Nonexocytotic noradrenaline release induced by pharmacological agents or anoxia in human cardiac tissue

In acute myocardial ischemia, noradrenaline is released locally from sympathetic varicosities by a Ca²⁺-independent nonexocytotic release mechanism that is effectively suppressed by inhibitors of the neuronal noradrenaline carrier (uptake₁). The purpose of the present study was to elucidate the significance of free axoplasmic amine concentration and disturbed neuronal sodium homeostasis for nonexocytotic noradrenaline release in the human heart by comparing the release induced by anoxia with that induced by reserpine, tyramine, or veratridine. The overflow of endogenous noradrenaline and dihydroxyphenylethyleneglycol was assessed in human atrial tissue incubated in calcium-free Krebs-Henseleit-solution to prevent interferences by exocytotic release. The overflow of dihydroxy-phenylethyleneglycol served as indicator of the free axoplasmic noradrenaline concentration.When vesicular uptake was blocked by the reserpine-like agent Ro 4-1284, high dihydroxyphenylethyleneglycol overflow was observed without concomitant noradrenaline overflow. If, however, Ro 4-1284 was combined with sodium pump inhibition (by omission of extracellular potassium) or with alteration of the transmembrane sodium gradient (by lowering the extracellular sodium concentration), both dihydroxyphenylethyleneglycol and noradrenaline were released. The indirectly acting sympathomimetic tyramine induced a marked increase in noradrenaline overflow which was accompanied by overflow of high amounts of dihydroxyphenylethyleneglycol, indicating interference of the drug with both vesicular catecholamine transport and amine transport via uptake₁. Likewise, veratridine induced an overflow of noradrenaline (which was prevented by blockade of uptake₁) and dihydroxyphenylethyleneglycol indicating a reserpine-like action of the drug. A disturbed energy status of the sympathetic neuron induced by cyanide intoxication or anoxia caused noradrenaline overflow which was suppressed by uptake, blockade. Blockade of sodium channels by tetrodotoxin effectively reduced noradrenaline overflow during cyanide intoxication but not during anoxia. Anoxia-induced noradrenaline release, however, was markedly suppressed by inhibition of Na⁺/H⁺ exchange with ethylisopropylamiloride, indicating the Na⁺/H⁺ exchange as the predominant pathway for sodium entry into the sympathetic neuron during anoxia.The results demonstrate that disturbed neuronal sodium homoeostasis and impaired vesicular storage function are critical conditions, causing nonexocytotic noradrenaline release in anoxic human cardiac tissue.     

Neuropeptide Y differentiates between exocytotic and nonexocytotic noradrenaline release in guinea-pig heart

Summary The overflow of neuropeptide Y (NPY; radioimmunoassay), noradrenaline and dihydroxyphenylethylenglycol (DOPEG; high pressure liquid chromatography) from guinea-pig perfused hearts was investigated in relationship to exocytotic and nonexocytotic release mechanisms. Exocytotic release: Electrical stimulation of the left stellate ganglion (12 Hz; 1 min) evoked a calcium-dependent overflow of noradrenaline and NPY, that was accompanied by a minor and prolonged increase in DOPEG overflow. This increase in DOPEG overflow was attenuated by blockade of neuronal amine re-uptake. In the presence of calcium, a closely related co-release of noradrenaline and NPY was also observed during administration of veratridine (10 M); it was completely prevented by tetrodotoxin (1 M). Nonexocytotic release: In the absence of extracellular calcium, veratridine (30 M) induced noradrenaline overflow only when combined with the reserpine-like agent Ro 4-1284 (10 M). This overflow was accompanied by efflux of DOPEG, but not of NPY. Similarily, tyramine (1–100 M) induced a calcium-independent concomitant overflow of both noradrenaline and DOPEG, but not of NPY. During anoxic and glucose-free perfusion a predominantly calcium-independent overflow of noradrenaline was observed; only in the presence of extracellular calcium was this overflow accompanied by a minor overflow of NPY. Noradrenaline overflow, induced by veratridine plus Ro 4-1284 (in the absence of calcium), by tyramine, or by anoxia, was suppressed by blockade of neuronal amine re-uptake, and was, therefore, mediated by reversed transmembrane amine transport by the neuronal uptake₁ carrier.The results indicate that NPY is co-released with noradrenaline only during calcium-dependent exocytosis. On the other hand, whenever, noradrenaline is released by non-exocytotic (calcium-independent and carrier-mediated) release mechanisms, no substantial NPY overflow is observed. The simultaneous determination of noradrenaline and NPY overflow, therefore, allows a differentiation between exocytotic and nonexocytotic noradrenaline release, and NPY may be utilized as a marker of exocytotic noradrenaline release.This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 320 — Herzfunktion und ihre Regulation)Presented in part at the 62nd Scintific Sessions of the American Heart Association, New Orleans/USA, November 1989     

The role of co-transported sodium in the effect of indirectly acting sympathomimetic amines

Summary The adrenergic nerve endings of vasa deferentia of either untreated or reserpine (R) and/or pargyline (P) pretreated rats were loaded with ³H-noradrenaline; COMT was inhibited by U-0521 (U). After 100 min of wash-out with Ca²⁺-free solution, the efflux of tritium (and of ³H-noradrenaline) from the tissue was largely of neuronal origin and remained constant with time (when expressed as fractional rate of loss; FRL). After 110 min of wash-out the effect of inhibition of the Na⁺,K⁺-ATPase (by low K⁺ or ouabain) on basal and on sympathomimetic amine-induced efflux of tritium (or ³H-noradrenaline, under the condition U) was studied in paired experiments.Inhibition of the Na⁺,K⁺-ATPase caused a time-dependent increase in the efflux of tritium (or ³H-noradrenaline) which was inhibited by desipramine.Inhibition of the Na⁺,K⁺-ATPase also caused a time-dependent reduction of the initial rate of neuronal uptake of ³H-noradrenaline.The effectiveness of the sympathomimetic amines tyramine and amphetamine in inducing release (i.e., outward-transport) of noradrenaline depended on the experimental condition: it was most pronounced under the condition RPU, followed by the condition PU and lowest under the condition U (i.e., in tissue of untreated rats). Inhibition of the Na⁺,K⁺-ATPase caused an early and transient enhancement of the release of noradrenaline induced by tyramine or amphetamine. This enhancement was seen already within the first min after inhibition of the ATPase, i.e., before a pronounced inhibition of uptake (of noradrenaline) and before a pronounced increase of the basal efflux was observed. It also depended on the experimental condition: RPU > PU > U; i.e., it was the more pronounced, the higher the free axoplasmic concentration of noradrenaline.In tissues of untreated rats, tyramine increased the rate of efflux of DOPEG, whereas amphetamine decreased it.1) Both, tyramine and amphetamine are transported by the Na⁺-dependent neuronal transport system; 2) the co-transported Na⁺ causes a local increase in the Na⁺ concentration at the inside of the neuronal plasma membrane and thereby contributes to the outward-transport of axoplasmic noradrenaline induced by indirectly acting sympathomimetic amines; however, this contribution is only of importance when the axoplasmic concentration of noradrenaline is high (RPU, PU).Some of the results were presented at the 4th Meeting on Adrenergic Mechanisms, Porto, Portugal (1981); Bönisch (1981)     

Simulation of outward transport of neuronal 3H-noradrenaline with the help of a two-compartment model

Summary In order to simulate the outward transport of ³H-noradrenaline induced by veratridine from adrenergic varicosities, a mathematical two-compartment model was developed in which the two compartments (representing axoplasm and storage vesicles) are arranged in series. Simulated results were compared with experimental results obtained with 100 mol/l veratridine + 1 mmol/l ouabain and rat vasa deferentia kept in calcium-free solution (Bönisch and Trendelenburg 1987). As in experiments, the time course of efflux of ³H-noradrenaline had a pronounced and early peak under RPU-conditions, a minor peak under PU-conditions, and solely a plateau under U-conditions (where R stands for pretreatment with reserpine, P for pretreatment with pargyline, and U for inhibition of COMT by U-0521). From the width of the peak of release, it was deduced that — under RPU-conditions — about 40% of neuronal ³H-noradrenaline are distributed into the axoplasm, about 60% into the storage vesicle. However, this estimate represents an average value; the results are compatible with the view that the ratio axoplasmic/vesicular ³H-noradrenaline is quite variable from rat to rat. Under U-conditions, calculations confirm that reserpinelike compounds induce an efflux of tritium that consists predominantly of deaminated ³H-metabolites. The stimulation of outward transport, on the other hand, causes an efflux of tritium that consists predominantly of ³H-noradrenaline; indeed, the efflux of deaminated ³H-metabolites declines (as it did in experiments). Simulations showed further that the highest rates of outward transport of ³H-noradrenaline were achieved when there was a simultaneous induction of outward transport of ³H-noradrenaline and a reserpine-like effect (as it is known to occur when tissues are exposed to veratridine; Bönisch and Trendelenburg 1987). While there was satisfactory agreement between simulated and experimental results under various conditions, there were also two discrepancies that may be caused by a) inhomogeneous labelling of the storage vesicles in individual varicosities (RPU < PU < U) and b) saturation of outward transport of ³H-noradrenaline when a reserpine-like compound greatly increases the axoplasmic level of total noradrenaline (under U-conditions).Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - FRL fractional rate of loss - MAO monoamine oxidase Supported by the Deutsche Forschunesgemeinschaft (SFB 176) Send offprint requests to E. Schömig     

The heterogeneity of the neuronal distribution of exogenous noradrenaline in the rat vas deferens

Summary After loading of the incubated rat vas deferens with 0.2 mol/l ³H-noradrenaline (followed by 100 min of wash-out with amine-free solution), the efflux of endogenous and exogenous compounds was determined by HPLC with electrochemical detection and by column chromatography with scintillation counting. Two different types of heterogeneity of labelling were found. The first one is due to the preferential labelling of varicosities close to the surface of the tissue, the second one to the preferential labelling of vesicles close to the surface of loaded varicosities. As diffusion distances within the tissue and within varicosities are then longer for endogenous than for exogenous amine and metabolites, the composition of spontaneous efflux of exogenous compounds differed from that for endogenous compounds. Because of preferential neuronal and vesicular re-uptake of endogenous noradrenaline, the percentage contribution by noradrenaline to overall efflux was: endogenous < exogenous. While ³H-DOPEG was the predominant exogenous metabolite, DOPEG and MOPEG equally contributed to the endogenous efflux.Desipramine abolished the consequences of the first heterogeneity of labelling, i.e., it increased the efflux more for endogenous than for exogenous noradrenaline; moreover it decreased the efflux of ³H-DOPEG, but increased that of ³H-MOPEG. The reserpine-like compound Ro 41284, on the other hand, abolished the consequences of the second type of heterogeneity; it reduced the specific activity of total efflux (i.e., of the sum of noradrenaline + DOPEG + MOPEG) to the specific activity of the tissue noradrenaline. The degree of heterogeneity of labelling was reduced after inhibition of monoamine oxidase and also when the tissues were loaded with 2 or 20 mol/l ³H-noradrenaline.It is proposed that the various compartments and pools of noradrenaline described in the literature reflect the two heterogeneities described here.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - FRL fractional rate of loss (= rate of efflux/tritium content of tissue measured at onset of collection period) - HPLC high performance liquid chromatography - MAO monoamine oxidase - MOPEG methoxyhydroxyphenylglycol - NMN normetanephrine - VMA vanillylmandelic acid Send offprint requests to E. Schömig at the above addressThis study was supported by the Deutsche Forschungsgemeinschaft (SFB 176, Gr 490/5 and Scho 383/1). Some of the results were presented to the German Pharmacological Society (Schönfeld 1990; Trendelenburg 1990)     

Veratridine-induced outward transport of 3H-noradrenaline from adrenergic nerves of the rat vas deferens

Summary 1. The neuronal release by 100 mol/l veratridine of preloaded ³H-noradrenaline was studied in the rat vas deferens, the MAO, COMT and vesicular uptake of which were inhibited. To prevent any exocytotic release of the ³-Hamine, all solutions were calcium-free. Veratridine induced an early and a late peak of tritium efflux. The early peak was abolished by the presence of 1 mol/l desipramine, the late peak was abolished by 1 mol/l tetrodotoxin (administered subsequently to the first peak). The administration of veratridine plus 1 mmol/l ouabain resulted in only the early peak of efflux. 2. The peak response to veratridine plus ouabain was increased by a very early administration of veratridine plus ouabain (after 40 min of wash-out instead of the usual 130 min) (i. e., when the relative size of the axoplasmic distribution compartment was increased). However, very high axoplasmic ³H-noradrenaline levels (after loading with 37 instead of the usual 0.2 mol/l) reduced the height of the peak (when expressed as a FRL). 3. Substantially similar responses to vcratridine plus ouabain were obtained after loading with ³H-noradrenaline, ³H-adrenaline or ³H-dopamine. 4. As the second peak of veratridine-induced release is ouabain-sensitive, it appears to be caused by exhaustion of neuronal ATP stores; this, in turn, raises the intravesicular pH and induces efflux of ³H-noradrenaline from the vesicles into the axoplasm. The first peak, on the other hand, represents outward transport of ³H-noradrenaline from the axoplasmic compartment. Evidently, a pronounced vesicular distribution of ³H-noradrenaline takes place even after inhibition by reserpine of the vesicular uptake. 5. In preparations with intact vesicular uptake (MAO and COMT inhibited) a plateauresponse was obtained; in the presence of 10 mol/l Ro 4-2184 (a reserpine-like compound) a peak response was restored after loading with 0.2 mol/l³H-noradrenaline, less so after loading with 37 mol/l. 6. It is confirmed that veratridine (plus ouabain) exerts a reserpine-like effect when applied to tissues with intact vesicular uptake and intact MAO.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxy mandelic acid - DOPEG dihydroxyphenylglycol - DOPAC dihydroxyphenylacetic acid - FRL fractional rate of loss - MAO monoamine oxidase - 5-HT 5-hydroxytryptamine with technical assistance of Marianne BablSupported by the Deutsche Forschungsgemeinschaft (Bo 521 and SFB 176) Send offprint requests to: H. Bönisch     

Effect of a reserpine-like agent on the release and metabolism of [3H]NA in cell bodies and terminals

1. The reserpine-like agent, Ro 4-1284 (2-hydroxy-2ethyl-3-isobutyl-9,10-dimethoxy-1,2,3,4,6,7-hexahydro-11b-[H]benzo (a)quinolizine) releases [³H]noradrenaline ([³H]NA) from prelabelled superior cervical ganglion (cell bodies) and nictitating membrane (nerve endings) of the cat.2. The potency of Ro 4-1284 29.0 μM was higher in the cell bodies than in the nerve endings.3. In both tissues, exposure to the reserpine-like agent Ro 4-1284 induced a selective increase in the spontaneous outflow of [³H]DOPEG, while the [³H]OMDA metabolites to the release induced by Ro 4-1284 was very small.4. The desamination is the preferential way of the metabolic inactivation of the [³H]NA released by the reserpine-like agent in both parts of the noradrenergic neuron.     

Effects of the noradrenaline metabolites on tyrosine hydroxylase activity in guinea-pig atria

Summary The effects of noradrenaline, its five metabolites and metanephrine, were studied on tyrosine hydroxylase activity in guinea-pig atria. The deaminated metabolite, (±)-3,4-dihydroxyphenylglycol (DOPEG), was equipotent with (±)-noradrenaline in its inhibitory action on tyrosine hydroxylase activity in the homogenates of guinea-pig atria. The inhibition by DOPEG was competitive with the cofactor, reduced pteridine. The deaminated acid, 3,4-dihydroxymandelic acid (DOMA) and the O-methylated deaminated acid, 3-methoxy, 4-hydroxymandelic acid (VMA) had 1/50th and 1/30th, respectively, the potency of noradrenaline in inhibiting tyrosine hydroxylase. The rest of the metabolites did not inhibit tyrosine hydroxylase in homogenates in concentrations up to 1.0 mM. In intact guinea-pig atria noradrenaline was considerably more potent than DOPEG in inhibiting tyrosine hydroxylase. Normetanephrine 1.4×10^–4 M inhibited tyrosine hydroxylase in the intact tissue but failed to inhibited the enzyme in the homogenate even in higher concentrations. The effect of normetanephrine in the intact tissue is related to the ability of this compound to release endogenous noradrenaline.A reserpine-like agent, Ro 4-1284, did not inhibit tyrosine hydroxylase activity in the homogenate but in the intact tissue the inhibition was more than 50%. This effect of Ro 4-1284 in the intact tissue appears to be related to the releasing effects of this agent and to an increase in the axoplasmic levels of DOPEG.Since the formation of the deaminated glycol, DOPEG, represents the main metabolic pathway for the neurotransmitter in adrenergic nerve endings, the present results are compatible with the view that, in addition to the pool of extravesicular noradrenaline, the cytoplasmic concentration of DOPEG could also participate in the regulation of the activity of tyrosine hydroxylase.     

Extracellular sodium and chloride depletion enhances nonexocytotic noradrenaline release induced by energy deficiency in rat heart

Summary The effect of either extracellular sodium or extracellular celoride reduction on the release of endogenous noradrenaline and its deaminated metabolite dihydroxyphenylglycol (DOPEG) has been studied in the isolated perfused rat heart under conditions of ischaemia and cyanide intoxication. The overflow of noradrenaline and DOPEG was determined by high pressure liquid chromatography. The efflux of DOPEG, the predominant neuronal noradrenaline metabolite, served as indicator of the free axoplasmic amine concentration. A calcium-free perfusion buffer was used to avoid exocytotic noradrenaline release. Sodium and chloride in the perfusion buffer were replaced by lithium and isethionate, respectively.(1) Reduction of extracellular sodium or chloride increased noradrenaline overflow in ischaemia. The release was suppressed by the uptake, blocker cocaine indicating carrier-mediated outward transport of noradrenaline. (2) In cyanide intoxication sodium or chloride reduction accelerated the onset of DOPEG efflux reflecting increased axoplasmic noradrenaline concentrations. This was accompanied by increased noradrenaline release. The ratio of noradrenaline/DOPEG overflow was increased by reduced sodium or chloride, indicating facilitation of carrier-mediated noradrenaline net outward transport. (3) In the presence of unaltered energy metabolism overflow of both, noradrenaline and DOPEG, was not enhanced by sodium or chloride reduction.The results demonstrate that reduction of extracellular sodium or chloride has two effects on noradrenaline release from the sympathetic neuron with reduced energy supply. First, reduced sodium or chloride induces increased axoplasmic noradrenaline concentrations by interference with vesicular storage function. Second, both interventions enhance carrier-mediated noradrenaline release.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 320) Send offprint requests to A. Schömig at the above address     

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     

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     

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)     

The mechanism of the 3H-noradrenaline releasing effect of various substrates of uptake1: role of monoamine oxidase and of vesicularly stored 3H-noradrenaline

Summary The mechanism of action of indirectly acting sympathomimetic amines was studied in vasa deferentia of unpretreated rats (COMT inhibited), preloaded with ³H-noradrenaline. 1. Concentration-release curves were obtained for 12 unlabelled indirectly acting amines. From differences between these results and those in an accompanying report (involving tissues from rats pretreated with reserpine and pargyline), it is concluded that a mobilisation of vesicular ³H-noradrenaline is required for high and sustained rates of outward transport of ³H-noradrenaline from intact adrenergic varicosities. 2. Experiments with a reserpine-like compound (Ro 4-1284) supported the view that a mobilisation of vesicular ³H-noradrenaline is required for substantial release. 3. An atypical time course of release and abnormally high rates of release were observed in the presence of excessive concentrations of (+)-amphetamine. Such atypical effects are ascribed to the ability of basic amines to increase the intravesicular pH. 4. Analysis of the ratio NA/DOPEGG (rate of efflux of ³H-noradrenaline/rate of efflux of ³H-DOPEGG) indicated that the inward transport (by uptake,) of substrates of MAO fails to achieve axoplasmic concentrations which saturate MAO. Inhibition (or saturation) of MAO is not a prerequisite for the initiation of outward transport. 5. A larger fraction of vesicular ³H-noradrenaline is accessible to equireleasing concentrations of (+)-amphetamine (an inhibitor of MAO) than of tyramine (a substrate of MAO). 6. From the present and the accompanying report it is concluded that substantial and sustained indirect sympathomimetic effects are to be expected for substrates of uptake₁ which additionally mobilise vesicular noradrenaline. However, this mobilisation does not seem to involve a change in intravesicular pH, except at excessive concentrations.Abbreviations: COMT catechol-O-methyl transferase - DOMAA dihydroxymandelic acid - DOPEGG dihydroxyphenylglycol - FRL fractional rate of loss (rate of efflux/tissue tritium content) - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase - OM-fraction sum of all O-methylated metabolites of noradrenaline, deaminated or not This study was supported by the Deutsche Forschungsgemeinschaft (Tr 96 and SFB 176). Some of the results were presented to the German Pharmacological Society (Langeloh 1986)The author was the recipient of a fellowship of the Humboldt-Foundation Send offprint requests to U. Trendelenburg     

Effects of nerve stimulation on enzyme secretion from the in vitro rat pancreas and 3H-release after preincubation with catecholamines

Summary In the presence of the cholinergic antagonist atropine, electrical field stimulation (FS) (5–20 Hz) caused a marked, reversible increase in the amylase output from superfused rat pancreatic segments. Adrenaline and noradrenaline evoked dose-dependent increases in amylase output which were similar to those produced by FS. The FS- and catecholamine-evoked amylase secretions were abolished by the -adrenergic antagonist propranolol. The FS-evoked secretion could be abolished by either the removal of external Ca²⁺ or the application of tetrodotoxin (TTX, 2×10^–6 M). FS also resulted in a reversible increase in the fractional efflux of tritium (³H) from rat pancreatic tissues preincubated with either ³H-noradrenaline or ³H-adrenaline. The effects of FS (5–20 Hz) on ³H efflux were abolished by TTX (2×10^–6 M). TTX had no effect on the enhancement of ³H efflux caused by elevation of external potassium concentration (high K⁺, 75 mM). Removal of superfusate Ca²⁺ completely abolished both the FS- and high K⁺-induced increases in ³H efflux. These observations suggest that intrinsic nerve stimulation (i.e. FS) results in the Ca²⁺-dependent release of sympathetic neurotransmitter, noradrenaline, which has a direct secretory action on the rat pancreas. Furthermore, the findings suggest that adrenaline can be taken up by nervous elements. This raises the possibility that uptake and re-release of circulating adrenaline might contribute to the control of rat pancreatic enzyme secretion by the adrenergic nervous system.     

Release of vesicular noradrenaline in the rat tail artery induced by cocaine

Summary The effects of cocaine on overflows of endogenous noradrenaline and DOPEG from isolated rat tail arteries were examined. 1. Both overflows increased progressively with increasing concentration of cocaine, while the (NA overflow)/(DOPEG overflow) ratio first increased and then decreased. The changes in the overflows induced by cocaine (0.1 mmol/l) appeared reversible. 2. Exposure of the tissue for 30 min to cocaine, 1 mmol/l, resulted in a significant decrease in the proportion of storage vesicles containing electron-dense cores. 3. The changes in overflows of noradrenaline and DOPEG induced by cocaine (0.1 mmol/l) were unaffected by the presence of desipramine (0.1 mol/l) or removal of extracellular Ca²⁺. The effect of cocaine on the overflow of noradrenaline was potentiated by prior inhibition of MAO with clorgyline. 4. Exposure of segments to a Ca²⁺-free, high K, low Na incubation medium was accompanied by increased overflow of noradrenaline. Cocaine (0.1 mmol/l) reduced the overflow of noradrenaline to about a half, and substantially increased the overflow of DOPEG. 5. The increase in the overflow of DOPEG from segments bathed in HEPES-buffered solutions, the pH of which ranged from 6.80 to 7.38, was approximately proportional to the calculated concentration of unprotonated (uncharged) cocaine. 6. Quantitatively similar changes in the overflows were observed when norcocaine was substituted for cocaine. Ecgonine methyl ester was much less potent than cocaine, and O-benzoyl ecgonine was ineffective. 7. The small increases in the overflow of noradrenaline observed at relatively low concentration (<30 mol/l) of cocaine can be attributed primarily to inhibition of reuptake of the released transmitter by the cocaine- and desipramine-sensitive amine carrier. The overflows of NA and DOPEG in the presence of higher concentrations of the alkaloid exhibit features compatible with the following hypothesis: (A) Cocaine is translocated across the axonal membrane mainly in the form of the unprotonated species, a large fraction of which is reprotonated upon the entry into the axon. (B) Cocaine releases noradrenaline from storage vesicles into the extravesicular space, where the bulk of the amine is converted to DOPEG. (C) Efflux of the remaining noradrenaline from the axon is not mediated by the Na⁺-dependent, cocaine- and desipramine-sensitive neuronal amine carrier. It seems to represent uncoupled efflux of the protonated form of noradrenaline.Abbreviations DOPEG 3,4-dihydroxyphenylethylene glycol - DOMA 3,4-dihydroxymandelic acid - HEPES N-(2-hydroxyethyl)piperazine-N-ethanesulfonic acid - MAO monoamine oxidase - MOPEG 3-methoxy-4-hydroxyphenylethylene glycol - NA (–)noradrenaline - pH_j pH in the extravesicular space of the axon - pH_o pH of the bathing solution - pK_a negative logarithm of the dissociation constant This study was supported by the British Columbia Heart Foundation Send of fprint requests to V. Palaty at the above address     

Metabolism of endogenous and exogenous noradrenaline in guinea-pig atria

Summary The outflow of noradrenaline, 3,4-dihydroxyphenylglycol (DOPEG) and 3,4-dihydroxymandelic acid (DOMA) from guinea-pig isolated atria was studied by chromatography on alumina followed by high pressure liquid chromatography with electrochemical detection. In the absence of drugs, the outflow of endogenous noradrenaline over a period of 3 h averaged 1.6 pmol×g^–1×min^–1 and the outflow of DOPEG 17 pmol×g^–1×min^–1. The outflow of DOMA was below the detection limit (<0.31 pmol×g^–1×min^–1). Tyramine greatly increased the outflow of noradrenaline and DOPEG, and the reserpine-like compound Ro 4-1284 selectively increased the outflow of DOPEG; DOMA remained below the detection limit. When atria were exposed to (–)-noradrenaline 1.7 or 17 M, the subsequent outflow of noradrenaline and DOPEG was enhanced. Moreover, substantial amounts of DOMA were now found. This outflow of DOMA was prevented when atria were exposed to (–)-noradrenaline in the presence of cocaine or after an initial incubation with amezinium. Exposure to (–)-noradrenaline 1.7 M mainly enhanced the formation of DOPGE, while exposure to (+)-noradrenaline 1.7 M mainly enhanced the formation of DOMA.Our experiments confirm some and qualify other conclusions drawn from studies in which exogenous ³H-noradrenaline had been used to examine the metabolism of noradrenaline in guinea-pig atria. In agreement with the isotope studies, DOPEG is a major metabolite of endogenous noradrenaline. In contrast to what the isotope studies had suggested, however, endogenous DOMA is a very minor product, at least as long as the neurones are at rest. DOMA is only formed when the tissue is exposed to high concentrations of exogenous noradrenaline. In further contrast to previous conclusions, DOMA is then formed intra- and not extraneuronally.     

Evidence for uptake1-mediated efflux of catecholamines from pulmonary endothelial cells of perfused lungs of rats

Previous pharmacological studies have demonstrated that pulmonary endothelial cells and noradrenergic neurones possess the same transporter for inward transport of catecholamines, uptake₁. In noradrenergic neurones, it has been shown that uptake₁ is also involved in the carrier-mediated outward transport, or efflux, of noradrenaline and dopamine. The aim of the present study was to examine the efflux of noradrenaline and dopamine from perfused lungs of rats to determine whether uptake₁, in addition to diffusion, mediates efflux of catecholamines from pulmonary vascular endothelial cells.The effects of reducing the cellular sodium gradient and of substrates and inhibitors of uptake₁ on the efflux of ³H-noradrenaline and ³H-dopamine from rat lungs were measured. Isolated; perfused lungs of rats (monoamine oxidase and catechol-0-methyltransferase inhibited) were loaded with ³H-(–)noradrenaline or ³H-dopamine for 10 min followed by perfusion with either (1) a low sodium, amine-free: Krebs solution, in which NaCl was replaced by either Tris.HCl or LiCl, for 15 or 10 min, respectively or (2) amine-free Krebs solution for 30 min in the absence or presence of a substrate or inhibitor of uptake₁ for the last 15 min. The rate constants for spontaneous efflux of noradrenaline and dopamine from the lungs were 0.0163 min^–1 and 0.0466 min^–1, respectively. When NaCl was replaced by Tris.HCl during efflux, the rate constants for efflux of noradrenaline and dopamine were increased 2.5-fold and 3-fold, respectively, whereas, when NaCl was replaced by LICl, the rate constants were increased 8-fold and 4-fold, respectively. The uptake₁ substrates, dopamine (1 and 3 mol/l) and adrenaline (40 mol/l), both caused a rapid and marked increase in the efflux of noradrenaline, while noradrenaline (4 mol/l) had a similar effect on the efflux of dopamine. The ^uptake 1 inhibitors, imipramine (3 and 10 mol/l) and nisoxetine (50 nmol/l), caused small and gradual increases in the efflux of noradrenaline and dopamine from rat lungs.These results demonstrate that efflux of noradrenaline and dopamine from rat lungs is affected by alterations in the normal sodium gradient across the cell and by drugs that interact with the uptake₁ transporter. Thus, it can be concluded that the spontaneous efflux of catecholamines from pulmonary vascular endothelial cells is mediated predominantly by uptake₁. In addition, efflux of catecholamines from the lungs has a diffusional component, which, combined with inhibition of reuptake, accounts for the small increase in amine efflux by inhibitors of uptake₁.Abbreviations COMT Catechol-O-methyltransferase - FRL Fractional rate of loss - K _m Michaelis or half-saturation constant - t _out rate constant for efflux - k _uptake rate constant for uptake - MAO monoamine oxidase - t _/12 half-time for efflux - U-0521 3,4-dihydroxy-2-methylpropiophenone - V _max maximal rate of uptake Preliminary results of this study were presented to the 1993 Meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (Scarcella et al. 1993).     

The effect of (±)-dobutamine on adrenergic nerve endings

Summary Possible effects of (±)-dobutamine on adrenergic nerve endings were determined in experiments with ghosts of bovine chromaffin granules, with rat phaeochromocytoma (PC-12) cells and with the rat vas deferens. Dobutamine inhibited the vesicular uptake of a mixture of 70% adrenaline + 30% ³H-noradrenaline into ghosts, with an IC₅₀ of 1.7 mol/l. Dobutamine inhibited uptake, of ³H-noradrenaline in PC-12 cells (with an IC₅₀ of 0.38 mol/l) without being a substrate. However, dobutamine easily entered PC-12 cells by diffusion. After inhibition of MAO, COMT and vesicular uptake dobutamine (15 and 45 mol/l) released tritium from rat vasa deferentia preloaded with ³H-noradrenaline. Equi-inhibitory concentrations of dobutamine and desipramine (against uptake₁) were equireleasing. On the other hand, when MAO and vesicular uptake were intact, dobutamine (15 mol/l) increased the efflux of tritium from preloaded vasa deferentia much more than did an equi-inhibitory concentration of desipramine. Most of the released tritium was then ³H-DOPEG.Dobutamine is a potent inhibitor of uptake₁ as well as of vesicular uptake; moreover, it easily diffuses into adrenergic nerve endings. Hence, it blocks the neuronal and the vesicular re-uptake of noradrenaline; consequently, when MAO and vesicular uptake are intact, dobutamine increases the net leakage of noradrenaline from the storage vesicles, thereby leading to an efflux of deaminated metabolites. However, dobutamine is virtually unable to release noradrenaline into the extracellular space.Abbreviations COMT catechol-O-methyl transferase - DOPEG dihydroxyphenylglycol - DOMA dihydroxymandelic acid - MAO monoamine oxidase Supported by the Deutsche Forschungsgemeinschaft (Gr 490/5 and SFB 176) Send offprint requests to P. Fischer at the above address     

Stereoselectivity of the distribution of labelled noradrenaline in rabbit aortic strips after inhibition of the noradrenaline-metabolizing enzymes

Rabbit aortic strips (nerv--free, reserpinepretreated or normal) whose noradrenaline-metabolizing enzymes were inhibited (by in vitro treatment with 0.5 mM pargyline for 30 min and by the presence of 0.1 mM U-0521) were exposed to 1.18 M labelled (-)- or (+)noradrenaline for 30 min. At the end of the incubation period some strips were used for analysis of radioactivity (i.e., of noradrenaline and its metabolites), while for others the efflux of radioactivity was determined during 250 min of wash out with amine-free solution. An estimate of the original distribution of the amine into the various extraneuronal and neuronal compartments of the tissue was obtained by compartmental analysis of the efflux curves.

Inhibition of N-methyl-d-aspartate (NMDA) and l-glutamate-induced noradrenaline and acetylcholine release in the rat brain by ethanol

Summary The influence of ethanol on stimulation-evoked ³H-transmitter release was examined in slices of the rat brain cortex and corpus striatum preincubated with ³H-noradrenaline and ³H-choline, respectively. ³H-Transmitter release was stimulated by NMDA, l-glutamate, electrical impulses, reintroduction of Ca²⁺ ions (Ca²⁺-evoked release; after superfusion with Ca²⁺-free, K⁺-rich solution) or veratridine. In cortical slices preincubated with ³H-noradrenaline and superfused with Mg²⁺-free, otherwise physiologically composed salt solution, ethanol inhibited the NMDA- or l-glutamate-induced tritium overflow (IC₅₀ 45 and 37 mmol/l, respectively). In contrast, the tritium overflow in response to electrical stimulation, reintroduction of Ca²⁺ ions or veratridine was not affected by ethanol at concentrations up to 320 mmol/l; these experiments were carried out in cortical slices superfused with solution containing a physiological Mg²⁺ concentration. Ethanol also failed to inhibit Ca²⁺-evoked release in the absence of Mg2⁺ ions. In the presence of 1 mol/l veratridine, but not in its absence, NMDA induced tritium overflow even when cortical slices were superfused with salt solution containing a physiological Mg²⁺ concentration; again, ethanol inhibited this NMDA-evoked tritium overflow (IC₅₀ 73 mmol/l). In striatal slices preincubated with ³H-choline and superfused with Mg²⁺-free physiological salt solution, the NMDA-evoked tritium overflow was also, although at lower potency, inhibited by ethanol (IC₅₀ 192 mmol/l).In spite of the differences between the IC₅₀ values of ethanol determined for the inhibition of cortical noradrenaline and striatal acetylcholine release, it may be concluded that the NMDA receptor-ion channel complex is one of the sites of action underlying the ethanol-induced inhibition of neurotransmitter release. Since in the brain cortex the NMDA-induced ³H-noradrenaline release appears to be mediated by an excitatory interneurone activated by NMDA, this neuronal system may be involved in the cortical actions of ethanol.