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

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

Extraneuronal uptake and O-methylation of 3H-adrenaline in the rabbit aorta

Summary The influence of uptake₂ inhibitors on the Omethylation and accumulation of ³H-adrenaline by the isolated rabbit aorta was studied. Strips were incubated with 0.05 mol/l ³H-(–)-adrenaline during 15 min. Monoamine oxidase and uptake, were inhibited and the ³H-adrenaline present in the tissue was measured as well as the metabolites found in the tissue and in the incubation fluid. In another series of experiments, monoamine oxidase, uptake₁ and catechol-O-methyl transferase (COMT) were inhibited, and tritium accumulation was measured in the tissue.When COMT was inhibited, inhibitors of uptake₂ produced a maximal reduction of ³H-adrenaline accumulation that did not exceed 50%. When COMT was in tact, inhibitors of uptake₂ diminished total ³H-removal and, more markedly, O-methylation and concomitantly increased the tissue content of ³H-adrenaline.Mineralocorticoids (corticosterone and deoxycorticosterone acetate) inhibited ³H-adrenaline uptake (when COMT was inhibited) and ³H-metanephrine formation (when COMT was functional) as effectively as did sexual steroids (17--oestradiol, progesterone and testosterone); hydrocortisone (hemisuccinate or phosphate) had no effect (for concentrations up to 120 mol/l).At the end of the incubation some strips were washed out with amine-free solution. Compartmental analysis of the efflux showed that the amine had distributed into three extraneuronal compartments (compartment I, II and III, with half times of 0.4, 4 and 15 min, respectively). Corticosterone (120 mol/l). decreased the amount of ³H-adrenaline in compartment III and simultaneously increased the amount of the amine in compartment I (extracellular space).The extraneuronal accumulation of ³H-adrenaline in rabbit aorta can be only partially ascribed to uptake₂, as already stated by other authors; our results clearly show that inhibition of uptake₂ increases the amount of ³H-adrenaline in the extracellular space, i. e., uptake₂ creates a concentration gradient for ³H-adrenaline in the extracellular space; elastin and collagen represent very probably the site of binding of ³H-adrenaline which is independent of uptake₂.Abbreviations COMT Catechol-O-methyltransferase - DOCA deoxycorticosterone acetate - DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - MAO monoamine oxidase - OMDA O-methylated and deaminated metabolites - MOPEG methoxyhydroxyphenylglycol - VMA methoxyhydroxymandelic acid A preliminary account of some of the results was presented to the 7th International Catecholamine Symposium in Amsterdam. Supported by Instituto Nacional de Investigação Cientffica (INIC, FmP1)PhD student with a grant from JNICT (Programa Ciência)Correspondence to F. Martel at the above address     

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)     

The uptake and O-methylation of 3H-(±)-isoprenaline in rat cerebral cortex slices

Summary The O-methylation and accumulation of ³H-isoprenaline in slices of the rat cerebral cortex were studied before and after inhibition of COMT. 1. Inhibition of COMT by mol/l U-0521 virtually abolished the O-methylation and increased the accumulation of ³H-isoprenaline; hence, there is evidence for the existence of a central O-methylating system (with a transport mechanism and intracellular COMT). 2. Experiments were carried out with selective uptake inhibitors for uptake, (cocaine and desipramine) or uptake2 (corticosterone and OMI), with phenoxybenzamine (known to inhibit both carriers) and with changes in the ionic composition of the incubation medium. They revealed that the central carrier differed from both, uptake, and uptake2, although exhibiting some resemblance with uptake₂ (lack of dependence on Na⁺ and Cl^–, sensitivity to K⁺ and phenoxybenzamine, ability to transport ³H-isoprenaline). 3. Although the central carrier was rather sensitive to inhibition by beta-adrenoceptor antagonists (propranolol, carteolol), the effect of propranolol was not stereoselective; hence, beta-adrenoceptors do not seem to be involved. 4. Virtually identical IC₃₀-values were obtained for inhibitors, when determined with or without inhibition of COMT. Only OMI was found to inhibit COMT as well as the central transport system; hence it was more potent in inhibiting the O-methylation than the accumulation of ³H-isoprenaline. 5. IC₅₀-values (against initial rates of accumulation of ³H-isoprenaline; COMT inhibited) were determined for various substrates and inhibitors of peripheral uptake₂. There was no correlation with the IC₅₀-values determined earlier for uptake2 in rat heart (Grohmann and Trendelenburg 1984). 6. Unlabelled catecholamines half saturated the intracellular COMT when slices were incubated with 0.22 mol/l [(±)-dobutamine] to 4.9 mol/l [(–)-noradrenaline]. As the presence of unlabelled catecholamines increased tissue levels of ³H-isoprenaline, catecholamines are substrates of the central carrier. 7. The carrier of the central O-methylating system differs from uptake₂ of peripheral organs, although it resembles the peripheral carrier in some respects.Abbreviations COMT catechol-O-methyl transferase - DOPEG dihydroxyphenylglycol - MAO monoamine oxidase - OMI 3-Omethyl-isoprenaline Supported by the Deutsche Forschungsgemeinschaft (Tr 96 and SFB 176) and by a scholarship of the Royal Society for V. G. Wilson. Some of the results were presented to the British Pharmacological Society (Trendelenburg and Wilson 1986)     

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     

Uptake of 3H-catecholamines by rat liver cells occurs mainly through a system which is distinct from uptake1 or uptake2

Isolated rat hepatocytes were incubated with 200 nmol/l ³H-(–)-noradrenaline or 50 nmol/l ³H(–)-adrenaline for 15 min, in Krebs-Henseleit solution at 37°C, gassed with 95% O₂ 5010 CO₂. Monoamine oxidase and catechol-O-methyl transferase were inhibited withpargyline (500 mol/l)and Ro 01-2812 (3,5-dinitropyrocatechol; 2 ol/l), respectively. Total radioactivity present in the cells, which corresponded mostly to intact ³H-amine, was measured.The content of ³H-noradrenaline increased with time of incubation, a plateau having been reached after 15 min of incubation. After 15 min of incubation,the cell: medium ratio for ³H-noradrenaline and ^3H-adrenaline was 0.6–0.7. Desipramine (an inhibitor of the neuronal uptake of catecholamines — uptake,; 1 mol/l) did not affect the uptake of either ³H-noradrenaline or ³H-adrenaline into hepatocytes. Corticosterone (an inhibitor of the extraneuronal uptake of catecholamines — uptake₂; 40 mol/l) slightly inhibited (by 28%) the uptake of ³H-adrenaline, and did not significantly reduce ³H-noradrenaline uptake. Probenecid (an inhibitor of the renal transport of organic anions; 100 mol/l) did not influence the amount of either ³H-noradrenaline or ³H-adrenaline in hepatocytes. Cyanine 863 (an inhibitor of the renal transport of organic cations; 10 mol/l) decreased by 62% the uptake of ³H-adrenaline into cells but did not significantly affect ³H-noradrenaline uptake. Bilirubin (a substrate of a hepatic transport for organic anions; 200 ol/l) produced a significant increase (50%) in the amount of ³H-noradrenaline and ³H-adrenaline present in the cells. When isolated hepatocytes were incubated in a sodium-free medium (sodium being replaced by choline or lithium) there was a very marked inhibition of ³H-noradrenaline and 3H-adrenaline uptake (by 85–97%). An increase in potassium content of the medium (from 6.6 to 50 mmol/l) did not affect the uptake of either ³H-amine into isolated cells.In conclusion, the uptake of catecholamines by isolated liver cells possesses characteristics that distinguish it from the classic uptake systems for catecholamines (uptake₁ and uptake₂): (1) it is sodium-dependent but not affected by desipramine; (2) it is only slightly sensitive to corticosterone and not affected by potassium-induced depolarization; (3) it is partially sensitive to cyanine 863. Moreover, the increase of ³H-amine content in the cells in the presence of bilirubin suggests the possibility of catecholamines being excreted from the hepatocytes through the bilirubin transporter.PhD student with a grant from JNICT (Programa Ciência)Abbreviations COMT catechol-O-methyl transferase - MAO monoamine oxidase - RTOC renal transport of organic cations Supported by Programa STRIDE (STRDA/P/SAU/259/92) Correspondence to: F. Martel at the above address     

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

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

The activity of the neuronal and extraneuronal catecholamine-metabolizing enzymes of the perfused rat heart

Summary In a comparative study the neuronal and extraneuronal metabolism of several ³H-catecholamines (all of which were tritiated in the C-7 position of the side chain only) was determined in isolated rat hearts perfused at a concentration of the 3H-amines of 50 nmol/1. While the neuronal MAO activity was determined after inhibition of extraneuronal uptake (100 mol/1 OMI) and COMT (10 mol/1 U-0521), the extraneuronal MAO activity was estimated after inhibition of neuronal uptake (30 mol/1 cocaine) and COMT. The extraneuronal COMT activity was determined under conditions of inhibition of both neuronal uptake and MAO (pretreatment with pargyline). Hearts were perfused with the 3H-catecholamines until the rate of appearance of the various ³H-metabolites in the venous effluent has reached a steady state. From these rates (v _st-st) and the steady-state content of the unchanged ³H-catecholamines in the tissue (S _i), the rate constants (V _max/K _m) for the unsaturated intracellular enzymes COMT (_COMT) and MAO (_MAO) were calculated. The _COMTvalues for all four catecholamines, (–)-noradrenaline, dopamine, (–)-adrenaline and (±)-isoprenaline exhibit a range from 0.24 to 0.78 min^–1; the metabolism of the catecholamines by the COMT differs: (-)-noradrenaline = dopamine < (–)-adrenaline < (±)-isoprenaline. The extraneuronal MAO activity was low for all three catecholamines, (–)-adrenaline, (–)-noradrenaline and dopamine (range of _MAOfrom 0.05 to 0.28 min^–1) and declined in the order: (–)-adrenaline < (–)-noradrenaline < dopamine. The neuronal MAO activity for (–)-adrenaline, (–)-noradrenaline and dopamine was slightly higher than that in the extraneuronal cells (range of kMAO from 0.08 to 0.35 min^–1), but the ranking order showed the same pattern: (–)-adrenaline < (–)-noradrenaline = dopamine.Abbreviations MAO monoamine oxidase - COMT catechol-Omethyltransferase - NMN normetanephrine - MN metanephrine - MT 3-methoxytyramine - OMI 3-O-methyl-isoprenaline - DOPEG dihydroxyphenylglycol - DOPET dihydroxyphenylethanol - DOMA dihydroxymandelic acid - DOPAC dihydroxyphenylacetic acid - U-0521 3,4-dihydroxy-2-methyl propiophenone     

The sensitivity of adrenergic varicosities to the 3H-noradrenaline-releasing effect of potassium

Summary After the loading of incubated, homogeneously innervated tissues with ³H-noradrenaline (monoamine oxidase and catechol-O-methyl transferase inhibited, calcium-containing solution) high K⁺ released the ³H-amine from adrenergic varicosities. In paired experiments the sensitivity of rat atria to high K⁺ exceeded that of vasa deferentia.In the rat vas deferens the releasing effect of high K⁺ was enhanced by drugs or procedures which induce a carrier-mediated outward transport of ³H-noradrenaline, i.e., by ouabain, by glucose deprivation and by hypoxia. — In the presence of extracellular calcium desipramine failed to affect the releasing effect of high K⁺ (except in the absence of glucose or during hypox1a), but in the absence of calcium desipramine reduced it. Apparently, whenever the axoplasmic levels of ³H-noradrenaline are increased, high K⁺ is able to induce some carrier-mediated outward transport of the 3H-amine.It is suggested that organ differences with respect to the sensitivity to high K⁺ may well be due to hypoxia (plus some lack of glucose) of those varicosities that had been loaded with ³H-noradrenaline. The risk of storage of ³H-noradrenaline in hypoxic varicosities appears to be greater in incubated than in perfused organs, and in the former it is greater in sparsely than in densely innervated tissues.Abbreviations COMT catechol-O-methyl transferase - FRL fractional rate of loss - MAO monoamine oxidase Supported by the Dr. Robert Pfleger-Stiftung and the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to U. Trendelenburg at the above address     

β-Endorphin-(1-27) is a naturally occurring antagonist of the reinforcing effects of opioids

Summary The influence of inhibitors of metabolism and uptake of noradrenaline on the ³H-noradrenaline removal from the perfusion fluid by the isolated rat liver was studied. Livers were perfused with 3 nmol/l ³H-noradrenaline and ³H-noradrenaline and ³H-metabolites were determined in effluent, liver and bile. After the perfusion with 14,900 ±920 dpm · g^–1 · min^–1 during 90 min, cumulative removal of tritium was 323,574 ± 63,103 dpm/g. ³H-metabolites recovered from the liver after 90 min perfusion represented 71.1 ± 9.0% of total metabolite formation. Only the OMDA-fraction appeared in the perfusate; its approach to steady state of efflux was slow. The inhibition either of MAO or COMT changed neither the total removal of tritium nor the ³H-metabolites recovered from the liver. Cocaine (10 mol/l) reduced the accumulation of ³H-noradrenaline in the liver. The uptake₂ inhibitor corticosterone (30 mol/l) diminished total removal of tritium and the ³H-metabolites recovered from the liver without changing the accumulation of ³H-noradrenaline. The hypothesis of two different compartments, one responsible for the metabolism and the other for the accumulation of the amine is discussed.Abbreviations NA noradrenaline - NMN normetanephrine - OMDA O-methylated deaminated metabolites - MAO monoamine oxidase - COMT catechol-O-methyl transferase Send offprint requests to M. C. Rubio     

A kinetic investigation of the pulmonary metabolism of dopamine in rats shows marked differences compared with noradrenaline

The aim of this study was to investigate the deamination of dopamine in the intact pulmonary circulation of isolated lungs of the rat. The first part of the study showed that dopamine is not converted to noradrenaline by dopamine--hydroxylase (DBH) when dopamine is perfused through isolated lung preparations with monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) inhibited. Hence, it was not necessary to inhibit DBH in subsequent experiments.The metabolite profile for deamination of dopamine in the lungs was examined by determining whether MAO and semicarbazide-sensitive amine oxidases (SSAO) contribute to the deamination of dopamine (and noradrenaline), and by determining the activity of MAO (k_MAO) for the metabolism of dopamine. Lungs were perfused with I nmol/l ³H-dopamine or ³H-noradrenaline with COMT inhibited and, in experiments to determine the contribution of SSAO to deamination, with MAO inhibited. Inhibition of MAO reduced the deamination of dopamine and noradrenaline by 99.8% and 98.6%, respectively, indicating that MAO, and not SSAO, was responsible for deamination of the catecholamines in the lungs. The k_MAO value for deamination of dopamine was 3.89 min^–1. Further experiments were carried out to determine the contributions of MAO-A and MAO-B to the deamination of dopamine in lungs perfused with 1 nmol/l ³H-dopamine and 100 nmol/1 lazabemide or 300 nmol/I Ro41-1049, respectively. The values of k_MAO-A and k_MAO-B were 3.05 min^–1 and 0.626 min^–1, respectively.It was concluded that, in rat lungs, MAO-A contributed 78–84% and MAO-B 16–22% to the total deamination of dopamine and SSAO had no significant role in its pulmonary metabolism. These relative contributions of MAO-A and MAO-B to the deamination of dopamine are very similar to those that have been determined previously for noradrenaline, but the rate constant for deamination of dopamine is 26-fold greater than that for noradrenaline in rat lungs.Abbreviations COMT Catechol-O-methyltransferase - DBH Dopamine-\-hydroxylase - DOPEG 3,4-dihydroxyphenylglycol - DOMA 3,4-dihydroxyman delic acid - DOPAC 3,4-dihydroxyphenylacetic acid - DOPET 3,4-dihydroxphenylethanol - ECS Extracellular space - K_m Michaelis or half-saturation constant - k_COMT Rate constant for O-methylation by COMT - k_deam Rate constant for total deamination - k_MAO Rate constant for deamination by MAO - MAO Monoamine oxidase - MB-COMT Membrane-bound COMT - SSAO Semicarbazidesensitive amine oxidases - S-COMT Soluble COMT - T/M Tissue to medium concentration ratio of dopamine or noradrenaline - V_max Maximal rate - V_{st - st} Steady-state rate of metabolite formation     

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

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

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

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

Mepiprazole,a new psychotropic drug: Effects on uptake and retention of monoamines in rat brain synaptosomes

The influence of mepiprazole (EMD 16,923), a new pyrazol-ylalkyl-piperazine derivative, on the uptake of ³H-norepinephrine (NE), ³H-dopamine (DA), and ³H-serotonin (5-HT) into rat brain synaptosomes from cerebral cortex, corpus striatum, and hypothalamus was investigated in comparison with several psychotropic drugs, including oxypertine, d-amphetamine, imipramine, desipramine, chlorimipramine, amitriptyline, and chlorpromazine in vitro. Mepiprazole was a relatively weak inhibitor of monoamine uptake and exhibited its strongest action on the hypothalamic 5-HT uptake, being almost equipotent with desipramine (IC₅₀=0.9 M). Furthermore, the influence of the drugs on the retention of ³H-amines previously taken up by whole rat brain synaptosomes was studied. Unlike the tricyclic antidepressants, mepiprazole as well as oxypertine and d-amphetamine markedly increased the efflux of radioactivity during a 20-min incubation at 37°C at low concentrations (10^–6 to 10^–5 M), whereas at 10^–4M all drugs greatly enhanced the efflux. The ability of mepiprazole to increase 5-HT concentration at the receptor level by a combination of neuronal uptake inhibition and release is discussed in relationship to the central actions of the drug.     

Dopamine and adrenaline,but not isoprenaline,are substrates for uptake and metabolism in isolated perfused lungs of rats

Summary The uptake and subsequent metabolism by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) of dopamine, adrenaline, isoprenaline and noradrenaline in isolated perfused lungs of rats has been examined. In lung preparations in which COMT and MAO were inhibited, the uptake of ³H-labelled dopamine, (–)-adrenaline and (–)-noradrenaline, but not (±)-isoprenaline, was reduced by cocaine (10 or 100 mol/l) The rank order of the Km values of the amines that were substrates for uptake in the lungs were: dopamine (0.246 mol/l) < noradrenaline (0.967 mol/l) < adrenaline (3.32 mol/l). These results are consistent with transport of catecholamines in rat lungs by Uptake₁.In lung preparations with COMT and MAO intact, dopamine and noradrenaline were removed from the circulation (50% and 32%, respectively) and mainly metabolized. There was very little (3.0%) removal of isoprenaline by the lungs and adrenaline was not included in this part of the study. In lung preparations in which only MAO was inhibited, the rank order of COMT activity for O-methylation of the amines was dopamine noradrenaline adrenaline (k_COMT values: 4.98 min^–1, 0.357 min^–1, and 0.234 min^–1, respectively).If dopamine or adrenaline are perfused through the pulmonary circulation in isolated lungs of the rat, they are taken up and then metabolized by COMT and MAO, as also occurs for noradrenaline. Isoprenaline is not a substrate for uptake in the lungs. There was less uptake of adrenaline than noradrenaline, indicating that uptake and metabolism in the lungs may not be a significant removal process for adrenaline in the circulation of rats in vivo. The more marked uptake of dopamine (than of noradrenaline) indicates that uptake and metabolism by the lungs, at least in the rat, may play an important role in the removal of dopamine from the circulation in vivo.Abbreviations COMT catechol-O-methyltransferase - DOMA 3,4-dihydroxymandelic acid - DOPAC 3,4-dihydroxyphenylacetic acid - DOPEG 3,4-dihydroxyphenylglycol - DOPET 3,4-dihydroxyphenyl ethanol - MAO monoamine oxidase - MN metanephrine - MTA 3-methoxytyramine - NMN normetanephrine - OMDA O-methylated deaminated metabolites - OMI 3-O-methylisoprenaline - U-0521 3,4-dihydroxy-2-methylpropiophenone Some of the results of this study were presented to the Australasian Society of Clinical and Experimental Pharmacologists (Bryan and O'Donnell 1987, 1988; Bryan et al. 1989; Bryan-Lluka 1990) Send offprint requests to L.J. Bryan-Lluka at the above address     

Autoradiographic study of the rat vas deferens incubated with 3H-noradrenaline

Summary Rat vasa deferentia were incubated with 0.2 mol/l ³H-noradrenaline for 60 min and then washed out with amine-free solution for 100 min. Autoradiography then revealed a preferential labelling of the varicosities in the immediate vicinity of the surface of the tissue. However, when tissues were obtained from reserpine-and pargyline-pretreated rats (to block vesicular uptake and monoamine oxidase), ³H-noradrenaline was able to penetrate more deeply into the tissue. These differences are in accordance with the view that the autoradiographs reflect the ³H-noradrenaline concentration gradient (within the extracellular space) generated by the neuronal uptake of the 3H-amine; the concentration gradient is steeper (and the heterogeneity of labelling is more pronounced) in tissues with intact vesicular uptake and monoamine oxidase than in tissues in which these mechanisms had been inhibited.Send offprint requests to I. Azevedo at the above adress     

The handling of five amines by the extraneuronal deaminating system of the rat heart

Summary The handling of five amines by the extraneuronal deaminating system was studied in perfused hearts of rats (pretreated with reserpine; COMT and neuronal uptake inhibited). Hearts were perfused with 50 nmol/l ³H-noradrenaline for 30 min, in the presence of increasing concentrations of unlabelled (–)-adrenaline, (–)-noradrenaline, dopamine, tyramine and 5-HT. IC₅₀'s were determined as those concentrations of unlabelled amines which halved the steady-state rate of deamination of ³H-noradrenaline. After correction for changes in the tissue/medium ratio for ³H-noradrenaline, half-saturating outside concentrations were obtained. They increased in the order (–)-adrenaline (15 mol/l) — tyramine — dopamine — noradrenaline —5-HT (53 mol/l). The V _max for extraneuronal deamination was determined for ³H-(–)-adrenaline, ³H-(–)-noradrenaline and ³H-dopamine, as well as (by HPLC and electrochemical detection) for tyramine and 5-HT. It was low for (–)-adrenaline, intermediate for (–)-noradrenaline, dopamine and 5-HT, high for tyramine. For the three catecholamines the half-saturating outside concentrations of the extraneuronal deaminating system clearly exceeded those for the extraneuronal O-methylating system of the same organ (see Grohmann and Trendelenburg 1985), although the two enzymes appear to co-exist in the same cells, so that the same transport system is involved.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase Supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to U. Trendelenburg     

Inhibition of 3H-noradrenaline accumulation by dopexamine hydrochloride in the isolated aorta of the rabbit

Summary The aim of the present work was to study the ability of dopexamine hydrochloride to interfere with the neuronal and extraneuronal uptake mechanisms by investigating the effect of dopexamine hydrochloride on ³H-noradrenaline accumulation by rabbit isolated aorta. Dopexamine hydrochloride (3 × 10^–9 – 10^–5 mol/l) reduced the accumulation of tritium by aorta incubated with ³H-noradrenaline (10^–8 mol/l). The effect of dopexamine was compared to cocaine, dopamine, dobutamine, ADTN [(+-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene], ouabain and isoprenaline. Dopexamine hydrochloride (3 × 10^–9 – 10^–7 mol/l) caused the same degree of inhibition irrespective of whether corticosterone (4 × 10^–5 mol/l) was present or not. The order of inhibitory potency was: desipramine > dopexamine hydrochloride > dopamine > ADTN cocaine > dobutamine > ouabain > isoprenaline. In the presence of desipramine (10^–6 mol/l), corticosterone (10^–6 – 10^–4 mol/l), but not dopexamine hydrochloride (10^–6 – 10^–4 mol/l), reduced the ³H-accumulation. It is concluded that dopexamine hydrochloride is a potent inhibitor of uptake-1 in rabbit isolated aorta. Dopexamine hydrochloride has no affinity for the uptake-2 mechanism in this tissue. Send offprint requests to O. A. Nedergaard at the above address     

Amezinium and debrisoquine are substrates of uptake1 and potent inhibitors of monoamine oxidase in perfused lungs of rats

Previous studies have resulted in the classification of amezinium as a selective inhibitor of neuronal monoamine oxidase (MAO), because it is a much more potent MAO inhibitor in intact tissues, in which it is accumulated in noradrenergic neurones by uptake₁, than in tissue homogenates. In the present study, the effects of amezinium on the deamination of noradrenaline were investigated in intact lungs of rats, since the pulmonary endothelial cells are a site where the catecholamine transporter is non-neuronal uptake₁. In addition, another drug that is both a substrate of uptake₁ and a MAO inhibitor, debrisoquine, was investigated in the study.The first aim of the study was to show whether amezinium and debrisoquine are substrates of uptake₁ in rat lungs. After loading of isolated perfused lungs with ³H-noradrenaline (MAO and catechol-O-methyltransferase (COMT) inhibited), the efflux of ³H-noradrenaline was measured for 30 min. When 1 mol/l amezinium or 15 mol/l debrisoquine was added for the last 15 min of efflux, there was a rapid and marked increase in the fractional rate of loss of ³H-noradrenaline, which was reduced by about 70% when 1 mol/l desipramine was present throughout the efflux period. These results showed that both drugs were substrates for uptake1 in rat lungs. In lungs perfused with 1 nmol/l ³H-noradrenaline (COMT inhibited), 10, 30 and 300 nmol/l amezinium caused 58%, 76% and 74% inhibition of noradrenaline deamination, respectively, and 30, 300 and 3000 nmol/l debrisoquine caused 56%, 89% and 96% inhibition of noradrenaline deamination, respectively. When MAO-B was also inhibited, 10 nmol/l amezinium caused 84% inhibition of the deamination of noradrenaline by MAO-A in the lungs. In contrast, in hearts perfused with 10 nmol/l ³H-noradrenaline under conditions where the amine was accumulated by uptake₂ (COMT, uptake₁ and vesicular transport inhibited), 10 nmol/l amezinium had no effect and 300 nmol/l amezinium caused only 36% inhibition of deamination of noradrenaline.The results when considered with previous reports in the literature show that amezinium is about 1000 times more potent and debrisoquine is about 20 times more potent for MAO inhibition in rat lungs than in tissue homogenates, and the reason for their high potencies in the intact lungs is transport and accumulation of the drugs in the pulmonary endothelial cells by uptake₁. Amezinium is much less potent as a MAO inhibitor in cells with the uptake₂ transporter, such as the myocardial cells of the heart. The results also confirmed previous reports that amezinium is highly selective for MAO-A.Abbreviations COMT catechol-O-methyltransferase - DOMA 3, 4-dihydroxy-mandelic acid - DOPEG 3, 4–'dihydroxyphenylglycol - ECS extracellular space - FRL fractional rate of loss - IC ₅₀ inhibitor concentration that causes 50% inhibition - K _{m uptake} Michaelis or half-saturation constant for uptake - k _{M AO} rate constant for deamination - k _{out NA} rate constant for efflux of noradrenaline - MAO monoamine oxidase - MAO-Aa type A monoamine oxidase - MAO-B type B monoamine oxidase - T/M _NA tissue to medium ratio of noradrenaline - U-0521 3, 4-dihydroxy-2-methylpropiophenone - V _max maximal rate - v _st–st steady-state rate of metabolite formation Preliminary results of this study were presented to the 1993 Meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (Bryan-Lluka 1993).     

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

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