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

After inhibition of extraneuronal uptake by corticosterone, isolated right atria and lengthwise halved vasa deferentia of the rat were incubated with 0.2 mumol/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 microns-1) than in the less densely innervated right atrium (0.011 microns-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 uptake1 during the incubation with this amine) largely depends on the density of the adrenergic innervation.

The force driving the extraneuronal transport mechanism for catecholamines (uptake2).

Recently, uptake2 was shown to exist in the clonal Caki-1 cell line. The aim of this study was two-fold: a) to determine, in Caki-1 cells, the intracellular fate of 3H-noradrenaline after its translocation by uptake2 and b) to analyse the force driving uptake2. Caki-1 cells have the characteristics of a "metabolizing system" in which the activity of catechol-O-methyl transferase (COMT) greatly exceeds that of monoamine oxidase (MAO). In all subsequent experiments these enzymes were inhibited. The determination of initial rates of uptake2 into Caki-1 cells at an extracellular pH between 6.9 and 7.9 indicated that the protonated species of 3H-noradrenaline is transported. Depolarization of Caki-1 cells (by three different procedures) inhibited the inward transport. Determination of the time course of the specific accumulation of 3H-noradrenaline in Caki-1 cells and of 3H-isoprenaline in the perfused rat heart (both mediated by uptake2) revealed that depolarization (by high K+) reduced the rate constant for inward transport (kIN) and increased that for outward movement (kOUT). Consequently, depolarization reduced the steady-state factor of accumulation. It is proposed that, as the protonated species of the substrates of uptake2 is transported, the membrane potential is likely to provide the driving force for uptake2. The fact that depolarization decreased kIN and increased kOUT agrees with this proposal, as do the magnitudes of the steady-state accumulation factors determined in Caki-1 cells and perfused rat heart.

Carrier-mediated outward transport of dopamine from adrenergic varicosities of the vas deferens of reserpine-pretreated rats.

In vasa deferentia of reserpine-pretreated rats a carrier-mediated (i.e., desipramine-sensitive) outward transport of endogenous dopamine was induced by either tyramine or ouabain. The dopamine taking part in the efflux induced by tyramine (and the concomitant efflux of DOPAC) was derived from ongoing synthesis of dopamine. Inhibition of MAO trebled the rate of spontaneous efflux of dopamine and reduced the spontaneous efflux of DOPAC by 90%. After inhibition of MAO, desipramine caused a further five-fold increase in the basal efflux of dopamine with no change in the basal efflux of DOPAC. Inhibition of COMT failed to affect the spontaneous efflux of dopamine but increased that of DOPAC. It is concluded that, after depletion of the noradrenaline stores by pretreatment with reserpine, an outward transport of axoplasmic dopamine is induced by the same mechanisms that (without any pretreatment with reserpine) are known to initiate an outward transport of noradrenaline.

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

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

The energy requirements for the basal efflux of 3H-noradrenaline from sympathetically innervated organs.

In the rat vas deferens (preloaded with 3H-noradrenaline, catechol-O-methyl transferase inhibited, calcium-free solution) ouabain, glucose deprivation or the combination of hypoxia plus presence of lactate were found to induce a carrier-mediated (desipramine-sensitive) outward transport of the 3H-amine. Glucose deprivation additionally increased the efflux of deaminated 3H-metabolites, as a consequence of an increased net leakage of vesicular 3H-noradrenaline; moreover, 3H-dihydroxymandelic acid then became the predominant neuronal metabolite. The simultaneous lack of oxygen and glucose resulted in a very pronounced release of the 3H-amine. Moreover, during spontaneous efflux more outward transport of 3H-noradrenaline was observed in the absence than in the presence of extracellular calcium. In rat atria (under the same experimental conditions) the contribution by carrier-mediated outward transport to the spontaneous efflux of tritium exceeded that in vasa deferentia. Moreover, the efflux of lactate (as an index of hypoxia of the tissue) exceeded that observed in vasa deferentia, under aerobic and anaerobic conditions. It is proposed that the greater contribution by outward transport of 3H-noradrenaline to spontaneous efflux in atria than in vasa deferentia does not reflect any basic difference between the varicosities in two different organs. It is likely that the less heterogeneous distribution of the 3H-amine in atria than in vasa deferentia is responsible for storage of the exogenous amine in atrial varicosities that are subject to some hypoxia, to an increased extracellular lactate level and to perhaps a minor degree of glucose deficiency; these factors may well be responsible for the difference with regard to outward transport of 3H-noradrenaline during spontaneous efflux. Thus, in addition to the heterogeneity of the distribution of 3H-noradrenaline, an additional heterogeneity with regard to the energy supply must be expected for incubated organs.

The TiPS lecture: functional aspects of the neuronal uptake of noradrenaline.

For various amine transmitters (noradrenaline, dopamine, 5-HT) re-uptake into the releasing varicosity limits the transmitter's life span in the biophase. In the second TiPS Lecture, given at this year's FASEB meeting in Atlanta, Georgia, Ullrich Trendelenburg summarized the evidence relating to the function of the neuronal noradrenaline carrier (uptake1), why it is absolutely dependent on Na+ and Cl- and how it functions as a metabolizing system, hand in hand with intraneuronal monoamine oxidase and vesicular storage. This carrier clears noradrenaline from the extracellular space very efficiently. Hence, loading of incubated organs with exogenous substrates of uptake1 results in a very heterogeneous distribution of the amine. Moreover, under certain experimental and pathophysiological conditions the carrier is able to transport axoplasmic noradrenaline out of the varicosity, a 'release' mechanism operating, for instance, in cardiac ischaemia.

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

The rat vas deferens was incubated with 0.2 mumol/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 autoradiographic 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. Tis decline amounted to 0.017 micron-1 or 0.124 varicosity-1, since the average distance between varicosities was calculated to be 7.4 microns. 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Energy requirements for the basal efflux of noradrenaline and its metabolites from adrenergic varicosities.

The combination of hypoxia plus glucose deprivation or of hypoxia plus lactate induces carrier-mediated outward transport of 3H-noradrenaline in the rat vas deferens. Lactate efflux is higher from atria than from vas deferens. Hence, the much lower contribution by outward transport to the spontaneous efflux of 3H-noradrenaline in vas deferens than atria is likely to be due to a better supply of oxygen (and perhaps also glucose) to the 3H-noradrenaline-storing varicosities in vas deferens than in atria.

Carrier-mediated outward transport of noradrenaline from adrenergic varicosities.

Normally, the carrier of the neuronal noradrenaline uptake mechanism (uptake1) is involved nearly exclusively in the inward transport of substrates. However, an outward transport is induced by either of two mechanisms: a. by the decrease (or reversal) of the Na+ concentration gradient across the neuronal membrane or b. by the "facilitated exchange diffusion" due to the inward transport of substrates of this carrier. In rat vasa deferentia (preloaded with 3H-noradrenaline; vesicular uptake, MAO and COMT blocked) all substrates of the carrier induced an outward transport of 3H-noradrenaline, in strict correlation with their Km for uptake1. The inward transport of these substrates increases the availability of the carrier on the inside of the neuronal membrane. However, additional factors contribute to this release: inhibition of re-uptake and co-transport of Na+ and Cl-. When vesicular uptake and MAO are intact, the strict correlation between Km and releasing effect was not seen. This is attributable to the very low axoplasmic concentration of 3H-noradrenaline under these conditions (i.e. to lack of substrate for outward transport). However, if substrates of uptake1 are also substrates of vesicular uptake, they are able to "mobilize" vesicularly stored 3H-noradrenaline and, hence, to induce substantial outward transport. These "good" releasers are identical with the "indirectly acting sympathomimetic amines" (i.e. they are tyramine-like).

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

After loading of the incubated rat vas deferens with 0.2 mumol/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 less than 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 4-1284, 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 mumol/l 3H-noradrenaline. It is proposed that the various "compartments" and "pools" of noradrenaline described in the literature reflect the two heterogeneities described here.

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

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

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

In the rat vas deferens, DMPP is a substrate of uptake1 (Km = 11.5 mumol/l). 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) mumol/l DMPP induced a hexamethonium-sensitive release of 3H-noradrenaline of short duration. Hence, in the presence of extracellular calcium, 100 mumol/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 mumol/l.

The interaction of transport mechanisms and intracellular enzymes in metabolizing systems.

The life span of extracellular catecholamines is limited by the combination of uptake and subsequent intracellular metabolism by either monoamine oxidase (MAO) and/or catechol-O-methyl transferase (COMT). Three such "metabolizing systems" are involved in the inactivation of noradrenaline: 1) Neuronal uptake (high-affinity uptake1) in association with neuronal MAO (and vesicular uptake), 2) extraneuronal uptake (low affinity uptake2) in association with intracellular COMT and MAO (in smooth muscles, myocardial cells, glands), and 3) uptake1 of non-neuronal cells in association with intracellular COMT and/or MAO (in vascular endothelium of rat lung). Such systems function as "pump and leak systems with enzyme(s) inside". The analysis of either uptake or enzyme fails to reveal the characteristics of such systems; they are determined by the interaction of both components. Because of the high activity of these intracellular enzymes, it is unlikely that either COMT or MAO is ever saturated in vivo. However, in vitro saturation of extraneuronal COMT and MAO reveals that extraneuronal COMT is a high-affinity, but extraneuronal MAO a low-affinity enzyme. Hence, membrane-bound COMT appears to be responsible for the extraneuronal O-methylation of noradrenaline. If intracellular enzymes remain unsaturated, the determination of the rate constants describing the unsaturated enzyme (KENZYME = Vmax/Km) is of particular interest. KENZYME can be determined for metabolizing systems, since this rate constant is not affected by the (usually unknown) fractional size of the metabolizing system.

Human Caki-1 cells are the first model for extraneuronal transport of noradrenaline (uptake2) which is based on a clonal cell line.

The neurotransmitter noradrenaline is inactivated by active transport out of the synaptic cleft--either back into the adrenergic neuron or into extraneuronal cells. Transport studies on isolated cells provide many advantages. However, an experimental model for the extraneuronal uptake of noradrenaline which is based on a clonal cell line was not known until now. The human renal carcinoma cell line Caki-1 is the first clonal cell line known to express the extraneuronal transport system for noradrenaline.

The efficiency of the neuronal re-uptake of noradrenaline-and the inhomogeneity of labelling of an incubated tissue.

In isolated vasa deferentia and atria of the rat, 90% of the spontaneous efflux of [(3)H]noradrenaline is subject to neuronal re-uptake, although the density of the adrenergic innervation is about seven times higher in the former than in the latter tissue. This surprising lack of any influence of the density of innervation on the efficiency of re-uptake is due to an inhomogeneous labelling of the tissue with [(3)H]noradrenaline. In the vas deferens the usual loading procedure results in two types of inhomogeneity: (1) autoradiography and an analysis of efflux indicate that a preferential labelling of surface-close varicosities takes place during loading. The consequences of this type of inhomogeneity are abolished by inhibition of uptake(1); desipramine enhances the spontaneous efflux much more for endogenous than for exogenous noradrenaline; (2) a second inhomogeneity involves a preferential labelling of the vesicles close to the surface of (labelled) varicosities. The consequences of this type of inhomogeneity of labelling are abolished by inhibition of vesicular uptake (by the reserpine-like compound Ro 4-1284). As a consequence of these inhomogeneities, the extent of neuronal re-uptake is much greater for endogenous than for exogenous noradrenaline.

The extent of neuronal re-uptake of 3H-noradrenaline in isolated vasa deferentia and atria of the rat.

After pretreatment of rats with reserpine and pargyline (to inhibit vesicular uptake and monoamine oxidase, respectively) and after inhibition of catechol-O-methyl transferase (by U-0521) and in calcium-free solution, the adrenergic neurones of isolated vasa deferentia and atria were loaded with 3H-noradrenaline. The spontaneous efflux of 3H-noradrenaline and 3H-dihydroxyphenylglycol was determined, as well as the steady-state effect of two concentrations of desipramine. On the basis of a mathematical model of the adrenergic nerve ending, fractional rates (FR = rate of flux divided by tissue tritium content) were calculated for unidirectional outward diffusion, for outward transport and for neuronal re-uptake (all for 3H-noradrenaline). Although the density of adrenergic innervation is lower in atria than in vasa deferentia, neuronal re-uptake amounted to about 90% of the spontaneous efflux of 3H-noradrenaline in both tissues. While the FR for unidirectional outward diffusion was virtually the same in both tissues, the FR for outward transport of 3H-noradrenaline was more than three times higher in atria than in vasa deferentia. There is, as yet, no explanation for this pronounced difference.

The release of 3H-noradrenaline by p- and m-tyramines and -octopamines, and the effect of deuterium substitution in alpha-position.

The 3H-noradrenaline-releasing effects of p- and m-tyramines and -octopamines, either deuterated or not, were studied in isolated vasa deferentia of the rat (COMT inhibited and calcium-free solution in all experiments). Km for uptake1 was higher for octopamines than for tyramines, but not increased by the introduction of deuterium in alpha-position, except for (probably contaminated) deuterated p-octopamine. Other tissues were preloaded with 3H-noradrenaline. After inhibition of vesicular uptake and MAO equi-releasing concentrations of the eight amines were strictly correlated with Km, they were 6 to 7 times higher for unsubstituted octopamines than for corresponding tyramines. When only MAO (but not vesicular uptake) was inhibited, this difference decreased to about 4-fold, but the releasing potency of the deuterated amines (relative to their parent amines) remained unchanged (except for p-octopamine). When vesicular uptake and MAO were intact, unsubstituted octopamines were only 1.5 to 2.2 times less potent than the corresponding tyramines. Analysis of the efflux of 3H-DOPEG confirmed that this gain in the relative potencies of octopamines is due to their increased ability to mobilize vesicular 3H-noradrenaline; moreover, deuterated amines as well were then better mobilizers than were their parent amines. It is concluded that, provided vesicular uptake is intact, the introduction of a beta-OH-group enhances the ability of indirectly acting sympathomimetic amines to mobilize vesicular noradrenaline; the introduction of deuterium in alpha-position, on the other hand, enhances this mobilizing effect exclusively when MAO is intact.