10 mol/l) did not alter ATP-evoked release. Removal of extracellular Ca²⁺ from the medium reduced ATP- and electrically induced overflow by about 95%. Tetrodotoxin (1 mol/l) abolished electrically evoked ³H-overflow but inhibited ATP-induced overflow by only 70%. The ₂-adrenoceptor agonist UK 14,304 at a concentration of 1 mol/l diminished both electrically and ATP-evoked tritium overflow by approximately 70%. These results indicate that activation of P₂-purinoceptors stimulates noradrenaline release from rat sympathetic neurons. The release resembles electrically induced transmitter release, but additional mechanisms may contribute. Correspondence to: S. Boehm at the above address     

Evidence for uptake2-mediated O-methylation of noradrenaline in the human amnion FL cell-line

Summary The uptake and metabolism of ³H-noradrenaline has been examined in the FL cell-line derived originally from human amnion. Cell cultures metabolised ³H-noradrenaline (1.0 mol/l) to ³H-normetanephrine and, to a lesser extent, to metabolites (not distinguished) of the O-methylated deaminated fraction; primary deaminated metabolites were not detected. ³(H-normetanephrine formation a) was not saturable in the noradrenaline concentration range 0.2–150 mol/l, b was decreased to 20%–30% of control levels by uptake₂ inhibitors (O-methylisoprenaline, 20 and 100 mol/l; cimetidine, 10 mol/l; hydrocortisone, 200 mol/l) and c, was almost insensitive to uptake₁ inhibitors (cocaine, 30 mol/l; desipramine, 3 mol/l).Uptake of noradrenaline was manifested after 30 minutes as a 6-fold increase in the cell content of the amine following inhibition of catechol-O-methyl transferase, either alone or in conjunction with inhibition of monoamine oxidase. Uptake was decreased maximally to 40% of control levels by O-methylisoprenaline. IC₅₀ values for inhibition of the O-methylisoprenaline-sensitive component of uptake were (in mol/l): corticosterone (0.3), papaverine (1.1), O-methylisoprenaline (3.0), cimetidine (6.0), (–)noradrenaline (460), and tetraethylammonium (2230). Except for the last agent, for which a comparative value is not available, the IC₅₀'s are in good agreement with those for inhibition of uptake₂ in the Caki-1 cell-line reported by other investigators.The component of uptake resistant to O-methylisoprenaline was depressed by papaverine (a 50% decrease at 50 mol/l), but was not affected by the other uptake₂ inhibitors or by cocaine (30 mol/l).It is concluded that the FL cell possesses an extraneuronal metabolising system very similar to the system in tissues such as heart and smooth muscle where transport of noradrenaline into the cell by uptake₂ is followed by rapid O-methylation via catechol-O-methyl transferase. The only difference appears to be the absence of saturation of ³H-normetanephrine formation in the FL cell at low micromolar concentrations of ³H-noradrenaline. The presence of a second uptake process is suggested by the inhibitory effect of papaverine on uptake resistant to O-methylisoprenaline; lack of effect of cocaine implies that this second process is not uptake,.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylethylene glycol - MAO monoamine oxidase - NMN normetanephrine - OMDA O-methylated and deaminated metabolite fraction - OMI 3-O-methylisoprenaline - TEA tetraethylammonium Correspondence to I. S. de la Lande at the above address     

Characteristics of the cocaine-sensitive accumulation and O-methylation of 3H-(−)-noradrenaline by rabbit endometrium

Summary 1. The extraneuronal uptake and O-methylation of 2,5,6 ³H-(–)-noradrenaline was studied in segments of uterine endometrium from rabbits pretreated with 17-oestradiol and progesterone. 2. The uptake of ³H-noradrenaline was measured in MAO- and COMT-inhibited tissues and obeyed Michaelis-Menten kinetics with an apparent K _m of 78 mol/1 and a V _max of 5.4 nmol/g min. Uptake was inhibited by low Na⁺ and by potential substrates in the order dopamine > (–)adrenaline > (–)isoprenaline = 5-hydroxytryptamine. 3. Following uptake at 1.2 mol/1, efflux of ³H-noradrenaline was slow and appeared to be from two compartments, of which the first (I) had a t1/2 of 53 min and a capacity of 1.8 nmol/g. The presence of the second compartment (II) was inferred from the tissue content of ³H after 60 min of efflux, which was 3–4 times greater than predicted if the ³H was present in compartment I only. Following incubation with ³H-noradrenaline in the presence of cocaine 30 mol/1 the ³H efflux was rapid and the combined capacities of compartments I and II were greatly decreased. 4. ³H-NMN formation, measured in MAO-inhibited tissues, obeyed Michaelis-Menten kinetics with a half-saturating outside concentration of 12 mol/1 and a V _max of 0.9 nmol/g · min. The formation was inhibited by the neuronal uptake inhibitors, desipramine 3 mol/1 and metaraminol 100 mol/1 (each by 80%), but was unaffected by the extraneuronal uptake inhibitor, NMN 100 mol/1, and by oxytetracycline 100 mol/1 and methoxamine 10 mol/1. 5. ³H-NMN formation was inhibited to a small extent (by 30%) by hydrocortisone in a concentration which inhibited ³H-NMN formation in the myometrium by 84%. 6. It is concluded that the extraneuronal uptake of exogenous noradrenaline in the endometrium (a) resembles uptake in sympathetic nerves in its sensitivities to sodium ions and uptake inhibitors, and (b) resembles corticosteroid-sensitive extraneuronal uptake in that it has a low affinity for noradrenaline, but is linked with O-methylation in a fashion which renders uptake and O-methylation a relatively high-affinity, low-capacity system for removing noradrenaline.Abbreviations DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - COMT catechol-O-methyl transferase - MAO monoamine oxidase - NMN normetanephrine - U-0521 3,4-dihydroxy-2-methyl propiophenone Send offprint requests to J. A. Kennedy at the above address     

Selective inhibition by amezinium of intraneuronal monoamine oxidase

Summary Intra- and extraneuronal compartments of rat hearts were selectively labelled by perfusion with ³H-noradrenaline in the presence of corticosterone 87 M or cocaine 30 M, respectively. The subsequent outflow of ³H-compounds was examined. As little as 1 nM amezinium diminished the outflow of intraneuronally formed ³H-DOPEG. This effect was antagonized by cocaine. Amezinium 1 M was necessary to diminish the outflow of extraneuronally formed ³H-DOPEG. This effect was not counteracted by corticosterone. The results indicate that amezinium is both a potent and, at low concentrations, selective inhibitor of intraneuronal MAO.Abbreviations used DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - MAO monoamine oxidase (EC 1.4.3.4) - NA noradrenaline - NMN normetanephrine - OMDA O-methylated deaminated metabolites     

Cyclic AMP facilitates the electrically evoked release of radiolabelled noradrenaline,dopamine and 5-hydroxytryptamine from rat brain slices

Summary The adenylate cyclase activator forskolin as well as 8-bromo-cyclic AMP enhanced the electrically evoked release of³H-noradrenaline and³H-5-hydroxytryptamine from superfused rat neocortical slices and that of³H-dopamine from neostriatal slices with comparable EC₅₀'s of about 0.5 and 50 M, respectively, without affecting spontaneous tritium efflux. The phosphodiesterase inhibitor ZK 62771 (3–100 M) also enhanced³H-noradrenaline and³H-dopamine release but slightly reduced³H-5-hydroxytryptamine release. However, this drug profoundly enhanced spontaneous tritium release in the latter case. The facilitatory effect of forskolin (0.3 M) on the release of the amine neurotransmitters was potentiated in the presence of ZK 62771 (30 M). Therefore, cyclic AMP appears to exert a general facilitatory effect on the release of these biogenic amines from central nerve terminals.     

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

Summary 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 -receptors is supported by the fact that mianserine failed to augment ³H-noradrenaline release further after blockade of the presynaptic -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 -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.It therefore appears that mianserine increases the concentration of noradrenaline in the synaptic cleft by blocking the presynaptic -receptors and inhibiting uptake. Whether or not this increase has functional consequences at postsynaptic noradrenergic receptor sites is unknown. It is possible, however, that postsynaptic receptor blockade counteracts the increase in available noradrenaline.A part of these results was presented at the 16th Spring Meeting of the German Pharmacological Society, Mainz, March 4–7, 1975     

Neuronal efflux of noradrenaline induced by tris or lithium as substitutes for extracellular sodium

Summary Vasa deferentia of either untreated or reserpine (R) and/or pargyline (P) pretreated rats were incubated with³H-noradrenaline and then washed with amine- and Ca²⁺-free solution until (after 100 min) the efflux of radioactivity largely originated from adrenergic nerve endings; COMT was inhibited by U-0521 (U).After 110 min of wash out, the sodium chloride in the wash-out solution was replaced by an equimolar concentration of either Tris-HCl or LiCl. This caused a despramine-sensitive (i.e., carrier-mediated) efflux of tritiated noradrenaline. The initial increase of the low Na⁺-induced efflux dependent on the experimental conditions: it was most pronounced when the axoplasmic concentration of noradrenaline was high (RPU) and relatively small when MAO and vesicular storage were intact (U). The effects of Li⁺ and Tris⁺ differed with regard to the time course of the efflux of tritium: under all three experimental conditions (RPU, PU, U), Tris⁺ caused the rate of efflux of tritium to increase gradually within the 30 min period of observation, while Li⁺⁺ either had a peak-effect (RPU, PU) or a plateau-effect (U). Under U-conditions Tris⁺ caused a slowly increasing, pronounced increase with time of the efflux of both,³H-noradrenaline and³H-DOPEG; whereas Li⁺ caused only a small and sustained increase of the efflux of³H-noradrenaline and a decrease in the efflux of³H-DOPEG.Conclusions: 1) The results are compatible with the view that the buffering agent Tris can diffuse into nerve endings and then also into storage vesicles, and, thus, increases the intravesicular pH; as a consequence of the elevated pH, the leakage of noradrenaline from the vesicles increases and, thus, more noradrenaline becomes available for deamination in and outward transport from the axoplasm. 2) A decrease of the sodium-gradient (brought about by e.g. low extracellular sodium) increases the availability of carrier sites on the internal face of the axonal membrane. This results in outward transport only when the axoplasmic concentration of noradrenaline is elevated (either due to inhibition of MAO or to increased vesicular efflux of noradrenaline).Supported by the Deutsche Forschungsgemeinschaft (Bo 521)     

Activation of presynaptic α2-adrenoceptors attenuates the inhibitory effect of μ-opioid receptor agonists on noradrenaline release from brain slices

Summary ³H-noradrenaline release from rat neocortical slices induced by 15 mM K⁺ was concentration-dependently inhibited by morphine, [D-Ala²-D-Leu⁵] enkephalin (DADLE) and the calcium entry blocker Cd²⁺. Blockade of presynaptic ₂-adrenoceptors with phentolamine, almost doubling K⁺-induced ³H-noradrenaline release, slightly enhanced the relative inhibitory effects of morphine and DADLE, whereas that of Cd²⁺ remained unaffected. In contrast, activation of presynaptic ₂-adrenoceptors with clonidine (1 M) or TL-99 (1 M), inhibiting release by about 50%, completely abolished the inhibitory effects of morphine and DADLE without affecting that of Cd²⁺. When in the presence of 1 M clonidine adenylate cyclase was activated with forskolin (10 M), which restored release to the drug-free control level, the opioids still did not display their inhibitory effects. Therefore, -opioid receptor efficacy appears to be dependent on the degree of activation of ₂-adrenoceptors in central noradrenergic nerve terminals, probably through a local receptor interaction within the nerve terminal membrane.     

The influence of 8-Br 3′,5′-cyclic nucleotide analogs and of inhibitors of 3′,5′-cyclic nucleotide phosphodiesterase,on noradrenaline secretion and neuromuscular transmission in guinea-pig vas deferens

Summary The effects of 3,5-cyclic nucleotide phosphodiesterase (PDE) inhibitors and of 8-Br 3,5-cyclic nucleotide analogs on nerve-muscle transmission were studied in the guinea-pig vas deferens preincubated with ³H-noradrenaline.8-Br cyclic AMP and the PDE inhibitors 3-isobutyl-1-methylxanthine (IBMX) and 3-propionyl-4-hydrazinopyrazolopyridine (SQ 20006) enhanced the secretion of ³H-NA evoked by transmural nerve stimulation. 8-Br cyclic GMP was without effect in this respect.The muscle contraction evoked by transmural nerve stimulation, high potassium or by application of exogenous noradrenaline was depressed by IBMX and SQ 20006. The contraction evoked by transmural nerve stimulation was enhanced by 8-Br cyclic AMP and depressed by 8-Br cyclic GMP.These findings suggest differential involvement of 3,5-adenosine- and guanosine-cyclic nucleotides in excitation-secretion-coupling in the noradrenergic sympathetic nerves, and in excitation-contraction-coupling in the smooth muscle, of guinea-pig vas deferens.     

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

Summary 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 M. Dopamine, 0.20 M, and (-)-noradrenaline, 0.18 M, inhibited ³H-noradrenaline release elicited by nerve stimulation at 4 or 10 Hz. Similar results were obtained with apomorphine 0.03 or 0.1 M. Chlorpromazine, 1 M, or pimozide, 1 M, antagonized selectively the reduction in ³H-noradrenaline release obtained with dopamine or apomorphine, without affecting the inhibition obtained with (-)-noradrenaline. Phentolamine, 1 M, antagonized more effectively the inhibitory effects of (-)-noradrenaline than those of dopamine. Phenoxybenzamine, 0.29 M, 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. In contrast to these results, block of -adrenoceptors by phentolamine or phenoxybenzamine resulted in an increase ³H-transmitter overflow during nerve stimulation. Inhibition by dopamine of ³H-transmitter overflow appears to be mediated through dopamine receptors probably located in the outer surface of adrenergic nerve endings. These dopamine receptors differ from the prejunctional -adrenoceptors that mediate the negative feed-back regulatory mechanism for 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.     

A comparative study of the distribution of tritiated and endogenous noradrenaline in the rat vas deferens and in the dog spleen capsule

Summary The aim of the present work was to study the influence of tissue morphological characteristics on the neuronal release (and by inference the distribution) of tritiated and endogenous noradrenaline.Rat vas deferens and dog spleen capsule were loaded with 0.2 mol/l ³H-noradrenaline, after inhibition of the noradrenaline metabolizing enzymes. Some preparations were washed out under control conditions (spontaneous efflux) and others were washed out in the presence of the releasing agents: 40 mol/l of Ro 4-1284 (a reserpine-like compound), 100 mmol/l potassium or 100 mol/l tyramine. The fractional rate of loss (efflux/tissue content) of each amine was determined and the ratio endogenous amine/³H-noradrenaline in the efflux and in the tissue were also calculated. The results showed no preferential release of one of the amines in the spleen capsule, whereas a preferential release of tritiated noradrenaline was observed in the vas deferens. The smooth muscle layer in the vas deferens was much thicker and more compact than that of the spleen capsule. The ³H-sorbitol space was smaller in the former than in the latter.We conclude that the morphological characteristics of the tissues contribute to the differences in ³H-noradrenaline distribution in the adrenergic varicosities of these preparations.Supported by INIC, FmP1 Correspondence to: M. Q. Paiva at the above address     

Differential effects of α-β-methylene ATP on responses to nerve stimulation in SHR and WKY tail arteries

Summary The effects of ,-methylene-adenosine triphosphate, (,-methylene ATP, a P₂-receptor desensitising agent) have been evaluated on vasoconstrictor responses elicited by exogenous agonists or electrical field stimulation in isolated perfused SHR or WKY tail arteries and on tritium release elicited by electrical field stimulation in SHR-tail arteries pre-labeled with ³H-noradrenaline.Exposure to ,-methylene ATP (0.1 mol/l) significantly inhibited vasoconstrictor responses to electrical field stimulation in SHR tail arteries. These inhibitory effects were not further increased at a higher concentration of ,-methylene ATP (1 mol/l). In WKY tail arteries, ,-methylene ATP (1 mol/l) failed to significantly inhibit vasoconstrictor responses to electrical stimulation.In SHR tail arteries prelabelled with ³H-noradrenaline, ,-methyleneATP (1 mol/l) did not inhibit the stimulation evoked release of tritium. However, at this concentration, ,-methylene ATP significantly antagonized the vasoconstrictor responses of SHR tail arteries induced by exogenous ATP (1 mol/l), ,-methylene ATP (30 mol/l), a stable agonist at P₂-receptors, or 60 mmol/l KCl. These effects of ,-methylene ATP on contractile responses to KCl were not observed in WKY-tail arteries.In tail arteries obtained from reserpine pretreated SHR, despite a 85–95% decrease in endogenous noradrenaline tissue content, the vasoconstrictor responses induced by periarterial field stimulation were greatly diminished, but not abolished. These residual responses to periarterial field stimulation were not antagonized by prazosin (0.1 mol/l), but were practically abolished by the addition of ,-methylene ATP (1 mol/l).In tail arteries from WKY rats pretreated with reserpine, exposure to prazosin (0.1 mol/l) further reduced the residual responses elicited by electrical field stimulation. In these WKY-tail arteries, addition of ,-methylene ATP (1 mol/l) did not further inhibit the remaining vasoconstrictor response obtained in the presence of prazosin.While our results suggest a significantly greater cotransmitter role for ATP with noradrenaline in tail arteries of SHR compared with control normotensive WKY rats, additional effects of ,-methylene ATP not involving P₂ receptors cannot be entirely excluded.     

Monoamines modulate the electrically-evoked efflux of 3H-choline from slices of guinea pig nucleus basalis magnocellularis

The influence exerted by monoamines on acetylcholine release was studied in electrically stimulated slices of guinea pig nucleus basalis magnocellularis (nbM) prelabelled with ³H-choline (3H-Ch).Noradrenaline, 30 M, and clonidine, 1 M, reduced the evoked ³H-Ch efflux by about 50%, but phenylephrine, 100 M. did not; idazoxan, 0.1 M. but not prazosin, 1 M, antagonized these effects. pointing to the involvement of alpha₂ receptors. Apomorphine, 1 or 30 M. reduced ³H-Ch efflux from nbM slices as well. The effect was shared by quinpirole, 1 or 10 M, but not by 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benz-azepine (SKF 38393). 10 M, and was antagonized by sulpiride, 1 M, but not by R-(+)-8-chloro-2,3,4,5-tetra-hydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol (SCH 23390). 1 M, suggesting the involvement of the D₂ receptor subtype.5-hydroxytryptamine (5-HT) 0.3–30 M, and alphamethyl-5-HT, 10 M, significantly increased ³H-Ch efflux from nbM slices; the 5-HT₂ antagonist ritanserin, 1 M. prevented this response. 2-methyl-5-HT, 1–30 M, inhibited the evoked ³H-Ch efflux and its effect was prevented by the 5-HT₃ antagonist 1H,3,5H-tropan-3-yl-3,5-dichlorobenzoate (MDL 72222). 1 M.These findings indicate that i) catecholamines inhibit nbM neurons through alpha₂ and D₂ receptors and that ii) a complex serotonergic modulation of cholinergic function exists in the nbM, involving the activation of various receptor subtypes. which can mediate opposite responses. Correspondence to: A. Siniscalchi at the above address     

Pre- and postsynaptic effects of yohimbine stereoisomers on noradrenergic transmission in the pulmonary artery of the rabbit

Summary Effects on noradrenergic neurotransmission of five stereoisomers of yohimbine and of the closely related compound yohimbol were studied in strips of the pulmonary artery of the rabbit. In some experiments the tissue was preincubated with ³H-noradrenaline. Three effects were observed. Firstly, antagonism to the contractile effect of noradrenaline and of sympathetic nerve stimulation; the antagonism reflected competitive blockade of postsynaptic -adrenoceptors. Secondly, an increase in the stimulation-evoked overflow of total tritium and ³H-noradrenaline; the increase appeared to be due to blockade of presynaptic -adrenoceptors. Thirdly, an increase in the basal outflow of ³H-3,4-dihydrophenylglycol, presumably by impairment of the vesicular storage of ³H-noradrenaline. According to their relative potencies in eliciting these effects, the drugs could be divided into three groups. Rauwolscine, -yohimbine and yohimbol preferentially blocked the presynaptic -adrenoceptor; rauwolscine and -yohimbine, like yohimbine, at low concentrations increased the contractile response to sympathetic nerve stimulation. Corynanthine preferentially blocked the postsynaptic -adrenoceptor. Pseudoyohimbine and 3-epi--yohimbine were very weak antagonists at either receptor; they mainly accelerated the basal outflow of ³H-3,4-dihydroxyphenylglycol.From these results and those of a previous study it is concluded that, in a series of twelve -adrenolytic drugs, rauwolscine shows the greatest preference for presynaptic and corynanthine the greatest preference for postsynaptic -adrenoceptors. In view of the chemical similarity of the two compounds these opposite properties are striking. Corynanthine and rauwolscine might be useful tools for the subclassification of -adrenoceptors.     

Alpha-receptor-mediated modulation of 3H-noradrenaline release from rat brain cortex synaptosomes

Summary The effects of oxymetazoline and noradrenaline (in the presence of desipramine) on the release of ³H-noradrenaline from rat brain cortex synaptosomes were studied using a superfusion technique. Both drugs (at 1M concentrations) were found to reduce the depolarization-induced (15 mM K⁺) release of ³H-noradrenaline. The release-modulating effect of noradrenaline was antagonized by phentolamine and yohimbine.The data provide direct evidence for the hypothesis that -receptors modulating the release of noradrenaline are localized on varicosities of central noradrenergic neurones.     

Postnatal development of vascular β-adrenoceptor-mediated responses and the increase in the adrenaline content of the adrenal gland have a parallel time course

The present study was undertaken to analyse the relationship between postnatal development of vascular ₂-adrenoceptor-mediated responses and the content of adrenaline in the adrenal gland and its concentration in plasma. Dog saphenous vein tissue from newborn, two-weeks old and adult animals were either preloaded with ³H-noradrenaline (or ³H-adrenaline) to study prejunctional -adrenoceptor-mediated effects or mounted in organ baths to determine isoprenaline-induced relaxation of preparations contracted by phenylephrine to about 65010 of the maximum. The adrenal glands and samples of blood from the same animals were taken for estimation of adrenaline and noradrenaline.At birth, there were no -adrenoceptor-mediated effects pre- or postjunctionally. At two weeks, while the results at the prejunctional level were not significantly different from those obtained in newborns, at the postjunctional level there was a relaxant response to isoprenaline, which antagonised about 35010 of the previous contraction to 1.75 mol·l^–1 phenylephrine. In adults, isoprenaline (50 nmol·l^–1) increased by 24% tritium overflow evoked by electrical stimulation of tissues preloaded with ³H-noradrenaline but not that of tissues preloaded with ³H-adrenaline. On the other hand, propranolol (1 mol·l^–1) reduced by 21% the overflow of tritium evoked by electrical stimulation of tissues preloaded with ³H-adrenaline but not that of tissues preloaded with ³H-noradrenaline; postjunctionally, the maximal response to isoprenaline antagonised 70% of the previous contraction to 1.75 mol·l^–1 phenylephrine.At birth the catecholamine content of the adrenals was relatively low (2.9 ol·g^–1) and the adrenaline/noradrenaline ratio was 0.26; two weeks later, the catecholamine content was 14.5 mol·g-1and the adrenaline/noradrenaline ratio was 0.74; in adults, the catecholamine content was 24.5 mol·g^–1 and the adrenaline/noradrenaline ratio was 2.3. In plasma, the highest concentration of adrenaline was observed at birth (11.8 nmol·l^–1); two weeks later it was 5.5 nmol·l^–1 and in adulthood it fell to 3.1 nmol·l^–1.On the basis of these results, it is concluded that some link between the postnatal increase in adrenaline adrenal content and the development of ₂-adrenoceptor-mediated pre- and postjunctional effects may exist. Additionally it is suggested that circulating adrenaline may trigger the development of ₂-adrenoceptor-mediated responses as well as some hypertensive states occurring as a consequence of an overreactivity of the sympathoadrenal system. Correspondence to: S. Guimarães at the above address     

Nicotine receptors do not modulate the 3H-Noradrenaline release from the isolated rat heart evoked by sympathetic nerve stimulation

Summary Isolated rat hearts with the right sympathetic nerves attached were perfused at a constant flow rate of 7 ml/min with Tyrode's solution. (-)-³H-Noradrenaline (final concentration 10–13.9 nM) was infused for 10 min to label the noradrenaline stores. After wash-out the sympathetic nerves were stimulated electrically (3 Hz, 180 impulses, 1 ms, 20–30 mA) three times (S1–S3) at intervals of 15 min. ³H-Noradrenaline and its metabolites were determined by liquid scintillation counting according to Graefe et al. (1973).Both, nicotine 50 M and p-aminophenethyltrimethylammonium (PAPETA) 30 M, enhanced the ³H-noradrenaline overflow in the absence of nerve stimulation. The effect of PAPETA was biphasic and was still observed in the presence of N-methylatropine 0.1 M. Hexamethonium 10 M abolished the first phase only, but cocaine 10 M antagonized both phases.The decline of the stimulation-evoked overflow of ³H-noradrenaline from the first to the third stimulation period was similar in the absence and in the presence of cocaine 10 M starting before S1 and perfused throughout. Cocaine 10 M added before S2, however, enhanced the evoked overflow by 77%.PAPETA 30 M increased the stimulation-evoked overflow by 67% in the absence, and by 73% of the respective control in the presence, of hexamethonium 10 M. PAPETA 30 M failed to enhance the evoked overflow in the presence of cocaine. Hexamethonium (added before S2) did not modify the effectiveness of nerve stimulation.Nicotine, neither when perfused from 6 min before S2, nor when added to the perfusion fluid simultaneously with the onset of nerve stimulation, caused changes in the ³H-noradrenaline output upon S2.Upon stimulation a rather discrete increase in ³H-DOPEG overflow was observed. This increase was abolished by cocaine and/or PAPETA.It is concluded that nicotine and PAPETA stimulate the output of ³H-noradrenaline from the rat heart sympathetic nerves by activation of nicotine receptors. However, the amount of transmitter released is small. Neither drug appeared to modulate the output of ³H-noradrenaline upon electrical nerve stimulation via nicotine receptors.PAPETA, like cocaine, appears to block the reuptake of released transmittsrs thereby enhancing the ³H-noradrenaline overflow and reducing the overflow of ³H-DOPEG (formed intraneuronally from recaptured noradrenaline after nerve stimulation).Abbreviations used DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - MOPEG 3-methoxy-4-hydroxy-phenylglycol - NA noradrenaline - NMN normetanephrine - OMDA O-methylated deaminated metabolites (sum of MOPEG and VMA) - PAPETA p-aminophenethyltrimethylammonium - VMA 3-methoxy-4-hydroxymandelic acid