Mechanisms of the release of 3H-noradrenaline by dimethylphenylpiperazinium (DMPP) in the rat vas deferens

Summary In the rat vas deferens, DMPP is a substrate of uptake, (K_rn = 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     

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

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

The mechanism of the 3H-noradrenaline releasing effect of various substrates of uptake1: multifactorial induction of outward transport

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     

The steady-state concentration gradient for 3H-noradrenaline generated by uptake1 in the extracellular space of the rat vas deferens incubated with this amine

Summary The rat vas deferens was incubated with 0.2 mol/l ³H-noradrenaline for 60 min, washed out with amine-free solution for 100 min and then prepared for autoradiography (same tissues as presented by Azevedo et al. (1990) Naunyn-Schmiedeberg's Arch Pharmacol 342: 245 – 248). The autoradiography images were then digitized, and grain density was determined as a function of the distance from the surface of the tissue. When neither monoamine oxidase nor vesicular uptake was impaired, i. e. under control conditions, grain density declined monophasically exponentially towards the centre of the tissue. This decline amounted to 0.017 m^–1 or 0.124 varicosity^–1, since the average distance between varicosities was calculated to be 7.4 m. After inhibition of monoamine oxidase and vesicular uptake the rate constant was significantly reduced, and the grain density in close proximity of the surface of the tissue was also reduced.It is proposed that the distribution of grain density observed in controls reflects the steady-state concentration gradient that is generated by uptake₁ during the incubation with ³H-noradrenaline.During spontaneous efflux of ³H-noradrenaline one has to distinguish between re-uptake of the ³H-amine into the leaking varicosity and uptake en passant (during diffusion through the extracellular space). On the basis of the present results, the extent of uptake en passant was calculated (with a computer-assisted model) for the spontaneous efflux of heterogeneously distributed ³H-noradrenaline (after wash-out). Uptake en passant into varicosities located between the source of efflux and the medium amounted to about 55% of the net leakage of ³H-noradrenaline from all varicosities. Earlier experiments had indicated that the sum of the two uptake processes was responsible for the neuronal uptake of 90% of the gross leakage of ³H-noradrenaline from varicosities. Hence, the following appears to take place: about 78% of the gross leakage of noradrenaline from a varicosity are subject to re-uptake into the same varicosity. During diffusion to the medium, about 55% of the ³H-noradrenaline escaping re-uptake is then subject to neuronal uptake en passant. Send offprint requests to E. Schömig at the above address     

Inhibition by dopamine of 3H-noradrenaline release elicited by nerve stimulation in the isolated cat's nictitating membrane

Summary After loading the isolated nerve-muscle preparation of the cat nictitating membrane with ³H-(±)-noradrenaline the effects of exogenous dopamine and (-)-noradrenaline were determined on ³H-transmitter overflow elicited by nerve stimulation in the presence of cocaine, 29 M. Dopamine, 0.20 M, and (-)-noradrenaline, 0.18 M, inhibited ³H-noradrenaline release elicited by nerve stimulation at 4 or 10 Hz. Similar results were obtained with apomorphine 0.03 or 0.1 M. Chlorpromazine, 1 M, or pimozide, 1 M, antagonized selectively the reduction in ³H-noradrenaline release obtained with dopamine or apomorphine, without affecting the inhibition obtained with (-)-noradrenaline. Phentolamine, 1 M, antagonized more effectively the inhibitory effects of (-)-noradrenaline than those of dopamine. Phenoxybenzamine, 0.29 M, prevented the inhibition of ³H-transmitter overflow obtained with (-)-noradrenaline, dopamine or apomorphine. In the absence of cocaine neither chlorpromazine nor pimozide were able to increase ³H-transmitter overflow during nerve stimulation. In contrast to these results, block of -adrenoceptors by phentolamine or phenoxybenzamine resulted in an increase ³H-transmitter overflow during nerve stimulation. Inhibition by dopamine of ³H-transmitter overflow appears to be mediated through dopamine receptors probably located in the outer surface of adrenergic nerve endings. These dopamine receptors differ from the prejunctional -adrenoceptors that mediate the negative feed-back regulatory mechanism for noradrenaline release by nerve stimulation. The prejunctional inhibitory dopamine receptors are not involved in an endogenously mediated regulatory mechanism for noradrenaline release by nerve stimulation under normal conditions. The possibility that these dopamine receptors are involved in the hypotension commonly observed in patients with chronic l-Dopa treatment is discussed.     

The influence of the density of adrenergic innervation on the extracellular steady-state concentration gradient for 3H-noradrenaline

Summary After inhibition of extraneuronal uptake by corticosterone, isolated right atria and lengthwise halved vasa deferentia of the rat were incubated with 0.2 mol/l ³H-noradrenaline for 60 min, washed out for 100 min and then prepared for autoradiography. The autoradiographic images were digitized, and silver grain density was determined as a function of the distance from the surface.Silver grain density declined towards the centre of the tissue; the decline was monophasic exponential and significantly steeper in the vas deferens (0.016 m^–1) than in the less densely innervated right atrium (0.011 m^–1). Silver grain density at the surface of the tissue was higher in vas deferens than in right atrium.The results show that the extracellular steady-state concentration gradient for ³H-noradrenaline (generated by uptake, during the incubation with this amine) largely depends on the density of the adrenergic innervation.Supported by the Deutsche Forschungsgemeinschaft (SFB 176), by the Alexander von Humboldt-Stiftung (AvH-Forschungskooperation Europa funded by the BMFT) and by INIC (FmP1) Send offprint requests to E. Schömig at the above address     

A kinetic study of the release of noradrenaline by tyramine

Summary Dog saphenous vein strips obtained from untreated or reserpine-pretreated animals were incubated with 1,4 mol/l ³H-(–)noradrenaline for 60 min after inhibition of the noradrenaline-metabolizing enzymes and of extraneuronal uptake. At the end of the incubation period the strips were perifused during 200 min. Some strips were exposed to tyramine from the 100th to the 200th min of perifusion. A compartment analysis of spontaneous or tyramine-induced efflux of ³H-noradrenaline was made. The spontaneous efflux of strips obtained from untreated animals had a long half time (t/2=269 min), and most of the ³H-noradrenaline which accumulated in the strips did not participate in the efflux (bound fraction, representing 85% of tissue activity at the 100th min of perifusion). The strips were exposed to five concentrations of tyramine, ranging from 0.49–3,240 mol/l. At all concentrations, tyramine mobilized only one noradrenaline compartment. The increase of tyramine concentration decreased the bound fraction, which became negligible for the highest concentration of tyramine. The half time of ³H-efflux induced by tyramine decreased from 278–106 min when the tyramine concentration was increased from 0.49–40 mol/l. However, further increases of the concentration of tyramine up to 3240 mol/l did not result in a significantly lower half time.Strips obtained from reserpine-pretreated animals were characterized by a low accumulation of ³H-noradrenaline (403 ng/g vs. 1,374 ng/g for strips obtained from unpretreated animals, at the 100th min of perifusion), the efflux had a half time of only 122 min and the bound fraction was smaller than in strips obtained from unpretreated animals (bound fraction representing 47% of tissue activity at the 100th min of perifusion). Reserpine-pretreated strips were exposed to 40 mol/l tyramine. Under these experimental conditions there was no significant bound fraction. The ³H-efflux induced by tyramine decayed according to a two-compartment model, with half times of 8 and 50 min.The results support the view that in preparations from unpretreated animals the size of the noradrenaline pool available for release by tyramine is dependent on the concentration of the latter amine. Furthermore, the half time of the efflux evoked by tyramine in strips with intact vesicular stores is dependent on a rate-limiting process, probably of vesicular location.Results presented to the 4th Meeting on Adrenergic Mechanisms (Porto 1980). This work was supported by a grant from Instituto Nacional de Investigação Cientifica (Centro de Farmacologia e Biopatologia Química da Universidade do Porto)     

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

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

Effects of a monoamine oxidase A inhibitor,FLA 668 (+) on the adrenergic mechanisms of the dog saphenous vein

Summary FLA 668(+)[(S)-(+)-4-amino-2, -dimethylethylphenethylamine (+)-hydrogen bitartrate] selectively inhibited MAO type A in homogenates of dog saphenous vein, with a IC₅₀ of 1 mol/l for MAO A and >1 mmol/l for MAO B.At concentrations of 1 mol/l and higher, FLA 668(+) progressively decreased the formation of deaminated metabolites by saphenous vein strips incubated with³H-noradrenaline, with a preferential action on DOPEG formation. Normetanephrine formation increased but this increase did not wholly compensate for the reduction in the formation of deaminated metabolites.FLA 668(+) caused increased reactivity of saphenous vein strips to noradrenaline and markedly reduced the accumulation of³H-noradrenaline in the incubated tissue; both effects were still evident in the presence of clorgyline. After cocaine, FLA 668(+) caused no further increase in sensitivity.It is concluded that FLA 668(+) is, in the saphenous vein of the dog, a selective inhibitor of MAO type A and that it exerts a cocaine-like effect.Part of the results were presented to the Amine Oxidases: a Cambridge workshop (Caramona et al. 1984). Supported by INIC (Centro de Farmacologia e Biopatologia Quimica da Universidade to Porto)with a grant from the Portuguese-Spanish Academic Exchange Service     

Alpha-receptor-mediated modulation of 3H-noradrenaline release from rat brain cortex synaptosomes

Summary The effects of oxymetazoline and noradrenaline (in the presence of desipramine) on the release of ³H-noradrenaline from rat brain cortex synaptosomes were studied using a superfusion technique. Both drugs (at 1M concentrations) were found to reduce the depolarization-induced (15 mM K⁺) release of ³H-noradrenaline. The release-modulating effect of noradrenaline was antagonized by phentolamine and yohimbine.The data provide direct evidence for the hypothesis that -receptors modulating the release of noradrenaline are localized on varicosities of central noradrenergic neurones.     

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

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

Low level chromium (VI) inhalation effects on alveolar macrophages and immune functions in Wistar rats

In inhalation chambers, 5-week-old male Wistar rats of the strain TNO-W-74 were continuously exposed to submicron aerosols of sodium dichromate in concentrations from 25 (low level) to 200 g/m³ Cr (high level). Subacute exposure (28 days) to 25 and 50 g/m³ Cr resulted in activated alveolar macrophages with stimulated phagocytic activities, and significantly elevated antibody responses to injected SRBC's. After subchronic (90 days) low level exposure there was a more pronounced effect on activation of the alveolar macrophages, with increased phagocytic activities. However, at high Cr (VI) exposure level (200 g/m³), inhibited phagocytic function of the alveolar macrophages was seen. In rats which were exposed to this chromium aerosol concentration for 42 days, the lung clearance of inert iron oxide was reduced significantly. The humoral immune system was still stimulated at subchronic low chromium aerosol concentrations of 100 g/m³, but significantly depressed at 200 g/m³ Cr. These results show that respiratory defence and immunologic functions were stimulated or inhibited depending on dose and time of chromium (VI) inhalation.This paper was presented in part at the International Seminar on the Immunological System as a Target for Toxic Damage, November 6–9, 1984, Luxembourg     

Facilitation by GABA of the potassium-evoked release of 3H-noradrenaline from the rat occipital cortex

Summary The effects of GABA were studied on the release of ³H-noradrenaline (³H-NA) evoked by potassium or by tyramine from slices of the rat occipital cortex and on the release of ³H-NA elicited by nerve stimulation from the cat nictitating membrane.GABA (30–1000M) facilitated the potassium-evoked release of ³H-NA in a concentration dependent manner. This facilitatory effect was not antagonized by bicuculline (1–100 M) or by picrotoxin (1–100 M). Exposure to the GABA agonist muscimol (1–100 M) did not affect either the spontaneous or the potassium-evoked release of ³H-NA.The facilitatory effect of GABA on the release of ³H-NA elicited by potassium was observed when the occipital cortex slices were exposed to 20 mM K⁺ during 1 min. When depolarization was induced by 35 mM K⁺ exposure to GABA failed to enhance the release of the neurotransmitter. GABA 300 M did not affect the release of the labelled neurotransmitter evoked by exposure to tyramine 0.6 M.In the cat nictitating membrane prelabelled with ³H-NA, the stimulation evoked release of the labelled neurotransmitter was not affected by GABA (10–300 M).In conclusion, GABA has a facilitatory effect on the calcium-dependent, potassium evoked release of ³H-NA when the depolarization is of moderate degree. This effect of GABA appears to be selective for the central nervous system.Some of the findings described in this paper have been presented at the Meeting of the British Pharmacological Society (March, 1978, Guildford, U.K.)     

The heterogeneity of the neuronal distribution of exogenous noradrenaline in the rat vas deferens

Summary After loading of the incubated rat vas deferens with 0.2 mol/l ³H-noradrenaline (followed by 100 min of wash-out with amine-free solution), the efflux of endogenous and exogenous compounds was determined by HPLC with electrochemical detection and by column chromatography with scintillation counting. Two different types of heterogeneity of labelling were found. The first one is due to the preferential labelling of varicosities close to the surface of the tissue, the second one to the preferential labelling of vesicles close to the surface of loaded varicosities. As diffusion distances within the tissue and within varicosities are then longer for endogenous than for exogenous amine and metabolites, the composition of spontaneous efflux of exogenous compounds differed from that for endogenous compounds. Because of preferential neuronal and vesicular re-uptake of endogenous noradrenaline, the percentage contribution by noradrenaline to overall efflux was: endogenous < exogenous. While ³H-DOPEG was the predominant exogenous metabolite, DOPEG and MOPEG equally contributed to the endogenous efflux.Desipramine abolished the consequences of the first heterogeneity of labelling, i.e., it increased the efflux more for endogenous than for exogenous noradrenaline; moreover it decreased the efflux of ³H-DOPEG, but increased that of ³H-MOPEG. The reserpine-like compound Ro 41284, on the other hand, abolished the consequences of the second type of heterogeneity; it reduced the specific activity of total efflux (i.e., of the sum of noradrenaline + DOPEG + MOPEG) to the specific activity of the tissue noradrenaline. The degree of heterogeneity of labelling was reduced after inhibition of monoamine oxidase and also when the tissues were loaded with 2 or 20 mol/l ³H-noradrenaline.It is proposed that the various compartments and pools of noradrenaline described in the literature reflect the two heterogeneities described here.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - FRL fractional rate of loss (= rate of efflux/tritium content of tissue measured at onset of collection period) - HPLC high performance liquid chromatography - MAO monoamine oxidase - MOPEG methoxyhydroxyphenylglycol - NMN normetanephrine - VMA vanillylmandelic acid Send offprint requests to E. Schömig at the above addressThis study was supported by the Deutsche Forschungsgemeinschaft (SFB 176, Gr 490/5 and Scho 383/1). Some of the results were presented to the German Pharmacological Society (Schönfeld 1990; Trendelenburg 1990)     

Selective inhibition by amezinium of intraneuronal monoamine oxidase

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

Differential effects of α-β-methylene ATP on responses to nerve stimulation in SHR and WKY tail arteries

Summary The effects of ,-methylene-adenosine triphosphate, (,-methylene ATP, a P₂-receptor desensitising agent) have been evaluated on vasoconstrictor responses elicited by exogenous agonists or electrical field stimulation in isolated perfused SHR or WKY tail arteries and on tritium release elicited by electrical field stimulation in SHR-tail arteries pre-labeled with ³H-noradrenaline.Exposure to ,-methylene ATP (0.1 mol/l) significantly inhibited vasoconstrictor responses to electrical field stimulation in SHR tail arteries. These inhibitory effects were not further increased at a higher concentration of ,-methylene ATP (1 mol/l). In WKY tail arteries, ,-methylene ATP (1 mol/l) failed to significantly inhibit vasoconstrictor responses to electrical stimulation.In SHR tail arteries prelabelled with ³H-noradrenaline, ,-methyleneATP (1 mol/l) did not inhibit the stimulation evoked release of tritium. However, at this concentration, ,-methylene ATP significantly antagonized the vasoconstrictor responses of SHR tail arteries induced by exogenous ATP (1 mol/l), ,-methylene ATP (30 mol/l), a stable agonist at P₂-receptors, or 60 mmol/l KCl. These effects of ,-methylene ATP on contractile responses to KCl were not observed in WKY-tail arteries.In tail arteries obtained from reserpine pretreated SHR, despite a 85–95% decrease in endogenous noradrenaline tissue content, the vasoconstrictor responses induced by periarterial field stimulation were greatly diminished, but not abolished. These residual responses to periarterial field stimulation were not antagonized by prazosin (0.1 mol/l), but were practically abolished by the addition of ,-methylene ATP (1 mol/l).In tail arteries from WKY rats pretreated with reserpine, exposure to prazosin (0.1 mol/l) further reduced the residual responses elicited by electrical field stimulation. In these WKY-tail arteries, addition of ,-methylene ATP (1 mol/l) did not further inhibit the remaining vasoconstrictor response obtained in the presence of prazosin.While our results suggest a significantly greater cotransmitter role for ATP with noradrenaline in tail arteries of SHR compared with control normotensive WKY rats, additional effects of ,-methylene ATP not involving P₂ receptors cannot be entirely excluded.     

Simulation of outward transport of neuronal 3H-noradrenaline with the help of a two-compartment model

Summary In order to simulate the outward transport of ³H-noradrenaline induced by veratridine from adrenergic varicosities, a mathematical two-compartment model was developed in which the two compartments (representing axoplasm and storage vesicles) are arranged in series. Simulated results were compared with experimental results obtained with 100 mol/l veratridine + 1 mmol/l ouabain and rat vasa deferentia kept in calcium-free solution (Bönisch and Trendelenburg 1987). As in experiments, the time course of efflux of ³H-noradrenaline had a pronounced and early peak under RPU-conditions, a minor peak under PU-conditions, and solely a plateau under U-conditions (where R stands for pretreatment with reserpine, P for pretreatment with pargyline, and U for inhibition of COMT by U-0521). From the width of the peak of release, it was deduced that — under RPU-conditions — about 40% of neuronal ³H-noradrenaline are distributed into the axoplasm, about 60% into the storage vesicle. However, this estimate represents an average value; the results are compatible with the view that the ratio axoplasmic/vesicular ³H-noradrenaline is quite variable from rat to rat. Under U-conditions, calculations confirm that reserpinelike compounds induce an efflux of tritium that consists predominantly of deaminated ³H-metabolites. The stimulation of outward transport, on the other hand, causes an efflux of tritium that consists predominantly of ³H-noradrenaline; indeed, the efflux of deaminated ³H-metabolites declines (as it did in experiments). Simulations showed further that the highest rates of outward transport of ³H-noradrenaline were achieved when there was a simultaneous induction of outward transport of ³H-noradrenaline and a reserpine-like effect (as it is known to occur when tissues are exposed to veratridine; Bönisch and Trendelenburg 1987). While there was satisfactory agreement between simulated and experimental results under various conditions, there were also two discrepancies that may be caused by a) inhomogeneous labelling of the storage vesicles in individual varicosities (RPU < PU < U) and b) saturation of outward transport of ³H-noradrenaline when a reserpine-like compound greatly increases the axoplasmic level of total noradrenaline (under U-conditions).Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - FRL fractional rate of loss - MAO monoamine oxidase Supported by the Deutsche Forschunesgemeinschaft (SFB 176) Send offprint requests to E. Schömig     

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

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

Peri‐marketing surveillance of lamotrigine in the Netherlands: Doctors'' and patients'' viewpoints

Purpose: Evaluation of daily clinical practice in prescribing lamotrigine in refractory epilepsy patients.Methods: A collaborative, retrospective, perimarketing study was performed in in and outpatients attending one of the three Dutch epilepsy centres. Analysis of both patients' and doctors' information was performed in 520 patients using questionnaires and medical files.Results:After one year of treatment 76% of patients maintained LTG treatment, an intentiontotreat analysis showed >= 50% seizure reduction in 23% of patients; 2050% seizure reduction in 23% of patients. Six percent of patients became at least three months seizure free. In about 20% of patients seizures became less severe and shorter of duration, while in 6% an increase was found.After three months a significant decrease in number of concomitant antiepileptic drugs was found (change from mean 1,8 to 1,5 AEDs) (p=< 0.01). After twelve months the mean number of AEDs was 1,4 per patient. Overall percentage of side effects appeared to be significantly higher if patients' questionnaire data were used. Epilepsy patients considered side effects as an important factor in the choice of medication and in withdrawal of medication. Future developments of new AEDs should take this into account. Conclusion: This perimarketing study gives insight information about longterm daily use of lamotrigine, with emphasis on effectiveness. Patients complained in the questionnaires much more about sideeffects, than was known according to the medical file. Therefore, it seems necessary to perform perimarketing studies more systematically.     

Opioid peptides decrease noradrenaline release and blood pressure in the rabbit at peripheral receptors

Summary Effects of dynorphin-(1–13), Leu⁵-enkephalin,d-Ala²,d-Leu⁵-enkephalin (DADLE), and for comparison bremazocine, on plasma noradrenaline concentration and mean arterial pressure (MAP) were studied in pithed rabbits. In the first series of experiments, the sympathetic outflow was stimulated electrically via the pithing rod at 2 Hz twice for 3 min each (S₁, S₂). Drugs were administered before S₂. Bremazocine 10 g/kg+2g/kg/h and 100 g/kg+20 g/kg/h, dynorphin 1 and 3 g/kg/min, Leu⁵-enkephalin 100 g/kg/min and DADLE 10 and 30 g/kg/min all diminished the electrically-evoked increase in plasma noradrenaline and MAP. The effects were antagonized by naloxone. In the second series, an infusion of noradrenaline (2 g/kg/min) was given twice for 3 min each (N₁, N₂). Drugs were administered before N₂. Bremazocine 100 g/kg+20 g/kg/h slightly enhanced the pressor effect of exogenous noradrenaline, whereas dynorphin 3 g/kg/min, Leu⁵-enkephalin 100 g/kg/min and DADLE 30 g/kg/min caused no significant change. In the third series, the sympathetic outflow was stimulated continuously at 2 Hz, and the interaction of dynorphin and DADLE was studied. Dynorphin 1 g/kg/min and DADLE 10g/kg/min initially decreased MAP to a similar extent. The effect of DADLE faded with time. When, during continuous infusion of DADLE 10 g/kg/min, and after return of MAP to the pre-DADLE level, dynorphin 1 g/kg/min or DADLE 10 g/kg/min was infused additionally, the effect of dynorphin was unchanged, whereas that of DADLE was almost abolished. We conclude that the opioid peptides as well as bremazocine decrease action potential-evoked release of noradrenaline and, secondarily, blood pressure. They act at peripheral sites, presumably prejunctional opioid receptors at postganglionic sympathetic axons. Dynorphin on the one hand, and Leu⁵-enkephalin and DADLE on the other hand, appear to act at different receptors, dynorphin probably at a - and DADLE and Leu⁵-enkephalin at a -receptor.