The in vitro iontophoretic release of radiolabelled histamine,Nτ-methylhistamine and GABA from 7-barrelled glass micropipettes

The release of [¹⁴C]histamine and [³H]N^τ-niethylhistamine into saline or tissue slices was studied under a variety of conditions using 7-barrelled glass micropipettes. For each compound, release was usually related linearly to the charge passed (positive currents) both into slices and into saline, but release was consistently greater into tissue than into saline. The ratio of the transport numbers into saline was similar to that into slices and so in vivo N^τ-methylhistamine would not be expected to be released more readily than histamine. Thus, the observation that N^τ-methylhistamine has a potency similar to that of histamin as a depressant on central neurones when applied by microiontophoresis, whereas it is virtually inactive on peripheral H₁ or H₂ receptors, cannot be attributed to a greater amount of this substance being released from the micropipettes.     

Effect of long-term administration of duloxetine on the function of serotonin and noradrenaline terminals in the rat brain

Duloxetine, an inhibitor of both 5-hydroxytryptamine (5-HT) and noradrenaline (NA) reuptake processes, has been developed as a potential antidepressant drug. The present study was initiated to investigate the functioning of multiple components of the 5-HT and NA systems following the long-term administration of duloxetine. In rats treated for 21 days with duloxetine (20 mg/kg/day), the recovery times of dorsal hippocampus CA₃ pyramidal neurons from microiontophoretic applications of 5-HT and NA were significantly increased, indicating ongoing reuptake blockade with the minipump in place delivering the drug. The remaining experiments were performed following a 48-h washout. Electrically evoked release of [³H]5-HT from preloaded slices was enhanced in the midbrain, presumably due to a desensitization of the somatodendritic 5-HT_1D and 5-HT_1A autoreceptors. In addition, evoked release of [³H]5-HT was increased in the hippocampus, which could have been due to the desensitization of the α₂-adrenergic heteroreceptors located on the 5-HT terminals. In contrast, there was no change in the evoked release of [³H]5-HT in the frontal cortex despite decreased functioning of the 5-HT transporter found in this brain region. Similar to changes in 5-HT release, electrically evoked release of [³H]NA was enhanced in the hippocampus and frontal cortex of rats treated chronically with duloxetine. These increases in [³H]NA release were most likely due to the desensitization of the α₂-adrenergic autoreceptor in the hippocampus and to the desensitization of the NA transporter in the frontal cortex, respectively. These data suggest that long-term administration of duloxetine is able to induce changes in the 5-HT and NA systems that lead to enhanced release of both 5-HT and NA in some limbic brain areas. Duloxetine, therefore, may be a useful antidepressant compound. Received: 15 December 1997 / Accepted: 12 March 1998     

Variation in noradrenaline output with changes in stimulus frequency and train length: Role of different noradrenaline pools

1 During adrenergic nerve stimulation the output/pulse of noradrenaline from the rabbit vas deferens and portal vein is not constant but increases as the stimulus frequency or train length is increased. Depending upon the stimulus frequency and train length the fractional release of noradrenaline may vary from less than 10^-7 to greater than 10^-4.

2 Endogenous tissue stores of noradrenaline were labelled by incubation with (-)-[³H]-noradrenaline and [¹⁴C]-tyrosine. The output/pulse of newly synthesized [¹⁴C]-noradrenaline remained constant as the train length was increased whilst the output/pulse of [³H]-noradrenaline increased under the same conditions. This phenomenon was independent of the stimulus frequency. Newly synthesized noradrenaline also appeared in the superfusate following nerve stimulation more rapidly than exogenously loaded noradrenaline.

3 Both [³H]-noradrenaline and [¹⁴C]-tyrosine were found to label an easily releasable store of noradrenaline. Mobilization from this store was the same at low and high frequencies of nerve stimulation.

4 It is concluded that at least two functional stores of noradrenaline exist within the adrenergic nerve ending. Newly synthesized noradrenaline is probably only a minor constituent of transmitter output under normal conditions of adrenergic nerve activity.

5 At least two mechanisms control the amount of noradrenaline released per pulse during nerve stimulation. Facilitation of release with increasing train lengths appears to be due to the mobilization of transmitter from a secondary store. Facilitation of release with increasing stimulus frequency is not dependent on mobilization from any particular store and at present there is no satisfactory explanation for this phenomenon.

     

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.     

Effect of veratridine on the fluxes of 3H-noradrenaline and 3H-bretylium in the rat vas deferens in vitro

Summary The effect of the depolarizing agent veratridine on the accumulation and release of ³H-noradrenaline and ³H-bretylium in the rat vas deferens in vitro was examined. Veratridine produced inhibition of the accumulation and induced marked release of the amines. in vas deferens from non-reserpinized rats the release of noradrenaline evoked by veratridine was partially antagonized by omission of Ca²⁺ in the incubation medium and partially inhibited by low concentrations of desipramine. In reserpinized vas deferens the release of noradrenaline like that of bretylium in normal vas was not affected by omission of Ca²⁺ but inhibited by low concentrations (3–5×10^–7 M) of desipramine. Tetrodotoxin and the local anesthetics millicaine and lidocaine antagonized the effect of veratridine on the accumulation and release of the amines, probably due to prevention of the depolarization. High concentration (3×10^–5 M) of desipramine had a similar effect on The release of noradrenaline in normal tissue in presence of external Ca²⁺. It is concluded that noradrenaline is released by veratridine from normal vas deferens by two mechanisms: 1) an exocytosis release, 2) a carrier mediated desipramine sensitive outward transport. In reserpinized tissue the second mechanism is solely responsible for the release of noradrenaline. Bretylium is only released by the second mechanism. It is suggested that the inhibition of the amine accumulation by veratridine is due to an equilibrium of the influx at a low tissue to medium ratio.     

Iontophoretic release of adrenaline, noradrenaline and 5-hydroxytryptamine from micropipettes

Estimations have been made of the amounts of adrenaline, noradrenaline and 5-hydroxytryptamine released by iontophoresis from micropipettes. In most experiments, the amount released was linearly related to the total electrical charge, but the transport numbers for different pipettes, filled with samples of the same solutions of adrenaline or of noradrenaline, varied substantially. Two pipettes containing noradrenaline failed to release any appreciable amounts of the drug. The transport number of 5-hydroxytryptamine was relatively constant (mean, 0.14).     

Pharmacological characterization of the inhibitory effects of neurotensin on the rabbit ileum myenteric plexus preparation.

1 [³H]-amezinium is taken up selectively into noradrenergic axons and their transmitter-storing vesicles and is released from these axons by action potentials. We used it as a non-α-adrenergic marker in order to study the α-adrenergic autoinhibition of noradrenaline release.

2 Rat occipitocortical slices were preincubated with [³H]-amezinium 0.03 μM and then superfused and stimulated electrically (3 Hz for 3 min). The stimulation-evoked overflow of tritium was measured in six groups of slices: from saline-pretreated rats; from saline-pretreated rats, the slices being exposed to exogenous noradrenaline before preincubation with [³H]-amezinium; from saline-treated rats, slices from which were exposed simultaneously to noradrenaline and cocaine before preincubation with [³H]-amezinium; from rats in which noradrenaline stores had been depleted by pretreatment with α-methyltyrosine (α-MT); from α-MT-treated rats, the slices being exposed to noradrenaline before preincubation with [³H]-amezinium; and from α-MT-treated rats, slices from which were exposed to noradrenaline plus cocaine before preincubation with [³H]-amezinium.

3 The stimulation-evoked overflow of tritium, expressed as a percentage of the tritium content of the tissue, was 1.15% in slices from saline-pretreated rats, and was similar in slices from saline-pretreated rats after exposure to noradrenaline or noradrenaline plus cocaine. It was 2.56% in slices from α-MT-treated rats, 1.20% from α-MT-treated rats after exposure to noradrenaline, and 2.88% from α-MT-treated rats after exposure to noradrenaline plus cocaine.

4 Yohimbine 0.1 and 1 μM increased the stimulation-evoked overflow of tritium in slices from all groups of saline-pretreated rats and in those slices from α-MT rats that had been in contact with exogenous noradrenaline. Yohimbine did not change the evoked overflow in slices from α-MT rats that had not been exposed to noradrenaline, or had been exposed to noradrenaline plus cocaine.

5 Clonidine 0.01-1 μM decreased the stimulation-evoked overflow of tritium moderately in slices from saline-pretreated rats, markedly in slices from α-MT-treated rats, and moderately again when the latter slices had been exposed to noradrenaline.

6 It is concluded that the action potential-evoked release of [³H]-amezinium as well as the modulation of this release by yohimbine and clonidine depend on the presence or absence of α-adrenergic autoinhibition caused by the co-secretion of noradrenaline. When there is co-secretion of noradrenaline, the evoked release of [³H]-amezinium is relatively small, yohimbine increases the release, and clonidine can cause only moderate inhibition. When there is no or very little co-secretion of noradrenaline, the evoked release of [³H]-amezinium is at least doubled, yohimbine causes no further increase and clonidine produces strong inhibition.

     

Noradrenaline uptake by synaptosomes and (Na+-K+) ATPase

The uptake of [³H]NA by synaptosomes prepared from rat cerebral cortex and brain stem was studied. The results indicate that there are two distinct systems for the uptake of noradrenaline. One system which predominates in cortical tissue has a sodium-dependent maximum rate of transport. This uptake system has a number of characteristics which are similar to the synaptic membrane (Na⁺-K⁺) ATPase. The activity of this enzyme was studied and the influence of a number of amines determined. Serotonin, tyrosine, l-Dopa, dopamine, noradrenaline and adrenaline were all stimulants of the (Na⁺-K⁺) ATPase. Fenfluramine, phentolamine, chlorpromazine and desipramine antagonized the amine stimulation of (Na⁺-K⁺) ATPase. Desipramine, which was a more potent inhibitor of noradrenaline uptake than was chlorpromazine, was less effective than chlorpromazine as an antagonist of the amine stimulation of (Na⁺-K⁺) ATPase. Although there was some similarity between the noradrenaline uptake system and the noradrenaline-stimulated (Na⁺-K⁺) ATPase, these results did not support the contention that synaptosomal uptake of noradrenaline was a primary active transport process.     

K+ Concentration influences methohexitone inhibition of K+-stimulated release of [3H]noradrenaline from minislices of rat cerebral cortex

Abstract— This study investigated the effect of methohexitone, a barbiturate anaesthetic, with variation of the K⁺ concentration used to stimulate [³H]noradrenaline release from superfused minislices of rat cerebral cortex. The amount of [³H]noradrenaline released by K⁺-stimulation depended on the concentration of K⁺ used (increasing release was seen at 10–50 Mm K⁺). Methohexitone (10^?7-3 × 10^?4 m ) added to the superfusing medium did not alter the basal efflux of [³H]noradrenaline, but high concentrations inhibited K⁺-stimulated release. Using 12·5 Mm K⁺, inhibition of release was observed only at 10^?4 m methohexitone (48% inhibition); the effect of 25 Mm K⁺ was reduced by 3 × 10^?5 m (72% inhibition) and higher concentrations while release stimulated by 50 Mm K⁺ was inhibited by 10^?5 m methohexitone (30% inhibition) and higher concentrations.     

Effect of pentobarbital on the noradrenaline release induced by drugs and field electrical stimulation from cerebral and femoral arteries of the cat

The present studies showed that field electrical stimulation, high potassium (K⁺), tyramine and ionophore X537A induced tritium release from cerebral and femoral arteries of cat prelabelled with [³H]noradrenaline. The secretion caused by K⁺ or field stimulation was Ca²⁺-dependent and was antagonized by high concentrations of pentobarbital (10^?4 and 10^?3M), whereas that induced by the rest of the drugs was unchanged in the same situations. The noradrenaline uptake by these arteries was reduced by pentobarbital (10^?3M and 10^?4M). These results suggest that this barbiturate interferes with Ca²⁺ entry to the adrenergic nerve endings, and therefore antagonizes the noradrenaline release by Ca²⁺-dependent processes (exocytosis).     

Presynaptic muscarinic receptor subtypes involved in the inhibition of acetylcholine and noradrenaline release in bovine cerebral arteries

Summary Experiments were performed in bovine cerebral arteries preincubated with [³H]-choline or [³H]-noradrenaline to analyze the presynaptic muscarinic receptors involved in inhibition of acetylcholine and noradrenaline release induced by electrical stimulation (4 Hz, 200 mA, 0.3 ms, 1 min). For this purpose, the actions of several muscarinic receptor antagonists on the ³H overflow and on the carbacol-induced inhibition of this overflow were assessed. The evoked [³H]-acetylcholine release and [³H]-noradrenaline release were markedly reduced by the presence of tetrodotoxin, Ca²⁺-free medium, and the inhibitor of both choline transport and choline acetyltransferase, AF64A. Chemical sympathetic denervation with 6-hydroxydopamine (6-OHDA) decreased the uptake of[³H]-noradrenaline, and AF64A reduced mainly the uptake of [³H]-choline, but also of [³H]-noradrenaline. Carbachol reduced the evoked [³H]-noradrenaline and [³H]-acetylcholine release; the IC₅₀ values were 0.37 and 0.43 mol/l, respectively.Atropine and 4-DAMP, but not AF DX 116, methoctramine or pirenzepine, increased the evoked [³H]-acetylcholine release. However, these muscarinic antagonists failed to modify the evoked [³H]-noradrenaline release. Carbachol inhibited the release of both acetylcholine and noradrenaline. The inhibition was blocked by the antagonists. The rank orders of potency (based on plC₅₀ values) were, in the case of [³H]-acetylcholine release, atropine > 4-DAMP >AF-DX 116 >- pirenzepine >- methoctramine, and, in the case of [³H]-noradrenaline release, atropine > 4-DAMP > AF-DX 116 >- methoctramine >-pirenzepine. These results suggest (1) that the prosynaptic receptors that modulate endogenous acetylcholine release are likely of the M₃ subtype, whilst those involved on the effect of the exogenous agonist Carbachol are of M₂ subtype, and (2) that those which inhibit noradrenaline release are probably a mixture of M₂ and M₃ subtypes as well. The autoinhibition of the acetylcholine release was funtionally active under our experimental conditions, while noradrenaline release does not appear to be modulated by muscarinic receptors in physiological conditions.Send offprint requests to G. Balfagón at the above address     

Retention of injected catechol amines by the mouse

The importance of tissue storage as a method of inactivation of circulating catechol amines has been assessed by measuring the amount of unchanged hormone remaining in the mouse 30 min after the injection of various doses of [³H]-adrenaline or [³H]-noradrenaline. The results show that this method of inactivation is quantitatively more important for noradrenaline than for adrenaline at all dose levels studied, and that for both hormones storage is relatively more important at physiological dose levels (3 to 30 μg/kg) than at higher dose levels (150 to 300 μg/kg). The results obtained after the simultaneous injection of various doses of [³H]-adrenaline and [¹⁴C]-noradrenaline show that under certain conditions the two hormones compete for entry into the tissue storage sites. The possible nature of the mechanisms by which circulating catechol amines enter the tissue stores is discussed in the light of previous findings on the uptake of catechol amines by tissues in vitro.     

Effect of treatment by clonidine on the synthesis of catecholamines in the heart, submaxillary and adrenal glands in the rat

The synthesis of catecholamines has been studied in the heart, sub-maxillary glands and adrenal of the rat by intravenous injection of [³H]tyrosine and evaluation, 30 min later, of the newly formed [³H]catecholamines. The synthesis rates of catecholamines have been estimated by calculation of the ratio: amount of [³H]catecholamine/ specific activity of [³H]tyrosine in the tissue. Clonidine (50 $\text{μg}\text{kg}$ i.p.) was administered 1 hr before the injection of [3H]tyrosine. Such treatment did not change significantly the endogenous levels of tyrosine or catecholamines in the organs studied. In clonidine treated rats, the synthesis rate of noradrenaline was reduced by 38 per cent in the heart and by 40 per cent in the sub-maxillary glands. In the adrenal, a 45 per cent reduction of the dopamine synthesis and a 60 per cent reduction of the adrenaline + noradrenaline synthesis were observed after clonidine treatment. The mechanisms by which clonidine reduces catecholamines synthesis are discussed.     

Prejunctional effects of cromakalim,nicorandil and pinacidil on noradrenergic transmission in rat isolated mesenteric artery

1 The present study investigated the effects of cromakalim, nicorandil and pinacidil on resting and stimulation-induced (S-I) effluxes of radioactivity from rat isolated mesenteric artery preparations in which the noradrenergic transmitter stores had been radiolabelled with [³H]-noradrenaline. The efflux of radioactivity evoked by field stimulation of peri-arterial sympathetic nerves (pulses at 2 Hz frequency in trains of 60 s duration) was taken as an index of transmitter noradrenaline release. 2 Cromakalim (1–100 μm ) and nicroandil (1–1000 μm ) produced minor effects on resting and S-I effluxes of radioactivity, but these did not exhibit concentration-dependency. 3 Pinacidil (1–1000 μm ) produced concentration-dependent increases, in both resting and S-I effluxes of radioactivity. With 1000 μm pinacidil, resting and S-I effluxes were increased to approximately 348% and 358% of their respective control values. 4 The effects of pinacidil on resting and S-I effluxes were unaltered when the neuronal amine transport system was inhibited by desipramine (1 μm ). 5 Inhibition of monoamine oxidase with pargyline (100μm ) treatment markedly reduced the enhancement of resting efflux by 1000 μm pinacidil but did not alter its effect on S-I efflux. It is proposed that the enhanced resting efflux produced by pinacidil without pargyline treatment consists of deaminated [³H]-noradrenaline metabolites formed from [³H]-noradrenaline displaced from transmitter storage vesicles by pinacidil. 6 The enhancement of S-I efflux by pinacidil does not appear to involve disruption of α₂-adrenoceptor auto-inhibition of transmitter release since equi-effective concentrations of phentolamine (1 μm ) and pinacidil (1000 μm ) produced additive effects on S-I efflux, whereas increasing the concentration of phentolamine from 1 to 2m produced no further increases in S-I efflux. 7 In conclusion this, study has provided no evidence of a prejunctional inhibitory effect of the potassium channel openers cromakalim, nicorandil and pinacidil on transmitter noradrenaline release. However, the findings with pinacidil suggest that, in high concentrations, pinacidil displaces noradrenaline from transmitter stores, such that deaminated noradrenaline metabolites are released from the nerve terminals. Furthermore, pinacidil enhances S-I transmitter noradrenaline release, possibly by blocking neuronal potassium channels.     

No evidence for reverse trans-synaptic regulation of neuronal uptake by beta-adrenoceptors in kitten atria

Kitten atria incubated with [³H]noradrenaline, 1.18 × 10^?7 M for 10 min or 3 × 10^?9 M for 30 min, actively accumulated the amine. Final tissue tritium concentrations were 2–4-fold and 8–12-fold higher, respectively, than those of the incubation fluid. Uptake was consistently greater in right than in left atria. The β₁-adrenoceptor antagonist, practolol, 10^?6 or 10^?5 M, and the β₁- and β₂-adrenoceptor antagonist, propranolol, 5 × 10^?8, 5 × 10^?7or 5 × 10^?6 M, did not affect noradrenaline uptake. Reverse trans-synaptic regulation of neuronal noradrenaline uptake by β-adrenoceptors therefore does not appear to operate in kitten atria as has been reported for rat atria and other tissues.     

Limited proteolysis of single-chain tetanus toxin by tissue enzymes,in cultured brain tissue and during retrograde axonal to the spinal cord

Summary Single-chain toxin was investigated in vitro and in vivo for limited proteolysis into the fully active two-chain toxin. Plasmin from serum, elastase and gelatinase from leucocytes, as well as clostripain from C. histolyticum cleaved single-chain toxin and increased by that way its ability to inhibit [³H]noradrenaline release in vitro. Cultured mouse brain generated fragments from ¹²⁵I-single-chain toxin which were cell-associated. Some of them comigrated in electrophoresis with light and heavy chain after mercaptolysis. When injected i. v. into rats, ¹²⁵I-single-chain-toxin disappeared from the blood with a half-life of about 11 h without signs of nicking. However, after its injection into the triceps surae muscle both single- and two-chain toxin were found in the ipsilateral ventral horn of the spinal cord. Thus single-chain toxin is subjected to limited proteolysis by enzymes involved in tissue damage, by cultured brain tissue, and during or after its retrograde axonal transport to the spinal cord. Limited proteolysis is necessary for the release of the light chain known to mediate the action of toxin on several systems.     

Propranolol inhibits nonexocytotic noradrenaline release in myocardial ischemia

Summary Ischemia induces a nonexocytotic noradrenaline release in the heart, which leads to high and potentially harmful interstitial noradrenaline concentrations. The effect of beta-adenoceptor antagonists on noradrenaline release in ischemia has been investigated in the present study. DL-Propranolol (1–100 mol/l) concentration-dependently reduced noradrenaline release during 20 min of global and total ischemia in the perfused rat heart. Other beta-adrenoceptor blocking agents such as atenolol, metoprolol, and timolol (10 mol/l each), however, did not share this effect. Moreover, both stereoisomers of propranolol were equipotent in suppression of ischemia-induced noradrenaline release, indicating a property of propranolol independent from interaction with beta-adrenoceptors. The well known local anesthetic action of propranolol was not likely to cause its inhibitory effect on ischemia-induced noradrenaline release, as lidocaine (10 mol/l) did not affect noradrenaline overflow in ischemia. The effect of propranolol was further examined in cyanide intoxication, an experimental model of energy depletion. In this experimental setting the release of dihydroxyphenylethyleneglycol - the major neuronal metabolite of noradrenaline - served as indicator of increased axoplasmic noradrenaline levels which are present during nonexocytotic noradrenaline release. In cyanide intoxication DL-propranolol also reduced noradrenaline overflow but did not affect release of dihydroxyphenylethylene glycol. The latter finding suggests an interaction of propranolol with the neuronal membrane transport of noradrenaline. In ischemia and cyanide intoxication, transport of noradrenaline across the plasma membrane is known to be driven by the noradrenaline carrier (uptake,) working in reverse of its normal direction - from inside to outside. Consequently, inhibitors of the noradrenaline carrier like desipramine were shown to suppress nonexocytotic noradrenaline release in ischemia and cyanide intoxication. In order to test the ability of propranolol to interact with the noradrenaline carrier a model of ³H-noradrenaline uptake was employed in normoxic rat heart. DL-Propranolol concentration-dependently (1–100 mmol/l) inhibited ³H-noradrenaline uptake, while atenolol and timolol did not interfere with ³H-noradrenaline uptake.In conclusion, the results indicate suppression of noradrenaline release in myocardial ischemia by propranolol. This action of propranolol is independent of its beta-adrenoceptor blocking properties and is rather due to an interaction of propranolol with the neuronal noradrenaline transport mechanism (uptake₁).The work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 320 - Cardiac Function and its Regulation)     

Subcellular distribution of [3H]amphetamine and [3H]guanethidine and their interaction with adrenergic neurons

The subcellular distribution of [³H]amphetamine and [³H]guanethidine and their interaction with each other and with noradrenaline binding sites have been examined. The ratio p/(p + s) × 100, an indication of affinity for noradrenaline storage particles, for [³H]amphetamine and [³H]guanethidine was 12% and 57% respectively. Protriptyline, a substance which inhibits amine transport mechanism at the level of the cell membrane, i.e. the membrane pump, and reserpine, an agent which impairs incorporation of amines into the storage particles in the adrenergic nerve fibre, inhibited the uptake and storage respectively, of [³H]guanethidine more than that of [³H]amphetamine. Retention of [³H]guanethidine by rat salivary glands was markedly decreased by sympathetic denervation of the glands while that of [³H]amphetamine was not. The results suggest that guanethidine possesses a much higher affinity for noradrenaline binding sites than amphetamine.     

Mathematical modelling and quantification of the autoinhibitory feedback control of noradrenaline release in brain slices

Summary Concentration-response curves, reflecting ₂-autoreceptor-mediated inhibition of [³H]-noradrenaline release by exogenous noradrenaline in rat cerebral cortex and rabbit hippocampus slices, were analysed in order to test the usefulness of a mathematical model describing the relation between the independent variable, exogenous noradrenaline, and the dependent variable, inhibition of release. This model was based on the assumption of direct proportionality between receptor occupation and response, implying that there is correspondence between the shape of a concentration-binding curve and a concentration-response curve. The experimental concentration-response curves were obtained by different approaches: noradrenaline release from brain slices prelabelled with [³H]-noradrenaline was elicited electrically either by pseudo-one-pulse (POP) stimulation or by stimulation with 36 pulses applied with a frequency of 3 Hz. POP stimulation avoids autoinhibition by released noradrenaline and, therefore, was a suitable touchstone for the applied mathematical model which evaluates by nonlinear regression analysis two primary parameters: the dissociation constant between noradrenaline and the ₂-adrenoceptor and the biophase concentration of noradrenaline which reflects the extent of autoinhibition and should be zero under POP conditions. In rat cerebral cortex tissue, the corresponding biophase concentration of endogenous noradrenaline was indeed estimated to be zero and the dissociation constant was K _d = 10^{–7.62±0.14} mol/l. With 3 Hz stimulation, the biophase concentration was 10^{–7.80±0.05} mol/l, which has to be interpreted with respect to a simultaneously estimated K _d of 10^{–7.63±0.12} mol/l. Since the K _d-values under POP or 3 Hz conditions were similar, the biophase concentration obviously had no influence on the estimate of the other primary parameter, K _d. With rabbit hippocampus, however, the main prerequisite of the mathematical model; i.e. direct proportionality between receptor occupation and response, was not established since the slope of the POP concentration-response curve (estimated as slope parameter c) did not correspond to that of a concentration-binding curve.In conclusion, mathematical modelling by nonlinear regression analysis of the autoinhibitory circuit of noradrenaline release allows the estimation of a parameter c of this feedback regulation which supports or rejects the assumption of direct proportionality between receptor occupation and functional response. When the given requirement of direct proportionality is shown to hold, this analysis allows the feedback circuit to be described quantitatively in terms of the affinity of noradrenaline for, and of the biophase concentration of noradrenaline at, the presynaptic ₂-adrenoceptors.Correspondence to T. J. Feuerstein     

Sympathomimetic Actions of Methylenedioxymethamphetamine in Rat and Rabbit Isolated Cardiovascular Tissues

Abstract— The aim of this study was to investigate the actions of methylenedioxymethamphetamine (MDMA) in several isolated cardiovascular tissues. In spontaneously beating rat atria, concentration-dependent positive chronotropic responses to MDMA and amphetamine were blocked by the neuronaluptake inhibitor desipramine (1 μm ) and the β-adrenoceptor antagonist propranolol (1 μm ). In atria incubated with [³H]noradrenaline to label transmitter stores, 10 μm MDMA and 1 μm amphetamine increased the resting outflow of radioactivity, while 1 μm desipramine had no effect on resting outflow. The MDMA- and amphetamine-induced release of radioactivity were blocked by 1 μm desipramine. MDMA, amphetamine and desipramine each enhanced the electrical stimulation-induced (2 Hz, 30-s train) release of radioactivity; the enhancing effects of MDMA and amphetamine were blocked by 1 μm desipramine. In rat isolated perfused hearts, MDMA (1 and 10 μm ) increased heart rate by a similar amount to the increase caused by noradrenaline (10 and 50 Nm ). MDMA also induced dysrhythmias in 7 out of 11 rat isolated perfused heart preparations. In rabbit isolated perfused and superfused ear arteries preloaded with [³H]noradrenaline, MDMA increased the resting release of radioactivity by 230 ± 18% (n = 6) of control resting release; the increase was accompanied by a rise in perfusion pressure of 17 ± 7 mmHg (n = 6). MDMA also facilitated the vasoconstrictor responses to noradrenaline (3–9 ng) and perivascular nerve stimulation (1–5 Hz, 10-s train). MDMA-induced vasoconstriction and the facilitation of vasoconstrictor responses to noradrenaline and electrical stimulation were blocked by 1 μm desipramine. These results suggest that MDMA has similar sympathomimetic activity to amphetamine on cardiovascular tissues. The sympathomimetic actions of MDMA could account for the cardiovascular side-effects associated with its use.