Medicine and humanism: clinical ethics and the community dimension of geriatrics

1. Rat type-C natriuretic peptide (CNP) has been studied for its effects on the neurogenically induced overflow of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), adenosine (ADO) and noradrenaline (NA) in endothelium-free segments of rat isolated tail artery. The overflow of each was evoked by electrical field stimulation (EFS) of 0.5 ms pulses at 8 Hz for 3 min and the amount of ATP, ADP, AMP and ADO was quantified by high-performance liquid chromatography (HPLC)-fluorescent detection, while the amount of NA was quantified by HPLC-electrochemical detection. 2. Type-C natriuretic peptide (100 nmol/L) was found to cause a significant reduction of the overflow of all adenine purines and NA. However, at lower concentrations (1 and 10 nmol/L), CNP caused a significant reduction of the overflow of NA but did not change ATP overflow. 3. The overflow of ADP, AMP and ADO was significantly reduced by either concentration of CNP, so that the ratio ATP:ADP was diminished from 1:2 in controls to 1:1 after 1 nmol/L CNP and to 1:1.2 after 10 nmol/L CNP. 4. The production of inorganic phosphate (Pi) in response to the exogenous application of ATP was significantly reduced by 1, 10 or 100 nmol/L CNP. 5. Type-C natriuretic peptide exerts neuromodulatory effects on the neurogenically induced release of the cotransmitters ATP and NA in rat tail artery, consisting of an inhibition of the release of both ATP and NA. This effect is accompanied by inhibition of the breakdown of ATP by ecto-ATPases. Either effect results in apparent CNP-induced differential modulation of the overflow of the cotransmitters ATP and NA.     

Local non-synaptic modulation of aldosterone production by catecholamines and ATP in rat: implications for a direct neuronal fine tuning

In addition to hypophyseal control, steroid synthesis and secretion in the adrenal cortex is also under direct local neural modulation. We obtained morphological and neurochemical evidence that a substantial proportion of the noradrenergic nerve endings lie in close proximity to zona glomerulosa cells without making synaptic contact, thus providing evidence for a direct local modulatory role of catecholamines in steroid secretion. These noradrenergic neurones, like other noradrenergic neurones in the central nervous system, are able to take up dopamine (DA), convert it partly into noradrenaline (NA) and to release both NA and DA together with the co-transmitter ATP when neuronal activity drives them to do so. These catecholamines and ATP may reach zona glomerulosa cells via diffusion in a paracrine way and modulate the synthesis of aldosterone. The presence of ecto-Ca-ATPases, enzymes that may terminate the effect of ATP, was demonstrated around the nerve profiles indicating that not only ATP but its metabolites (ADP, AMP, adenosine) can also influence the production of aldosterone. These data strongly support the possibility of a paracrine, non-synaptic modulatory role of catecholamines and ATP in the regulation of adrenocortical steroid secretion.     

Beta-adrenoceptor mediated facilitation of noradrenaline and adenosine 5'-triphosphate release from sympathetic nerves supplying the rat tail artery

1. The effects of prejunctional beta-adrenoceptor activation on electrically evoked noradrenaline (NA) and adenosine 5'-triphosphate (ATP) were studied by use of continuous amperometry and conventional intracellular recording techniques. Excitatory junction potentials (e.j.ps) were used as a measure of ATP release, and NA-induced slow depolarizations and oxidation currents as measures of NA release, from postganglionic sympathetic nerves innervating the rat tail artery in vitro. 2. Isoprenaline (0.1 microM) increased the amplitude of e.j.ps, slow depolarizations and oxidation currents evoked by short trains of stimuli at 1 to 4 Hz. The facilitatory effect of isoprenaline on e.j.ps and oxidation currents was most pronounced on responses evoked by the first stimulus in a train. 3. Isoprenaline (0.1 microM) did not detectably alter the amplitude-frequency distribution of spontaneous e.j.ps. 4. The facilitatory effect of isoprenaline on e.j.ps, slow depolarizations and oxidation currents was abolished by the beta-adrenoceptor antagonist, propranolol (0.1 microM). Propranolol alone had no effect on e.j.ps, slow depolarizations or oxidation currents. 5. Thus, activation of prejunctional beta-adrenoceptors increases the release of both NA and ATP from postganglionic sympathetic nerves. The findings are consistent with the hypothesis that NA and ATP are released from the same population of nerve terminals and presumably from the same vesicles.     

Noradrenaline and ATP: cotransmitters and neuromodulators

Adenosine 5'-triphosphate (ATP) is a cotransmitter with noradrenaline (NA) in sympathetic nerves supplying the vas deferens and a number of blood vessels. ATP is responsible for the excitatory junctional potentials (EJPs) in response to single nerve impulses and the initial twitch responses of the smooth muscle, while NA produces the longer-lasting tonic contractions. The proportions of ATP to NA vary between different sympathetic nerves; they also change during development and in some pathological conditions, including hypertension. Prejunctional neuromodulation of release of the two cotransmitters appears to involve independent mechanisms and is frequency dependent; this raises the question of whether ATP and NA are stored in separate vesicles or whether there are subpopulations of sympathetic nerves with a predominance of ATP or NA. ATP and NA have synergistic postjunctional actions, whether excitatory (as in the vas deferens and most blood vessels) or inhibitory (as in rabbit coronary vessels). It is suggested that use of the term 'adrenergic nerves' as a synonym for sympathetic nerves is no longer appropriate, although 'adrenergic transmission' or 'purinergic transmission' are still useful terms.     

Some biochemical properties of the new potential antidepressant agent N-methyl-N-propargyl-2-(1-methyl-5-methoxyindolyl)methylamine

The biochemical properties of the new methyl indole derivative IM-24 (N-methyl-N-propargyl-2(1-methyl-5-methoxyindolyl)methylamine HCl) have been investigated. The activity on both forms of monoamine oxidase MAO was tested in several nervous and non nervous tissues ex vivo after chronic administration. IM-24 is mainly an inhibitor of the activity of MAO A without any effect on intestinal MAO B at the doses studied. IM-24 was compared with tricyclic antidepressants in tests for serotonin (5HT), noradrenaline (NA) and dopamine (DA) uptake inhibition in vitro. IM-24 is mainly an inhibitor of the 5HT uptake mechanism but is less active than paroxetine and chlorimipramine which are very potent 5HT-uptake inhibitors. Radioligand binding techniques in rat brain ex vivo showed that IM-24 after chronic administration (21 days) produces no change in the number or the affinity of the alpha 2-adrenoceptors. IM-24 reduces by 70% the number of 5HT2 receptors but does not modify the affinity for the ligand. IM-24 is thus an interesting compound which combines monoamine oxidase inhibition with inhibition of 5HT uptake. Both these actions will lead to an increase of the availability of serotonin at the synaptic site.     

Effect of 8-sulfophenyl theophylline on endogenous noradrenaline release from sympathetic nerves of the rabbit ear artery

1. The release of endogenous noradrenaline (NA) and adenyl purine (ATP, ADP, AMP and adenosine) from the rabbit ear artery, evoked by electrical stimulation (ES; 16 Hz), was examined. 2. ES evoked a significant release of NA and purine; the ratio of the amount of total purine released to NA released was approximately 180 on a molar base. 3. ES-evoked purine release was significantly reduced by the denudation of the endothelium and abolished by the alpha 1-adrenoceptor antagonist, prazosin (1 mumol/L). 4. ES-evoked NA release was significantly reduced by a P1-purinoceptor antagonist, 8-sulfophenyl theophylline (8SPT). Purine release was slightly reduced by 8SPT. 5. These results suggest that endogenous NA released by ES results in the release of a large amount of purine, which may, in turn, increase the release of NA by acting on prejunctional purinoceptors on sympathetic nerve terminals.     

Changes in levels of monoamines and their metabolites in incompletely ischemic brains of spontaneously hypertensive rats

In order to investigate changes in levels of monoamines and their related substances together with those of other neurotransmitters (acetylcholine and GABA), choline and substances related to energy metabolism (ATP, lactate and glucose) accompanying incomplete cerebral ischemia, a bilateral common carotid artery occlusion model of spontaneously hypertensive rats (SHR) was utilized. Animals were subjected to 1 or 2 h ischemia. Then the concentrations of substances were measured in the cerebral cortex, hippocampus and striatum and compared with control values. Due to the incomplete ischemia, ATP showed a moderate decrease, while lactate and choline increased remarkably, and GABA underwent a moderate increase. With regard to monoamines, both noradrenaline and serotonin levels were reduced in the cerebral cortex and hippocampus, whereas dopamine levels increased in the hippocampus. All monoamine metabolites, i.e. metabolites by monoamine oxidase (MAO), metabolites by catechol-O-methyltransferase (COMT), and metabolites by both MAO and COMT, underwent increases. The 3-methoxytyramine level in particular showed marked increases. Furthermore levels of precursor amino acids as well as 5-hydroxytryptophan rose. Acetylcholine decreased moderately only in the cerebral cortex. Among these changes, sustained increases in all the monoamine metabolites were characteristic of changes in the incompletely ischemic brain, suggesting that both COMT and MAO retain their activities in the incompletely ischemic brain.     

Endothelin in perivascular nerves. An electron-immunocytochemical study of rat basilar artery

The endothelium is recognized as a major source of endothelin in the vasculature. In this report we show for the first time that endothelin can be demonstrated with immunoelectronmicroscopy in perivascular nerves. About 36% of the axon profiles (varicosity/intervaricosity) examined in the rat basilar artery showed immunolabelling with a polyclonal antibody to endothelin-1. Endothelin-labelled axon varicosities were characterized by the presence of small spherical agranular vesicles (42 +/- 1 nm); some varicosities also contained large granular vesicles (92 +/- 5 nm) with labelled cores. Immunolabelling was mostly distributed in the axoplasm and in association with the membrane of synaptic vesicles (the lumen of the small agranular vesicles was immuno-negative). The presence of endothelin in perivascular nerves of the basilar artery suggests a neuronal as well as endothelial role in the physiological control of the vessel wall.     

Graft-derived cholinergic reinnervation of the hippocampus prevents a lasting increase of hippocampal noradrenaline concentration induced by septohippocampal damage in rats

Long-Evans female rats sustained aspirative lesions of the septohippocampal pathways and, 2 weeks later, received into the dorsal hippocampus grafts prepared from the septal area (rich in cholinergic neurons; Group Sep) or from the mesencephalic raphe (poor in cholinergic neurons; Group Rap) of rat fetuses. Lesion-only (Group Les) and virtually intact (Group Sham) rats served as controls. Between 9.5 and 10.5 months after grafting surgery, we found the lesions to decrease choline acetyltransferase activity (ChAT), high affinity synaptosomal uptake of [3H]choline (HACU) and serotonin concentration ([5-HT]), as well as to increase the noradrenaline concentration ([NA]) in the dorsal hippocampus. Raphe grafts increased [5-HT] to 456% of normal, but had only weak or no effects on the other lesion-induced modifications in brain neurochemistry. Septal grafts dramatically increased ChAT activity and HACU, enhanced [5-HT], and reduced [NA] to near-normal levels. We also found a significant negative correlation between HACU and [NA] in rats with lesions, whether grafted or not. These data show that grafts providing the denervated hippocampus with a new cholinergic innervation might be able to exert inhibitory effects on the lesion-induced increase of [NA]. Since such an increase is indicative of sympathetic sprouting, the finding of reduced [NA] in rats with graft-derived cholinergic reinnervation of the hippocampus is in line with the hypothesis that hippocampal cholinergic denervation plays a crucial role in the induction of sympathetic sprouting. However, our data do not allow to distinguish whether grafts rich in cholinergic neurons inhibited the sympathetic sprouting itself, or rather reduced the NA content of sprouted fibers.     

The serotonin receptor agonist 5-methoxy-N,N-dimethyltryptamine facilitates noradrenaline release from rat spinal cord slices and inhibits monoamine oxidase activity

1. The influences of the purported serotonergic agonist 5-methoxy-N,N-dimethyltryptamine (MeODMT) on noradrenaline release and metabolism were investigated in a rat spinal cord release model and a monoamine oxidase (MAO) assay. 2. MeODMT inhibited the basal outflow of tritium from rat spinal cord slices preincubated with [3H]noradrenaline and enhanced the electrically-evoked overflow. 3. Effects on basal outflow were not observed, when monoamine oxidase (MAO) was inhibited by pargyline. Effects on the evoked overflow were not observed in the presence of metitepine or phentolamine. 4. Preferential inhibition by MeODMT of MAO A-type enzyme activity was found in a direct assay. 5. The results provide evidence for two different effects by which MeODMT reinforces noradrenergic neurotransmission in the rat spinal cord: facilitation of stimulation-evoked noradrenaline release and inhibition of noradrenaline metabolism by MAO inhibition.     

Geometry, kinetics and plasticity of release and clearance of ATP and noradrenaline as sympathetic cotransmitters: roles for the neurogenic contraction

The paper compares the microphysiology of sympathetic neuromuscular transmission in three model preparations: the guinea-pig and mouse vas deferens and rat tail artery. The first section describes the quantal release of ATP and noradrenaline from individual sites. The data are proposed to support a string model in which: (i) most sites (> or = 99%) ignore the nerve impulse and a few (< or = 1%) release a single quantum of ATP and noradrenaline; (ii) the probability of monoquantal release is extremely non-uniform; (iii) high probability varicosities form 'active' strings; and (iv) an impulse train causes repeated quantal release from these sites. Analogy with molecular mechanisms regulating transmitter exocytosis in other systems is proposed to imply that coincidence of at least two factors at the active zone, Ca2+ and specific cytosolic protein(s), may be required to remove a 'fusion clamp', form a 'fusion complex' and trigger exocytosis of a sympathetic transmitter quantum, and that the availability of these proteins may regulate the release probability. The second section shows that clearance of noradrenaline in rat tail artery is basically > or = 30-fold slower than of co-released ATP, and that saturation of local reuptake and binding to local buffering sites maintain the noradrenaline concentration at the receptors, in spite of a profound decline in per pulse release during high frequency trains. The third section describes differences in the strategies by which mouse vas deferens and rat tail artery use ATP and noradrenaline to trigger and maintain the neurogenic contraction.     

Physiological and induced neuronal death are not affected in NSE-bax transgenic mice

The intracortical organization of the noradrenaline (NA) and vasoactive intestinal polypeptide (VIP) systems provides ample opportunity for functional convergence, and accumulated evidence indicates that NA and VIP share certain cellular actions upon both neuronal and nonneuronal cortical elements. In the present study, a double immunolabeling method was combined with a silver-gold intensification procedure to examine the ultrastructural relationships of the NA coeruleocortical afferents and the intrinsic VIP neurons with three main constituents of the cortex: neurons, astrocytes, and blood vessels. Electron microscopy of singly or doubly labeled material indicated that NA and VIP boutons are engaged in a variety of anatomical relationships with both neuronal and nonneuronal elements. Dendritic shafts and perikarya of nonpyramidal neurons, some of which are VIP positive, receive combined NA and VIP synapses. A significant number of cortical microvessels are in intimate contact with NA or VIP profiles. NA axons often form perivascular loops, and VIP dendritic shafts of large diameter are frequently observed to bend around the vessel circumference. Serial section examination demonstrates that some NA boutons are directly apposed to the capillary wall at sites of glial end-feet discontinuities, whereas VIP boutons contact astrocytic sleeves of capillaries but never cross the perivascular astroglial barrier. Some VIP dendrites containing coated vesicles make intimate contact with the capillary basal lamina. Astrocytic perikarya, mainly in the supragranular layers, are also directly apposed to NA and/or VIP elements. These complex anatomical relationships provide a structural basis for the known interactions between NA and VIP in the control of cortical metabolism and function.     

An analysis of the postjunctional component of denervation supersensitivity in the isolated vas deferens of the guinea-pig

An attempt was made to devise evidences for a role of calcium ions in the postjunctional component of denervation supersensitivity. The evidences obtained are: chronic postganglionic denervation increases sensitivity and maximum response of the vas deferens to cumulative concentrations of calcium and alters the pattern of response to low-calcium, potassium-rich Krebs solution; denervation supersensitivity could not be demonstrated after depolarization, and in KC1-Ringer or in Ca2+/--free Krebs solution the rate of loss of responsiveness to acetylcholine was delayed after denervation whereas the rate of loss of responsiveness to noradrenaline was unaffected. It is suggested that the postjunctional component of denervation supersensitivity in the isolated vas deferens of the guinea-pig is due, at least partially, to an increased availability of a membrane-bound calcium store(s) associated to an enhanced cell membrane permeability to the ion.     

Association of a cellular myosin II with anionic phospholipids and the neuronal plasma membrane

Myosin II has been observed in close proximity to the neuronal plasma membrane, suggesting the possibility that at least one isoform of neuronal myosin II may be capable of direct association. Here, we demonstrate that a significant fraction (> 30%, saturable around 90%) of brain myosin II, but not myosins from skeletal or cardiac muscle, can bind to lipid vesicles composed of the anionic phospholipid L-alpha-phosphatidyl-L-serine but not with vesicles made from the neutral phospholipid L-alpha-phosphatidylcholine. Binding to lipid vesicles made from L-alpha-phosphatidyl-L-serine is enhanced in the presence of millimolar amounts of free calcium. ATPase activity remains unimpaired after vesicle association. Myosin II was also shown to remain in tight association with purified plasma membranes, even after depletion of actin. The above observations suggest that mechanisms involving membrane-bound myosin II are required to facilitate metazoan cell motility.     

Vas deferens desensitization by noradrenaline and other drugs

Desensitization of the isolated rat vas deferens was demonstrated by using noradrenaline, 5-hydroxytryptamine, carbachol, acetylcholine or barium chloride as a test concentration after a large concentration (concentration producing a contraction that was 80% of the maximum) of the same agonist. The large concentration of noradrenaline caused desensitization in response to 5-hydroxytryptamine and acetylcholine but not to carbachol and barium chloride. Desensitization is not altered by reserpine--in pretreatment of rats with reserpine or reserpine in Tyrode solution--imipramine and 6-hydroxydopamine. Surgical denervation or modification of the sodium and calcium concentration of the Tyrode solution did not either alter desensitization. Results show that in the rat isolated vas deferens desensitization is not mediated by an action of noradrenaline on the adrenergic neuron.     

Rapid and sensitive determination of catecholamines and the metabolite 3-methoxy-4-hydroxyphen-ethyleneglycol using HPLC following novel extraction procedures

In the present study assays were improved for the determination of free catecholamines and 3-methoxy-4-hydroxyphenethyleneglycol (MHPG), the major metabolite of peripheral and central noradrenaline. The compounds were extracted by a fluid phase extraction: a diphenyl boric acid method for the purification of catecholamines and an ethyl acetate extraction for MHPG were used, respectively. High-performance liquid chromatography with electrochemical detection was employed for quantitative analysis. In previous studies, significant differences between plasma concentrations of these substances in normal volunteers and hospital patients were demonstrated. Therefore, we established valid reference values for a hospital population. Blood and urine samples of 59 patients without disorders and medication affecting catecholamine synthesis and metabolism or the activity of the sympatho-adrenal system were collected and analyzed for free and total (free plus conjugated) MHPG, noradrenaline (NA), adrenaline (A) and dopamine (DA); total MHPG was assayed after enzymatic hydrolysis of conjugates. Our data clearly demonstrate that these methods are sensitive, specific, rapid, and can easily be standardized. The intra- and inter-assay precision were high (CV 2.6-5.3% and 4.3-6.9% for plasma, CV 3.8-4.9% and 5.1-8.2% for urine, respectively). For plasma, the mean concentrations +/- SD were determined to be 20.82+/-4.70 pmol/ml for free MHPG, 68.43+/-16.21 pmol/ml for total MHPG, 2.11+/-0.24 pmol/ml for NA and 0.31+/-0.08 pmol/ml for A. For 24h-urine the mean concentrations +/-SD were determined to be 0.44+/-0.13 mmol/24h for free MHPG, 8.79+/-2.13 mmol/24h for total MHPG, 169.8+/-58.25 nmol/24h for NA, 62.19+/-21.79 nmol/24h for A and 757.2+/-382.6 nmol/24h for DA. In summary, these novel and rapid methods can clearly be employed in a routine clinical setting.     

Ca2+-dependence of vasoconstriction mediated by alpha1A-adrenoceptors in perfused rat hindlimb: a pharmacological approach

We investigated the source of Ca2+ for the vasoconstriction mediated by alpha1a-adrenoceptors in perfused rat hindlimb in functional studies. The noradrenaline (NA)-induced maximum response was decreased by 92% following perfusion with Ca2+-free medium. Depletion of intracellular Ca2+-stores with repeatedly application of caffeine and NA in Ca2+-free medium resulted in complete abolishment of NA-response. Nifedipine concentration-dependently inhibited NA-contraction with a maximum inhibition of 65%. The residual nifedipine-insensitive response was further inhibited by Cd2+. Following depletion of Ca2+ stores with cyclopiazonic acid in Ca2+ free medium for 30 min, the NA-response obtained by re-admission of Ca2+ was decreased by 80%. However, re-introduction of Ca2+ to NA-treated tissues in Ca2+-free medium without prior treatment with cyclopiazonic acid normalizes the NA-response. These results suggest that the NA-contraction in this preparation is mediated largely via an influx of extracellular Ca2+, of which the majority utilizes L-type calcium channels. Only a small portion of the contractile response to NA is derived from intracellular stores, which probably also play a modulatory role on Ca2+ influx.     

Prostate carcinoma: transrectal US after cryosurgical ablation

The effects of denervation of central noradrenergic system on the interpartner relationships of adult cats were examined in a predatory test in the competitive situation for paired animals. Direct administration of the noradrenaline neurotoxin, N-2-chloroethyl-N-ethyl-2-bromobenzylamine (DSP-4 12 microg) into the medial forebrain bundle (MFB) of submissive cats changed previously established dominant-submissive relationship. Biochemical analysis demonstrated a significant reduction of noradrenaline (NA) concentration in the hypothalamus (AH), amygdala (AM), hippocampus (HC), and frontal cortex (CTX), and elevation of NA content in the midbrain central gray matter (CG) in MFB-lesioned cats. Simultaneously, DSP-4-induced lesions exerted significant decrease of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) content in AH, CG, HC and CTX, and increased GABA level in AH, CG, AM, and HC. These results suggest that a coincident decrease of NA metabolism and increase of GABA metabolism led to fear drive reduction.     

Changes in plasma dopamine-beta-hydroxylase activity during the perioperative period of cardiac surgery: an index of sympathetic nerve activity

To evaluate the usefulness of plasma dopamine-beta-hydroxylase (DBH) activity as an index of sympathetic nerve activity during cardiac operations, we examined the serial changes in plasma DBH activity, in relation to the plasma noradrenaline (NA) level and hemodynamic parameters, in patients who underwent cardiac surgery. The plasma DBH activity decreased significantly after cardiopulmonary bypass, and remained low during dopamine (DA) infusion until 72 h after the operation. However, recovery of the hemodynamic parameters, being the mean arterial pressure, heart rate and cardiac index, was seen as early as 1-3 h postoperatively. It was therefore assumed that the plasma DBH activity takes a long time to recover after an operation. The time-course changes in the plasma NA level were quite different from the changes in DBH activity, with an apparent negative correlation being observed between them. Thus, there is a possibility that exogenously administered DA, as well as increased plasma NA, might inhibit DBH activity during cardiac surgery. Moreover, since catecholamines are often administered upon completion of cardiac surgery, measurement of the plasma catecholamine level would be inappropriate for evaluating real sympathetic nerve activity. From the results of this study, it is surmised that measurement of the plasma DBH activity could be useful for estimating the intrinsic sympathetic nerve activity of patients who have undergone cardiac surgery.     

The effect of inhibitors of extraneuronal uptake on the distribution of 3H-(+/-)noradrenaline in nerve-free rabbit aortic strips

1. Nerve-free rabbit aortic strips were exposed to 1.18muM 3H-(+/-)noradrenaline for 30 min. When either MAO or COMT was inhibited, far more O-methylated (MAO inhibited) than deaminated metabolites (COMT inhibited) were formed during the incubation. The accumulation of unchanged amine in the extraneuronal stores was inversely related to the rate of metabolism. 2. After inhibition of both metabolizing enzymes, nerve-free strips were first incubated with the amine and then washed out with amine free solution. Compartmental analysis of the efflux curves showed that two extraneuronal compartments were involved in accumulation (with half times of efflux of 3 and 11 min, respectively). 3. 86muM corticosterone or 30muM phenoxybenzamine greatly decreased the accumulation of noradrenaline in these two compartments. 4. When corticosterone or phenoxybenzamine was added to the wash out solution only, the half time of the efflux from both compartments was greatly increased. However, this effect was seen only after inhibition of COMT and not after inhibition of MAO only (the filling of the extraneuronal stores with unchanged noradrenaline being better after inhibition of COMT than when this enzyme was intact). The effect of corticosterone appeared to be reversible, that of phenoxybenzamine irreversible. 5. Analysis of the efflux of metabolites (in experiments in which only one enzyme was inhibited) indicated that corticosterone affected the efflux of noradrenaline but not that of the metabolites. 6. When either COMT or MAO was inhibited throughout the experiment, very little or no metabolism of noradrenaline occured during prolonged wash out. On the other hand, dis-inhibition of COMT during wash out (by the omission of U-0521 from the wash out solution after it had been present during the initial incubation) revealed that noradrenaline, stored extraneuronally during the initial incubation, is quickly O-methylated during wash out, especially when the efflux of the parent amine is inhibited by corticosterone. 7. The results show that COMT is the major extraneuronal noradrenaline-metabolizing enzyme of rabbit aorta, that inhibition of COMT is a pre-requisite for any corticosterone-sensitive accumulation of noradrenaline, that there are two important extraneuronal compartments (compartments III and IV; Henseling et al., 1976a), and that inhibitors of extraneuronal uptake inhibit both, influx and efflux of noradrenaline.