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

Naunyn-Schmiedeberg's Arch Pharmacol (1996) 354:7-16 © Springer-Verlag 1996

Thomas Kurz • Gert Richardt • Melchior Seyfarth Albert SchiJmig

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

Received: 27 September 1995/Accepted: 1 March 1996

Abstract 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 uptake0

T. Kurz • G. Richardt • M. Seyfarth • A. Sch6mig 1. Medizinische Klinik, Technische Universit/it, Munich, Germany

T. Kurz (N:~) Medizinische Universitiit zu Liibeck, Medizinische Klinik II, Ratzeburger Allee 160, D-23538 L/ibeck, Germany

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.

Key words Calcium-independent noradrenaline release • Uptakel-carrier • Myocardial ischemia • Human atrium • Tyramine • Veratridine

Introduction

Myocardial ischemia is associated with the release of massive amounts of noradrenaline within the ischemic myocardium. This results in excess sympathetic stimulation which not only contributes to the development of lethal ventricular arrhythmias (Penny 1984) but also accelerates the development of irreversible cellular damage (Rona 1985). The release of noradrenaline from cardiac sympathetic nerve endings, which is not due of central sympathetic stimulation, represents the predominant source of the presence of high amounts of amine in the extracellular space of the underperfused myocardium (Sch~Smig et al. 1984; Abrahamsson et al. 1985). This local, metabolically mediated release is independent of extracellular calcium and inhibited by blockade of the neuronal uptakel-carrier for

noradrenaline, both are characteristics of a carrier- mediated release (Sch6mig et al. 1984, 1987; Carlsson et al. 1986). It has been suggested that the release mechanism constitutes a two-step process induced by energy deficiency in the sympathetic nerve ending (Sch6mig et al. 1988). In the first step, noradrenaline is set free from the vesicular stores resulting in increased axoplasmic noradrenaline concentrations. In the second step, axoplasmic noradrenaline passes through the cytoplasmic membrane into the extracellular space via the uptakel-carrier reversing its normal transport direction.

The mechanisms of ischemia-induced catecholamine release have been demonstrated to differ markedly in different species and tissues. In bovine adrenal chromaffin cells, catecholamine release elicited by anoxia or metabolic inhibition is independent of extracellular calcium, but, in contrast to the release in ischemic rat hearts, cannot be inhibited by uptake1 blockers (Dry et al. 1991). In rat heart, brief myocardial ischemia causes a reduction in stimulation-induced noradrenaline overflow, whereas the overflow is increased in guinea pig heart (Seyfarth et al. 1993). Is- chemia-induced noradrenaline release has been demonstrated to be effectively inhibited by bradykinin in the rat heart (Chahine et al. 1993). In contrast, bradykinin increases ischemia-induced noradrenaline release in the guinea pig heart (Imamura and Levi 1995). The species- related differences in the mechanism of ischemia-induced catecholamine release strongly indicate the need for studies in human cardiac tissue.

In a previous study, we provided evidence for carrier-mediated release of noradrenaline from sympathetic neurons in the anoxic human myocardium (Kurz et al. 1995). Previously, we could rule out potential differences of atrial versus ventricular myocardium and perfusion versus incubation by comparing results obtained in incubated rat atria with those obtained in perfused rat hearts (Kurz et al. 1995). The present study was designed to identify conditions that induce outward transport of noradrenaline from sympathetic neurones of human cardiac tissue. The inward transport of catecholamines into the neuron via uptakes- carrier depends on the presence of extracellular sodium (Sammet and Graefe 1979; Graefe et al. 1984). Therefore, we focused our interest on the role of the sodium concentration gradient across the cell membrane in causing a reversal of the carrier's net transport direction. The second aim was to provide evidence for the significance of the free axoplasmic amine concentration and disturbed neuronal sodium homoeostasis for nonexocytotic noradrenaline release in anoxic myocardium.

study was approved by the Local Ethics Committee, and all patients gave their informed consent. Immediately after excision, specimens of atrial appendage were placed in ice-cold Krebs-Henseleit solution and transferred to the laboratory. Experiments were carried out with corresponding controls after dividing the tissue into two to four samples (weight 50 100mg). Each sample was preincubated for 25 min in tubes containing 3 ml of modified Krebs-Henseleit solution (in mmol/l: NaC1 125, NaHCO 3 16.9, Na2HPO e 0.2, KC1 4.0, CaC12 0, MgC12 1.0, glucose 11, Na2EDTA 0.027), gassed with 95% 0 2 and 5% CO 2 (pH 7.4) and placed in a 37.5 °C warm water bath. In experiments with reduced sodium concentration or potassium- free medium, the ionic composition of the incubation buffer was altered already during the pre-incubation period. Reduction of sodium or potassium-free medium involved equimolar replacement with lithium or sodium chloride. After the preincubation period, the tissues were subjected to the interventions (addition of drugs, anoxia, blockade of energy metabolism). Throughout the experiments, tissues were transferred to a subsequent tube every 5 rain.

Anoxia experiments. Tissues were incubated in tubes containing 1 ml calcium- and glucose-free solution. Anoxia was induced by gassing the medium with a N2(95%)/CO2(5% ) mixture (pH adjusted to 7.4) and by adding the reducing agent, sodium dithionite, at a final concentration of 3 mmol/1 (Sch6mig et al. 1987; Kurz et al. 1995). Anoxia was confirmed by measuring the pO 2 with a pH/blood gas analyzer. Interventions were started 10 min prior to anoxia and continued throughout anoxia. Determination of noradrenaline was performed in the medium during each 5-min incubation period before and after anoxia and in the incubation medium during the anoxic period.

Blockade of energy metabolism. For experiments involving energy depletion, human atrial tissues were placed in calcium-free incubation buffer which was deficient of glucose and contained 1 mmol/1 cyanide to inhibit oxidative phosphorylation. If drugs were used, they were added 10 min prior to the blockade of energy metabolism and were present throughout the experiment. Noradrenaline and D O P E G were determined in the medium during each 5-min incubation period before and during blockade of energy metabolism.

Determination of endogenous noradrenaline and dihydroxyphenyl- glycol (DOPEG). Samples for estimation of endogenous noradrenaline and D O P E G were cooled on ice, and stabilized by the addition of Na2EDTA (10 mmol/1). Samples were stored at - 60 °C until assayed. Noradrenaline and D O P E G were quantified by high-pres- sure liquid chromatography (HPLC) and electrochemical detection as described by Sch6mig et al. (1987). The drugs used here did not interfere with the extraction, separation or detection of noradrenaline and DOPEG.

Agents used. Cocaine HC1 (Merck, Darmstadt, Germany), desipramine HC1 (CIBA-Geigy, Basle, Switzerland), Ro 4-1284 (2-hy- droxy-2-ethyl-3-isobutyl-9,10-dimethoxy- 1,2,3,4,6,7-hexahydro- 1 lb-H-benzo (a)-quinolizine; Hoffmann-La Roche, Basle, Switzer- land), sodium cyanide (Merck), sodium dithionite (Merck), tetrodotoxin and veratridine (Sigma, Munich, Germany). Ethylisop- ropylamiloride was synthesized according to Cragoe et al. (1966). The agents not soluble in water were dissolved in ethanol with a final concentration less than 0.01%. Corresponding control experiments with identical solvent concentration were performed. All other agents were dissolved primarily in distilled water and diluted in Krebs Henseleit solution.

Methods

Human atrial tissue was obtained during routine cannulation of the right atrium in patients undergoing coronary artery surgery. The

Statistical methods. The results stated in text and figures are expressed as means _+ SEM. The significance of differences was assessed by Student's t-test. A P-value of less than 0.05 was con- sidered significant.

overflow of noradrenaline

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Fig. I Effect of Ro 4-1284 on overflow of noradrenaline (upper panel) and D O P E G (lower panel) from human atrial tissue incubated in calcium-free Krebs-Henseleit buffer. Noradrenaline and D O P E G overflow was determined in the absence (n = 6) and in the presence (n = 6) of Ro 4-1284 (10 gmol/1; 5-50 min). Mean ± SEM


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Fig. 2 Effect of Ro 4-1284 combined with alteration of the transmembrane sodium gradient on noradrenaline (upper panel) and D O P E G (lower panel) overflow. Human atrial tissues were incubated in calcium-free Krebs-Henseleit solution containing either 142 mmol/1 sodium (n = 9) or 50 mmol/1 sodium (n = 9; 0-50 min). In both series, tissues were exposed to 10gmol/1 Ro 4-1284 (5-50min). Mean ___ SEM

Results

Effect of altered transmembrane sodium gradient or inhibition of sodium-potassium ATPase on noradrenaline overflow in the presence of high axoplasmic noradrenaline concentrations

The effect of Ro 4-1284 (10 gmol/1), an inhibitor of the vesicular amine carrier, on the time course of D O P E G and noradrenaline overflow is demonstrated in Fig. 1. Despite loss of noradrenaline from storage vesicles and increased noradrenaline concentrations within the neuron, as reflected by the increase in D O P E G overflow, no increase in noradrenaline overflow was observed (cumulative overflow during 45 min Ro 4-1284: noradrenaline 70 ± 24pmol/g, D O P E G 847 _ 78 pmol/g vs. control: 40 _+ 18 and 101 4-39 pmol/g, respectively; P < 0.01 for DOPEG). The noradre- nal ine/DOPEG ratio decreased from 0.393 (control) to 0.082 during Ro 4-1284.

However, when the transmembrane sodium gradient was altered by incubation of the tissue in medium containing 50 mmol/1 sodium, Ro 4-1284 (10 ~tmol/1)

induced a simultaneous release of noradrenaline and D O P E G (Fig. 2) (cumulative overflow after 45 min of Ro 4-1284 exposure: low sodium = noradrenaline 639 4- 40 pmol/g and D O P E G 811 _+ 46 pmol/g vs. normal sodium: 116 __+ 22 and 1030 _+ 135 pmol/g, respectively; P < 0.01 for noradrenaline). The noradrena- l ine/DOPEG ratio increased from 0.093 at normal sodium to 0.788 at low sodium. Likewise, inhibition of sodium-potassium ATPase by extracellular potassium depletion induced a marked but transient noradrenaline release when cytoplasmic noradrenaline levels were raised by Ro 4-1284 (Fig. 3) (cumulative overflow after 45 min of Ro 4-1284 exposure: no potassium = noradrenaline 301 + 4 8 p m o l / g and D O P E G 718 _+ 70pmol/g vs. normal potassium: 69_+ 18 and 998 ± 100 pmol/g, respectively; P < 0.01 for noradrenaline, P < 0.01 for DOPEG). The noradrena- l ine/DOPEG ratio increased from 0.078 at normal potassium to 0.419 in the absence of potassium. Neither lowering of the extracellular sodium concentration nor potassium-free medium induced noradrenaline release

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Fig. 3 Effect of Ro 4-1284 combined with inhibition of Na +, K +- ATPase on noradrenaline (upper panel) and D O P E G (lower panel) overflow. Human atrial tissues were incubated in calcium-free Krebs-Henseleit solution containing either 4 mmol/1 potassium (n = 6) or no potassium (n = 6; 0-50 min). In both series, tissues were exposed to 10 gmol/1 Ro 4-1284 (5-50 min). Mean _+ SEM


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Fig. 4 Effect of tyramine on overflow of noradrenaline (upper panel) and D O P E G (lower panel) from human atrial tissue incubated in calcium-free Krebs-Henseleit buffer. Noradrenaline and D O P E G overflow was determined in the absence (n = 6) and presence (n = 6) of the indirectly acting amine tyramine 10 gmol/l; (5-50 min). Mean + SEM

prior to elevation of the axoplasmic noradrenaline concentration by Ro 4-1284.

Effect of the indirectly acting amine tyramine on noradrenaline and DOPEG overflow

The effect of tyramine on the time course of noradrenaline and DOPEG overflow is demonstrated in Fig. 4. Tyramine (10 ~tmol/1) induced a marked increase in noradrenaline overflow (cumulative overflow during 45rain tyramine: 384-t-35pmol/g versus control: 26 +_ 11 pmol/g; P < 0.01). Noradre- naline overflow was accompanied by a significant increase in the overflow of DOPEG (cumulative release at 40 min tyramine: 367 +_ 45 pmol/g versus control: 79 _+ 28 pmol/g; P < 0.01). The noradrenaline/DOPEG ratio increased from 0.325 (control) to 1.105 during tyramine. The tyramine-induced noradrenaline overflow was sensitive to inhibition by 0.1 gmol/1 desipramine (data not shown).

Reserpine-like effect of the sodium channel opener veratridine

Veratridine (100 gmol/1) elicited a marked overflow of DOPEG (cumulative overflow during 40 rain veratridine: 317_+ 27pmol/g versus control: 68 +_ 30 pmol/g, P < 0.05). Veratridine also induced a progress- ive overflow of noradrenaline starting 5 min after addition of the drug (cumulative overflow during 40 min veratridine: 1406 _+ 113 pmol/g versus control: 16 _+ 12 pmol/g; P < 0.01) (Fig. 5). The noradrena- l ine/DOPEG ratio increased from 0.242 (control) to 4.439 during veratridine. Though less effectively, veratridine at concentrations of 10 and 1 gmol/1, respectively, induced the overflow of both DOPEG and noradrenaline (data not shown). Veratridine-induced noradrenaline overflow was sensitive to blockade of the uptake: carrier. Desipramine (0.1 ~tmol/1) suppressed noradrenaline overflow by about 50% (cumulative overflow after 40min of exposure to veratridine: 953 + 83 pmol/g in the absence versus 401 __ 84 pmol/g in the presence of desipramine; P < 0.05).


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Fig. 5 Effect of veratridine on overflow of noradrenaline (upper panels) and D O P E G (lower panels) from human atrial tissue incubated in calcium-free Krebs-Henseleit buffer. Left panels: Norad- renaline and D O P E G overflow was determined in the absence (n = 6) and in the presence of (n = 6) of veratridine 100 Itmol/1; 10 50 min). Right panels: Effect of the uptake 1 inhibitor desipramine (0.1 pmol/1) on veratridine-induced overflow of noradrenaline and DOPEG. Human atrial tissue was exposed to veratridine (100 lamol/1; 1(~50 min) in the absence (n = 6) and presence (n = 6) of desipramine (DMI; ~ 5 0 rain). Mean _+ SEM

Conversely, inhibition of veratridine-induced noradrenaline overflow by desipramine tended to increase the overflow of DOPEG (cumulative overflow after 40min of exposure to veratridine: 144 _+ 35 pmol/g versus 271 -t- 54 pmol/g in the presence of

desipramine; n.s.). The noradrenaline/DOPEG ratio decreased from 6.59 (veratridine) to 1.48 in the presence of desipramine.

Effect of cyanide intoxication on noradrenaline and DOPEG overflow

The time course of noradrenaline and DOPEG overflow induced by cyanide intoxication and glucose deprivation is shown in Fig. 6. Starting 30 min after addition of cyanide and withdrawal of glucose from the

incubation medium, noradrenaline overflow progress- ively increased. The addition of the uptake~ blocker cocaine (10 pmol/1) significantly attenuated noradrenaline overflow induced by cyanide intoxication (cumulative overflow after 60 min of cyanide intoxication: 620_+ 80pmol/g in the absence versus 105_+ 11 pmol/g in the presence of cocaine; P < 0.01). Norad- renaline overflow was accompanied by an increase in the overflow of DOPEG, which preceded the increase in noradrenaline overflow by about 30 min (cumulative overflow of DOPEG after 60 min of cyanide intoxication: 299_+ 109pmol/g in the absence versus 630 + 108 pmol/g in the presence of cocaine; n.s.). The noradrenaline/DOPEG ratio decreased from 2.07 (cyanide) to 0.17 in the presence of cocaine. Blockade of sodium channels with tetrodotoxin (1 pmol/1) effectively suppressed noradrenaline overflow induced by cyanide intoxication (cumulative overflow after 60 min of cyanide intoxication: 458 _+ 73 pmol/g in the absence versus 101 _+ 27 pmol/g in the presence oftetrodotoxin; P < 0.01; Fig. 6). DOPEG overflow was enhanced when noradrenaline overflow was inhibited by tetrodotoxin (cumulative overflow after 60 min of cyanide intoxication: 339 _+ 88 pmol/g in the absence versus 607 _+ 73 pmol/g in the presence of tetrodotoxin; P < 0.05). The noradrenaline/DOPEG ratio decreased from 1.35 (cyanide) to 0.17 in the presence of tetrodotoxin.

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Fig. 6 Overflow of endogenous noradrenaline (upper panels) and DOPEG (lower panels) from calcium-free incubated human atrial tissue induced by blockade of energy metabolism (glucose-free medium containing cyanide 1 retool/l, 20-100 min). Left panels: Inhibi- tion of noradrenaline overflow by the uptakerinhibitor cocaine. Tissues were incubated without (n = 6) or with cocaine (10 gmol/1; n = 6) starting 10 rain prior to blockade of energy metabolism. Right panels: Role of sodium influx via tetradotoxin (TTX)-sensitive channels for noradrenaline release induced by energy deficiency. The time course of overflow was studied in the absence (n = 9) and presence (n = 9) of TTX (1 gmol/1) starting 10 min prior to blockade of energy metabolism. Mean _+ SEM

Effect of manipulation of neuronal sodium channels or sodium-proton exchange on noradrenaline overflow in myocardial anoxia

Simulation of myocardial ischemia by 60 min of anoxic and glucose-free incubation of tissue resulted in a marked overflow of noradrenaline (Fig. 7). Inhibition of uptake1 by cocaine (10 gmol/1) prior to anoxic incubation reduced noradrenaline overflow. Blockade of sodium channels by tetrodotoxin (1 gmol/1) had no effect on noradrenaline overflow induced by 60 min of anoxic incubation. In contrast, inhibition of transmembrane sodium-proton exchange with ethylisopropylamiloride (10gmol/1) reduced anoxia-induced noradrenaline overflow. During 60 min of anoxic incubation, only minor amounts of DOPEG were found in the incubation medium.

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Fig. 7 Overflow of noradrenaline induced by 60 rain of anoxia pro- duced by gassing the medium with a N2/CO2-mixture and addition of sodium dithionite. Human atrial tissues were exposed to anoxia in the absence of drugs and in the presence of cocaine (10 gmol/1), tetrodotoxin (TTX; 1 gmol/l), or ethylisopropylamiloride (EIPA; 10 gmol/1). Drugs were added to the incubation medium 10 min prior to the beginning of anoxia. Each column: n = 6; mean + SEM. Statistical significance (P < 0.05) is denoted by an asterisk

Discussion

Increased axoplasmic noradrenaline concentration in the sympathetic neuron

The capability of neuronal storage vesicles to retain catecholamines is established by an intact proton

13

gradient across the membrane of storage vesicles; this is the driving force for vesicular noradrenaline uptake via a specific carrier located within the vesicular membrane (Beers et al. 1982; Phillips 1982; Winkler et al. 1986). Inhibition of the vesicular amine carrier induces a loss of noradrenaline from the vesicles, resulting in an increase in the axoplasmic amine concentration (Schul- diner et al. 1995). Since axoplasmic noradrenaline concentrations cannot be directly assessed, the overflow of DOPEG, the predominant, intraneuronally formed metabolite of noradrenaline, served as an indirect measure of the axoplasmic noradrenaline concentration in the present study (Stute and Trendelenburg 1984; Sch6mig et al. 1987). The lipophilic DOPEG easily diffuses across the axonal membrane and, therefore, the overflow of this noradrenaline metabolite re- flects axoplasmic noradrenaline concentrations as long as intraneuronal monoamine oxidase is not saturated. In the present study, inhibition of the vesicular amine carrier by the reserpine-like agent Ro 4-1284 resulted in a marked increase in the overflow of DOPEG, indicating elevated axoplasmic noradrenaline concentrations.

Despite of increased axoplasmic noradrenaline concentrations, there was no increase in the overflow of noradrenaline. In contrast to the Iipophilic DOPEG, diffusional efflux of the hydrophilic noradrenaline, which is ionized at physiological pH, is very low (Tren- delenburg et al. 1980). Therefore, noradrenaline release across intact axonal membranes depends on the opera- tion of a specific carrier system.

Nonexocytotic noradrenaline release induced by interference with neuronal sodium homeostasis in the presence of elevated axoplasmic noradrenaline

Noradrenaline transport across the sarcolemma of the sympathetic nerve terminal is brought about by the neuronal amine carrier (uptake1) associated with the axonal membrane. Uptake1 is coupled to the electrochemical sodium gradient across the axonal membrane (Sammet and Graefe 1979; Friedrich and B6nisch 1986). It is independent of extracellular calcium (Graefe et al. 1984) and specifically inhibited by cocaine and various antidepressants such as desipramine (B6nisch and Br/iss 1994). Sodium ions increase the accessibility of the carrier at that side of the plasma membrane where the sodium concentration is high by forming an immobile sodium-carrier complex and by enhancing the affinity of the uptakel-carrier for noradrenaline (Graefe et al. 1984). Consequently, at physiological sodium concentrations, noradrenaline transport pre- dominantly occurs in the inward direction, and carrier- mediated outward transport of noradrenaline is prevented. Any condition, however, under which the sodium gradient is reduced or reversed will induce outward transport and bring about a release of ex- travesicular noradrenaline.

In the present study, the transmembrane sodium gradient was altered either by lowering the extracellular sodium concentration or by inhibiting the sodium- potassium ATPase by potassium free medium. Al- though sodium concentrations within the sympathetic neuron were not directly assessed, microelectrode studies of papillary muscle and Purkinje fibers as well as nuclear magnetic resonance studies of perfused rat hearts demonstrated a twofold to threefold rise in intracellular sodium activity when the sodium-potassium ATPase was inhibited by omission of extracellular potassium (Lee and Fozzard 1975; Ellis 1977; Brill and Wasserstrom 1986; Pike et al. 1985). In human atrial tissue, no increase in noradrenaline overflow was observed either by lowering of the extracellular sodium concentration or by inhibiting the sodium-potassium ATPase when the axoplasmic amine concentration was normal. In the presence of high axoplasmic noradrenaline concentrations (i.e. in the presence of Ro 4-1284), however, alterations of the transmembrane sodium gradient induced a marked increase in noradrenaline overflow, whereas DOPEG overflow was reduced. There are basically two pathways which allow axoplasmic noradrenaline to leave the sympathetic nerve terminal: first, deamination to DOPEG, resulting in DOPEG overflow, and second, outward transport of unchanged noradrenaline mediated through the opera- tion of uptake1. The ratio of noradrenaline/DOPEG overflow provides information about the change of the rate constant for noradrenaline net outward transport as long as the rate constants for DOPEG formation and diffusion remain unaltered (Sch6mig and Tren- delenburg 1987). Extracellular sodium reduction or potassium depletion increased the ratio of noradrena- l ine/DOPEG overflow 7- or 6-fold, indicating a massive efttux of noradrenaline mediated by uptake1. These experiments demonstrate for the first time, that inhibition of the vesicular amine-carrier combined with inhibition of sodium-potassium ATPase serves as a basically efficient mechanism to trigger nonexocytotic noradrenaline release in human cardiac tissue.

In previous experiments in the rat perfused heart, potassium-flee perfusion caused an increase in noradrenaline overflow lasting as long as intraneuronal noradrenaline concentration was increased (Sch6mig et al. 1988) and was suggested to be due to inhibition of sodium/potassium ATPase since ouabain induced a similar effect. In human atrial tissue, however, the increase in noradrenaline overflow caused by the absence of potassium was transient i.e. there was a time- dependent decline of the increased overflow of noradrenaline despite increased axoplasmic noradrenaline concentration. Since we did not use ouabain in human atrial tissue, there is still the possibility that the effect of potassium-free incubation on noradrenaline release is not solely due to the inhibition of sodium/potassium ATPase but a consequence of the lack of potassium. As demonstrated by Harder and B6nisch (1985) internal

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potassium enhances uptake and accumulation of 3H- noradrenaline in plasma membrane vesicles isolated from PC-12 pheochromocytoma cells. According to their hypothetical scheme for the transport of noradrenaline potassium increases the turnover rate by formation of a mobile potassium-carrier complex. Thus, in the time course of potassium-free incubation noradrenaline outward transport via uptake, could be limited by decreased mobility of the amine carrier due to the lack of potassium.

Carrier-mediated neuronal noradrenaline transport induced by pharmacological agents

It is well known that indirectly acting sympathomimetic amines, such as tyramine, release noradrenaline through a calcium-independent mechanism that is blocked by inhibitors of uptake1 (Langeloh and Tren- delenburg 1987). Thus, this type of drug-induced noradrenaline release has all the characteristics of being mediated by uptake1. The mechanism of tyramine- induced noradrenaline release can be assigned to a dual-type of action. After being transported into the neurone, tyramine mobilizes vesicular noradrenaline by competing with noradrenaline for vesicular uptake resulting in leakage of noradrenaline out of the storage vesicles and an increase in axoplasmic noradrenaline. At the same time, the inward transport of tyramine into the neurone provides the uptakej carrier at the inner face of the membrane, required for outward transport of axoplasmic noradrenaline. In accordance with this mode of action, the indirectly acting sympathomimetic tyramine caused the release of both, DOPEG and noradrenaline in human cardiac tissue, comparable to the release induced by the combined inhibition of the vesicular amine carrier and the sodium-potassium ATPase.

By opening the fast sodium channels of sympathetic neurons, veratridine induces calcium-dependent, exocytotic noradrenaline release (Kirpekar and Prat 1979). However, veratridine is also known to induce neurotransmitter release even in the absence of extracellular calcium (Ross and Kelder 1976; B6nisch et al. 1983). To explain this phenomenon according to the calcium hypothesis, it was suggested that the aug- mented release of neurotransmitters might be due to an increased calcium efflux from mitochondria elicited by the accumulation of sodium and activation of sodium- calcium exchange in the nerve endings (Schoffelmeer and Mulder 1983). However, Okada et al. (1990) demonstrated that the release of noradrenaline after stimulation with veratridine showed no correlation with the change of intracellular calcium. In accordance with these studies, veratridine induced a pronounced increase in the overflow of noradrenaline from human atrial tissue in the absence of extracellular calcium. The

present study like B6nisch et al. 1983, however, provides evidence, that the noradrenaline-releasing effect of veratridine under calcium-free conditions is mediated by uptake1 and due to an additional reserpine- like effect of the drug. Firstly, as it is expected of a reserpine-like action, veratridine induced a marked increase in the overflow of DOPEG, indicating elevated axoplasmic noradrenaline concentrations due to the loss of noradrenaline from storage vesicles. Second- ly, the veratridine-induced noradrenaline overflow was effectively attenuated by uptake~ blockade, indicating a nonexocytotic carrier-mediated release. The effect of veratridine on noradrenaline and DOPEG overflow in human atrial tissue is in accordance with findings in the rat was deferens preloaded with 3H-noradrenaline (B6nisch and Trendelenburg 1987). In this tissue, veratridine (plus ouabain) induced a desipramine-sensitive outward transport of 3H-noradrenaline accompanied by the effiux of 3H-DOPEG and 3H-dihydroxy man- delic acid.

Nonexocytotic noradrenaline release induced by energy deficiency

Energy depletion of the nerve terminal is a necessary and sufficient cause for noradrenaline release to occur during myocardial ischemia (Sch6mig et al. 1987). Con- firming previous results in rat hearts (Sch6mig et al. 1987, 1988), the present study demonstrates carrier- mediated release of noradrenaline from sympathetic neurons induced by impairment of energy metabolism in human atrial tissue. As previously shown with desipramine (Kurz et al. 1995), noradrenaline release induced by energy deficiency in human atrial tissue was sensitive to uptake1 blockade by cocaine in the present study. Similar to the pharmacologically-induced release of noradrenaline by the combined action of disturbed vesicular storage function and neuronal sodium homeostasis, energy depletion by cyanide intoxication induced a calcium-independent release of noradrenaline. This is not surprising, since energy depletion ulti- mately reduces the activity of two ATPases demonstrated to be critically involved in nonexocytotic noradrenaline release. Firstly, inhibition of the vesicular proton ATPase causes loss of noradrenaline from storage vesicles, leading to a rise of the axoplas- micamine concentration. Secondly, inhibition of the sodium-potassium ATPase induces a rise of neuronal sodium levels, thus enabling carrier-mediated outward transport of axoplasmic noradrenaline. Noradrenaline release induced by energy depletion in human cardiac tissue was preceded by an increase in the overflow of DOPEG, indicating elevated axoplasmic noradrenaline concentrations prior to carrier-mediated noradrenaline release. Consequently, the high susceptibility of storage vesicles to energy deficiency causes a rapid increase in the axoplasmic noradrenaline concentration,

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whereas carrier-mediated noradrenaline release is inhibited during the early phase of energy depletion, pre- sumably as a result of the delayed rise of intraneuronal sodium. Noradrenaline release was markedly inhibited by tetrodotoxin during cyanide intoxication, but not under conditions of anoxia, indicating that in the first instance the increase in axoplasmic sodium concentration is due to sodium influx via tetrodotoxin-sensitive sodium channels (Weiss and Horn 1986). Comparable to findings in the isolated rat heart (Sch6mig et al. 1988), noradrenaline release induced by anoxia in human atrial tissue is effectively suppressed by inhibition of the sodium-proton exchange with ethylisopropylamiloride (Vigne et al. 1983), suggesting that the sodium-proton exchange in the ischemic human myocardium constitutes the predominant pathway for sodium entry into the sympathetic nerve endings.

Acknowledgement We would like to thank Maike Liiking for her excellent technical assistance. Human atrial tissue was generously pro'~ided by the Deutsches Herzzentrum, Munich. This work was supported by a grant of the Deutsche Forschungsgemeinschaft.

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