214
Neuronal Sodium Homoeostasis and
Axoplasmic Amine Concentration Determine
Calcium-Independent Noradrenaline Release
in Normoxic and Ischemic Rat Heart
Albert Schomig, Thomas Kurz, Gert Richardt, and Edgar Schomig
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Calcium-independent noradrenaline release was studied in the isolated perfused rat heart under
conditions of normoxia, cyanide intoxication, and ischemia. The release of endogenous
noradrenaline and dihydroxyphenylglycol were determined by high-performance liquid chroma tography. The release of dihydroxyphenylglycol, the main neuronal noradrenaline metabolite, was used as an indicator of the free axoplasmic amine concentration. When storage function
of neuronal vesicles was disturbed by Ro 4-1284 or trimethyltin, high dihydroxyphenylglycol
release was observed without concomitant overflow of noradrenaline. If, however, these agents
were combined with inhibition of Na + ,K + -ATPase or with veratridine-induced entry of sodium
into the neuron, both dihydroxyphenylglycol and noradrenaline were released. Noradrenaline
release was independent of extracellular calcium and was suppressed by blockade of neuronal
catecholamine uptake (uptake,), indicating nonexocytotic noradrenaline liberation from the
sympathetic nerve ending. This release critically depended on two conditions: 1) increased
cytoplasmic concentrations of noradrenaline within the sympathetic neuron and 2) intraneuronal sodium accumulation. Both conditions together were required to induce noradrenaline efflux
across the plasma membrane using the uptake, carrier in reverse of its normal transport
direction. A disturbed energy status of the sympathetic neuron, induced by cyanide intoxication
or ischemia, likewise caused calcium-independent noradrenaline release by interfering with
both vesicular storage function and neuronal sodium homoeostasis. Again, release was sensitive
to uptake, blockade. Since neuronal sodium accumulation++ was the rate-limiting step, release
was further accelerated when residual N a , K - A T P a s e activity was inhibited. N a - H
exchange was identified as the predominant pathway of sodium entry into the sympathetic nerve
ending hi ischemia, and its inhibition by amiloride and etbylisopropylamiloride markedly
suppressed ischemia-induced noradrenaline release. (Circulation Research 1988;63:214-226)
M
yocardial ischemia is associated with the
accumulation of high catecholamine concentrations within the extracellular space
of the underperfused area.1-4 Deleterious consequences, such as the occurrence of fatal arrhythmias 36 and the acceleration of cell damage, 78 have
been attributed to an adrenergic excess stimulation
of the ischemic myocardium. The ischemia-induced
catecholamine accumulation is predominantly due
to noradrenaline release from local sympathetic
From the Department of Cardiology, University of Heidelberg, Heidelberg, Germany.
Supported by the Deutsche Forschungsgemeinschaft (SFB
320—Herzfunktion und ihre Regulation).
Address for correspondence: Dr. Albert Schomig, Department
of Cardiology, University of Heidelberg, Im Neuenheimer Feld
326, 69 Heidelberg, FRG.
Received June 18, 1987; accepted January 15, 1988.
nerve endings that is not influenced by central
sympathetic activity.3-4-9 Such local metabolically
mediated release of noradrenaline has been shown
to be independent of extracellular calcium and has
been characterized as nonexocytotic release in myocardial ischemia, 3 4 9 -" anoxia, 9 " and cyanide
intoxication." The release has been proposed to be
a two-step process induced by energy deficiency in
the sympathetic nerve ending." First, noradrenaline is lost from the storage vesicles, resulting in
increased axoplasmic concentrations. Second, the
amine is transported across the plasma membrane
into the extracellular space using the neuronal catecholamine uptake (uptake,) carrier in reverse of its
normal transport direction.
The first purpose of this study was to identify
conditions that induce outward transport of noradrenaline across the cell membrane of the sympa-
Schomig et al
Calcium-Independent Noradrenaline Release In Ischemia
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thetic neuron. The inward transport of catecholamines into the neuron via uptake, depends on the
extracellular presence of sodium.1213 Therefore, we
focused our interest on the role of intraneuronal
sodium in causing a reversal of the carrier's net
transport direction. The second aim was to establish the significance of free axoplasmic amine concentration and disturbed neuronal sodium homoeostasis for nonexocytotic noradrenaline release in
ischemia and cardiac energy deficiency caused by
cyanide intoxication.
The experiments were performed in isolated rat
hearts using a modified Langendorff preparation,14
and the overflow of endogenous noradrenaline and
3,4-dihydroxyphenylglycol (DOPEG) were measured. DOPEG was chosen because it is the main
product of intraneuronal oxidative deamination of
noradrenaline by monoamine oxidase, and it has been
demonstrated to reflect axoplasmic noradrenaline concentrations that cannot be detected directly." To
avoid exocytosis, experiments were done in the
absence of calcium in the perfusion buffer.
In the experiments with undisturbed energy metabolism, vesicular storage function was impaired pharmacologically, and the release of endogenous noradrenaline and DOPEG were measured. In these
experiments, we investigated the effects on noradrenaline release of increased sodium influx into
the sympathetic neuron, sodium pump inhibition,
and depolarization of the plasma membrane.
In the two experimental models of energy depletion, neuronal sodium balance was modulated by
interfering with various pathways of transmembrane sodium movement. The energy metabolism of
the sympathetic nerve ending was disturbed by
either inhibition of oxidative phosphorylation with
cyanide in combination with glucose-free perfusion
or by global ischemia of the heart.
Materials and Methods
Male Wistar rats (180-250 g; Ivanovas, Kislegg,
FRG) were anesthetized with thiobutabarbital (50
mg/kg i.p.). After injection of 500 IU heparin, the
thorax was opened, the heart was rapidly removed
and weighed, and the ascending aorta was cannulated for retrograde coronary perfusion.M Hearts
were perfused at a constant flow of 5 ml/min/g heart
wt using a multichannel peristaltic pump. Initial
perfusion was done with a modified KrebsHenseleit solution (KHS; composition in mmol/1:
NaCl 125, NaHCOj 16.9, Na2HPO4 0.2, KC1 4.0,
CaCl2 1.85, MgCl2 1.0, glucose 11, and EDTA
0.027). The buffer was gassed with 95% O2-5% CO2
and the pH adjusted to 7.4. The temperature of the
chamber and of the perfusate at the point of entry
into the aorta were adjusted to 37.5° C. After 20
minutes of initial perfusion, in most series the
perfusion was continued with calcium-free KHS,
and the hearts were subjected to the experiments.
Experimental series with blockade of energy
metabolism were done at constant flow (5 ml/min/g
215
heart wt). The perfusion buffer was deficient of
glucose, and oxidative phosphorylation was inhibited by 1 mM sodium cyanide. Samples for highperformance liquid chromatography (HPLC) determination of noradrenaline and DOPEG were taken
cumulatively for 2-minute periods.
For ischemia experiments, the hearts were subjected to global ischemia by stopping perfusion
flow, and they were subsequently reperfused at the
initial flow rate. If drugs were used, they were
added 10 minutes prior to ischemia and were continued to the end of the experiment. Samples for
noradrenaline estimation were taken throughout the
last minute before ischemia and during the first and
second minute of reperfusion.
Samples for the estimation of endogenous noradrenaline and DOPEG were taken from the coronary venous effluent, put on ice, and stabilized by
the addition of sodium EDTA to a final concentration of 10 mM. The samples were stored at -80° C
until assayed by an HPLC method.
Agents used in this study were amiloride (Sigma,
Munich, FRG), desipramine (CIBA-Geigy, Basle, Switzerland), monensin (Sigma), ouabain (Merck, Darmstadt, FRG), reserpine (Sigma), Ro 4-1284 (2-hydroxy2-ethyl-3-isobutyl-9,10-dimethoxy-l,2,3, 4,6,7hexahydro-11 b-H-benzo(a)-quinolizine; HoffmannLa Roche, Basle, Switzerland), sodium cyanide
(Merck), and trimethyltin (Merck). Ethylisopropylamiloride was synthesized according to Cragoe et al. l5
The agents not soluble in water were dissolved in
ethanol, withfinalconcentrations of ethanol being less
than 0.05%. For control experiments, identical solvent and ion concentrations were used.
Assay of Noradrenaline and DOPEG
Endogenous noradrenaline and DOPEG were measured using an HPLC method as described by
Schomig et al." Briefly, the pH of the sample was
adjusted to 8.5 by the addition of NH4CI-NH4OH
buffer (2 M, pH 8.5) containing 0.5% sodium EDTA
and 0.2% diphenylborate. Catecholamines and
DOPEG were extracted into a 5-ml organic phase
consisting of 99% n-heptane and 1% octanol in the
presence of 0.25% tetraoctylammonium bromide.
Back-extraction of catecholamines into an aqueous
phase was performed by shaking the organic phase
with 0.15 ml 0.2 M phosphoric acid. To increase the
recovery of DOPEG, the same step was repeated
after the addition of 1 ml octanol. One hundred
microliters of the aqueous phase was injected into
the HPLC system (Latek, Heidelberg, FRG). Separation was performed using a 5 /im C18 reversed
phase column (Latek) with a flow of 0.8 ml/min. The
solvent was 0.2 M phosphate buffer (pH 3.0) containing sodium EDTA (40 jtM) and octylsulphate (25
piM). Quantitative analysis was performed by electrochemical detection (LC 4B, Bioanalytic Systems,
West Lafayette, Indiana) at 0.6 V. Retention times
for noradrenaline and DOPEG were 4.5 and 6.2
minutes. Recovery was 98% for noradrenaline and
216
Circulation Research
Vol 63, No 1, July 1988
92% for DOPEG, the limits of detection were 0.1 and
0.2 nmol/1, and the coefficients of variation were
5.9% and 5.8%, respectively. None of the drugs used
in the experiments interfered with extraction, separation, or detection of noradrenaline and DOPEG.
overflow of
noradranalln*
and DOPEQ
600,
Statistical methods
In text and figures, results are expressed as
mean±SEM. The significance of differences was
assessed by Student's t test or analysis of variance.
Results
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Increased Cytoplasmic Noradrenaline
Concentration in Sympathetic Neuron Induced by
Pharmacological Agents
In this study, DOPEG release was used to reflect
free axoplasmic noradrenaline concentrations because previous studies have shown that DOPEG
formation is related to the concentration of noradrenaline within the cytoplasm of the sympathetic
neuron. 1116
Inhibition of the vesicular catecholamine transporter by reserpine17 or Ro 4-128418 resulted in a
concentration-dependent overflow of DOPEG and
no significant noradrenaline release (Figure 1, upper
panels). At 1 fiM, the effect of Ro 4-1284 was
maximal, and this concentration was utilized in
further experiments. Likewise inhibition of vesicular H + -ATPase by trimethyltin19 resulted in a
concentration-dependent overflow of DOPEG, and
only high concentrations, which were supramaximal concerning DOPEG release, resulted in a measurable overflow of noradrenaline (Figure 1, lower
left panel). In the concentration of 30 /xM, which
was used in further experiments, trimethyltin
induced maximal DOPEG overflow without major
noradrenaline release. Addition of the ionophore
monensin, which directly interferes with2021 the vesicular protone gradient,- resulted in the overflow
of high amounts of DOPEG. The release reached
approximately one third of total cardiac noradrenaline content within 10 minutes at a concentration of
1 /uM monensin. DOPEG release was accompanied
by noradrenaline release only when monensin concentrations higher than 1 jtM were applied. This effect
may be related to a sodium influx into the22 neuron
induced by high concentrations of monensin.
Effect of Increased Neuronal Sodium Influx on
Noradrenaline Release in Presence of High
Axoplasmic Noradrenaline Concentrations
The effect of 1 fiM Ro 4-1284 on the time course
of DOPEG and noradrenaline overflow is demonstrated in the left panel of Figure 2A. Despite loss of
noradrenaline from storage vesicles and increased
cytoplasmic noradrenaline concentrations within the
neuron, reflected by high DOPEG overflow, no
significant amounts of noradrenaline were released.
In the absence of extracellular calcium, veratridine
(30 fiM) resulted only in a minor noradrenaline
0 0.001 0.01 0.1
1800
1
O 0.01 0.1
1
10
|trimethyltin|
pmol/fl
/10mln
1200.
800.
400.
8_100
0
0.1
10 fiM
FIGURE 1. Effect of various agents interfering with
vesicular catecholamine storage on the overflow of endogenous noradrenaline and DOPEG from rat hearts perfused with calcium-free Krebs-Henseleit buffer. Upper
panels: Effect of increasing concentrations of two inhibitors of the vesicular catecholamine carrier, reserpine (1
nM to 1 uM; n=7, left panel) and Ro 4-1284 (0.01 \iM to
10 fiM; n=7; right panel). The hearts were exposed to
each concentration for 10-minute periods. Lower panels:
Effect of various concentrations of the inhibitor of H+ATPase trimethyltin (1, 10, 30, and 100 fjM; left panel)
and of the monovalent ionophor monensin (0.1, 1, and 10
liM, right panel). Four to seven hearts were exposed to
one concentration for a 10-minute period. Mean±SEM.
release from the heart (Figure 2A, middle panel).
After enhancement of cytoplasmic noradrenaline
levels by Ro 4-1284, however, stimulation of sodium
influx by veratridine caused major noradrenaline
release at the expense of DOPEG release (Figure
2A, right panel). In the presence of 1 jtM tetrodotoxin, veratridine following Ro 4-1284 did not induce
any noradrenaline release (Figure 2B, left panel).
The data indicate that sodium influx via tetrodotoxinsensitive channels is necessary for veratridineinduced noradrenaline release.
Noradrenaline release induced by the combined
action of Ro 4-1284 and veratridine could be attenuated by 80% when uptakei was specifically inhibited
by 0.1 fiM desipramine23 (Figure 2B, middle panel). In
Schomig et al
overflow of
Calcium-Independent Noradrenaline Release in Ischemia
I Ro 4 - 1 2 8 4
217
Ro 4 - 1 2 8 4 I
noradrenaflne
Iverat.l
IveraL I
and DOPEG
80
pmot
l<3
/rrdn
60.
40.
20
10
20
30
rrdn
noradrenalne
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overflow of
10
0
30
min
10
20
30
o DOPEQ
I Ro 4 - 1 2 8 4 I
I Ro 4 - 1 2 8 4 I
I verat. I
Iverat.l
noradrenallrte
I tetrodotoxtn I
and DOPEQ
20
- dsslpramlne
I Ro 4 - 1 2 8 4 I
Iverat.l
1
I—
EIPA
—|
80
pmol
/0
/miri
60.
FIGURE 2. Effect of stimulated neuronal
sodium influx on overflow of endogenous noradrenaline and DOPEG from rat hearts perfused with calcium-free Krebs-Henseleit
buffer. Upper panels (A): The hearts were
exposed to the reserpine-like agent Ro 4-1284
(1 fiM, n=7, left panel), to 30 /xM veratridine
(verat., n=4, middle panel), and to 1 fj.M Ro
4-1284 and 30 fiM veratridine, which was
added 10 minutes after Ro 4-1284 (n=7, right
panel). Lower panels (B): In addition to
perfusion with 1 YM RO 4-1284 and 30 \iM
veratridine (verat.), sodium channels were
blocked by 1 \LM tetrodotoxin (n=7, left
panel), uptake, was inhibited by 0.1 yJ4 desipramine (n=7, middle panel), and Na-H
exchange was suppressed by 10 yM ethylisopropylamiloride (EIPA, n=7, right panel).
Time courses of drug exposures are indicated
in the figure. Mean±SEM.
40.
20.
0J
3
10
20
• noradrenallne
10
min
0
10
o
20
10
rrdn
0
10
20
10
rrdn
DOPEG
contrast, blockade of plasmalemmal Na + -H + exchange
by 10 tiM ethylisopropylamiloride did not influence
noradrenaline release induced by Ro 4-1284 and veratridine (Figure 2B, right panel), indicating that that
ethylisopropylamiloride has neither tetrodotoxin-like
nor desipramine-like properties.
Release of Noradrenaline and DOPEG Induced
by Inhibition of Vesicular H+- ATPase and
Plasma Membrane Na+,K+-ATPase
Inhibition of vesicular H+-ATPase by 1 jtM
trimethyltin19 resulted in a marked release of
DOPEG, the deaminated metabolite of noradrenaline, that was not accompanied by any release of
noradrenaline itself. However, if the hearts had
been preperfused with 1 mM ouabain for 30 minutes
to suppress the outward transport of sodium by
Na + ,K + -ATPase, identical concentrations of trimethyltin induced the simultaneous release of noradrenaline and DOPEG in a proportion of 1 to 2
(Figure 3A). The combined overflow of the catecholamine and its metabolite did not exceed the
DOPEG overflow found in the absence of ouabain.
Blockade of the uptake, carrier by 0.1 pM desipramine prior to the addition of trimethyltin almost
completely suppressed the noradrenaline release
that was found after ouabain and trimethyltin.
DOPEG overflow was not reduced by uptake, blockade (Figure 3A, lower panel).
Extracellular potassium depletion, used instead
of ouabain to reduce Na + ,K + -ATPase activity,
resulted in a similar eflFect on noradrenaline release
(Figure 3B). Following 30 minutes of potassiumfree perfusion, the noradrenaline release induced by
trimethyltin amounted to one quarter of the concomitant DOPEG overflow.
Effect of Membrane Depolarization by Increased
Potassium Concentrations on the Overflow of
Noradrenaline and DOPEG
In the absence of extracellular calcium, stepwise
rise of potassium concentrations in the perfusion
buffer had no eflFect on the release of noradrenaline
and DOPEG (Figure 4). If the axoplasmic noradren-
218
Circulation Research
ouabaln
overflow of
noradrenaline
100,
• desipramine -
overflow of
noradrenaline
50,
trimethyltin ouabaln
mM
pmol
/g
Vol 63, No 1, July 1988
80
/mln
60.
•
0
D
1
a
1
desipramine
nM
trimethyltln
potassium
mM
pmol
/g
40,
/min
o
0
•
4
30,
40.
20,
20.
10.
OJrffe-rfb—ft— *fe—tb-n^m—S
50
DOPEQ
l=g
0J
50
min
o—•
0
DOPEQ
250,
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
pmol
/g
/mln
potassium
•> — «>-•'*-•
10 20 30 40
50
50
mln
50
50
min
250.
pmol
/g
/min
200
200.
150.
150.
100
100.
50.
50.
0.
*B— ft)— rffc-
0
10 20 30 40
50
50
mln
«D
0
+
10
«•
• — '
20
+
30
+
40
FIGURE 3. Effects of combined inhibition of vesicular H -ATPase and plas male mmal Na ,K -ATPase on noradrenaline
(upper panels) and DOPEG release (lower panels). Left panels (A): Rat hearts perfused with calcium free KrebsHenseleit buffer were exposed to 1) the inhibitor of vesicular H+-A TPase trimethyltin (30 fiM; n=7), 2) 30 \x.M trimethyltin
and 1 mM ouabain (n=7), and 3) 30 \iM trimethyltin, 1 mM ouabain, and 0.1 pM desipramine (n=7). Right panels (B):
Rat hearts were perfused with calcium free Krebs-Henseleit buffer containing either 4 mM potassium (n=7) or no
potassium (n=7). In both series hearts were exposed to 30 fiM trimethyltin. The time course of electrolyte variation and
drug exposure are indicated in the figure. Mean±SEM.
aline level was increased by Ro 4-1284, indicated by
a high DOPEG overflow, potassium induced a
concentration-dependent release of noradrenaline.
In comparison with the noradrenaline release obtained
by Na + ,K + -ATPase inhibition or sodium-channel
opening, the release caused by high extracellular
potassium was small. Blockade of sodium channels
by 1 fiM tetrodotoxin further reduced significantly
the potassium-induced noradrenaline release without
interfering with the concomitant DOPEG overflow
(Figure 4). Comparable effects were observed when
the potassium concentration was increased from 4 to
40 mM in one step (without figure).
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 Figures 5-7. Starting
10 minutes after addition of cyanide and withdrawal
of glucose, noradrenaline was progressively released.
Peak values were reached after 30 minutes. With
progressing noradrenaline depletion of the hearts
the overflow continuously decreased (Figure 5A,
upper panel). Addition of calcium to the perfusate
attenuated and retarded the noradrenaline overflow
(Figure 5B, upper panel). Irrespective of calcium
content of the perfusion buffer, the release of
DOPEG consistently preceded that of noradrenaline by about 5 minutes (Figures 5, 6, and 7A).
Blockade of sodium channels with tetrodotoxin24
resulted in a concentration-dependent inhibition of
noradrenaline release that was more pronounced in
the absence (Figure 5A, upper panel) than in the
presence (Figure 5B, upper panel) of extracellular
calcium. The overflow components insensitive to tetrodotoxin were not influenced by the presence or absence
of calcium. Tetrodotoxin had no effect on the overflow of DOPEG irrespective of the presence of calcium in the perfusion buffer (Figure 5, lower panels).
Inhibition of transmembrane Na + -H + exchange
with 10 fiM ethylisopropylamiloride25 did not influ-
Schdmig et al
Calcium-Independent Noradrenaline Release in Ischemia
release disappeared (Figure 7B, left panel). Under
these experimental conditions desipramine was again
effective in suppressing noradrenaline release (Figure
7B, right panel).
potassium mM
overflow of
noradrenaline
10,
pmol/g/mln
DOPEQ
10
15
20
25
mln
10
15
20
25
min
80_
pmol/g/min
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60.
40.
20.
0J
219
••o
FIGURE 4. Effect of stepwise depolarisation on overflow
of endogenous noradrenaline (upper panel) and DOPEG
(lower panel)/rom rat hearts. The Krebs-Henseleit buffer
was free of calcium, and sodium was substituted stepwise
by potassium to final potassium concentrations of 10, 20,
30, and 40 mM. Catecholamine overflow was determined
in the absence of drugs (n=7), in the presence of I fiM Ro
4-1284 (Ro; n = 7; 5-25 minutes) and in the presence of1
fj.M tetrodotoxin (TTX; 0-25 minutes) and 1 fiM Ro 4-128
(n=7). Mean±SEM.
ence the overflow of noradrenaline and DOPEG
(Figure 6). Blockade of the uptake, carrier with 0.1
/AM desipramine markedly suppressed noradrenaline release but had no effect on the overflow of
DOPEG (Figure 6).
Figure 7A (left panel) demonstrates the delay of
noradrenaline overflow following cyanide intoxication relative to the overflow of DOPEG. The latter
reflects the preceding rise of cytoplasmic noradrenaline concentration.1116 Inhibition of Na + , K+-ATPase
with 1 mM ouabain 30 minutes prior to cyanide
intoxication abolished this delay and both DOPEG
and noradrenaline werereleasedconcomitantly (Figure
7A, right panel). The effect of 30 minutes potassiumfree perfusion prior to cyanide poisoning was comparable and the lag between DOPEG and noradrenaline
Effect of Interference With Neuronal Sodium
Handling on Noradrenaline Overflow in Ischemia
Ischemic periods up to 10 minutes were not
accompanied by noradrenaline overflow during subsequent reperfusion. When Na + ,K + -ATPase was
inhibited by 1 mM ouabain 30 minutes prior to
ischemia a marked overflow of noradrenaline was
observed during reperfusion after 5 and 10 minutes
ischemia (Figure 8, left panel).
Ischemic periods of 20 minutes were associated
with an overflow of 118 ± 15 pmol/g (Figure 8, right
panel). Blockade of uptake, by 0.1 /iM desipramine
10 minutes prior to ischemia reduced noradrenaline
release to 19.7 ±2.8 pmol/g (p<0.01; Figure 9). Blockade of sodium channels with 1 fiM and 10 ^.M
tetrodotoxin (10 minutes prior to ischemia) had no
significant effect on noradrenaline release induced by
20 minutes ischemia. In contrast, inhibition of transmembrane sodium protone exchange with 1 mM
amiloride markedly suppressed noradrenaline release
(Figure 8; 23.9 ±7 pmol/g; p<0.0\). The more specific and potent derivate ethylisopropylamiloride25-26
reduced ischemia-induced noradrenaline release in
micromolar concentrations (Figure 8; 1 /iM: 23.3 ±5.5
pmol/g, p<0.01; IOJAM: 16.9±4.2 pmol/g, p<0.0\).
Following 10- or 20-minute ischemic periods, only
minor amounts of DOPEG were found in the reperfusate. The oxygen-dependent deamination of
noradrenaline may be assumed to occur predominantly during the reperfusion period, which is accompanied by the reoxygenation of the hearts.
Discussion
Calcium-independent, nonexocytotic noradrenaline release has been demonstrated to critically
depend on two conditions: 1) increased cytoplasmic
concentrations of noradrenaline within the sympathetic neuron and 2) enhanced intraneuronal concentrations of sodium ions (scheme in Figure 9).
Increased Cytoplasmic Noradrenaline
Concentration in the Sympathetic Nerve Ending
By accumulating protons within the neuronal
catecholamine storage vesicles the H + -ATPase
located in the vesicular membrane generates a transmembrane H+ electrochemical potential of close to
200 mV that is composed of the vesicular membrane
potential (inside positive) and a proton gradient
(inside pH 5.5)."-» The proton potential is the
driving force of vesicular noradrenaJine uptake, and
amine inward transport is coupled with proton
outward transport by a specific carrier located within
the vesicular membrane.27-29
In this study, the capability of neuronal storage
vesicles to retain catecholamines was experimentally disturbed by agents interfering either with the
220
Circulation Research
overflow of
Vol 63, No 1, July 1988
overflow of
cyanide 1mM, no substrate -
noradrenaline
noradrenaline
,100_
pmol
80.
pmol100'
/g
/min
80.
60.
60.
40.
40.
20.
20.
/win
-cyanide 1mM, no substrate -
TTX
Ca+
0.
DOPEG
10
20
30
40
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50
min
DOPEG
20.
10
20
30
40
50
min
10
20
30
40
50
min
20.
pmol
/g
/min
pmol
/g
/min
10.
10.
0J
0J
10
20
30
40
50
min
FIGURE 5. Role of sodium influx via tetrodotoxin (TTX) sensitive channels for noradrenaline release induced by energy
deficiency. Blockade of energy metabolism was induced by glucose-free perfusion and the addition of 1 mM cyanide
(10-50 minutes). Left panels (A): Overflow of endogenous noradrenaline (upper panel) and DOPEG (lower panel) from
rat hearts perfused with calcium free Krebs-Henseleit buffer. The time course of overflow was studied in the absence of
tetrodotoxin (TTX; n=14), in the presence of 10 nM TTX (n = 7), and 1 pM TTX (n=7). Right panels (B): Overflow of
endogenous noradrenaline (upper panel) and DOPEG (lower panel) from rat hearts perfused with buffer containing 1.8
mM calcium. The time course of overflow was studied in the absence of TTX (n=7) and in the presence of I fiM TTX
(n = 7). Mean±SEM.
vesicular amine transport or with vesicular proton
potential. Inhibition of the H+-ATPase by trimethyltin results in a progressive collapse of the H + potential
at first affecting the electrical component.19 Consequently, the capability of maintaining the high intravesicular amine concentrations is severely hampered,
and catecholamines are lost from the storage vesicles
into the axoplasm. The trimethyltin concentrations
necessary to induce vesicular noradrenaline loss in
the rat heart are without effect on Na+,K+-ATPase
and are in the same range as those used to inhibit
H+-ATPase in isolated vesicular membranes.19-27 A
similar release could be achieved by the monovalent
ionophore monensin, which causes a proton and
sodium ion leak22 and thus directly interferes with
vesicular H + potential.20 At high concentrations, this
agent induces swelling and damage of vesicles.21
Inhibition of the vesicular amine carrier by
reserpine'617 or Ro 4-12841618 likewise results in a
net loss of noradrenaline from the vesicles and a less
pronounced axoplasmic accumulation of the amine.
Axoplasmic noradrenaline concentrations cannot
be assessed directly. The release of DOPEG had to
serve as an indirect measure of cytoplasmic noradrenaline. Outside the storage vesicles, neuronal
catecholamines are substrates of monoamine oxidase located at the outer mitochondrial membrane.
In the presence of oxygen, noradrenaline is deaminated and DOPEG is the predominant metabolite
formed in the sympathetic neuron.3031 In contrast
to the hydrophilic amine, the more lipophilic DOPEG
easily diffuses through the plasma membrane with
an efflux/content ratio of 0.35/min.32 Hence, DOPEG
release reflects axoplasmic noradrenaline concentrations as long as deamination is not saturated or
inhibited. The Km oxygen value of the oxidative
deamination of noradrenaline is 50 mm Hg oxygen
pressure, and therefore, oxygen limits the reaction
only under hypoxic conditions11 such as ischemia.
Schomig et al
overflow of
Calcium-Independent Noradrenaline Release in Ischemia
cyanide, no substrate
noradrenaline
100,
control
desipramine
O.I JJM
80,
pmol
GPA 1QuM
/min
60.
40.
20.
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
0J
10
20
10
20
4 0 min
DOPEQ
20,
pmol
/min
10.
0J
0
30
40 min
6. Effect of blockade of uptake, and Na+-H+
exchange on overflow of endogenous noradrenaline (upper
panel) and DOPEG (lower panel) from rat hearts perfused with calcium-free Krebs-Henseleit buffer. Energy
metabolism was inhibited by glucose-free perfusion and
the addition of 1 mM cyanide (10-50 minutes). The time
course of overflow was studied in the absence of additional agents (n = 7), in the presence of 10 pM ethylisopropylamiloride (EIPA; n=7), and in the presence of O.I
desipramine (n = 7). Mean±SEM.
FIGURE
Increased axoplasmic noradrenaline concentrations by themselves were not sufficient to cause a
release from the nerve ending into the extracellular
space where the transmitter can interact with its
receptors. In contrast to its lipophilic metabolite
DOPEG,32 unfacilitated transmembrane diffusion is
negligible in the case of the hydrophilic amine,
which is ionized at physiological pH. Thus, catecholamine transport across intact membranes is not
conceivable without a specific carrier system.
Carrier-Mediated Neuronal Noradrenaline
Transport and Role of Intraneuronal Sodium
The inward transport of noradrenaline from the
extracellular space into the sympathetic neuron via
uptake, has been extensively studied,l213-33-34 and
has been demonstrated to be a cotransport with
sodium ions energetically depending on the physio-
221
logical electrochemical sodium gradient. Furthermore, the transport depends on the extracellular
presence of chloride,33-34 is independent from extracellular calcium, and is specifically inhibited by various antidepressants such as desipramine and
cocaine.12-23
A reversed transport direction of uptake, had first
been proposed by Paton to potentially act as a
release mechanism under conditions of extracellular
sodium depletion or inhibition of Na + ,K + -ATPase in
the rabbit atrium.35 These observations were confirmed by other investigators, who measured the
release of tritium36 or 3H-noradrenaline13-37 from rat
vas deferens labeled with 3H-noradrenaline. Either
extracellular sodium depletion13-37 or veratridine36
induced a calcium-independent release, which was
suppressed by inhibitors of uptake, such as cocaine
or desipramine. In all of these studies, the tissues
were pretreated with reserpine to inhibit vesicular
function and pargyline to block monoamine oxidase
activity. In cultured sympathetic nerve cells38 and
PC-12 cells34 prelabeled with 3H-noradrenaline, a
comparable efflux of tritium was observed when the
transmembrane sodium gradient was altered. Using
rat vas deferens with active monoamine oxidase,
Stute and Trendelenburg measured the release of
3
H-noradrenaline and 3H-DOPEG.16 In the presence
of reserpine, inhibition of Na + ,K + -ATPase or stimulation of sodium influx led to release of 3Hnoradrenaline at the expense of 3 H-D0PEG. From
these results it was concluded that a reduced transmembrane sodium gradient is a precondition for
reversed amine transport by uptake,.
In the present study, the release of endogenous
noradrenaline and endogenous DOPEG, its deaminated metabolite, were measured. Thus, we were
able to avoid problems potentially associated with
the use of radiolabeled catecholamines, such as
isotope effects and inhomogenous distribution of the
labeled amine and its metabolites in various compartments. In studies using labeled amines, therefore,
the quantification of the liberated amine is hampered
because of undefined and variable ratios between the
labeled and the endogenous compound during release.
Three interventions have been utilized to increase
cytoplasmic sodium concentrations: stimulation of
sodium influx by veratridine and inhibition of
Na+,K+-ATPase by ouabain or potassium-free perfusion. With methods currently available, sodium
concentrations within the sympathetic neuron cannot be quantified. Our considerations, therefore,
have to rely on microelectrode studies of papillary
muscle39 and Purkinje fibers40-42 and on nuclear
magnetic resonance studies of perfused rat hearts.43
In Purkinje fibers, inhibition of Na + ,K + -ATPase
using high concentrations of digitalis glycosides or
potassium-free perfusion resulted in a twofold to
threefold rise in intracellular sodium activity from
basal levels in the range of 5 to 8 mM.40-41 Similar
effects were achieved using micromolar concentrations of veratridine.42
222
Circulation Research
Vol 63, No 1, July 1988
overflow of
norsdrsnaline
overflow of
DOPEQ
ouabaln
.40
1OO_
pmol
/g/ntin
pmol
/g/rrtn
noradrenaline
80.
DOPEQ
.30
60.
FIGURE 7.
.20
40.
.10
20.
Lo
OJ
10
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
overflow of
noradrenaline
mln 20
0
10
trtn
20
overflow of
DOPEQ
potassium free
deslpr amine
potassium free
.40
pmol
/g/rr*i
100_
pmol
/g/mln
• noradrenaline
80.
o DOPEQ
.30
60.
.20
Significance
ofNa+,K+-ATPase
activity for noradrenaline release induced by
cyanide intoxication. Overflow of endogenous noradrenaline and DOPEG was determined in rat hearts perfused with calciumfree Krebs-Henseleit buffer. Blockade of
energy metabolism was induced by glucosefree perfusion and the addition of I mM
cyanide (0-20 minutes). Upper panels (A):
Time course of overflow was studied in the
absence of additional agents (left panel; n=7)
and in the presence of I mM ouabain (right
panel; n=7). Ouabain was added to the perfusate 30 minutes prior to blockade of energy
metabolism. Lower panels (B): Calcium and
potassium were substituted by sodium 30
minutes prior to blockade of energy metabolism. Time course of overflow was studied in
the absence of additional agents (left panel;
n=7) and in the presence of 0.1 /JM desipramine (DMI; right panel; n = 7). Mean±SEM.
40.
.10
20.
0J
o—of
L0
10
mln 20
0
10
rrrin
20
In our experiments, neither ouabain nor potassium depletion caused a significant noradrenaline
release when the axoplasmic concentrations of the
amine were not increased. Veratridine, however,
induced a measurable noradrenaline overflow sensitive to tetrodotoxin. The mechanism of this sodiummediated release remains unclear, and since intracellular liberation of calcium cannot be excluded,
residual exocytosis may be the cause.
When the rise in neuronal sodium concentration
was combined with high axoplasmic noradrenaline
levels, a brisk noradrenaline overflow was observed,
whereas DOPEG release was reduced. Two processes are thought to compete for noradrenaline in
the axoplasmic pool: deamination of noradrenaline
by monoamine oxidase resulting in DOPEG formation and DOPEG release on the one side, and the
outward transport of unchanged noradrenaline on
the other side. The ratio NA/DOPEG overflow
reflects the ratio between the rate constants of both
processes. Assuming that the rate constants for
DOPEG formation and diffusion remain unaltered,
the relative increase of the rate constant of noradrenaline outward transport44 is quantified by
NA/DOPEG ^ ^ ^ I NA/DOPEG(low uM
where NA is noradrenaline. The rate constants of
noradrenaline efflux were increased by 30 /nM veratridine by a factor of 72, by 1 mM ouabain by a
factor of 80, and by extracellular potassium deficiency
by a factor of 26. Thus, a presumably threefold
increase of intracellular sodium activity gives rise to a
drastic efflux of noradrenaline from sympathetic nerve
cells mediated by the uptake, carrier.
In comparison with the transmembrane sodium
gradient, the membrane potential has been demonstrated to be of minor significance for noradrenaline
efflux. High potassium concentrations resulted only
in a moderate noradrenaline release and tetrodotoxin blockade of the sodium channel further reduced
this release.
The presented data are compatible with the uptake,
model by Graefe who proposed a complex interaction
of sodium with the amine transport.13 In agreement
Schomig et al
Calcium-Independent Noradrenaline Release in Ischemia
223
overflow of
noradrenaline
30 _
5 min
10 min
*
*
pmol/g
20 min Ischemia
*
«
*
T
20
100.
50,
0 J
I
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
I
oj
I
I
no drug
l__j
tetrodotoxin 1j
ouabatn 1mM
E3
EPA
desipramine 0.1pM
iHi
amiloride 1mM
10PM
with this hypothesis, sodium ions not only increase
the affinity of noradrenaline to the carrier but also
enhance the availability of the carrier at that side of
the plasma membrane where the sodium concentration is high by forming an immobile sodium-carrier
complex (transinhibition). In the presence of a physiological sodium gradient, number and affinity of
transport sites are low at the cytoplasmic side and
high at the outside of the plasma membrane, thus
facilitating inward and preventing outward amine transport. With increasing axoplasmic sodium concentrations, the barrier of transinhibition is lost, and driven
by high axoplasmic noradrenaline concentrations, an
outward amine flux is found, even against the residual
inward-directed electrochemical sodium gradient.
Neuronal Sodium Balance and Nonexocytotic
Noradrenaline Release Induced by Energy
Deficiency
Inhibition of two ion pumps in the sympathetic
nerve cell has been shown for the first time to be a
sufficient cause for nonexocytotic noradrenaline
release. Blockade of H+-ATPase led to noradrenaline
loss from storage vesicles and cytoplasmic amine
accumulation. Inhibition of Na + ,K + -ATPase, by
increasing neuronal sodium levels, induced carriermediated efflux of the amine. It is not surprising,
therefore, that energy deficiency, which ultimately
reduces ATPase activities, caused amine release from
sympathetic neurons with the same characteristics as
those found after pharmacological inhibition of both
ATPases: independence from extracellular calcium
and inhibition by uptake, blockade, both being incompatible with the assumption of exocytosis.
The rate-limiting step of metabolically induced
noradrenaline release is the carrier-mediated amine
transport across the plasma membrane. Following
cyanide intoxication and glucose depletion, the rise
in axoplasmic noradrenaline, reflected by D0PEG
overflow, preceded the noradrenaline release from
*
FIGURE 8. Cumulative overflow of endogenous
noradrenaline from perfused rat hearts during
2-minute reperfusion following 5-, 10-, and 20minute periods of total ischemia in the absence
of calcium in the perfusion buffer. Left panel:
Noradrenaline overflow induced by 5 and 10
minute ischemia in the absence and presence of
1 mM ouabain 30 minutes prior to ischemia.
Right panel: Noradrenaline overflow induced by
20 minutes of ischemia in the absence of drugs
and in the presence of 0.1 fiM desipramine, 1
fiM tetrodotoxin, 10 y.M ethylisopropylamiloride (EIPA), and 1 mM amiloride. Addition of the
agents began 10 minutes prior to ischemia and
lasted to the end of the experiments. Each
column n=7; mean±SEM.
the nerve ending by 5 minutes. This delay of noradrenaline release disappeared when the Na + -K +
pump had been inhibited prior to cyanide intoxication. Similarly, blockade of Na + ,K + -ATPase prior
to ischemia resulted in a marked acceleration of
noradrenaline release. These data demonstrate a
high sensitivity of storage vesicles to energy deficiency that resulted in fast axoplasmic noradrenaline accumulation. However, ongoing activity of the
Na + -K + pump prevented a major rise of intraneuronal sodium concentration and thereby prevented
noradrenaline release for the first 10 minutes of
energy deficiency.
Virtually no direct information is available concerning intraneuronal sodium accumulation during
myocardial ischemia, anoxia, or cyanide intoxication, and we are aware of the reluctance to apply
knowledge about ischemic processes in the myocyte
to the sympathetic nerve cell. In principle, however,
the alterations of transmembrane ion gradients that
are caused by energy deficiency are likely to be
comparable in both cell types. In rat myocardium,
intracellular sodium has been shown to increase
fivefold within 10 to 15 minutes of anoxia43 and more
than threefold within 12 minutes of total ischemia.46
Intracellular sodium accumulation is determined not
only by decreasing activity of the Na + ,K + -ATPase
but also by changes of passive sodium influx, which
may involve various pathways of entry.
The pathways of neuronal sodium entry have
been demonstrated to differ in ischemia and cyanide
intoxication. Blockade of sodium channels by tetrodotoxin effectively reduced noradrenaline overflow
during cyanide intoxication in the absence of calcium. When calcium was present in the perfusion
buffer, noradrenaline release was reduced in comparison with calcium-free perfusion, and its further
inhibition by tetrodotoxin was less pronounced. In
ischemia, blockade of sodium channels did not reduce
noradrenaline release at all. These observations may
224
Circulation Research
Vol 63, No 1, July 1988
H* ATPase
sympathetic
neuron
Na* Ca**
exchange
Ca**
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
extracellular
space
FIGURE 9. Hypothetical scheme of nonexocytotic, calcium-independent noradrenaline (NA) release (left part) and its
relation to neuronal sodium homoeostasis (right part). Various interventions used in the experiments and their specific
sites of action are indicated. Four different agents have been used to interfere with vesicular catecholamine storage. The
inhibitor of vesicular W-ATPase trimethyltin (1) and the ionophore monensin (2) disturb intravesicular pH and
transmembrane potential, which are essential for vesicular catecholamine transport and storage. Reserpine (3) and Ro
4-1284 (4) have been used to directly block vesicular noradrenaline transport. Application of any of the four agents
resulted in a loss of noradrenaline from storage vesicles that could be detected by markedly increased DOPEG release.
Increased axoplasmic noradrenaline concentrations did not cause relevant noradrenaline release from the nerve ending
as long as neuronal sodium homoeostasis was undisturbed. Only the combination of increased noradrenaline and sodium
within the axoplasm resulted in a major noradrenaline release via uptake,, which reversed its normal transport direction
under those conditions. Blockade of uptake, by desipramine (5) inhibited nonexocytotic noradrenaline release, but not
DOPEG overflow. Several interventions have been used to interfere with neuronal sodium homoeostasis (right part) and
to modify nonexocytotic noradrenaline release thereby. Inhibition of Na+,K+-ATPase by ouabain (6) or potassium-free
perfusion (7) on the one hand and increased sodium influx induced by veratridine (8) on the other hand caused
noradrenaline release if high cytoplasmic noradrenaline concentrations were present. A disturbed energy status, induced
by ischemia or cyanide intoxication, of the sympathetic nerve ending interfered with both vesicular storage function and
sodium homoeostasis and therefore resulted in nonexocytotic noradrenaline release. Blockade of sodium channels by
tetrodotoxin (9) and inhibition ofNa-H
exchange by amiloride (10) and ethylisopropylamiforide (11) have been used to
identify Na*-H* exchange to be major route of neuronal sodium influx in early ischemia.
be explained by the direct effects of calcium ions and
protons on sodium channels. Protons, as well as
calcium ions, have been shown to inhibit the sodium
flux through tetrodotoxin-sensitive channels.47-49
Thus, in ischemia, the marked intracellular and extracellular acidosis may be assumed to reduce the
permeability of sodium channels47-48 and to prevent
major sodium influx via this pathway. The absence
of extracellular calcium, on the other hand, facilitates this route of sodium entry,47-49 as shown in
cyanide intoxication.
Acidosis activates another pathway for sodium
entry into the nerve ending: the Na + -H + exchange.
This carrier-mediated transport system has been
identified in plasma membranes of various cell
types, including nerve cells.50 The exchange plays a
critical role in the regulation of intracellular pH and
is maximally activated by intracellular acidosis.26-50
The extrusion of protons is coupled with a sodium
entry, which leads to intracellular sodium accumulation, especially when Na + ,K+-ATPase activity is suppressed. The Na + -H + exchange is inhibited by amiloride and analogues.26-50 One of the most potent
derivatives is ethylisopropylamiloride,25 which is selective for Na + -H + exchange in comparison with other
sodium coupled transport systems such as Na+-Ca2+
exchange.26 Furthermore, it has been demonstrated
not to interfere with tetrodotoxin-sensitive sodium
channels or with uptake, (Figure 2) in concentrations
that were applied in this study.
In ischemia, but not during cyanide intoxication,
noradrenaline release was markedly inhibited by
amiloride and ethylisopropylamiloride, indicating
that in ischemia Na + -H + exchange is the predomi-
Schomig et al
Calcium-Independent Noradrenallne Release in Ischemia
nant pathway for sodium entry into the sympathetic
nerve ending. This activated, neuronal sodium influx
(in combination with reduced activity of plasmalemmal Na + -K + pump) and disturbed vesicular storage
function are necessary and sufficient conditions to
cause calcium-independent noradrenaline release in
myocardial ischemia.
Acknowledgment
We would like to thank Anette Bruckner, Michaela
Oestringer, and Peter Stefan for excellent technical
assistance.
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KEY WORDS • calcium-independent noradrenaline release •
uptake, • vesicular H+-ATPase • Na + ,K + -ATPase •
Na + -H + exchange • amiloride • ischemia
Neuronal sodium homoeostatis and axoplasmic amine concentration determine
calcium-independent noradrenaline release in normoxic and ischemic rat heart.
A Schömig, T Kurz, G Richardt and E Schömig
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Circ Res. 1988;63:214-226
doi: 10.1161/01.RES.63.1.214
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
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