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1.
Lesley J. Bryan-Lluka Hayley Seers Iain Sharpe 《Naunyn-Schmiedeberg's archives of pharmacology》1996,353(5):536-544
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). 相似文献
2.
H. Russ M. Gliese J. Sonna E. Schömig 《Naunyn-Schmiedeberg's archives of pharmacology》1992,346(2):158-165
Summary The corticosterone-sensitive extraneuronal transport mechanism for noradrenaline (uptake2) removes the neurotransmitter from the extracellular space. Recently, an experimental model for uptake2 has been introduced which is based on tissue culture techniques (human Caki-1 cells). The present study describes some properties of uptake2 in Caki-1 cells and introduces a new substrate, i.e., 1-methyl-4-phenylpyridinium (MPP+).Experiments on Caki-1 cells disclosed disadvantages of tritiated noradrenaline as substrate for the investigation of uptake2. The initial rate of 3H-noradrenaline transport [kin = 0.58 l/(mg protein · min)] was low compared with other cellular transport systems and intracellular noradrenaline was subject to rapid metabolism (kO-methylation = 0.54 min–1). The neurotoxin MPP+ was found to be a good substrate of uptake2. Initial rates of specific 3H-MPP+ transport into Caki-1 cells were saturable, the Km being 24 mol/l and the Vmax being 420 pmol/(mg protein · min). The rate constant of specific inward transport was 34 times higher [19.6 mol/l (mg protein · min)] than that of 3H-noradrenaline. The ratio specific over non-specific transport was considerably higher for 3H-MPP+ (12.6) than for 3H-noradrenaline (3.0). 3H-MPP+ transport into Caki-1 cells was inhibited by various inhibitors of uptake2. The highly significant positive correlation (p < 0.001, r = 0.986, n = 7) between the IC50's for the inhibition of the transport of 3H-noradrenaline and 3H-MPP+, respectively, proves the hypothesis that MPP+ enters Caki-1 cells via uptake2. 3H-MPP+ is taken up via uptake2 not only by Caki-1 cells but also by the isolated perfused rat heart which is another established model of uptake2.Tritiated MPP+ is a new and convenient tool for the investigation of uptake2. The rate constant for inward transport, the factor of accumulation and the ratio specific over non-specific transport are considerably higher for 3H-MPP+ than for 3H-noradrenaline. In uptake studies with 3H-MPP+ inhibition of intracellular noradrenaline-metabolizing enzymes is not necessary. In tissues and tissue cultures which possess fewer uptake2 carriers than Caki-1 cells or the rat heart, the identification and characterization of uptake2 can be expected to be greatly facilitated by the use of 3H-MPP+.Supported by the Deutsche Forschungsgemeinschaft (SFB 176)
Send offprint requests to H. Russ at the above address 相似文献
3.
V. Marino I. S. de la Lande M. Newlyn D. A. S. Parker 《Naunyn-Schmiedeberg's archives of pharmacology》1993,347(4):371-378
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 相似文献
4.
Summary This study describes for the first time an experimental system for the extraneuronal transport mechanism of noradrenaline (uptake2) which is based on a clonal cell line (Caki-1). Caki-1 cells were originally derived from a human renal cell carcinoma. The conclusion that these cells express uptake2 is supported by several experimental findings. (1) The initial rate of 3H-noradrenaline uptake in Caki-1 cells is saturable, the K
m being 450 mol/l. (2) Inhibitors of uptake2 such as corticosterone (1 mol/l) and O-methyl-isoprenaline (100 Emol/l) largely inhibit 3H-noradrenaline uptake in Caki-1 cells. Whereas inhibitors of the neuronal transport mechanism for noradrenaline (uptake1) such as desipramine (1 mol/l) and cocaine (10 mol/l) do not reduce it. (3) Depolarization of Caki-1 cells by the elevation of extracellular potassium inhibits 3H-noradrenaline uptake. (4) There is a highly significant correlation between the IC50's of various compounds for the inhibition of 3H-noradrenaline uptake in Caki-1 cells and rabbit aorta known to possess uptake2.Interestingly enough, uptake2 in Caki-1 cells and rabbit aorta is inhibited by cimetidine, quinidine and procainamide which are substrates of the renal transport mechanism for organic cations. Moreover, 3H-cimetidine is shown to be a substrate of uptake2 in the isolated perfused rat heart. These results indicate a striking similarity between uptake2 and the renal transport mechanism for organic cations.
Send offprint requests to E. Schömig at the above addressSupported by the Deutsche Forschungsgemeinschaft (SFB 176, Scho 373) and the Dr. Robert Pfleger Stiftung 相似文献
5.
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: kCOMT 0.70±0.15 min–1 and kMAO 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 相似文献
6.
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 相似文献
7.
C. -L. Schönfeld U. Trendelenburg 《Naunyn-Schmiedeberg's archives of pharmacology》1989,339(4):433-440
Summary The3H-noradrenaline-releasing effects of p- and m-tyramines and -octopamines, either deuterated or not, were studied in isolated vasa deferentia of the rat (COMT inhibited and calcium-free solution in all experiments).
K
m, for uptake1 was higher for octopamines than for tyramines, but not increased by the introduction of deuterium in -position, except for (probably contaminated) deuterated p-octopamine. Other tissues were preloaded with3H-noradrenaline. After inhibition of vesicular uptake and MAO equi-releasing concentrations of the eight amines were strictly correlated withK
m, they were 6 to 7 times higher for unsubstituted octopamines than for corresponding tyramines. When only MAO (but not vesicular uptake) was inhibited, this difference decreased to about 4-fold, but the releasing potency of the deuterated amines (relative to their parent amines) remained unchanged (except for p-octopamine). When vesicular uptake and MAO were intact, unsubstituted octopamines were only 1.5 to 2.2 times less potent than the corresponding tyramines. Analysis of the efflux of3H-DOPEG confirmed that this gain in the relative potencies of octopamines is due to their increased ability to mobilize vesicular 3H-noradrenaline; moreover, deuterated amines as well were then better mobilizers than were their parent amines.It is concluded that, provided vesicular uptake is intact, the introduction of a \-OH-group enhances the ability of indirectly acting sympathomimetic amines to mobilize vesicular noradrenaline; the introduction of deuterium in -position, on the other hand, enhances this mobilizing effect exclusively when MAO is intact.Abbreviations used here COMT
catechol-O-methyl transferase
- DOMA
dihydroxymandelic acid
- DOPEG
dihydroxyphenylglycol
- MAO
monoamine oxidase
- OM-fraction
column chromatographic fraction containingall O-methylated metabolites
Supported by the Deutsche Forschungsgemeinschaft (SFB 176) 相似文献
8.
P. Fischer A. Burger K. H. Graefe U. Trendelenburg 《Naunyn-Schmiedeberg's archives of pharmacology》1989,339(1-2):79-84
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 相似文献
9.
R. Bals-Kubik A. Herz T. S. Shippenberg 《Naunyn-Schmiedeberg's archives of pharmacology》1988,337(4):392-396
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 相似文献
10.
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 相似文献
11.
Summary In the rat vas deferens, DMPP is a substrate of uptake, (Krn = 11.5 mol/I). After block of vesicular uptake, monoamine oxidase and catechol-O-methyl transferase, after loading of the tissue with 3H-noradrenaline, and in calcium-free solution (i. e., when axoplasmic 3H-noradrenaline levels were high and when depolarization-induced exocytotic release was impossible), DMPP induced a pronounced outward transport of 3H-noradrenaline. On the other hand, when, in similar experiments, vesicular uptake and monoamine oxidase were intact (i.e., when axoplasmic 3H-noradrenaline levels were low), DMPP induced very little outward transport of 3H-noradrenaline. This discrepancy indicates that DMPP has little ability to mobilize vesicularly stored 3H-amine.When the medium contained calcium (catechol-O-methyl transferase inhibited, all other mechanisms intact), 100 (but not 10) mol/l DMPP induced a hexamethonium-sensitive release of 3H-noradrenaline of short duration. Hence, in the presence of extracellular calcium, 100 mol/l DMPP elicits exocytotic release via activation of hexamethonium-sensitive nicotinic acetylcholine receptors.DMPP inhibits the monoamine oxidase of rat heart homogenate with an IC50 of about 100 mol/l.Abbreviations COMT
catechol-O-methyl transferase
- DMPP
dimethylphenylpiperazinium
- DOMA
dihydroxymandelic acid
- DOPEG
dihydroxyphenylglycol
- MAO
monoamine oxidase
- NMN
normetanephrine
- OM-fraction
column chromatographic fraction containing all O-methylated 3H-metabolites
- OMDA
fraction containing O-methylated and deaminated metabolites
Supported by the Deutsche Forschungsgemeinschaft (SFB 176)
Send offprint requests to U. Trendelenburg at the above address 相似文献
12.
Lesley J. Bryan-Lluka 《Naunyn-Schmiedeberg's archives of pharmacology》1995,351(4):408-416
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 Vmax uptake Vmax 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
- Km uptake
Half-saturation constant for uptake
- kCOMT
Rate constant for O-methylation
- kMAO
Rate constant for deamination
- kout 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/MNA
Tissue to medium ratio of noradrenaline
- U-0521
3,4-dihydroxy-2-methylpropiophenone
- Vmax
Maximal rate of uptake or O-methylation
- Vst-st
Steady-state rate of metabolite formation
- Vuptake
Rate of uptake
Preliminary results of part of this study were presented to the Seventh Meeting on Adrenergic Mechanisms, Porto, Portugal (Bryan 1990) 相似文献
13.
V. Marino I. S. de la Laude D. A. S. Parker J. Dally S. Wing 《Naunyn-Schmiedeberg's archives of pharmacology》1992,346(2):166-172
Summary The extraneuronal removal and disposition of noradrenaline in rabbit dental pulp was examined in view of earlier evidence that the tissue possessed an extra-neuronal uptake process resembling neuronal uptake1. Pulp, which had been depleted of sympathetic nerves by homolateral superior cervical ganglionectomy, was incubated in vitro with 3H-noradrenaline in low concentrations (0.025 or 0.18 mol/l). When the metabolising enzymes (monoamine oxidase, catechol-O-methyl transferase) were active, 3H retention by the denervated pulp, as indicated by the 3H content after the tissue had been washed for 30 min following incubation with 3H-noradrenaline, was less than 30% of that of the innervated pulp. When the enzymes were inhibited, retention rose to approximately 30% of that of the innervated pulp. Analysis of the time course of the 3H efflux indicated that the 3H-noradrenaline in the denervated pulp had accumulated in a single compartment characterised by a t1/2 for efflux of several hours. Accumulation did not occur under Na+-free conditions, and was inhibited by desipramine (IC50 < 0.03 mol/l) and by substrates of neuronal uptake1. Mean IC50, values of the latter were very similar to those for inhibition of neuronal uptake1 and comprised (in mol/l): (+)amphetamine (0.29), dopamine (0.31), tyramine (0.39), (–)noradrenaline (0.70), (–)adrenaline (1.50), 5-hydroxytryptamine (20) and bretylium (35). Uptake2 inhibitors were less active (O-methyl isoprenaline, IC50 = 60 mol/l) than uptake1 inhibitors, or were without inhibitory effects at the concentrations tested (hydrocortisone, 210 mol/l; 2-methoxy oestrone, 10 mol/l).The effects of Na+ omission, of (+)amphetamine, and of O-methylisoprenaline on 3H-normetanephrine formation (measured in the absence of catechol-O-methyl transferase inhibition) matched their effects on 3H-noradrenaline accumulation. The results provide strong support for the presence in rabbit dental pulp of extraneuronal uptake1 which is linked with catechol-O-methyl transferase in the removal of noradrenaline.
Send offprint requests to D. A. S. Parker at the above address 相似文献
14.
U. Trendelenburg 《Naunyn-Schmiedeberg's archives of pharmacology》1989,339(3):293-297
Summary The accumulation and metabolism of 3H-catecholamines were studied in cerebral cortex slices obtained from rats pretreated with reserpine, during 30 min of incubation with 50 nmol/l of the 3H-amines. In some experiments neuronal uptake (uptake,) was inhibited by the presence of 0.3 mol/l desipramine, in others COMT was inhibited by 30 mol/l U-0521. When both MAO and COMT were intact, most of the metabolism of 3H-noradrenaline was neuronal (i. e., desipramine-sensitive). For 3H-adrenaline rates of neuronal metabolism were much lower than for 3H-noradrenaline, non-neuronal O-methylation accounting for about 50% of total metabolism. Rates of metabolism of 3H-dopamine were similar to those of 3H-noradrenaline, but with a predominance of non-neuronal metabolism, which involved O-methylation and deamination. Under these conditions, very little 3H-catecholamine was recovered from the tissues; moreover, desipramine tended to increase tissue levels. Hence, tissue content then appears to partly reflect extracellularly distributed 3H-amines. After block of MAO rates of metabolism of 3H-noradrenaline and 3H-dopamine were greatly reduced, and tissue levels were increased. Desipramine now antagonized the accumulation of 3H-amines in the tissue, while U-0521 increased it. Rates of O-methylation (in the presence of desipramine) increased in the order 3H-noradrenaline < 3H-dopamine. It is concluded that neuronal uptake is associated with MAO only, and rates of neuronal deamination increased in the order: 3H-adrenaline < 3H-dopamine « 3H-noradrenaline. Non-neuronal uptake is associated with both, COMT and MAO, and rates of non-neuronal metabolism increased in the order: 3H-adrenaline < 3H-noradrenaline « 3H-dopamine.Abbreviations COMT
catechol-O-methyl transferase
- DOMA
dihydroxymandelic acid
- DOPAC
dihydroxyphenylacetic acid
- DOPEG
dihydroxyphenylglycol
- DOPET
dihydroxyphenylethanol
- HVA
homovanillic acid
- MAO
monoamine oxidase
- MN
metanephrine
- MOPEG
methoxyhydroxyphenylglycol
- MOPET
methoxyhydroxyphenylethanol
- NMN
normetanephrine
- 3-OMT
3-O-methyl-tyramine
- VMA
vanillylmandelic acid
Supported by the Deutsche Forschungsgemeinschaft (SFB 176)
Send offprint requests to U. Trendelenburg at the above addresswith the technical assistance of M. Babl 相似文献
15.
Fátima Martel Isabel Azevedo Walter Osswald 《Naunyn-Schmiedeberg's archives of pharmacology》1993,347(4):363-370
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 相似文献
16.
A. Langeloh H. Bönisch U. Trendelenburg 《Naunyn-Schmiedeberg's archives of pharmacology》1987,336(6):602-610
Summary The mechanism of action of indirectly acting sympathomimetic amines was studied in the rat vas deferens, after inhibition of vesicular uptake (by reserpine), of MAO (by pargyline) and of COMT (by U-0521). 1. K
m-values for the neuronal uptake of 12 substrates were determined as the IC50 of the unlabelled substrate inhibiting the initial rate of neuronal uptake of 0.2 mol/l 3H-(–)-noradrenaline. The IC50 ranged from 0.35 mol/l (for (+)-amphetamine) to 44.3 mol/l (for 5-HT). The V
max (determined for 8 substrates) was substrate-dependent. 2. Tissues were loaded with 0.2 mol/l 3H-(–)-noradrenaline and then washed out with amine-free solution. All 12 substrates of uptake1, induced an outward transport of 3H-noradrenaline, and equieffective concentrations were positively correlated with K
m. Moreover, the EC50 for release greatly exceeded K
m. It is proposed that this discrepancy between EC50 and K
m is indicative of the fact that at least four factors (each one in strict dependence on K
m) contribute to the initiation of outward transport of 3H-noradrenaline: a) the appearance of the carrier on the inside of the axonal membrane (facilitated exchange diffusion), b) the co-transport of Na+, c) the co-transport of Cl– (both lowering the K
m for 3H-noradrenaline at the inside carrier), and d) inhibition of the re-uptake of released 3H-noradrenaline (through competition for the outside carrier). 3. At least for amezinium, V
max. appears to limit the maximum rate of outward transport. 4. For some substrates (especially for the highly lipophilic ones) bell-shaped concentration-release curves were obtained. Apparently, inward diffusion of the substrates can lead to partial saturation of the inside carrier. Moreover, if release is expressed as a FRL (fractional rate of loss), loading with 37 mol/l 3H-(–)-noradrenaline decreased the releasing effect of various substrates. In this case the inside carrier appears to be partially saturated by the high axoplasmic concentration of 3H-noradrenaline. 5. Very high concentrations (especially of highly lipophilic substrates) were able to induce an additional intraneuronal release mechanism, presumably by increasing the pH inside storage vesicles.Abbreviations COMT
catechol-O-methyl transferase
- DOMAA
dihydroxymandelic acid
- DOPEG
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 (Bo 521, Tr 96 and SFB 176). Some of the results were presented to the German Pharmacological Society (Langeloh 1986)A. L. was the recipient of a fellowship of the Humboldt-Foundation
Send offprint requests to: U. Trendelenburg 相似文献
17.
C. L. Schönfeld U. Trendelenburg 《Naunyn-Schmiedeberg's archives of pharmacology》1991,344(5):520-526
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 相似文献
18.
Nicola N. Westwood Deborah L. Scarcella Lesley J. Bryan-Lluka 《Naunyn-Schmiedeberg's archives of pharmacology》1996,353(5):528-535
Previous pharmacological studies have demonstrated that pulmonary endothelial cells and noradrenergic neurones possess the same transporter for inward transport of catecholamines, uptake1. In noradrenergic neurones, it has been shown that uptake1 is also involved in the carrier-mediated outward transport, or efflux, of noradrenaline and dopamine. The aim of the present study was to examine the efflux of noradrenaline and dopamine from perfused lungs of rats to determine whether uptake1, in addition to diffusion, mediates efflux of catecholamines from pulmonary vascular endothelial cells.The effects of reducing the cellular sodium gradient and of substrates and inhibitors of uptake1 on the efflux of 3H-noradrenaline and 3H-dopamine from rat lungs were measured. Isolated; perfused lungs of rats (monoamine oxidase and catechol-0-methyltransferase inhibited) were loaded with 3H-(–)noradrenaline or 3H-dopamine for 10 min followed by perfusion with either (1) a low sodium, amine-free: Krebs solution, in which NaCl was replaced by either Tris.HCl or LiCl, for 15 or 10 min, respectively or (2) amine-free Krebs solution for 30 min in the absence or presence of a substrate or inhibitor of uptake1 for the last 15 min. The rate constants for spontaneous efflux of noradrenaline and dopamine from the lungs were 0.0163 min–1 and 0.0466 min–1, respectively. When NaCl was replaced by Tris.HCl during efflux, the rate constants for efflux of noradrenaline and dopamine were increased 2.5-fold and 3-fold, respectively, whereas, when NaCl was replaced by LICl, the rate constants were increased 8-fold and 4-fold, respectively. The uptake1 substrates, dopamine (1 and 3 mol/l) and adrenaline (40 mol/l), both caused a rapid and marked increase in the efflux of noradrenaline, while noradrenaline (4 mol/l) had a similar effect on the efflux of dopamine. The
uptake
1 inhibitors, imipramine (3 and 10 mol/l) and nisoxetine (50 nmol/l), caused small and gradual increases in the efflux of noradrenaline and dopamine from rat lungs.These results demonstrate that efflux of noradrenaline and dopamine from rat lungs is affected by alterations in the normal sodium gradient across the cell and by drugs that interact with the uptake1 transporter. Thus, it can be concluded that the spontaneous efflux of catecholamines from pulmonary vascular endothelial cells is mediated predominantly by uptake1. In addition, efflux of catecholamines from the lungs has a diffusional component, which, combined with inhibition of reuptake, accounts for the small increase in amine efflux by inhibitors of uptake1.Abbreviations COMT
Catechol-O-methyltransferase
- FRL
Fractional rate of loss
-
K
m
Michaelis or half-saturation constant
-
t
out
rate constant for efflux
-
k
uptake
rate constant for uptake
- MAO
monoamine oxidase
-
t
/12
half-time for efflux
- U-0521
3,4-dihydroxy-2-methylpropiophenone
-
V
max
maximal rate of uptake
Preliminary results of this study were presented to the 1993 Meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (Scarcella et al. 1993). 相似文献
19.
V. G. Wilson M. Grohmann U. Trendelenburg 《Naunyn-Schmiedeberg's archives of pharmacology》1988,337(4):397-405
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) 相似文献
20.
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 相似文献