Neuronal Noradrenaline Carrier Research Articles

Chloride-dependence of the potency of inhibitors of the neuronal noradrenaline carrier in the rat vas deferens.

(1) Vasa deferentia obtained from reserpine-pretreated rats were exposed to 0.15 mumol l-1 3H-(-)noradrenaline (with monoamine oxidase and catechol-O-methyltransferase being inhibited) and initial rates of the neuronal 3H-noradrenaline uptake as well as IC50 values for inhibition of uptake by desipramine, cocaine or (-)metaraminol determined at various external Cl- concentrations (0-145 mmol l-1) and a fixed high Na+ concentration (145 mmol l-1). (2) When the Cl- concentration in the medium was decreased neuronal uptake fell. As far as Cl- concentrations ranging from 10 to 145 mmol l-1 are concerned, the dependence of uptake on Cl- obeyed Michaelis-Menten kinetics with an apparent Km and Vmax of 6.2 mmol l-1 and 116 pmol g-1 min-1, respectively. At Cl- concentrations below 10 mmol l-1, uptake was higher than expected from the values of Km and Vmax, and even in the nominal absence of Cl- from the medium a remainder of neuronal uptake was still detectable. Evidence is presented to show that, on incubation at Cl- concentrations below 10 mmol l-1, intracellular Cl- leaks out, so that the actual Cl- concentrations in the extracellular fluid are probably higher than in the medium. (3) The potencies of desipramine and cocaine for inhibition of neuronal uptake were markedly dependent on the Cl- concentration in the medium, but the type of Cl- -dependence differed. While the IC50 for desipramine decreased, that for cocaine increased with increasing Cl- concentration (2-145 mmol l-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Nonexocytotic noradrenaline release induced by pharmacological agents or anoxia in human cardiac tissue.

In acute myocardial ischemia, noradrenaline is released locally from sympathetic varicosities by a Ca(2+)-independent nonexocytotic release mechanism that is effectively suppressed by inhibitors of the neuronal noradrenaline carrier (uptake1). The purpose of the present study was to elucidate the significance of free axoplasmic amine concentration and disturbed neuronal sodium homeostasis for nonexocytotic noradrenaline release in the human heart by comparing the release induced by anoxia with that induced by reserpine, tyramine, or veratridine. The overflow of endogenous noradrenaline and dihydroxyphenylethyleneglycol was assessed in human atrial tissue incubated in calcium-free Krebs-Henseleit-solution to prevent interferences by exocytotic release. The overflow of dihydroxyphenylethyleneglycol served as indicator of the free axoplasmic noradrenaline concentration. When vesicular uptake was blocked by the reserpine-like agent Ro 4-1284, high dihydroxyphenylethyleneglycol overflow was observed without concomitant noradrenaline overflow. If, however, Ro 4-1284 was combined with sodium pump inhibition (by omission of extracellular potassium) or with alteration of the transmembrane sodium gradient (by lowering the extracellular sodium concentration), both dihydroxyphenylethyleneglycol and noradrenaline were released. The indirectly acting sympathomimetic tyramine induced a marked increase in noradrenaline overflow which was accompanied by overflow of high amounts of dihydroxyphenylethyleneglycol, indicating interference of the drug with both vesicular catecholamine transport and amine transport via uptake1. Likewise, veratridine induced an overflow of noradrenaline (which was prevented by blockade of uptake1) and dihydroxyphenylethyleneglycol indicating a reserpine-like action of the drug. A disturbed energy status of the sympathetic neuron induced by cyanide intoxication or anoxia caused noradrenaline overflow which was suppressed by uptake1 blockade. Blockade of sodium channels by tetrodotoxin effectively reduced noradrenaline overflow during cyanide intoxication but not during anoxia. Anoxia-induced noradrenaline release, however, was markedly suppressed by inhibition of Na+/H+ exchange with ethylisopropylamiloride, indicating the Na+/H+ exchange as the predominant pathway for sodium entry into the sympathetic neuron during anoxia. The results demonstrate that disturbed neuronal sodium homoeostasis and impaired vesicular storage function are critical conditions, causing nonexocytotic noradrenaline release in anoxic human cardiac tissue.

Nonexocytotic noradrenaline release and ventricular fibrillation in ischemic rat hearts.

In myocardial ischemia, nonexocytotic noradrenaline release has been identified as underlying mechanism of ischemia-evoked noradrenaline release. Nonexocytotic noradrenaline release can be suppressed by inhibitors of the neuronal noradrenaline carrier (uptake), such as desipramine. Utilizing this pharmacological intervention the role of local noradrenaline release in the genesis of ischemia-induced ventricular arrhythmias was studied. Regional ischemia was induced in rat isolated perfused hearts by ligature of the left anterior descending coronary artery, and the venous effluent obtained during the first 2 min of reperfusion was used to measure the release of endogenous noradrenaline by high-performance liquid chromatography methods. Coronary occlusion caused ventricular fibrillation in a well reproducible manner with an incidence of 70 to 80% during a 30 min observation period. Blockage of uptake1 by desipramine decreased the occurrence of ischemia-induced ventricular fibrillation to 60% (0.01 mumol/l) or 20% (0.1 mumol/l), and ventricular fibrillation was completely suppressed by 1 mumol/l desipramine. Likewise, desipramine (0.01-1 mumol/l) concentration-dependently reduced endogenous noradrenaline release during 30 min of regional myocardial ischemia. Nisoxetine, a structurally unrelated inhibitor of uptake1, also suppressed ischemia-evoked ventricular fibrillation. In contrast to its antifibrillatory effect during regional myocardial ischemia, desipramine precipitated arrhythmias when ventricular fibrillation was induced by perfusing normoxic hearts with exogenous noradrenaline. Combination of desipramine (0.1 mumol/l) with exogenous noradrenaline (0.01 to 1 mumol/l) increased the incidence of ventricular fibrillation compared to noradrenaline perfusion alone. Under these conditions, uptake1-blockade is known to increase the extracellular concentration of the perfused noradrenaline. Finally, in the isolated, spontaneously beating papillary muscle of the left rat heart, desipramine (0.1 and 1.0 mumol/l) had no effect on the upstroke velocity of action potentials, the action potential duration and the effective refractory period. In conclusion, the findings demonstrate that nonexocytotic noradrenaline release is an important mediator of ischemia-induced ventricular fibrillation in isolated hearts of the rat. It is also documented that uptake1 inhibitors such as desipramine reveal their effects on ventricular fibrillation secondary to their action on transmembrane noradrenaline transport.

Cardiac Noradrenaline Release Accelerates Adenosine Formation in the Ischemic Rat Heart: Role of Neuronal Noradrenaline Carrier and Adrenergic Receptors

In the present study the hypothesis was tested that local noradrenaline release contributes to adenosine formation in myocardial ischemia. Therefore, in ischemic non-working rat hearts either adrenergic receptors or ischemia-evoked noradrenaline release were blocked. Noradrenaline and adenosine were determined in the effluent using HPLC-methods. Following 20 min of stop of perfusion flow both the β-adrenergic receptor antagonist bisoprolol (91.6 ± 10.5 nmol/g) and the inhibitor of ischemia/induced noradrenaline release desipramine (108.5 ± 12.5 nmol/g) caused a suppression of adenosine release (control: 140.9 ± 7.3 nmol/g). To examine the time-course of the release, further experiments were performed at constant perfusion flow with energy metabolism blocked by cyanide together with removal of glucose from the perfusion buffer. This condition resulted in a nearly simultaneous release of adenosine and noradrenaline from the hearts. The β-adrenoceptor blocking agents atenolol and bisoprolol postponed the release of adenosine, whereas the α-antagonists prazosin and yohimbine had no effect on adenosine release induced by cyanide. None of the adrenergic receptor blockers affected the release of noradrenaline. The inhibitors of the neuronal noradrenaline carrier (uptake), desipramine, oxaprotiline, and cocaine suppressed the release of noradrenaline during cyanide administration, indicating a carrier-mediated efflux of noradrenaline. Reduction of extracellular noradrenaline by these agents coincided with a delay of adenosine release (cumulative release within 20 min—control: 251.2 ± 13.9, desipramine: 172.1 ± 15.3, oxaprotiline 36.5 ± 5.8, cocaine: 111.8 ± 23.6 nmol/g). Desipramine and cocaine were also used during administration of exogenous noradrenaline in normoxic hearts, to confirm specificity of their action. Under these condition, desipramine and cocaine increased the concentration of extracellular noradrenaline, which paralleled by an augmentation of adenosine release into the effluent of the hearts (control: 32 ± 8, desipramine: 103 ± 15, cocaine: 79 ± 9 nmol/g). Comparable to the findings in energy depleted hearts, only the administration of β-antagonists suppressed adenosine formation during noradrenaline infusion in normoxic hearts. In conclusion, in ischemic hearts adenosine formation is accelerated by the release of endogenous noradrenaline acting primarily via β-adrenergic receptor stimulation.

Preferential location of N-methyl-D-aspartate (NMDA) receptors on postsynaptic membranes and on non-noradrenergic nerve terminals of the rat brain cortex.

The effect of DSP4-induced destruction of noradrenergic neurones on 3H-3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (3H-CPP) binding to N-methyl-D-aspartate (NMDA) receptors and on 3H-desipramine (3H-DMI) binding to the neuronal noradrenaline carrier was investigated in rat brain cortex buffy coat membranes. 3H-DMI bound with high affinity to a single site at the neuronal noradrenaline carrier (KD = 5.26 +/- 1.67 nmol/l) whereas the binding of 3H-CPP to the NMDA receptor was of intermediate affinity (KD = 274 +/- 45 nmol/l). Fourteen days after a single-dose treatment with DSP4 (1) the Bmax value for 3H-DMI binding was reduced by 74%, (2) the Bmax value for 3H-CPP binding only tended to be decreased (by 24%; not statistically significant), (3) the endogenous noradrenaline content was reduced by 70% compared to untreated controls and, (4) the absolute amount of the NMDA-evoked 3H-noradrenaline overflow but not the fractional release was reduced by 55%. It is concluded that in the rat cerebral cortex presynaptic NMDA-receptors on noradrenergic nerve endings, which have previously been detected in release experiments with NMDA on cortical synaptosomes preincubated with 3H-noradrenaline, cannot be identified in radioligand binding experiments. Obviously, the cerebral cortical NMDA receptors are predominantly located on postsynaptic neuronal membranes and potentially on non-noradrenergic nerve terminals as well.

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 (uptake 1), 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 uptake 1 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.

Inhibition of neuronal noradrenaline transport (uptake1) and desipramine binding by amiloride and ethylisopropylamiloride

The diuretic amiloride and its N-5 substituted analogue ethylisopropylamiloride (EIPA) inhibit both the specific high affinity desipramine binding to isolated plasma membranes of PC12 rat phaeochromocytoma cells and the carrier-mediated neuronal uptake of noradrenaline into PC12 cells. The inhibition by EIPA of both desipramine binding (Ki = 5.6 mumol/l) and noradrenaline uptake (Ki = 24 mumol/l) inversely depend on the extracellular sodium concentration. The degree of inhibition increased with decreasing sodium concentration. A more detailed analysis of the mode of interaction revealed a competitive interaction between EIPA and desipramine binding but an "uncompetitive" interaction between EIPA and noradrenaline uptake. EIPA is the first inhibitor of uptake1 known so far, which reduces both Km and Vmax of neuronal noradrenaline transport. Extracellular alkalinization from pH 7.4 to 7.9 during incubation with EIPA markedly increased the effects on the kinetics of noradrenaline transport. A model has been proposed to explain the kinetic phenomena. It is based on the hypothesis that EIPA diffuses through the plasma membrane and binds to the inward facing sodium binding site of the neuronal noradrenaline carrier.

Kinetic evidence for a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier.

The neuronal noradrenaline uptake mechanism (uptake1) has been further characterized. For a number of substrates of uptake1 the half-saturating concentration (Km) and the maximal initial transport rate (Vmax) were determined. Furthermore, the dissociation constants (KD) for binding of these substrates to the desipramine binding site of the neuronal noradrenaline carrier were measured. The uptake experiments were done on rat phaeochromocytoma cells (PC12 cells), the binding experiments on purified plasma membranes of PC12 cells. The substrates differed markedly in respect of Vmax, Km, and KD. Neither Km and Vmax nor KD and Vmax were found to be correlated. However, the discrepancy between Km and KD expressed as the ratio, Km/KD, was negatively correlated with Vmax (r = -0.9315, n = 7, p less than 0.01). For the interpretation of these results a model on the basis of the steady-state assumption has been proposed for uptake1. From the mathematics of that model the following conclusions can be drawn. (1) The half-saturating substrate concentration (Km) is not identical with the dissociation constant for the binding of a substrate to the substrate recognition site (KD). (2) The discrepancy between Km and KD is expected to be negatively correlated with the maximal initial transport rate of the substrate (Vmax). The experimental results are in good agreement with the proposed model for uptake1. Especially the negative correlation between Km/KD and Vmax supports the hypothesis that desipramine inhibits uptake1 via binding to the substrate recognition site of the neuronal noradrenaline carrier.

Inhibition of neuronal noradrenaline uptake (uptake1) and desipramine binding by N-ethylmaleimide (NEM).

The inhibition of N-ethylmaleimide (NEM) of uptake1 and desipramine binding was studied on clonal rat phaeochromocytoma cells (PC12 cells) in different experimental settings: (1) 3H-noradrenaline uptake into intact PC12 cells; (2) 3H-noradrenaline uptake into isolated PC12 plasma membrane vesicles; (3) 3H-desipramine binding to isolated PC12 plasma membrane vesicles. In plasma membrane vesicles, NEM inhibited 3H-desipramine binding and 3H-noradrenaline uptake with similar potency (the IC50's were 1.36 mmol/l and 1.04 mmol/l, respectively). However, in intact cells, NEM was about 75 times more potent in inhibiting 3H-noradrenaline uptake (IC50 = 0.014 mmol/l). The increased potency of NEM in intact cells is probably due to an inhibition of the Na+/K+-ATPase and not to a direct interaction with the noradrenaline carrier. The inactivation by NEM of 3H-desipramine binding to PC12 plasma membrane vesicles was irreversible. Both an inhibitor (cocaine, 1 mmol/l) and a substrate of uptake1 (amezinium, 1 mmol/l) protected desipramine binding from inactivation. These results are compatible with the hypothesis of a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier.

Failure of K+ to affect the potency of inhibitors of the neuronal noradrenaline carrier in the rat vas deferens.

To examine whether K+ affects the potency of inhibitors of neuronal uptake, experiments were carried out in the rat vas deferens after pretreatment of the animals with reserpine and after inhibition of monoamine oxidase and catechol-O-methyltransferase. Initial rates of the neuronal uptake of 3H-noradrenaline and IC50 values for uptake inhibition by desipramine, cocaine and (-)metaraminol were determined in the presence of various concentrations of external K+ (5-45 mmol/l), both at 100 mmol/l Na+ and 50 mmol/l Na+. When measured at the 3H-noradrenaline concentration used to determine IC50 values (0.024 mumol/l), neuronal uptake was progressively impaired by increasing K+ concentrations at 50, but not at 100 mmol/l Na+. Neither at 100 mmol/l Na+ nor at 50 mmol/l Na+ was there any consistent, concentration-dependent effect of K+ on the IC50 values of desipramine, cocaine and (-)metaraminol. The analysis of the saturation kinetics of 3H-noradrenaline uptake (determined in the presence of 50 mmol/l Na+ at 5 mmol/l K+ or 45 mmol/l K+) showed that high K+ concentrations inhibit neuronal uptake by decreasing Vmax without any change in Km. The results indicate that K+ does not competitively interact with Na+ at sites on the noradrenaline carrier which mediate the transport-stimulating properties of Na+. Hence, the inhibition of neuronal uptake produced by high K+ concentrations is probably due to membrane depolarization which simply reduces Vmax.

Sodium-dependence of the potency of inhibitors of the neuronal noradrenaline carrier in the rat vas deferens.

Vasa deferentia obtained from reserpine-pretreated rats were incubated (monoamine oxidase and catechol-O-methyltransferase inhibited) in media containing various concentrations of 3H-(-)noradrenaline and Na+ and initial rates of the neuronal uptake of 3H-noradrenaline measured both in the absence and presence of uptake inhibitors after 1 min of incubation. When rates of uptake were determined at various 3H-noradrenaline (1.0-12.2 mumol/l) and two fixed Na+ concentrations (25 and 140 mmol/l), the inhibition of uptake produced by (+)amphetamine, (-)metaraminol, desipramine, nomifensine and cocaine was competitive with respect to 3H-noradrenaline at both Na+ concentrations. While the Ki for (+)amphetamine, (-)metaraminol desipramine and nomifensine increased when the Na+ concentration was lowered, that for cocaine decreased. When the Na+ concentration was varied (10-140 mmol/l) and the 3H-noradrenaline concentration held constant (1.2 mumol/l), (+)amphetamine, (-)metaraminol, nomifensine and desipramine acted as mixed-type inhibitors with respect to Na+, and the inhibition of uptake produced by these drugs was the more pronounced, the higher the Na+ concentration. On the other hand, cocaine was competitive with Na+ and the inhibition produced by this drug was the more pronounced, the lower the Na+ concentration. It is concluded that the inhibitors of neuronal uptake tested here act in dependence on the external Na+ concentration. Desipramine and nomifensine resemble alternative amine substrates in being more potent at high than at low Na+ concentrations. On the other hand, cocaine is more potent at low than at high Na+ concentrations.

Binding of 3H-desipramine to the neuronal noradrenaline carrier of rat phaeochromocytoma cells (PC-12 cells).

The specific (i.e., nisoxetine-sensitive) binding of 3H-desipramine was studied in purified plasma membranes of PC-12 cells (rat phaeochromocytoma cells). 3H-desipramine bound reversibly and with high affinity (KD = 4.5 nmol/l) to a single, non-interacting site (Hill coefficient = 1.04); the maximal number of binding sites (Bmax) was 19.6 pmol/mg protein. Like the uptake of noradrenaline (by uptake1), the binding of 3H-desipramine was dependent on both sodium and chloride. The stimulation of binding by chloride and sodium was characterized by a Hill coefficient of about 1 and 2, respectively. Both, chloride and sodium, slowed the rate of dissociation of bound 3H-desipramine. Increasing concentrations of sodium decreased the KD of 3H-desipramine binding without altering the Bmax. The binding of 3H-desipramine was inhibited by tricyclic antidepressants and other noradrenaline uptake blockers. There was a highly significant correlation between the potencies of a series of drugs for the inhibition of 3H-desipramine binding and for the inhibition of 3H-noradrenaline uptake into intact PC-12 cells. Both, binding of 3H-desipramine and uptake of 3H-noradrenaline, were stereoselectively inhibited by the enantiomers of cocaine and oxaprotiline. However, for most of the substrates of uptake1 the IC50 for inhibition of 3H-desipramine binding was much higher than that for inhibition of 3H-noradrenaline uptake. Nevertheless, noradrenaline competitively inhibited 3H-desipramine binding and unmasked dissociation of bound 3H-desipramine. Thus, 3H-desipramine probably binds to the substrate recognition site.(ABSTRACT TRUNCATED AT 250 WORDS)

Choline+: A substrate of the neuronal noradrenaline carrier in the rat vas deferens

The effects of choline+ (10-40 mmol/l) on 3H-noradrenaline uptake by, and 3H-noradrenaline efflux from, noradrenergic neurones were studied in vasa deferentia of reserpine-pretreated rats at an external Na+ concentration of 100 mmol/l. Monoamine oxidase and catechol-O-methyltransferase were inhibited. Choline+ (20 and 40 mmol/l) competitively inhibited the neuronal uptake of 3H-noradrenaline. From the choline+-induced changes in the apparent Km for 3H-noradrenaline transport, a Ki of 35 mmol/l was obtained. Choline+ (10, 20 and 40 mmol/l) accelerated the neuronal efflux of 3H-noradrenaline in a concentration-dependent manner. This acceleration of efflux was greatly reduced in the presence of 1 mumol/l desipramine, indicating that choline+ is capable of eliciting "accelerative exchange diffusion". Choline+ (40 mmol/l) and (-)noradrenaline (4.5 mumol/l) (i.e., concentrations about equivalent to the Ki and Km for choline+ and (-)noradrenaline, respectively) produced virtually identical increases in the neuronal efflux of 3H-noradrenaline. Choline+ (3-300 mmol/l) inhibited the specific binding of 3H-desipramine to plasma membranes derived from cultured rat phaeochromocytoma (PC-12) cells. The Ki for this interaction was 48 mmol/l. This results suggest that choline+ acts as alternative substrate of the neuronal noradrenaline transport system and should, therefore, not be used in transport studies with noradrenaline as substitute for Na+ in Na+-deficient media.

The transport of (+)-amphetamine by the neuronal noradrenaline carrier.

PC-12 cells (a clonal line of rat phaeochromocytoma cells) take up noradrenaline by a transport system which is identical with the neuronal amine transport system ("uptake1"). The uptake of 3H-noradrenaline into reserpine-pretreated PC-12 cells (monoamine oxidase inhibited) was saturable (Km = 0.6 +/- 0.1 mumol/l), dependent on sodium and chloride, and competitively inhibited by (+)-amphetamine (Ki = 0.18 +/- 0.04 mumol/l), cocaine (Ki = 0.55 +/- 0.15 mumol/l) and desipramine (Ki = 4.3 +/- 0.6 nmol/l). The uptake and accumulation of 3H (+)-amphetamine showed characteristics comparable to those of 3H-noradrenaline, since the uptake of 3H (+)-amphetamine (0.1 mumol/l) was reduced by omission of sodium or chloride from the incubation medium. The sodium-sensitive component of uptake and accumulation of 3H (+)-amphetamine was fully inhibited by cocaine and desipramine. The IC50 of desipramine for inhibition of the sodium-sensitive component of the 1-min uptake of 3H (+)-amphetamine (20 nmol/l) was about 2 nmol/l, i.e., identical with the Ki for inhibition of uptake of 3H-noradrenaline. At concentrations above 1 mumol/l, desipramine additionally caused an inhibition of the sodium-independent permeation of 3H (+)-amphetamine into PC-12 cells. Hence, by using a homogeneous population of cells endowed with "uptake1", it is possible to demonstrate - besides a pronounced lipophilic entry - a carrier-mediated uptake of 3H (+)-amphetamine.