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

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

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

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

Possible physiological significance of the initial step in the catabolism of noradrenaline in the central nervous system of the rat

Summary The hypothalamus, the cerebral cortex and the cerebellar cortex of the rat were labelled in vitro with ³H-noradrenaline (³H-NA) and the metabolism of the tritiated transmitter was studied during spontaneous outflow and under conditions of release elicited by exposure to 20 mM K⁺.In the three areas of the central nervous system of the rat, ³H-NA accounted for approximately 40% of the total radioactivity in spontaneous outflow while the ³H-O-methylated deaminated fraction (³H-OMDA) and ³H-3,4-dihydroxyphenylglycol (³H-DOPEG) were the main metabolites. Exposure to the reserpine-like agent, Ro 4-1284 induced a selective increase in the spontaneous outflow of ³H-DOPEG, while the contribution of the ³H-OMDA metabolites to the release induced by Ro 4-1284 was very small.During ³H-transmitter release elicited by exposure to 20 mM K⁺, approximately 80% of the radioactivity was collected as unmetabolized ³H-NA, while ³H-DOPEG was the main metabolite formed under these experimental conditions. Exposure to cocaine prevented ³H-DOPEG formation from ³H-NA released by K⁺, indicating that ³H-DOPEG was formed after neuronal reuptake of the transmitter released by K⁺.After in vitro labelling with ³H-NA, the unmetabolized transmitter represented approximately 70% of the total radioactivity retained in the tissue. However, when ³H-NA was administered in vivo, by intraventricular injection, only 30% of the total radioactivity retained by the tissue was accounted for by ³H-NA, and 60% of the radioactivity corresponded to the ³H-OMDA fraction, most of which was retained as ³H-MOPEG sulfate.When the rats were pretreated with pyrogallol, free ³H-DOPEG accounted for nearly 50% of the radioactivity retained in the three areas of the central nervous system after in vivo labelling with ³H-NA. When monoamine oxidase was inhibited by pargyline and ³H-NA was administered by intraventricular injection, ³H-NMN accounted for approximately 50% of the total radioactivity retained in the three areas of the central nervous system of the rat.The results obtained are compatible with the view that formation of the deamined glycol is the first step in the metabolism of ³H-NA in the rat central nervous system. In addition, it is concluded that the determination of the levels of some NA metabolites retained in the central nervous system does not necessarily represent an accurate reflection of the degree of central noradrenergic activity or of selective metabolic pathways. Consequently, in studies on the metabolism of NA it is important to take into account not only the transmitter and its metabolites in the tissue but also in the outflow from the structures studied either under in vivo or in vitro conditions.     

Inhibition of adrenergic neuroeffector transmission in rabbit pulmonary artery and aorta by adenosine and adenine nucleotides

The effect of adenosine and adenine nucleotides on sympathetic neuroeffector transmission in the rabbit isolated pulmonary artery and aorta was studied. Adenosine (10(-5)-3 x 10(-4)M) decreased the contractile response of pulmonary artery and aorta evoked by electrical-field stimulation. The decrease was reversible. No tachyphylaxis developed. Inhibition of either adenosine deaminase by deoxycoformycin (3.6 x 10(-6)M) or of adenosine transport by dilazep (3 x 10(-6)M) did not alter the inhibitory effect of adenosine on the neurogenic contractions in the pulmonary artery. However, deoxycoformycin plus dilazep markedly enhanced the inhibitory effect of adenosine. The calcium antagonists nifedipine (1.5 x 10(-8)M) and nimodipine (1.3 x 10(-8)M) had no effect on the adenosine-induced inhibition. This was also the case with theophylline (5 x 10(-5)M), atropine (10(-7)M), and the prostaglandin synthetase inhibitors indomethacin (5 x 10(-5)M and suprofen (3 x 10(-5)M). The contractile response of the pulmonary artery elicited by exogenous (-)-noradrenaline (NA; 10(-9)-3 x 10(-4)M) was essentially not altered by adenosine (10(-5)-3 x 10(-4)M). Adenosine (10(-4)M) did not alter the spontaneous 3H-outflow from rabbit aorta preloaded with 3H-(-)-noradrenaline (3H-NA). Adenosine (10(-5)-3 x 10(-4)M), ADP (10(-4)M), ATP (10(-5)M), and inosine (10(-4)M) diminished the overflow of tritium from pulmonary artery and aorta preloaded with 3H-NA. The spontaneous outflow of tritium from aorta preloaded with 3H-NA consisted of 3H-NA (17%), 3H-dihydroxyphenylglycol (3H-DOPEG; 30%), 3H-dihydroxymandelic acid (3H-DOMA, 4%), 3H-O-methylated and deaminated metabolites (3H-OMDA; 42%), and 3H-normethanephrine (3H-NMN; 2%). Adenosine (10(-5) and 10(-4)M) enhanced 3H-DOPEG and 2H-NMN, decreased 3H-NA, and did not alter 3H-DOMA and 3H-OMDA. The stimulation-evoked overflow of tritium for aorta preloaded with 3H-NA consisted of 3H-NA (31%), 3H-DOPEG (18%), 3H-DOMA (2%), 3H-ONDA (46%), and 3H-NMN (3%). Adenosine (10(-5) and 10(-4)M) enhanced 3H-NA and 3H-DOPEG, decreased 3H-OMDA and did not alter 3H-DOMA and 3H-NMN. Adeosine (10(-6)-10(-4)M) did not alter the accumulation of 3H-NA (10(-8)M) by aorta. It is concluded that adenosine inhibits vascular sympathetic neuroeffector transmission by diminishing the release of transmitter from the nerve terminals.     

Effect of chlorpromazine on the metabolism of 3H-noradrenaline released from rabbit aorta

The effect of chlorpromazine on the metabolism of 3H-noradrenaline (3H-NA) released spontaneously or by electrical-field stimulation was studied on the rabbit isolated aorta preloaded with 3H-NA. Chlorpromazine (10(-6) M) neither altered the spontaneous outflow of total tritium nor had any major effect on the distribution of the 3H-outflow on 3H-NA and its 3H-metabolites. Chlorpromazine (10(-6) M) altered the distribution in stimulation-evoked 3H-overflow. Thus, 3H-NA and 3H-NMN were increased, 3H-DOPEG was markedly decreased and 3H-DOMA and 3H-OMDA were unchanged. It is concluded that chlorpromazine is an inhibitor of uptake-1.     

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

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

Frequency-Dependence of the Metabolism of 3H-Noradrenaline Released from Rabbit Isolated Aorta: Effects of a Combination of Cocaine and Corticosterone or Hydrocortisone

Abstract The effect of cocaine (neuronal uptake inhibitor) in combination with either hydrocortisone or corticosterone (extraneuronal uptake inhibitors) on the metabolism of ³H-noradrenaline (³H-NA) released spontaneously or by electrical-field stimulation was studied on the isolated rabbit aorta preloaded with ³H-NA. In the spontaneous outflow ³H-O-methylated and deaminated metabolites (³H-OMDA) accounted for 41% of the total radioactivity whereas ³H-NA represented only 22%. Cocaine (3 × 10^-5M) + hydrocortisone (10^-4 M) neither changed the spontaneous outflow of total tritium nor the distribution of the ³H-outflow on ³H-NA and its ³H-metabolites. However, cocaine (3 × 100^-5M) + corticosterone (4 × 10^-5M) enhanced the passive ³H-outflow, increased the percentage recovered as ³H-3,4-dihydroxyphenylglycol (³H-DOPEG), and reduced the percentage of ³H-OMDA + ³H-NMN. The effect of field-stimulation was investigated during and ofter stimulation at two frequencies: 3 and 10 Hz. The stimulation-induced ³H-overflow consisted mainly of unmetabolized ³H-NA and ³H-OMDA in the sample collected during stimulation. The ratio between ³H-NA and ³H-OMDA was strongly dependent on the frequency of stimulation, Thus it was 1:2 at 3 Hz, but 3:1 at 10 Hz. These ratios were about the same during the poststimulation period. On the other hand, whereas the formation of ³H-DOPEG from ³H-NA released during stimulation at both frequencies was small, it increased rapidly in the poststimulation sample. At 3 Hz, inhibition of neuronal and extraneuronal uptake by cocaine + steroids did not change the stimulation-induced ³H-overflow, but the distribution on ³H-NA and ³H-metabolites was markedly altered. Thus the percentage of ³H-N A was doubled, ³H-OMDA was halved, and ³H-DOPEG was almost abolished. At 10 Hz, however, cocaine + corticosterone decreased the stimulation-induced ³H-overflow, but did not change the proportions of ³H-NA and ³H-OMDA. Only the formation of ³H-DOPEG was markedly reduced. It is concluded that the distribution of stimulation-induced ³H-overflow on ³H-NA and its ³H-metabolites and the effect of neuronal and extraneuronal uptake inhibition on this is strongly influenced by the frequency of stimulation. Furthermore, inhibition of both neuronal and extraneuronal uptake does not fully prevent metabolism of ³H-NA released by electrical-field stimulation.     

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     

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

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

A thin-layer chromatographic procedure for the assay of labelled noradrenaline and its metabolites in tissues and in incubation medium.

A method for the extraction, separation and quantitative determination of [3H]noradrenaline [3H-NA] and its five major metabolites has been devised using thin layer chromatography. This procedure was used to study the pattern of 3H-NA and its metabolites in the total radioactivity of the tissues and that released spontaneously from the rat isolated vas deferens. Whereas in tissues 3H-NA represented almost all of the total radioactivity (94-8+/-0.47%), in the samples of spontaneous outflow it represented only 16-8+/-2.1%. The rest was mostly accounted for by the five metabolites, primarily 3H-DOPEG and 3H-MOPEG. These findings show that in the rat vas deferens 3H-NA is preferentially metabolized via the two glycol derivatives, i.e. 3H-DOPEG and 3H-MOPEG.     

Influence of MAO A and MAO B on the inactivation of noradrenaline in the saphenous vein of the dog

Specific inhibitors of MAO A (clorgyline 0.1 mumol/l) and of MAO B [(-)deprenyl 10 mumol/l] were used in dog saphenous vein strips in order to study the relative influence of the two types of MAO on: 1. Termination of contractile response to exogenous noradrenaline (NA); 2. Metabolism and accumulation of exogenous (3H)-NA; 3. Metabolism of 3H-NA released by electrical stimulation. To study the termination of contractile response to exogenous NA, the oil immersion technique was used to determine the time for half-relaxation. The experiments were performed on strips with or without treatment with cocaine 10 mumol/l (to inhibit neuronal uptake) plus U-0521 100 mumol/l (to inhibit catechol-O-methyl transferase) before and after exposure to MAO inhibitors. Clorgyline, but not (-)deprenyl enhanced significantly the time for half-relaxation of the strips, whether cocaine was present or not. To study the metabolism and accumulation of exogenous 3H-NA, the strips were incubated (with or without preincubation with cocaine) with 3H-NA 0.23 mumol/l and 2.3 mumol/l in the presence and in the absence of MAO inhibitors. The formation of deaminated metabolites was significantly reduced by clorgyline, but not by deprenyl. To study the metabolism of 3H-NA released by electrical stimulation, the strips were incubated with 3H-NA 1.4 mumol/l. In the presence of cocaine and U-0521, field stimulation was applied during two periods of 5 min (10 Hz, 100 V, 2 ms), in the absence or presence of MAO inhibitors. Under these experimental conditions clorgyline, but not deprenyl, abolished DOPEG formation, without affecting the other metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

β-ADRENOCEPTORS IN CIRCULAR AND LONGITUDINAL MYOMETRIAL MEMBRANES AND IN LUNG MEMBRANES FROM DIOESTROUS AND POST-PARTUM GUINEA-PIGS

1. We have examined the binding of (-)[125]-cyanopindolol ((-)[125I]-CYP) to membranes prepared from uterus and lung of dioestrous and post-partum (1-6 days) guinea-pigs. 2. The densities of beta-adrenoceptor binding sites in circular and longitudinal myometrium from dioestrous animals were similar, and approximately one-eight of those in the lung. Ascorbate and ethylenediaminetetraacetic acid in the incubation medium did not affect binding. 3. The numbers and affinities of beta-adrenoceptor binding sites in both myometrial layers and in lung parenchyma from post-partum animals were similar to those in corresponding tissues from dioestrous animals. 4. The distribution of beta-adrenoceptor binding sites in the post-partum uterus was examined using receptor autoradiography. Binding to circular and longitudinal muscle layers and to the endometrium was inhibited by (-)-propranolol (1 mumol/l), by the beta 2-adrenoceptor selective antagonist ICI 118,551 (70 nmol/l), but not by the beta 1-adrenoceptor selective antagonist CGP 20712A (100 nmol/l), indicating that the beta-adrenoceptor present was of the beta 2-subtype. 5. The ability of isoprenaline to compete for (--)[125]-CYP binding sites in uterine membranes from post-partum animals was approximately twice that in corresponding preparations from dioestrous animals. 6. Changes in the numbers or affinity of beta 2-adrenoceptors cannot account for the marked and selective enhancement of the actions of sympathomimetic amines at beta-adrenoceptors previously observed in longitudinal myometrium taken from post-partum guinea-pigs. It is suggested that enhancement of later steps in the chain of events between beta-adrenoceptor occupancy and uterine relaxation, and/or a decrease in the contribution of alpha-adrenoceptors to the net effect of the amine might provide alternative explanations.     

Predominance of oxidative deamination in the metabolism of exogenous noradrenaline by the normal and chemically denervated human uterine artery

Summary Longitudinal strips were prepared from human uterine arteries obtained at hysterectomy. The artery had a low content of noradrenaline and dopamine, contrasting with a high content of the deaminated catechols, dihydroxyphenylglycol (DOPEG) and dihydroxymandelic acid (DOMA), which together represented 98% of endogenous catechols.When incubated with ³H-noradrenaline (0.1 mol/l), the uterine artery removed, accumulated and metabolized noradrenaline. Deaminated metabolites predominated, DOMA being the most abundant metabolite.Cocaine markedly reduced the accumulation of ³H-noradrenaline and abolished ³H-DOPEG formation, but did not change ³H-DOMA. Selective monoamine oxidase (MAO) inhibitors (clorgyline, selegiline and 2-amino ethyl carboxamide derivatives) caused a marked decrease in the amounts of ³H-DOPEG, ³H-DOMA and ³H-O-methylated and deaminated metabolites (OMDA) formed by the tissue and an increase in ³H-normetanephrine (NMN) formation. Inhibition of catechol-O-methyltransferase suppressed NMN formation and reduced that of OMDA; hydrocortisone slightly depressed the formation of DOMA and OMDA.Homogenates of the uterine artery deaminated ³H-5-HT, ¹⁴C-phenylethylamine and ³H-tyramine; inhibition curves of the deamination of ³H-tyramine by clorgyline and selegiline were compatible with the presence of both MOA A and MOA B.Exposure of the strips to 6-hydroxydopamine (1.5 mmol/l for 20 min; 3 exposure periods followed by washout periods of 15,15 and 30 min) resulted in complete and selective chemical denervation of the arterial tissue. This chemical denervation had effects which were similar to those of cocaine. The 2-amino ethyl carboxamide derivatives markedly reduced the formation of deaminated metabolites by the denervated strips.The semicarbazide-sensitive amine oxidase inhibitor semicarbazide reduced the formation of ³H-DOMA and ³H-DOPEG in intact strips, but was devoid of action in the denervated ones.It is concluded that, in the human uterine artery, deamination predominates over O-methylation and that extraneuronal deamination, leading to the formation of DOMA (and of OMDA) plays a major role in the metabolism even of low concentrations of exogenous noradrenaline.Abbreviations COMT Catechol O-methyltransferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - HPLC-ED high pressure liquid chromatography with electrochemical detection - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase - NMN normetanephrine - 6-OHDA 6-hydroxydopamine - OMDA O-methylated and deaminated metabolites of noradrenaline (3-methoxy-4-hydroxyphenylglycol and 3-methoxy-4-hydroxymandelic acid) - Ro 01-2812 3,5-dinitropyrocatechol - Ro-19-6327 N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide hydrochloride - Ro 41-1049 N-(2-aminoethyl)-5-(mfluorophenyl)-4-thiazole carboxamide hydrochloride Supported by Instituto Nacional de Investigação Científica (INIC, FmP1) and Junta Nacional de Investigação Científica e Tecnológica (JNICT). Fatima Martel is a PhD student with a grant from JNICT Send offprint requests to W. Osswald at the above address     

Kinetics of the O-methylating system for isoprenaline in the trachea and aorta of rabbit

Segments of tracheal smooth muscle or aorta from rabbits pretreated with reserpine (1 mg/kg) were incubated in 3H-isoprenaline (0.5-60 mumol/l). Steady-state rates of O-methylation were determined by measuring the formation of 3-O-methylisoprenaline (OMI) after incubation of tracheal and aortic tissues for 30 min and 10 min, respectively. The steady-state O-methylation of isoprenaline in rabbit trachea was saturable, at least up to 60 mumol/l isoprenaline. In rabbit aorta, the O-methylation appeared to be saturable up to 30 mumol/l isoprenaline, but the rate of O-methylation increased for higher concentrations. The Km values for the saturable component of O-methylation were 11.8 mumol/l in trachea and 3.03 mumol/l in aorta. The Vmax values were 0.51 nmol X g-1 X min-1 in trachea and 0.56 nmol X g-1 X min-1 in aorta. In tissues incubated in 0.5 mumol/l isoprenaline, 100 mumol/l corticosterone caused 78% inhibition of OMI formation in trachea and 86% inhibition in aorta. There was no inhibition of OMI formation by 100 mumol/l corticosterone in tracheal or aortic tissues incubated in 60 mumol/l isoprenaline. Model calculations showed that the experimental results in trachea and aorta (3. above) were consistent with (a) entry of isoprenaline into the cells in the tissues by extraneuronal uptake and diffusion, and (b) exposure of the isoprenaline to intracellular catechol-O-methyltransferase with Vmax enzyme much less than Vmax uptake.     

PREJUNCTIONAL ACTIONS OF TACRINE ON AUTONOMIC NEUROEFFECTOR TRANSMISSION IN RABBIT ISOLATED PULMONARY ARTERY AND RAT ISOLATED ATRIA

1. This study investigated the effects of tacrine (1,2,3,4-tetrahydro-9-aminoacridine) on the resting and stimulation-induced (SI) release of radioactive substances from isolated preparations of rat atria and rabbit pulmonary artery in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline, and from rat atrial preparations in which cholinergic transmitter stores had been labelled with [3H]-acetylcholine. In addition, the effect of tacrine on the uptake of [3H]-noradrenaline by noradrenergic nerves in rat atria was determined. 2. Tacrine produced concentration-dependent increases in the resting efflux of radioactivity from both the [3H]-noradrenaline-loaded artery and atrial preparations. Blockade of neuronal amine transport with desipramine reduced the release of radioactivity evoked by tacrine from atria but not that evoked from artery preparations. Inhibition of monoamine oxidase by pargyline pretreatment markedly reduced the tacrine-evoked release of radioactivity in both atrial and artery preparations. 3. The radioactivity released from [3H]-noradrenaline-labelled rat atrial preparations by 30 mumol/L tacrine consisted entirely of the deaminated metabolite [3H]-DOPEG. The evoked release of [3H]-DOPEG from atria was reduced by approximately 50% by desipramine (1 mumol/L). When atrial monoamine oxidase had been inhibited by pargyline treatment in vivo and in vitro, 30 mumol/L tacrine evoked the release of [3H]-noradrenaline instead of [3H]-DOPEG. However, the amounts of [3H]-noradrenaline released by tacrine when monoamine oxidase was inhibited were only about 25% of the amounts of [3H]-DOPEG released in untreated atria. 4. Tacrine, in concentrations of 1 and 10 mumol/L, enhanced the release of radioactivity evoked by field stimulation of [3H]-noradrenaline-loaded rabbit pulmonary artery preparations. This effect was unaltered by desipramine or pretreatment with pargyline. However, in artery preparations pretreated with pargyline, a high concentration of tacrine (100 mumol/L) markedly reduced SI efflux. In contrast to the findings with artery preparations, tacrine (1-30 mumol/L) did not alter SI efflux in rat atrial preparations. 5. It is concluded that tacrine displaces noradrenaline from intraneuronal transmitter stores of sympathetically-innervated tissues, and that the displaced amine is totally metabolized by monoamine oxidase before leaving the nerve terminals. When deamination of neuronal cytoplasmic noradrenaline is prevented, only a portion of the noradrenaline displaced from storage vesicles passes to the extracellular space. It is likely that the transfer of cytoplasmic noradrenaline out of the terminals is limited by the activity of the amine transport mechanism.     

INTERACTION OF CHOLINE WITH NICOTINIC AND MUSCARINIC CHOLINERGIC RECEPTORS IN THE RAT BRAIN IN VITRO

The ability of choline to interact with nicotinic receptors was investigated by measuring its ability to inhibit the specific binding of [3H]-nicotine in rat brain. Choline, with an IC50 of 241 mumol/l, was three times more potent than its analogue deanol and almost 1000-fold less potent than acetylcholine. Choline also inhibited the binding of the antagonist [3H]-quinuclidinyl benzilate (IC50 = 2.5 mmol/l) and of the agonist [3H]-oxotremorine-M (IC50 = 165 mumol/l) to muscarinic cholinergic receptors. These results indicate that choline is able to interact directly, in vitro with brain cholinergic receptors of both the nicotinic and muscarinic type.     

Histamine H3 receptors are not involved in the regulation of rat gastric secretion.

The effects of histamine H3 receptor activation [(R)alpha-methylhistamine] and blockade (thioperamide) on rat gastric secretion were determined in vivo and in vitro. (R)alpha-Methylhistamine (0.1-5 mumol/kg i.p.) did not modify secretory volume and acidity in pylorus-ligated rats; it did not affect basal acid secretion and the secretion stimulated by histamine, pentagastrin and 2-deoxy-D-glucose in the lumen-perfused stomach of anaesthetized rats, when administered by continuous infusion (0.01-1 mumol/kg/h) or bolus injection (0.05-25 mumol/kg). In this preparation, the H3 agonist increased acid secretion at doses of 3-25 mumol/kg i.v., the effect being antagonized by famotidine. In the isolated gastric fundus from immature rats both (R)alpha-methylhistamine (0.01-10 mumol/l) and thioperamide (0.01-1 mumol/l) were totally ineffective against both spontaneous and stimulated gastric secretion. These results suggest that histamine H3 receptors are unlikely to have a role in regulating gastric acid secretion in the rat.     

α1- AND α2-ADRENOCEPTORS OF THE CIRCULAR MYOMETRIUM FROM THE DIOESTROUS GUINEA-PIG

1. In order to investigate the nature of the alpha-adrenoceptors mediating contraction of circular myometrium from dioestrous guinea-pigs, the effects of several adrenoceptor antagonists upon log concentration curves to noradrenaline and phenylephrine have been examined. 2. In the presence of ICI 118,551 and nisoxetine both phenylephrine and noradrenaline produced concentration-dependent contractures of circular myometrium from virgin dioestrous guinea-pigs. Noradrenaline was the more potent and produced larger maximal contractions. Indomethacin (1 mumol/l) decreased the maximum effects of phenylephrine but not those of noradrenaline. Xylazine produced indomethacin-sensitive contractions which were not dose-related and which never exceeded 40% of those evoked by noradrenaline. Responses to xylazine and to noradrenaline, but not those to phenylephrine, were reduced in a low calcium solution. 3. Prazosin produced competitive antagonism of the effects of phenylephrine upon preparations of circular myometrium from virgin and parous dioestrous animals; pA2 values were both 8.1. Phentolamine also competitively antagonized the effects of phenylephrine (virgin animals, pA2=7.7). 4. Both prazosin and phentolamine antagonized the effects of noradrenaline upon preparations from virgin dioestrous animals, however, Schild plot analysis did not indicate a simple bimolecular interaction between agonist and receptors. In the presence of prazosin the alpha 2-adrenoceptor antagonist idazoxan produced dose-dependent parallel shifts in the positions of the log concentration-response curves to noradrenaline; the pA2 was 7.7. In the presence of idazoxan or of indomethacin prazosin competitively antagonized the effects of noradrenaline; the pA2 values were 8.5 and 8.2 respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of [3H]-nitrendipine and [3H]-diltiazem to rat myocardial sarcolemma

The binding properties of two major and chemically distinct calcium antagonists, [3H]-nitrendipine and [3H]-diltiazem, were investigated in highly purified rat cardiac sarcolemma. In the case of [3H]-nitrendipine, there appeared a single set of high affinity binding sites with a B max of approximately 0.9 pmol X mg-1 protein and a KD of approximately 0.15 nmol/l. Because of the extremely high value obtained for KD (29 mumol/l), the specificity of [3H]-diltiazem binding seemed questionable. The specific binding of 0.1 nmol/l [3H]-nitrendipine to cardiac sarcolemma was inhibited by nitrendipine and nifedipine (1 mumol/l), only partly inhibited by verapamil (1 mumol/l), and was enhanced by diltiazem (0.1-10 mumol/l). The stimulation of [3H]-nitrendipine binding by diltiazem was associated with an increase in the number of binding sites, Bmax, but with no change in the KD or the Hill coefficient. An enantiomer of diltiazem (1-cis) neither stimulated nor inhibited the [3H]-nitrendipine binding. These results strongly suggest that major prototype calcium antagonists have distinct and variously interacting sites of action in the rat cardiac sarcolemma.     

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)