The role of MAO-A and MAO-B in the metabolic degradation of noradrenaline in human arteries

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Selective inhibition by amezinium of intraneuronal monoamine oxidase

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

Effect of ovarian steroids on the metabolism of noradrenaline in rabbit uterus

The metabolism of (-)-3H-noradrenaline was examined in uterine slices from ovariectomized rabbits which were either untreated or treated with 17 beta-oestradiol, alone or in combination with progesterone. 17 beta-oestradiol caused uterine enlargement which was not accompanied by a change in the formation per g of O-methylated metabolites (3H-NMN, 3H-VMA, 3H-MOPEG). Accumulation of unchanged 3H-noradrenaline and the formation of deaminated catechols (3H-DOMA and 3H-DOPEG) were decreased per g tissue, but increased per uterine horn. Progesterone produced further enlargement of the oestrogen-dominated uteri which was accompanied by (a) a decrease in deaminated catechol formation and (b) an increase in 3H-NMN formation per unit mass of tissue. In all uteri (control and hormone-treated), cocaine inhibited the formation of deaminated catechols, but not that of the O-methylated metabolites. It is suggested, therefore, that, per unit of uterine mass, the neuronal deamination of (-)-3H-noradrenaline is decreased by 17 beta-oestradiol and further decreased by progesterone, and that these changes reflect failure of the intraneuronal deaminating system in the whole uterus to increase in proportion to the increase in uterine mass. Since other agents which decreased the deamination of (-)-3H-noradrenaline (cocaine and nialamide) did not affect 3H-NMN formation in oestrogen-dominated uteri, it is suggested that stimulation of 3H-NMN formation represents a direct effect of progesterone on the extraneuronal O-methylation of noradrenaline.     

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

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

Cocaine-sensitive O-methylation of noradrenaline in dental pulp of the rabbit: Comparison with the rabbit ear artery

Summary Incisor pulp from the rabbit metabolises exogenous noradrenaline in concentrations between 0.12 and 1.2 mol/l mainly to NMN.Effects of chronic sympathetic denervation indicated that in incisor pulp the NMN is extraneuronal in origin, and that DOPEG and DOMA formation, as well as a major part of the noradrenaline which accumulates in the tissue, are associated with the sympathetic nerves.NMN formation was unaffected by hydrocortisone 210 mol/l, but was strongly inhibited by cocaine 30 mol/l. These effects contrasted with those in the rabbit ear artery, where NMN formation was increased by cocaine 30 mol/l and decreased by hydrocortisone 210 mol/l.In COMT-inhibited denervated pulp, cocaine inhibited the accumulation of noradrenaline.Monoamine fluorescence histochemistry of pulp exposed to noradrenaline 50 mol/l indicated that cocaine-sensitive uptake occurred in fibroblasts.It is concluded that O-methylation of noradrenaline in dental pulp involves prior uptake of the amine by a process resembling uptake, but which is distinguished from uptake₁ by its extraneuronal location.Abbreviations DOMA 3,4-dihydroxy mandelic acid - DOPEG 3,4-dihydroxyphenylethyleneglycol - NMN normetanephrine - OMDA O-methyl deaminated metabolite fraction, comprising vanillyl-mandelic acid (VMA) plus the 3-methoxy derivative of DOPEG (MOPEG) - MAO monoamine oxidase - COMT catecholO-methyl transferase Send offprint requests to I. S. de la Lande at the above address     

Effect of progesterone on the metabolism of noradrenaline in rabbit uterine endometrium and myometrium

The metabolism of (-)-3H-noradrenaline was examined in the endometrium and the myometrium from rabbits which had received 17 beta-oestradiol, either alone (oestrogen-dominated) or with progesterone (progesterone-dominated). The progesterone treatment resulted in a 2.5-fold increase in 3H-NMN formation in the endometrium, with no change in 3H-DOPEG, 3H-DOMA or 3H-OMDA formation. In the myometrium, progesterone caused a 5-fold increase in 3H-NMN formation and a 2.5-fold increase in 3H-OMDA formation, but did not affect 3H-DOPEG or 3H-DOMA formation. In the progesterone-dominated endometrium, both 3H-NMN and 3H-OMDA formation were strongly inhibited by cocaine 30 mumol/l. When O-methylation was inhibited by a COMT inhibitor, cocaine prevented the resultant increases in deamination of noradrenaline to 3H-DOPEG and in the accumulation of 3H-noradrenaline by the tissue. The 3H-noradrenaline which accumulated in endometria, in which both MAO and COMT were inhibited, was firmly bound; desipramine 3 mumol/l and (+)-amphetamine 10 mumol/l were equieffective with cocaine 30 mumol/l in inhibiting the accumulation. Cocaine 30 mumol/l was without effect on 3H-NMN and 3H-OMDA formation in the progesterone-dominated myometrium, nor did it prevent the increase in 3H-DOPEG formation produced by COMT inhibition. Fluorescent histochemical analysis of the endometrium indicated that the epithelial cells of the endometrial glands were the site of cocaine-sensitive noradrenaline accumulation. It is concluded that progesterone stimulates O-methylation in the endometrium and myometrium in different ways.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of inhibition of catechol-O-methyl transferase or of monoamine oxidase on the extraneuronal metabolism of 3H-(−)-noradrenaline in the rat heart

Summary The extraneuronal metabolism of ³H-(–)-noradrenaline (1 nmol/l) was determined in rat hearts obtained from reserpine-pretreated animals (in the presence of 30 mol/l cocaine).Inhibition of monoamine oxidase (MAO) (by pretreatment of the animals with pargyline) increased the formation of O-methylated metabolites by nearly that amount by which the formation of deaminated metabolites declined; hence, catechol-O-methyl transferase (COMT) seemed to be able to nearly fully compensate for the loss of MAO activity. However, when COMT was inhibited (by the presence of either 1 or 10 mol/l U-O521), the increase in the formation of deaminated metabolites was smaller than the decrease in the formation of O-methylated metabolites; hence, MAO seemed to be unable to fully compensate for the loss of COMT activity.These results are discussed with regard to the hypothesis that the two extraneuronal enzymes co-exist in one compartment. As inhibition of COMT causes a much greater increase in the steady-state tissue/medium ratio for ³H-(–)-noradrenaline than does inhibition of MAO, it is suggested that it is this increase in the intracellular concentration of ³H-(–)-noradrenaline which-by promoting an efflux of the unchanged amine that is proportional to the tissue/medium ratio-actually decreases the net removal of ³H-(–)-noradrenaline from the perfusion fluid.The results are compatible with (but no evidence for) the hypothesis that the two enzymes co-exist in the same extraneuronal compartment.The following abbreviations are used here NMN normetanephrine - DOPEG dihydroxyphenylglycol - DOMA dihydroxymandelic acid - MOPEG methoxyhydroxyphenylglycol - VMA methoxyhydroxymandelic acid - OMDA MOPEG+VMA Supported by the Deutsche Forschungsgemeinschaft     

The neuronal and extraneuronal uptake and metabolism of 3H-(−)-noradrenaline in the perfused rat heart

1. Hearts were obtained from reserpine-pretreated rats and perfused with 0.95 micron 3H(-)-noradrenaline. The rate of removal of 3H-noradrenaline from the perfusion fluid was measured (from the arterio-venous difference) as well as the rate at which the 3H-metabolites appeared in the venous effluent. 2. When either 30micron corticosterone was added under steady-state conditions during perfusion with 3H-noradrenaline (to inhibit neuronal and extraneuronal uptake, respectively), each inhibitor reduced the removal of noradrenaline by about 50%; in the presence of both inhibitors removal was abolished. 3. Dihydroxymandelic acid (DOMA) was of neuronal, normetanephrine (NMN) of extraneuronal origin; dihydroxyphenylglycol (DOPEG) and the OMDA fraction (containing methoxyhydroxyphenylglycol-MOPEG-and methoxyhydroxymandelic acid-VMA) were formed both neuronally and extra-neuronally. 4. The extraneuronal metabolism of 3H-noradrenaline was in quick equilibrium with the 3H-noradrenaline in the perfusion fluid; most of the total formation of DOPEG, MOPEG and NMN was recovered from the venous effluent. 5. Extraneuronally formed DOPEG, MOPEG and NMN distributed in the tissue with half times corresponding to their half time for efflux. 6. Inhibition of monoamine oxidase (MAO) by pargyline increased the extraneuronal formation of NMN; MAO and catechol-O-methyl transferase (COMT) appear to be contained in the same extraneuronal compartment. 7. The extraneuronal accumulation of 3H-noradrenaline required 30 min or more to reach a steady state; inhibition of one or both enzymes slowed this process. Inhibition of MAO increased the extra-neuronal accumulation of 3H-noradrenaline; inhibition of COMT failed to do so, since the enzyme inhibitor (U-0521) was a weak inhibitor of extra-neuronal uptake. 8. The rate constants for the efflux of the metabolites of noradrenaline decreased in the order of MOPEG greater than DOPEG greater than NMN greater than DOMA greater than VMA.     

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

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

Release and disposition of 3H-noradrenaline in the saphenous vein of neonate and adult dogs

Summary Release of ³H-noradrenaline and formation of ³H-metabolites were studied in the saphenous vein of newborn (mean age, 18 h) and adult dogs. Vein strips were incubated with 0.23 mol/l of ³H-noradrenaline during 1 h and washed out for 110 min; thereafter, the perifusion fluid was collected in 5-min samples. Electrical stimulation was applied at 120 min (1 Hz, 2 ms, 100 V, for 5 min). In some experiments the tissues were preincubated with 1 mmol/l pargyline (to inhibit monoamine oxidase). In these experiments, 12 mol/l cocaine (to inhibit uptake₁), 41 mol/l hydrocortisone (to reduce uptake₂) and 50 mol/l U-0521 (to inhibit COMT) were present during the perifusion. ³H-noradrenaline, ³H-DOPEG, ³H-NMN, ³H-DOMA and ³H-OMDA were separated by column chromatography. The noradrenaline content of the tissue was estimated by HPLC followed by electrochemical detection. A morphological study was also carried out by light and electron microscopy.The endogenous noradrenaline content of the saphenous vein was 4.3 times higher in adults than in neonates. The number of varicosities was similar in adults and newborns but the number of vesicles per varicosity profile was 5 times higher in adults. Hence, the endogenous noradrenaline content per vesicle was about the same in adults and newborns. The accumulation of ³H-noradrenaline per vesicle was about 5 times higher in newborns than in adults. On the other hand, the vein wall media of neonates was about 3 times thinner than that of adults. The evoked fractional release of tritium was about 10 times higher in neonates than in adults, whether the inactivation pathways were blocked or not. This difference in the evoked fractional release therefore cannot be ascribed to any difference in the efficacy of the inactivation pathways between neonates and adults. On the other hand, the difference also cannot be ascribed to a different alpha₂-adrenoceptor-mediated inhibition of ³H-noradrenaline release since, as previously shown, this mechanism is as effective in newborns as in adults.It is concluded that the evoked fractional release of ³H-noradrenaline is higher in neonates than in adults because in neonates the majority of varicosities (and vesicles) is situated closer to the surface of the tissue such that both uptake from the surrounding media into the stores and release from the stores into the surrounding media become easier in the neonates.Abbreviations COMT catechol-O-methyl transferase - DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - HPLC high pressure liquid chromatography - MAO monoamine oxidase - NMN normetanephrine - OMDA 3-methoxy-4-hydroxyphenylglycol (MOPEG) plus 3-methoxy-4-hydroxymandelic acid (VMA) - U-0521 3,4-dihydroxy-2-methyl propiophenone Correspondence to D. Moura at the above address     

Deamination of released 3H-noradrenaline in the canine saphenous vein

Summary Experiments were designed to determine the effect of monoamine oxidase (MAO) inhibitors on the release and the metabolism of noradrenaline in the canine saphenous vein. Helical strips were incubated with ³H-noradrenaline and mounted for superfusion and measurement of the efflux of labelled transmitter and its metabolites; in certain experiments the tissue content of ³H-noradrenaline and its metabolites was also determined. The MAO-A inhibitor clorgyline, and the non-specific inhibitor pargyline, but not the MAO-B inhibitor deprenyl decreased the appearance of deaminated and O-methylated deaminated metabolites under basal conditions and during electrical stimulation. The MAO-A and the non-specific MAO inhibitor did not decrease the efflux of VMA to the same extent as that of the other deaminated metabolites. During superfusion with etidocaine, an agent causing increased leakage of stored transmitter, clorgyline abolished the appearance of DOPEG. Addition of semicarbazide in preparations treated with pargyline did not affect the efflux of deaminated and O-methylated deaminated metabolites. From the measurement of tissue VMA, it appeared that the efflux of VMA poorly reflects quick changes in the rate of its formation but that formation is abolished by pretreatment with pargyline. These experiments indicate that in the canine saphenous vein: (1) DOPEG is formed mainly in intraneuronal sites, while DOMA, MOPEG and VMA are formed extraneuronally; (2) VMA is retained in the tissue after its formation; and (3) the only subtype of MAO involved in the metabolism of ³H-noradrenaline released from adrenergic nerve endings can be classified as MAO-A.Supported in part by grant HL-05883     

The aziridinium derivative of DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) accelerates the beating rate of isolated rat atria by enhancing the spontaneous release of noradrenaline

Summary The aziridinium derivative of the compound N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (az-DSP4) depletes endogenous noradrenaline stores and exerts neurotoxic actions on noradrenergic neurons. These effects are persistent in the central nervous system and transient in the periphery. To determine if transmitter release plays a role in the noradrenaline depletion caused by az-DSP4, the action of the compound was studied in isolated and spontaneously beating rat atria. 1. az-DSP4 enhanced atrial beating rate when present in the incubation medium at concentrations ranging from 10^–3 M to 10^–4 M but at 10^–3 s M decreased that rate below basal levels. 2. Preincubation of atria for 30 min with the noradrenaline uptake blocker desimipramine (DMI, 10^–6 M) or with the betablocker propranolol (10^–7 M), abolished the positive chronotropic action of az-DSP4. 3. The rate-accelerating effect of az-DSP4 could be prevented by pretreating the rats with reserpine (5 mg/kg i. p. 24 h) or enhanced by pargyline pretreatment (100 mg/kg i. p. 18 h). 4. az-DSP4 stimulated the spontaneous efflux of tritium from the isolated atria previously labeled with ³H-noradrenaline (4 × 10^–7 M), an increase that was mainly accounted for by DOPEG. 5. COMT and MAO activities in atria homogenates were inhibited by az-DSP4 in a concentration-dependent manner. However, MAO inhibition did not result in a change of the metabolic pattern as could be expected. 6. The results obtained indicate that az-DSP4 enhances the rate of spontaneous beating of isolated rat atria. The positive chronotropic effect of az-DSP4 requires the interaction of the compound with the noradrenaline uptake system. The mechanism of the accelerating effect of az-DSP4 most probably involves the release of noradrenaline from adrenergic nerve terminals in the atria and its subsequent interaction with adrenergic beta-receptors.Abbreviations DSP4 N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride - az-DSP4 aziridinium derivative of DSP4 - NA noradrenaline - DOMA 3,4-dihydroxy mandelic acid - DOPEG 3,4-dihydroxyphenylethyleneglycol - NMN normetanephrine - OMDA O-methyl deaminated metabolite fraction, comprising vanillyl-mandelic acid (VMA) plus the 3-methoxy derivative of DOPEG (MOPEG) - COMT catechol-O-methyltransferase - MAO monoamineoxidase Send offprint requests to M. E. Landa     

Effects of clorgyline and (−)deprenyl on the deamination of normetanephrine and noradrenaline in strips and homogenates of the canine saphenous vein

Summary The influence of specific inhibitors of MAO A (clorgyline) and MAO B [(–)deprenyl] on the metabolism of normetanephrine (NMN), in strips of canine saphenous vein was studied, both in the absence and in the presence of inhibitors of neuronal (cocaine) and extraneuronal (hydrocortisone) uptake. Moreover, the formation of metabolites of noradrenaline and of NMN by saphenous vein homogenates and the influence of clorgyline or (–)deprenyl on this formation are described.Clorgyline reduced to the same degree (by about 70%) the formation of methoxy-hydroxy-phenylglycol (MOPEG) and of vanillylmandelic acid (VMA) in strips incubated with NMN, whereas (–)deprenyl reduced by about 50% the formation of MOPEG and had no effect on VMA production. Hydrocortisone had effects very similar to those of (–)deprenyl.Saphenous vein, homogenates ()-methylation inhibited), deaminated both noradrenaline and NMN; clorgyline and (–)deprenyl reduced the formation of metabolites of both noradrenaline and NMN.It is concluded that both MAO A and B are able to deaminate noradrenaline and NMN, but that in the intact tissue the former has no access to MAO B. Even in intact tissues MAO B may play a role in the metabolism (but not in the inactivation) of noradrenaline by deaminating the NMN formed from noradrenaline and giving preferentially origin to MOPEG.Supported by Instituto Nacional de Investigação Científica (INIC, FmPl)On leave from Faculdade de Farmácia, Universidade de Coimbra, with a grant from Instituto Nacional de Investigação Científica     

Evidence for saturation of catechol-0-methyltransferase by low concentrations of noradrenaline in perfused lungs of rats

Previous studies on the pulmonary removal and metabolism of catecholamines in rat lungs have shown that, when the lungs are perfused with a low concentration (1 nmol/1) of noradrenaline, the amine is metabolized by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), but is predominantly O-methylated, and the activities of COMT and MAO are 0.357 min^–1 and 0.186 min^–1, respectively. The aim of the present study was to examine the changes in the metabolic profile of noradrenaline in rat lungs over a range of concentrations, and to examine the kinetics of the pulmonary O-methylation of noradrenaline and adrenaline.In isolated lungs perfused with ³H-noradrenaline, there was a progressive decrease in the proportion of O-methylated metabolites and a corresponding increase in the proportion of deaminated metabolites, as the noradrenaline concentration in the perfusion solution was increased from 1 to 10 to 100 to 1000 nmol/l. Experiments designed to determine the rate of uptake of noradrenaline in lungs perfused with 1 nmol/l ³H-noradrenaline, under conditions of MAO inhibited, COMT inhibited and COMT and MAO inhibited, showed that the results were compatible with co-existence of COMT and MAO in the pulmonary endothelial cells. Hence, it appeared that the changing metabolic profile with amine concentration in the previous series of experiments was not due to saturation of noradrenaline uptake into cells that contained COMT but not MAO.Further experiments to examine the kinetics of O-methylation of noradrenaline and adrenaline (MAO inhibited) showed that the O-methylation of these amines in the lungs was predominantly saturable, with half-saturation occurring at concentrations (9.8 nmol/I and 19.4 nmol/l, respectively) that were two orders of magnitude lower than those required to half-saturate uptake₁ of the amines. Saturation of O-methylation by these low concentrations of noradrenaline (1) provides the explanation for the change in the metabolic profile of noradrenaline described above and (ii) appears to occur because V_{max uptake} V_{max COMT} for the metabolizing system consisting of non-neuronal uptake₁ + COMT in the lungs, as has been described previously for the system consisting of uptake₂ + COMT in extraneuronal sites in rat heart. The results show that the metabolic profile of catecholamines in the pulmonary circulation will reflect that occurring at physiological levels only if studies are carried out with very low amine concentrations.Abbreviations COMT Catechol-O-methyltransferase - DOMA 3,4-dihydroxymandelic acid - DOPEG 3 4-dihydroxyphenylglycol - ECS Extracellular space - HSOC Half-saturating outside concentration - K_{m uptake} Half-saturation constant for uptake - k_COMT Rate constant for O-methylation - k_MAO Rate constant for deamination - k_{out NA} Rate constant for efflux of noradrenaline - MAO Monoamine oxidase - MB-COMT Membrane-bound - COMT NMN Normetanephrine - OMDA O-methylated deaminated metabolites - S-COMT Soluble COMT - T/M_NA Tissue to medium ratio of noradrenaline - U-0521 3,4-dihydroxy-2-methylpropiophenone - V_max Maximal rate of uptake or O-methylation - V_st-st Steady-state rate of metabolite formation - V_uptake Rate of uptake Preliminary results of part of this study were presented to the Seventh Meeting on Adrenergic Mechanisms, Porto, Portugal (Bryan 1990)     

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

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

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     

Cocaine and neuronal uptake in the canine saphenous vein

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

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

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

Effect of Cocaine and Corticosterone on the Metabolism of 3H-Noradrenaline Released from Rabbit Isolated Aorta and Adventitia

Abstract: The effect of cocaine and corticosterone (neuronal and extraneuronal uptake inhibitors, respectively) on the metabolism of ³H-noradrenaline (³H-NA) released spontaneuosly or by electrical-field stimulation was studied in the rabbit isolated aorta and tunica adventitia preloaded with ³H-NA. The spontaneous outflow of tritium from untreated aorta consisted of ³H-NA (25%); ³H-3,4-dihydroxyphenylglycol (³H-DOPEG; 24%); ³H-3,4-dihydroxymandelic acid (³H-DOMA; 8%); ³H-O-methylated plus deaminated compounds (³H-OMDA; 45%); and ³H-normetanephrine (³H-NMN; 1%). The percentage distribution of ³H-NA and its ³H-metabolites in this outflow was not altered by either cocaine (3 × 10^?5 M), corticosterone (4 × 10^?5 M), or cocaine (3 × 10^?5 M) + corticosterone (4 × 10^?5 M). The spontaneous ³H-outflow from untreated tunica adventitia did not differ from that of aorta. Cocaine (3 × 10^?5 M) + corticosterone (4 × 10^?5 M) did not alter the composition of ³H-NA and its ³H-metabolites in the spontaneous outflow from adventitia. The stimulation-evoked ³H-overflow from untreated rabbit aorta preloaded with ³H-NA consisted of ³H-NA (40%); ³H-DOPEG (15%); ³H-DOMA (2%); ³H-OMDA (50%); and ³H-NMN (3%). Cocaine (3 × 10^?5 M) decreased the percentage recovered as ³H-DOPEG, increased ³H-NMN and had no effect on the other ³H-metabolites. Corticosterone (4 × 10^?5 M) had no effect on the percentage distribution of ³H-NA and its ³H-metabolites. Cocaine (3 × 10^?5 M) + corticosterone (4 × 10^?5 M) increased ³H-NA, decreased ³H-DOPEG and had no effect on the percentage distribution of the other ³H-metabolites. In the case of adventitia, cocaine (3 × 10^?5 M) + corticosterone (4 × 10^?5 M) only decreased the percentage recovered as ³H-DOPEG, without any significant effect on ³H-NA and its other ³H-metabolites. It is concluded that inhibition of neuronal plus extraneuronal uptake of H-NA by cocaine plus corticosterone, respectively does not fully prevent metabolism of ³H-NA released either spontaneously or by electrical-field stimulation.     

Diet-induced atherosclerosis inhibits release of noradrenaline from sympathetic nerves in rabbit arteries

Contractile responses to sympathetic nerve stimulation and exogenous noradrenaline were compared in aortas and pulmonary arteries of control rabbits and rabbits fed a cholesterol-rich diet (0.3%) for 16 or 30 weeks. The diet-induced atherosclerosis reduced the contractions to increasing concentrations of exogenous noradrenaline (0.1 nM to 10 μM) in both arteries, and the reduction was more pronounced after 30 weeks of the hypercholesterolemia. The contractions produced with increasing frequencies of electrical stimulation (1–32 Hz) were nearly abolished in the atherosclerotic arteries. Labeling of the aorta and the pulmonary arteries with [³H]noradrenaline resulted in accumulation of radioactivity in both control and atherosclerotic blood vessels. After mounting the labeled blood vessels for superfusion, a basal efflux of [³H]noradrenaline and of ³H-metabolites was detected. In the atherosclerotic arteries, a decreased efflux of the intraneuronal deaminated metabolites 3,4-dihydroxyphenyl glycol (DOPEG) and 3,4-dihydroxymandelic acid (DOMA) was detected. Electrical stimulation at 1 Hz (pulmonary artery) or 2 Hz (aorta) caused an augmented efflux of total ³H from the control arteries; this was mostly due to release of intact [³H]noradrenaline. The electrical imp ulses evoked significantly less (16 weeks) or no (30 weeks) release of [³H]noradrenaline in the atherosclerotic arteries. These data illustrate that diet-induced atherosclerosis exerts an inhibitory action on the sympathetic nerve terminals in the aorta and the pulmonary artery of the rabbit. This effect, together with an inhibitory effect at the postjunctional level results in a loss of the responsiveness to nerve stimulation. The atherosclerotic process also inhibits the intraneuronal deamination of the sympathetic transmitter.