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     

Effect of catechol-O-methyl-transferase (COMT) inhibition on the vascular and metabolic responses to noradrenaline,isoprenaline and sympathetic nerve stimulation in canine subcutaneous adipose tissue

Summary The effect of COMT-inhibitors (U-0521, H 22/54) on lipolysis and on vascular responses induced by noradrenaline, isoprenaline and nerve stimulation in canine adipose tissue in situ has been studied.COMT inhibition, before or after -adrenoceptor blockade, did not influence the -adrenoceptor mediated vasoconstriction due to nerve stimulation (2–5 Hz). Vasoconstriction due to close i.a. noradrenaline was unaffected at a dose of 2×10^–10 moles but at a dose of 10^–9 moles the peak vasoconstriction was enhanced but the duration of the response was not changed. Prior -adrenoceptor blockade did not alter the responses to U-0521.After -adrenoceptor blockade with Hydergine (120–300 g i.a.) a vasodilatation was induced by noradrenaline 2×10^–10 mole. This response and that produced by isoprenaline (2×10^–11 or 10^–10 moles) were enhanced after U-0521-treatment, but the vasodilating response to nerve stimulation (2 Hz) after -adrenoceptor blockade was unaffected.Lipolysis induced by nerve stimulation or noradrenaline in vivo was increased 50% and that induced by isoprenaline was increased 100% by COMT inhibition. U-0521 did not alter basal lipolysis in vitro but enhanced lipolysis induced by 0.1 M noradrenaline significantly, both in the presence and in the absence of theophylline.The present results shows that COMT influences the effective concentration of isoprenaline more than that of noradrenaline. In the vasculature the effects of exogenous noradrenaline is influenced to a larger degree than those induced by nerve stimulation. However, COMT blockade increased lipolysis induced by equieffective doses of exogenous noradrenaline and nerve stimulation to an equal extent. These results are in agreement with the idea that COMT is of physiological importance mainly at receptor sites that are not in close contact with sympathetic nerve endings and supports the idea that vascular -adrenoceptors are innervated receptors. Vascular -adrenoceptors on the other hand seem to be humoral receptors unrelated to sympathetic nerve endings and preferentially stimulated by circulating noradrenaline. The -adrenoceptors on the fat cells may be of both types, i.e. innervated and humoral.     

The influence of oestrogen and oestrogen metabolites on the sensitivity of the isolated rabbit aorta to catecholamines

Summary 1. In the present study the influence of oestradiol, catechol oestrogens, and O-methylated oestrogens was determined on the contractile responses of the isolated rabbit aorta to (–)-adrenaline. 2. Oestradiol (40 mol/l), 2-hydroxyoestradiol (2OHE₂) (20 mol/l), and 2-methoxyoestradiol (2MeOE₂) (20 mol/l) all sensitized the rabbit aorta to contractile responses to (–)-adrenaline. Similarly, the 2-hydroxy and 2-methoxy derivatives of oestrone and oestriol also sensitized the aorta to (–)-adrenaline-induced contractions. The largest degree of sensitization was seen in the presence of the 2-methoxysteroids. 3. Oestradiol and 2OHE₂ did not increase responses of the aorta to (–)noradrenaline, while slight potentiation of contraction was seen in the presence of 2MeOE₃. 4. The potentiating effect of 2OHE₂ on contractile responses to (–)-adrenaline was abolished by prior treatment of the tissue with a COMT inhibitor (U-0521, 55 mol/l). Conversely, pretreatment of the tissue with 2OHE₂ prevented the augmented aortic contraction to (–)-adrenaline usually seen after inhibition of COMT. The non-additive nature of the sensitization seen after combined treatment with 2OHE₂ and U-0521 was qualitatively similar to that seen following combined exposure to maximally effective concentrations of U-0521 and an inhibitor of extraneuronal uptake (hydrocortisone 100 mol/l). 5. Oestradiol and 2MeOE₂ reduced the formation of both the ³H-O-methylated, ³H-deaminated and the ³H-O-methylated deaminated metabolites of ³H-(–)-adrenaline (0.15 mol/l) during exposure of the aorta to the tritiated catecholamine. Treatment of the aorta with the extraneuronal uptake inhibitor phenoxybenzamine (33 mol/l) produced a similar inhibition of formation of all ³H-metabolites of ³H-(–)-adrenaline. 6. While preincubation with 2OHE₂ markedly reduced the formation of ³H-O-methylated and ³H-O-methylated deaminated metabolites from ³H-(–)-adrenaline (0.15 ol/l), the formation of ³H-deaminated adrenaline metabolites was considerably enhanced by exposure to 2OHE₂. Pretreatment of the aorta with the COMT inhibitor U-0521 produced a similar effect on ³H-adrenaline metabolism.7. Oestradiol, 2OHE₂, and 2MeOE₂ did not alter the tritium contents retained in segments of aorta incubated with ³H (±)-isoprenaline (0.15 ol/l). In contrast, the three steroids reversed the enhanced retention of tritium in the aorta induced by prior inhibition of COMT (U-0521 55 mol/l).8. It is concluded that the biotransformation of oestradiol to its 2-hydroxy and 2-methoxy metabolites does not diminish the potentiating actions of the steroids on adrenaline-induced aortic contractions. Oestradiol and 2MeOE₂ appear to produce their effects through inhibition of the extraneuronal amine uptake process, while 2OHE₂, a substrate for COMT, probably exerts is sensitizing through a combination of inhibition of the extraneuronal uptake process and competitive inhibition of COMT.Abbreviations 2OHE₁ 2-hydroxyoestrone - 2OHE₂ 2-hydroxyoestradiol - 2OHE₃ 2-hydroxyoestriol - 2MeOE₁ 2-methoxyoestrone - 2MeOE₂ 2-methoxyoestradiol - 2MeOE₃ 2-methoxyoestriol - U-0521 34-dihydroxy-2-methylpropiophenone Supported by the part in West Virginia University Biomedical Fund; American Heart Association, West Virginia Affiliate; and NIH grants HL30351 and K04-HL01228Selected aspects of this study were presented to the American Society for Pharmacology and Experimental Therapeutics (Barone et al. 1985; Panek et al. 1986) Send offprint requests to R. E. Stitzel at the above address     

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)     

Two distinct adrenoceptor-biophases in the vasculature: One for α-and the other for β-agonists

Summary Strips of two canine vessels with different patterns of sympathetic innervation were used: the mesenteric artery which has an adventitio-medial plexus and the saphenous vein in which nerve terminals are distributed throughout the media.The pD₂ of (–)-adrenaline for -and -adrenoceptors was determined for both vessels (in the presence of 0.5 M propranolol and 7 M phentolamine, respectively; in the latter case the strips were contracted by 0.28 M prostaglandin F2) and found to be very similar: 6.96 and 7.01 in the saphenous vein and 6.77 and 6.91 in the mesenteric artery, respectively. The similarity of pD₂ for adrenaline acting on -and -adrenoceptors in both preparations allowed us to compare the effect of inhibition of neuronal uptake by 12 M cocaine with that of inhibition of COMT by 50 M dihydroxy-2-methyl propiophenone (U-0521) on: a) the potency of adrenaline (noradrenaline was used in the saphenous vein only) acting on -and -adrenoceptors and b) the time required by the strips to recover 50% in oil (t ₅₀) after contractions or relaxations caused by 0.23 M adrenaline. In the saphenous vein cocaine increased the potency of both adrenaline and noradrenaline for -effects more than for -effects (2.8 times vs. no increase for adrenaline; 7.1 vs. 1.9 times for noradrenaline) and U-0521 increased the potency of both adrenaline and noradrenaline for -effects more than for -effects (4.1 vs. 2.6 times for adrenaline; 1.8 times vs. no increase for noradrenaline); regarding the termination of action of adrenaline, cocaine prolonged the t ₅₀ 1.6 times after contraction and did not change it after relaxation, whereas U-0521 prolonged the t ₅₀ 6.9 times after relaxation and only 1.8 times after contraction.In the mesenteric artery only sensitivity experiments were done. Cocaine increased the potency of adrenaline by a factor of 2.1 for the -effects while there was no influence on its potency with regard to its -effects, and U-0521 increased the potency of adrenaline for the -effects more than for -effects (3.9 vs. 1.8 times, respectively).These results show that there are two different biophases for sympathomimetic agonists in the vasculature: one for -adrenoceptors which is more under the influence of the neuronal uptake and one for -adrenoceptors which is more under the influence of COMT activity. We conclude that -adrenoceptors are situated close to the nerve endings and -adrenoceptors close to COMT sites. Since these results do not differ qualitatively in two vessels with different patterns of innervation, we conclude that this asymmetry in the distribution of -and -adrenoceptors may be due to either an uneven distribution of cells with only one type of receptors each or due to an uneven distribution of receptors on the same cell.     

The kinetic characteristics of the extraneuronal O-methylating system of the dog saphenous vein and the supersensitivity to catecholamines caused by its inhibition

Summary The half-saturating outside concentration and the V _max of the extraneuronal O-methylating system of the dog saphenous vein were determined in vitro for 3 catecholamines: isoprenaline, adrenaline and noradrenaline.Strips pretreated with 1 mmol/l pargyline were exposed to 30 mol/l cocaine for 30 min before and during the 30 min incubation with the amines.Two methods were used to reach our aims: a) the classical one in which the ³H-O-methylated metabolites formed from a mixture of unlabelled and labelled amine were determined by using final concentrations of the substrate ranging from 0.2 and 12.8 mol/l and b) an indirect one in which 0.2 mol/l ³H-(±)-isoprenaline was used to assess the extraneuronal O-methylation of the tracer amine, and then those concentrations of unlabelled amines were determined which reduce the O-methylation of ³H-(±)-isoprenaline by 50% (IC₅₀).The half-saturating outside concentrations and the V _max obtained by the first method were: 1.3, 2.5 and 3.4 mol/l and 241, 317 and 294 pmol/g/min for ³H-(±)-isoprenaline, ³H-(±)-adrenaline and ³H-(-)-noradrenaline, respectively. The IC₅₀s obtained by the second method used were: 1.1, 0.6, 0.7 and 1.4 mol/l for (±)-isoprenaline, (-)-isoprenaline, (-)-adrenaline and (-)-noradrenaline, respectively.It was observed that the contraction of the strips caused by adrenaline and noradrenaline distorted IC₅₀ values. In the presence of 1 mol/l phentolamine the IC₅₀ for adrenaline and noradrenaline was about 2.5 times higher than in its absence.The relationship between the ratio of half-saturating outside concentration/ED₅₀ for -adrenoceptor-mediated responses for the three amines and the supersensitivity to them caused by inhibition of COMT was also assessed. It was observed that the ratio was 37, 8 and 1.3 for isoprenaline, adrenaline and noradrenaline, respectively and the supersensitivity caused by inhibition of COMT was 11.8-, 8.9- and 1.8-fold, respectively.     

The saturability of a site of loss and the degree of supersensitivity to agonists which are substrates of this site of loss

Summary The present investigation was undertaken to test the hypothesis that the experimentally determined degree of supersensitivity to an agonist caused by inhibition of a site of loss depends on the ratio K _m of the site of loss/ED₅₀ of the agonist.The influence of inhibition of neuronal uptake by cocaine on the -adrenoceptor-mediated effect of noradrenaline was studied on the dog saphenous vein; the influence of inhibition of COMT by U-0521 on the -adrenoceptor-mediated effect of isoprenaline was studied on the dog saphenous vein and on the guinea-pig trachea; the influence of inhibition of acetylcholinesterase by physostigmine on the effect of acetylcholine was studied on the guinea-pig ileum. To further extend the range of values of the ratio K _m/ED₅₀, several concentrations of phentolamine, propranolol or atropine were used to increase the ED₅₀ of the respective agonist. It was thus possible to determine the degree of supersensitivity caused by inhibition of a site of loss over a range of K _m/ED₅₀ values of 0.02 to 2,307.In seven situations the ratio K _m/ED₅₀ was higher than 10. In all of these cases the supersensitivity caused by inhibition of the site of loss was maximal. In eleven situations the ratio K _m/ED₅₀ was less than 10 and higher than 0.1 and the supersensitivity obtained was sub-maximal but was closer to the maximum, the closer the ratio was to 10. In two situations the ratio was less than 0.1 and no supersensitivity was obtained.The results confirm the hypothesis.     

Characteristics of the cocaine-sensitive accumulation and O-methylation of 3H-(−)-noradrenaline by rabbit endometrium

Summary 1. The extraneuronal uptake and O-methylation of 2,5,6 ³H-(–)-noradrenaline was studied in segments of uterine endometrium from rabbits pretreated with 17-oestradiol and progesterone. 2. The uptake of ³H-noradrenaline was measured in MAO- and COMT-inhibited tissues and obeyed Michaelis-Menten kinetics with an apparent K _m of 78 mol/1 and a V _max of 5.4 nmol/g min. Uptake was inhibited by low Na⁺ and by potential substrates in the order dopamine > (–)adrenaline > (–)isoprenaline = 5-hydroxytryptamine. 3. Following uptake at 1.2 mol/1, efflux of ³H-noradrenaline was slow and appeared to be from two compartments, of which the first (I) had a t1/2 of 53 min and a capacity of 1.8 nmol/g. The presence of the second compartment (II) was inferred from the tissue content of ³H after 60 min of efflux, which was 3–4 times greater than predicted if the ³H was present in compartment I only. Following incubation with ³H-noradrenaline in the presence of cocaine 30 mol/1 the ³H efflux was rapid and the combined capacities of compartments I and II were greatly decreased. 4. ³H-NMN formation, measured in MAO-inhibited tissues, obeyed Michaelis-Menten kinetics with a half-saturating outside concentration of 12 mol/1 and a V _max of 0.9 nmol/g · min. The formation was inhibited by the neuronal uptake inhibitors, desipramine 3 mol/1 and metaraminol 100 mol/1 (each by 80%), but was unaffected by the extraneuronal uptake inhibitor, NMN 100 mol/1, and by oxytetracycline 100 mol/1 and methoxamine 10 mol/1. 5. ³H-NMN formation was inhibited to a small extent (by 30%) by hydrocortisone in a concentration which inhibited ³H-NMN formation in the myometrium by 84%. 6. It is concluded that the extraneuronal uptake of exogenous noradrenaline in the endometrium (a) resembles uptake in sympathetic nerves in its sensitivities to sodium ions and uptake inhibitors, and (b) resembles corticosteroid-sensitive extraneuronal uptake in that it has a low affinity for noradrenaline, but is linked with O-methylation in a fashion which renders uptake and O-methylation a relatively high-affinity, low-capacity system for removing noradrenaline.Abbreviations DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - COMT catechol-O-methyl transferase - MAO monoamine oxidase - NMN normetanephrine - U-0521 3,4-dihydroxy-2-methyl propiophenone Send offprint requests to J. A. Kennedy at the above address     

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

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

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)     

Effects of Δ8-THC,and Δ9-THC on the acquisition of a discriminative positional habit in rats

Using a reversal learning paradigm the dissociative effects of two tetrahydrocannabinols (THC) on the acquisition and reversal of a discriminative positional habit in rats were studied. A T-shaped water maze was used. From these experiments it is concluded that learning under the influence of ⁸-THC (10 and 20 mg/kg), and ⁹-THC (5 mg/kg) is state-dependent (StD) in the rat.Numbering system according to IUPAC rules.Parts of the results were presented at the Symposium on the chemistry and biological activity of cannabis, Stockholm, October 26–28, 1971 and at the Symposium on medical plants of Brazil, Sao Paulo, April 17–20, 1972.     

Inward transport of 3H-MPP+ in freshly isolated rat hepatocytes: evidence for interaction with catecholamines

1-Methyl-4-phenylpyridinium (MPP⁺), the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is efficiently taken up and accumulated by rat hepatocytes. However, the nature of the mechanism(s) involved in the hepatic uptake of MPP⁺ remains partially unknown. The aim of the present study was to further characterize the hepatic uptake of ³H-MPP⁺, namely by investigating the interactions of catecholamines (which are also efficiently taken up by rat hepatocytes) with MPP¹ transport.The accumulation of ³H-MPP⁺ in isolated rat hepatocytes occurred through saturable and non-saturable mechanisms. The kinetics of the saturable component of ³H-MPP⁺ uptake was as follows: V_max = 181.3 ± 11.1 pmol mg protein^–1 min^–1 and K_m = 47.1 M (27.9, 66.3) (n = 5). The diffusion constant (in ml mg protein^–1 min^–1) for the non-saturable uptake of ³H-MPP⁺ was 0.00068 (0.00052, 0.00083) (n = 5). From the analysis of the time course of ³H-MPP⁺ accumulation at a substrate concentration of 100 nM ³H-MPP⁺, it was found that the rate constant of inward transport of ³H-MPP⁺ into hepatocytes (k_in) was 15.7 ± 3.8 l mg protein^–1 min^–1, the rate constant of outward transport of ³H-MPP⁺ from hepatocytes (k_out) was 0.077 ± 0.023 min^–1 and the equilibrium accumulation (A_max) of ³H-MPP⁺ was 20.2 ± 2.0 pmol mg protein^–1 (n = 36). Decynium22 (1,1-diethyl-2,2-cyanide; 1 M) significantly reduced k_in to 6.1 ± 1.8 l mg protein^–1 min^–1 (P < 0.05) and the equilibrium accumulation (A_max) of ³H-MPP⁺ to 9.6 ± 1.3 pmol mg protein^–1 (P < 0.005) (n = 36). ³H-MPP⁺ accumulation (in cells incubated with 200 nM ³H-MPP⁺) was sensitive to (–)-adrenaline, (–)-isoprenaline, (–)-dopamine, (±)-adrenaline and (–)-noradrenaline. The most potent catecholamine in inhibiting ³H-MPP⁺ uptake was (–)-adrenaline, with an IC₅₀ of 99 (22, 449) M (n = 6). (–)-Adrenaline competitively inhibited ³H-MPP⁺ uptake, as it significantly increased the K_m value of ³H-MPP⁺ uptake (to 125.4 M (63.6; 187.1); P < 0.02; n = 3) but did not change the V_max value. The cyanide-derivatives decynium22 and cyanine863 (1-ethyl-2-([1,4-dimethyl-2-phenyl-6-pyrimidinylidene]methyl)quinolinium), which inhibit uptake₂ as well as the apical type of the renal transporter for organic cations, potently inhibited ³H-MPP⁺ uptake with IC₅₀'s of 1.4 (0.4–5.3) (n = 6) and 6.5 (2.6–16) (n = 4) M, respectively. Under conditions of monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) inhibition with either pargyline (500 M + Ro01-2812) (3,5-dinitropyrocatechol; 2 M) or pargyline (500 M) + U-0521(3,4-dihidroxy-2-methyl-propiophenone; l2 M)), (–)-adrenaline (up to 1 mM) had no inhibitory effect on the uptake of ³H-MPP⁺. Moreover, the uptake of ³H-MPP⁺ in the presence of pargyline + Ro 01-2812 was significantly lower (66.9 ± 30.4%; P < 0.05; n = 4) than in the absence of these compounds. Therefore, the effect of these MAO and COMT inhibitors on ³H-MPP⁺ uptake was examined. Interestingly enough, pargyline, Ro 01-2812 and U-0521 were found to inhibit the uptake of ³H-MPP⁺ (in cells incubated with 200 nM ³H-MPP⁺): 500 M pargyline, 2 M Ro 012812 and 100 M U-0521 decreased the accumulation of ³H-MPP⁺ to 38.1 ± 6.8 (n = 5), 60.5 ± 10.1(n = 7) and 71.3 ± 14.5 (n = 7) % of control, respectively.It is concluded that ³H-MPP⁺ is efficiently taken up by rat hepatocytes by a carrier-mediated mechanism sensitive to catecholamines, decynium22 and cy anine863, and to the enzyme inhibitors pargyline, Ro 01-2812 and U-0521.     

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     

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

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

Dopamine D1 and D2 mediation of the discriminative stimulus properties ofd-amphetamine and cocaine

Evidence suggests that stimulants such asd-amphetamine and cocaine act presynaptically by increasing the amount of dopamine (DA) available to stimulate postsynaptic DA receptors. Since two subpopulations of DA receptors (D₁ and D₂) exist, we investigated the role of both of these receptor subtypes in mediating the internal state produced by these stimulants. Two groups of rats (N=8/group) were trained to discriminate intraperitoneal (IP) injections of eitherd-amphetamine (1 mg/kg) or cocaine (10 mg/kg) from saline in a two-lever, water-reinforced, drug discrimination task. After stable performance was established (i.e., more than 85% correct under each training condition), substitution and combination tests were conducted with selective D₁ and D₂ agonists and antagonists. The D₂ agonist quinpirole (0.0313–0.125 mg/kg) mimicked both stimulant cues while the D₁ agoinst SKF 38393 (5–20 mg/kg) substituted partially for cocaine but notd-amphetamine. Combination tests with DA antagonists indicated that both the D₁ antagonist SCH 23390 (0.0063–0.25 mg/kg) and the D₂ antagonist haloperidol (0.125–0.5 mg/kg) attenuated the effects of both stimulants; in addition, the substitution of cocaine (20 mg/kg) ford-amphetamine was blocked by both DA antagonists. The ability of both D₁ and D₂ antagonists to attenuate the stimulus effects ofd-amphetamine and cocaine raises the possibility that a synergistic (enabling) interaction between D₁ and D₂ receptors may modulate stimulant cues.     

Comparative invstigations on the duration of action of two synthetic corticotropins

Summary The duration of the adrenocorticotrophic effects of two synthetic ACTH preparations was studied in healthy volunteers. One of the hormones, ^1–24 corticotropin, was adsorbed on zinc; the other, a D-serine¹-norleucine⁴-valinamide²⁵- ^1–25 corticotropin, was thought to be more stable, owing to the replacement of the three most susceptible amino acids in the ACTH molecule. — ^1–24 corticotropin, administered intramuscularly in a dose of 1 mg, showed biological activity lasting for 28 h, an effect equal to that which may be expected of depot preparations of pituitary extracts. D-serine¹-norleucine⁴-valinamide²⁵- ^1–25 corticotropin, administered intra muscularly in a dose of 0.16 mg, had only a weak adreno corticotrophic effect, whereas intravenously the same dose produced a greater effect that persisted for nearly 8 h. After intramuscular injection of both preparations a significant difference was found between the maximum responses shown by men and women.     

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     

Zinc,among a ‘cocktail’ of metal pollutants,is responsible for the absence of the terrestrial isopod Porcellio scaber from the vicinity of a primary smelting works

Porcellio scaber Latreille (Crustacea: Isopoda) of one month in age were reared for a year on leaf litter of field maple (Acer campestre) contaminated in the laboratory with a range of concentrations of cadmium, copper, lead or zinc. The metals were applied topically to the leaves as nitrates. Growth and survival, numbers of live offspring produced by females that matured, and concentrations of metals in adult isopods at the end of the experiment were measured.Critical concentrations of metals in food at which all the isopods died before producing offspring were 100 g Cd g^–1, 100 g Cu g^–1, 2000 g Pb g^–1 and 1000 g Zn g^–1 (on a dry weight basis). The relative toxicities of the four metals in the laboratory were compared with concentrations of cadmium, copper, lead and zinc in surface leaf litter in the vicinity of a primary smelting works at Avonmouth, South West England. The results support the hypothesis that the absence of Porcellio scaber from sites in the immediate vicinity of the factory is due to zinc poisoning. Although cadmium is approximately ten times more toxic to isopods than zinc in the laboratory, zinc is most likely to be killing isopods in the field because its concentration is always at least 30 times higher than cadmium in Avonmouth leaf litter, and more than 100 times higher at most sites.Populations of Porcellio scaber survive in field sites where surface leaf litter contains up to 5000 g Zn g^–1. This is at least five times higher than the critical concentration in laboratory experiments. Thus, the methodology for assessing metal toxicity described in this paper, exaggerates the potential effects of metals to isopods in the field. Such differences between laboratory and field toxicities of metals should be taken into account when environmental protection levels for metals are being proposed for soil invertebrates based on ecotoxicological tests conducted in the laboratory.     

A study of the adrenoceptor-mediated feedback mechanisms by using adrenaline as a false transmitter

Summary After incubation with 2.3 M ³H-(±)-adrenaline(³H-AD) or ³H-(-)-noradrenaline(³H-NA) for 60 min in the presence of hydrocortisone and U-0521, dog saphenous vein strips were perifused (the fluid containing cocaine+hydrocortisone+U-0521) and electrically stimulated. Tritium fractional release per shock was calculated for 1 and 5 Hz.Phentolamine (3 M) enhanced the overflow of tritium evoked by electrical stimulation at both frequencies but at 1 Hz the enhancement was higher for strips loaded with ³H-AD than for strips loaded with ³H-NA.Propranolol (1 M) reduced the overflow evoked by electrical stimulation at 1 Hz from the strips loaded with ³H-AD but not from those loaded with ³H-NA.Isoprenaline (0.04 M) increased the overflow of tritium evoked by electrical stimulation at 1 Hz from the strips loaded with ³H-NA but did not change that from strips loaded with ³H-AD.It is concluded that: a) the -adrenoceptor-mediated feedback mechanism is also present in the dog saphenous vein; b) this feedback mechanism also functions with the false transmitter AD; c) the use of ³H-AD as false transmitter revealed the existence of a -adrenoceptor-mediated positive feedback mechanism in this tissue.     

Does the carrier of chromaffin granules transport the protonated or the uncharged species of catecholamines?

Summary By osmotic lysis in the presence of urea ghosts (60–100 nmol catecholamine/mg prot.) were prepared from chromaffin granules (4–6 mol catecholamine/mg prot.) of the bovine adrenal medulla. In the presence of 1–300 mol/l³H-catecholamine and ATP-Mg²⁺, ghosts show a net uptake of catecholamine. The net uptake is sensitive to reserpine or agents (uncouplers and ammonium) which diminish the electrochemical potential difference for protons at the granule membrane (p). The same uptake was found by³H-counting or by fluorimetric measurements. At various pH-values (pH 6.2–82.) theK _m andV _max of the ATP-stimulated rate of uptake of³H-catecholamine into ghosts was determined (at 30°C) to identify the species of catecholamine (protonated, uncharged, or anionic) which is the substrate for the granule carrier. The pH difference (pH=pH_out-pH_in) and the electrical potential difference () were determined to calculate p under conditions of³H-catecholamine uptake. When the pH_out was increased (pH 6.2, 7.4, 8.2), the apparentK _m of uptake decreased (50, 5, 1–2 mol/l), showing a linear relation between pH and logarithm ofK _m. TheK _m was calculated for the uncharged catecholamine (with pK₁=8.8 and pK₂=10.0); it was nearly pH-independent and amounted to about 0.2 mol/l. TheV _max declined only in the extreme pH-range. Between pH 6.6 and 7.8V _max and p showed a slight increase from 16 to 20 nmoles/(mg prot.·min) and from 110 to 140 mV, resp. In the same pH-range the pH_in inside ghosts increased from pH 5.2 to 5.7, whereas was constant (30 mV). At constant pH_out (=7.3) ammonium (0–30 mmol/l) caused an increase of pH_in from 5.5 to 6.6. The increase of pH_in was accompanied by an increase ofK _m from 5 to 20 mol/l³H-catecholamine and by a decrease of bothV _max and p from 20 to 5 nmoles/(mg prot.·min) and from 123 to 85mV, respectively. From the dependence of theK _m of uptake on pH_out is concluded that the uncharged species of catecholamine is transported, whereas the dependence ofK _m on pH_in suggests that the translocation of the catecholamine-carrier complex across the granule membrane is not the rate-limiting step of catecholamine uptake.A preliminary account was presented to the Deutsche Pharmakologische Gesellschaft (Kobold and Burger 1983)