Effects of flow-stop on the metabolism of noradrenaline released by nerve stimulation in the perfused spleen

Summary The metabolic pathway of ³H-noradrenaline released spontaneously and by nerve stimulation was studied in the isolated perfused spleen of the cat. The deaminated glycol, DOPEG, (3,4 dihydroxyphenylglycol) was the main metabolite in spontaneous outflow, accounting for 62.5±1.6% of the total radioactivity (n=13). Of the total increase in radioactive products elicited by nerve stimulation at 5 Hz or 10 Hz around 30% was accounted for by the noradrenaline metabolites, particularly DOPEG and the O-methylated fraction. In the presence of 2.9×10^–6 M of cocaine the total overflow of radioactivity induced by stimulation was unchanged but DOPEG formation from released noradrenaline was abolished. These findings indicate that DOPEG formation results from the recapture of the released transmitter by adrenergic nerve endings and subsequent intraneuronal deamination. The total overflow of noradrenaline was reduced by flow-stop while the metabolism of the released transmitter was increased significantly. Cocaine, 2.9×10^–6 M, prevented the increase in DOPEG when stimulation was applied under flow-stop conditions. The decrease in noradrenaline overflow induced by flow-stop is partly due to the increase in the metabolism of the released transmitter.     

Metabolic fate of 3H-noradrenaline released from the mouse hypothalamus

Summary In slices of mouse hypothalamus labelled in vitro with ³H-noradrenaline (³H-NA), the deaminated metabolite ³H-3,4-dihydroxyphenylglycol (³H-DOPEG), represented 40.2±2.6% of the total outflow of radioactivity and was the main fraction in the sponteneous efflux. Inhibition of neuronal monoamine oxidase by exposure to 60 M bretylium, reduced the outflow of ³H-DOPEG to 9.7±0.3%. At the same time, the proportion of ³H-normetanephrine (³H-NMN) was significnatly increased. On the other hand, an increased outflow of ³H-DOPEG and a lower proportion of ³H-NMN was obtained in the presence of 2.9 M of the reserpine like agent Ro 4-1284.It is suggested that in the mouse hypothalamus, the deaminated metabolite, DOPEG, is formed inside the nerve terminals, while the O-methylated metabolite, NMN, might result from the activity of extraneuronal catechol O-methyltransferase.     

Mechanism of the enhancement in transmitter release from central and peripheral noradrenergic nerve terminals induced by the purified scorpion venom,tityustoxin

Summary In the isolated nerve-muscle preparation of the cat nictitating membrane exposure to 0.04 M of the scorpion venom tityustoxin (TsTX) increased significantly the overflow of ³H-noradrenaline and the responses elicited by postganglionic nerve stimulation (1200 pulses, 0.5 ms duration, supramaximal voltage). Concentration effect curves to exogenous (-)-noradrenaline were not affected in the presence of this concentration of TsTX.The enhanced release of ³H-noradrenaline obtained during nerve stimulation as well as the increase of the postsynaptic responses observed during exposure to TsTX were more pronounced at 4 Hz than at 20 Hz. The increase in the overflow of noradrenaline observed with the toxin was selective for nerve stimulation since the release evoked by tyramine was not affected by TsTX.TsTX did not increase further the enhancement of ³H-noradrenaline release obtained in the presence of 18 mM tetraethylammonium (TEA). On the other hand, both TsTX and TEA were able to increase further the overflow of ³H-noradrenaline after block of the presynaptic alpha-adrenoceptors with phenoxybenzamine 0.29 or 2.9 M.In slices of rat cerebral cortex, TsTX 0.04 M increased ³H-noradrenaline release induced by 10 mM and by 20 mM KCl. The increased release evoked by the toxin was more pronounced for the lower concentration of K⁺.An increased release of ³H-noradrenaline in the presence of the toxin was also observed in rat hypothalamic slices stimulated with 20 mM K⁺. The K⁺ stimulated induced release of ³H-noradrenaline was also increased by 1.8 mM TEA. As shown for the peripheral nervous, system the simultaneous addition of TEA and TsTX did not result in additive effects when compared with the effects of the two agents added separately. Tityustoxin did not modify the metabolic pattern of the neurotransmitter released by K⁺ from rat hypothalamic slices.It is concluded that TsTX increases the stimulation-induced release of ³H-noradrenaline from both peripheral and central noradrenergic nerve terminals. Tityustoxin appears to act on the nerve terminal by a mechanism similar to that of TEA, an agent known to enhance the amount of noradrenaline released by nerve stimulation by increasing the duration of the action potentials.     

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.     

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     

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.     

Plasma normetanephrine for examination of extraneuronal uptake and metabolism of noradrenaline in rats

The importance of neuronal reuptake for terminating the actions of noradrenaline is well established, but the role of extraneuronal uptake is less clear. This study used plasma concentrations of the extraneuronal noradrenaline metabolite, normetanephrine, to estimate rates of extraneuronal removal of noradrenaline in rats. Animals received infusions of ³H-noradrenaline,. with and without inhibition of catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), to examine the extraneuronal removal of noradrenaline and formation of normetanephrine from infused and endogenous noradrenaline. Infusions of ³H-normetanephrine were also carried out to examine the plasma kinetics of normetanephrine before and after inhibition of MAO.Normetanephrine was cleared rapidly from the circulation and had a short plasma halflife (1 min). Spillover of normetanephrine into plasma (79 pmol kg^–1min^–1) was a third that of noradrenaline, but increased 2.8-fold after inhibition of MAO; noradrenaline spillover remained unchanged. Combined inhibition of MAO and COMT decreased the plasma clearance of ³H-noradrenaline by 38070, reflecting removal of ³H-noradrenaline by extraneuronal uptake. Division of the rate of extraneuronal removal of ³H-noradrenaline by the specific activity of plasma ³H-normetanephrine during the ³H-noradrenaline infusion indicated that the rate of extraneuronal removal of endogenous noradrenaline was 250 pmol kg^–1min^–1; this was close to the spillover of normetanephrine into plasma after inhibition of MAO (219 pmol kg^–1 min^–1).Forty-five % of plasma normetanephrine was derived from circulating noradrenaline and 55% from noradrenaline before entry into the circulation. Assuming that these proportions reflected the sources of noradrenaline metabolized extraneuronally indicated that the rate of extraneuronal metabolism of noradrenaline before entry into the circulation was 138 pmol kg^–1min n^–1. Comparison of this with the rates at which noradrenaline was recaptured by sympathetic nerves (2540 pmol kg^–1min^–1) or spilled over into plasma (228 pmol kg^–1min^–1), indicated that 87% of the noradrenaline released by sympathetic nerves was recaptured, 5% was metabolized extraneuronally and 8% escaped into plasma. Thus, extraneuronal uptake removes much less of the noradrenaline released by sympathetic nerves than neuronal reuptake.     

On the role of the adrenergic receptors for extraneuronal amine uptake and retention in rat salivary glands in vitro

Summary The effect of an -receptor blocking agent, phenoxybenzamine, and a series of -receptor blocking agents on extraneuronal uptake and retention of radioactivity after incubation of rat salivary gland slices with ³H-noradrenaline or ³H-isoprenaline has been investigated. In some experiments with ³H-noradrenaline as substrate, neuronal uptake was prevented by adding protriptyline to the incubation medium. Phenoxybenzamine reduced extraneuronal accumulation of radioactivity after both ³H-noradrenaline and ³H-isoprenaline in a concentration-dependent manner, whereas after propranolol the levels of extraneuronally retained radioactive material were markedly increased, the efflux from the slices not being diminished. Some other nonselective and selective -receptor blocking agents with and without intrinsic activity were found to produce the same effect as propranolol upon the retention of radioactivity after incubation with ³H-noradrenaline and ³H-isoprenaline. When both phenoxybenzamine and propranolol were present in the incubation medium the effect of extraneuronally retained radioactivity after ³H-noradrenaline as well as after ³H-isoprenaline was the same as when only phenoxybenzamine was used. The metabolic pattern of the radioactivity in the slices revealed that the extraneuronally retained radioactive compounds consisted mainly of tritiated catabolites, especially 3-O-methylated ones. The relationship between the extraneuronal accumulation and adrenergic receptor mechanisms is discussed.     

Electrical stimulation releases 3H-betaxolol from rat atrial slices: Possible role of noradrenergic nerves

Summary Under in vitro conditions, ³H-betaxolol was accumulated in rat atrial slices, reaching a tissue-medium ratio of 12.3±1.8 ml/g. This process was temperature-dependent, but ouabain-resistant. ³H-Betaxolol accumulated in atrial slices was subsequently released by electrical stimulation. The electrically-evoked release of ³H-betaxolol was abolished in the absence of calcium, and reduced in the presence of bretylium 10 M. After surgical sympathetic denervation by stellate ganglionectomy there was a marked reduction in the endogenous content of noradrenaline and in the retention of ³H-noradrenaline in atrial slices. The concomitant decrease in the amount of ³H-noradrenaline released by electrical stimulation following denervation was modest, although statistically significant. Following sympathetic denervation, the tissue retention of ³H-betaxolol was not significantly affected, but the release of ³H-betaxolol by electrical stimulation was considerably reduced. After pretreatment with reserpine the amount of radioactivity released by electrical stimulation from slices labelled with ³H-betaxolol or ³H-noradrenaline was markedly reduced. The tissue retention of ³H-noradrenaline was reduced to a larger extent than that of ³H-betaxolol following the administration of reserpine. The simultaneous release of noradrenaline and betaxolol by nerve stimulation observed under our experimental conditions may represent a mechanism through which betaxolol can reach selectively the postsynaptic ₁-adrenoceptors and reinforce -adrenoceptor blockade in the heart.     

Pharmacological characterization of B-HT 933 (2-amino-6-ethyl-4,5,7,8-tetrahydro-6H-oxazolo-[5,4-d]-azepindihydrochloride) as a hypotensive agent of the “Clonidine-type”

Summary The contractile responses to transmural stimulation of, and the overflow of tritium from the rat portal vein prelabelled with ³H-noradrenaline were studied.The contractile responses of the rat portal vein were sustained throughout the period of stimulation. The tension developed did not decline when two consecutive periods of stimulation were compared. In contrast, the tritium overflow decreased during the second period of stimulation.Preincubation with 3 M phenoxybenzamine during 30 min increased 3-fold the tritium overflow during stimulation.Phentolamine and phenoxybenzamine were nearly equipotent in reducing the vascular response to stimulation. In contrast, phentolamine was less potent than phenoxybenzamine in increasing the ³H-noradrenaline overflow elicited by stimulation.The results obtained with phentolamine are interpreted in terms of a different potency of phentolamine to produce blockade of prejunctional and postjunctional -adrenoceptors in the rat portal vein.ATP inhibited by 70% the tritium overflow induced by stimulation. The potency of ATP in inhibiting the overflow increased when the prejunctional -adrenoceptors were blocked.The purine compounds ATP, ADP, AMP and adenosine were roughly equipotent in inhibiting stimulation-induced tritium overflow. The tritium released by stimulation decreased when uptake and metabolism of adenosine were inhibited. Under physiological conditions, a prejunctional purinergic inhibition of noradrenaline release might be involved in an endogenously mediated negative feed-back regulatory mechanism. It is possible that the purinergic inhibition of the noradrenaline liberation elicited by stimulation plays a physiological role in tissues with both purinergic and adrenergic innervation.     

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

Summary 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 M 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.     

Transmitter release patterns of noradrenergic,dopaminergic and cholinergic axons in rabbit brain slices during short pulse trains,and the operation of presynaptic autoreceptors

Summary Slices of rabbit brain were field-stimulated either by single electrical pulses or by trains of 4 or 8 pulses at 1 or 100 Hz in order to study transmitter release patterns and the autoinhibition of transmitter release. The slices were preincubated with ³H-noradrenaline (cortex), ³H-dopamine (caudate nucleus) or ³H-choline (caudate nucleus).Slices preincubated with ³ H-noradrenaline were superfused with medium containing desipramine 1 gmol/l. The overflow of tritium elicited by single pulses amounted to 0 .19% of the tritium content of the tissue. The overflow elicited by 4 pulses/1 Hz was similar, whereas that elicited by 4 pulses/100 Hz was 5.1-fold higher. Yohimbine 101000 nmol/l increased up to 2.5-fold the overflow evoked by 4 pulses/1 Hz but did not change the overflow evoked by single pulses or 4 pulses/100 Hz. - Slices preincubated with ³ H-dopamine were superfused with medium containing nomifensine 1 mol/l. The overflow of tritium elicited by single pulses was 0.39% of the tritium content of the tissue. The overflow elicited by 4 pulses/1 Hz was 1.3-fold and the overflow elicited by 4 pulses/100 Hz 1.4-fold higher. Domperidone 1–100 nmol/l and sulpiride 10–1000 nmol/1 increased up to 2.4-fold the overflow evoked by 4 pulses/ 1 Hz but increased only slightly the overflow evoked by single pulses or 4 pulses/100 Hz. - Slices preincubated with ³ H-choline were superfused either with physostigmine-free medium or with medium containing physostigmine 1 mol/l. In physostigmine-free medium, atropine did not increase the evoked overflow of tritium at any stimulation condition. In physostigmine-containing medium, the overflow elicited by single pulses was 0.18% of the tritium content of the tissue. The overflow elicited by 8 pulses/1 Hz was 2.0-fold and the overflow elicited by 8 pulses/100 Hz 2.2-fold higher. Atropine 2–200 nmol/1 increased up to 2.4-fold the overflow evoked by 8 pulses/1 Hz but increased only slightly the overflow evoked bysingle pulses or 8 pulses/100 Hz. In physostigmine-free medium, sulpiride 10–1000 nmol/1 did not change the single-pulse-evoked overflow of tritium in the absence but increased it in the presence of nomifensine 1 mol/l.Single pulses elicit a large release of ³H-noradrenaline, ³H-dopamine and ³H-acetylcholine under the conditions of these experiments. Release elicited by single pulses is not subject to autoinhibition except for a small inhibition by spontaneously released transmitter in the case of dopaminergic and cholinergic axons. When 3 or 7 further pulses follow the first one at intervals of 1 s, they elicit much smaller release. At least a great part of the fall is due to autoreceptor mediated inhibition (for 3H-acetylcholine release in the presence of physostigmine only). When 3 or 7 further pulses follow at intervals of 10 ms, they elicit release that is either similar to that evoked by the first pulse (³H-noradrenaline) or much smaller (³H-dopamine, ³H-acetylcholine). However, the fall is not due to stimulation-dependent, auto-receptor-mediated inhibition; autoinhibition does not develop in these short high-frequency trains. Overall, the results are in accord with the autoreceptor theory. They demonstrate the role of autoinhibition in determining the transmitter release patterns of central noradrenergic, dopaminergic and cholinergic neurones. Send offprint requests to N. Limberger at the above address     

Frequency-Dependence of the Metabolism of 3H-Noradrenaline Released from Rabbit Isolated Aorta: Effects of a Combination of Cocaine and Corticosterone or Hydrocortisone

Abstract The effect of cocaine (neuronal uptake inhibitor) in combination with either hydrocortisone or corticosterone (extraneuronal uptake inhibitors) on the metabolism of ³H-noradrenaline (³H-NA) released spontaneously or by electrical-field stimulation was studied on the isolated rabbit aorta preloaded with ³H-NA. In the spontaneous outflow ³H-O-methylated and deaminated metabolites (³H-OMDA) accounted for 41% of the total radioactivity whereas ³H-NA represented only 22%. Cocaine (3 × 10^-5M) + hydrocortisone (10^-4 M) neither changed the spontaneous outflow of total tritium nor the distribution of the ³H-outflow on ³H-NA and its ³H-metabolites. However, cocaine (3 × 100^-5M) + corticosterone (4 × 10^-5M) enhanced the passive ³H-outflow, increased the percentage recovered as ³H-3,4-dihydroxyphenylglycol (³H-DOPEG), and reduced the percentage of ³H-OMDA + ³H-NMN. The effect of field-stimulation was investigated during and ofter stimulation at two frequencies: 3 and 10 Hz. The stimulation-induced ³H-overflow consisted mainly of unmetabolized ³H-NA and ³H-OMDA in the sample collected during stimulation. The ratio between ³H-NA and ³H-OMDA was strongly dependent on the frequency of stimulation, Thus it was 1:2 at 3 Hz, but 3:1 at 10 Hz. These ratios were about the same during the poststimulation period. On the other hand, whereas the formation of ³H-DOPEG from ³H-NA released during stimulation at both frequencies was small, it increased rapidly in the poststimulation sample. At 3 Hz, inhibition of neuronal and extraneuronal uptake by cocaine + steroids did not change the stimulation-induced ³H-overflow, but the distribution on ³H-NA and ³H-metabolites was markedly altered. Thus the percentage of ³H-N A was doubled, ³H-OMDA was halved, and ³H-DOPEG was almost abolished. At 10 Hz, however, cocaine + corticosterone decreased the stimulation-induced ³H-overflow, but did not change the proportions of ³H-NA and ³H-OMDA. Only the formation of ³H-DOPEG was markedly reduced. It is concluded that the distribution of stimulation-induced ³H-overflow on ³H-NA and its ³H-metabolites and the effect of neuronal and extraneuronal uptake inhibition on this is strongly influenced by the frequency of stimulation. Furthermore, inhibition of both neuronal and extraneuronal uptake does not fully prevent metabolism of ³H-NA released by electrical-field stimulation.     

Early intraneuronal mobilization and deamination of noradrenaline during global ischemia in the isolated perfused rat heart

Summary Isolated rat hearts were perfused according to the Langendorff technique and both extraneuronal uptake of noradrenaline and COMT were inhibited. The noradrenergic neurones were first prelabelled with ³H-(–)-noradrenaline (13 nmol/1). Thereafter the hearts were submitted to global ischemia (perfusion rate reduced from 5 up to 0.5 ml/min) for 60 min and subsequently reperfused for 5 min. The coronary effluent was continuously collected and analyzed for the appearance of ³H-noradrenaline and its metabolites. 1. Global ischemia was associated with an early release of ³H-noradrenaline. At reperfusion a brisk increase in the FRL of ³H-noradrenaline was observed which may indicate that, on severe restriction in coronary flow, perfusion of the tissue became heterogenous and thus partially masked the amount of ³H-noradrenaline released from the noradrenergic nerve terminals. Gradual reduction in coronary flow also progressively reduced (but did not abolish) the total formation of ³H-DOPEG. 2. The maximal efflux of ³H-noradrenaline was observed during the 1st min of reperfusion whereafter the efflux declined rapidly, indicating a wash-out of transmitter trapped in the extracellular space. The efflux of the lipophilic metabolite ³H-DOPEG, on the other hand, continuously increased during the reperfusion. This was due to both new formation and wash-out of ³H-DOPEG retained and/or distributed into the tissue during the period of restricted flow. 3. Neither a reduction of the extracellular calcium concentration (from 2.6 mmol/l to 0.1 mmol/1) nor the presence of the calcium entry blocker verapamil (250 nmol/l) reduced the efflux of ³H-noradrenaline seen during ischemia and reperfusion. 4. Desipramine (100 nmol/l) markedly reduced the ischemia-induced release of ³H-noradrenaline and simultaneously attenuated the formation of ³H-DOPEG. 5. A moderate reduction in the ischemia-induced mobilization of ³H-noradrenaline was seen in hearts perfused with 1ol/l reserpine, whereas the formation of ³H-DOPEG from such hearts was markedly higher than in corresponding controls. Only minor deviations from this pattern was observed when desipramine was present in addition to reserpine. It is concluded that a severe restriction in myocardial perfusion rate is associated with an enhanced net leakage of vesicular noradrenaline. This results in a rise of the free axoplasmic noradrenaline concentration which, in combination with an altered transmembrane sodium gradient, induces an increased local release of noradrenaline partly mediated by a calcium-independent, carrier-mediated outward transport. Desipramine, which inhibits this transport mechanism, may have, in addition to its effect on the membrane carrier, an additional effect in reducing the net leakage of transmitter from storage vesicles. Furthermore, despite severe restriction in coronary flow, and thus oxygen delivery, DOPEG is still formed, possibly as a consequence of the elevated axoplasmic noradrenaline concentration which may in part compensate for a reduced monoamineoxidase activity.Abbreviations DOPEG dihydroxyphenylglycol - DOMA dihydroxymandelic acid - MAO monoamineoxidase - COMT catechol-O-methyltransferase - OMI 3-O-methoxy-(±)-isoprenaline - FRL fractional rate of loss This study was supported by the Deutsche Forschungsgemeinschaft (Gr 490/5-1) and AB Hässle, Mölndal, Sweden     

The influence of uptake2 on the inhibition by unlabelled noradrenaline of the stimulation-evoked overflow of 3H-noradrenaline in rabbit aorta with regard to surface of amine entry

The release by electrical field stimulation of 3H-noradrenaline from the adrenergic nerve endings in the rabbit aorta was studied in a special double-chambered organ bath. Independently of the frequency (1-10 Hz) and the number of pulses used (300-3,000 pulses), only 10-20% of the stimulation-evoked overflow of tritium (total evoked overflow) left the aortic wall via the intimal surface (intimal evoked overflow). The corresponding percentage value for the basal efflux was twice that for the evoked overflow, thus indicating that part of the radioactivity in the basal efflux originated from an extraneuronal compartment. The radioactivity in the total evoked overflow (in response to stimulation at 10 Hz) originated from at least two compartments (compartment I and II) with half times for efflux of about 2 and 6 min, respectively. In the intimal evoked overflow, compartment II (but not compartment I) was involved. When uptake1 was inhibited, 70% of the radioactivity in the intimal evoked overflow (stimulation at 1 Hz) consisted of metabolites, while unchanged amine prevailed by far in the total and adventitial evoked overflow, respectively. Additional inhibition of uptake2 thus had a striking effect only on the composition of the radioactivity in the intimal evoked overflow. The intimal surface was exposed to unlabelled noradrenaline in order to inhibit the evoked overflow of tritium (stimulation at 1 Hz; uptake1 inhibited). When uptake2 was inhibited additionally, the dose-response curve for the inhibitory effect of noradrenaline was shifted to the left by a factor of 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of labetalol (AH 5158) on metabolism of 3H(-)-noradrenaline released from the cat spleen by nerve stimulation

The competitive α and β adrenoceptor antagonist labetalol, in concentrations up to 10^?4 M, produced a dose dependent increase in overflow of ³H and [³H]noradrenaline in the isolated blood perfused cat spleen following stimulation of the splenic nerves at a frequency of 10 Hz. Labetalol had no effect on the pattern of overflow of label following stimulation. In experiments in which the metabolism of [³H]noradrenaline released on nerve stimulation was examined, labetalol produced a concentration dependent increase in the percentage of [³H]noradrenaline and a decrease in the percentage of [³H]DOPEG in the venous blood following nerve stimulation. Production of [³H]COMT metabolites and [³H]DOMA was not affected. It is suggested that in the isolated blood perfused cat spleen labetalol produces the elevation of overflow and effects on noradrenaline metabolism by inhibition of neuronal uptake of noradrenaline. The drug has no detectable effects on the enzymes MAO or COMT or on extraneuronal uptake.     

Uptake and metabolism of 3H-adrenaline and 3H-noradrenaline by isolated hepatocytes and liver slices of the rat

Summary Isolated rat hepatocytes were incubated with 0.05 mol/l or 0.2 mol/l ³H-(–)-noradrenaline or 0.05 mol/l ³H-(–)-adrenaline for 15 min and the content of amines as well as the formation of metabolites was measured.The removal Of both amines from the incubation medium was quantitatively similar, and mainly due to metabolism (which represented 96% of the removal of ³H-adrenaline and 98% of the removal of ³H-noradrenaline). O-methylation predominated for ³H-adrenaline: O-methylated and deaminated metabolites (³H-OMDA) and ³H-metanephrine (³H-MN) were the most abundant metabolites, accounting for 63% and 34% of total metabolite formation, respectively. Deamination predominated for ³H-noradrenaline: ³H-OMDA and ³H-dihydroxymandelic acid (³H-DOMA) were the most abundant metabolites, representing respectively 56% and 36% of total metabolite formation. The following activities of monoamine oxidase and catechol-O-methyl transferase were determined for ³H-noradrenaline: k_COMT 0.70±0.15 min^–1 and k_MAO 2.27±0.14 min^–1 In experiments with ³H-noradrenaline, inhibition of monoamine oxidase reduced the formation of ³H-OMDA and deaminated metabolites [³H-dihydroxyphenylglycol (³H-DOPEG) and ³H-DOMA] and increased the formation of ³H-normetanephrine (³H-NMN). Inhibition of catechol-O-methyl transferase, On the Other hand, decreased ³H-NMN and increased ³H-DOPEG formation. When both enzymes were inhibited, the formation of all metabolites was strongly reduced but surprisingly there was no accumulation of ³H-amines in the cells, as the cell: medium ratio for ³H-noradrenaline or ³H-adrenaline was about unity. In experiments with either ³H-noradrenaline or ³H-adrenaline, specific inhibitors of either uptake, or uptake₂ produced discrete effects, slightly decreasing the formation of ³H-OMDA and ³H-NMN or ³H-MN, and having no effect on ³H-amine content of the cells. Additional experiments were carried Out with rat liver slices incubated for 15 min with ³H-noradrenaline 0.2 mol/l. The pattern of metabolism of ³H-noradrenaline (³H-OMDA and ³H-DOMA were the most abundant metabolites) as well as the degree of metabolism of the amine removed from the incubation medium (91% of the removal) were similar to those of the isolated cells. Likewise, there was no accumulation of intact ³H-noradrenaline in the tissue. Moreover, the results obtained with enzyme inhibitors as wells as with uptake inhibitors were similar to those obtained with hepatocytes.In conclusion, isolated hepatocytes remove and metabolize catecholamines very efficiently, being one of the most active systems studied in this respect. Uptake₁ and uptake₂ are responsible for part of the removal of catecholamines by hepatocytes; the system(s) involved in the remaining removal seem(s) to be active, but possess(es) characteristics that do not allow us to characterize it (them) either as uptake₁ or uptake₂.Abbreviations COMT catechol-O-methyl transferase - DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MAO monoamine oxidase - MN metanephrine - NMN normetanephrine - OMDA O-methylated and deaminated metabolites (i.e., MOPEG = 4hydroxy-3-methoxyphenylglycol and VMA = 4-hydroxy-3-methoxymandelic acid) Supported by Programa STRIDE (STRDA/P/SAU/259/92)PhD student with a grant from JNICT (Programa Ciência) Correspondence to: F. Martel at the above address     

Histamine H3 receptor-mediated inhibition of noradrenaline release in pig retina discs

Summary Discs of pig retina were preincubated with ³H-noradrenaline, ³H-dopamine or ³H-serotonin and then superfused. Electrical field stimulation increased the outflow of tritium from discs preincubated with ³H-noradrenaline or ³H-dopamine, but no from discs preincubated with ³H-serotonin. The tritium content at the end of superfusion was similar in discs preincubated with ³H-noradrenaline or ³H-dopamine but about tenfold lower in discs preincubated with ³H-serotonin. The tritium content in discs preincubated with ³H-noradrenaline was markedly reduced when desipramine was present during preincubation but was not affected by selective inhibitors of dopamine and serotonin uptake. The tritium content in discs preincubated with 3Hdopamine and ³H-serotonin, in contrast, was reduced or tended to be reduced by a selective dopamine and serotonin uptake inhibitor, respectively.The electrically evoked overflow of tritium from discs preincubated with ³H-noradrenaline was abolished by tetrodotoxin or omission of Ca²⁺. In discs superfused with desipramine, the electrically evoked overflow was enhanced by phentolamine but not affected by histamine. When both desipramine and phentolamine were present in the superfusion medium, histamine inhibited the evoked overflow (pIC₁₅ 6.85). This effect was mimicked by the histamine H₃ receptor agonist R-(–)--methylhistamine as well as by its S-(+)-enantiomer (pIC₁₅ 7.85 and 5.30, respectively) but not by the H₁ receptor agonist 2-(2-thiazolyl)ethylamine and the H₂ receptor agonist dimaprit (each 10 mol/l). The inhibitory effect of histamine was abolished by the H₃ receptor antagonist thioperamide 0.32 mol/l and attenuated by impromidine 3.2 mol/l but not affected by the H₁ receptor antagonist dimetindene 3.2 mol/l and the H₂ receptor antagonist ranitidine 10 mol/l.The results suggest that, in the pig retina, noradrenaline is taken up into, and released from, noradrenergic neurones (most likely vascular postganglionic sympathetic nerve fibres, less probably tissue-specific noradrenergic neurones of the retina) and that noradrenaline release is subject to modulation via H₃ receptors and probably also a-adrenoceptors.Send offprint requests to E. Schlicker at the above address     

Cocaine and neuronal uptake in the canine saphenous vein

Summary This study was designed to investigate the effects of the neuronal uptake inhibitor, cocaine on the adrenergic neuroeffector interaction in the canine saphenous vein. Tissues were incubated with ³H-noradrenaline in control solution or in presence of the cocaine. The tissue content of ³H-noradrenaline and its metabolites was determined after the incubation. As the concentration of cocaine in the incubation medium increased gradually less ³H-noradrenaline and DOPEG were detected in the tissue, while the content of DOMA, NMN, MOPEG and, in particular that of VMA increased; comparable results were obtained with high concentrations of cocaine and desmethylimipramine (DMI). Helical strips of canine saphenous veins were incubated with ³H-noradrenaline and mounted for isometric tension recording and for measurement of the efflux of labelled transmitter and its metabolites. Cocaine, but not DMI, slightly increased the spontaneous efflux of DOPEG, suggesting that cocaine enters the nerve terminals and displaces noradrenaline from its storage sites. During electrical stimulation, cocaine at 3×10^–5 mol/l increased the contractile response and the overflow of ³H-noradrenaline, DOMA, NMN and MOPEG and decreased the appearance of DOPEG. Similar results were obtained with DMI (10^–6 mol/l) except that it did not increase the overflow of DOMA and MOPEG. During electrical stimulation in presence of DMI, cocaine did not affect the contractile response and decreased the appearance of intact labelled transmitter. Electrical stimulation, cocaine and DMI did not affect the overflow of VMA. The present experiments indicate that in the canine saphenous vein: (1) DOPEG is formed intraneuronally, but DOMA, MOPEG, NMN and VMA extraneuronally; (2) VMA is retained in the tissue much longer than the other metabolites; (3) determination of total ³H-content after incubation with ³H-noradrenaline in presence of inhibitors of neuronal uptake underestimates the degree of inhibition of the neuronal amine carrier; and (4) the quantification of the effect of cocaine on the neuronal uptake of released transmitter is complicated by several other actions of the drug (local anesthetic properties, displacement of stored transmitter, activation of effector cells) and that of the effect of DMI by its inhibitory effect on monoamine oxidase, in particular at extraneuronal sites.Supported in part by grant HL 05883 from the National Institutes of Health     

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